Transgenic Strategies for Developing Crops Resistant to Geminiviruses Student Chairman D. Raghu (II Ph.D., Biotechnology) Dr. D. Sudhakar 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 virusDNA virus Plant pathogenic viruses species dsRNAssRNA (-)ssRNA (RT)ssRNA (+) ssDNA dsDNA

crops Plant virus and shape

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

Geminivirus Geminivirus disease complex WhiteflyPlant

Group Group II (ssDNA) Family Geminiviridae Genera Mastrevirus Curtovirus Begomovirus topocuvirus Geminivirus Taxonomy

Host and Vector GenusType memberHost rangeVectorGenome MastrevirusMaize streak virus (MSV)Monocot and a few dicots LeafhoppersMonopartite CurtovirusBeet curly top virus (BCTV)DicotsLeafhoppersMonopartite BegomovirusBean golden mosaic virus (BGMV) DicotsWhitefliesMono and bipartite TopocuvirusTomato pseudo-curly top virus (TPCTV) DicotsTreehopperMonopartite

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 Monopartite genome 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 Monopartite genome 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

Pathogen-derived resistance through the expression of viral proteins 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

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)

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

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

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

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)

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

Conclusion