Mutational analysis of begomovirus-associated, betasatellite-encoded βC1 gene and infectivity of betasatellite related molecules

Abstract

Most of the economically important viruses of the family Geminiviridae fall in the genus Begomovirus. Begomoviruses may have either monopartite or bipartite single-stranded DNA genomes and are transmitted by the whitefly Bemisia tabaci. With a single exception (Tomato leaf deformation virus), begomoviruses native to the New World (NW) are bipartite, consisting of two components known as DNA A and DNA B. In the Old World (OW) most begomoviruses are monopartite, with a single component genome that is a homolog of the DNA A of the bipartite begomoviruses. The majority of the monopartite begomoviruses are associated with single-stranded DNA satellites. Three classes of satellites have been identified, the most important of which are the betasatellites. Betasatellites are approximately half the size of their helper begomoviruses (~1350 bp) and are, in most cases, required by the begomovirus for efficient infection of host plants. The begomovirus-associated betasatellites encode a single gene product, known as βC1 that is a pathogenicity determinant, a suppressor of post-transcriptional gene silencing and may be involved in virus movement in plants. The third class of satellites consists of molecules that are approximately one quarter (~650 bp) the size of their helper begomoviruses, are non-coding and are believed to have evolved from betasatellites. Recently this type of satellite has been identified in sweetpotato plants in association with sweepoviruses (phylogenetically distinct begomoviruses that generally infect sweetpotato). This class of satellites includes the first begomovirus-associated satellite identified (known as the Tomato leaf curl virus-satellite). The study described here was designed to identify possible functional domains of the βC1 protein of the cotton leaf curl disease-associated Cotton leaf curl Multan betasatellite (CLCuMuB) and to identify amino acid sequence motifs involved in βC1 function. Additionally the aim was to examine the possible effects of non-coding satellites on the pathogenicity of their helper viruses in planta. Mutants of CLCuMuB βC1 gene were produced and cloned in a Potato virus X (PVX) expression vector for expression in plants. The expression constructs were examined for the effects of mutations of βC1 on pathogenicity and suppression of post-transcription gene silencing (PTGS) activity. The study produced site-directed mutants of the CLCuMB βC1and also included some N- and C-terminal deletion xiv mutants that were produced earlier. The results indicated that the C-terminus of βC1 is important for pathogenicity, whereas amino acid sequences towards the N-terminus have a role in PTGS. The results showed that the pathogenicity and PTGS activities of βC1 can be uncoupled, yielding a protein with suppressor activity which does not induce symptoms in plants. Suppressor proteins lacking pathogenicity are potentially useful in biopharming. The results are discussed in light of the recent findings concerning the functions and interactions of betasatellites and βC1. Constructs for the infectivity of several non-coding satellites found in sweetpotato plants were produced and examined for the ability of the satellites to be maintained by various begomoviruses and for their effects on helper virus DNA levels and symptoms induced in plants. The non-coding satellites were trans-replicated and maintained by SPLCLaV and two heterologous begomoviruses (Tomato yellow leaf curl virus and Tomato yellow leaf curl Sardinia virus), in a number of plant species. The satellites strongly affected the levels of accumulation of the heterologous viruses in N. benthamiana and tomato. One of the non-coding satellites was also shown to be transmissible by the Mediterranean species of the B. tabaci complex. Furthermore, in common with the beta- and alphasatellites, the non-coding satellites were shown to modulate the symptom severity of their helper begomoviruses, including SPLCLaV. The described study was conducted with the aim of a better understanding of satellite molecules associated with begomoviruses. A more in-depth understanding of the functions and interactions of satellites may allow better control methods to be designed. The results are discussed with this aim in mind.