vdsnf1, the sucrose nonfermenting protein kinase gene of

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MPMI Vol. 24, No. 1, 2011, pp. 129–142. doi:10.1094 / MPMI -09-09-0217. © 2011 The American Phytopathological Society

VdSNF1, the Sucrose Nonfermenting Protein Kinase Gene of Verticillium dahliae, Is Required for Virulence and Expression of Genes Involved in Cell-Wall Degradation

Aliki K. Tzima,1 Epaminondas J. Paplomatas,1 Payungsak Rauyaree,2 Manuel D. Ospina-Giraldo,2 and Seogchan Kang2 1Laboratory of Plant Pathology, Agricultural University of Athens, 75 Iera Odos, 11855 Athens, Greece; 2Department of Plant Pathology, The Pennsylvania State University, University Park 16802, U.S.A.

Submitted 25 September 2009. Accepted 1 September 2010.

Verticillium dahliae is a soilborne fungus causing vascular wilt in a diverse array of plant species. Its virulence has been attributed, among other factors, to the activity of hy-drolytic cell wall–degrading enzymes (CWDE). The su-crose nonfermenting 1 gene (VdSNF1), which regulates catabolic repression, was disrupted in V. dahliae tomato race 1. Expression of CWDE in the resulting mutants was not induced in inductive medium and in simulated xylem fluid medium. Growth of the mutants was significantly re-duced when grown with pectin or galactose as a carbon source whereas, with glucose, sucrose, and xylose, they grew similarly to wild-type and ectopic transformants. The mutants were severely impaired in virulence on tomato and eggplant (final disease severity reduced by an average of 87%). Microscopic observation of the infection behavior of a green fluorescent protein (gfp)-labeled VdSNF1 mutant (70ΔSF-gfp1) showed that it was defective in initial coloni-zation of roots. Cross sections of tomato stem at the cotyle-donary level showed that 70ΔSF-gfp1 colonized xylem ves-sels considerably less than the wild-type strain. The wild-type strain heavily colonized xylem vessels and adjacent parenchyma cells. Quantification of fungal biomass in plant tissues further confirmed reduced colonization of roots, stems, and cotyledons by 70ΔSF-gfp1 relative to that by the wild-type strain.

Verticillium dahliae Kleb. is a destructive fungus that causes wilt disease in a wide range of annual herbaceous plants, per-ennials, and woody angiosperms (Armengol et al. 2005; Bhat et al. 2003; Levin et al. 2003; Pegg 1989; Vallad and Subbarao 2008). Existing control strategies have proven insufficient due to the wide distribution of the pathogen, its broad host speci-ficity, the long-term survival of microsclerotia in the soil, and the confinement of the pathogen in the xylem (Tjamos 1989). V. dahliae enters the plant through the root and reaches the

vascular tissue by crossing the endodermis (Schnathorst 1981; Vallad and Subbarao 2008). Shortly after hyphal invasion of the vessel, conidia are formed, carried by the xylem fluid and trapped at vessel end walls. They then grow toward adjacent pits in the vessel walls; upon contact, hyphae enlarge to form bulbous tips, and the fungus produces hydrolytic enzymes (Cooper and Wood 1975) that erode the pectin-containing pit membrane (Beckman 1989; Bishop and Cooper 1983a and b; Fradin and Thomma 2006; Pegg et al. 1976; Schnathorst 1981). During colonization, hyphae also grow from one vessel to another through pits; and, at later stages, they may colonize the middle lamella intercellularly (Green 1981). Some of the vessels may be clogged by mycelium, spores, or polysaccha-rides produced by the fungus. Clogging becomes aggravated by gels and gums formed by the accumulation of breakdown products of the primary cell wall and middle lamella by fungal enzymes (Agrios 2005; DeVay 1989).

Hydrolytic enzymes produced by V. dahliae, particularly pectinases and cellulases, have been considered to be impor-tant for the expression of disease symptoms and pathogenesis (Pegg 1981; Pegg and Brady 2002; Puhalla and Bell 1981). In several studies, such enzymes have been isolated from Verticil-lium culture and characterized for their role in plant cell-wall degradation and symptom induction (Bidochka et al. 1999; Cooper and Wood 1973; Huang and Mahoney 1999; Mussel and Strause 1972; Wang and Keen 1970). Through chemical muta-genesis, V. dahliae pectinase-deficient mutants were obtained; the mutants caused normal symptoms on cotton (Howell 1976; Pegg 1989; Puhalla and Howell 1975). In a later study, mutants of V. albo-atrum deficient in one or more cell wall–degrading enzymes (CWDE) showed reduced virulence on tomato. Mu-tants deficient in pectinases retained the ability to colonize the host. One secretory mutant that produced low levels of pecti-nases and other CWDE was hardly virulent and showed low levels of vessel colonization (Cooper and Durrands 1989; Durrands and Cooper 1988a and b). This suggests that Verticil-lium spp. employ several CWDE that act synergistically during host colonization (Durrands and Cooper 1988b; Fradin and Thomma 2006).

Due to functional redundancy, it has been very difficult to determine the role of CWDE in pathogenicity. Because these enzymes are often encoded by multigene families, disruption of a single gene may have no effect due to the masking of its loss by other genes (Apel-Birkhold and Walton 1996; Dobinson et al. 2004; García-Maceira et al. 2000; Idnurm and Howlett 2001; Wu et al. 1997). For example, deletion of a gene encod-ing trypsin protease had no effect on pathogenicity or growth

Corresponding author: A. K. Tzima; E-mail: [email protected]

Current address for P. Rauyaree: Biotechnology Research and Develop-ment Office, Department of Agriculture, Rangsit-Nakhonnayok Road,Thunyaburi district, Pathumthani 12110, Thailand.

Present address of M. D. Ospina-Giraldo: Biology Department, LafayetteCollege, 29 Kunkel Hall, Easton, PA 18042, U.S.A.

*The e-Xtra logo stands for “electronic extra” and indicates that four sup-plementary figures are published online and that Figures 4, 5, 6, and 7 appear in color online.

e-Xtra*

130 / Molecular Plant-Microbe Interactions

of V. dahliae in culture (Dobinson et al. 2004). Similarly, mu-tants of an exopolygalacturonase-encoding gene (pgx4) in another vascular wilt fungus (Fusarium oxysporum) retained their ability to cause disease (García-Maceira et al. 2000).

In many microorganisms, including fungi, CWDE are sub-ject to catabolite repression, a mechanism that controls the preferential use of easily fermentable carbon sources, such as glucose, by repressing genes that are used to metabolize other carbon sources, such as sucrose, galactose, pectin, and xylose (Aro et al. 2005; Ronne 1995; Walton 1994). Thus, manipula-tion of the genes controlling the catabolite-repression mecha-nism could serve as a means for indirectly assessing the role of CWDE in fungal biology (Kahman and Basse 2001; Tonukari et al. 2000). One such gene is sucrose nonfermenting 1 (SNF1), which is required for the derepression of catabolite repressed genes under glucose-limiting conditions (Celenza and Carlson 1984a and b; Hardie et al. 1998). SNF1 mediates glucose re-pression of multiple genes in yeast and is involved in invasive and filamentous growth (Palecek et al. 2002). Disruption of SNF1 homologs in plant-pathogenic fungi resulted in reduced expression of a large number of CWDE genes (Ospina-Giraldo et al. 2003; Tonukari et al. 2000).

Studies in yeast have shown that the SNF1 kinase complex is a heterotrimer composed of one α subunit (encoded by the SNF1 gene), one β subunit, and one γ subunit (Celenza and Carlson 1986; Celenza et al. 1989; Jiang and Carlson 1997). The SNF1 protein (α subunit) contains the catalytic kinase do-main at the N-terminal, an autoinhibitory sequence C-terminal to the kinase domain, and the regulatory domain at the C-ter-minal (Crute et al. 1998).

Subcellular localization of the SNF kinase complex is con-trolled by the three β subunits, which control the formation of the three different SNF1 isoforms. Under high glucose condi-tions, the kinase complex is inactive and all three SNF1 hetero-

trimer isoforms are located in the cytoplasm. Under glucose limitation, autoinhibition is relieved upon binding of the γ sub-unit to the regulatory domain and SNF1 is phosphorylated by one of the three SNF1-activating kinases. Upon activation, one SNF1 isoform translocates to the nucleus, another relocalizes to the vacuolar membrane, and the third remains cytoplasmic (Jiang and Carlson 1996, 1997; McCartney et al. 2005; Vincent et al. 2001).

SNF1 phosphorylates a number of substrates, including transcription factors and metabolic enzymes. MigI is a DNA-binding transcriptional repressor that is negatively regulated by SNF1. Phosphorylation of MigI abolishes its binding to GC-box motifs that are present in the promoters of many glucose-repressed genes. One such gene is SUC2, encoding an inver-tase that hydrolyzes sucrose to glucose and fructose (Aro et al. 2005; Carlson et al. 1981; Lutfiyya and Johnston 1996; Ronne 1995; Treitel et al. 1998). Moreover, SNF1 phosphorylates and inhibits the activity of acetyl CoA carboxylase (AccI) that is involved in fatty acid biosynthesis (Schneiter et al. 1999; Woods et al. 1994) and a regulator of INO1 transcription (Shirra and Arndt 1999; Shirra et al. 2001). Differently from its function in yeast, in the filamentous fungus Hypocrea je-corina, SNF1 seems not to phophorylate the MigI homolog, CRE1, but an unknown 36-kDa protein. SNF1 from H. jeco-rina also failed to phophorylate a peptide from Sclerotinia sclerotiorum containing a potential SNF1 target site, whereas it phophorylated a S. cerevisiae MigI target site peptide (Cziferszky et al. 2003). Moreover, CRE1 from S. sclerotiorum could complement glucose repression in a CREA mutant in Aspergillus nidulans but not in an MigI mutant in S. cerevisiae (Vautard et al. 1999).

Despite the above-mentioned differences in downstream pathways of SNF1 between yeasts and filamentous fungi, SNF1 subunits seem to be functionally conserved in fungi, be-cause expression of SNF1 homologs from the plant pathogens Cochliobolus carbonum (CcSNF1) and S. sclerotiorum (ScSNF1) in yeast complemented the deficiency of SNF1 mu-tants to grow on sucrose (Tonukari et al. 2000; Vacher et al. 2003). Disruption of CcSNF1 in C. carbonum resulted in re-duced transcription of a large set of CWDE genes. As a possi-ble consequence of this reduction, these mutants were im-paired in their ability to grow on certain simple and complex carbon sources and to cause disease on maize (Tonukari et al. 2000). Similarly, in F. oxysporum, FoSNF1 mutants showed reduced expression of several genes encoding CWDE. The ability of FoSNF1 mutants to cause wilt disease in Arabidopsis thaliana and cabbage was considerably reduced compared with that of the wild-type strain (Ospina-Giraldo et al. 2003). Attenuated virulence was also observed in MoSNF1 mutants of Magnaporthe oryzae. However, expression of CWDE-encod-ing genes in MoSNF1 mutants was induced at wild-type levels under glucose-limiting conditions, indicating that MoSNF1 is not required for release from carbon catabolite repression (Yi et al. 2008).

In V. dahliae, CWDE are subject to catabolite repression (Cooper and Wood 1975). Several studies have been undertaken to assess the role of CWDE in virulence using conventional and molecular approaches; however, conclusive evidence for their role in virulence has not been established (Dobinson et al. 2004; Howell 1976; Pegg 1989; Puhalla and Howell 1975). To gain insight into the role of the SNF1 homolog in virulence of V. dahliae, the VdSNF1 gene was disrupted, and resulting mu-tants were examined for their growth on different carbon sources, the expression of CWDE, and the ability to cause dis-ease on different hosts. Fluorescent microcopy was employed to compare the colonization ability of VdSNF1 mutants with that of wild-type strains during infection.

Fig. 1. Phylogenetic relationships among sucrose nonfermenting proteinkinases from fungi and other organisms. The phenogram was generated onthe basis of evolutionary distances inferred from amino acid sequences,implementing the unweighted pair group method using arithmetic averagemethod in the TreeconW software. Accession numbers for the sequencesare: Fusarium oxysporum (AAN32715), Hypocrea jecorina(AAK69560.2), Neurospora crassa (CAD70761), Magnaporthe oryzae(ABF48563.1), Sclerotinia sclerotiorum (CAB40826), Cochliobolus carbonum (AAD43341.1), Schizosaccharomyces pombe (CAA20833),Saccharomyces cerevisiae (P06782.1), Candida glabrata (AAB48642),Kluyveromyces lactis (CAA61235), Candida tropicalis (BAA75889),Candida albicans (AAB48643), Homo sapiens (AAB32732), and Oryza sativa (BAA36298). Sequence of VdSNF1 (VDAG_08621.1) was takenfrom the Verticillium dahliae genome database (Broad Institute of MITand Harvard).

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RESULTS

Isolation of the VdSNF1 gene in V. dahliae. A conserved region among SNF1 homologs in multiple

fungi was used to design degenerate primers. Amplification of V. dahliae genomic DNA by polymerase chain reaction (PCR) with these primers produced a 450-bp product that exhibited a high degree of sequence similarity to SNF1 genes in other fungi, particularly F. oxysporum, H. jecorina, and C. carbo-num, with 92, 93, and 89% identity, respectively.

In order to clone the whole gene for performing gene dis-ruption, the sequence of the 450-bp amplicon was used as a query for searching the V. dahliae genome database at the Broad Institute of MIT and Harvard. VdSNF1 is a single-copy gene with a coding sequence of 2.447 bp that contains an open reading frame (ORF) of 2.139 bp interrupted by three introns (104, 88, and 51 bp in length, located at positions 461, 641, and 1,402, respectively). The ORF of VdSNF1 is predicted to encode a protein of 733 amino acids. The deduced amino acid sequence of VdSNF1 displayed 67.7% identity with FoSNF1 of F. oxysporum (Ospina-Giraldo et al. 2003), 55.9% identity with MoSNF1 from M. oryzae (Yi et al. 2008), 54.8% with SsSNF1 from S. sclerotiorum (Vacher et al. 2003), and 42.4% identity with CcSNF1 from C. carbonum (Tonukari et al. 2000). Homology to SNF1 proteins from yeast was lower (37.1 and 35.4% identity with SNF1 proteins from S. cerevisiae and Schizosaccharomyces pombe, respectively). A phylogenetic

analysis of SNF1 homologs showed that VdSNF1 clustered together with SNF1 proteins from filamentous fungi, while yeast homologs formed a separate group (Fig. 1).

Disruption and complementation of the VdSNF1 gene in V. dahliae.

A VdSNF1 mutant allele was constructed by replacing a por-tion of the ORF with a geneticin resistance gene cassette (Fig. 2A). Four of nine geneticin-resistant transformants of V. dahl-iae 70wt-r1 (tomato race 1 isolate) analyzed by PCR revealed a homologous recombination event at the VdSNF1 locus (Fig. 2B). The absence of the VdSNF1 transcript in three mutants (70ΔSF4, 70ΔSF20, and 70ΔSF22) was confirmed also by quantitative real-time PCR (qPCR) analysis (Fig. 2C). A func-tional copy of VdSNF1, including promoter and terminator sequences, was successfully reintroduced in mutants 70ΔSF4 and 70ΔSF20 for complementation (Fig. 2D). VdSNF1 tran-scription was restored in the complemented strains 70C3ΔSF4 and 70C4ΔSF20 (Supplementary Fig. 1).

Expression of CWDE-encoding genes is reduced in VdSNF1 mutants.

Two VdSNF1 mutants, (70ΔSF4 and 70ΔSF20), two comple-mented strains (70C3ΔSF4 and 70C4ΔSF20), wild-type 70wt-r1, and ectopic strain 70EctSF1 were analyzed for the expres-sion of several genes encoding CWDE. After germinating co-nidia of these strains in a noninducing glucose-containing me-

Fig. 2. Disruption of the VdSNF1 gene in Verticillium dahliae. A, Construction of a mutant allele for VdSNF1 gene disruption. The middle line shows the re-striction map of the 2.100-bp VdSNF1 fragment (black box) of clone pSF2kb, the open reading frame of the gene (white arrow), and the 234-bp OliI/EcoRV region (small black arrows) that was replaced by the geneticin resistance gene cassette (white box, top line). The bottom line illustrates the resulting mutant allele construct. B, Confirmation of the gene replacement event by polymerase chain reaction (PCR). In gene replacement mutants 70ΔSF4, 70ΔSF20, and 70ΔSF22, primers VdSNF1_H-up and VdSNF1-dn amplified 3.340 bp of the mutant allele whereas, in ectopic transformant 70EctSF1 and wild-type strain 70wt-r1, the endogenous 2.100-bp VdSNF1 fragment was amplified. C, Loss of VdSNF1 transcription in 70ΔSF mutants. Transcript levels of VdSNF1 were measured in mutants 70ΔSF4, 70ΔSF20, and 70ΔSF22; wild-type 70wt-r1; and ectopic transformant 70EctSF1 relative to the level of the constitutively expressed β-tubulin gene, as determined by quantitative real-time reverse-transcription PCR. D, Evidence for the successful reintroduction of a VdSNF1 copy in two disruption mutants. D1, In gene replacement mutants 70ΔSF4 and 70ΔSF20, primers VdSNF1_H-up and VdSNF1-dn amplified 3.340 bp of the mu-tant allele whereas, in wild-type strain 70wt-r1, ectopic transformant 70EctSF1 and complemented strains 70C3ΔSF4 and 70C4ΔSF20, the endogenous 2.100-bp VdSNF1 fragment was amplified. D2, The geneticin resistance gene was amplified from mutants 70ΔSF4 and 70ΔSF20, ectopic transformant 70EctSF1, and ectopically complemented strain 70C4ΔSF20, whereas it was not amplified in 70wt-r1 and complemented strain 70C3ΔSF4, providing evi-dence for a homologous recombination event that re-replaced the replacement allele.


Fig. 3. Induction of cell-wall-degrading enzyme genes in VdSNF1 mutants compared with the wild-type, ectopic transformants, and complemented strains, grown in A, pectin-containing medium and B, simulated xylem fluid medium. Levels of gene transcripts were measured relative to the level of the constitutively expressed β-tubulin gene, as determined by quantitative real-time reverse-transcription polymerase chain reaction. Error bars indicate standard errors calculated from three replicates. Accession numbers (Broad Institute of MIT and Harvard) for the sequences of the genes are: VdPL1 (pectate lyase B, VDAG_05344.1), VdPL3 (pectate lyase, VDAG_07566.1), VdPL4 (pectate lyase, VDAG_05402.1), VdPL7 (pectate lyase B, VDAG_08067.1), VdPL9(pectate lyase, VDAG_02709.1), VdPL11 (pectin lyase, VDAG_07144.1), and VdXyl1 (endo-1,4-β-xylanase A, VDAG_06165.1).

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dium, they were transferred to a pectin-containing medium or the simulated xylem fluid medium (SXM) (Neumann and Dobinson 2003) and cultured for 20 h. When grown in the pres-ence of pectin, pectate lyase genes (VdPL1-VDAG_05344.1, VdPL3-VDAG_07566.1, VdPL4-VDAG_05402.1, VdPL7-VDAG_08067.1, VdPL9-VDAG_02709.1, and VdPL11-VDAG_07144.1, were strongly induced in wild-type, ectopic, and complemented strains but not in disruption mutants (Fig. 3A). Representative xylanase and cellulase genes were not in-duced under the same conditions in all strains analyzed (data not shown). Moreover, when grown in SXM, the expression of rep-resentative CWDE genes (VdPL1, VdPL3, VdPL11, and VdXyl1) was lower in VdSNF1 VDAG_06165 mutants than that in the wild-type, ectopic, and the complemented strains (Fig. 3B).

Growth of VdSNF1 mutants on different carbon sources. The SNF1 homolog in other plant-pathogenic fungi has been

shown to be involved in the uptake or utilization of specific carbon sources (Ospina-Giraldo et al. 2003; Tonukari et al. 2000). To investigate the role of VdSNF1 in carbon utilization, radial growth rates of VdSNF1 mutants 70ΔSF4, 70ΔSF20, and 70ΔSF22, ectopic transformant 70EctSF1, and wild-type strain 70wt-r1 on different carbon sources were compared. All strains grew at similar levels on glucose, sucrose, and xylose whereas, on pectin and galactose, growth of all three VdSNF1 mutants was significantly reduced compared with 70wt-r1 and 70EctSF1 (Fig. 4). VdSNF1 mutants also grew considerably more slowly than 70wt-r1 in liquid medium containing pectin (Supplementary Fig. 2). Moreover, the VdSNF1 mutants were

Fig. 4. Growth of VdSNF1 mutants compared with the wild type and ectopic transformant on different carbon sources. A, Diagram presenting radial growth rates of 70ΔSF4, 70ΔSF20, and 70ΔSF22 mutant strains; wild-type 70wt-r1; and ectopic transformant 70EctSF1. Error bars indicate standard errors calcu-lated from four replicates. B, Colony appearance of strains 70ΔSF4, 70ΔSF20, and 70ΔSF22; ectopic transformant 70EctSF1; and wild-type strain 70wt-r1. Photographs were taken after 24 days of incubation from the bottom of the plates.

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impaired in forming microsclerotia regardless of carbon sources. This mutant phenotype was more prominent during growth on glucose and sucrose than galactose and xylose (Fig. 4). When grown on fructose, the VdSNF1 mutants grew at the same rate as 70wt-r1 and 70EctSF1 but did not produce microsclerotia (data not shown). Complementation of mutants 70ΔSF4 and 70ΔSF20 with a functional VdSNF1 copy restored growth on pectin to wild-type levels (Supplementary Fig. 3).

VdSNF1 is required for virulence. To evaluate the role of VdSNF1 in pathogenicity, three mu-

tants were evaluated for their ability to cause disease on to-mato and eggplant. In tomato plants, the VdSNF1 mutants did not cause visible symptoms, whereas 70wt-r1 and 70EctSF1 caused severe wilting symptoms and leaf necrosis (Fig. 5A). Tomato plants inoculated with the latter two strains showed first wilt symptoms at 15 days postinoculation (dpi) and reached disease incidence of 58 and 54%, respectively, at 28 dpi (Fig. 5B).

At 28 dpi, tomato plants inoculated with the mutants showed only mild chlorosis in a very few leaves. Interestingly, the aver-age height of plants inoculated with the mutants (ranging from 33 to 75 cm over time) was greater than that inoculated with 70wt-r1 (ranging from 23 to 49 cm over time) and the uninocu-lated plants (ranging from 28 to 62 cm over time) (Fig. 5C).

Reintroduction of a functional VdSNF1 copy in mutant 70ΔSF4 through homologous recombination and in 70ΔSF20 through ectopic integration restored their virulence (Fig. 5D). At early stages of infection, the ectopically complemented strain 70C4ΔSF20 was less virulent (Fig. 5D) and caused sta-tistically less disease (15.75% relative area under the disease progress curve [AUDPC]) compared with the wild-type, ectopic, and complemented strain 70C3ΔSF4 (18.88, 18.92, and 17.5% relative AUDPC, respectively) (Fig. 5D2).

For the infection assay with eggplant, plants were inoculated with mutant strain 70ΔSF4 and 70wt-r1. Eggplant inoculated with 70wt-r1 showed typical symptoms of chlorosis and leaf epinasty at 14 dpi and disease progressed rapidly, causing leaf necrosis and wilting, up to 63%, at 26 dpi. In contrast, at 26 dpi, eggplant inoculated with 70ΔSF4 exhibited only mild interveinal chlorosis of leaves which never became necrotic (Fig. 5E). Disease progressed slowly up to 11% leaf chlorosis at 31 dpi (Fig. 5F).

The infection process of roots by VdSNF1 mutants is impaired.

To facilitate microscopic observation of the VdSNF1 mu-tants’ infection process of root tissues, we used transformants of mutant 70ΔSF4 and 70wt-r1 that constitutively express the enhanced green fluorescent protein (EGFP). Nicotiana bentha-miana seedlings were used to monitor early stages of root attachment and infection, because the small size of N. bentha-

miana made inoculation on glass slides and direct microscopic observation easy. After 24 h of incubation, both wild-type and mutant spores had germinated; however, clear differences in hyphal elongation and root attachment were observed between them. Wild-type (70wt-gfp) spores became elongated and grew along the root surface; and, in some cases, a large number of hyphae surrounded the root and appeared to penetrate the root and root tip (Fig. 6A and B). In contrast, no germ tube elonga-tion and hyphal growth by 70ΔSF-gfp1 was observed; instead, conidia had formed short germ tubes that got attached at the root surface and appeared as spidery structures (Fig. 6C and D).

To monitor fungal colonization of the vascular system of tomato plants, seedlings at the second leaf stage were inocu-lated by root-dipping with 70ΔSF-gfp1 and 70wt-gfp. At 16 dpi, plants inoculated with 70wt-gfp showed typical wilt symptoms (35% disease incidence), while plants inoculated with 70ΔSF-gfp1 showed no wilting but mild chlorosis in a very small number of leaves (5% disease severity) (Fig. 7A and B). Plants were uprooted and horizontal cuttings were made at the cotyle-donary node to observe fungal colonization of the vascular sys-tem by fluorescence microscopy. Approximately three to four sections from each plant were observed. Xylem vessels and adjacent parenchyma cells infected with 70wt-gfp exhibited intense gfp fluorescence, compared with the considerably weaker fluorescence in xylem vessels infected with 70ΔSF-gfp1 (Fig. 7C). The uninoculated control plants showed no green fluorescence.

To examine whether the reduced gfp fluorescence in 70ΔSF-gfp1-infected xylem vessels correlated with reduced in planta

Fig. 5. Disease severity and progression in tomato and eggplant infected with Verticillium dahliae wild-type and VdSNF1 disruption mutants. A, Disease symptoms (arrows) and stunting of tomato plants (cv. Ailsa Craig) caused by wild-type isolate 70wt-r1 and ectopic transformant 70EctSF1 compared with asymptomatic plants inoculated with mutants 70ΔSF4, 70ΔSF20, and 70ΔSF22. Plants were imaged 22 days after inoculation. B, Disease progress curves of tomato plants inoculated by root dipping with spores of 70ΔSF4, 70ΔSF20, 70ΔSF22, 70wt-r1, and 70EctSF1. C, Heights of tomato plants inoculated with 70ΔSF4, 70ΔSF20, 70ΔSF22, 70wt-r1, and 70EctSF1 and uninoculated control plants were measured over time. D, Complementation of VdSNF1 restored pathogenicity of disruption mutants D1, Disease progress curves of tomato plants inoculated with VdSNF1 mutants and wild-type, ectopic, and complemented strains. D2, Disease severity expressed as relative area under the disease progress curve (AUDPC) caused by VdSNF1 mutants and wild-type, ectopic, and complemented strains. Relative AUDPC values calculated for each treatment were subjected to analysis of variance and means were separatedby Duncan’s multiple range test. Columns with different letters are statistically different at P ≤ 0.05. E, Leaves of eggplant inoculated with 70wt-r1 (1) showing necrosis, with mutant 70ΔSF4 (2) showing chlorotic symptoms, compared with the uninoculated plant (3). Plants were imaged 31 days afterinoculation. F, Disease progress curves in eggplant inoculated by root injection with mutant 70ΔSF4 and 70wt-r1. To create disease progress curves, disease severity at each observation was expressed by the percentage of leaves showing wilting. Error bars indicate standard errors calculated from 12 replicates in diagrams of tomato disease and height progression (B and C), from 16 replicates in complementation experiments (D), and from 24 replicates in the diagramof disease progression in eggplant (F).

Fig. 6. Observation of early stages of infection by Verticillium dahliae A and B, wild-type 70wt-gfp and C and D, mutant 70ΔSF-gfp1 on Nicotiana benthamiana roots. A, Wild-type spores on the root surface germinated and infect the root tip (arrow). B, Wild-type hyphae massively infecting a root tip (arrow). C and D, Mutant spores germinated, producing small hyphae. Images were taken 24 h postinoculation (×100 magnification).


biomass of the mutant, fungal biomass was determined by quan-titative reverse-transcription (RT)-PCR at the cotyledonary level, as well as in roots and stems of infected plants. Biomass of 70ΔSF-gfp1 was significantly lower in roots and stems com-pared with that of 70wt-gfp. In contrast to 70wt-gfp, the mu-tant was hardly detectable at the cotyledonary level (Fig. 7D).

DISCUSSION

Numerous classical and biochemical studies with V. dahliae have provided circumstantial evidence for the involvement of hydrolytic enzymes in symptom expression (Cooper and Durrands 1989; Pegg 1989). Nevertheless, little is known about molecular mechanisms underpinning Verticillium wilt

and controlling the production of CWDE (Dobinson et al. 2004; Fradin and Thomma 2006). In the present study, the VdSNF1 gene was characterized for its role in virulence and hydrolytic enzyme production in V. dahliae.

Phylogenetic analysis with SNF1 proteins from diverse fungi showed that homologs from filamentous fungi and yeasts formed separate clusters. Among proteins from filamentous fungi, VdSNF1 was more closely related to homologs from the wilt fungus F. oxysporum and the biocontrol agent H. jecorina than SNF1 proteins from M. oryzae, the sclerotia-forming fun-gus Sclerotinia sclerotiorum, and C. carbonum (Fig. 1).

Similar to functional studies of SNF1 homologs in other plant-pathogenic fungi (Ospina-Giraldo et al. 2003; Tonukari et al. 2000; Yi et al. 2008), disruption of VdSNF1 impaired the

Fig. 7. Disease severity and colonization of tomato plants by wild-type 70wt-gfp and mutant strain 70ΔSF-gfp1, 16 days after inoculation. A, Disease symp-toms of tomato plants inoculated with 70wt-gfp compared with plants inoculated with 70ΔSF-gfp1 that show no visible symptoms. B, Disease severity of tomato plants inoculated by root dipping with 70ΔSF-gfp1 and 70wt-r1. C, Colonization of tomato xylem vessels at the cotyledonary node by 70ΔSF-gfp1(2) and 70wt-gfp (3), as revealed by enhanced green fluorescent protein fluorescence relative to the uninoculated control (1). Horizontal cuttings and micro-scopic observations were performed 16 days after inoculation. Numbers at the base of photographs indicate the shutter speed. D, Detection of fungal biomass in infected plant tissues. Quantification of 70ΔSF-gfp1 and 70wt-r1 in root and stem sections and at the cotyledonary section. Vertical bars indicate standard errors calculated from 10 replicates for the disease incidence diagram and 5 replicates for the fungal quantification diagram at the base. In diagrams of fungal quantification at the root and the cotyledon, standard errors were calculated from two replicates.

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ability of V. dahliae to cause wilt and necrosis symptoms in tomato and eggplant. Interestingly, VdSNF1 mutants seemed to stimulate plant growth because tomato plants inoculated with VdSNF1 mutants grew taller than uninoculated controls. A growth-promoting effect upon inoculation with V. dahliae has been observed in wilt-resistant tomato cultivars (Schnathorst 1981). Moreover, tomato plants infected with V. albo-atrum have been reported to display increased indole 3-acetic acid production, a process known as hyperauxiny (Pegg 1989). Thus, VdSNF1 mutants might be able to trigger hyperauxiny, resulting in the promotion of plant growth. SNF1 mutants of M. oryzae caused no visible symptoms on rice, whereas mutants of F. oxy-sporum and C. carbonum caused delayed and reduced disease symptoms compared with the wild-type strains (Ospina-Giraldo et al. 2003; Tonukari et al. 2000; Yi et al. 2008).

The reduced virulence of SNF1 mutants in F. oxysporum and C. carbonum was explained by their reduced ability to utilize complex carbon sources and the reduced expression of CWDE (Ospina-Giraldo et al. 2003; Tonukari et al. 2000). In agreement with these results, in VdSNF1 mutants, transcripts of several pectin lyase genes (VdPL1, VdPL3, VdPL4, VdPL7, VdPL9, and VdPL11) remained repressed whereas, in the wild-type strain, these genes were induced.

Pectin lyase enzymes have long been implicated in symptom expression by V. dahliae, because the fungus grows through the pectin-containing pit membranes and, at later stages, colo-nizes the pectinaceous middle lammela (Fradin and Thomma 2006; Pegg 1989). Nevertheless, the importance of pectin lyase for virulence of V. dahliae could not be established by the use of pectin lyase-deficient mutants (Howell 1976; Pegg 1989; Puhalla and Howell 1975). Moreover, a V. albo-atrum mutant deficient in pectin lyase and polygalacturonase produc-tion and with low conidiation ability was able to colonize plants at wild-type levels but caused delayed symptoms. The same mutant displayed higher cellulase activity than the wild type, which might have complemented the pectate lyase defi-ciency during plant colonization. In the same study, a secretory mutant (with extremely low production in pectate lyase, poly-galacturonase, and cellulase and a threefold reduction in β-ga-lactosidase, β-glucosidase, and amarylidase activity compared with the wild-type strain) was not able to colonize inoculated plant and cause disease (Cooper and Durrands 1989; Durrands and Cooper 1988a and b). Thus, in VdSNF1 mutants, the regu-lation of other CWDE genes might be affected similarly to SNF1 mutants of C. carbonum that showed significantly re-duced expression of many xylanase and pectate lyase genes (Tonukari et al. 2000). However, it cannot be excluded that, in addition to the defect in the induction of CWDE, the VdSNF1 knock-out affected other cellular processes important for viru-lence. MoSNF1 mutation in M. oryzae did not affect the ex-pression of CWDE genes under derepressible conditions, indi-cating that carbon catabolite derepression might be regulated by other unknown pathways in this plant pathogen (Yi et al. 2008). The impaired virulence of M. oryzae MoSNF1 mutants was explained by their reduced conidiation ability and other growth defects during germination and appressoria formation. However, in the mutants in which only the regulatory subunit was deleted, lower virulence but no growth and conidiation de-fects were observed (Yi et al. 2008). Similar to SNF1 mutants of C. carbonum (Tonukari et al. 2000), conidiation of VdSNF1 mutants was not impaired. In fact, VdSNF1 mutants produced approximately 50% more conidia than the wild-type, ectopic, and complemented strains (Supplementary Fig. 4).

Radial growth rates of VdSNF1 mutants on different carbon sources varied significantly compared with growth patterns of SNF1 mutants of F. oxysporum and C. carbonum. Growth on galactose was reduced at different levels (approximately 30%

for VdSNF1, 40% for CcSNF1, and 60% for FoSNF1) in mu-tants of all three fungi. On pectin, VdSNF1 and CcSNF1 mutants showed reduction in growth (approximately 40% for VdSNF1 and 60% for CcSNF1) while FoSNF1 mutants grew as fast as the wild-type strain (Ospina-Giraldo et al. 2003; Tonukari et al. 2000). On xylose, growth of VdSNF1 mutants was similar to that of wild-type strains whereas both CcSNF1 and FoSNF1 mutants displayed impaired growth (approximately 60% reduc-tion) compared with wild-type strains. Growth on sucrose was similar to the wild-type strains for both VdSNF1 mutants and CcSNF1 mutants, indicating that invertase (which hydrolyzes sucrose to glucose and fructose) is not subject to SNF1-medi-ated catabolite repression in both fungi. Mutant strains of all three fungi grew at wild-type levels on glucose whereas MoSNF1 mutants showed a 50 to 60% reduction in growth on all the above-mentioned carbon sources, including glucose (Yi et al. 2008). Thus, in V. dahliae, similar to F. oxysporum and C. carbonum, the SNF1 gene is required for derepression of catabolite repression whereas this is not the case for M. oryzae (Ospina-Giraldo et al. 2003; Tonukari et al. 2000; Yi et al. 2008).

These differences in carbon utilization by SNF1 mutants of the aforementioned fungal pathogens probably reflect different requirements for specific carbon sources and distinct mecha-nisms during plant colonization. Interestingly, SNF1 seems to control pectin and xylose utilization differently in the wilt-causing fungi F. oxysporum and V. dahliae, although they colo-nize the hosts and induce symptoms in a similar manner. VdSNF1 mutants were able to utilize xylose whereas FoSNF1 mutants were impaired in the utilization of this sugar. More-over, FoSNF1 mutants were able to utilize pectin (Ospina-Giraldo et al. 2003), indicating that additional pectate lyase genes, which are probably not subject to catabolite repression, might still be functional. Pectate lyase genes have been shown to be important for virulence in F. solani f. sp. pisi (Rogers et al. 2000), whereas their role in Verticillium spp. pathogenesis has not been unequivocally established (Fradin and Thomma 2006; Pegg 1989).

During growth of VdSNF1 on different carbon sources, it became apparent that they were impaired in their ability to produce microsclerotia. VdSNF1 produced almost no micro-sclerotia on glucose, sucrose, and fructose whereas, on xylose and galactose, VdSNF1 formed some but less microsclerotia than wild-type strains. Several studies have suggested that for-mation of microsclerotia by V. dahliae is linked to virulence of V. dahliae and its long-term survival in the soil (Fradin and Thomma 2006; Schnathorst 1981). Reduced microsclerotia formation was observed in hydrophobin and mitogen-activated protein (MAP) kinase mutants of V. dahliae (Klimes and Dobinson 2006; Rauyaree et al. 2005). Similar to VdSNF1 mu-tants, MAP kinase mutants were not able to cause disease on several hosts (Rauyaree et al. 2005). In contrast, disruption of components of the cAMP signaling pathway reduced virulence significantly but enhanced microsclerotia formation (Tzima et al. 2010). In yeast, a network of signaling pathways appears to control response to changes in carbon source (Gray et al. 2004; Hedbacker et al. 2004; Hubbard et al. 1992; Palecek et al. 2002). Such a network probably functions in V. dahliae, con-trolling virulence, microsclerotia formation, and other devel-opmental processes in response to nutrient availability.

During the initial interaction of fungal plant pathogens with their hosts, concentrations of free sugars are probably low, causing the activation of SNF1 and subsequent induction of CWDE. VdSNF1 was shown to be important for utilization of simple sugars such as galactose and for growth on pectin-con-taining medium. Thus, VdSNF1 mutants are probably deficient in early infection processes necessary to successfully colonize


the plant root. Indeed, a gfp-transformed VdSNF1 mutant was deficient in successfully colonizing N. benthamiana roots; mu-tant spores formed short hyphae, whereas wild-type hyphae elongated and successfully penetrated the root surface. CcSNF1 mutants were also impaired in their ability to penetrate the plant surface (Tonukari et al. 2000). Moreover, high expression of CgSNF1, an SNF1 homolog of Colletotrichum gloeosporoides f. sp malvae, was observed during appressorium formation (Goodwin and Chen 2002). In F. oxysporum, FoSNF1 was found to be important not only for root penetration but also for subsequent colonization of vascular tissues (Ospina-Giraldo et al. 2003).

VdSNF1 mutants were also impaired in their ability to effi-ciently colonize the vascular system at later stages of infection. A gfp-transformed VdSNF1 mutant was hardly observed at the cotyledonary node of infected tomato plants, whereas the wild-type strain successfully colonized vascular vessels and adja-cent parenchyma cells. This observation was further confirmed by quantification of fungal biomass in roots, stems, and cotyle-dons, which showed that VdSNF1 mutants are mostly confined in the root system and are deficient in the ability to colonize the plant systemically. In agreement with these results, FoSNF1 mutants could be reisolated only from roots and not from stems of infected plants (Ospina-Giraldo et al. 2003). Experiments on SXM that resemble in planta growth of V. dahliae (Neumann and Dobinson 2003) showed that the expression of pectate lyase and endo-1,4-β-xylanase A genes was lower in VdSNF1 mutants than in wild-type, ectopic, and complemented strains. Thus, the limited colonization ability of VdSNF1 mutants can be explained, at least partially, by the low production of CWDE that are important for fungal proliferation from vessel to vessel through pit membranes. Moreover, VdSNF1 mutants were defective in the utilization of released carbon sources, probably restricting their growth within the vascular system.

The findings of the present study provide evidence for a role of VdSNF1 in catabolite repression, production of CWDE, and virulence of the soilborne, microsclerotia-forming fungus V. dahliae.

MATERIALS AND METHODS

Fungal isolates, media, and culture conditions. A V. dahliae isolate from tomato in Greece (70wt-r1), be-

longing to race 1, was used in this study. This isolate and its transformants were stored at –80°C as a microconidial suspen-sion in 25% glycerol and cultures were activated on potato dextrose agar (PDA) medium.

Growth experiments on different carbon sources were per-formed using a basal medium containing mineral salts and trace elements, as in minimal medium (Puhalla and Mayfield 1974). Carbon sources were added individually to the basal medium (at a concentration of 2% [wt/vol]) and the media were adjusted to pH 6.5. Radial growth assays were performed on basal medium containing 2% agar, supplemented with 2% xylose (Merck, Darmstadt, Germany), galactose (Serva, Hei-delberg, Germany), pectin (Sigma, St. Louis), glucose, and su-crose. After placing a 10-μl conidial suspension (107 conidia/ml) of each strain at the center of each plate, plates were incubated at 23°C. Colony diameter was recorded at intervals of 2 to 3 days, and colony morphology was observed and recorded. For gene expression analysis, liquid basal medium supplemented with 2% glucose was inoculated with four 4-mm plugs of 8-day-old cultures of mutant, wild-type, ectopic, and comple-mented strains. After incubation for 5 days at 23°C, fungal cul-tures were passed through several layers of cheesecloth (under sterile conditions), conidia were harvested by centrifugation and washed with sterile water, and the conidial concentration

was adjusted to approximately 1 × 107 conidia/ml of liquid basal medium supplemented with 2% pectin or per milliliter of a SXM (Neumann and Dobinson 2003). After 20 h of incuba-tion at 23°C, fungal cultures were harvested by centrifugation, washed with sterile water, lyophilized, and used for RNA ex-traction.

For conidia production, fungal isolates derived from single spores were grown on PDA. After 11 to 14 days of incubation, 4 ml of sterilized water was added to each plate to harvest co-nidia. Plates were shaken gently and 10 μl of the suspension was placed on a hematocytometer to count the number of conidia.

Nucleic acid manipulations and enzyme reactions. For genomic DNA isolation, a cetyltrimethylammonium bro-

mide protocol was used as previously described (O’Donnell et al. 1997). Fungal isolates were grown in potato dextrose broth for 5 to 7 days with shaking at 100 rpm, and the resulting myce-lium was harvested by filtering through Whatman no. 1 filter paper. The collected mycelium was stored at –80°C until DNA was extracted. Fungal cultures on PDA plates for 7 to 10 days were also used for DNA extraction; mycelium (0.1 to 0.15 g) was scraped off, placed in 1.5-ml tubes, and lyophilized. The mycelium was pulverized using a bead beater (MM103; Retsch, RETSCH GmbH & Co., Haan, Germany), and DNA was extracted according to Lee and Taylor (1990).

Total RNA was extracted from lyophilized mycelia in the presence of liquid nitrogen using TRI reagent (Molecular Re-search Center, Cincinnati, OH. U.S.A.) according to the manu-facturer’s instructions. Total RNA (1 μg) treated with RNase-free DNase (Fermentas) was reverse-transcribed into cDNA with SuperScript II Reverse Transcriptase (Invitrogen, Carls-bad, CA, U.S.A.) using a mixture of oligo dT as primers.

Escherichia coli DH5α was used for bacterial transforma-tion and plasmid propagation. Plasmid DNA isolation, DNA restriction digestion, ligation reaction, and E. coli transforma-tion were performed using standard procedures (Sambrook et al. 1989). For blunt-ending restricted DNA fragments, T4 DNA polymerase (Fermentas, Vilnius, Lithuania) was used, follow-ing the manufacturer’s instructions. PCR amplification of DNA or cDNA fragments used for cloning was performed with DNAzyme EXT DNA polymerase (Finzymes, Espoo, Finland) whereas, in all other PCR analysis reactions, Taq DNA poly-merase was used.

Isolation, cloning, and sequencing of VdSNF1. A portion of VdSNF1 was amplified from V. dahliae using a

PCR-based strategy. Three degenerate primers were designed based on conserved regions from SNF1 homologs of other fungi: SNF1F (5′-CAYCCNCAYATHATHAA-3′, forward primer), SNF1R2 (5′-ARRAARTTNCCRTCNGTCAT-3′, 3′-end internal nested primer), and SNF1R (5′-TCNGGNGCNG CRTARTT-3′, reverse primer). A single PCR fragment of ap-proximately 450 bp was amplified and cloned into pGemT-Easy Vector (Promega Corp., Madison, WI, U.S.A.) to generate pSK591.

The DNA sequence of the cloned PCR product showed strong sequence similarity to SNF1 genes in other fungi. This PCR fragment was labeled with [32P]dCTP using the Ma-gaprime DNA labeling system (Amersham Pharmacia, Buck-inghamshire, U.K.) as recommended by the manufacturer to isolate clones carrying the whole VdSNF1 gene by screening a lambda genomic library of V. dahliae (Rauyaree et al. 2005). DNA of positive clones was digested with BamHΙ, EcoRΙ, and SacΙ restriction enzymes. A 2.4-kb SacΙ fragment hybridizing to the probe was subcloned into pBluescriptΙΙ SK to generate pSK911. Sequencing of pSK911 confirmed cloning of the VdSNF1 gene.

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Multiple sequence alignment and phylogenetic tree construction.

Amino acid sequences corresponding to SNF1 proteins from 15 fungi were obtained from public databases and aligned using ClustalW (Larkin et al. 2007). Subsequently, a file in phylip in-terleaved format was generated as input for Treeconw software (Van de Peer and De Wachter 1994) to infer phylogenetic rela-tionships using the unweighted pair group method with arithme-tic average. Branch strength was tested by 100 bootstrap values.

Vector construction and fungal transformation. A VdSNF1 mutant allele for gene disruption was prepared as

follows: primers VdSNF1_H-up (5′-AAGCTTACCATTTCCC TCTCTCCATCC-3′) and VdSNF1-dn (5′-TTGTCTCCCATC GCACCTTGATGT-3′) were designed to amplify a 2.1-kb fragment covering almost the entire VdSNF1 gene from V. dahliae tomato race 1 (70wt-r1). The VdSNF1_H-up primer contained the HindIII cleavage sites (underlined) at the 5′ end to facilitate subcloning of the resulting PCR product and mu-tant allele. The resulting 2.1-kb VdSNF1 fragment was cloned in pGEM-T (Promega Corp.), creating pSF2kb. A mutant allele was generated by replacing the 234-bp OliI/EcoRV fragment of pSF2kb, located at 1.186 nucleotides downstream from the start codon, with the 1.480-bp geneticin resistance gene cassette (Fig. 2A). The geneticin resistance gene cassette was excised from vector pSK666 using EcoRI and BamHI, and cloned between the OliI/EcoRV sites of pSF2kb after blunt ending both the vector and the insert. The mutant allele was subcloned between the HindIII and PstI sites of Agrobacterium binary vector pGKO2 (Khang et al. 2005), that carries the HSVtk (herpes simplex virus thymidine kinase) gene as a negative se-lection marker against ectopic transformants. The resulting clone, which was named pGSFGen, was introduced into Agro-bacterium AGL1 for fungal transformation.

Agrobacterium tumefaciens–mediated transformation (ATMT) of V. dahliae 70wt-r1 with pGSFGen was performed as de-scribed previously (Mullins et al. 2001), with the following modifications. i) Co-cultivation of V. dahliae spores and A. tu-mefaciens cells was performed on Hybond membranes (Amer-sham Pharmacia); ii) PDA amended with geneticin (Invitro-gen) (for transformants selection) at 50 μg/ml, moxalactam at 100 μg/ml, and 200 μM cefotaxime (to inhibit A. tumefaciens growth) was used as selection medium. V. dahliae transformants were subjected to single-spore isolation on PDA supplemented with geneticin at 50 μg/ml and 50 μM 5-fluoro-2-deoxyuridine (F2dU) (Sigma). The nucleoside analog F2dU selects against ectopic transformants (Khang et al. 2005).

For complementation, a 4.635-bp fragment—including 939 bp upstream of the start codon, the VdSNF1 coding sequence (2.139 bp; 2,447 bp including introns) and 1.249 bp down-stream of the stop codon—was amplified from genomic DNA of strain 70wt-r1 using primers SNF4kbLG_B-up (5′-GAGGA TCC CGGCGCCAGTGAGGTAAGAGG-3′) and SNF4kbLG_ H-dn (5′-GAGAAGCTT AGGGGCGTGCGATGATTGTG-3′) and cloned into pGEM-T, creating vector pSNF4kb. The 4.635-bp fragment was subcloned from vector pSNF4kb into the BamHI/HindIII sites of Agrobacterium binary vector pSK561, carrying the hygromycin resistance gene cassette. The VdSNF1 complementation clone was introduced via ATMT in 70ΔSF4 and 70ΔSF20 mutants. Transformants resistant to hygromycin were subjected to single-spore isolation.

Confirmation of VdSNF1 disruption or complementation. Screening of transformants for the disruption of the VdSNF1

gene and the reintroduction of a functional copy of the gene in disruption mutants was done by PCR using primers VdSNF1_ H-up and VdSNF1-dn. Transcription of the VdSNF1 gene was

examined by quantitative RT-PCR analysis using 1 μl of V. dahliae cDNA as template and primers SNFqif-up (5′-CCCAA CCAGGCCATCAAGAAGA-3′) and SNFqif-dn (5′-GCACGG CACCGACTCCAGA-3′).

Quantitative real-time RT-PCR reaction. Transcription levels of CWDE were examined in VdSNF1

mutants and wild-type strains grown in liquid basal medium supplemented with pectin or in SXM (Neumann and Dobinson 2003). RNA extraction and cDNA synthesis was described above. A qPCR reaction was performed using first-strand cDNAs as a template and the QuantiFast SYBR Green PCR (Qiagen, Valencia, CA, U.S.A.) master mix. Gene-specific prim-ers were designed for VdPL1, VdPL3, VdPL4, VdPL7, VdPL9, VdPL11, and VdXyl1 (Table 1). qPCR reactions were performed in duplicates and, for data analysis, average threshold cycle val-ues were calculated for each gene of interest (Pfaffl 2001) on the basis of three independent biological samples.

Reactions with no cDNA verified the absence of contamina-tion and primer dimer formation while the absence of nonspe-cific products was confirmed by analysis of melting curves. Transcription levels of the target genes were quantified relative to the constitutively expressed β-tubulin gene (DQ266153) that was amplified with primers VdBt-up (5′-TTCCCCCGTC TCCACTTCTTCATG-3′) and VdBt-dn (5′-GACGAGATCGT TCATGTTGAACTC-3′).

qPCR was performed in a Stratagene Mx3005P thermocy-cler. The results were analyzed with the MxPro qPCR software. PCR cycling started with an initial step of denaturation at 95°C for 5 min, followed by 40 cycles of 95°C for 10 s and 60°C for 30 s.

Infection assays. Virulence of 70wt-r1 and its mutants was evaluated using

tomato (susceptible near-isogenic lines of cv. Ailsa Craig) and eggplant (cv. Black Beauty). To prepare inocula, fungal cul-tures grown for 5 to 7 days in SSN medium (Sinha and Wood 1968) were passed through several layers of cheesecloth (to remove mycelia), and the conidial concentration was adjusted to approximately 1 × 107 conidia/ml. For the virulence assay on tomato, six plants were inoculated for each isolate at the fourth true-leaf stage by immersing their roots in the conidial suspension for 20 min. In virulence assays including the com-plemented strains, eight plants were inoculated for each iso-late. For the eggplant assay, 12 plants (per isolate) at the third true-leaf stage were inoculated by injecting 10 ml of conidial suspension of each isolate into the soil at three points around the stem. Virulence assays on tomato and eggplant were performed twice.

Table 1. Primers designed for quantitative polymerase chain reaction ampli-fication of transcript levels of cell wall–degrading enzyme genes in VdSNF1disruption mutants and wild-type, ectopic, and complemented strains

Primer Sequence

VdPL1q-up 5′-CGAGGGCACCGGCACTACC-3′ VdPL1q -dn 5′-GCGGCGACGACCTTGGAGAT-3′ VdPL3q-up 5′-CGACGGCCCTCTGACCATC-3′ VdPL3q –dn 5′-GGGACCGCGGCTGAAAAC-3′ VdPL4q-up 5′-TCCGATAACAACGCCGCAGA-3′ VdPL4q-dn 5′-GCCAGCCACGCCGACAAC-3′ VdPL7q-up 5′-GGGCGGCGACCTTGTTGA-3′ VdPL7q-dn 5′-GCGCGAGTTGACGTTGATGAA-3′ VdPL9q-up 5′-TGACAACTTGGCTGAGGCACACTT-3′ VdPL9q-dn 5′-GTCCGGGCTTCTCTTGGTTCTTCT-3′ VdPL11q-up 5′-GCGTGCGAGGTCAACAAGG-3′ VdPL11q-dn 5′-GCCGACAGACGAGGAGATGA-3′ VdXYL1q-up 5′-GCGGCGAGTACAGCGTGAGC-3′ VdXYL1q-dn 5′-GGCGGTCCAGGCGTTGAAG-3′


Disease severity at each observation was expressed by the percentage of leaves that showed wilting symptoms. Subse-quently, disease ratings were plotted over time to generate the disease progress curves. In virulence assays including the complemented strains, the AUDPC was calculated by the trapezoidal integration method (Campbell and Madden 1990). Disease severity was expressed as a percentage of the maximum possible AUDPC for the whole period of the experiment and is referred to as relative AUDPC (Korolev et al. 2001). Relative AUDPC values calculated for each treatment were subjected to analysis of variance and means were separated by Duncan’s multiple range test. In the tomato experiment, the plant height measured from the soil line was recorded over time.

Microscopic observation of infection. To observe the infection process of mutant and wild-type

strains in infected plants, GFP-labeled strains were employed. Mutant strain 70ΔSF4 was transformed via ATMT with the EGFP gene under the control of the Cochliobolus heterstro-phus GAPD promoter, as described above. Fungal transfor-mants were selected using hygromycin B at 50 μg/ml of PDA. Transformant 70ΔSF-gfp1 exhibiting high levels of gfp fluo-rescence was used for histological studies together with wild-type strain 70wt-gfp (Paplomatas et al. 2005).

N. benthamiana plants were used to study the early infection process of the VdSNF1 mutants. Seed of N. benthamiana were surface sterilized with 0.5 ml of 70% ethanol for 30 s and washed three times for 30 s with sterile water. Subsequently, an aqueous solution containing 10% bleach was added and seed were agitated for 10 min. Four washes of sterile deionized water were performed to remove residual bleach. Seed were spread on Whatman paper that was soaked in nutrient solution (2 mM Ca(NO3)2 · 4H2O, 5 mM KNO3, 2.5 mM KH2PO4, 2 mM MgSO4, Fe-EDTA at 20 mg/liter, H3BO4 at 5.4 mg/liter, MnCl2 at 1.7 mg/liter, ZnSO4 at 161 μg/liter, CuSO4 at 80 μg/liter, NaMo7O4 at 44 μg/liter, NaCl at 650 μg/liter, and CoCl2 at 13 μg/liter) and placed on petri plates.

At 2 to 4 days after germination, approximately 10 to 12 N. benthamiana seedlings per isolate were transferred to a 1-cm cavity of sterile glass slides that were placed on petri plates. Subsequently, approximately 200 μl of V. dahliae 70ΔSF-gfp1 and 70wt-gfp conidial suspension (5 × 105 conidia/ml) in half-strength nutrient solution was pipetted into the cavity. After incubation at 25°C and a 16-h photoperiod for 24 h, fungal conidia germination and initial interaction with the root surface was observed with a fluorescence microscope using a UV filter.

To examine colonization of VdSNF1 mutants during later stages of infection, 10 tomato plants at the second true-leaf stage were inoculated by root dipping with 70ΔSF-gfp1 and 70wt-gfp as described above. At 16 dpi, five plants were up-rooted and horizontal cuttings were performed at the level of cotyledon and observed with a fluorescence microscope using a UV filter. Furthermore, tomato tissue samples from the root, the stem base (above soil line up to the cotyledon point), and the cotyledon segment were stored at –80°C for fungal bio-mass quantification.

qPCR quantification of fungal biomass in plant tissue. To investigate more accurately the colonization ability of the

VdSNF1 mutant compared with the wild-type strain, biomass of V. dahliae was estimated by qPCR in tomato plants infected with 70ΔSF-gfp1 and 70wt-gfp. DNA was extracted from roots (pool of five plants), stem (above soil line up to the cotyledon point, stem sections from five plants), and the cotyledon point (pool of five plants) from tomato plants inoculated with 70ΔSF-gfp1 and 70wt-gfp. For DNA isolation, tissue samples were ground to a fine powder using an autoclaved mortar and

pestle in the presence of liquid nitrogen, and total DNA was extracted according to Dellaporta and associates (1983). DNA was quantified by spectrophotometry and 100 ng of DNA of each sample was used for qPCR reaction. For detection of fun-gal strains in tomato plants, primer pairs Vd-F (5′-CCG CCG GTC CAT CAG TCT CTC TGT TTA TAC-3′) and Vd-R (5′-CGC CTG CGG GAC TCC GAT GCG AGC TGT AAC-3′), designed based on the internal transcribed spacer (ITS)1 and ITS2 regions of ribosomal RNA genes (Z29511) of V. dahliae, were used. qPCR reactions were performed as described above. To normalize differences in DNA template amounts, the Lycopersicon. esculentum β-tubulin gene (DQ205342), which was amplified using the primer pair LeTUB-F (5′-GATTTGC CCCACTAACCTCTCGT-3′) and LeTUB-R (5′-ACCTCCTT TGTGCTCATCTTACCC-3′), was used as the internal standard. The primer specificity and the formation of primer-dimers were monitored by dissociation curve analysis.

ACKNOWLEDGMENTS

This work was supported by a bilateral project founded by the Greek General Secretariat of Research and Technology. A. Tzima acknowledges a grant from the State Scholarship Foundation of Greece, and P. Rauyaree was supported by a fellowship from the Thai Government and a Storkan-Hanes Foundation Fellowship. We thank D. Tsitsigiannis for critically reviewing the manuscript and M. Typas for providing assistance for gene expression analysis.

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