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Gerardo R. Argüello Astorga 1 Curriculum Vitae

Curriculum vitae Nombre: Gerardo Rafael Argüello Astorga Domicilio particular: Nardos No. 360, Col. Residencial La Florida San Luis Potosí, S.L.P., C.P. 78438, México Teléfono: (444) 835-00-59 Correo electrónico: grarguel @ ipicyt.edu.com Registro Federal de Causantes: AUAG-580701-SB9 Clave única de registro de población: AUAG580701HDGRSR00 Sistema Nacional de Investigadores- SNI 12605- Nivel 1 Datos laborales Cargo actual: Profesor Investigador Titular “B” División de Biología Molecular Instituto Potosino de Investigación Científica y

Tecnológica, A.C. Domicilio laboral: Camino a la Presa San José 2055 Lomas 4a Sección C.P. 78216, San Luis Potosí, S.L.P. Tel.: (444) 8-34-20-00 ext. 2079 Fax: (444) 8-34-20-10

Formación Académica Licenciatura: Título: Biólogo

Institución: Facultad de Ciencias Biológicas, Universidad Autónoma de Nuevo León.

Fecha de graduación: Junio 1983 Tutor: Dr. Carlos Argüello López (CINVESTAV, IPN) Tesis: “Evolución del metabolismo energético: fotosíntesis y respiración” Maestría:

Especialidad: Biología Molecular Institución: Depto. de Genética y Biología Molecular Centro de Investigación y de Estudios Avanzados, I.P.N., Zacatenco.


Tutores: Dra. June K. Simpson Williamson y Dr. Patricio Gariglio Vidal. Fecha de Obtención del Grado: 27 de Febrero de 1992

Tesis: “Identificación de factores nucleares que interactúan con elementos de secuencia dentro del promotor SS3.6 de chícharo (Pisum sativum)”.

Doctorado : Especialidad: Biotecnología de Plantas Institución: Depto. de Ingeniería Genética de Plantas Centro de Investigación y de Estudios Avanzados, I.P.N. Unidad Irapuato Tutor: Dr. Luis Herrera Estrella Obtención del Grado: 16 de Junio de 1996 Tesis: “Delimitación teórica y experimental de módulos de regulación

transcripcional y replicativa en plantas: identificación de unidades mínimas de fotorrespuesta”.

Estancias Postdoctorales

Laboratorio de la Dra. Linda Hanley-Bowdoin Department of Molecular and Structural Biochemistry North Carolina State University, Raleigh, N.C. (Octubre 1999-Octubre 2001) Laboratorio del Dr. Rafael F. Rivera Bustamante Depto. de Ingeniería Genética CINVESTAV, IPN, Unidad Irapuato (Mayo 1998 -Septiembre 1999) Laboratorio del Dr. Luis R. Herrera Estrella Depto. de Ingeniería Genética CINVESTAV, IPN. Unidad Irapuato. (Julio 1996-Diciembre 1997)

Docencia Cursos Impartidos: “Tecnología del DNA Recombinante”, Programa de Maestría en BM (Desde 2002 a la fecha). Coordinador


“Tópicos Selectos en Biología Molecular I”, Postgrado en BM, IPICYT. (2004-2008) Participante. “Tópicos Selectos en Biología Molecular I”, Postgrado en BM, IPICYT. (2003) Participante y Coordinador. “Biología Celular y Molecular”, Programa de Maestría en BM (2002-2004) Participante. “Métodos de Investigación en Biología Molecular”, Postgrado en BM, IPICYT (2003-2004-2008). Participante. “Curso Propedéutico” del Postgrado en Biología Molecular, IPICYT (2006-2008) Participante “Temas Selectos en Ciencias Biológicas”, Postgrado en Ciencias Aplicadas, IPICYT (2002-2008) Participante. “Tópicos selectos en Virología Vegetal”, Postgrado en BM, IPICYT (2003-2004) Coordinador. “Curso Propedéutico” del Postgrado en Ciencias Aplicadas, IPICYT (2002) Participante Curso de “Genética General” Centro de Estudios Superiores del Estado de Sonora-Escuela Superior de Acuacultura. Profesor único- 1 semestre (1989). “Fisiología Animal” Centro de Estudios Superiores del Estado de Sonora-Escuela Superior de Acuacultura. Profesor único. 1 semestre (1989). “Botánica Acuática” Centro de Estudios Superiores del Estado de Sonora-Escuela Superior de Acuacultura. Profesor único. 1 semestre (1986) “Zoología Acuática” Centro de Estudios Superiores del Estado de Sonora- Unidad Novojoa, Profesor único. 1 semestre (1985). “Biología General” Centro de Estudios Superiores del Estado de Sonora-Escuela Superior de Acuacultura, Profesor único. 2 semestres (1984-85).


Formación de Recursos Humanos

Tesistas de Maestría (Tesis concluídas)

Q.F.B. Rosa Gpe. Gómez Castañón. “Generación de vectores de expresión para el análisis experimental de secuencias reguladoras de los genes tardíos del virus Huasteco del chile”. Director único. Agosto del 2004. Q.B.P. Clara Teresa Monreal Vargas “Desarrollo de métodos de diagnóstico molecular de enfermedades virales, bacterianas y fúngicas en hortalizas”. Co-dirección con el Dr. Angel Alpuche Solís. Julio del 2005. Q.F.B. Astrid García Moreno Rublí “Desarrollo de métodos basados en la PCR para la generación rápida de casetes de expresión con promotores geminivirales”. Co-dirección con el Dr. Sergio Casas Flores. Diciembre 2005. Q.F.B. Armando Mauricio Castillo. “Métodos moleculares que potencian el descubrimiento de nuevas especies de begomovirus y la detección de infecciones mixtas.” Director único. Mayo 2006. Q.F.B. Alejandro Juárez Reyes. “Delimitación de Secuencias Involucradas en el Silenciamiento y Transactivación de los Genes Tardíos del Virus Huasteco del Chile “ Director único. Enero 2007.

Tesistas de Doctorado (todos en formación)

Doctorado Directo LC. Josefat Gregorio Jorge “Mecanismos de evolución viral: Análisis del proceso de reversión rápida de mutaciones en geminivirus” Fecha tentativa de Graduación: Agosto del 2009


L.C. Bernardo Bañuelos Hernández Identificación de los determinantes de especificidad de la proteína iniciadora de la replicación de geminivirus por mutagénesis dirigida” Fecha tentativa de Graduación: Agosto del 2009 Q.F.B. Mariana Cantú Iris “Estudio del promotor AC2 y secuencias que responden al transactivador TrAP en begomovirus”. Fecha tentativa de Graduación: Diciembre del 2010 L.C. Yair Cárdenas Conejo “Análisis de la evolución genómica de geminivirus utilizando enfoques heurísticos y bioinformáticos” Co-dirección con la Dra. Lina Riego Fecha tentativa de Graduación: Diciembre del 2010 Doctorado Tradicional M.C. Aurora Londoño Avendaño “Análisis Filogenético-Funcional de Regiones de control Transcripcional y Replicativo en Virus de ssDNA.” Co-dirección con la Dra. Lina Riego Fecha tentativa de Graduación: Julio del 2009 M.C. Jorge Armando Mauricio Castillo “”Desarrollo de nuevos métodos moleculares para la caracterización de begomovirus y curtovirus en infecciones mixtas”. Fecha tentativa de Graduación: Octubre del 2009 Sandra Iliana Torres Herrera “Análisis de la diversidad y evolución de begomovirus del linaje del SLCV (Squash leaf curl virus) en México” Fecha tentativa de Graduación: Diciembre del 2009


Producción científica

Publicaciones en revistas internacionales indexadas

(16 artículos “in extenso” y 9 artículos cortos o “Disease Notes”). Citas totales (excluyendo autocitas): 422

Artículos “in extenso” 1.- López-Ochoa, L., Acevedo-Hernández, G., Martinez-Hernández, A., Arguello-Astorga, G., Herrera-Estrella, L. (2007) “Structural relationships between diverse cis-acting elements are critical for the functional properties of a rbcS minimal Light-regulatory unit”. Journal of Experimental Botany . 58: 4397- 4406. 2007. 2. Arguello-Astorga G, Ascencio-Ibanez JT, Dallas MB, Orozco BM, Hanley-Bowdoin L. (2007) "High Frequency Reversion of Geminivirus Replication Protein Mutants During Infection." Journal of Virology, 81 (20): 11005-11015. 3. Flores-Benítez, S., Jiménez-Bremont, J.F., Rosales-Mendoza, S., Argüello-Astorga, G.R., Castillo-Collazo, R. y Alpuche-Solís, A.G. (2007) "Genetic transformation of Agave salmiana by Agrobacterium tumefaciens and particle bombardment. ". Plant cell tissue and organ culture. 91 (3): 215-224. 4. Rosales-Mendoza S., Soria-Guerra R E, Olivera-Flores M Tde J, López-Revilla R, Arguello-Astorga G R, Jiménez-Bremont J F, García-de La Cruz, R F, Loyola-Rodríguez, Alpuche-Solis, AG. (2007) "Expression of Escherichia coli heat-labile enterotoxin β subunit (ltb) in carrot (Daucus carota.)". Plant Cell Reports. 26(7) :969-976. 5.- Ibarra-Junquera V, Torres LA, Rosu HC, Argüello G, Collado-Vides J (2005). Non-linear software sensor for monitoring genetic regulation processes with noise and modeling errors. Physical Review E 72: 011919. 6.- Argüello-Astorga GR, López-Ochoa L, Kong L-H, Orozco B, Seetlage S., Hanley-Bowdoin L. (2004) A novel motif in geminivirus replication proteins interacts with the plant retinoblastoma homolog RBR. Journal of Virology. 78: 4817-4826. 7.- Ramos PL, Guevara-González R, Peral R, Ascencio-Ibañez JT, Polston JE, Argüello-Astorga GR, Rivera-Bustamante R (2003) Tomato mottle Taino virus pseudorecombines with PYMV but not with ToMoV: Implications for the delimitation of cis-and trans-acting replication specificity determinants. Archives of Virology . 148: 1697-1712


8.- Martínez-Hernández A, López-Ochoa L, Argüello-Astorga GR, Herrera-Estrella L (2002). Functional properties and regulatory complexity of a minimal rbcS light-responsive unit activated by phytochrome, cryptochrome and plastid signals. Plant Physiology 128: 1223-1233. 9.- Argüello-Astorga GR, Ruiz-Medrano R (2001). An iteron-related domain is associated to Motif 1 in the replication proteins of geminiviruses: Identification of potential DNA-protein contacts by a comparative approach. Archives of Virology.146: 1465-1485. 10.- Ruiz-Medrano R, Guevara-González R, Argüello-Astorga GR, Monsalve-Fonnegra Z, Herrera-Estrella LR, Rivera-Bustamante RF. (1999) Identification of a sequence element involved in AC2-mediated transactivation of the pepper huasteco virus coat protein gene. Virology 253: 162-169. 11.- Argüello-Astorga G, Herrera-Estrella L. (1998) Evolution of light-regulated plant promoters. Annual Review of Plant Physiology and Plant Molecular Biology 49: 525-555. 12.- Argüello-Astorga GR, Herrera-Estrella L (1996) Ancestral multipartite units in light-responsive plant promoters have structural features correlating with specific phototransduction pathways. Plant Physiology, 112: 1155-1166. 13.- Argüello-Astorga GR, Guevara-Gonzalez RG, Herrera-Estrella LR, Rivera-Bustamante RF. (1994) Geminivirus replication origins have a group-specific organization of iterative elements: a model for replication. Virology, 203: 90-100. 14.- Argüello-Astorga GR., Herrera-Estrella, LR, Rivera-Bustamante RF. (1994) Theoretical and experimental definition of geminivirus origin of replication. Plant Molecular Biology, 26: 553-556. 15.- Guevara-García, A, Mosqueda-Cano G, Argüello-Astorga GR, Simpson J, Herrera-Estrella L. (1993) Tissue-specific and wound inducible pattern of expression of the mannopine-synthase promoter is determinated by the interaction between positive and negative cis-regulatory elements. The Plant Journal, 4: 495-505. 16.- Argüello G, García-Hernández E, Sánchez M, Gariglio P, Herrera-Estrella L and Simpson J. (1992) Characterization of DNA sequences that mediate nuclear protein binding to the regulatory region of the Pisum sativum chlorophyl a/b binding protein gene AB80: Identification of a repeated heptamer motif. The Plant Journal, 2: 301-307.


Artículos cortos (“Disease Notes”)

(Todas en la revista Plant Disease-(Factor Impacto: 1.79) publicada por The American Phytopathological Society) 1. Mauricio-Castillo, J.A., Argüello-Astorga, G.R., Ambriz-Granados, S., Alpuche-Solis, A.G., Monreal-Vargas, C.T. (2007) "First Report of Tomato golden mottle virus on Lycopersicon esculentum and Solanum rostratum in Mexico". Plant Disease. 91: 1513. 2.- Mauricio-Castillo, J.A., Arguello-Astorga, G.R., Alpuche-Solis, A., Monreal-Vargas, C, de la Torre-Almaraz, R. (2006). "First Report of Tomato severe leaf curl virus in México". Plant Disease. 90: 1116. 3.- Méndez-Lozano,J., Perea-Araujo, L., Leyva-Lopez, N., Mauricio-Castillo, A., Argüello-Astorga, G. (2006) "A begomovirus isolated from chlorotic and stunted soybean plants in Mexico, is a new strain of Rhynchosia golden mosaic virus.". Plant Disease. 90: 972. .4.- Holguin-Peña, R., Arguello-Astorga, G.R., Rivera-Bustamante, R., Brown, J.K. (2006) "A new strain of Tomato Chino La Paz virus associated with leaf curl disease of tomato in Baja California Sur, Mexico.". Plant Disease. 90: 973. 5.- De la Torre-Almaraz R, Monsalvo-Reyes A, Romero-Rodríguez A., Argüello-Astorga GR, Ambriz-Granados S (2006) A new begomovirus inducing yellow mottle in okra crops in México is related to Sida yellow vein virus. Plant Disease. 90: 378. 6.- Méndez-Lozano J, Quintero-Zamora E, Barbosa-Jasso MP, Leyva-López NE, Garzón-Tiznado JA., Argüello-Astorga GR. (2006) A Begomovirus associated with leaf curling and chlorosis of soybean in Sinaloa, Mexico, is related to Pepper golden mosaic virus. Plant Disease. 90:109. 7.- Ramos PL, Fernández A, Castrillo G, Díaz L, Echemendía AL, Fuentes A, Peral R, Pujol M, Ascencio-Ibañez JT, Rivera-Bustamante R, Argüello-Astorga G (2002) Macroptilium yellow mosaic virus, a New Begomovirus Infecting Macroptilium lathyroides in Cuba. Plant Disease. 86: 1049. 8.- Ascencio-Ibáñez JT, Argüello-Astorga GR, Méndez-Lozano J, and Rivera-Bustamante RF (2002) First Report of Rhynchosia golden mosaic virus (RhGMV) infecting tobacco in Chiapas, Mexico. Plant Disease 86 : 692.


9.- Ascencio-Ibañez JT, Diaz-Plaza R, Mendez J, Monsalve-Fonnegra Z, Argüello-Astorga GR, and Rivera-Bustamante RF (1999) First report of tomato yellow leaf curl geminivirus in Yucatan, Mexico. Plant Disease 83: 1178.

Capítulos en libros Monsalve-Fonnegra Z, Argüello-Astorga GR, Rivera-Bustamante RF (2002) Geminivirus Replication and Gene Expression. In: Viruses as molecular plant pathogens (Ed.) Khan, J.A. and Dijkstra, J. Haworth Press, Inc., New York, p.257-277. Ruiz-Medrano R, Guevara-González RG., Argüello-Astorga GR, Herrera-Estrella LR, y Rivera-Bustamante RF. (1996). Análisis de la expresión de los principales promotores del geminivirus huasteco del chile. En: Galindo E (ed.) Fronteras en Biotecnología y Bioingeniería. Sociedad Mexicana de Biotecnología y Bioingeniería, A.C., México. p. 389-396. Argüello-Astorga GR, Herrera-Estrella LR. (1995) Theoretical and experimental definition of minimal photoresponsive elements in cab and rbcS genes. In: Terzi, A. et al. (eds.) Current Issues in Plant Molecular and Cellular Biology. Kluwer Academic Publishers, Dordrecht, Netherlands. p. 501-511. Argüello-Astorga GR y Herrera-Estrella LR. (1994) Métodos de transformación y vectores de expresión en plantas. En: Vicente, M. (ed) Avances en Ingeniería Genética. 2a. Edición. Consejo Superior de Investigaciones Científicas, Madrid, España. p. 41-70. Argüello-Astorga GR, Guevara-García A, y Herrera-Estrella LR. (1991). Introducción a la Ingeniería Genética de Plantas. en: Rivera-Bustamante et al. (Eds). Introducción a la Biología Molecular e Ingeniería Genética de Plantas. CINVESTAV-SARH, México. p. 44-93.

Patentes

(en trámite de registro)

Casas Flores, J.S., Arguello Astorga, G.R., Salas Marina, M.A., Herrera Estrella, A.H.

“Cepas transformantes del hongo micoparasítico Trichoderma spp. promotoras del crecimiento y resistencia a enfermedades fúngicas y bacterianas en plantas solanáceas, composiciones que las contienen, procedimiento de aplicación y uso de las mismas.”

Expediente MX/a/2008/005371- Instituto Mexicano de la Propiedad Industrial


Proyectos obtenidos en Convocatorias Proyecto 42639-Q (CONACYT-Ciencias Básicas) Delimitación experimental de los determinantes de especificidad de la proteína de replicación del Virus Huasteco del Chile y geminivirus relacionados. (2004-2007) Monto $ 1, 774, 000. 00 Proyecto FMSLP-2008-C01-86994 “Identificación de virus que afectan cultivos agrícolas en el estado de San Luis Potosí: desarrollo de técnicas moleculares más eficientes para la detección oportuna de enfermedades.” (Julio 2008- Junio 2010) Monto: $ 544,125.00 Proyecto 84004 (CONACYT-Ciencias Básicas-2007) “Delimitación y análisis funcional de elementos silenciadores y activadores de la transcripción de los genes tardíos del virus del mosaico de Euphorbia (EuMV) y el virus Huasteco del chile (PHYVV).” (Octubre 2008- Octubre 2011) Monto: $ 1,061,000.00

Participación en Congresos Internacionales .A. Mauricio-Castillo, C.T. Monreal-Vargas, J. Gregorio-Jorge, A.G. Alpuche-Solís, and G.R. Argüello-Astorga. "A novel molecular method that greatly enhances detection of mixed infections of begomoviruses and discovery of new species" en 5th. International Geminivirus Symposium. Ouro Preto, Brasil. Mayo 2007 Argüello-Astorga G, Gomez-Castañón R, Alpuche-Solís AG. Ancestral arrays of cis-acting elements are conserved in promoters from geminivirus: clues for the evolution of American begomoviruses. 4th. International Geminivirus Symposium. Sudáfrica, 2004. Argüello-Astorga G, Monreal-Vargas C, Holguin R, Rivera Bustamante R, Ambriz-Granados S, Solís-Alpuche AG. Using evolutionary PCR to analyze begomovirus diversity and produce gene expression cassettes with viral promoters. 4th International Geminivirus Symposium. Sudáfrica, 2004. Argüello-Astorga GR, Ruiz-Medrano R. Theoretical delimitation of amino acid residues determining the DNA-binding specificity of geminivirus replication proteins. 3rd. International Geminivirus Symposium. Norwich, Norfolk, U.K., 2001. Argüello-Astorga GR, Kong L-J, Orozco BM, Hanley-Bowdoin L. A deleterious mutation in the retinoblastoma-binding domain of AL1 is reverted at high frequency in infected plants. 3rd. International Geminivirus Symposium. Norwich, Norfolk, U.K., 2001.


Argüello-Astorga GR, Hanley-Bowdoin L. Identification of residues of a viral protein that are critical for interaction with a cell cycle regulator. Annual Meeting of the PEW Program in the Biomedical Sciences. Costa Rica, 2001. Kong L, Argüello-Astorga GR, Orozco BM, Hanley-Bowdoin L. Interaction between geminivirus replication protein and a plant cell cycle regulator. 6th Congress of Plant Molecular Biology. Quebec, Canada, 2000. López-Ochoa L, Argúello-Astorga G, Herrera-Estrella L. Structural Analysis of the minimal light-responsive RbcS I-G Unit. 6th Congress of Plant Molecular Biology. Quebec, Canada, 2000. Ramos-González PL, Argüello-Astorga GR, Ascencio-Ibañez JT, Oramas Frenes P, Rivera-Bustamante RF. Análisis de complementación replicativa entre geminivirus con iterones diferentes o idénticos. Taller Internacional de Biotecnología Vegetal. Ciego de Avila, Cuba, 1999. Martínez A, Argüello-Astorga GR, Herrera-Estrella L. A Conserved Modular Arrangement of rbcS promoters functions as a Light-Regulatory Unit.. 5th International Congress of Plant Molecular Biology. Singapur, 1997. Argüello-Astorga GR, Herrera-Estrella L. An evolutionary conserved region of the rbcS gene promoters functions as a light-responsive element. 4th International Congress of Plant Molecular Biology. Amsterdam, Holanda. 1994. Ruiz-Medrano R, Guevara-Gonzalez RG, Argüello-Astorga GR, Herrera-Estrella L, Rivera-Bustamante RF. Tissue specificity of a geminivirus promoter. 4th International Congress of Plant Molecular Biology. Amsterdam, Holanda. 1994. Argüello-Astorga GR, Guevara-Gonzalez RG, Herrera-Estrella L., Rivera-Bustamante, R.F. Phylogenetic and structural analysis of the intergenic region of geminiviruses. 4th International Congress of Plant Molecular Biology. Amsterdam, Holanda. 1994. Argüello-Astorga GR, Herrera-Estrella L. Determination of Minimal Photoresponsive Elements in cab and rbcS genes. Workshop on identification, mapping, expression and regulation of plant genes. Guaruja, Brasil. 1993. Argüello-Astorga GR, Simpson J. Analysis of transacting factors binding to the chlorophyll A/B gene AB80 from pea. Fifteenth EMBO Annual Symposium “Molecular Communication in Higher Plants”. Heidelberg, Alemania. 1989.

Participación en Congresos Nacionales

Miembro del Comité Organizador del V Congreso Nacional de Virología. Sociedad Mexicana de Bioquímica Querétaro, Qro., México. octubre 2007

Londoño-Avendaño A., Riego-Ruiz, L., Arguello-Astorga, GR Delimitación teórica de los determinantes de especificidad replicativa de circovirus, nanovirus y replicones relacionados. (Presentación Oral).


V Congreso Nacional de Virología. Sociedad Mexicana de Bioquímica Querétaro, Qro., México. octubre 2007 Gregorio-Jorge, J. y Arguello-Astorga, GR. Análisis del proceso de reversión rápida de mutaciones en el gen que codifica la proteína Rep de geminivirus (Presentación Oral). V Congreso Nacional de Virología. Sociedad Mexicana de Bioquímica Querétaro, Qro., México. octubre 2007

Bañuelos-Hernández B., Shimada H, Rivera-Bustamante R, Arguello-Astorga, GR. Los primeros 10 aminoácidos de la proteína Rep determinan el reconocimiento específico del origen de replicación de los geminivirus (Presentación Oral). V Congreso Nacional de Virología . Sociedad Mexicana de Bioquímica Querétaro, Qro., México. octubre 2007 Bernal-Alcocer A., Virgen Calleros G., Alpuche Solis, A.G., Arguello Astorga, G.R., Frías Treviño, G "Caracterización molecular de un aislado de Euphorbia mosaic virus con un Ori diferente y un promotor críptico potencialmente involucrado en la síntesis de mRNA. " (Poster). V Congreso Nacional de Virología. Sociedad Mexicana de Bioquímica Queretaro, Qro., México. octubre 2007

Mauricio-Castillo, J.A., Londoño Avendaño A, Arguello Astorga, G.R. "Aislamiento y caracterización de un nuevo curtovirus en Mexico. " (Poster). V Congreso Nacional de Virología. Sociedad Mexicana de Bioquímica Queretaro, Qro., México. octubre 2007

Cárdenas Conejo, Y., Riego Ruiz, L., Arguello Astorga, G.R. "Identificación de arreglos modulares conservados (CMAs) en la región intergénica del componente B de begomovirus." (Poster) V Congreso Nacional de Virología. Sociedad Mexicana de Bioquímica Querétaro, Qro., México. octubre 2007 Arguello-Astorga, GR, Ascensio-Ibañez, J.T., Hanley-Bowdoin, L. (2006) Mutaciones en el dominio de union a pRBR de la proteina Rep de geminivirus revierten con una frecuencia de 100 por ciento en plantas infectadas (Presentación Oral). IV Congreso Nacional de Virología, Sociedad Mexicana de Bioquímica, Veracruz, Ver. Marzo 2006. De la Torre Almaraz R., Monsalvo Reyes, A, Arguello-Astorga G, Ambriz-Granados, S. (2006) Una nueva especie de begomovirus aislada de okra presenta iterones atipicos y una proteina de replicación con un dominio similar al de un virus de Tailandia. (Presentación Oral). IV Congreso Nacional de Virología. Sociedad Mexicana de Bioquímica. Veracruz, Ver. Marzo 2006. Bañuelos-Hernández B, Shimada-Beltrán H, Rivera-Bustamante R, Argüello-Astorga G. (2006) Delimitación experimental del dominio de unión específica al DNA de la proteína iniciadora de la replicación de geminivirus. (Poster) IV Congreso Nacional de Virología, Sociedad Mexicana de Bioquímica, Veracruz, Ver. Marzo 2006. Gregorio-Jorge J, Argüello-Astorga G. Análisis del proceso de reversión fenotípica de mutaciones en el dominio de unión a retinoblastoma de la proteína Rep de geminivirus. (Poster) IV Congreso Nacional de Virología. Sociedad Mexicana de Bioquímica. Veracruz, Ver. Marzo 2006


Juárez-Reyes A, Gómez-Castañón RG, Argϋello-Astorga G. Delimitación de secuencias involucradas en el silenciamiento y transactivación de los genes tardíos del virus huasteco del chile (phv).(Poster) IV Congreso Nacional de Virología. Sociedad Mexicana de Bioquímica. Veracruz, Ver. Marzo 2006. Mauricio-Castillo A, Argϋello-Astorga G. Desarrollo de un método molecular para amplificar el genoma A de begomovirus pertenecientes al linaje del Squash leaf curl virus. IV Congreso Nacional de Virología. Sociedad Mexicana de Bioquímica. Veracruz, Ver. Marzo 2006. Mauricio-Castillo A, Méndez-Lozano J, Argϋello-Astorga G. Caracterización de genomas B de geminivirus por un nuevo metodo molecular. IV Congreso Nacional de Virología. (Poster) Sociedad Mexicana de Bioquímica. Veracruz, Ver. Marzo 2006. Bernal-Alcocer, A., Virgen-Calleros G, Alpuche-Solis AG, Arguello Astorga GR, Zarate-Chavez V, Frias Treviño GA (2006) Incidencia de virosis e identificacion de begomovirus en cultivos de chile y jitomate en el sur de Jalisco (Poster). IV Congreso Nacional de Virología. Sociedad Mexicana de Bioquímica. Veracruz, Ver. Marzo 2006. Monreal Vargas C, Alpuche Solís AG, Argüello Astorga GR. Diagnóstico basado en la reacción en cadena de la polimerasa (PCR), de enfermedades del jitomate y chile producidos en el Altiplano Potosino. (Presentación oral). XXXII Congreso Nacional de la Sociedad Mexicana de Fitopatología, Chihuahua, Chih. Sept 2005. Monreal Vargas C, Argüello Astorga GR, Alpuche Solís AG. Nuevo método basado en la PCR para el diagnóstico y la identificación de begomovirus. (Presentación oral). XXXII Congreso Nacional de la Sociedad Mexicana de Fitopatología, Chihuahua, Chih. Sept 2005. Mauricio-Castillo JA, Méndez-Lozano J, Argüello-Astorga G. Desarrollo de un método molecular para amplificar el genoma B de geminivirus. (Poster). XXXII Congreso Nacional de la Sociedad Mexicana de Fitopatología, Chihuahua, Chih. Sept 2005. Mauricio-Castillo JA, Perea-Araujo L, Méndez-Lozano J, Argüello-Astorga G. Una nueva especie de geminivirus aislada de una planta con infección viral mixta. (Presentación oral). XXXII Congreso Nacional de la Sociedad Mexicana de Fitopatología, Chihuahua, Chih. Sept 2005. Algara-Suárez P, Arguello-Astorga G. Delimitación experimental de los determinantes de especificidad de la proteína de replicación del virus huasteco del chile y geminivirus relacionados. XXV Congreso de la Sociedad Mexicana de Bioquímica. Ixtapa, Gro, 2004. García-Moreno Rublí A, Arguello-Astorga G. Desarrollo de una técnica molecular para la generación de múltiples cartuchos de expresión génica con promotores virales.(Poster) XXV Congreso de la Sociedad Mexicana de Bioquímica. Ixtapa, Gro, 2004. Monreal-Vargas C, Arguello-Astorga GR, Alpuche-Solís AG, Rivera-Bustamante R. Desarrollo de un método de diagnóstico molecular que facilita el análisis de la diversidad de geminivirus en agroecosistemas. III Congreso Nacional de Virología. Morelia, Mich, 2004.


Arguello-Astorga GR, Gómez-Castañón AG, Alpuche-Solís AG. El análisis comparado de la región intergénica de geminivirus revela una insospechada conexión evolutiva entre linajes de Asia y América. (Presentación Oral). III Congreso Nacional de Virología. Morelia, Mich, 2004. Argüello-Astorga GR, Monreal-Vargas C, Holguín-Peña JR, Méndez-Lozano J, Ambriz-Granados S, Rivera-Bustamante R, Alpuche-Solis AG. A PCR-based method to discover new begomovirus species and produce gene expression cassettes with viral promoters. (Poster). XI Congreso Nacional de Bioquímica y Biología Molecular de Plantas-5o. Simposio México-EUA. Acapulco, Gro. (Nov 2003). Hernández-Rico E, Monreal-Vargas C, Argüello-Astorga GR, Castillo-Collazo R, Moreno-Chavez G, Alpuche-Solis A. Evaluation of molecular, serological, biochemical and microbial methods of diagnostic for the detection of tomato and pepper diseases. (Poster). XI Congreso Nacional de Bioquímica y Biología Molecular de Plantas-5o. Simposio México-EUA. Acapulco, Gro. (Nov 2003). Argüello-Astorga GR. Genómica comparada de virus vegetales. Simposium de Genomas en el Siglo XXI. Saltillo, Coah, 2003. López Ochoa L, Argüello Astorga G, Herrera Estrella L. Analysis of the minimal light-responsive I-G Unit: a combined site. IX Congreso Nacional de Bioquímica y Biotecnología Molecular de Plantas. III Simposio México-Estados Unidos. Mérida, Yuc., 1999. López Ochoa L, Argüello Astorga G, Herrera Estrella L. Molecular Analysis of the minimal light-responsive I-G Unit: a combined site?.VIII Congreso Nacional de Bioquímica y Biotecnología Molecular de Plantas. II Simposio México-Estados Unidos. Guanajuato, Gto., 1998. López Ochoa L, Argüello Astorga G, Herrera Estrella L. Análisis funcional de unidades mínimas de respuesta a luz de promotores Cab y Rubisco. XXI Congreso Nacional de Bioquímica. Manzanillo, Col., 1996. Ruiz Medrano R, Guevara González RG, Argüello Astorga GR, Rivera Bustamante RF. Especificidad de tejido de un promotor geminiviral. XX Congreso Nacional de Bioquímica. Zacatecas, Zac., 1994. Argüello Astorga GR, Herrera Estrella L. Delimitación teórica y experimental de una Unidad Mínima de fotorrespuesta en genes rbcS. XX Congreso Nacional de Bioquímica (Presentación Oral). Zacatecas, Zac.,1994 Argüello Astorga GR, Herrera Estrella L. Determination of Minimal Units of transcriptional photoresponse in cab and rbcS gene promoters. XIX Congreso Nacional de Bioquímica (VII PAABS Congress) Ixtapa, Gro.,1992. Argüello Astorga GR, Ochoa N, Herrera Estrella L. Análisis comparado de la respuesta bioquímica al agobio hídrico y salino en ecotipos de Nicotiana glauca. (Presentación Oral). XVIII Congreso Nacional de Bioquímica. San Luis Potosí, S.L.P., 1990


Argüello Astorga GR, Herrera Estrella L, Simpson J. Factores proteicos involucrados en la regulación de genes por luz. XVII Congreso Nacional de Bioquímica Oaxaca, Oax., 1988.

Distinciones: -Reconocimiento de Agrobio-Mexico como asesor de la Tesis de Maestria que obtuvo el Primer Lugar en su categoría, Premio Agrobio 2005 -Becario del Programa de Repatriación de Investigadores Mexicanos (CONACYT), Noviembre del 2001 a Octubre del 2002 - Reconocimiento del Institute for Scientific Information (ISI-Thomson Scientific) como primer autor de uno de los artículos mexicanos más citados en la década de los 90's, Area de Virología (Septiembre del 2000). - Becario Postdoctoral 1999-2001 de The PEW Latin American Fellow Program in Biomedical Sciences. -Investigador Nacional Nivel 1 Sistema Nacional de Investigadores, SNI (1996 al presente) -Candidato a Investigador Nacional, S.N.I. (1992-1996) -Beca para Estudios de Doctorado, CONACYT (1992-1995) Firma:

Dr. Gerardo Rafael Argüello Astorga

e-mail: [email protected] Tel. (444) 8 35 00 59

(444) 8 34 20 00 ext. 2079

San Luis Potosí, S.L.P a 30 de Marzo del 2009

G.Arguello-Astorga Reseña de Logros 1

Reseña de Logros Académicos Dr. Gerardo R. Argüello Astorga

1.- Delimitación teórica y experimental de “Unidades Mínimas de Fotorrespuesta” en genes de plantas. En el curso de mi doctorado desarrollé una metodología para el análisis comparativo de secuencias no-codificantes (“Método de Análisis Filogenético-Estructural”o MAFE). Utilizando este enfoque identificamos más de 30 potenciales LREs en diversas familias de genes asociados a la fotosíntesis, y postulamos algunas hipótesis acerca del origen y función de algunas unidades complejas conservadas (“CMAs”) conservadas en ellos (Plant Physiol. 112: 1155. 1996; Ann. Rev. Plant Mol. Biol.. 49: 525. 1998). Uno de los potenciales LREs identificados, la llamada “Unidad I-G” de promotores rbcS, fue caracterizada funcionalmente por varios colegas y yo en el laboratorio del Dr. Luis Herrera, y logramos demostrar que este elemento complejo de solo 52 pb. es capaz de mediar la respuesta transcripcional a señales provenientes del cloroplasto y de varios fotorreceptores (Plant Physiology 128: 1223. 2002; J. Exp.Bot. 58: 4397. 2007). 2.- Identificación por MAFE de los determinantes de especificidad replicativa (DERs) de virus de plantas. Utilizando el mismo enfoque comparativo identificamos una serie de elementos iterativos organizados de manera característica en linajes geográficos de geminivirus, y. postulamos la hipótesis de que esos “iterones” funcionan como sitios de unión de la proteína de replicación viral (Rep). Esta noción ha sido experimentalmente confirmada. El trabajo en el que presentamos la hipótesis (Virology 203: 90-100. 1994) recibió una distinción del Institute for Scientific Information como el artículo mexicano más citado en la década de los 90´s en el Area de Virología. 3.- Identificación de DERs en trans en geminivirus, circovirus, nanovirus, y satélites de tipo nanoviral. Para delimitar el dominio de la proteína de replicación (Rep) que interactúa con los iterones, desarrollé con el Dr. Ruiz-Medrano un método comparativo basado en el agrupamiento de proteínas homólogas en conjuntos “isoespecíficos”, usando a los iterones como herramientas heurísticas. De este modo identificamos los residuos que funcionan potencialmente como los determinantes de especificidad de esa proteína viral, que se localizan en un “dominio relacionado al iterón” (IRD) (Archives of Virology.146: 1465. 2001). La hipótesis del IRD ha recibido fuerte apoyo experimental en estudios realizados por diversos grupos, y en mi propio laboratorio (Bañuelos-Hernández et al., en preparación). Recientemente utilizamos el mismo enfoque metodológico al análisis de los DERs de Circovirus, Nanovirus y satélites de tipo nanoviral, con resultados sorprendentes y notables, que permiten establecer la existencia de una conexión evolutiva indudable entre todas esas familias virales (Londoño-Avendaño et al, en preparación). 4.- Delimitación de elementos involucrados en la regulación de los genes tardíos de begomovirus. Por medio del MAFE identificamos en la región intergénica de geminivirus un elemento que denominamos el CLE (“Conserved Late Elements”), del cual postulamos, y demostramos en un caso particular, su participación en el proceso de transactivación de la expresión de genes tardíos por la proteína viral TrAP (Virology 253: 162. 1999). La hipótesis del CLE ha sido sustentada por diversos trabajos experimentales, pero sigue siendo un tema controversial. A la fecha hemos demostrado de manera fehaciente que ese elemento es reconocido por factores transcripcionales del huésped (Cantú-Iris, datos no publicados). 5.- Evolución experimental de geminivirus: análisis de la reversión rápida de mutaciones.

Como parte de un análisis de mutagénesis dirigida del dominio de unión a Retinoblastoma de la proteína Rep de dos begomovirus, descubrimos que ciertas mutaciones revierten en el 100% de los casos, y en el curso de unos pocos días. Este fenómeno, inesperado en un virus de DNA con tasas de mutación presumiblemente bajas, lo hemos analizado en 5 begomovirus diferentes, y los datos han llevado a definir un papel de la planta en la frecuencia

G.Arguello-Astorga Reseña de Logros 2 relativa de ciertos mutantes (J. Virol. 81: 11005. 2007), y en la preponderancia de mutaciones supresoras en ciertos virus, las cuales nos han permitido mapear muy finamente un dominio de Rep que es crítico para el proceso de replicación del virus (Gregorio-Jorge et al., en preparación). 6.- Análisis Multifactorial de regiones codificantes y no-codificantes de genomas virales para la reconstrucción de su historia evolutiva . Hemos desarrollado una metodología que combina el análisis MAFE de regiones no-codificantes, la identificación de “indels” en el genoma y el análisis de “motifs” en las proteínas virales asociados a linajes geográficos, que nos han permitido hacer una reconstrucción plausible de la historia evolutiva de los cuatro géneros de la familia Geminiviridae, lo que estamos sustentando experimentalmente con el aislamiento de nuevas especies virales con las características predichas (Londoño-Avendaño et al., en preparación). Estas metodologías pueden extenderse fácilmente al análisis de otros grupos virales.

Propuesta de Programa de Investigación

Dr. Gerardo R. Argüello Astorga El programa de investigación que me propongo realizar en los próximos tres años incluye los siguientes tópicos que abordan cuestiones evolutivas de virus y plásmidos, así como aspectos de regulación transcripcional en begomovirus. 1) Delimitación teórica y experimental de los determinantes de especificidad replicativa de plásmidos de la familia pMV158 y de plásmidos RCR de cianobacterias. La superfamilia de proteínas iniciadoras de la replicación por Círculo Rodante (RCR) a la que pertenecen las proteínas Rep de circovirus, nanovirus y geminivirus, incluye a un gran número de proteínas codificadas por plásmidos, tanto de eucariontes (v.gr. plásmidos del alga roja Porphyra pulcra) como de procariontes (Streptococcus sp., Bacillus sp. y otros Gram+; cianobacterias como Nostoc sp. y Synechocystis sp., entre otros). Por esta razón nos proponemos examinar la cuestión de si los dominios de unión al DNA de esas proteínas plasmídicas son derivados evolutivamente de los dominios análogos de proteínas virales. La respuesta a esta cuestión tendrá importantes implicaciones para nuestro entendimiento de la evolución de virus y plásmidos, que ha sido un campo de fuertes debates en el que no existen todavía consensos. Nosotros ya hemos iniciado el análisis comparativo de los plásmidos de la familia pMV158, y los datos obtenidos al presente nos permiten diseñar experimentos que pueden conducir a conclusiones relevantes. 2) Caracterización funcional de elementos que regulan la transcripción de genes tardíos en begomovirus. Los mecanismos de control de la expresión de los genes begomovirales que se expresan en las etapas media y tardía de la infección continúan siendo poco claros, y se sabe que difieren entre los miembros de distintos subgrupos de los geminivirus. Por ejemplo, en los begomovirus existe un potente silenciador que actúa en forma independiente de distancia y orientación, pero que es específico de promotores tardíos. Por otra parte, la proteína viral TrAP que regula principalmente la actividad de los promotores tardíos CP. BC1 y BV1, no tiene la capacidad de unirse al DNA por sí sola y parece mediar su efecto a través de interacciones con una o más proteínas del huésped, las cuales sí reconocen y unen secuencias específicas en los promotores virales. Más aún, la evidencia indica que TrAP actúa como un supresor de la represión en tejido vascular, y como un transactivador en mesófilo. Para abordar este complejo sistema de regulación estamos generando promotores sintéticos que incorporan módulos pequeños compuestos por dos o tres “phylogenetic footprintings”, en diferentes arreglos, y estamos generando plantas transgénicas de tabaco y Arabidopsis con estas construcciones. Contamos con un amplio repertorio de begomovirus pertenecientes a diferentes linajes para explorar las diferencias en los mecanismos de control subyacentes. 3) Caracterización de promotores crípticos en la región intergénica del DNA-B de begomovirus. Uno de los hallazgos más interesantes del Análisis Filogenético-Estructural del componente genómico B de algunos begomovirus recientemente aislados en nuestro laboratorio (v.gr: Euphorbia mosaic virus-Jalisco) es la inclusión de segmentos intermedios de 85-110 pb que son altamente similares al promotor CP del componente genómico A, y cuya caja TATA

putativa queda muy distante (más de 150 pb) del gen más proximo, BC1, lo que constituye una observación intrigante. Más aún, estos presuntos “promotores crípticos” se localizan inmediatamente arriba de secuencias de 50 pb que presentan una elevada identidad (mayor al 90%) con secuencias codificantes del componente genómico complementario. Estas observaciones sugieren la posibilidad de que exista un mecanismo de regulación mediado por RNAi a través del cual el DNA-B de un virus controle la expresión de ciertos genes del componente genómico homólogo, de lo cual no existe ningún antecedente. Para explorar esta posibilidad examinaremos la actividad transcripcional de estos presuntos promotores crípticos, y caracterizaremos los transcritos generados in vivo a partir de ellos, usando técnicas moleculares apropiadas. 4) Aislamiento y caracterización de nuevos virus de ssDNA transportados por insectos homópteros relacionados con vectores de geminivirus, utilizando la técnica de amplificación isotermica por RCA. En los últimos meses se han aislado y caracterizado en el Viejo Mundo a dos geminivirus con una organización genómica tan diferente respecto a todos los conocidos que se ha propuesto crear dos géneros nuevos en la familia Geminiviridae. De acuerdo a nuestros análisis evolutivos, muchos de los grupos de geminivirus actuales han surgido de eventos de recombinación entre virus muy divergentes y evolutivamente distantes, algunos de los cuales no se conocen en su forma nativa, no recombinante.. Esto conduce a postular la probable existencia en la naturaleza de linajes geminivirales todavía no descritos, que serían transmitidos por insectos lejanamente relacionados con los vectores conocidos de geminivirus (mosquitas blancas, chicharritas, chicharritas saltadoras). En colaboración con entomólogos expertos en homópteros, y utilizando la técnica de amplificación de moléculas circulares por CR, que hemos estandarizado en el laboratorio para amplificar DNA viral a partir de tejidos de insectos, nos proponemos explorar la existencia de virus ssDNA que podrían ser representativos de linajes ancestrales, y que nos proporcionarán valiosa información sobre la coevolución de virus fitopatógenos con sus insectos vectores. 5) Otros temas de investigación potenciales. Sin entrar en detalles, y dependiendo de las colaboraciones que se están estableciendo actualmente con otros grupos de investigación, tenemos la intención de desarrollar provectores virales de tipo “Magnifección”, que combinan elementos virales diversos y vectores de Agrobacterium tumefaciens, para la producción a gran escala de proteínas antigénicas o de valor biofarmacéutico en plantas. Por otra parte, pretendemos colaborar con grupos de expertos en nanotecnología para utilizar cápsides virales y diatomeas (microorganismos fotosintéticos con cápsulas de sílice) para diversos fines tecnológicos.

Investigadores a los que pueden solicitarse referencias sobre mi persona:

1.- Dra. June Kilpatrick Simpson Williamson Investigadora Titular del Departamento de Ingeniería Genética CINVESTAV, Unidad Irapuato. e-mail: [email protected] 2.- Dr. Luis Herrera Estrella Director del Laboratorio Nacional de Genómica para la Biodiversidad (LANGEBIO)-Irapuato, Gto. e-mail: [email protected] 3.- Dra. Linda Hanley-Bowdoin William Neal Reynolds Distinguished Professor Department of Molecular and Structural Biochemistry Department of Genetics North Carolina State University Email: [email protected]

JOURNAL OF VIROLOGY, Oct. 2007, p. 11005–11015 Vol. 81, No. 200022-538X/07/$08.00�0 doi:10.1128/JVI.00925-07Copyright © 2007, American Society for Microbiology. All Rights Reserved.

High-Frequency Reversion of Geminivirus Replication ProteinMutants during Infection�

Gerardo Arguello-Astorga,† J. Trinidad Ascencio-Ibanez, Mary Beth Dallas,Beverly M. Orozco,‡ and Linda Hanley-Bowdoin*

Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, North Carolina 27695-7622

Received 30 April 2007/Accepted 24 July 2007

The geminivirus replication protein AL1 interacts with retinoblastoma-related protein (RBR), a key regu-lator of the plant division cell cycle, to induce conditions permissive for viral DNA replication. Previous studiesof tomato golden mosaic virus (TGMV) AL1 showed that amino acid L148 in the conserved helix 4 motif iscritical for RBR binding. In this work, we examined the effect of an L148V mutation on TGMV replication intobacco cells and during infection of Nicotiana benthamiana plants. The L148V mutant replicated 100 times lessefficiently than wild-type TGMV in protoplasts but produced severe symptoms that were delayed compared tothose of wild-type infection in plants. Analysis of progeny viruses revealed that the L148V mutation revertedat 100% frequency in planta to methionine, leucine, isoleucine, or a second-site mutation depending on thevaline codon in the initial DNA sequence. Similar results were seen with another geminivirus, cabbage leaf curlvirus (CaLCuV), carrying an L145A mutation in the equivalent residue. Valine was the predominant aminoacid recovered from N. benthamiana plants inoculated with the CaLCuV L145A mutant, while threonine was themajor residue in Arabidopsis thaliana plants. Together, these data demonstrated that there is strong selectionfor reversion of the TGMV L148V and CaLCuV L145A mutations but that the nature of the selected revertantsis influenced by both the viral background and host components. These data also suggested that high mutationrates contribute to the rapid evolution of geminivirus genomes in plants.

Geminiviruses constitute the largest and most diverse andeconomically important family of plant DNA viruses (56).They infect a broad range of plants and cause devastating cropdiseases, particularly in tropical and subtropical regions of theworld (36, 39, 41). Geminiviruses are characterized by twinicosahedral capsids and small, single-stranded DNA (ssDNA)genomes (56) that display high levels of genetic variability (62).Several studies have indicated that recombination contributesto geminivirus diversity (48, 60, 71). However, unlike double-stranded DNA (dsDNA) viruses, there is also mounting evi-dence that ssDNA viruses are subject to high nucleotide mu-tation rates similar to the levels reported previously for RNAviruses (34, 35, 53). Thus, geminiviruses represent a uniqueopportunity to examine the processes that contribute to thegenetic variation of ssDNA viruses as well as the mechanismsunderlying virus evolution in plants.

The family Geminiviridae is classified into four genera, Be-gomovirus, Curtovirus, Topocuvirus, and Mastrevirus, based ontheir genome organization, host range, and insect vectors (19).The largest genus corresponds to the begomoviruses, whichhave one- or two-genome components (designated DNA-Aand DNA-B), infect dicots, and are transmitted by Bemisiatabaci. Over the past 20 years, there has been a significant

increase in the frequency and severity of begomovirus diseases.During this time, agricultural intensification and changes in theinsect vector facilitated the expansion of begomovirus popula-tions and their movement into new plant hosts and contributedto the emergence of new, more virulent viruses. Sequenceanalysis of emerging viruses implicated recombination and re-assortment in begomovirus evolution. Both processes dependon the formation of mixed infections and the presence ofmultiple viral genome components in a single plant cell (54).Recombinant begomoviruses have been associated with se-vere epidemics in cassava, cotton, and tomato (22, 23, 27, 40,43, 60, 71) and divergence of the viruses indigenous to theIndian subcontinent (58). Reassortment is a contributingfactor to cassava mosaic disease (50), and there are exam-ples of monopartite begomoviruses acquiring DNA-B com-ponents (61). In addition, many begomoviruses are associ-ated with DNA satellites that increase virulence and alterhost range (7, 37). The satellite DNAs can recombine withthemselves and viral genome components (1), further in-creasing variability.

Nucleotide misincorporation during viral DNA replicationalso contributes to genome diversity. Studies of bacterial andanimal systems indicated that the mutation rates of dsDNAand ssDNA viruses differ significantly. The mutation rates fordsDNA phages range from 10�7 to 10�8, while ssDNA phagesdisplay rates of approximately 10�6 (15, 53). Like dsDNAphages, polyomavirus and papillomavirus genomes display lowmutation rates (10�8 to 10�9), similarly to their hosts (26). Incontrast, high mutation rates (ca. 10�4) have been reported forparvoviruses (35, 64, 65) and circoviruses (6, 21). Like gemi-niviruses, these viruses have ssDNA genomes that replicate viarolling-circle mechanisms. Thus, the high levels of sequence

* Corresponding author. Mailing address: Department of Molecularand Structural Biochemistry, North Carolina State University, Raleigh,NC 27695-7622. Phone: (919) 515-6663. Fax: (919) 515-2047. E-mail:[email protected].

† Present address: Division de Biologıa Molecular, Instituto Poto-sino de Investigaciones Cientıficas y Tecnologicas, 78216 San LuisPotosı, SLP, Mexico.

‡ Present address: Talecris Biotherapeutics, Clayton, NC 27520.� Published ahead of print on 1 August 2007.

11005

heterogeneity reported for begomoviruses and mastreviruses(9, 24, 28, 44, 59) may reflect replication errors.

Tomato golden mosaic virus (TGMV) and cabbage leaf curlvirus (CaLCuV) are begomoviruses with two-component ge-nomes. Both viruses encode a replication protein designatedAL1 (also named AC1, C1, or Rep), which is required for theinitiation and termination of viral DNA synthesis (20, 32, 45)and acts as a DNA helicase (11, 12). The AL1 protein alsoreprograms mature plant cells to create a permissive environ-ment for viral replication through interactions with the hostretinoblastoma-related protein (RBR), which regulates cell di-vision and differentiation in plants (14, 17, 31). TGMV andCaLCuV AL1 interact with RBR via a unique 11-amino-acidsequence (2, 31). Alanine substitutions across the helix 4 se-quence of TGMV AL1 differentially impacted RBR binding inyeast two-hybrid studies and suggested that residue L148 in themiddle of the motif provides a critical binding contact (2, 31).In the experiments reported here, we examined the impact ofvarious amino acid substitutions at TGMV AL1 L148 and theequivalent CaLCuV AL1 L145 on viral replication and infec-tivity. These studies showed that some mutations reverted at100% frequency during infection and provided evidence forthe capacity of geminivirus populations to evolve rapidly toamend deleterious changes in their genomes.

MATERIALS AND METHODS

AL1 mutants and PCR. The construction of the TGMV AL1 L148A, L148V,L148M, L148G, and L148I mutations was described previously (2). The muta-tions are designated by the wild-type residue and its position number followed bythe mutant amino acid. TGMV AL1 L148V* and E146A L148V were generatedusing pNSB148 (46) and primers 5�-TAATTATCTGaAcGGCTTCTTCTTTGGAAGAAGCATTTAAC and 5�-ATTATCTGCAcGGCcgCTTCTTTGGAAGAAGCATTTAA, respectively (lowercase type indicates mutant nucleotides.).TGMV A replicons encoding the mutant AL1 proteins were generated by sub-cloning SalI/NheI fragments corresponding to AL1 amino acids 120 to 312 fromthe mutagenesis clones into the same sites of the wild-type replicon pMON1565(45) to give pNSB919 (E146A/L148V), pNSB979 (L148V), pNSB997 (L148A),pNSB1000 (L148G), and pNSB1031 (L148V*).

TGMV AL1 mutants were also constructed from variants generated duringinfection with TGMV AL1 L148V, E146A/L148V, or L148V* mutants. TotalDNA from systemically infected, symptomatic leaves was amplified by PCR usingprimers 5�-CGACAAAGACGGAGATACTC and 5�-GTCTCATCTCGTCTGGCACG to give a 281-bp fragment corresponding to TGMV A positions 2006 to2287. The PCR products were digested with SalI/NcoI and subcloned into thesame sites of a modified pBlueScript SK(�) plasmid (Stratagene, Inc.) to gen-erate the intermediate plasmids pNBS1076, pNSB1111, pNSB1077, andpNSB1078. SalI/NheI fragments from these plasmids were then subcloned intothe same sites of pMON1565 to generate replicons carrying the AL1 mutationsL148I (pNSB1082), L148M (pNSB1113), C128W L148V (pNSB1083), andR125G L148V I155L (pNSB1084).

Wild-type CaLCuV replicons pCpCLCV A.003 and pCpCLCV B.003 contain1.6 copies of the A and B genomes, respectively (68). The CaLCuV AL1 muta-tion CaL145A in pNSB1097 (2) was subcloned as an AatII/NsiI fragment (AL1amino acids 132 to 332) into the equivalent sites of pCpCLCVA.003 to generatethe corresponding replicon pNSB1101. Mutant CaLCuV replicons were alsogenerated by PCR of variants produced during infection with the CaLCuVL145A mutant. Total DNA from systemically infected symptomatic leaves wasamplified by PCR using primers 5�-GTGAATCCGGGCAGTACAAGGTGTC-3� and 5�-CCCAGATAAAAACGGAATTCTCTGCC-3 to give an 854-bpfragment between positions 1425 and 2279. The PCR products were digestedwith AatII/EcoRI and subcloned into the same sites of pCpCLCV A.003 toproduce replicons carrying the CaLCuV L145V (pNSB1104), L145A/I167L(pNSB1005), and L145T (pNSB1008) mutations.

Replication and infectivity assays. Transient replication assays were per-formed using protoplasts isolated from Nicotiana tabacum (BY2) suspensioncells, electroporated, and cultured as described previously (20). Cells were trans-fected with 5 �g of wild-type or mutant A component DNA from TGMV or

CaLCuV and 25 �g of sheared salmon sperm DNA. Total DNA was extracted72 h after transfection, digested with either DpnI/XhoI (TGMV) or DpnI/EcoRI(CaLCuV), and examined for double- and single-stranded viral DNA accumu-lation by agarose gel blot analysis using 32P-radiolabeled virus-specific probesagainst A-component DNA. Double-stranded viral DNA was quantified by phos-phorimager analysis. Each replication assay was performed in at least threeindependent experiments.

Nicotiana benthamiana plants were infected by bombardment or agroinocula-tion (16, 42), while Arabidopsis thaliana Col-0 rosettes were infected by agroin-oculation (66). For bombardment, wild-type or mutant replicon DNA (10 �g) foreither TGMV A or CaLCuV A was precipitated onto 1-mm gold microprojec-tiles in the presence of the corresponding wild-type B-replicon DNA. The wild-type TGMV A and B plasmids were pMON1565 (45) and pTG1.4B (20), whilethe wild-type CaLCuV A and B plasmids were pCPCBLCVA.003 andpCPCbLCVB.002 (68). For agroinoculation, Agrobacterium tumefaciens culturescarrying a wild-type CaLCuV A (pNSB1090) or a mutant A replicon were mixedwith a culture carrying a wild-type CaLCuV B replicon (pNSB1091) and syringeinoculated immediately below the plant apex. Total DNA was extracted fromyoung leaf tissue of individual plants at the indicated times after bombardment(13) and linearized with XhoI (TGMV) or EcoRI (CaLCuV). Total DNA (2.5�g/lane) was resolved on 1% agarose –Tris-acetate-EDTA gels, transferred ontonylon, and hybridized with a 32P-radiolabeled probe specific for A-componentDNA.

Total DNA from infected plants was also amplified by PCR using the primersdescribed above, and the AL1 coding region was sequenced directly. The DNAsequencing chromatograms were examined directly to assess the heterogeneity ofthe population sequence at individual nucleotide positions.

RESULTS

Virus replication is differentially affected by substitutions atL148. Previously, we showed that valine and glycine substitu-tions at TGMV AL1 amino acid L148 in the helix 4 motif (Fig.1A) reduce RBR binding activity to 31 and 36% of wild-typebinding in yeast two-hybrid assays (2). To better understandthe role of L148 in AL1 function in planta, we examined theimpact of valine and glycine mutations on TGMV replicationand infectivity assays. We compared the replication of an ala-nine (E146A L148A), a glycine (L148G), and three valine(L148V, L148V* and E146A L148V) mutants to that of awild-type replicon (Fig. 1B). L148V and L148V* encode iden-tical proteins but contain one or two nucleotide changes incodon 148, respectively. E146A L148A and E146A L148V aredouble mutants that also carry alanine substitutions at positionE146. E146A L148V has a single nucleotide change at codon148, like L148V. These mutations were subcloned into the AL1open reading frame of a replicon plasmid carrying a partialtandem copy of TGMV A with two common regions (55). Toensure maintenance of the mutations, they were subclonedinto the unique copy region of the plasmid.

We analyzed the transient replication of wild-type TGMV Aand the mutant replicons in N. tabacum BY-2 protoplasts at72 h posttransfection on agarose gel blots probed with radio-labeled TGMV A DNA. The double-stranded form of theE146A L148A mutant (Fig. 1C, lane 2) accumulated to 12% ofwild-type levels (lane 1), similar to the 13% level reportedpreviously for an L148A mutant (2). The L148G mutant (Fig.1C, lane 3) failed to replicate to detectable levels (Fig. 1C, lane3). All three valine mutants (Fig. 1C, lanes 4 to 6) were se-verely impaired for replication, accumulating to ca. 1% ofwild-type TGMV A DNA levels. The same TGMV DNA ac-cumulation patterns were observed when tobacco protoplastswere cotransfected with a TGMV B replicon and plant expres-sion cassettes corresponding to the mutant AL1 proteins andwild-type AL3 (data not shown).

11006 ARGUELLO-ASTORGA ET AL. J. VIROL.

The L148 valine mutants develop symptoms and accumulateviral DNA at variable times after inoculation. Previous studiesshowed that N. benthamiana plants infected with the TGMVAL1 helix 4 mutants KEE146 and L148A develop mild chlo-rosis along the veins 2 to 3 weeks later than plants inoculatedwith wild-type virus, which showed severe stunting, chlorosis,and leaf curling (2, 31). We asked if the glycine and valinesubstitutions at position L148 also impact symptoms in infec-tivity assays. N. benthamiana plants were cobombarded withwild-type TGMV B replicon DNA and either wild-type ormutant A-component DNA, and symptoms were monitoreduntil the plants flowered and set seed at ca. 45 days postinocu-lation (dpi).

Plants bombarded with wild-type TGMV began to show

symptoms at 4 to 5 dpi, with all of the plants displaying severesymptoms by 6 to 7 dpi (Fig. 2). In contrast, none of the plantsinoculated with the glycine or valine mutants showed symp-toms at 6 dpi in three independent experiments. Consistentwith its inability to replicate in tobacco protoplasts, the L148Gmutant did not induce any disease symptoms by 45 dpi (datanot shown). The three valine mutants were infectious but dis-played different kinetics of symptom appearance (Fig. 2).Plants infected with the L148V mutant developed symptomsbetween 8 and 14 dpi, while plants infected with the E146AL148V mutant began to show symptoms between 9 and 21 dpi.Plants infected with the L148V* mutant showed the greatestdelay, with symptoms appearing between 13 and 27 dpi. Theaverage time of symptom appearance was 11.8 dpi for L148V,15.5 dpi for E146A L148V, and 23.2 dpi for L148V*. In allcases, plants inoculated with the valine mutants eventuallydeveloped severe symptoms that were indistinguishable fromthose induced by wild-type TGMV.

To determine if the time of symptom appearance corre-sponded to DNA accumulation for the valine mutants, totalDNA was isolated from newly emerging leaves of inoculatedplants at 7, 14, and 19 dpi and analyzed on agarose gel blotsusing a radiolabeled TGMV A probe. At 7 dpi, high levels ofviral DNA were observed in all plants inoculated with wild-type TGMV (Fig. 3A, lanes 1 and 2), while only one plantinfected with the L148V mutant contained detectable levels ofTGMV A DNA (lanes 3 to 5), and no plants inoculated withL148V* (lanes 8 to 12) or E146A L148A (lanes 13 to 17) had

FIG. 1. L148 mutants are impaired for TGMV AL1 replication.(A) Schematic of the TGMV AL1 protein. Solid boxes mark thelocations of the three motifs conserved among rolling-circle replicationinitiator proteins, the oval indicates a predicted pair of �-helices, andthe stippled box shows the location of the ATP binding motif. Helix 4residues (E146 and L148) that were mutated are indicated. (B) Thesequence between TGMV AL1 amino acids 144 and 154 (helix 4) isshown. E146 and L148 substitutions are shown for the five AL1 mu-tants below the sequence. The codons specifying residues E, A, and Lof helix 4 and the mutations introduced into the three codons areshown on the right (modified nucleotides are indicated by lowercasetype). Mutants L148V and L148V* differ only in the third position ofthe codon. (C) Replication of TGMV AL1 mutants was analyzed intobacco protoplasts by agarose gel blot hybridization. Lanes 1 to 6 aretransfections with TGMV A replicons with either wild-type (wt) (lane1) or mutant AL1 genes corresponding to E146A L148A (lane 2),L148G (lane 3), L148V (lane 4), E146A L148V (lane 5), and L148V*(lane 6). The positions of double-stranded (ds) and single-stranded (ss)forms of TGMV A DNA are marked on the left. An overexposedimage (magnification, �20) of lanes 4 to 6 is shown on the right. Thelevels of replication of the different mutants relative to wild-typeTGMV (100) are indicated at the bottom.

FIG. 2. Symptom appearance is delayed in plants infected withTGMV L148 valine mutants. N. benthamiana plants cobombardedwith either wild-type or mutant TGMV A and wild-type TGMV Breplicons were examined daily for the appearance of symptoms in newgrowth. The dpi when plants displayed unequivocal symptoms (yellowveins and leaf curling) are plotted for each construct. The symbolsrepresent when individual plants displayed symptoms for wild type(�), L148V (E), L148V* (�), and E146A L148V (†). The total num-ber of plants was 12 for the wild type, 8 for L148V, 10 for L148V*, and9 for E146A L148V. The arrows indicate the average time of symptomappearance for the plants infected with each construct. The data sum-marize results from two independent experiments.

VOL. 81, 2007 AL1 REVERTANTS 11007

detectable viral DNA. By 14 dpi, all plants bombarded with theL148V mutant accumulated wild-type levels of viral DNA (Fig.3B, lanes 3 to 7). At 14 dpi, plants infected with the E146AL148V mutant exhibited variable levels of TGMV DNA intheir tissues, ranging from higher-than-wild-type levels (Fig.3B, lanes 15 and 16) to barely detectable levels (lanes 13 and14). In contrast, only one of five plants inoculated with theL148V* mutant accumulated substantial amounts of viralDNA at 14 dpi (Fig. 3B, lane 9), although TGMV A DNA wasdetected at very low levels in two other plants (lanes 8 and 12).At 19 dpi, all plants infected with the E146A L148V mutant(Fig. 3C, lanes 13 to 17) and four of five plants inoculated withthe L148V* mutant exhibited high levels of viral DNA inleaves (lanes 8 to 12). In general, plants with detectableTGMV DNA were symptomatic, with the only exception beingplants with very low DNA levels (Fig. 3B, lanes 12 and 14).These results were unexpected because of the low replicationefficiencies observed for the valine mutants in protoplasts. Theresults also differed significantly from those reported previ-ously for the KEE146 and L148A mutants, which never accu-mulated high levels of viral DNA in infected plants over time(2, 31).

The L148 valine mutations are unstable in infected plants.The variability in symptom appearance and viral DNA accu-mulation and the subsequent development of severe symptomsand high viral DNA levels in plants infected with the L148

valine mutants are consistent with the selection and propaga-tion of a more fit viral variant. We tested this idea by examiningthe mutated region of the AL1 open reading frame in totalDNA extracts from symptomatic young leaves isolated fromplants infected with the mutant viruses. A 280-bp fragmentencoding TGMV AL1 amino acids 106 to 198, which encom-passes the RBR-binding domain (31), was amplified from eightplants infected with the L148V mutant and sequenced directly(Fig. 4A). The L148V codon was modified in all eight plants. Insix plants, a G-A transition resulted in a methionine codon atposition 148 (Fig. 4A). In the remaining two plants, a G-C orG-T transversion was associated with a reversion of the L148Vmutation to leucine.

The bias towards methionine substitutions at L148V wasalso seen in plants inoculated with the E146A L148V mutant(Fig. 4A). In 12 of 13 plants, sequencing uncovered a transitionevent in which GTG was changed to ATG. Reversion of theL148V mutation to leucine as a consequence of a G-T trans-version was seen in only one plant. Interestingly, the E146Amutation in the double mutant was unaltered in all 13 plants(Fig. 4A), indicating that variant selection was highly specificfor the L148V codon.

The L148V* codon was also altered at high efficiency during

FIG. 3. Viral DNA accumulation is delayed in plants infected withTGMV L148 valine mutants. N. benthamiana plants were bombardedwith DNA corresponding to TGMV A and B replicons. The AL1 geneeither was the wild type (wt) (lanes 1 and 2) or carried the L148V(lanes 3 to 7), L148V* (lanes 8 to 12), or E146A L148V (lanes 13 to 17)mutation. For each construct, total DNA was isolated from youngleaves of the same five plants at 7, 14, and 19 dpi and analyzed byagarose gel blot hybridization. The positions of single-stranded (ss)and double-stranded (ds) forms of TGMV A DNA are marked on theleft. ND, not determined.

FIG. 4. TGMV L148 valine mutants revert at high frequency. TotalDNA was isolated from symptomatic leaves of N. benthamiana plantsinfected with mutant TGMV A and wild-type TGMV B replicons at 19dpi. The AL1 coding region between amino acids 120 and 180 wasamplified from individual plants and sequenced directly. (A) Modifi-cations recovered at codon 148 for L148V, L148V*, and E146A L148Vmutants. The original mutations are designated by lowercase type, andthe nucleotide changes in the revertants are shown by uppercase,boldface type. The resulting amino acid, the number of plants, andtime of symptom appearance (dpi) are shown on the right for each typeof revertant. The dagger corresponds to the second-site revertants inB. The total number of plants analyzed for each TGMV mutant isindicated. (B) The TGMV AL1 sequence between amino acids 115and 156 is shown, with the locations of the predicted �-helices and aconserved sequence marked. Mutations in the second-site revertantsare listed on the left, and the amino acid changes are shown below thecorresponding positions.


infection, but the sequence changes differed from those de-tected for the L148V codon (Fig. 4A). The G-A transitionfound at high frequency in the viral progeny of the L148V andE146A L148V mutants occurred only twice in the 10 L148V*mutant-infected plants. In this case, the GTT codon was al-tered to ATT to specify an isoleucine. In six plants, a G-Ctransversion in the first nucleotide of codon 148 took place,resulting in a reversion to leucine (Fig. 4A). Surprisingly, twophenotypic revertants maintained the original L148V* muta-tion but were altered at other nucleotides in adjacent se-quences. One of these second-site revertants displayed a pointmutation at codon 128 that substituted a cysteine residue fortryptophan, whereas the other phenotypic revertant with anintact L148V* codon had two changes that generated the sub-stitutions R125G and I155L (Fig. 4B).

Because only a minority of the valine mutants reverted tothe wild-type leucine residue, we asked if the mutations in thesequenced region of the recovered TGMV AL1 mutants wereresponsible for the observed revertant phenotypes. A fragmentencoding TGMV AL1 amino acids 119 to 180 from cloned andsequenced PCR products corresponding to each revertant classwas subcloned in place of the homologous region in the wild-type TGMV A replicon. The mutant replicons were tested intransient replication assays (Fig. 5). The L148M mutant (Fig.5A, lane 2) replicated more efficiently than wild-type TGMV A(lane 1) in tobacco protoplasts, resulting in four times moreviral DNA. Viral DNA levels corresponding to the L148I (Fig.5A, lane 4) and C128W L148V (lane 5) mutations were lower(32% and 8%, respectively) than those of the wild type (lane 3)but significantly higher than those of the original L148V* mu-tant (Fig. 1C, lane 6). The R125G L148V I155L triple mutant(Fig. 5A, lane 6) did not replicate to readily detected levels inprotoplasts, suggesting that other compensatory mutationsoutside of the subcloned region were responsible for the re-vertant phenotype.

The revertant replicons were also assayed for symptom pro-duction in N. benthamiana infectivity assays (data not shown).Plants inoculated with the L148M and L148I mutants dis-played severe symptoms at 6 dpi. One plant infected with theC128W L148V mutant also showed symptoms at 6 dpi, whilethree plants displayed symptoms by 8 dpi. Plants inoculatedwith the R125G L148V I155L mutant developed wild-typesymptoms at variable times ranging from 13 to 31 dpi, like theother L148V* mutants (Fig. 2). To verify that the observedsymptoms corresponded to viral DNA accumulation, totalDNA was isolated from plants at 7 dpi and analyzed on agarosegel blots using a radiolabeled TGMV A probe. High levels ofdsDNA and ssDNA were seen in plants inoculated with wild-type TGMV (Fig. 5B, lanes 1 to 3) and the L148M mutant(lanes 4 to 6), while lower amounts of viral DNA were detectedfor the L148I (lanes 7 to 9) and C128W L148V (lanes 10 to 12)mutants. None of the plants inoculated with the R125G L148VI155L mutant (Fig. 5B, lanes 13 to 15) had detectable amountsof viral DNA at this time point. Together, these results showedthat the L148M, L148I, and C128W L148V mutations re-stored, at least partially, virus replication and infectivity to thevaline mutants. The absence of detectable viral DNA early ininfection and the development of delayed severe symptoms incombination with the protoplast data suggested that the

R125G L148V I155L mutant replicates poorly and undergoesreversion during infection.

The CaLCuV L145A mutant also reverts at high frequency.CaLCuV is a begomovirus that is distantly related to TGMV.A previous study showed that the mutation of L145 in the helix4 motif of the CaLCuV AL1 protein also impairs RBR inter-actions (2). We asked if an alanine substitution at positionL145 negatively impacts CaLCuV infectivity (Fig. 6A), as re-ported previously for the equivalent TGMV L148A mutation(2). N. benthamiana plants were cobombarded with a wild-typeCaLCuV B replicon and either a wild-type CaLCuV A repli-con or a mutant A component carrying the L145A substitution.By 4 to 5 dpi, plants inoculated with wild-type virus developedclear symptoms that included leaf curling, vein yellowing, andstunting of new growth (data not shown). The symptoms weremore severe than those observed for TGMV-infected plants.In contrast, the five plants inoculated with the CaLCuV L145Amutant did not exhibit any sign of disease at that time. Instead,symptoms appeared in two plants at 15 to 16 dpi, and theremaining plants showed symptoms after 21 dpi. There was a

FIG. 5. Replication analysis of TGMV revertants. (A) Replicationof TGMV A replicons encoding AL1 revertants was analyzed in to-bacco protoplasts by agarose gel blot hybridization. Lanes 1 to 6 aretransfections with TGMV A replicons with either wild-type (wt) (lanes1 and 3) or mutant AL1 genes corresponding to L148M (lane 2), L148I(lane 4), C128W L148V (lane 5), and R125G L148V I155L (lane 6).The position of the double-stranded (ds) TGMV A DNA is marked onthe left, and levels of replication relative to wild-type TGMV (100) ateach exposure are indicated at the bottom of each lane. (B) N.benthamiana plants were cobombarded with wild-type or mutantTGMV A and wild-type TGMV B DNA. At 7 dpi, total DNA wasisolated from three individual plants infected with TGMV B and eitherwild-type TGMV A (lanes 1 to 3) or mutant replicons carrying theL148M (lanes 4 to 6), L148I (lanes 7 to 9), C128W L148V (lanes 10 to12), or R125G L148V I155L (lanes 13 to 15) mutations. DNA accu-mulation was monitored by agarose gel blot hybridization. The posi-tions of single-stranded (ss) and double-stranded forms of TGMV Aare marked on the left.


statistically significant reduction in the overall height of CaLCuV-infected plants (Fig. 6B) compared to mock-inoculated plants.Plants inoculated with CaLCuV L145A were less stunted thanthose infected with wild-type virus but displayed shorter inter-nodes than mock-inoculated plants (Fig. 6B).

The delay observed for the CaLCuV L145A mutant is rem-iniscent of the TGMV L148A mutant, but unlike TGMVL148A, all of the CaLCuV L145A-inoculated plants eventuallydeveloped strong symptoms. To better understand this differ-ence, we examined viral DNA accumulation in plants infectedwith the CaLCuV L145A mutant on agarose gel blots using aradiolabeled CaLCuV A probe (Fig. 6C). At 7 dpi, high levelsof viral DNA were detected in plants inoculated with wild-typevirus (Fig. 6C, lane 1), while none of the plants infected withthe L145A mutant contained detectable levels of viral DNA(lanes 3 to 7). By 16 dpi, three plants infected with the L145Amutant contained detectable levels of viral DNA (Fig. 6C,lanes 8, 9, and 11), and all of the plants were positive for viral

DNA by 25 dpi (lanes 13 to 17). The level of viral DNA at 16 and25 dpi was variable, ranging from high amounts to trace amounts.These results resembled the TGMV DNA accumulation patternsseen for the L148 valine substitutions (Fig. 3), suggesting that theCaLCuV L145A mutation is not stable in plants.

To determine if the L145A mutation is stable, we amplifiedan 854-bp fragment comprising part of the CaLCuV AL1 andAL2 genes out of total DNA from symptomatic young leaves offive plants infected with the mutant. Sequencing of the PCRproducts revealed that the original alanine codon was nolonger present in four plants (Fig. 6D). In three plants, a C-Ttransition had occurred to give a valine codon at position 145.In the fourth plant, a GG-AA double transition produced asynonymous mutation at alanine codon 144 and a nonsynony-mous mutation at position 145, resulting in a threonine substi-tution (Fig. 6D). In a fifth plant, the L145A mutation wasmaintained, but an A-C transversion changed L167 to isoleu-cine (Fig. 6A and D).

FIG. 6. Reversion of the CaLCuV L145A mutation in N. benthamiana. (A) The sequence of wild-type CaLCuV AL1 from amino acids 141 to177 is shown. The location of helix 4 and the L145A mutation is indicated. The † symbol marks the amino acid modified in the second-site revertantL145A I167L in D and E. (B) Comparison of the heights of mock-, CaLCuV (wild-type [wt])-, or L145A (mutant)-inoculated N. benthamiana plantsat 25 dpi. Each point represents an individual plant, with the mean height for each treatment shown at the top. The means of the three treatmentswere statistically different (P � 0.01 in a two-tailed Student’s t test). (C) N. benthamiana plants were cobombarded with a wild-type CaLCuV Breplicon and wild-type CaLCuV A (lanes 1 and 2) or an L145A mutant replicon (lanes 3 to 17). Total DNA from the same five plants at 7, 16,and 25 dpi was analyzed by DNA blot hybridization. The positions of single-stranded (ss) and double-stranded (ds) forms of CaLCuV A DNA aremarked on the left. Viral DNA was detected only in plants displaying symptoms. (D) The AL1 coding region between amino acids 132 and 349was amplified from individual plants and sequenced directly. The original mutation is designated by lowercase type, and the nucleotide changesin the revertants are shown by uppercase, boldface type. Numbers of plants are shown on the right for each type of revertant for bombardment(gun) and agroinoculation (agro) experiments. The total number of plants analyzed for each inoculation protocol is indicated below. (E) Repli-cation of CaLCuV A mutants in tobacco protoplasts was analyzed by agarose gel blot hybridization. Lanes 1 to 6 correspond to transfections withCaLCuV A replicons with either wild-type (lane 1 and 3) or mutant AL1 genes corresponding to L145A (lane 2), L145V (lane 4), L145A I167L(lane 5), and L145T (lane 6). The relative accumulation of viral DNAs is given at the bottom of each lane, with the wild type set at 100 for eachexposure.


The preferential reversion of CaLCuV L145A to valine wasunexpected because a valine substitution at L148 in TGMV AL1severely impaired replication and infectivity. The CaLCuV andTGMV AL1 proteins show significant divergence in the resi-dues flanking the helix 4 motif, and substitutions at the con-served leucine may differentially impact the replication of thetwo viruses. To test this possibility, a fragment containingCaLCuV AL1 amino acids 132 to 349 derived from PCR prod-ucts of each revertant class was subcloned in place of thehomologous region in the wild-type CaLCuV A replicon andsequenced prior to transient replication assays (Fig. 6E). TheCaLCuV L145V (Fig. 6E, lane 4) and L145A I167L (lane 5)mutants replicated similarly in tobacco protoplasts, accumulat-ing to ca. half of wild-type DNA levels (lane 3). The CaLCuVL145T mutant (Fig. 6E, lane 6) replicated less efficiently, re-sulting in only 8% of wild-type levels. The L145A (Fig. 6E, lane6) mutant was the most severely impaired for replication, ac-cumulating at 1% of wild-type levels. This value is significantlyless than that reported previously for TGMV L148A, whichreplicates to 14% of wild-type levels (2), and more similar tothat seen for the TGMV L148 valine mutations (Fig. 1C).Consistent with their replication phenotypes, the subclonedCaLCuV L145V, L145A I167L, and L145T replicons wereinfectious on plants (data not shown).

N. benthamiana plants agroinoculated with wild-typeCaLCuV developed severe symptoms indistinguishable fromthose seen with bombardment (data not shown). The timing ofsymptom appearance differed between the two protocols, withthe agroinoculated plants displaying symptoms 10 to 12 dpi,compared to 4 to 5 dpi for bombarded plants. However, like

the bombardment experiments, plants agroinoculated with theCaLCuV L145A mutant were delayed relative to the wild-typecontrol. None of the L145A-inoculated plants exhibited symp-toms at 12 dpi, two plants showed signs of disease at 21 dpi,four additional plants displayed symptoms at 23 to 25 dpi, andall nine plants exhibited severe symptoms by 33 dpi.

PCR amplification of viral DNA followed by direct sequenc-ing revealed that the engineered alanine codon was altered inmost plants (Fig. 6D). In four plants, the original GCC alaninecodon was changed to a GCT valine codon, while a mixture ofthe two codons was detected in two plants. One plant con-tained a mixture corresponding to the original GCC codon anda new GTC codon specifying threonine. Another plant re-tained the original GCC codon but had a second-site L167Imutation, the same second-site reversion found in the bom-bardment experiment. These results indicated that CaLCuVL145A reversion is not dependent on the inoculation protocolfor N. benthamiana.

Different CaLCuV L145A reversions are recovered fromArabidopsis plants. Although CaLCuV infects N. benthamiana,its natural hosts are members of the family Brassicaceae. Wetook advantage of the ability of CaLCuV to infect Arabidopsisthaliana to ask if the plant host can influence the frequency orthe nature of reversion. Arabidopsis plants were agroinoculatedwith a wild-type CaLCuV B replicon and either a wild-typeCaLCuV A replicon or a mutant A component carrying theL145A substitution. At 12 to 15 dpi, all of the plants infectedwith wild-type CaLCuV developed strong symptoms character-ized by yellowing, leaf curling, and severe stunting of newgrowth. In contrast, none of the 12 plants inoculated with the

FIG. 7. Reversion of the CaLCuV L145A mutation in Arabidopsis plants. A. thaliana plants agroinoculated with a wild-type CaLCuV B repliconand wild-type CaLCuV A or an L145A mutant replicon are shown. (A) Mock (left), wild-type (middle), and L145A (right) symptoms at 20 dpi.(B) L145A symptoms at 29 dpi. (C) The AL1 coding region between amino acids 132 and 349 was amplified from individual plants and sequenceddirectly. The original mutations are designated by lowercase type, and the nucleotide changes in the revertants are shown by uppercase, boldfacetype. The altered amino acid and the numbers of plants are shown on the right for each type of revertant. The total number of plants analyzedis indicated below. (D) Total DNA was isolated from plants at 29 dpi and analyzed by agarose gel blot hybridization. The reversion at L145A isindicated at the top of each lane. The position of double-stranded (ds) CaLCuV A DNA is marked on the left.


L145A mutant exhibited symptoms at this time. By 20 dpi, oneplant showed mild symptoms, and three additional plants dis-played signs of disease by 25 dpi (Fig. 7A). Over the next 8days, all of the L145A-inoculated plants developed strongsymptoms in leaves and flowers (Fig. 7B). These results sug-gested that the CaLCuV L145A mutation is also unstable inArabidopsis plants.

An 854-bp viral fragment was amplified from total DNAisolated from symptomatic young leaves of CaLCuV L145A-inoculated Arabidopsis plants at 29 dpi, and the PCR productswere sequenced directly. In six plants, a G-A transition at thefirst nucleotide position changed the engineered alanine codon(GCC) to a threonine codon (ACC) (Fig. 7C). In one plant, aC-T transition at the second nucleotide position resulted in avaline codon (GTC). A double substitution changing GCC tothe leucine codon CTC was recovered from a single plant. Infour cases, the recovered sequence was a mixture of the engi-neered GCC codon and the revertant ACC codon, indicativeof the presence of two viral variants in the same plant. To-gether, these results demonstrated a preference for the A145Treversion (10 out 12), with the A145V and A145L reversionsoccurring at reduced frequency (Fig. 7C). This is in contrast todata for N. benthamiana (Fig. 6D) showing that the A145Vreversion occurs more frequently (9/14) than the A145T event(3/14).

The same DNA samples were analyzed by agarose gel blot-ting using a radiolabeled CaLCuV A-specific probe. A bandcorresponding to the double-stranded form of CaLCuV A wasobserved in 10 of the 12 plants (Fig. 7D), while no single-stranded DNA was detected (data not shown). The levels ofdouble-stranded viral DNA varied between plants and did notcorrelate with revertant type. CaLCuV replication assays incultured Arabidopsis cells were not successful (data notshown), so it was not possible to distinguish the impact ofreplication efficiency and the time of reversion on the fre-quency and accumulation of the revertants. However, theCaLCuV data demonstrated that the plant host influencesthe outcome of the reversion process but not the overallfrequency, which was 100% in all instances.

DISCUSSION

It is generally thought that nucleotide misincorporation doesnot contribute significantly to the genomic variation of smallDNA viruses that are replicated by cellular DNA polymerases(15). This assumption is supported by long-term mutation ratesfor dsDNA viruses, which are low and comparable to thosemeasured for cellular genes (5). There are numerous reports ofthe emergence of geminivirus strains with altered pathogenic-ity (62), indicative of rapid genetic change that has been at-tributed to recombination or reassortment among differentviral genomes. However, our finding that mutations in the helix4 motif of the AL1 gene of two distantly related begomovirusesrevert at 100% frequency suggests that nucleotide substitutionsoccur with high incidence and are under strong selective pres-sure during geminivirus infection. Thus, in agreement withrecent reports of high mutation rates for other ssDNA virusesinfecting vertebrates and bacteria (51, 64, 65), nucleotide sub-stitution events are likely to contribute to the diversity andrapid evolution of geminivirus ssDNA genomes.

Analysis of TGMV and CaLCuV variants with mutations atthe equivalent L148 and L145 residues in their respective helix4 motifs revealed several features of the nucleotide substitu-tion process during geminivirus infection. First, the process ishighly efficient, with reversions occurring in 100% of plantsinfected with either one of the TGMV L148 valine mutants(L148V, L148V*, and E146A L148V) or the CaLCuV L145Amutant. Second, the process is selective for mutations thatimpair protein function. The E146A mutation, which has nodetectable impact on AL1 function (2), was stable even whenthe valine residue reverted in the E146A L148V mutant. Last,the frequency of a reversion event reflected the number ofnucleotide changes required to generate a given amino acidcodon. L148V, which has a GTG valine codon, reverted to anATG methionine codon via a single nucleotide change in 18 of21 events. The generation of an ATG codon from L148V*,with a GTT valine codon, would have required two changesand was not recovered. Instead, the most common change (6 of10 events) was to a CTT leucine codon, which also involved asingle nucleotide change. Similarly, the low recovery of leucineor methionine revertants (1 of 25) from CaLCuV L145A-in-fected plants most likely reflected the need for multiple nucle-otide changes to generate the requisite codons.

Comparison of TGMV and CaLCuV revertants also uncov-ered some important differences. The CaLCuV L145A mutantwas unstable during infection, while the equivalent L148A mu-tant of TGMV AL1 was stable (2). Valine was the most fre-quent reversion recovered from N. benthamiana plants inocu-lated with the CaLCuV L145A mutant, while TGMV L148Vmutants were unstable during infection. These differences can-not be attributed to host effects because N. benthamiana servedas the host for both viruses. CaLCuV AL1 is representative ofa small group of replication proteins in the SLCV group thatlack the conserved element DGRSARGG(C/Q)Q (3). Inter-estingly, the second-site mutation C125W mapped to this se-quence in TGMV AL1. The CaLCuV L145V mutant repli-cated efficiently in cultured cells (46% of wild-type levels),while a TGMV L148V mutant replicated poorly (1% of wild-type levels). An I167L second-site revertant of CaLCuVL145A, which was recovered twice, also replicated efficiently inculture. Leucine occurs at the equivalent position in TGMVand is the most common residue at this site in other begomo-virus replication proteins except for members of the SLCVgroup, which have branched aliphatic residues (our unpub-lished observation). However, a TGMV L148A mutant accu-mulates to only 13% of the wild-type level in cultured cells,while a CaLCuV L145A I167L mutant accumulates to 47% ofwild-type levels, suggesting that different sequence constraintson TGMV and CaLCuV AL1 impact virus stability and rever-tant selection during infection.

The different fates of the CaLCuV L145A mutant in N.benthamiana and Arabidopsis indicated that the plant host alsoinfluences the reversion outcome. Like the experiments with N.benthamiana plants, the CaLCuV L145A mutant was unstablein Arabidopsis and reverted at 100% frequency. However, mostrevertants (10 of 12) contained a threonine substitution atcodon 145 in CaLCuV AL1, in contrast to the valine revertantsisolated from N. benthamiana plants. The L145T mutant accu-mulated to 7% of wild-type levels in BY-2 protoplasts, suggest-ing that the threonine mutant does not replicate efficiently in


Nicotiana species, consistent with it being isolated only oncefrom N. benthamiana. The preferential isolation of CaLCuVL145T from Arabidopsis may be indicative of efficient replica-tion in this host. The selection of a valine or a threoninerevertant of CaLCuV L145A may reflect different sequencerequirements for an AL1/host protein interaction in N.benthamiana versus Arabidopsis. Although no studies havelinked the begomovirus AL1 protein to host range (49, 52), thereversion of a tomato yellow leaf curl virus C4 mutant intomato but not in N. benthamiana has been attributed to dif-ferent host constraints on systemic movement (29). Mastrevi-rus replication proteins have also been implicated in hostadaptation (33). Interestingly, partial reversion of a three-nu-cleotide mutation in the RepA coding sequence also occurs at100% frequency during maize streak virus (MSV) infection(67). Unlike the TGMV and CaLCuV reversions, the MSVreversion was restricted to a single nucleotide transversionevent that restored nucleic acid folding but not RepA function.

The 100% reversion frequencies for TGMV, CaLCuV, andMSV mutants and the isolation of second-site revertants implythat the family Geminiviridae is subject to high rates of nucle-otide substitution. The high rates may reflect a failure of gemi-nivirus infection to activate the mismatch repair system, whichis responsible for the excision and replacement of misincorpo-rated nucleotides during chromosomal replication. Methylatedviral DNA is not a good template for replication and transcrip-tion (8, 18), and geminiviruses actively interfere with DNAmethylation pathways in infected cells (70). As a consequence,viral DNA is undermethylated, making it difficult for the mis-match repair machinery to distinguish between parental andnascent DNA strands. In addition, gene profiling experimentsindicated that although other DNA repair pathways are up-regulated during geminivirus infection, the expression of mis-match repair machinery is not increased in CaLCuV-infectedArabidopsis plants (J. T. Ascencio-Ibanez and L. Hanley-Bow-doin, unpublished result). Together, these results indicatedthat geminivirus replication products are not corrected by mis-match repair, increasing the likelihood that a mutation will befixed. Greater genetic variability might facilitate geminivirusadaptation to new hosts and changing environments, ultimatelyleading to increased viral fitness (57). The failure to recoverthe TGMV L148V and CaLCuV L145A mutants from mostplants suggested that the less fit mutant A component is lostrandomly during viral movement, ultimately leading to its dis-appearance from the population. The 7 of 26 plants carryingmixtures of the CaLCuV L145A mutant and various revertantsmay reflect intermediates in this process.

TGMV L148 is located in an 80-amino-acid region of AL1known to mediate oligomerization and binding to AL3, RBR,and other host factors (4, 10, 30, 31, 47, 63). There was noobvious correlation between the effects of the various L148mutations on RBR binding and viral replication, as illustratedby comparisons of the relative RBR binding (25, 31, and 36%)and replication (13, 1, and 0%) activities of L148A, L148V andL148G, respectively (2). In addition, AL1 oligomerization ac-tivity, which is required for viral replication, is only moderatelyreduced for the L148A, L148V, and L148G mutants (2). Thus,the instability of the valine mutants is not due to reducedoligomerization or RBR binding and instead may reflect thedestabilization of the AL1 protein or impaired interactions

with a host factor required for viral replication. Strikingly, onlyleucine, methionine, and isoleucine revertants were recovered,indicating that only a few amino acids are permissible at theL148 position. All three amino acids have large hydrophobicside chains and high probabilities of occurring in �-helices.TGMV AL1 proteins carrying either leucine or methioninedisplay similar functional properties, while the isoleucine vari-ant is moderately reduced in activity. Interestingly, leucine andmethionine are the only amino acids found at the equivalentposition in the helix 4 motif of all characterized begomovirusand curtovirus replication proteins (data not shown).

An important consequence of high mutation and recombi-nation rates is the continuous production of genetic variationin geminivirus populations. This variability is balanced by acomplex set of selection pressures including those associatedwith intrinsic properties of the virus, such as the maintenanceof essential nucleotide structures and replication signals, andselection pressures to maintain crucial interactions with planthosts and insect vectors. Thus, despite their variation potential,geminiviruses populations exhibit significant genetic stabilityover time and space, as has been documented for plant RNAviruses that also display high mutation rates (25, 57). None-theless, the evolutionary potential of geminiviruses needs to beconsidered in long-term control strategies, because any diseasemanagement effort will result in selective pressure on the viruspopulation to adapt to new circumstances (38, 57). A recentmathematical analysis of the potential impact of disease con-trol strategies concluded that the use of resistant cultivars withreduced within-plant virus titers puts pressure on the targetvirus to evolve towards a higher multiplication rate (69). Theresults reported here demonstrated experimentally that gemi-nivirus variants with residual replication capabilities are understrong selective pressure to generate variants that replicate tohigh titers. Given the large size and genetic heterogeneity ofgeminivirus populations and their capacity to rapidly changetheir genomes by recombination and mutation, it will be nec-essary to devise resistance strategies that prevent virus repli-cation and not simply reduce it because of the risk of gener-ating more harmful variants that overcome resistance.

ACKNOWLEDGMENTS

This research was supported by a grant from the National ScienceFoundation (MCB-0110536 to L.H.-B.) and a postdoctoral fellowshipfrom the PEW Foundation (P0291SC to G.A.-A.).

We thank Marilyn Roossinck (The Samuel Roberts Noble Founda-tion), Dominique Robertson, Sharon Settlage, and Luisa Lopez-Ochoa(all at NCSU) for their critical comments on the manuscript.

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JOURNAL OF VIROLOGY, May 2004, p. 4817–4826 Vol. 78, No. 90022-538X/04/$08.00�0 DOI: 10.1128/JVI.78.9.4817–4826.2004Copyright © 2004, American Society for Microbiology. All Rights Reserved.

A Novel Motif in Geminivirus Replication Proteins Interacts with thePlant Retinoblastoma-Related Protein

Gerardo Arguello-Astorga,† Luisa Lopez-Ochoa, Ling-Jie Kong,‡ Beverly M. Orozco,§Sharon B. Settlage, and Linda Hanley-Bowdoin*

Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, North Carolina 27695-7622

Received 22 October 2003/Accepted 9 December 2003

The geminivirus replication factor AL1 interacts with the plant retinoblastoma-related protein (pRBR) tomodulate host gene expression. The AL1 protein of tomato golden mosaic virus (TGMV) binds to pRBRthrough an 80-amino-acid region that contains two highly predicted �-helices designated 3 and 4. Earlierstudies suggested that the helix 4 motif, whose amino acid sequence is strongly conserved across geminivirusreplication proteins, plays a role in pRBR binding. We generated a series of alanine substitutions across helix4 of TGMV AL1 and examined their impact on pRBR binding using yeast two-hybrid assays. These experimentsshowed that several helix 4 residues are essential for efficient pRBR binding, with a critical residue being aleucine at position 148 in the middle of the motif. Various amino acid substitutions at leucine-148 indicatedthat both structural and side chain components contribute to pRBR binding. The replication proteins of thegeminiviruses tomato yellow leaf curl virus and cabbage leaf curl virus (CaLCuV) also bound to pRBR in yeastdihybrid assays. Mutation of the leucine residue in helix 4 of CaLCuV AL1 reduced binding. Together, theseresults suggest that helix 4 and the conserved leucine residue are part of a pRBR-binding interface inbegomovirus replication proteins.

Geminiviruses are DNA viruses that replicate their single-stranded genomes through double-stranded intermediates innuclei of plant cells (reviewed in reference 28). Because oftheir small genomes, geminiviruses provide only the factorsrequired to initiate rolling circle replication and depend onplant nuclear DNA polymerases to amplify their genomes.Many geminiviruses replicate in differentiated cells that haveexited the cell cycle and no longer contain detectable levels ofhost DNA polymerases and associated factors (10, 44, 47).Geminiviruses are thought to overcome this barrier by inter-acting with a host protein, the retinoblastoma-related protein(pRBR), to induce transcription of genes encoding host repli-cative enzymes (17, 18, 38). Hence, geminiviruses are valuabletools for studying how the cell division cycle and differentiationare regulated by pRBR in plants (26).

Geminiviruses are a diverse family of plant-infecting virusesthat fall into four genera based on their genome structure,insect vector, and host range (6, 58). Tomato golden mosaicvirus (TGMV) is a typical begomovirus with a bipartite ge-nome that encodes seven proteins (4). AL1 (also designatedAC1, C1, or Rep) is the only viral protein that is essential forreplication (19). AL1 recognizes the origin by binding to aspecific DNA sequence (21), catalyzes DNA cleavage and li-gation to initiate and terminate rolling circle replication (40,

52), and may act as a DNA helicase during the elongationphase of replication (24, 54). AL1 also plays a central role inreprogramming mature plant cells to support DNA replicationand recruiting host replication machinery to the origin (38, 45).

Many functions of AL1 involve protein-protein interactions.Oligomerization is essential for origin recognition and may berequired for other catalytic functions in vivo (51, 60). AL1binds to the viral replication enhancer protein, AL3 (60),which in turn binds to proliferating cell nuclear antigen(PCNA), the processivity factor for DNA polymerase � (8).Geminivirus replication proteins also interact with componentsof the host replication apparatus, like PCNA and the replica-tion factor C complex, the clamp loader that transfers PCNAto the replication fork (8, 45). These interactions are likely torepresent early steps in the assembly of a DNA replicationcomplex on the geminivirus origin. AL1 also interacts with hostfactors involved in cell division and differentiation. AL1 bindsto a mitotic kinesin and a protein kinase associated with earlyleaf development (37), but the roles of these interactions dur-ing infection have not been determined. In contrast, there isevidence that binding of AL1 to pRBR alters host transcrip-tional controls to induce the synthesis of the plant DNA rep-lication machinery (17, 18, 38).

In animals, the retinoblastoma protein (pRb) is part of asmall family that also includes p107 and p130 (30). pRb familymembers negatively regulate cell cycle progression and pro-mote differentiation, in part, through their interactions withE2F transcription factors and histone deacetylase (61). Theseinteractions repress transcription of genes encoding proteinsrequired for entry into S phase and DNA replication (41) andfacilitate maintenance of a differentiated state (29). In late G1,phosphorylation of pRb by cyclin-dependent kinases disruptsits association with E2F and allows expression of genes re-quired for S phase (35). Mammalian DNA tumor viruses by-

* Corresponding author. Mailing address: North Carolina StateUniversity, Department of Molecular and Structural Biochemistry, 128Polk Hall, Box 7622, Raleigh, NC 27695. Phone: (919) 515-6663. Fax:(919) 515-2047. E-mail: [email protected].

† Present address: Departamento de Biología Molecular, Institutode Potosino de Investigaciones Científicas y Tecnologicas, 78216 SanLuis Potosí, SLP, Mexico.

‡ Present address: Department of Genetics, Howard Hughes Med-ical Institute, Duke University Medical Center, Durham, NC 27710.

§ Present address: Bayer Corporation, Clayton, NC 27520.

4817

pass this phosphorylation requirement by binding directly topRb and disrupting its interaction with E2F (34). Several linesof evidence argue that the pRb/E2F regulatory network isconserved in plants. pRBR and E2F proteins have been iden-tified in a variety of plant species (for reviews, see references12, 15, and 27). pRBR is a substrate of cyclin-dependent ki-nases (5, 48), and its levels are high in differentiated tissues(33). E2F consensus sites have been found in the promoters ofa number of plant genes (55) and shown to modulate tran-scription during the cell division cycle and development (7, 9,11, 17, 39, 62). Begomoviruses are thought to overcome E2F-mediated repression of PCNA transcription in mature leaves(18), most likely through the interaction of AL1 with pRBR(38).

pRb family members display strong sequence homologyacross a large central domain known as the A/B pocket (46).This region is involved in a variety of protein interactions (63),including those with simian virus 40 large T antigen, Adeno-virus E1A and human papillomavirus E7 (42). Each of theseDNA tumor virus proteins binds to pRb through a conservedLXCXE motif (16). LXCXE motifs have also been identifiedin pRBR-binding proteins, like cyclin D (49), the mastrevirusRepA protein (25, 32, 43, 66), and the nanovirus Clink protein(3). However, there are plant proteins, including some E2Ftranscription factors (11, 14) and chromatin-remodeling pro-teins (2, 56, 57), that interact with pRBR through other se-quences. TGMV AL1 does not have an LXCXE motif and fallsinto this second group of pRBR binding proteins. A previousstudy mapped the pRBR binding domain of AL1 betweenamino acids 101 to 180 and showed that mutations in thisregion impact AL1/pRBR interactions and tissue specificity ofinfection (38). In the experiments described here, we asked if

a highly conserved motif within this region plays a role inpRBR binding and virus infection.

MATERIALS AND METHODS

AL1 mutants. The plasmid pNSB148, which contains the TGMV AL1 codingsequence in a pUC118 background, was used as the template for site-directedmutagenesis as described previously (51). The oligonucleotide primers and re-sulting clones are listed in Table 1. Viral DNA fragments containing the muta-tions were verified by DNA sequence analysis. Viral replicons encoding themutant AL1 proteins were generated by subcloning SalI/NheI fragments corre-sponding to ALI amino acids 120 to 232 from the mutant clones into the samesites of the wild-type replicon, pMON1565 (52), to give pNSB954 (K144),pNSB1032 (EE146), pNSB999 (A147Y), pNSB997 (L148), pNSB979 (L148V),pNSB1000 (L148G), and pNSB998 (II151). Alanine substitution mutants aredesignated with the wild-type amino acid(s) and the final position number. Whenthe mutation was not to alanine, the mutant amino acid is given after the positionnumber.

Site-directed mutagenesis of cabbage leaf curl virus (CaLCuV) AL1 was per-formed by using a PCR-based method. Complementary oligonucleotides Cb-M1and Cb-M2 (Table 1) were used as primers in combination with the M13 uni-versal and reverse primers, respectively, in two separate amplification reactionswith pNSB1085 as a template. pNSB1085 contains a CaLCuV A DNA fragment(positions 1499 to 33) carrying the AL1 coding sequence in a pUC118 back-ground. The PCR products of Cb-M1/M13-universal and Cb-M2/M13-reverseamplification reactions were isolated from agarose gels and pooled for a newamplification reaction using M13-universal and reverse primers. The PCR prod-uct was digested with BamHI and BglII and cloned into the same sites of amodified pUC118 to give pNSB1097.

Yeast two-hybrid assays. Yeast cassettes with the Gal4 DNA binding domain(DBD) were generated with pAS2-1 (Clontech, Palo Alto, Calif.). Zm214Cincludes a truncated maize RBR1 coding sequence consisting of the pocket andC-terminal domains fused to the DBD (1). pNSB736 contains a full-length,wild-type TGMV AL1-DBD fusion (53).

Yeast cassettes containing the Gal4 activation domain (AD) were generatedby using pACT2 (Clontech). Cassettes for wild-type TGMV AL1 (pNSB809) andthe mutants KEE146 (pNSB894) and REK154 (pNSB759) have been describedelsewhere (53). Cassettes for the TGMV mutants K144 (pNSB916), E145(pNSB917), E146 (pNSB975), EE146 (pNSB1040), A147Y (pNSB1003), L148

TABLE 1. Constructs

Protein and mutation Mutagenesis oligonucleotidesaYeast dihybrid vector

Viral repliconGal4 AD-AL1 Gal4 BD

TGMV AL1 pNSB809 pNSB736 pMON1565KEE146b pNSB894K144 TCTGCAGGGCTTCTTCcgcGGAAGAAGCATTTAA pNSB916 pNSB954E145 TCTGCAGGGCcTCTgCTTTGGAAGAAGCATTTAA pNSB917E146 CTGCAGGGCTgCTTCTTTGGAAGAAGCA pNSB975EE146 TCTGCAGGGCggCcgCTTTGGAAGAAGCATTTAAC pNSB1040 pNSB1032A147Y TAATTATCTGaAGGtaTTCTTCTTTGGAAGAAGCATTTAA pNSB1003 pNSB999L148 TAATTATCTGCgcaGCTTCTTCTTTGGAAGAAGCATTTAA pNSB1001 pNSB997L148V ATTATCTGCAcGGCcTCTTCTTTGGAAGAAGCATTTAA pNSB980 pNSB979L148M pNSB979 revertant pNSB1112L148G CTAATTATCTGgccGGCTTCTTCTTTGGAAGAAGCATTTA pNSB1004 pNSB1000L148I pNSB1030 revertant pNSB1079I1151 TTTCTCTCTAgcTgcCTGaAGGGCTTCTTCTTTGGAAGA pNSB1002 pNSB998REK154b pNSB759

CbLCV AL1 pNSB901 pNSB909Cb1-207 pNSB974Cb1-207, L145A GTGTGGAAGAGGCGgccGCAATTATAAGGGC pNSB1114

TYLCV-DR C1 pTYLC102 pTYLC103

Maize RBR1 214C

a Lowercase letters designate mutated residues.b Data from reference 53.


(pNSB1001), L148V (pNSB980), L148G (pNSB1004), and II151 (pNSB1002)were made by replacing the AatII-BamHI fragment of pNSB735 (53) with theequivalent fragments from pNSB946, pNSB945, pNSB968, pNSB1033,pNSB995, pNSB993, pNSB969, pNSB996, and pNSB994, respectively. Cassettesfor L148M (pNSB1112) and L148I (pNSB1079) were generated by the samestrategy using revertant replicons that were cloned out of plants inoculated witha wild-type B replicon and a mutant A replicon corresponding to pNSB979 orpNSB1030, respectively.

The C1 open reading frame was amplified from a tomato yellow leaf curl virus(TYLCV-DR [Dominican Republic isolate]) genomic clone (a gift from Mon-santo) using the oligonucleotides, 5�-GGACACCGATTggaTcCAgCATGCCTCand CCACAGTCgAatTCCCCggGCTTACGC. (The lowercase letters indicatemutated nucleotides.) The resulting PCR product (positions 1686 to 12 of theTYLCV-DR genome) was digested with BamHI and SmaI and cloned intopUC119 to give pTYLC78. The corresponding yeast AD vector was constructedby cloning the BamHI (trimmed)/SmaI C1 fragment from pTYLC78 into pACT2digested with SmaI to give pTYLC102.

pCPCaLCuVA.001, which contains a single copy of CaLCuV A DNA (64), wasmodified by PCR mutagenesis using the oligonucleotide 5�-CCTAAATAagatcTACAAGgATcCCACGAAACCCTA to introduce a BamHI site at the 5� end ofthe AL1 open reading frame. The resulting clone, pNSB900, was digested withBamHI, and the fragment containing the full-length AL1 sequence was clonedinto the BamHI site of pACTII to give pNSB958. The NcoI fragment frompNSB958 encoding amino acids 2 to 178 of AL1 was then subcloned into pACTIIto give pNSB974. The L145A mutation was introduced into the CaLCuV AL1coding sequence of pNSB900 by using the oligonucleotide 5�-GTGTGGAAGAGGCGgccGCAATTATAAGGGC for PCR mutagenesis. A mutant frag-ment from the resulting plasmid (pNSB1097) with AatII/NcoI (repaired) endswas subcloned into pNSB974 with AatII/XhoI (repaired) ends to give pNSB1114.

Interactions between the Gal4 fusion proteins were evaluated in Saccharomy-ces cerevisiae strain Y187 by measuring �-galactosidase activity as describedpreviously (53). Protein concentrations were measured by Bradford assays (Bio-Rad, Hercules, Calif.). The enzyme specific activity (1 U � 1.0 �M product/minat pH 7.3 and 37°C) was determined by using purified �-galactosidase (Sigma, St.Louis, Mo.) as the standard. The different constructs were tested in a minimumof four independent transformants in at least two experiments. The relativeactivities of the mutant proteins were normalized against wild-type AL1, whichwas set to 100%.

Replication and infectivity assays. TGMV A replicons carrying mutant AL1coding sequences were made using pMON1565, a pUC-based plasmid that con-tains 1.5 copies of wild-type TGMV A DNA (52). The mutant replicons in Table1 were generated by replacing the SalI/NheI fragment encoding AL1 amino acids120 to 313 of pMON1565 with the equivalent fragment of mutant AL1 openreading frames described above. pTG1.4B, which includes 1.4 copies of wild-typeTGMV B, has been described previously (21).

For replication assays, protoplasts were isolated from Nicotiana tabacum(BY-2) suspension cells, electroporated, and cultured according to publishedmethods (20). Cells were transfected with 5 �g of wild-type or mutant TGMV Areplicon DNA and 25 �g of sheared salmon sperm DNA. Total DNA wasextracted 72 h after transfection, digested with DpnI and XhoI, and examined fordouble- and single-stranded viral DNA accumulation by DNA gel blot analysisusing a TGMV A specific probe (21). Viral DNA was quantified by phospho-rimage analysis. Each protoplast assay was performed in at least three indepen-dent experiments.

For infectivity assays, Nicotiana benthamiana plants at the six-leaf stage wereinoculated with a biolistics device as described previously (47). Wild-type ormutant TGMV A replicon DNA (10 �g) was precipitated onto 1-�m micro-projectiles in the presence of a wild-type TGMV B replicon (pTG1.4B) and usedto bombard plants. Total DNA was extracted from leaf tissue 14 days afterbombardment (13), linearized with XhoI, and analyzed on DNA gel blots.

RESULTS

Mutations in the KEE sequence of AL1 differentially alterpRBR binding. The geminivirus TGMV interacts with pRBR,a plant homolog of the mammalian Rb protein, via a novelmechanism. An earlier study showed that mutations inKEE146 in the pRBR binding domain of TGMV AL1 impairinteraction (38). The KEE146 mutant contains three consecu-tive alanine substitutions, which may act alone or together toconfer the mutant phenotype. To better understand the basis

of the phenotype, we generated four additional alanine substi-tution mutants: K144, E145, E146, and EE146 (Fig. 1A). Themutant AL1 open reading frames were fused to the codingsequence of the Gal4 AD and expressed in yeast. Mutantproteins accumulated to levels comparable to those of an AD–wild-type AL1 fusion (data not shown), consistent with previ-ous experiments showing that mutations in this region do notimpact AL1 fusion protein stability in yeast (53). The AL1oligomerization properties of the mutant proteins were as-sessed in two-hybrid assays using a wild-type TGMV AL1protein fused to the Gal4 DBD (53). The influence of the

FIG. 1. Mutations in the helix 4 motif of AL1 impair interactionswith pRBR. (A) Schematic of TGMV AL1. Solid boxes mark thelocation of the three motifs conserved among RCR initiator proteins,the oval indicates a predicted set of �-helices, and the stippled boxshows the location of the ATP binding domain. The AL1 sequencebetween amino acids 132 to 156 is shown, with the locations of thepredicted �-helices 3 and 4 indicated. Vertical lines mark the positionsof the alanine substitutions. Mutations are designated by the corre-sponding wild-type sequence and the position of the last amino acidthat was modified. (B) Yeast was cotransformed with an expressioncassette (Zm214C) encoding amino acids 214 to the C terminus ofZmRBR fused to the Gal4 DBD and cassettes for either wild-type ormutant AL1 fused to the Gal4 AD. Protein interactions were assayedby measuring �-galactosidase activity in total protein extracts andnormalized to wild-type values (100%). Filled bars indicate mutantsstrongly impaired in pRBR binding, grey bars mark moderately im-paired mutants, and open bars indicate mutants with activity similar toor greater than that of wild-type AL1. Error bars correspond to 2standard errors. The effects of the mutations on AL1/AL1 interactions(oligomerization activity) are indicated on the right.

VOL. 78, 2004 AL1/pRBR INTERACTIONS 4819

mutations on AL1/AL1 interactions was minimal (Fig. 1B) andnot statistically significant, indicating that the mutations do nothave a global effect on AL1 function. However, technical con-straints precluded testing the mutants in AL1/AL3 bindingassays.

The impact of the mutations on AL1/pRBR interactions wasanalyzed in two hybrid assays. Previous studies established thatTGMV AL1 interacts with maize and Arabidopsis RBR pro-teins similarly but that binding to maize pRBR is stronger inyeast assays (1, 38). As a consequence, we used a truncatedversion (Zm214C) of maize pRBR1 from amino acid 214 tothe C terminus fused to the Gal4 DBD (1, 38) for these ex-periments. This region contains the A/B pocket domain andthe C-terminal domain of pRBR. The E145, E146, and EE146mutations did not alter AL1/pRBR binding significantly (Fig.1B), suggesting that the E residues are not essential for wild-type binding activity. In contrast, the K144 mutation reducedpRBR interactions to 42% of the wild-type levels (Fig. 1B),indicating that this residue is required for full binding activity.However, the reduction in pRBR binding activity was less forK144 than KEE146 (16% of wild-type levels; Fig. 1B), uncov-ering a role for one or both of the E residues in combinationwith K144 in pRBR interaction.

Mutations in the helix 4 motif of AL1 impair pRBR inter-actions. The KEE sequence constitutes the first three residuesof an 11-amino-acid motif designated helix 4 (Fig. 1A; also, seeFig. 5). The helix 4 motif is conserved across all begomovirus,curtovirus, and topocuvirus replication proteins and mastrevi-rus RepA/Rep proteins with respect to both amino acid se-quence and predicted �-helical structure (53). Because of thisstrong conservation, we hypothesized that other residues inhelix 4 also contribute to pRBR binding. This hypothesis issupported by previous results showing that the pRBR bindingactivity of an REK154 mutant, which contains three alaninesubstitutions in the last three amino acids of the motif, isreduced twofold (Fig. 1B) (38). To further explore the role ofhelix 4 in pRBR binding, we generated alanine substitutions atthe conserved L148 and II151 residues and a tyrosine substi-tution at the invariant A147 position to mimic the conservedtyrosine at the equivalent position in mastrevirus RepA/Repproteins. The Q149 position, which is highly variable, was notmutated. The mutant AL1 coding sequences were fused to theGal4 AD coding sequence and analyzed in yeast two-hybridassays as described above. The A147Y and II151 mutants dis-played significantly lower pRBR binding activities than wild-type AL1 (Fig. 1B). However, these mutations also reducedAL1 oligomerization activity (Fig. 1B, right), indicating thattheir effects were not specific for pRBR binding. In contrast,the L148 mutation reduced the AL1/pRBR interactions to25% of wild-type levels without a concomitant loss in AL1oligomerization activity (Fig. 1B), establishing the specificity ofthe mutation for pRBR binding. Together, these results indi-cated that several of the amino acid residues in the helix 4motif are important for both AL1/pRBR and AL1/AL1 inter-actions in yeasts and that both the KEE146 and the L148residues contribute to pRBR binding. It was not technicallyfeasible to verify the impact of the mutations on AL1/pRBRinteractions in infected plant cells or tissues. The AL1 proteinis expressed at very low levels and is not extracted efficiently

under native conditions required to maintain protein com-plexes.

The L148 mutation reduces viral DNA accumulation andsymptom severity. The KEE146 mutation alters the level ofTGMV DNA accumulation in cultured cells and the tissuespecificity and symptoms of TGMV infection in plants (38). Todetermine if mutations in other helix 4 residues also have animpact on these viral processes, we examined the newly gen-erated mutants in transient-replication and infectivity assays.The mutations were transferred into the AL1 open readingframe of a TGMV A replicon, and viral DNA accumulation inN. tabacum BY-2 protoplasts was assessed on DNA gel blots.The levels of double-stranded and single-stranded DNA thataccumulated for the K144 (Fig. 2A, lane 2) and EE146 (lane 3)mutants were essentially wild type (lane 1). A similar result wasobserved with the E145 and E146 mutants (data not shown).The L148 mutant (Fig. 2A, lane 5) also supported viral DNAsynthesis but at levels significantly lower than that for wild-typeTGMV A (lane 1) and similar to those previously reported forKEE146 (38). The reduction in L148 DNA accumulation wasobserved for both double- and single-stranded forms of TGMVDNA. In contrast, the A147Y (Fig. 2A, lane 4) and II151 (lane6) mutants, both of which were severely impaired in AL1oligomerization (Fig. 1B), failed to replicate to detectable lev-els in cultured cells. The same viral DNA accumulation pat-terns were observed when BY-2 protoplasts were cotransfectedwith a TGMV B replicon and plant expression cassettes forAL1 and AL3 (data not shown).

Plant infection experiments were carried out by cobombard-ing either wild-type or mutant A component DNA with aTGMV B replicon onto N. benthamiana plants. Plants inocu-lated with the wild-type virus developed clear symptoms by 6 to7 days postinoculation, exhibiting leaf curling, general chloro-sis, and stunting of new growth (Fig. 2B, wt). The K144, E145,E146, and EE146 mutants caused symptoms that were indis-tinguishable from those caused by the wild type, indicating thatthese mutations do not visibly alter the infection process. Incontrast, plants inoculated with the L148 mutant only devel-oped chlorosis along the veins (Fig. 2B) between 14 and 21days postinfection and never displayed stunted growth or leafcurling. These symptoms, which resemble those reported forKEE146-infected plants (38), were observed in all 12 inocu-lated plants and were maintained over a 5-week infection pe-riod. The A147Y and II151 mutants produced no detectablesymptoms even at 5 weeks postinoculation, consistent withtheir inability to replicate in tobacco protoplasts.

We also examined TGMV DNA accumulation in the N.benthamiana plants inoculated with either wild-type or mutantvirus. Total DNA was isolated from systemically infectedleaves 14 days postinoculation and analyzed on DNA gel blotsby using a TGMV A probe. Viral DNA was detected in ex-tracts of plants infected with all mutant viruses that producedsymptoms (Fig. 2C) but not in asymptomatic plants inoculatedwith the A147Y (lanes 8 to 10) and II151 (lanes 14 to 16)mutants. Plants infected with the K144 (Fig. 2C, lanes 2 to 4)or EE146 (lanes 5 to 7) mutant contained essentially wild-typelevels of single- and double-stranded DNA (lane1). Again, thesame results were obtained with E145- and E146-infectedplants (data not shown). In contrast, both DNA forms werereduced in L148-inoculated plants relative to wild-type-inocu-


lated plants (Fig. 2C, cf. lane 1 and lanes 11 to 13). DNA gelblot analysis at 7, 14, and 21 days postinoculation showed thatthe differences in TGMV DNA levels between L148- and wild-type-infected plants are stable over time (data not shown).Reduced accumulation of viral DNA (3 to 4% of wild-typevalues) was previously observed for KEE146-infected plants(38). In these earlier experiments, an E- -N140 mutant accu-mulated to 1 to 2% of wild-type levels but caused wild-typesymptoms, establishing that there is no direct relationship be-tween the altered symptoms and reduced viral DNA accumu-lation during infection (38). Hence, the altered symptomscaused by the L148 mutation are likely to be due to reducedpRBR binding, as has been hypothesized for KEE146.

AL1/pRBR binding activity is differentially affected by sub-stitutions at L148. Because of the strong phenotypic effect ofthe L148 mutation, we examined the role of L148 in AL1/pRBR interactions in greater detail. The leucine residue may

facilitate pRBR binding by contributing molecular contactsand/or by stabilizing the predicted structure of the helix 4motif. To address these possibilities, we substituted a series ofamino acids at position L148 with different side chains andtendencies to occur in �-helices. The impact of the differentmutations on the pRBR binding and oligomerization activitiesof TGMV AL1 was analyzed in yeast two-hybrid assays (Fig.3). An L148 M mutation had no detectable effect on pRBRbinding activity, whereas an L148I substitution resulted in amoderate reduction. Like the L148 mutant, the L148V andL148G mutants displayed significantly less pRBR binding ac-tivity than wild-type AL1. In general, the binding activities ofthe mutants declined with the decreasing probability of thesubstituted amino acid to occur in an �-helix. However, the lowactivity of the L148 mutant, which is predicted to readily forman �-helical structure, supports the involvement of side chaincontacts. Together, these results suggest that L148 contributes

FIG. 2. The L148 mutation reduces replication and symptom severity. (A) DNA replication of TGMV AL1 mutants was analyzed in tobaccoprotoplasts. DNA was isolated 72 h posttransfection, and 10 �g of total DNA was analyzed by DNA gel blot hybridization by using a radiolabeledTGMV A probe. Lanes 1 to 6, transfections with TGMV A replicons with either wild-type (wt) or mutant AL1 open reading frames correspondingto the indicated mutations. The positions of double-stranded (ds) and single-stranded (ss) forms of TGMV A DNA are indicated. (B) N.benthamiana plants infected with the pRBR binding mutant L148 developed chlorosis along the veins but no leaf curling or stunting characteristicof wild-type TGMV infection. These milder symptoms were maintained over a 5-week infection period. (C) N. benthamiana plants werecobombarded with DNAs corresponding to TGMV A and B replicons. The AL1 open reading frames of the A components were wild type (wt)or carried the indicated mutations. Total DNA (2.5 �g/lane) was isolated from young leaves from three plants for each construct at 14 dayspostinfection and analyzed on DNA gel blots. Viral DNA was detected with a radiolabeled probe specific for TGMV A. In panels A and C, theaverage relative accumulation of double- and single-stranded DNA for three independent samples is given below each lane, with the wild-typevalue set at 100. No signals were detected in mutants A147Y and II51 even with longer exposures.


both structural and specific contacts to pRBR binding. None ofthe mutations had a strong effect on AL1 oligomerizationactivity, indicating that the reduced pRBR binding activitiesare not due to a general destabilization of AL1. It was notpossible to use the L148V and L148G mutants to assess furtherthe impact of AL1/pRBR interactions on the disease processdue to instability or lack of functionality of these mutationsduring infection (unpublished data).

The replication proteins of TYLCV and CaLCuV bind topRBR. To date, TGMV AL1 is the only begomovirus replica-tion protein that has been shown to interact with pRBR (1).The begomovirus genus includes many viruses with distincthost ranges, symptoms, and tissue tropisms, and it not knownif pRBR binding is a general property of begomovirus repli-cation proteins. As a consequence, we asked if the C1/AL1proteins of other begomoviruses interact with pRBR. Forthese experiments, we chose two geminiviruses—TYLCV(TYLCV-DR) and CaLCuV (also referred to as CbLCV)—that are evolutionarily distant from TGMV and each other.TYLCV-DR, which has a single genome component, is repre-sentative of Old World begomoviruses (59). CaLCuV is from asmall group of New World begomoviruses whose AL1 proteinslack a highly conserved sequence of unknown function be-tween the DNA cleavage motif III and the predicted helix 3(31, 53). We generated Gal4 AD fusions corresponding tofull-length TYLCV C1 and CaLCuV AL1 and tested the fu-sions for interaction with a DBD-pRBR fusion in yeast. ThepRBR binding activity of TYLCV C1 was similar to that de-tected for TGMV AL1 in parallel assays (Fig. 4A). In contrast,we were unable to recover colonies carrying the expressioncassette corresponding to the full-length CaLCuV AL1 fusion.We have encountered similar expression problems in bacterialand insect cell systems with full-length CaLCuV AL1, indicat-ing that its accumulation is detrimental. To overcome thisproblem, we generated a Gal4 AD fusion corresponding toamino acids 2 to 178 of CaLCuV AL1. Using this fusion, we

detected a reduced but significant level of pRBR binding bythe CaLCuV AL1 N terminus (Fig. 4A).

We compared the AL1/C1 sequences from 78 begomovi-ruses of both Old and New World descent to derive a consen-sus sequence for the helix 4 motif (Fig. 4B). These comparisonsrevealed that the motif consists of a conserved hydrophobiccore flanked by charged residues. The core includes an invari-ant alanine residue followed by a leucine in 67 of the examinedAL1/C1 proteins and methionine in the remaining 11 proteins.The L/M position corresponds to L148 in TGMV and is rep-resented by a leucine in both TYLCV and CaLCuV. We askedif an alanine substitution at residue L145 in CaLCuV AL1impairs pRBR binding analogous to the TGMV L148 mutant.The L145 mutation was introduced into the Gal4 AD fusionvector carrying amino acids 2 to 178 of the CaLCuV AL1 codingsequence and tested for pRBR binding in yeast two-hybrid assays.As shown in Fig. 4A, the CaL145 mutation caused a significantreduction in pRBR binding activity. The CaLCuV L145 andTGMV L148 mutations reduced pRBR binding to 23% (Fig. 4A)and 25% (Fig. 1B) of their respective wild-type controls. To-gether, these data show that diverse begomovirus replication pro-teins interact with pRBR through a conserved motif.

DISCUSSION

Geminiviruses do not encode the DNA polymerases andaccessory factors required for their replication; instead, they

FIG. 3. The pRBR binding activity of AL1 is differentially affectedby substitutions at position 148. Yeast was cotransformed with theZmRBR 214C cassette fused to the Gal4 DBD and Gal4 AD cassettesfor either wild-type (L148) or mutant AL1 coding sequences (on theleft) and analyzed as described for Fig. 1B. The arrow indicates de-creasing �-helical tendency of the amino acid substitutions (50). Barsare as defined for Fig. 1. The effect of the mutations on AL1 oligomer-ization activity is indicated on the right.

FIG. 4. pRBR interacts with the replication proteins of CaLCuVand TYLCV. (A) Yeast was cotransformed with the ZmRBR 214Ccassette fused to the Gal4 DBD and Gal4 AD cassettes for TGMVAL1, TYLCV C1, CaLCuV AL1, or mutant CaLCuV AL1 (CaL145).Protein interactions were assayed by measuring �-galactosidase activ-ity in total protein extracts and normalized to wild-type TGMV AL1(100%). The error bars correspond to 2 standard errors. (B) Helix 4motifs of TGMV AL1 (amino acids 144 to 156), TYLCV to DR C1(amino acids 142 to 154), and CaLCuV AL1 (amino acids 141 to 153).The conserved leucine residue in the helix center that was mutated toan alanine in CaL145 is marked with a dot. A consensus for begomo-virus AL1/C1 proteins is shown at the bottom.


rely on plant DNA replication machinery. Earlier studiesshowed that TGMV and CaLCuV alter host transcriptionalcontrols to induce PCNA synthesis, a component of the hostDNA replication complex (17, 18). Interaction of TGMV AL1with pRBR plays a key role in this induction process (38), butit was not known if other begomovirus replication proteins alsobind to pRBR. In this paper, we show that like TGMV AL1,CaLCuV AL1 and TYLCV C1 bind to pRBR. Mutationalstudies established that the helix 4 motif, which is distinct fromthe canonical LXCXE motif (Fig. 5), contributes several im-portant contacts for pRBR binding. Together, these studiesdemonstrated conservation of the pRBR binding activity ofbegomovirus AL1/C1 proteins through the helix 4 motif.

The helix 4 motif is an 11-amino-acid sequence that is lo-cated within residues 140 to 170 of geminivirus replicationproteins and is predicted to form an �-helix with more than90% probability (53). It contains clusters of charged aminoacids at its ends separated by a conserved hydrophobic core(Fig. 4B). These properties are characteristic of many proteinbinding surfaces that rely on a combination of ionic bonds andburied hydrophobic residues to stabilize protein interactions(22). Earlier studies showed that the helix 4 motif in TGMVAL1 is located within the overlapping oligomerization andpRBR binding domains and indicated that the charged resi-dues at the edges of the motif are involved in protein interac-tions (38, 53). The C-terminal residues REK154 contribute toAL1/AL1 and AL1/pRBR interactions equally, while the N-terminal residues KEE146 are more important for pRBR bind-ing. Analysis of individual mutations in the KEE sequencerevealed that pRBR binding is more dependent on K144 thanon E145 and E146. The wild-type phenotype of the doublemutant EE146 ruled out the possibility that the individual E

residues are redundant with each other. The KEE146 mutationwas stronger than any of the individual or double mutants,indicating that at least two residues in the sequence are essen-tial for wild-type pRBR binding. The involvement of multiplecharged residues is consistent with the presence of two or threecharged amino acids at the N-terminal edge of helix 4 in mostgeminivirus replication proteins (Fig. 4B).

Analysis of the hydrophobic core of helix 4 further sup-ported its involvement in pRBR binding and oligomerization.Mutation of A147Y and II151 affected both activities ofTGMV AL1. Both mutations were significantly more detri-mental to oligomerization activity, suggesting that pRBR bind-ing is not strictly dependent on AL1/AL1 interactions as pre-viously proposed (38, 53). In contrast, substitutions at TGMVL148 specifically reduced pRBR binding without having a sig-nificant impact on oligomerization. The importance of theleucine residue was underscored by the reduced pRBR bindingactivity of the equivalent CaLCuV L145 mutant. Differentamino acid substitutions for TGMV L148 indicated that theleucine residue contributes both side chain contacts and to themaintenance of structural integrity of the helix 4 motif. Re-duction of pRBR binding activity by the alanine replacementmost likely reflected the loss of a key side chain contact, whilethe lower �-helical tendencies of the isoleucine and valinesubstitutions may have induced local structural changes thatinterfered with pRBR binding. The only substitution at L148that was phenotypically normal is methionine, which has alarge hydrophobic side chain and a high probability of occur-ring in an �-helical region. Phylogenetic analysis showed thatmethionine is the only naturally occurring alternative toleucine at this position in geminivirus replication proteins (Fig.4B).

FIG. 5. Sequence conservation in the pRBR binding domain of geminivirus proteins. Geminivirus AL1/C1 proteins are aligned with theequivalent regions of mastrevirus RepA/Rep proteins. Residues shaded in blue are conserved in AL1/C1 proteins of begomoviruses (TGMV,TYLCV, CaLCuV, Sri Lankan cassava mosaic virus [SLCMV], and tomato leaf curl virus [ToLCV]), curtoviruses (beet curly top virus [BCTV]),and topocuviruses (tomato pseudo-curly top virus [TPCTV]). Residues shaded in yellow are also conserved in the RepA/Rep proteins ofmastreviruses (BeYDV and maize streak virus [MSV]). Together, these geminiviruses represent all four genera and five continents. The alignedregion corresponds to amino acids 143 to 200 of TGMV AL1. The helix 4 (blue bar) and LXCXE (red bar and red type) motifs are marked. Thehelix 4 A and Y residues that have diverged between mastreviruses and other geminivirus genera are in bold type. The LXCXE motif includes abulky hydrophobic residue in the 8th position (relative to L) that also directly contacts pRb (42). The oligomerization core of TGMV AL1 (greenbar) is also marked (53). Accession numbers for the viruses in alignment are as follows: TGMV, K02029; TYLCV, X15656; CaLCuV, U65529;SLCMV, AJ314737; ToLCV, S53251; BCTV, AF379337; TPCTV, X84735; BeYDV, Y11023; and MSV, K02026.


Helix 4 mutations differentially altered virus replication andinfectivity. The A147Y and II151 mutations blocked both viralreplication in protoplasts and infection. Both of these mutantsare severely impaired in TGMV AL1 oligomerization, which isessential for origin recognition, the first step in viral replication(51). The results with K144 and EE146, both of which werewild type for replication and infection, differed from the mu-tant phenotype reported previously for KEE146 (38). In con-trast, the L148 mutant closely resembled KEE146 in that itshowed reduced replication in protoplasts and chlorosis onlyalong the veins. It was proposed that the altered tissue speci-ficity of KEE146 infection is a result of reduced pRBR bindingand reflected a lower threshold for induction in vascular versusmesophyll and epidermal cells (38). The correlation betweenpRBR binding and symptoms observed for K144, EE146, andL148 further supports this hypothesis and provides insight intothe nature of the proposed threshold. K144 retained 42% ofwild-type pRBR binding activity, while KEE146 and L148 had16 and 25% of the wild-type activity, respectively. These resultssuggested that 42% but not 25% pRBR binding activity issufficient to allow infection of mesophyll and epidermal cells.Our data also indicated that pRBR binding may play a role invirus replication in protoplasts isolated from mid-log-phaseBY-2 cells. pRBR binding mutants of WDV RepA also displayreduced replication in cultured maize cells (67). These resultsdiffer significantly from those obtained with mammalian DNAtumor viruses, which do not require pRb binding activity toreplicate efficiently in actively cycling cells (23), and suggest aunique role for pRBR during the plant cell division cycle.

The helix 4 motif is highly conserved across all geminivirusgenera and is likely to be involved in interaction with pRBR bybegomoviruses, curtoviruses, and topocuviruses, whoseAL1/C1 proteins lack LXCXE motifs. In contrast, the role ofthe helix 4 motif in mastrevirus RepA and Rep proteins is lessclear because of the presence of the canonical LXCXE motifthat is required for efficient pRBR binding in vitro (43, 67).However, recent studies in animal systems showed that thecontacts between pRb and LXCXE-containing proteins areextensive and that other protein domains are necessary forfunctional interaction. Simian virus 40 large T antigen contactspRb through two �-helices in addition to the LXCXE se-quence (36). A nonapeptide containing the LXCXE motif ofhuman papillomavirus binds to pRb but is not sufficient tointerfere with E2F/pRb interactions (42, 65). Recent crystallo-graphic studies showed that E2F, which lacks an LXCXE mo-tif, contacts pRb through an extended region including themarked box and the transactivation domain (65). The idea thatgeminiviruses also interact with pRBR through a larger proteindomain is supported by the observations that the pRBR bind-ing domain of TGMV AL1 encompasses 80 amino acids andthat mutations outside of helix 4 impact binding (38). Hence, itis likely that the mastrevirus pRBR-binding domain extendsbeyond the LXCXE motif and may encompass the conservedhelix 4 motif. The involvement of a more extensive domain thatstabilizes the RepA/pRBR complex in vivo may explain whyLXCXE mutations have no obvious phenotypic effect on beanyellow dwarf virus (BeYDV) infection (43).

The evolutionary conservation of the LXCXE motif in plantand animal systems suggests that it is an ancient pRb/pRBRbinding motif that arose before divergence of the kingdoms. In

contrast, a search of the Arabidopsis and human protein data-bases did not uncover any known or potential pocket domain-binding proteins with homology to the helix 4 consensus se-quence (unpublished result), indicating that this motif is ofmore recent origin and unique to geminiviruses. As a conse-quence, we hypothesize that the ancestral geminivirus pRBRbinding domain contained the LXCXE motif and that the helix4 sequence evolved before divergence of the genera. Later ingeminivirus evolution, begomoviruses, curtoviruses, and to-pocuviruses lost the LXCXE motif but retained the helix 4motif as their primary pRBR binding site. Sequence compar-ison across the different geminivirus genera supports this hy-pothesis. In addition to the conservation of the helix 4 motif,the C-terminal half of the LXCXE motif is conserved in allgenera (Fig. 5). In begomoviruses, curtoviruses, and topocuvi-ruses, the N-terminal half of the LXCXE motif appears to haveundergone concerted evolution away from the consensus. Inaddition, helix 4 may have evolved by the replacement of atyrosine residue conserved in mastrevirus RepA/Rep proteinswith an invariant alanine in other geminivirus genera, leadingto enhanced pRBR binding through this motif. Recent studies(32) of the pRBR binding properties of the RepA and Repproteins may provide insight into the basis of the selectivepressure for these changes. Even though these proteins aresplicing variants that share the same 200-amino-acid N termi-nus and include the LXCXE and helix 4 motifs, only RepA isable to bind to pRBR. It was proposed that the inability of Repto interact with pRBR reflects steric hindrance induced by itsC-terminal domain that is absent in RepA but shared by theAL1/C1 proteins of other geminivirus genera. Hence, it ispossible that when the ancestral C1-C2 gene rearranged andlost the ability to produce two proteins, there was strong pres-sure to maintain the ability to bind to pRBR and alter plantcell cycle controls, leading to evolution of the pRBR bindingdomain of the AL1/C1 proteins of begomoviruses, curtovi-ruses, and topocuviruses.

ACKNOWLEDGMENTS

This research was supported by a grant from the National ScienceFoundation (MCB-0110536 to LHB) and a postdoctoral fellowshipfrom the PEW Foundation (P0291SC to GAA).

We thank Dominique Robertson and Gerardus Dambrauskas forcritical reading of the manuscript.

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Arch Virol (2001) 146: 1465–1485

An iteron-related domain is associated to Motif 1 in the replicationproteins of geminiviruses: identification of potential interacting

amino acid-base pairs by a comparative approach

G. R. Argüello-Astorga1,∗ andR. Ruiz-Medrano2

1Departamento de Ingenierıa Genética, Centro de Investigación y de EstudiosAvanzados del IPN, Unidad Irapuato, México

2Departamento de Biotecnologıa, Centro de Investigación y de EstudiosAvanzados del IPN, México, D.F., México

Accepted April 12, 2001

Summary.Geminiviruses encode a replication initiator protein, Rep, which bindsin a sequence-specific fashion to iterated DNA motifs (iterons) functioning as es-sential elements for virus-specific replication. By using the iterons of more thanone hundred geminiviruses as heuristic devices, we have identified a Rep subdo-main 8 to 10 residues in length, whose primary structure varies among virusesharboring different iterons, but which is similar among viruses with identicaliterons, regardless of their differences in host range, insect vector, geographicalorigin or genome structure. Close analysis of this iteron-related domain (IRD)revealed consistent correlations between specific Rep residues and defined nu-cleotides of its cognate iteron, thus providing important insights about the molec-ular code which dictates the Rep preference for specific DNA sequences. A modelof potential Rep-iteron contacts is proposed. The identified IRD is adjacent to aconserved motif characteristic of a superfamily of rolling-circle (RC) replicationproteins, and secondary structure predictions suggest that those Rep subdomainsform together the core of a novel DNA-binding domain possessing ab-sheet asrecognition subdomain, which is apparently conserved in the replication proteinsof nanoviruses, circoviruses, microviruses, and a variety of ssDNA plasmids ofeubacteria, archaebacteria and red algae. The evolutionary implications of thesefindings are discussed.

∗Present address: Department of Molecular and Structural Biochemistry, North CarolinaState University, Raleigh, N.C. 27695-7622, USA.

1466 G. R. Argüello-Astorga and R. Ruiz-Medrano

Introduction

Geminiviruses are plant pathogens characterized by twin icosahedral capsids andsmall genomes consisting of one or two single-stranded circular DNA moleculesof around 2.5 to 3 kb in length [28, 42, 55]. They cause economically impor-tant diseases in a variety of cereal, fiber and vegetable crops worldwide [9].Besides their importance as pathogens, geminiviruses have attracted the attentionof plant molecular biologists because along with nanoviruses, they are the onlyknown plant viruses with true DNA replication cycles. This feature, in additionto their simple genomes and extensive reliance on the host synthetic machinery,makes geminiviruses ideal model systems for the study of plant DNA replication[6, 25, 26, 28, 66].

The geminivirus family has been divided into four genera (i.e.Mastrevirus,Curtovirus, TopocuvirusandBegomovirus) on the basis of virus vector species,host range and genome organization [67]. All geminiviruses replicate in the nu-cleus of infected plant cells via a double-stranded DNA intermediate througha rolling-circle (RC) mechanism [31, 60, 63]. The virus multiplication reliesmostly on the host DNA replication apparatus, with only one virus-encoded pro-tein, Rep, being indispensable for the process [19]. Geminivirus Rep, also namedAC1 or AL1 in begomoviruses and C1 in curtoviruses, belongs to a superfam-ily of RC replication initiator proteins characterized by a coherent arrangementof three conserved motifs, N-1-2-3-C [39, 40]. These proteins are encoded bya variety of systems including archaebacterial and eubacterial ssDNA plasmids,bacteriophages (e.g.,fX174), plant ssDNA viruses, and vertebrate-infecting cir-coviruses [7, 34, 48]. As far as is known, all these proteins possess DNA nicking-closing activities and bind double-stranded DNA in a sequence-specific manner[16, 37, 40, 65].

Besides its key role in viral RC replication, Rep is involved in the inductionof at least one host gene, PCNA [28, 47] as well as in the control of viral geneexpression, downregulating its own gene promoter in begomoviruses [17, 18,27, 33, 64] and transactivating the capsid protein gene promoter in mastreviruses[15, 32, 68]. The Rep-dependent transcriptional repression is virus-specific, and ispartially mediated by the same DNA elements that are essential for virus-specificreplication [17, 18].

Two different DNA elements in the geminivirus origin of replication(ori) arefunctional targets of Rep: 1) the nonameric motif 5′-TAATATTAC-3 ′, invariablylocated at the loop of a conserved “hairpin” element, where Rep introduces asite-specific nick to initiate virus replication via a RC mechanism [41], and 2) atandemly repeated motif located at variable distances from the conserved hairpin,which is bound specifically by its cognate Rep protein and functions as a majorrecognition element of the replication origin in begomoviruses and curtoviruses[4, 14, 21, 22]. Analogous repeated DNA elements, albeit arranged in a differentmanner, are apparently bound by Rep/RepA proteins of mastreviruses [11, 45].Thesecis-acting elements belong to a series of iterated DNA motifs (iterons)which display a virus-specific nucleotide sequence and a similar arrangement

Specificity determinants of geminivirus Rep 1467

among the members of a defined geminivirus lineage. It was anticipated on atheoretical basis that theseori-associated iterons are actually Rep-binding sites[2].

The trans-acting determinants of geminivirus replication specificity, on theother hand, have been mapped coarsely in recent years. The virus-specific recog-nition domain of Rep proteins encoded by tomato yellow leaf curl virus (TYLCV),tomato golden mosaic virus (TGMV) and bean golden mosaic virus (BGMV),were mapped to amino acids 1–116 [24, 35]. The analogous domain in the Repprotein from a curtovirus, beet curly top virus (BCTV), was mapped to the regionencompassing amino acids 3–89 [13].

In an early attempt to define potential Rep DNA-bindingspecificity deter-minants (SPDs) more closely, the predicted Rep proteins from∼40 geminiviruseswere analyzed by a comparative approach, using the iterons as a heuristic device.As a result, a Rep N-terminal domain 8–10 residues in length whose primarystructure correlates with the nucleotide sequence of its homologous iterons, wasidentified, and proposed to be a major DNA-binding SPD of that viral protein[1]. Here we present the results of an updated, more extensive analysis that in-cluded sequence data from more than 120 geminiviruses. This new study hasnot only confirmed the conclusions of an earlier analysis but it also revealed aconsistent protein-DNA sequence correlation which allowed the prediction ofRep-iteron interactions at the level of specific amino acid-base pair contacts. Fur-thermore, it provided insights about the molecular code ruling the preference ofgeminivirus Rep for specific DNA sequences, and led to the finding that Motif 1 ofRC replication proteins is, plausibly, the conserved core element of a previouslyunrecognized DNA-binding domain.

Materials and methods

General approach

Several assumptions were used as heuristic hypotheses in the present study: a) the iterons,as previously defined [2], constitute the specific DNA-binding sites for Rep; b) Rep has adiscrete DNA-binding domain in which certain amino acid residues determine preference fora specific DNA-sequence element, and will be termed specificity determinants (SPDs) of Rep;c) Rep proteins from viruses possessing iterons with different nucleotide sequence shoulddiffer in their DNA-binding SPDs and, conversely, proteins from viruses harboring identicaliterons should be similar, if not identical, in the same polypeptide segment, regardless of theirphylogenetic relationship.

Based on these simple assumptions, a three-step strategy to tentatively map the gemi-nivirus Rep SPDs was derived: 1) comparison of the predicted Rep proteins of selected pairsof closely related geminiviruses with different iterons; 2) comparison of computer-generatedalignments of a number of these pairs in a segment-by-segment fashion, in order to identifyshort protein regions where variations in amino acid sequence occur in all cases; 3) compar-ison of the proteins from selected pairs of distantly related geminiviruses (e.g. New Worldand Old World begomoviruses) displaying similar iterons, within the “hypervariable” Repdomains identified in the previous analyses. The domain for which a significant convergencein amino acid sequence was found in all, or most, of the compared pairs of proteins, wasidentified as a potential component of the Rep DNA-recognition domain.


Geminivirus sequences and computer analysis

Sequences of geminiviruses were obtained directly from GenBank databases; virus names,acronyms and GenBank accession numbers have been recently summarized by Fauquet et al.[20]. Sequences were assembled and analyzed using the Geneworks 2.2 (Intelligenetics, Inc.)and the LaserGene software packages. Phylogeny trees were produced with the Geneworkssoftware package which uses the UPGMA (Unweighted Pair Group Method with ArithmeticMean) procedure. The relative evolutionary distances between geminiviruses were also basedon data from several published phylogenetic studies of this virus family (e.g. [53, 57]).

Results

Identification of an iteron-correlated Rep region

The putative Rep-binding sites of eight mastreviruses, one curtovirus, and 21 be-gomoviruses were previously identified [2]. By using the structural rules definedin that work, the potential Rep cognate sequences from other three curtoviruses,one topocuvirus, six mastreviruses, and∼80 begomoviruses were identified. Ingeneral, the arrangement of the iterons in these viruses display the same group-specific organization originally described (data not shown). However, a few atyp-ical cases were found. For example, the only two known mastreviruses infectingdicots, namely, tobacco yellow dwarf virus, TYDV [46] and bean yellow dwarfvirus, BeYDV [43], display two different, overlapping sets of iterons, one show-ing the typical arrangement of the members of this genus, and another reminiscentof that found in begomoviruses (not shown, but see below).

In order to identify potential DNA-binding SPDs of geminivirus Rep, thepredicted proteins from∼30 different pairs of closely related viruses possessingdifferent iterons were compared. The overall analysis of the diverse Rep single-pair alignments (see Materials and methods) led to the identification of a smallhypervariable domain in the protein N-terminus.

Systematic comparisons of Rep proteins encoded by distantly related virusesharboring identical iterons revealed that the identified hypervariable region issimilar in amino acid sequence between most of them, as anticipated for a domaininvolved in iteron recognition (see Materials and methods). These observationsstrongly suggested that the identified protein region (which will be referred fromnow on asI teron-RelatedDomain, IRD) could be actually a component of theRep domain involved in recognition of its cognate DNA elements.

Mapping of specificity determinants by an alternative approach

Since the specific-recognition domain of begomovirus/curtovirus Rep lies in itsfirst 89–116 aa [12, 23, 35], an alternative, more direct method to map potentialSPDs is to compare this region between highly similar proteins differing in speci-ficity. Theoretically, it is evident that potential SPDs correspond only to thoseresidues differing between compared proteins. If on further analysis differentialresidues are compared with their counterparts in proteins with the same speci-ficity, it is possible to discard as potential SPDs all those aa residues whose identityis not conserved within an isospecific group (i.e., with similar cognate iterons).


Using this approach, a number of begomovirus Rep proteins displaying 85–94%identity were examined. The analysis showed that the aa residues which meet theSPD criteria indicated above are always located within positions 3–11 of the Nterminus (i.e., within the previously defined IRD) regardless of the phylogeneticdistance of compared viruses. Some examples illustrating this kind of analysisare shown in Fig. 1. Interestingly, in the case of ToLCNdV strains (Fig. 1A), theAsn/Asp residue in position 10 has been experimentally defined as atrans-actingreplication SPD [12], thus confirming the predictive value of this comparativeapproach.

The IRD overlaps a conserved motif of Rep proteins

A general analysis of the Rep IRDs showed that, in spite of their remarkablesequence variability, some aa residues are entirely conserved both within andnext to the IRD itself. This observation prompted a closer examination of theregion adjacent to the IRD in order to define whether the latter is a part of alarger conserved domain. Alignments of the N-termini of Rep proteins encodedby ∼90 dicot-infecting geminiviruses revealed the existence of a strong consen-sus, FXL∗X(A/S)(K/R)N(Y/I)FLTYPq∗C (L∗ = Leu, Ile or Val; q∗ = Gln, Lys,Arg), which includes the so called Motif 1 (FLTYP) of RC replication initiatorproteins [34]. The FXL∗X amino acid stretch of this “extended Motif 1 con-sensus” is a part of the identified IRD. Analysis of the homologous region ofMastrevirus Rep proteins showed that they display a more degenerate consensus,namely, F-7X-FLTYPq∗C. Together, these observations led to the important sug-gestion that Motif 1 and the IRD might be components of a same Rep functionaldomain, because the IRD conserved Phe residue is invariably spaced by 7 residuesfrom Motif 1, in spite of their variable position within Rep (i.e., they apparentlyconstitute a structural unit).

Structural correlation between Rep IRDs and their cognate iterons

With the aim of defining a possible significant correlation between the Rep IRDprimary structure and the nucleotide sequence of its cognate iteron, a systematicanalysis of these elements was performed. For the sake of clarity, the IRD residueswere numbered taking the invariant Phe residue as the reference point. Conse-quently, the Rep IRD was represented as X−n . . .X−2 X−1 F X1 X2 X3, whereX−n is the first residue of the protein. On the other hand, the representation of theiterons was simplified to include only a 5-bp core sequence, GGN1N2N3, that isboth the most conserved sequence within iterons from a given virus, and the mostvariable segment of those elements between geminiviruses [2]. Furthermore, thisis the DNA core sequence that has been experimentally defined as critical for thespecific recognition by Rep [14, 21, 22].

Using this framework, the iterons from the dicot-infecting geminiviruses weregrouped on the basis of their similarities in the GGN1N2N3 core, and the IRDfrom their Rep proteins examined in detail. As a result, a clear and consistentcorrespondence between these viral elements was found. Figure 2 presents the


Fig. 2 (continued)


Fig. 2. Correlation between the iteron core sequence GGN1N2N3 and the amino acid se-quence of Rep IRD in 108 dicot-infecting geminiviruses. Enclosed in rectangles are thecorrelative DNA-protein sequence motifs. The TGGTGTCC iterons from the members ofthe SLCV cluster have been separated from other iterons with a GGTGT core because theycorrelate with different Rep IRD sequences. Degenerate iteron core sequences represent theconsensus of all iterons present in a virus. Most virus names are as in [20]. An asteriskindicates viruses that are not included in that review, and their GeneBank accesion num-bers are as follow: EYVV (AB007990); HYVMV (AB020781); LBGMV (U92531); OkEV(AF155064); SCrLV (AB020977); TbLCCV-Y1 (AF240675); TbLCCV-Y2 (AF240673);TbLCCV-Y3 (AF240674); TbLCJV-JP2 (AB028604); ToLCV-IN (L11746); ToLCV-LK(AF274349); ToLCV-MM (AF206674); ToLCSinV-CR (AF131213); ToVEV (AF026464).

Symbols: Y= C,T; R= A,G; K = T,G; W= A,T; M = A,C; V = A,C,G; N= A,C,G,T

relevant data from all examined dicot-infecting geminiviruses, which may besummarized as follows.

i) Geminiviruses harboring iterons with a GGAGN core encode Rep proteinswhose IRD display a (P/T)XXF(R/K)L∗Q motif. They include two curtoviruses,two dicot-infecting mastreviruses and∼20 begomoviruses from America, Africa,Australia and Asia.

ii) Viruses with a GGTA(C/G/A) iteron display Rep-IRDs with a PKRFQI mo-tif, which is not found in any other known member ofGeminiviridae. They com-prise several OW begomoviruses, a single American begomovirus (TGMV), andtomato pseudocurly top virus (TPCTV), the only known treehopper-transmittedgeminivirus [8].

iii) With the exception of some strains of EACMV, the viruses harboringiterons with a GGGGG core display a similar Rep IRD sequence, MPRXGXFSI∗K.They include begomoviruses from 3 continents.


iv) Viruses whose iterons display GGTGG, GGGG(T/A), GG(T/G)GT,GG(T/A)GT, and GGGT(C/A) core sequences encode Rep proteins with the cor-relative IRD amino acid motifs XGRF(N/S)L∗N, XKRFKVS, PXXF(R/K)L∗N,APNRFRL∗N, and K(K/R)FL∗L∗N, respectively.

v) Viruses from the SqLCV-cluster harboring GGTGTCY iterons have Rep-IRDs with a PXSFRL∗(A/T) amino acid sequence, which is different to the cor-responding IRD motif found in other begomoviruses with an analogous GGTGTiteron core. Viruses from this American lineage encode Rep proteins whose N-terminal half significantly differ of the homologous proteins of other whitefly-transmitted geminiviruses (our unpublished data).

vi) A few viruses harboring similar iterons differ, sometimes greatly, in theirRep IRD sequences (e.g., compare MGMV-JM2 with other viruses having similariterons).

vii) Viruses possessing iterons whose nucleotide sequence is uniqueamong theGeminiviridaeencode Rep proteins with IRD sequences that are alsounique.

Correlation between specific Rep IRD and iteron residues

In order to refine the search for structural correlations between iterons and Rep-IRDs at the level of specific nucleotide-amino acid pairs, the corresponding el-ements from viruses differing in only one iteron nucleotide were systematicallycompared. The results of this new analysis can be summarized as follows: 1) dif-ferences in the iteron N1 base consistently correlate with changes in the identityof the IRD X3 amino acid residue (Fig. 3A); 2) differences in the iteron N2 basecorrelate with variations in the IRD X1 residue (Fig. 3B); 3) the iteron N3 basedoes not correlate with the same IRD residue in all cases; rather, it correlateswith the IRD residue X−4 in the case of viruses harboring iterons with GGTAC,GGTAG, GGTAA, GGGTC, and GGGTAcore sequences (Fig. 3C), but in gem-iniviruses having GGGGGor GGTGGiterons, the N3 base apparently correlateswith the IRD X−2 residue (not shown, but see Fig. 2).

Analysis of the Rep IRD of mastreviruses

The iterons of mastreviruses display a different arrangement to those present inother geminiviruses [2]. Evidence supporting the hypothesis that those elementsalso function as specific Rep-binding sites was recently reported [11], althougha direct, unequivocal interaction between iterons and Rep has not been experi-mentally demonstrated in a mastrevirus to date. Taking into account the peculiarfeatures of mastreviruses, their unique iteron arrangement and the considerabledivergence of their Rep proteins, it was important to establish whether there isalso a clear structural correlation between iterons and the Rep IRD region.

Close analysis of mastrevirus sequences revealed that MSV, DSV, PanSV,SSV, and the recently described SSEV and SSMV [5] encoded Rep proteinswith a conserved HRN(A/V)NT sequence in the immediate vicinity of Motif 1(Fig. 4). The predicted Rep cognate sequences of MSV and its relatives (with


Fig. 3. Correlations between amino acid residues of Rep IRD and specific nucleotides ofits cognate iterons.A Comparisons of Rep IRDs from closely-related geminiviruses whoseiterons differ in the N1 base. Because changes in the identity of the X3 amino acid residue areobserved in all cases, it is inferred that this IRD residue probably interacts with the formeriteron nucleotide.B, C Comparisons of Rep-IRDs from geminiviruses differing in the iteronN2 and N3 bases, respectively. The IRD residues whose chemical nature varies in a correlative

fashion with the former iteron nucleotides are indicated

the exception of PanSV) contain a pentanucleotide DNA core [YGCGC] whichis not present in the iterons of other mastreviruses (Fig. 4A), hence suggestinga functional connection between this iteron sequence and the former Rep motif.This presumption was strongly supported by the analysis of numerous MSVsequences available through GenBank databases. Indeed, it was found that only2 out∼30 MSV strains/isolates display a divergent sequence in the Rep X1-X7region and, correspondingly, they are the only MSV strains harboring differentiterons (Fig. 4B). The elements of MSV-Raw contain the [CGCGC] core andclear differences are observed only in the 3′ end of the TATA-associated iteron.In contrast, the iterons of MSV-Set contain a different pentameric core, CTCGC[55], and its Rep IRD displays a HRSPNT sequence instead of the HRN(A/V)NTmotif which is conserved in proteins of other MSV strains (Fig. 4B).

The relevance of these differences between MSV-Set and other mastrevirusesfrom the so-called African streak virus cluster [57] is highlighted by the obser-vation of considerable divergence outside the IRD-homologous segment amongproteins encoded by those viruses (Fig. 4B), which is in agreement with both theirevolutionary distance and the IRD hypothesis. On the other hand, analysis of pro-teins encoded by the dicot-infecting members of this genus (BeYDV and TYDV),which display two different, overlapping arrays of iterons, led to the presumptionthat the elements actually recognized by Rep are those with the begomovirus-like


Fig. 4. Structural correlations between iterative DNA elements and the Rep IRD-homologousregion of mastreviruses.A Putative Rep-binding sites and N-termini of replication proteinsfrom Mastrevirusspecies. The pentanucleotide motif which is common to iterons of virusesrelated to MSV, as well as a conserved amino acid stretch adjacent to Motif 1, are indicated.B Correlative variation of the iteron core and the amino acid sequence in the vicinity ofRep Motif 1 in viruses of the African streak group.C Comparison of the iteron arrangementdisplayed by the dicot-infecting mastreviruses TYDV and BeYDV with the homologouselements of a begomovirus (BGMV-PR) and two monocot-infecting mastreviruses (DSVand WDV). Note the remarkable similarities in nucleotide sequence between BeYDV andWDV within this region. The CA-motif is acis-acting element involved in geminivirus

replication [49]


arrangement, because these proteins display the IRD’s FRL∗Q motif character-istic of geminiviruses harboring iterons with a GGAGN core (Fig. 4A; see alsoFig. 2). This conclusion agrees well with the results of a phylogenetic-structuralanalysis of the intergenic region of TYDV and BeYDV, which revealed that thoseiterons, but not the ones displaying the typical mastrevirus arrangement, are thetrue evolutionary counterparts of begomovirus and curtovirus iterons (Fig. 4C).

Taken together, the data derived from this analysis of mastrevirus sequencesindicate that the amino acid stretch preceding Motif 1 is, plausibly, an importantcomponent of the Rep DNA-recognition domain, in close agreement with conclu-sions derived from the study of curtoviruses, topocuviruses and begomoviruses.

Discussion

By using a comparative approach based on a series of heuristic hypotheses, wehave identified a Motif 1-associated subdomain of geminivirus Rep proteins dis-playing two significant features: a) its primary structure differs among virusesharboring distinctori-associated iterons, and b) it is generally similar amongviruses with identical iterons, regardless of their differences in host range, in-sect vector, geographical origin or genome structure. Close examination of thisiteron-related subdomain revealed a consistent correlation between its amino acidsequence and its predicted cognate DNA elements, hence suggesting that it is,plausibly, a major component of the specific DNA recognition domain of gemi-nivirus Rep.

Several lines of experimental and indirect evidence support the latter con-clusion. 1) The IRD is located within the Rep region where the trans-actingreplication specificity determinants of geminiviruses have been mapped [12, 13,23, 35, 61]. 2) The deletion or mutation of the IRD-Motif 1 region of TGMV Repeliminated its specific DNA-binding capability [50, 51]. 3) A truncated ACMVRep containing only the first 57 aa residues, could suppress the expression of itsown gene as effectively as the intact, full-length protein [33]. 4) The divergencein IRD sequences among proteins encoded by geminiviruses whose genomes arevery similar, and are generally considered as “strains” of a virus, consistentlycorrelates with differences in their iterons (data from this study; see Fig. 2). 5)Geminiviruses with a defined iteron GGN1N2N3 core encode proteins with acharacteristic IRD sequence consensus not found in viruses harboring differentiterons. Such a similarity in Rep IRD cannot be explained satisfactorily by merephylogenetic relationships, host-or vector-adaptation processes, random sequenceconvergence or recombination events, because viruses from an isospecific groupinfect a variety of plant species, are indigenous to different continents and/or be-long to different genera. 6) The∼25 known geminiviruses whose iteron N1 baseis an Adenine encode Rep proteins whose IRD residue X3 is Gln, but none of the∼95 remaining members of this virus family display a similar IRD X3 residue;similarly, the only two geminiviruses with an N1 = C (i.e. ToLCNdV-Mld andToLCNdV-Luf) are also the only ones displaying an IRD X3 = Asp; these andother analogous observations are consistent with the hypothesis of a functional


relationship between specific IRD residues and iteron nucleotides. 7) Finally, thepredicted functional relationship between the IRD X3 residue and the iteron N1nucleotide has been experimentally verified in the case of two strains of a bego-movirus, whose iterons differ only in N1. Indeed, it was shown that the exchange,by site-directed mutagenesis, of amino acid residue 10 of the Rep proteins en-coded by ToLCNdV-Sev and ToLCNdV-Mld (Fig. 1A), resulted in the exchangeof its replication specificity [12].

A simple model of Rep-iteron interactions

The DNA/protein correlation found in this study suggests a defined polarity inthe interactions between Rep and its cognate elements. Thus, nucleotides withinthe 3′ end of the iteron are apparently recognized by aa residues located withinthe IRD N-terminus (e.g. X−4), while N1 interacts with the X3 residue in the IRDC-terminus. Figure 5A summarizes these correspondences in a general “model”,which stresses the coherence of data derived from multiple viral sequence compar-isons. Since the GG dinucleotide conserved in the iterons of all dicot-infectinggeminiviruses apparently correlate with the also conserved (A/S)(K/R)N(Y/I)motif, it is possible that this is involved in its recognition. Two additional ob-servations support this presumption: a) an analogous motif is not present in theequivalent Rep region from the monocot-infecting geminiviruses, the iterons ofwhich lack a GG dinucleotide in the analogous position (see Fig. 4); b) the AKNYmotif is part of a predicted amphipathicb-sheet structural element that includesresidue X3 (see below), which has been implicated in the recognition of the iteronnucleotide adjacent to the GG dinucleotide in ToLCNdV [12].

Is the DNA-binding specificity of Rep proteins determinedby a combinatorial molecular code?

The data assembled in this work suggest that the X1 and X3 IRD residues playa pivotal role in the control of Rep DNA-binding specificity. Indeed, in somecases the nature of the X3 and X1 amino acid apparently dictate the preferencefor specific bases in the iteron N1 and N2 nucleotides, respectively. For example,viruses whose Rep have a Gln in any one of these two IRD positions, bear almostinvariably an Adenine in the corresponding iteron nucleotides (see Figs. 2 and 3).

Nonetheless, several observations strongly suggest that recognition of theiteron by Rep is not dependent on a simple one amino acid-one base correspon-dence code, but on one that is combinatorial in nature. For example, a few viruseswith similar iterons display different IRD sequences (Fig. 2). This is the case ofSACMV and EACMV-TZ, whose iterons display a GGGGG core; since theseviruses have a Rep IRD only differing in the X1 and X3 positions (Fig. 5B), aplausible conclusion is that different X1−X3 combinations can determine a sim-ilar DNA specificity. On the other hand, a few viruses encoding proteins witha common FX1−L∗−X3 motif display divergent iterons; in these cases a clearvariation in the X−3 to X−1 positions is observed (e.g., see the ToLCTZV andEACMV-TZ elements compared in Fig. 5B), suggesting that those aa residues,


Fig. 5. A Model of Iteron-Rep interactions derived from data of this study. The hypotheticalfunctional interactions between the IRD X3, X1 and (X−4/X−2) residues, with the N1, N2and N3 iteron nucleotides, respectively, were inferred from consistent correlations betweeniteron sequences and the primary structure of the former protein domain. It is suggested thatthe (A/S)(K/R)N(Y/I) sequence is involved in recognition of the GG dinucleotide conservedin the iterons from all dicot-infecting geminiviruses.B Examples illustrating the notion thata combinatorial code dictates the Rep preference for specific DNA sequences. The cases inwhich different combinations of X1–X3 IRD residues apparently determine a Rep preference

for iterons with identical N1–N2 bases are indicated with brackets


although themselves not correlating with specific iteron nucleotides, could sig-nificantly alter the relative affinity of the FX1−L∗−X3 segment by a given DNAelement.

The comparisons illustrated in Fig. 5B also indicate that the number of dif-ferences between iteron cores do not correlate linearly with differences betweenRep IRDs, and even a single substitution in certain IRD positions can, apparently,determine significant changes in DNA preferences (e.g. compare the TYLCTHVand PYMV-TT elements). A corollary of all these observations is that the pre-dictive value of the model illustrated in Fig. 5A, however significant, is clearlylimited. In effect, that model can anticipate that a Rep protein displaying a MPPP-KRFRLQ sequence in its N-end (corresponding to a cognate GGAGN iteron)would change its DNA preference for a GGTAC, GGGGA or GGTGT iteron,if the FX1X2X3 core is mutated to FQIN, FSVN or FRLN, respectively. How-ever, it cannot predict the DNA preferences of mutant Rep proteins displayingan identical FX1X2X3 core but a different X−4X−3X−2X−1 motif, neither it cananticipate what would be the IRD sequence of a Rep protein whose cognateiteron is not found in any known geminivirus. It is important to bear in mind,when experiments aimed to test the former predictions were designed, that thepostulated interaction between iterons and Rep IRD is simply a specific protein-DNA binding relationship, required but not sufficient for Rep-mediated replica-tion.

Does Rep recognize more than one iteron core?

The number of iterons in theori of geminiviruses varies from lineage to lineage,being typically 3 in curtoviruses and NW begomoviruses, 4 in OW begomoviruses,and 5–6 in begomoviruses from the SqLCV-cluster [2]. Although in most casesthe iteron core is well conserved among all the elements in a given virus, asignificant number display iterons with a degenerate sequence (see Fig. 2). Theseobservations suggest the intriguing possibility that Rep may be able to recognizeand bind different DNA sequences, albeit with different affinity. Two corollariescan be derived from this notion: 1) geminiviruses might replicate, under certainconditions, heterologous genomic components with different iterons; 2) althoughrare, viruses with different iterons but identical IRDs might exist (e.g. CPGMV-BZ and BGMV-BZ; see Fig. 2).

The scant reports of cross-replication between geminiviruses harboring dif-ferent iterons, such as MSV-Kom and MSV-Setaria [55], or the apparent abilityof the A component of certain bipartite geminiviruses to support, at low but sig-nificant levels, the replication of B components displaying divergent iterons [12,54, 56] represent important evidence that, at least in some conditions, Rep mightbe able to recognize different iterons in vivo. Analogous situations have been ob-served among RCR plasmids of bacteria, whose Rep proteins are usually highlyspecific but may drive the replication from a heterologous origin if they are ex-pressed at high levels, hence suggesting that they can bind a range of sequenceswith different affinity ([37, 38], and references therein).


Is the Rep Motif 1 the core element of an evolutionarilyconserved DNA-binding domain?

An important finding of this work is that a putative Rep specificity determinantis closely associated to a conserved motif of RC replication proteins [34]. Todate, no precise function has been assigned to Motif 1, although a recent study ofTGMV Rep showed that mutations in this motif abolished both Rep DNA bindingand cleavage activities [51].

The assembled data allow us to speculate that Motif 1 could be a structuralcore of a yet unrecognized DNA-binding domain, a notion supported by the ob-servation that conserved IRD residues and Motif 1 are spaced in an invariant way,as above noted (see Results). Furthermore, analysis of homologous proteins froma number of RC-replicating systems related to geminiviruses, including the plant-infecting nanoviruses [7, 10, 36, 58], the mammal- and bird-infecting circoviruses[3, 44, 48], three plasmids of the red algaPorphyra(D. A. Moon and L. G. Goff, un-published; Accession No. AF106326-8), and bacterial plasmids belonging to thegeminivirus-related pMV158 family [16, 39] and the microvirus-related pC194and pUB101 families [37], display an analogous structural arrangement, with ashort, hypervariable amino acid stretch adjacent to Motif 1, which is similar onlyamong replicons harboring identical (actual or putative) Rep binding sites (ourunpublished data).

Secondary structure analysis of∼50 geminivirus Rep proteins using the Pro-tean program available with the LaserGene package revealed that the protein N-terminal arm (1–52 region) forms an amphipathicbaa structure, in which Motif1 and IRD together constitute theb-sheet component (Fig. 6). Thisbaa struc-tural arrangement was also predicted by a different program (PHDSEC) used ina comparative analysis of replication proteins encoded by nanoviruses and gem-iniviruses [24]. Preliminary analysis of homologous proteins from diverse RCviruses and plasmids have revealed a strong structural conservation in thebaarrangement in the Motif 1 region, but a less stringent conservation or absence ofa seconda-helix (our unpublished data).

The conservation in geminivirus Rep proteins of twoa-helices adjacentto Motif 1 was previously noticed [51], and mutational analysis showedthat their integrity is important for both Rep DNA binding and cleavageactivities [50]. Interestingly, a similarbaa structure is displayed by theDNA-binding domain of MetJ, Arc and Mnt, three well characterizedprokaryotic repressor proteins. Moreover, structural and genetic studies haveestablished that theb-sheet is involved in specific DNA recognition in allthese proteins [30, 52], as suggested for geminivirus Rep by our comparativeanalysis.

Given that Rep acts as a potent repressor of its own gene promoter via inter-actions with the iterons [17, 18], the findings in the present study and the modelof iteron-IRD interactions proposed here may well serve as basis to the develop-ment of new strategies for the protection of crops against geminivirus infections.Finally, this work suggests a simple and novel theoretical method which, experi-


Fig. 6. Sequence alignment for 15 geminivirus Rep proteins shows a predictedb-strandseparated by 5–6 amino acids from two predicteda-helices. The region potentially forminga b-strand encompasses both Motif 1 (FLTY) and the IRD. The shaded box shows theamphipathic segment of this structural element which does not include Motif 1. The sequencesare grouped according to well-defined geminivirus lineage: I- Old World begomoviruses, II-New World begomoviruses, III- curtoviruses, IV- begomoviruses of the SLCV-cluster, V-Mastreviruses. A hexapeptide motif (RELHED) conserved in the seconda-helice of proteinsencoded by members of all geminivirus lineage, with the exception of the SLCV cluster,

is underlined

mental data pending, will be useful to predict interactions between DNA-bindingproteins and its cognate sequences.

Acknowledgements

We thank Drs. Gabriela Olmedo-Alvarez, Ramón Guevara-González, Rafael Rivera-Bustamante, Trino Ascencio-Ibañez, and Luis Herrera-Estrella for critical reading of themanuscript and many helpful suggestions. We are also grateful to Dr. Octavio Martinez de laVega and Dr. Irineo Torres-Pacheco for assistance with the phylogenetic analysis. The authorswere supported by a fellowship from the Sistema Nacional de Investigadores (SNI, México).

We dedicate this work to the memory of Dr. Carlos Argüello-López, a lucid scientist andan exceptional human being.

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Author’s address: Dr. G. R. Argüello-Astorga, NCSU/Biochemistry, Box 7622, Raleigh,NC 27695-7622, U.S.A.; e-mail: [email protected]

Received December 14, 2000

P1: PSA/nny/kdg P2: ARS/plb QC: ARS/anil T1: ARS

March 30, 1998 10:27 Annual Reviews AR060-21

Annu. Rev. Plant Physiol. Plant Mol. Biol. 1998. 49:525–55Copyright c© 1998 by Annual Reviews. All rights reserved

EVOLUTION OF LIGHT-REGULATEDPLANT PROMOTERS

Gerardo Arguello-Astorga and Luis Herrera-EstrellaDepartamento de Ingenier´ıa Genetica de Plantas, Centro de Investigaci´on y de EstudiosAvanzados del IPN, Apartado Postal 629, 36500 Irapuato, Guanajuato, M´exico

KEY WORDS: gene evolution, plant evolution, plastid-nuclear interactions, photoreceptors

ABSTRACT

In this review, we address the phylogenetic and structural relationships betweenlight-responsive promoter regions from a range of plant genes, that could explainboth their common dependence on specific photoreceptor-associated transductionpathways and their functional versatility. The well-known multipartite light-responsive elements (LREs) of flowering plants share sequences very similar tomotifs in the promoters of orthologous genes from conifers, ferns, and mosses,whose genes are expressed in absence of light. Therefore, composite LREs haveapparently evolved fromcis-regulatory units involved in other promoter functions,a notion with significant implications to our understanding of the structural andfunctional organization of angiosperm LREs.

CONTENTS

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 526

LIGHT REGULATION OF GENE TRANSCRIPTION: A BRIEF OVERVIEW. . . . . . . . . . 527Control of Gene Expression by Photoreceptors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 527Light-Responsive Promoters. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 527The Phylogenetic-Structural Sequence Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 528

EVOLUTION OF LREs INLhcb PROMOTERS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 529The Proximal LRE of Lhcb 1 Genes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 529Evolution of the Proximal Region of Lhcb1 Promoters. . . . . . . . . . . . . . . . . . . . . . . . . . . . 531LREs in the Central Region of Lhcb1 Promoters. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 532Evolution of the Central LRE of Lhcb1 Promoters. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 532Delimitation of a LRE in Lhcb2 Promoters. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 533Evolution of Lhcb2 Promoters. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 533

EVOLUTION OF LREs INrbcS PROMOTERS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 534The Light-Responsive Box II-III Region. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 534Evolution of the Box II-III Homologous Regions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 536

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The Light-Responsive I-G Unit. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 538Evolution of the I-G Region. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 538

EVOLUTION OF THE LIGHT-REPRESSED PHYTOCHROME A PROMOTER. . . . . . . . 539Functional Organization of phyA Promoters. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 539Evolution of phyA Promoters. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 539Evolution of chs Promoters. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 540

EVOLUTION OF LIGHT-REGULATED PARALOGOUS GENE PROMOTERS. . . . . . . . . 542Differential Activity of Paralogous Gene Promoters. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 542Do Paralogous Promoters Evolve by Nonrandom Processes?. . . . . . . . . . . . . . . . . . . . . . 543

LRE-ASSOCIATED CMAs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 544CMAs in Additional Plant Genes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 544Structural Analogies Between LRE-Associated CMAs. . . . . . . . . . . . . . . . . . . . . . . . . . . . 544Common Structural Features of Composite LREs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 545Evolution of LREs: The Chloroplast Connection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 546

CONCLUDING REMARKS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 549

INTRODUCTION

Photosynthesis is an ancient biochemical process that probably evolved 3500million years ago (111). The nuclear genome of higher plants encodes proteinshomologous to cyanobacterial proteins of photosynthesis; because these bac-teria are the living descendants of the original photosynthetic organisms, plantgenes encoding such proteins are among the oldest genes of eukaryotes.

To date, molecular evolution studies have concentrated on the coding se-quences of gene families. The evolution of regulatory sequences, which de-termine where, when, and the level at which genes are transcribed, has beenlargely neglected. In the case of the photosynthesis-associated nuclear genes(PhANGs) from higher plants, interesting evolutionary aspects of the molec-ular mechanisms by which transcription is activated by light receptors (e.g.phytochrome) could be addressed through the comparative analysis of pro-moter sequences. For instance, why does light profoundly affect transcriptionof PhANGs in monocotyledonous and dicotyledonous plants, while PhANGpromoters in conifers, ferns, and mosses are either light insensitive or, at most,weakly photoresponsive (4, 71, 89, 99, 129). The systematic comparisons ofangiosperm and nonflowering plant PhANGs promoter sequences provide aunique opportunity to explore how a new regulatory function, light respon-siveness, was incorporated into the promoters of a wide range of genes whoseexpression is coordinately regulated.

Besides its obvious relevance for evolutionary studies, a comparative analysisof the structure of photoregulated promoters can be useful to address otherimportant issues in plant gene expression. Comparative analysis of PhANGupstream sequences may contribute to reduce the apparent diversity of light-responsive elements (LREs) by revealing concealed phylogenetic and structural

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PROMOTER EVOLUTION 527

relationships between dissimilar promoter regions with analogous functions.Other important issues concerning the composition and functional organizationof LREs in plant genes could also be uncovered by their analysis from anevolutionary perspective.

The purpose of this review is to address the phylogenetic and structuralrelationships between light-responsive promoter regions from orthologous andparalogous plant genes. We review LREs from a wide range of genes, exploretheir common dependence on specific phototransduction pathways, and analyzecorrelations between the composition of multipartite LREs and their overallfunctional properties.

LIGHT REGULATION OF GENE TRANSCRIPTION:A BRIEF OVERVIEW

Control of Gene Expression by PhotoreceptorsThe responses of plants to light are complex: seed germination, de-etiolation ofseedlings, chloroplast development, stem growth, pigment biosynthesis, flow-ering, and senescence (67). Most of these responses require changes in bothchloroplast and nuclear gene expression, which are mediated by three majorclasses of photoreceptors: phytochromes, blue/UV-A light receptors, and UV-Blight receptor(s) (2, 61, 100). Light-regulated genes may respond to more thanone photoreceptor, thus allowing a finely tuned control of their expression whenstimulated by light (123).

The best characterized light receptor is phytochrome (PHY), which exists intwo photochemically interconvertible forms, Pr and Pfr, and is encoded by asmall family of genes in angiosperms (42, 99, 100). PHY controls the expres-sion of diverse genes at the transcriptional, posttranscriptional, and translationallevels (43, 112, 123). Gene expression is regulated by at least three differentsignal transduction pathways activated by PHY: one depends on cyclic GMP(cGMP), which regulates genes such as those involved in anthocyanin biosyn-thesis in some species; a second pathway depends on calcium/calmodulin,which activates a subset of chloroplast-associated nuclear genes (17, 85); and athird signal pathway, which requires both calcium and cGMP, activates a subsetof genes necessary for chloroplast development (e.g. the gene encoding ferre-doxin NADP+ oxidoreductase) (18), and represses transcription ofphy-Aandthe gene encoding asparagine synthetase (91).

Light-Responsive PromotersIn flowering plants, light regulation of nuclear genes occurs mainly at the tran-scriptional level, as demonstrated by nuclear run-on transcription assays and

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promoter-reporter gene fusions (112, 122, 126). The most extensively studiedlight-responsive genes are those encoding the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcS) and the chlorophylla/b bindingproteins (Lhc, formerly calledCab), both of which are a paradigm for PhANGscontrolled by the calcium/calmodulin phototransduction pathway (17). Chal-cone synthase (chs) genes, on the other hand, have been the model for genesinvolved in the anthocyanin biosynthetic pathway in some dicots and, hence, forgenes targeted by the phytochrome signal pathway dependent on cGMP (17, 85).More recently, PHY-A genes have become a model for the diverse genes whosetranscription is down-regulated by light and which are controlled by the thirdphytochrome signaling pathway, dependent on calcium and cGMP (91).

A plethora ofcis-acting elements and protein factors presumably involvedin transcriptional light responses have been identified (13, 16, 122, 126); how-ever, conclusive evidence for an essential role in light responsiveness has beenobtained for only a few of them (5, 41, 66, 136). Several general conclusionsare possible: 1. No single conserved sequence element is found in all light-responsive promoters. 2. The smallest native promoter sequences, sufficient toconfer light inducibility on heterologous minimal promoters, are multipartiteregulatory elements that contain different combinations ofcis-acting sequences.3. Even the smallest known photoresponsive promoter regions, when exam-ined in a heterologous context, display fairly complex responses, often retainingthe dependence on light wavelength, developmental stage of chloroplasts, andtissue specificity of the native promoter. 4. Numerous protein factors bindsequences within photoregulated promoters, although their actual contributionto light-activated transcription remains uncertain (13, 122, 126, 128).

The Phylogenetic-Structural Sequence AnalysisA commonly used approach to identify regulatorycis-elements is to search forconserved DNA motifs within the promoter region of orthologous genes. Suchanalyses have been successful in identifyingcis-acting elements involved inthe light responsiveness of PhANGs, such as the G-box and I-box elementsfrom rbcSgenes (48) and the GATA motifs ofLhcb1genes (44). However,regulatory elements showing sequence degeneracy are not easily recognized incomparative analyses.

Sequence heterogeneity of regulatory elements may be functionally over-come if multiprotein regulatory complexes facilitate binding to imperfect targetsites (86, 135). Because conventional computer programs for DNA sequencecomparisons can fail to detect evolutionarily related but structurally variablepromoter regions with analogous functions, alternative approaches have beendeveloped, such as the “phylogenetic-structural method” of sequence analy-sis (8, 10). This method is based on the search of “homologous” (rather than

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“similar”) DNA sequences of a functionally characterized promoter. Two se-quences are homologous when they share common ancestry, regardless of thedegree of similarity between them (37). In this sense, the G-box elements ofrbcS, chs, andLhcb1genes are not homologous but only similar because theyhave different evolutionary origins, whereas therbcSI-G unit of solanaceousspecies is probably homologous to therbcSX-Y promoter region ofLemna,an aquatic monocotyledonous plant, despite their rather low overall sequencesimilarities (10).

The individual elements found within a multipartitecis-regulatory regionare termed phylogenetic footprints (PFs); they share high conservation overa segment of 6 contiguous base pairs in alignments of orthologous upstreamsequences and represent potential binding sites for transcription factors (53).A cluster of PFs whose arrangement (combination, spacing, and relative ori-entation) is conserved through a phylogenetic series of homologous promoterregions is termed a conserved modular arrangement (CMA). If a given CMAconsistently correlates with experimentally defined light-responsive promoterregions, then it is called an LRE-associated CMA. In this method, sequencecomparisons are made in a phylogenetically ordered fashion, because the over-all structure of multipartite regulatory units tends to diverge in evolution. Thus,homologous genes from species belonging to the same plant taxon should becompared first. Comparisons with other taxons follow. This procedure allowsus to discern how an ancestral multipartite regulatory module has changed inan evolutionary context and can establish phylogenetic relationships betweenpromoter regions that are apparently unrelated by structural criteria. An exam-ple of this analysis applied toLhcb1 light-responsive regions is presented inFigure 1a.

EVOLUTION OF LREs INLhcbPROMOTERS

The Proximal LRE of Lhcb 1 GenesThree types of chlorophylla /b binding proteins are found in the major light-harvesting complex of photosystem II, encoded byLhcb1, Lhcb2, andLhcb3(52, 60). MostLhcbpromoters that have been functionally analyzed are fromtheLhcb1gene family and typically lack introns. InLhcb1genes from dicotyle-dons, an LRE is located in the proximal promoter region of these genes. ThisLRE is characterized by three conserved GATA motifs spaced by 2 and 6 bp,respectively, which are located between the CCAAT and TATA boxes (44, 87).In ArabidopsisLhcb1∗1 (formerlyCab2), the−111 to−33 region is sufficientto confer a pattern of expression dependent on both PHY and a circadian clock,to a heterologous minimal promoter (6). A nuclear factor that specifically inter-acts with the triple GATA repeats found in this LRE was identified and named

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CGF-1 (6). If the three GATA motifs are altered by site-directed mutagenesis,the interaction of CGF-1 is disrupted and responsiveness to PHY and part ofthe light-induced circadian oscillation ofCab2expression are comprised (5).Mutational analysis of the ArabidopsisLhcb1∗3 (Cab1) gene promoter iden-tified additionalcis-acting motifs, that could be involved in the functionalityof this LRE. Mutations in a 27-bp region upstream the CCAAT box, whichis bound by the CA-1 factor (75a), drastically reduced the overall promoteractivity and also eliminated PHY responsiveness (68). More recently, a Myb-related transcription factor that specifically interacts with a conserved sequencemotif [AA(C/A)AAATCT] within the CA-1 region was cloned (131). Trans-genicArabidopsisexpressing an antisense mRNA for this factor (called CCA-1)showed reduced PHY induction of the endogenousLhcb1∗3 gene, whereas ex-pression of other PHY-regulated genes, such asrbcS, was not affected; thusCCA-1 has a specific role inLhcb1photoregulation (131).

Evolution of the Proximal Region of Lhcb1 PromotersAt least five PFs are found within the first 110–130 bp of theLhcb1promotersequences from dicotyledons. All these PFs bind defined proteins. The mostdistal PF is the CUF-1 binding site, a G-box-related sequence which functionsas a general activating element (6). The second PF is the CCA-1 bindingsite (131). The third is the CCAAT-box, which is part of the binding site ofTac, a protein factor proposed to be involved in the regulation ofArabidopsiscab2by the circadian clock (22). The fourth and fifth PFs are located in theCGF-1 binding region, comprising the three GATA motifs located upstream tothe TATA box. Comparative analyses indicate that these GATA elements aredistinct from both the phylogenetic and the structural point of view. 1. GATA Iis part of a PF whose consensus is GATAAGR (the I-box motif) (48), whereasGATA II/III form a single PF with a GATANNGATA consensus in dicotyledons.2. Apparently GATA I is a more ancient element than GATA II/III, inferredfrom the fact that the former, but not the latter, element is present inLhcbpromoters of gymnosperms (11, 71).

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−Figure 1 (a) Lhcb1promoter regions that are homologous but dissimilar. The indicated CGF-1binding site was defined by Teakle & Kay (121). The Z-DNA element of theArabidopsis Lhcb1∗3promoter has been functionally characterized by Ha & An (54) and by Puente et al (98). Theillustrated region corresponds to the cabCMA3 of the genes indicated in the figure. (b) Hypotheticalarrangement of the upstream LRE ofLhcb1promoters in the common ancestor of Dicotyledons.Two putative LREs derived from the ancestor, which have been previously identified as cabCMA-3and cabCMA-2 (10), are shown. GATA-containing sequences of maize genes that are inverted withrespect to dicot homologous motifs areunderlined. Notice that in some cases, a CCA-1 motif isthe evolutionary counterpart of an I-box element.

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Within dicots only the CCAAT and GATA motifs are invariably present inthe TATA-proximal region. The CUF-1 and CCA-1 binding sites are foundin some but not allLhcb1genes. Only one tomato gene (Lhcb1∗5) containsboth CUF-1 and CCA-1 binding sites, four (Lhcb1∗1, ∗2, ∗3, and∗4) containthe CUF-1 element but lack the CCA-1 site, and two (Lhcb1∗6 and∗7) lack adiscernible CUF-1 site but contain an inverted CCA-1 binding site. Althoughit is probable that these structural differences determine qualitative differencesin the regulation of these paralogous promoters, transcript abundance in tomatoleaves is not correlated with the composition of their proximal region (97).

Most of the knownLhcb1genes of monocotyledons have promoters clearlydistinct from those of dicots, with two exceptions: theLemna Lhcb1∗1 (AB30)and the maizeLhcb1∗2 (cab-1) genes (69, 118), both containing a (CUF-1site)-(CCAAT-box)-(GATA II)-(I-box) arrangement homologous to that of dicotpromoters (10). This arrangement inLhcb1promoters may either predate thedivergence of monocots and dicots or result from convergent evolution. Supportfor an ancient origin comes from an orthologous gene of a conifer (Pinuscontorta) that has a promoter (CCAAT-box)-(I-box)-(TATA-box) arrangementthat is similar to that found in angiosperms (see Figure 6). The pine gene lacksthe GATA III, CCA-1, and CUF-1 elements, suggesting that these elementsprobably evolved after the divergence of the lineages leading to modern conifersand angiosperms.

LREs in the Central Region of Lhcb1 PromotersLhcb1genes contain additional LREs, besides those found in the TATA-proxi-mal region. Castresana et al (23) demonstrated by gain-of-function experimentsthe photoresponsiveness of the−396 to−186 region of the tobaccoCab-Egene. A light-responsive region has also been mapped to the−347 to−100promoter region of the peaLhcb1∗2 (AB80) gene. This 247-bp fragment wasshown to function as a light-responsive, tissue-specific enhancer in gain-of-function experiments (113). Two nuclear factors binding this regulatory regionwere identified, one of which (ABF-1) is found only in green tissues (7). Bysystematic deletion theAB80enhancer light-responsive core is located in the−200 to−100 region (Arg¨uello-Astorga & Herrera-Estrella, manuscript inpreparation). Evidence for LREs in the−240 to−100 region of theArabidopsisCab2promoter has been also reported (121).

Evolution of the Central LRE of Lhcb1 PromotersThe comparative analysis of the tobaccoCabEand pea AB80 upstream LREs,with the corresponding promoter segments from otherLhcb1genes from di-cots, suggests a structure of this promoter region in the common ancestor ofdicotyledons. An extensive ancestral CMA encompassing at least five PFs is

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inferred (Figure 1b). This ancestral organization is still conserved in somegenes of leguminous species [e.g. peaCab-8(3)]. The central PF of this CMAis a sequence similar, but not identical, to the G-box fromrbcSgenes. In to-baccoCabEthis G box–like element (G∗box) is associated with a Box II–likeelement corresponding to a PF with a GATA core motif (23). Homologous Gbox–like and 5′ associated GATA-motif elements (I∗box) exist in members ofthe Lhcb1gene family in plants belonging to four orders of dicotyledons. Athird PF (YCCACART) is found immediately upstream of the G-box elementin severalLhcb1genes of legumes and the tobaccoCab-Epromoter but notin other dicot genes. Interestingly, this PF is also found in the homologousregion of two maizeLhcb1genes (Figure 1b), suggesting a very ancient originof this PF. A fourth PF with the consensus CATTGGCTA closely precedes aPF encompassing an I-box motif located 15–20 bp downstream of the G-boxelement. The arrangement of thisLhcb1region (cabCMA2) is highly analo-gous to that of the G-, I-box region of dicotrbcSpromoters, with a very similarPF associated 5′ to the I-box motif. The resemblance in some cases is so strik-ing that aLhcb1promoter (e.g. peaAB80) can display more similarity in thisregion with the analogousrbcSpromoter segment (e.g. pearbcS 3A) than withcertain homologousLhcb1 regions (10). The analogies between the overallstructural organization of the I∗-G∗-I promoter region ofLhcb1genes and theI-G-I unit of rbcSpromoters are intriguing and suggest either a common ances-try or convergent evolution of these regulatory promoter modules.

Delimitation of a LRE in Lhcb2 PromotersAlthough comparatively few members of theLhcb2 gene family have beenstudied, the promoter of one, theLhcb2∗1 (formerly AB19) gene fromLemnagibba, has been characterized in great detail. Deletion analysis, linker scanning,and site-directed mutagenesis identified two 10-bp elements in the−134 to−105 region that are critical for light responsiveness. One of them containsthe I box–related GATAGGG motif and the other a CCAAT motif. Mutationof the latter element led to high levels of expression in the dark, suggestingthat it binds a repressor in the absence of light. Mutations in the I-box motifled to complete loss of red-light responsiveness, suggesting that this region isinvolved in PHY- mediated light activation of transcription (66). More detailedsite-directed mutagenesis of those 10-bp regions allowed the identification oftwo shorter sequences (REα = AACCAA and REβ = CGGATA) that arecritical for AB19light regulation (31).

Evolution of Lhcb2 PromotersGenomic clones ofLhcb2genes have been isolated from the mossPhyscomi-trella patens (79), the fernPolystichum munitum(96), the coniferPinus

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thunbergii(71), the monocotLemna gibba(64), and three dicotyledonous plants(pea,Petunia, and cotton) (39, 103, 115). These promoters contain a number ofPFs, two of which correspond to theLemnaREα and REβ elements (Figure 2).Only a few of the identified PFs are present in all species. Among the PFscommon to most if not all the knownLhcb2promoters are theLemnaREα ele-ment, generally recognized as a conserved CCAAT box, and a G(G/A)AAATCTmotif, which is similar to the CCA-1 binding site of theLhcb1genes.

An interesting observation is that the gene harboring sequences with thehighest similarity to theLemnaREα and REβ light-responsive elements isthat ofP. thunbergii(see Figure 2), whose promoter directs light-independentgene expression in both its native context (71) and in transgenic angiosperms(70, 138). This paradoxical observation is discussed below. Another relevantobservation is thatLhcb2genes from dicotyledons lack a REβ element, insteaddisplaying three lineage-specific PFs, two of which include inverted GATAmotifs (Figure 2). It would be interesting to determine whether those elementsare functionally equivalent to REβ.

Upstream sequences ofLhcb2andLhcb1genes are probably derived froma common ancestral promoter, because they display a similar overall struc-tural organization, with a (G Box/CUF-1 site)-(CCA-1 element)-(CCAAT-box)-(I-box)-(TATA box) basic arrangement. Spacing between these elements is,however, very different in the two gene families.

EVOLUTION OF LREs INrbcSPROMOTERS

The Light-Responsive Box II-III RegionThe pearbcS-3Agene has been used as a paradigm for the study of light-regulated gene expression in dicotyledons. Analysis of therbcS-3Apromoterhas uncovered three independent regions that contain an LRE (47). The LRElocated in the−166 to−50 region has been characterized in most detail (45).This region includes two elements called Box II and Box III, both of which arebinding sites for a nuclear factor named GT-1 (49). This factor binds in vitro tosequences related to the degenerate consensus (A/T) GTGPu (T/A) AA (T/A)(50). A synthetic tetramer of the pea Box II element (GTGTGGTTAATAATG)conferred light-responsive transcriptional activity to the−90 CaMV 35S pro-moter (75, 98) but was unable to enhance transcription when fused to eitherthe−46 CaMV 35S or the−50 rbcS-3Aminimal promoters (28). This ele-ment appears necessary, but not sufficient, for light-regulated transcriptionalactivation. Arabidopsisand tobacco cDNAs encoding GT-1, a Box II DNAbinding proteins, have been cloned and partially characterized (46, 58, 95). In-terestingly, GT-1 is closely related to the GATA-binding nuclear factors CGF-1and IBF-2b and can bind to similar cognate DNA sequences (121). Two other

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Fig

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proteins called 3AF-3 and 3AF-5 probably act together with GT-1 to conferrbcS-3Alight-regulated expression; they bind inverted GATA motifs locatedat each end of Box III. Mutations in these GATA sequences severely reducedpromoter activity (104).

Evolution of the Box II-III Homologous RegionsUpstream sequences of many angiospermrbcSgenes are known. However,only two rbcS promoters are known from nonflowering plants, the coniferLarix laricina (59) and the homosporous fernPteris vittata(56). Comparisonof the Pteris and Larix proximal promoter regions (−130/−1) reveals onlyfour PFs, two of which are inverted I box–related motifs: one overlappingthe putative TATA-box of theLarix gene, and the second, with the sequenceGTTATCC, found∼70 bp upstream. In theLarix promoter, this PF is foundas an imperfect direct repeat, flanked by two additional PFs, one of whichencompasses a CCAAT motif. The relative position of this CMA in the coniferpromoter (i.e.∼25 bp downstream to the I-box element) is practically identicalto that of the Box II–3AF3 region in dicotrbcSpromoters. Comparison of theseCMAs uncovers several interesting characteristics:

1. The Box II element seems to be evolutionarily derived from two separatePteris/Larix PFs (Figure 3). One of them is the most 5′ GTTATCC motifin Larix, which is homologous to the 3′ half of Box II. This relationshipis especially clear inrbcS promoters of the Brassicaceae (see Figure 6).Therefore, Box II could be a composite element bound in vivo by two pro-tein factors, one of them being a GATA-binding factor.

2. The second repeat of the GTTATCC motif ofLarix is homologous to a con-served sequence immediately downstream to Box II, which in ArabidopsisandBrassicagenes is almost identical to the so-called LAMP binding site(51), an inverted I-box element. In nonbrassicaceous dicots the sequencesimmediately downstream of Box II do not resemble the LAMP motif or thePteris/LarixPF, but their structural relationship is easily recognizable in aphylogenetic series (not shown, but see 82).

3. ThePteris/Larix CCAAT-box is apparently homologous to the pea 3AF3binding site (104). In the conifer promoter the LAMP-like motif and theCCAAT box are close, but in dicots the 3AF3 element is separated fromthe LAMP-related motif by 10–24 bp. Sequences in this intermediate DNAare functionally relevant, encompassing in pearbcS-3Athe Box III and the3AF5 elements (104) (Figure 3).

The Box II–containing CMA is absent in all knownrbcSgenes of mono-cotyledons (∼10), and in the orthologous genes of a dicot species, the common

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Fig

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ice plant (Azoideae) (35). Our interpretation is that these groups have lost BoxII, which we consider an ancestral feature ofrbcSgenes.

The Light-Responsive I-G UnitrbcSpromoters usually contain two closely associated elements, the I and Gboxes. Mutations of either the G-box or the two flanking I-box elements ofthe ArabidopsisrbcS-1Apromoter almost abolished its activity (36). A simi-lar drastic drop of transcriptional activity was observed in the spinachrbcS-1promoter when the G-box element was mutated (80). In spite of its functionalrelevance in dicots, deletion of a G-box in therbcSZm1gene of maize had nosignificant effects on promoter activity. Deletion of the associated I-box motifreduced expression in light 2.5-fold (106).

Lemna rbcSpromoters lack a typical G-box element but contain a canonicalI-box motif within the so-called X-box element, which is part of the binding siteof LRF-1, a light-regulated nuclear factor (21). This I-box motif is included ina 30-bp region of theLemna rbcS SSU5Bpromoter necessary for PHY regula-tion (31).

Gain-of-function experiments showed that the region of therbcSpromotersencompassing the I- and G-box elements functions as a composite LRE, able todirect a tissue-specific pattern of expression almost identical to the nativerbcSpromoter, including the dependence on the developmental stage of plastids (9).Mutation of either the I-box or the G-box eliminated detectable transcription(9). The I-G region functions as a complex regulatory unit, similar to that ofthe light-responsive Unit 1 of the parsleychsgene (108).

Evolution of the I-G RegionThe two I-box elements of the I-G-I CMA display a different sequence con-sensus and are flanked by different conserved sequence motifs. The moreupstream I-box has the consensus GATAAGAT (A/T) and is adjacent 3′ to aPF with the consensus (A/T) ARGATGA; the second I-box has the consensusATGATAAGG and is 5′ flanked by a PF with the consensus TGGTGGCTA(Figure 3). The I-box-associated PFs are conserved in genes of plants belong-ing to five orders of dicotyledons. A homologous I-G-I arrangement is found insome maizerbcSgenes but not in other genes from monocotyledons. However,CMAs that are probably derived from an ancestral I-G-I structure are presentin all these promoters (Figure 3).

The finding that the I-G (-I) arrangement is also found in the homologous pro-moters of a conifer and a fern (Figure 3) is unexpected because these promotersare presumably light-insensitive, whereas the I-G unit is apparently involvedin responses to light signals in angiosperms. Based on the persistence of theI-G-I arrangement since at least the divergence of ferns and seed plants, 395mya (116), we propose that it has played, and probably still does, one or several

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important functional roles, different to light-regulation, in the control ofrbcSgene transcription.

EVOLUTION OF THE LIGHT-REPRESSEDPHYTOCHROME A PROMOTER

Functional Organization of phyA PromotersPhytochrome (PHY) is encoded by small gene families in angiosperms; inArabidopsisthe PHY apoprotein is encoded by five genes (99). Each PHY isproposed to have a different physiological role (100). PHYs have been classifiedinto two types. The type I, or “etiolated-tissue” PHY, is most abundant in dark-grown plants, and its Pfr form is rapidly degraded in light by an ubiquitin-mediated proteolytic process. Type II or “green-tissue” PHY are present inmuch lower levels, but their Pfr form is stable in light (99, 100). The onlyknown type I PHY is that encoded by thephyAgene, whose mRNA abundancealso decreases in light. This inhibition ofphyAgene activity is autoregulatory(PHY-dependent) and operates at the transcriptional level (19, 65, 77).

ThephyApromoters of two monocotyledons, oat and rice, have been func-tionally characterized. A combination of deletion analysis and linker-scan mu-tagenesis identified in oat threecis-regulatory elements designated PE1 (positiveelement-1), PE3 (positive element-3), and RE1 (repressor element-1) (19, 20).PE1 and PE3 act synergistically to support maximal expression under dere-pressed conditions (i.e. low Pfr levels); mutation of either element decreasesexpression to basal levels. In contrast, mutation of the RE1 element results inmaximal transcription under all conditions, suggesting that Pfr repressesphyAtranscription through this negatively acting element (19).

The ricephyApromoter has no element similar to PE1. Instead, this promotercontains a triplet of GT-elements that have been shown, in transient expressionassays, to be functionally equivalent to the oat PE1 element (32, 99). Theseelements, related in sequence to the GT-1 binding sites ofrbcSpromoters (65),are bound by a transcription factor, named GT-2 (32, 34).

In addition tophyA, several other genes are down-regulated by light (88, 92,123), including the genes encoding asparagine synthetase (127). Recently a17-bp element was identified that is both necessary and sufficient for the PHY-mediated repression of the pea asparagine synthetase gene. This sequence is verysimilar to thephyARE1 element and is the target for a highly conserved PHY-generated repressor, whose activity is regulated by both calcium and cGMP (91).

Evolution of phyA PromotersPhytochrome genes have been found in phototropic eukaryotic organisms rang-ing from algae to angiosperms (42, 99), and genomic clones ofphygenes havebeen isolated from angiosperms and several lower plants. The latter include

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species representative of lineages dated to the Silurian and Devonian, and whichdiverged more than 400 million years ago (27, 110, 116).

The cereal PE3-RE1 region is conserved in thephyA promoters of plantspecies belonging to three different orders of dicotyledons (1, 33, 105), butno obvious counterpart of the monocot GT-boxes region was detected. Theconservation of the PE3-RE1 arrangement in dicots suggests a conserved reg-ulatory activity of these elements (33). ThephyAupstream sequences of bryo-phytes (the mossesPhyscomitrellaandCeratodon; 107, 124), a lycopodiophyta(Selaginella; 57), and a psilotophyta (Psilotum; 107) contain a number of PFs.Two clusters of these PFs or CMAs are common to all reported lower plantphyApromoters (Figure 4). The CMA nearest the start codon of the mossesand lower vascular plant genes includes sequences similar to the PE3 core ele-ment and other flanking sequence elements. The central, most conserved DNAmotif of the lower plant CMA is nearly identical to a sequence element in theArabidopsisPE3–RE1 region, immediately upstream of the RE1 motif. Nocanonical RE1 element is found in lower plantphypromoters.

Interestingly, the second, more distal lower plantphyA-CMA seems to be theevolutionary counterpart of the region encompassing the GT boxes in monocotphyApromoters (Figure 4). Thus, thecis-regulatory elements of monocotyle-donphyAgenes seem to have evolved fromcis-acting sequences already presentin orthologous genes of the common ancestor of land plants. What the regula-tory function was of such elements in the primitive land plants is an intriguingquestion that could be partially solved by the functional characterization of theidentifiedphyACMAs in mosses and vascular cryptogams.

Evolution of chs PromotersIn some species, induction ofchs gene expression by light is required forflavonoid accumulation, which provides a protective shield against potentiallyharmful UV irradiation (55).

The promoters of parsley and mustardchsgenes have been functionally an-alyzed, and a 50-bp light-responsive conserved region, named light-regulatoryunit 1 (LRU1), has been studied in great detail (41, 62, 63). LRU1 is sufficient toconfer light- responsiveness to heterologous minimal promoters (63, 102, 132)and consists of at least two distinctcis-acting elements, ACEchs and MREchs(formerly Box II and Box I, respectively) (108, 109). ACEchs contains a G-box element that interacts in vivo and in vitro with bZIP regulatory factors(40, 132). MREchs was originally identified as a 17-bp in vivo DNA footprint(109) with a conserved sequence motif called the H-box core (78), which isrecognized by PcMYB1, a Myb-related transcription factor of parsley (41).Recently, it was shown thatchsLRU1 activity is controlled by PHY throughthe cGMP-dependent transduction pathway (136). Comparative analysis of

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Fig

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orthologouschspromoters showed that LRU1 homologous sequences are pre-sent only in genes of cereals and brassicaceous plants (10). Homologous genesof legumes, snapdragon, and carrot contain, in the same relative position asLRU1, a CMA comprising G-box and H-box elements as in LRU1, but spaceddifferently (6–8 bp). Several regulatory functions have been assigned to thisG-H box CMA, including transcriptional activation by p-coumaric acid (78)and tissue-specific transcription (38). Although this CMA is found withinlight-responsive regions of somechsgenes (e.g.Anthirrinum chs; 76), there isno direct evidence of a role in light regulation. Because of both their proximalposition to the TATA box and their PF composition, LRU1 and the G-H CMAare clearly homologouscis-regulatory regions. Nonetheless, evolutionary di-vergence ofchsgenes harboring LRU1 and G-H CMA, respectively, date backto an era before the divergence of lines that gave rise to monocotyledons anddicotyledons, as inferred from the phylogenetical distribution of LRU1 (10, 26).

EVOLUTION OF LIGHT-REGULATEDPARALOGOUS GENE PROMOTERS

Differential Activity of Paralogous Gene PromotersSeveral light-regulated genes such asLhcb, rbcS, andchsare found in multiplecopies in most genomes. Members of these gene families frequently displayquantitative and/or qualitative differences in expression reflecting in part tran-scriptional regulation (29a, 30, 82, 117, 133). Some of these functional differ-ences correlate with differences in promoter architecture. For example, amongthe eightrbcSgenes of Petunia, only the two most highly expressed (SSU301andSS611) contain the I-G-box arrangement (29a). Insertion of an 89-bp frag-ment of the SSU301 promoter containing the I-G unit into the equivalent regionof the weakly expressedSSU911gene increased its expression 25-fold (29).

Tomato has fiverbcSgenes (117). Three have promoters with the I-G unit.The mRNAs from all five tomatorbcSgenes accumulate to similarly high levelsin leaves and light-grown cotyledons; however, only the genes containing theI-G unit are coordinately expressed in dark-grown cotyledons, water-stressedleaves, and developing fruits (12). Interestingly, the spacer DNA sequencebetween the I-box and the G-box elements is highly divergent in tomatorbcSgenes but conserved in orthologous genes from other plant species. A fruit-specific factor (FBF) specifically interacts with the I-G spacer DNA sequenceof the tomatorbcS-3Apromoter, which correlates with its reduced activity indeveloping tomato fruit (84). The tomatorbcS-2and tobaccorbcS 8.0displaya very similar sequence to the pearbcS-3AI-G spacer element, which is boundin vitro by GT-1 (50). It is conceivable that these “paralogous gene-specific

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motifs” modulate environmental or tissue-specific effects on the structurallyinvariant I-G regulatory unit.

Do Paralogous Promoters Evolve by Nonrandom Processes?Paralogous gene promoters often diverge in discrete segments, displaying non-random patterns of structural variation. For example, in therbcS family ofArabidopsisthe paralogous 1B, 2B, and 3B gene promoters clearly differ fromthat ofrbcS-1Aby a 60-bp internal deletion. This molecular event juxtaposedthe photoresponsive Box II–LAMP element with downstream conserved mo-tifs, creating a new combination ofcis-regulatory elements. In therbcS-1Bpromoter an additional deletion event removed a 44-bp region encompassingthe I-G-I Unit, juxtaposing the two I boxes originally flanking the G-box se-quence (72). This gene is the only member of theArabidopsis rbcSgene familyunable to respond to light pulses (30).

The evolution of discrete, short regulatory elements interspersed along thepromoters of paralogous genes also seems to occur, and this is exemplified bythe maizerbcSZm1and rbcZm3genes. Their promoter sequences are verysimilar but differ in the presence of small insertions, which are distributed ina discontinuous pattern (Figure 5a). Some of these paralogous gene-specificmotifs arecis-regulatory elements (106).

Figure 5 Paralogous gene promoters. (a) Schematic comparisons of two maizerbcSpromotersmainly differing by small insertion/deletions events; some changes createcis-acting elements [basedon data from Sch¨affner & Sheen (106)]. (b) Schematic comparison of two functionally distinctLhcb1 promoters of pea. Segments of high sequence divergence coinciding with relevantcis-regulatory elements are shown.Clear rectanglesrepresent blocks of conserved sequences, whosesimilarity is indicated as the ratio between the number of identical nucleotides and the overalllongitude (in bp) of the compared promoter segments.

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A probable example of evolution of differential function by loss of specificcis-acting elements is available with theLhcb1genes of pea. Two of thesegenes (Cab-8 and AB96) showed significant transcript accumulation after ared light pulse, whereas the other threeLhcb1genes (AB80, AB66, andCab-9)require continuous red light for significant expression (133, 134). The peaCab-8gene encodes a mature protein 99.5% similar to that of the AB80 and AB66genes (3, 125) and it displays significant similarity in the 350-bp proximalpart of the promoter.Cab-8 has a consensus dicotLhcb1 promoter, with adistal I∗-G∗-I structural unit and a proximal arrangement of elements (CUF-1)-(CCA-1)-(CCAAT-box)-(CGF-1 site). In the AB80 and AB66 promoters theoriginal CCAAT box sequence seems to have been eliminated by an 8 nt internaldeletion, the CUF-1 binding site is lost by multiple nucleotide substitutions,and the conserved GATA motif, upstream to the G-box, is mutated in the Gresidue and in a few additional upstream nucleotides (Figure 5b). Because theCab-8 promoter has the organization of the hypothetical, ancestral dicotLhcb1promoter, the architecture of the AB80 and AB66 promoters could be consideredas derived, by mutation and deletion of specificcis-regulatory elements, fromaCab-8-like promoter. The cause of the mutations and the selective forces thatfixed them in the species remain unknown.

LRE-ASSOCIATED CMAs

CMAs in Additional Plant GenesPhotoresponsive regions have been defined in other genes by diverse approaches(13, 122). Somecis-regulatory elements different to those identified inrbcS,chs, and Lhcb promoters have been proposed for light-regulated transcrip-tion (14, 81, 93). Discrete arrays of sequence motifs in light-responsive re-gions of those genes are conserved between phylogenetically distant plantspecies (10). Some of these LRE-associated CMAs are present in orthologousgenes from both monocots and dicots, indicating a very remote evolutionaryorigin. These ancient CMAs are found in genes encoding ferredoxin, andthe pyruvate–orthophosphate dikinase, sedoheptulose-bisphophatase, and theA subunit of chloroplast glyceraldehyde 3-phosphate dehydrogenase. LRE-associated CMAs were also identified in dicot genes encoding plastocyanin,subunits of the chloroplast ATPase, a 10-kDa protein of photosystem II,4-coumarate:CoA ligase, and phenylammonia-lyase (10).

Structural Analogies Between LRE-Associated CMAsMost of the∼30 identified CMAs (10) can be grouped in a small set of structuraland phylogenetic types.

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1. The (I-box)-(G-box)-(I-box) arrangement. Included are the I-G and G-I unitsof rbcSpromoters and their evolutionary variants, including the (X-box)-(Y-box) region ofLemnagenes (Figure 3); the I∗-G∗-I ancestral arrangementof Lhcb1genes found in legumes, and their evolutionary derivatives such asthe (GATA-motif)-(Z-DNA) region ofArabidopsisLhcb1∗3, the (Box II)-(G-box) arrangement of tobaccoCabE, and the (G-box)-(CCA-1 element)region of some spinach, tobacco, andArabidopsisgenes (Figure 1); also inthis CMA group are included the I∗-G-(I) array ofFedpromoters; and theinverted I-G-I unit of dicot and monocotsbpgenes.

2. The (GT-1)-(LAMP-site)-(GT-1) arrangement, observed in both the BoxII-3AF3 region ofrbcSgenes (rbcSCMA-3) and in the CMA-1 ofLSgenes

3. The (CCAAT-motif)-(GATA/I-box) combination, found in several light-responsive promoters, including the REα-REβ unit of Lemna Lhcb2∗1 (i.e.cabCMA4), the (CCAAT)-(GATA I-III) arrangement fromLhcb1promot-ers (i.e. cabCMA1), the (CCAAT box)-(motif 15) from solanaceousrbcSgenes (i.e.rbcSCMA-2), and the CMA containing the PC2 region fromplastocyanin gene promoters (i.e.PcCMA-2).

4 The (LAMP-site)-(TATA-box) arrangement, characteristic of all of the sola-naceousrbcSpromoters (82) and found in a modified form in the−50/+15light-responsive region of pearbcS-3A(73). This arrangement is also ob-served in the LRE-associated CMA ofatpC, in plastocyanin gene promoters(i.e. PcCMA-1), and ingapACMA-1.

5. The (G-box)-(H-box) arrangement, found in the three identified CMAs ofchsgenes, including the photoresponsive units 1 and 2 of parsleychsgene(108, 109).

Common Structural Features of Composite LREsDo all, or most, of the genes whose transcription is dependent on PHY harbora commoncis-regulatory element in their promoters? This appealing idea hasnot been confirmed by the sequence data from the dozens of PHY-dependentgenes. The identification of LRE-associated CMAs provides a new opportunityto assess whether the regulatory regions of those genes share structural featuresthat could explain their common dependence on such a photoreceptor. Thecomparative analysis of∼30 of these natural combinations of sequence motifsled to two important findings. 1. All of the LRE-associated CMAs present inPhANGs include at least one sequence identical, or related, to either the I-boxcore motif or its inverted version, the LAMP-site. 2. All of the CMAs foundin genes encoding enzymes involved in the metabolism of phenylpropanoids

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(PhEMAGs) share a conserved module related to thechsH-box core motif,ACCTA(A/C) C (A/C) (10).

Because PHY regulates expression of its target genes by three differenttransduction pathways (85), the superfamily-specific conserved motifs couldbe binding sites for transcription factors targeted by specific PHY-signalingpathways (10). Based on the knowledge of the genes that are activated or re-pressed by these phototransduction pathways, it has been proposed that the I-box/GATA-binding factors are direct or indirect targets of the Ca2+/calmodulin-dependent transduction pathway, whereas transcription factors binding at theH-box motifs in PheMAG CMAs would be affected by the cGMP-dependentphototransduction pathway (10).

A general model of LRE function proposes that LREs are multipartitecis-regulatory elements with two general components: “light-specific” elementsand “coupling elements.” The former are bound by transcription factors targetedby the light-signal transduction pathways (i.e. I-box/GATA-binding factors andHBFs), which confer photoresponsiveness. Coupling elements are bound byeither cell-specific factors or regulatory proteins targeted by other signalingsystems; consequently, the light stimulus to transcription is coupled to otherendogenous and exogenous signals.

Using microinjection into single cells of the tomatoaureamutant, Wu et al(136) established that constructs containing either 11 copies of therbcSBox IIelement or 4 copies ofchsUnit 1 are activated by different PHY signaling path-ways. Box II is affected by the calcium-dependent pathway and thechsUnit 1 isactivated by the cGMP pathway. Taking into account that GT-1, the factor thatbinds Box II, is highly related to the nuclear factors CGF-1 and IBF-2b, bothof which bind I-box/GATA motifs (121) the work by Wu et al (136) supportsthe hypothesis that factors interacting with I-box-related sequences are targetsfor the Ca2+/calmodulin PHY activated pathway. Moreover, Feldbr¨ugge et al(41) recently determined that the light-responsive core of parsleychsLRU1 isindeed the H-box, making it the most likely target of the cGMP pathway.

Evolution of LREs: The Chloroplast ConnectionThe finding that LREs from angiosperm gene promoters are very similar toputative regulatory units present in promoters from conifers and lower plants(Figure 6) is unexpected, because it is generally assumed that such promoters areeither light insensitive or, at most, weakly photoresponsive. Such physiology is

−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→Figure 6 Similarities of light-responsive elements of angiosperm with homologous promotersequences in nonflowering plants. Notice that PetuniaCab 22RandP. thunbergiiCab-6 belong todifferent gene families (i.e.Lhcb1andLhcb2, respectively) and thatrbcSBox II contains two PFs.Asterisks denote mismatches between aligned sequences.

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well established in conifers (4, 71, 89, 94, 137), although to our knowledge, nodetailed molecular studies have been carried out in pteridophytas and bryophy-tas. Conifers and other nonflowering plants (Gingko bilobabeing an exception;24, 25) develop chloroplasts when grown in darkness (83, 129, 130), indicatingthat their PhANG promoters are active in the dark. A central question emerges:did light-responsiveness evolve by changes incis-regulatory elements or trans-acting factors, or by both.

Because I-box/LAMP elements, which are critical for PHY responsiveness inangiosperms, are also components of ancestral, presumably non-photorespon-sive, regulatory units ofLhcbandrbcSgenes, it is probable that factors bind-ing these motifs became direct or indirect targets of light-signaling pathwaysin organisms preceding flowering plants. This notion is in agreement withthe available experimental evidence, including the recent demonstration thatsynthetic pairwise combinations of I-box sequences with diverse conserved el-ements function as complex LREs (98). However, the presence of an I-boxin combination with other conserved sequence motifs is not necessarily suf-ficient for light regulation. ThePinus thunbergii cab-6promoter containingReα and Reβ (an I-box motif) elements identical to those of theLemna AB19gene (Figure 6) directs a light-independent and tissue-specific expression of areporter gene in both dicots and monocots transgenic plants (70, 138). There-fore, othercis-acting signals in addition to the I-box core seem to be necessaryfor proper light control in flowering plants.

Our data suggest that composite LREs evolved from regulatory units that per-formed functions other than light-regulation. What could these functions havebeen? In the case of PhANGs, we hypothesize that these functions were relatedto nuclear gene regulation by chloroplast-derived signals. This possibility issupported by the finding that even the smallest light-responsive PhANG pro-moter segments display a tissue-specific and chloroplast-dependent pattern ofexpression similar to that of entire promoters; to date no evidence has been ob-tained that these two functions can be separated (9, 14, 15, 74, 98, 114, 120).Because coordination of gene expression between nuclear and chloroplastgenomes should have evolved a long time before terrestrial plants (101), itis plausible that PhANGcis-acting promoter elements targeted by plastid sig-nal transduction pathways evolved before LREs. Therefore, it is possible thatphotoreceptor-mediated transcriptional regulation was produced during evo-lution by targeting, either directly or through new regulators (i.e. via protein-protein interactions), the same transcription factors andcis-regulatory elementsthat mediated the influence of chloroplasts on PhANGs transcription. This pos-sibility is attractive because it suggests a simple mechanism by which differentgene families whose expression is coordinate could simultaneously acquire anew, coordinated pattern of regulation.

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CONCLUDING REMARKS

Much remains to be learned about the structure, mode of action, and evolu-tion of LREs. It is clear that LREs are complex, composed of at least twocis-acting elements, that can be targeted by different photoreceptor-activatedsignal transduction pathways. The composite and variable structure of LREscould explain the specific properties of individual light-responsive promoters.Phylogenetic analysis of LREs clearly indicates that they have evolved fromancient regulatory elements, whose original, primary function was probablynot light regulation. Transformation of mosses and other nonflowering plantsin which the expression of photosynthesis-associated genes is not regulated bylight could help in answering some questions concerning the evolution of LREsand the different signal transduction pathways that activate them. It will be ofgreat interest to explore how a new mode of regulation, affecting many genefamilies involved in photosynthesis, arose during evolution.

ACKNOWLEDGMENTS

We are grateful to June Simpson for critical reading of this manuscript. Thiswork was supported by a grant from the HHMI to LH-E (75 197-526902).

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Annual Review of Plant Physiology and Plant Molecular Biology Volume 49, 1998

CONTENTSTHEMES IN PLANT DEVELOPMENT, Ian Sussex 0GENETIC ANALYSIS OF OVULE DEVELOPMENT, C. S. Gasser, J. Broadhvest, B. A. Hauser 1

POSTTRANSLATIONAL ASSEMBLY OF PHOTOSYNTHETIC METALLOPROTEINS, Sabeeha Merchant, Beth Welty Dreyfuss 25

BIOSYNTHESIS AND FUNCTION OF THE SULFOLIPID SULFOQUINOVOSYL DIACYLGLYCEROL, Christoph Benning 53

SPLICE SITE SELECTION IN PLANT PRE-mRNA SPLICING, J. W. S. Brown, C. G. Simpson 77

PROTEIN TARGETING TO THE THYLAKOID MEMBRANE, Danny J. Schnell 97

PLANT TRANSCRIPTION FACTOR STUDIES, C. Schwechheimer, M. Zourelidou, M. W. Bevan 127

LESSONS FROM SEQUENCING OF THE GENOME OF A UNICELLULAR CYANOBACTERIUM, SYNECHOCYSTIS SP. PCC6803, H. Kotani, S. Tabata

151

ELABORATION OF BODY PLAN AND PHASE CHANGE DURING DEVELOPMENT OF ACETABULARIA : How Is the Complex Architecture of a Giant Unicell Built, Dina F. Mandoli

173

ABSCISIC ACID SIGNAL TRANSDUCTION, Jeffrey Leung, Jérôme Giraudat 199

DNA METHYLATION IN PLANTS, E. J. Finnegan, R. K. Genger, W. J. Peacock, E. S. Dennis 223

ASCORBATE AND GLUTATHIONE: Keeping Active Oxygen Under Control , Graham Noctor, Christine H. Foyer 249

PLANT CELL WALL PROTEINS, Gladys I. Cassab 281 MOLECULAR-GENETIC ANALYSIS OF PLANT CYTOCHROME P450-DEPENDENT MONOOXYGENASES , Clint Chapple 311

GENETIC CONTROL OF FLOWERING TIME IN ARABIDOPSIS, Maarten Koornneef, Carlos Alonso-Blanco, Anton J. M. Peeters, Wim Soppe

345

MEIOTIC CHROMOSOME ORGANIZATION AND SEGREGATION IN PLANTS, R. Kelly Dawe 371

PHOTOSYNTHETIC CYTOCHROMES c IN CYANOBACTERIA, ALGAE, AND PLANTS, Cheryl A. Kerfeld, David W. Krogmann 397

BRASSINOSTEROIDS: Essential Regulators of Plant Growth and Development, Steven D. Clouse, Jenneth M. Sasse 427

NUCLEAR CONTROL OF PLASTID AND MITOCHONDRIAL DEVELOPMENT IN HIGHER PLANTS, P. Leon, A. Arroyo, S. Mackenzie

453

BORON IN PLANT STRUCTURE AND FUNCTION, Dale G. Blevins, Krystyna M. Lukaszewski 481

HORMONE-INDUCED SIGNALING DURING MOSS DEVELOPMENT, Karen S. Schumaker, Margaret A. Dietrich 501

EVOLUTION OF LIGHT-REGULATED PLANT PROMOTERS, Gerardo Argüello-Astorga, Luis Herrera-Estrella 525

GENES AND ENZYMES OF CAROTENOID BIOSYNTHESIS IN PLANTS, F. X. Cunningham Jr., E. Gantt 557

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RECENT ADVANCES IN UNDERSTANDING LIGNIN BIOSYNTHESIS, Ross W. Whetten, John J. MacKay, Ronald R. Sederoff

585

DESATURATION AND RELATED MODIFICATIONS OF FATTY ACIDS, John Shanklin, Edgar B. Cahoon 611

PHYTOREMEDIATION, D. E. Salt, R. D. Smith, I. Raskin 643MOLECULAR BIOLOGY OF CATION TRANSPORT IN PLANTS, Tama Christine Fox, Mary Lou Guerinot 669

CALMODULIN AND CALMODULIN-BINDING PROTEINS IN PLANTS, Raymond E. Zielinski 697

FROM VACUOLAR GS-X PUMPS TO MULTISPECIFIC ABC TRANSPORTERS , Philip A. Rea, Ze-Sheng Li, Yu-Ping Lu, Yolanda M. Drozdowicz, Enrico Martinoia

727

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gerardo r. argüello astorga 1 curriculum vitae · annual review of plant physiology and plant...

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