ANALYSIS OF THE VIRUS- ENCODED GENES REQUIRED FOR THE MAINTENANCE OF BETASATELLITES BY GEMINIVIRUSES

Abstract

Viruses of the family Geminiviridae are phytopathogens with circular single-stranded DNA genome encapsidated in characteristic geminate particles that are transmitted by insects. Economically the most important are the geminiviruses of the genus Begomovirus which are transmitted exclusively by the whitefly Bemisia tabaci that occur in both the Old World (OW) and New World (NW). Bipartite begomoviruses, with components known as DNA A and DNA B, are native to the NW. However, in the OW, the bipartite begomoviruses are out-numbered by the monopartite begomoviruses, with genomes consisting of a homolog of the DNA A component of the bipartite viruses. Many monopartite begomoviruses are associated with two classes of single-stranded DNA satellites, designated as alphasatellite and betasatellite. Betasatellites are, in many cases, essential for the helper begomovirus to infect, and induce typical disease symptoms in, the host from which they were isolated. For many begomovirus-betasatellite pairs the betasatellite encodes the major pathogenicity/symptom determinant of the complex. The study described here was designed to investigate the effects of mutation of selected begomovirus genes on infectivity, symptoms and the ability to maintain betasatellites. The coat protein (CP), V2, C2 and C4 genes, the products of which have in previous studies of monopartite begomoviruses been implicated in virus movement and/or pathogenicity, of two betsatellite-associated monopartite begomoviruses, Pedilanthus leaf curl virus (PedLCV) and Cotton leaf curl Kokhran virus (CLCuKoV) were mutated. Mutant viruses were inoculated to Nicotiana benthamiana in both the presence and absence of the cognate betasatellites, Tobacco leaf curl betasatellite (TbLCB) and Cotton leaf curl Multan betasatellite (CLCuMB), respectively. Mutation of the CP abolished infectivity of monopartite begomoviruses. The mutation could be complemented by transient expression of the CP at the point of inoculation-albeit without inducing symptoms. These results are consistent with previous studies, and suggest that the protein is required for virus spread/movement in plants. Also the results show for the first time that the CP is important for the maintenance of betasatellites. Mutation of the V2 genes of PedLCV and CLCuKoV lead to reduced and asymptomatic infections. Betasatellite fully restored the infectivity of CLCuKoV and partially restored the infectivity of PedLCV, although for xviiCLCuKoV infections were asymptomatic whereas PedLCV infections exhibited very mild symptoms. These findings, for the most part, agree with earlier studies indicating that the V2 is a pathogenicity determinant, has a role in virus movement and V2 plays a part in the maintenance of betasatellites by monopartite begomoviruses. Mutation of the C2 genes of both monopartite begomoviruses resulted in reduced and asymptomatic infections. Co-inoculation with betasatellite resulted in a greater number of plants in which virus spread systemically, but for the CLCuKoV/CLCuMB combination, viral DNA levels were higher than for mutant in the absence of the betasatellite. These findings agree with earlier studies, indicating that C2 is not essential for infectivity but affects symptom expression, and are consistent with C2 being a suppressor of post-transcriptional gene silencing, as well as showing that C2 plays a part in the maintenance of betasatellites. The results for the mutation of the C4 for the two viruses differed. For PedLCV mutation of C4 resulted in significantly reduced infectivity. For plants inoculated with TbLCB and the C4 mutant, fewer plants ultimately showed systemic movement of the betasatellite. For CLCuKoV mutation of the C4 had no discernible effect on infectivity of the virus or the maintenance of the betasatellite by the virus. The effects on symptoms are consistent with earlier results indicating that the C4 of monopartite begomoviruses plays a part in symptoms induced by the virus. Betasatellites are increasingly being identified in the field with bipartite begomoviruses. It was therefore of interest to examine the requirements for betasatellite maintenance by a bipartite virus such as Tomato leaf curl New Delhi virus (ToLCNDV). For the majority of bipartite begomoviruses, including ToLCNDV, both components are essential for symptomatic infection. As for the monopartite viruses, four mutants (of the CP, AV2, AC2 and AC4 genes encoded on DNA A) of ToLCNDV were produced. In the presence of the DNA B none of the mutations affected infectivity or symptoms. The betasatellite was not efficiently maintained by ToLCNDV (both in the presence and absence of the DNA B) but mutations of the AC2 and AC4 abolished the ability of the virus to maintain the satellite, indicating that these proteins are important in maintenance of the satellite by the virus. The earlier finding that betasatellites can complement DNA B functions of bipartite begomoviruses led to the suggestion that begomovirus movement is constrained by an RNAi-based resistance in plants. To investigate this hypothesis ToLCNDV DNA A was inoculated together with three well characterised suppressor xviiiproteins from heterologous RNA viruses. Transient expression of each of the three suppressors (the Tobacco etch virus [genus Potyvirus, family Potyviridae] HC-Pro, the Cymbidium ringspot virus [genus Tombusvirus, family Tombusviridae] p19 and the Turnip crinkle virus [genus Carmovirus, family Tombusviridae] coat protein) at the point of inoculation with ToLCNDV DNA A resulted in more plants in which systemic movement of the virus was evident. These findings support the contention that begomovirus movement is countered by a plant RNAi-based defence. In addition to being of academic interest, the studies described here were conducted in the belief that a better understanding of virus-satellite interactions could lead to novel means of reducing agricultural losses due to these pathogens; possibly identifying new targets for engineered resistance that interfere with virus-satellite interactions. Advances made in this regard are discussed.