Transgenic Strategies for Developing Crops Resistant to Geminiviruses

1. 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

2. Virus “virus is an obligate intracellular parasites that cannot reproduce independently” • 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

3. Plant Pathogenic Virus RNA virus DNA virus Plant pathogenic viruses - 450 species

4. Plant virus and shape crops

5. Why to study Geminivirus? (Vanderschuren et al., 2007)

6. Geminivirus disease complex Geminivirus Whitefly Plant

8. Host and Vector

9. 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

10. Genome organization of Geminiviridae

11. The Geminivirus DNA replication cycle RCR-Rolling Circle Replication mechanism of virus

12. Interference of geminivirus in the host

13. Genus I. MastrevirusMaize streak virus • Monopartite genome • Transmitted by leafhopper vectors to monocotyledonous plants • H-Maize, Sugarcane, wheat, Bajra, Chickpea, Millets, Bean leafhopper

14. 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

15. Genus II. CurtovirusBeet curly top virus • Monopartite genome • Transmitted by leafhoppers to dicotyledonous plants • Ambisense nature • Host: pepper, melons, beans, tomato, spinach and ornamentals

16. 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

17. Genus III.TopocuvirusTomato pseudo-curly top virus • Monopartite genome • Transmitted by tree hoppers to dicotyledonous plants

18. 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

19. Genus IV. BegomovirusBean golden yellow mosaic virus Bipartite • 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 A B whiteflies

20. Transgenic strategies • Pathogen-derived resistance through the expression of viral proteins • Replication associated protein • Coat protein-mediated protection • Movement protein • Pathogen-derived resistance without protein expression • Gene silencing • Antisense RNA • Resistance due to the expression of non-pathogen derived antiviral agents • Virus-induced cell death • DNA binding protein • GroEL-mediated protection • Peptide aptamers • InPAct

21. Pathogen-derived resistance through the expression of viral proteins Replicationassociated 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

22. Pathogen-derived resistance through the expression of viral proteins Coat protein-mediated resistance • Systemic infection by monopartitegeminiviruses(Rojas et al., 2001) • Tomato plants expressing CP of the monopartitebegomovirus (TYLCV) • exhibited delayed symptom Development • CP of bipartite geminiviruses is not absolutely necessary , as NSP can • substitute (Poomaet al., 1996) • CP-mediated strategy against bipartite geminiviruses will not produce • a high level of resistance

23. Pathogen-derived resistance through the expression of viral proteins 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)

25. Pathogen-derived resistance without protein expression Gene silencing - mediated resistance

26. Pathogen-derived resistance without protein expression Antisense RNA - mediated resistance

28. 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

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

30. Resistance due to the expression of non-pathogen - derived antiviral agents GroEL – mediated resistance • Chaparon • Homologue of GroELproduced by endosymbioticbacteria 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. tabaciGroEL gene expressed in transgenic tomatoes under phloem specific promoter, protected the plants from the TYLCV infection (Rudolph et al., 2003

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

33. Resistance due to the expression of non-pathogen - derived antiviral agents InPAct system

34. Conclusion