transgenic strategies for developing crops resistant to geminiviruses student chairman d. raghu (ii...

Transgenic Strategies for Developing Crops Resistant to

Geminiviruses

Student Chairman

D. Raghu (II Ph.D., Biotechnology) Dr. D. Sudhakar

08-807-002 Professor, DPMB&B,CPMB

Virus

• Latin – “toxin or poison”

• Nucleocapsid

• Enveloped viruses – possess an envelop around the

protein coat

• Virus core – additional protein layer between capsid and

the nucleoid

• Replicate inside the cells of another organism

• Electron microscope

“virus is an obligate intracellular parasites that

cannot reproduce independently”

Plant Pathogenic Virus

RNA virus DNA virus

Plant pathogenic viruses - 450 species

cropsPlant virus and shape

Why to study Geminivirus?

(Vanderschuren et al., 2007)

Geminivirus

Geminivirus disease complex

Whitefly Plant

Host and Vector

Genus Type member Host range Vector Genome

Mastrevirus Maize streak virus (MSV) Monocot and

a few dicots

Leafhoppers Monopartite

Curtovirus Beet curly top virus (BCTV) Dicots Leafhoppers Monopartite

Begomovirus Bean golden mosaic virus

(BGMV)

Dicots Whiteflies Mono and bipartite

Topocuvirus Tomato pseudo-curly top

virus (TPCTV)

Dicots Treehopper Monopartite

General characteristics

• Genome comprised of one or two circular ss-DNA molecules

• each of which is 2.5–3.0 kb: Total ∼genome size 2.5–5.0 kb

• The smallest known genome for an independently replicating virus

• Bidirectional transcription and overlapping genes for efficient coding of proteins

• Distinguishing feature is their twinned icosahedral virions

The Latin “geminus” meaning twin

Genome organization of Geminiviridae

The Geminivirus DNA replication cycle

RCR-Rolling Circle Replication mechanism of virus

Interference of geminivirus in the host

Genus I. Mastrevirus Maize streak virus

• Monopartite genome

• Transmitted by leafhopper vectors to monocotyledonous plants

• H-Maize, Sugarcane, wheat, Bajra, Chickpea, Millets, Bean

leafhopper

Maize streak virus

• One of the oldest known plant viral diseases

• Economically it is the most damaging disease in maize in sub-Saharan Africa resulting in up to 100% yield loss

• Endemic in Africa where wild grasses are its natural hosts

Cicadulina mbila, the leafhopper vector of Maize streak virus

Genus II. Curtovirus Beet curly top virus


• Transmitted by leafhoppers to dicotyledonous plants

• Ambisense nature

• Host: pepper, melons, beans, tomato, spinach and ornamentals

Beet curly top virus

• Symptoms - vein clearing, curling, general malformations and become leathery and brittle

• Stunted, turn yellow, and the phloem shows necrosis, early infection usually results in early death (Brunt et al.,1996)

• In the late 1990s BCTV emerged as a serious problem of chilli cultivation in southern New Mexico and destroyed nearly 80% of the crop

Beet

chilli

Genus III.Topocuvirus Tomato pseudo-curly top virus


• Transmitted by tree hoppers to dicotyledonous plants

Tomato pseudo-curly top virus

• Virus is transmitted in a semi-persistent manner,retained when the vector moults

• Symptoms - vein-clearing, leaf curling and cupping and shoot proliferation

• Stunted and set few fruit

• Host - Ambrosia sp., Solanum nigrum

Genus IV. Begomovirus Bean golden yellow mosaic virus

• Transmitted by whiteflies

• Dicotyledonous plants

• Bipartite genomes (A and B components)

• With some exceptions (e.g.,

Tomato yellow leaf curl virus, Cotton leaf curl virus, Tomato leaf curl virus…) for which no B components have been found

whiteflies

Bipartite

A B

Transgenic strategies

1. Pathogen-derived resistance through the expression of viral proteins

• Replication associated protein• Coat protein-mediated protection• Movement protein

2. Pathogen-derived resistance without protein expression• Gene silencing• Antisense RNA

3. Resistance due to the expression of non-pathogen derived antiviral agents

• Virus-induced cell death• DNA binding protein• GroEL-mediated protection• Peptide aptamers• InPAct

Pathogen-derived resistance through the expression of viral proteins

Replication associated protein (Reps)

• Viral gene transcrioption regulation

• Initiation & termination of viral replication

• Regulation of host gene expression

Eg. Interaction of geminiviral Rep with host pRBR induce viral

DNA synthesis

• Driving cells into “S” phase

• Activating the expression of “S” phase specific factors


Coat protein-mediated resistance

•Systemic infection by monopartite geminiviruses (Rojas et al., 2001)

•Tomato plants expressing CP of the monopartite begomovirus

(TYLCV)

exhibited delayed symptom Development

•CP of bipartite geminiviruses is not absolutely necessary , as NSP can

substitute (Pooma et al., 1996)

•CP-mediated strategy against bipartite geminiviruses will not produce

a high level of resistance


Movement protein (MP) - mediated resistance

•Cell-to-cell and long distance systemic spread

•Used to engineer resistance to various begomoviruses

•Transgenic plants expressing the defective movement protein were

resistant to both ToMoV & CaLCuV

(Shepherd et al., 2009)

Pathogen-derived resistance without protein expression

Gene silencing - mediated resistance

Pathogen-derived resistance without protein expression

Antisense RNA - mediated resistance

Resistance due to the expression of non-pathogen - derived antiviral agents

Virus - induced cell death

•Death of infected cells and their neighbours induced by host innate

defensive hypersensitive (Shepherd et al., 2009)

•Transgenic plant shows resistance to geminivirus by combined action

of

the barnase & barstar proteins of B. amyloliquefaciens

•Barnase – viral “V” sense promoter (expressed during virus infection)

•Barstar – viral “C” sense promoter (repressed during virus infection)

•Absence of geminivirus infection, barnase & barstar equally expressed

•Presence of infection

• Barnase is over expressed

•Cell die before infecting virus can replicate & move


DNA binding proteins

•Zinc finger proteins are high affinity for the “Rep–specific direct repeats

“

in the “virion-ori “ of different geminiviruses

•Block the binding of “Rep” to “virion- ori” of geminivirus

•Transgenically expressed artificially designed Zinc finger protein

provide

resistant against geminiviruses


GroEL – mediated resistance

•Chaparon

•Homologue of GroEL produced by endosymbiotic bacteria from B.

tabaci

•Higher affinity to TYLCV coat protein

•Vector – virus interaction protect the virus from distruction during its

passage through insect haemolymph

Eg. B. tabaci GroEL gene expressed in transgenic tomatoes under

phloem specific promoter, protected the plants from the TYLCV infection (Rudolph et al., 2003


Peptide aptamers

•Short recombinant protein, ~ 20 amino acid length

•Strongly binds with target protein and destructs the function

•Transgenic N. benthamiana - nucleoprotein of the Tomato spotted wilt

Virus

(Lopez et al., 2006)

Tansgenic virus resistance strategies (Table)


InPAct system

Conclusion

transgenic strategies for developing crops resistant to geminiviruses student chairman d. raghu (ii...

Documents

tomato leaf curl virus

tomato yellow leaf curl

cotton leaf curl virus

species cropsplant virus

ornamentalsbeet curly

curtovirus beet curly

virusmonopartite genome

b components