Identification Of The Suppressor Activity Of The Rep Protein Encoded By Alphasatellite Associated With Begomovirus/Betasatellite Complex And The Study Of Interaction Among The Complex

Alphasatellites are circled single-stranded molecules which have been shown to associate with begomovirus/betasatellite complexes. An increasing number of alphasatellites have been reported to associate with begomovirus/betasatellite complexes to infect crops and weeds. Three types of alphasatellites were selected in this study:type1is Y10alphasatellite (Y10DNA1) associates with Tomato yellow leaf curl China virus/Tomato yellow leaf curl China betasatellite complex (TYLCCNV/TYLCCNB), type2is Y35alphasatellite (Y35DNA1) associates with Tobacco curly shoot virus/Tobacco curly shoot betasatellite complex (TbCSV/TbCSB), and type3is Y132alphasatellite (Y132DNA1) associates with TbCSV/Ageratum yellow vein betasatellite complex (TbCSV/AYVB). The suppressor activity of the Rep proteins encoded by these three alphasatellites and the interaction among begomovirus, betasatellite and alphasatellite were investigated.1. Identification of the suppressor activity of the Rep protein encoded by alphasatelliteThe post-transcriptional gene silencing (PTGS) suppressor activity of the Rep proteins encoded by three alphasatellites were tested. Recombinant pCHF3vectors (pCHF3-Y10DNA1Rep, pCHF3-Y35DNAl Rep, pCHF3-Y132DNAl Rep) were constructed, and transformed into agrobacterium strain C58C1, then co-inoculated with a plasmid carrying35S-GFP segment into leaves of16c N.benthamiana plants. These three Rep proteins were identified as weak local PTGS suppressor by UV inspection, northern blot and western blot after3to5days post inoculation (dpi). In addition, systemic PTGS suppressor activity of these three Rep proteins were observed by UV inspection after30dpi.The transcriptional gene silencing (TGS) suppressor activity of the Rep proteins were detected. Recombinant PVX vectors (PVX-Y10DNA1Rep, PVX-Y35DNA1Rep, PVX-Y132DNA1Rep) were constructed and inoculated into methylated16-TGS N.benthamiana plants. Results of UV inspection, northern blot and western blot showed that these three Rep proteins had TGS suppressor activity after14dpi. The one encoded by Y10alphasatellite has the strongest PTGS and TGS suppressor activity among these Rep proteins. The accumulation of begomovirus was increased when co-infiltration with the helper begomovirus (TYLCCNV) and one of these three Rep proteins into N.benthamiana plants.2. Study of the interaction between monopartite begomovirus and its’ satellitesThe widespread begomovirus complex in China, TYLCCNV/TYLCCNB/Y10alphasatellite (Y10A/Y10β/Y10DNA1) and TbCSV/TbCSB/Y35alphasatellite (Y35A/Y35β/Y35DNA1), were chosen for study. After co-inoculation of alphasatellites with the homologous or heterologous helper viruses (Y35A and Y10A) and betasatellites (Y35(3and Y10β) into N.benthamiana plants, symptoms inspection and Southern blot was utilized to analysize the interaction among begomovirus, betasatellites and alphasatellites.Our results demonstrated that the accumulation of both the helper begomovirus and betasatellite were decreased in the presence of the alphasatellite (Y35DNA1or Y10DNA1), and the degree of reduction relied on the kinds of helper begomovirus and alphasatellite, while the symptoms were attenuated when the accumulation of helper begomovirus and betasatellites decreased significantly. Meanwhile, the accumulation of both helper begomovirus and alphasatellite significantly increased in the presence of the betasatellites followed severe symptoms. Results of yeast two-hybrid assay revealed that the Rep of Y10alphasatellite could interact with the Rep of TYLCCNV. It suggested that the trilateral interaction existing in the begomovirus, betasatellite and alphasatellite complexes.