Studies Of Suppression Of Transcriptional Gene Silencing By The Tomato Yellow Leaf Curl Virus Encoded V2Protein

Tomato yellow leaf curl virus (TYLCV) is a monopartite geminivirus belonging to the genus Begomovirus, causing severe chlorosis, downward leaf curling symptoms and stunting of tomato plants. In China, TYLCV infection of tomato was firstly described in Shanghai in1996, and now it is one of the most devastating viruses that occurs in more than10provinces causing severe losses in tomato production. TYLCV encoded V2protein is shown to be a post-transcriptional gene silencing (PTGS) suppressor, its function on suppression of transcriptional gene silencing (TGS) has not been reported. To better understand the biological roles of V2protein during viral infection, suppression of TGS by TYLCV V2has been investigated.16-TGS plants were inoculated with a TYLCV infectious clone, pBinPLUS-TYLCV-SH2-1.4A, and suppression of TGS was observed after TYLCV infection. To identify the TGS suppressor(s) encoded by TYLCV, a Potato virus X (PVX) expression vector was employed to express individual TYLCV open reading frames (ORFs) and16-TGS plants were inoculated with various PVX vectors expressing different TYLCV ORFs. We found that, like TYLCV itself, V2can reverse the TGS of the GFP transgene. Results of bisulfite sequencing and methylation-sensitive restriction endonuclease-PCR showed that the cytosine methylation levels of35S promoters in TYLCV or V2inoculated16-TGS plants were reduced. To further explore the mechanism for suppresson of TGS by V2, we expressed TYLCV V2in Arabidopsis thaliana through stable transformation. Flowering of the V2transgenic Arabidopsis was delayed when compared with the wild type (WT) plants and the DNA methylation levels were interfered at AtSNl and MEA-ISR loci, which are known to be transcriptional silenced in WT Arabidopsis through methylation.A yeast two hybrid (Y2H) screen was performed to identify host proteins from a Nicotiana benthamiana cDNA library that interact with TYLCV V2, using TYLCV V2as bait. The plasmid pGBKT7-V2and N. benthamiana cDNA library were co-transformed into Saccharomyces cerevisiae strain Y2HGold, and the double transformants were assayed for histidine prototrophy and α-galactosidase activity. One cDNA clone designated as NbHDA6was selected. The full-length cDNA of NbHDA6has1,368nucleotides encoding a protein of456amino acids (about51kD). NbHDA6is closely related to HDA6in Nicotiana tomentosiformis, Solanum lycopersicum, Vitis vinifera, Morus notabilis, Theobroma cacao and A. thaliana. Putative functional domains in NbHDA6were deduced. NbHDA6belongs to the type I (RPD3-like) superfamily HDACs, N terminal contains a conserved histonc deacetylase kinase domain, middle region contains active sites necessary for histone deacetylase activity, and C terminal contains a polyglycine region and a aspartate-rich region. The V2-NbHDA6interaction was also confirmed by bimolecular fluorescence complementation (BiFC) analysis. The fluorescence showed that these two proteins interacted in nuclear or cytoplasm in living N. benthamiana epidermal cells. GST pull down assays further verified the interaction between V2and NbHDA6in vitro using the purified V2and NbHDA6proteins.Semi-quantitative reverse transcription (RT)-PCR was performed to check the specific expression of NbHDA6mRNA in different tissues of N. benthamiana plant. Highest accumulation level of NbHDA6mRNA was found in root with intermediate in flower, and lower in leaf and stem. NbHDA6-GFP fusion gene driven by the35S promoter was created and inoculated into N. benthamiana epidermal cells. Results showed that NbHDA6-GFP fusion protein localized in the nucleus. Agrobacterium-mediated floral dip method was then performed to transform NbHDA6into Arabidopsis HDA6mutant, axel-5. We found that NbHDA6complemented both late flowering phenotype and hyperacetylation level of H3, H4in axe1-5. These foundings reveal that NbHDA6can complement the biological function of AtHDA6in Arabidopsis.To illustrate the biological role of the V2-NbHDA6interaction, real time RT-PCR and semi-quantitative RT-PCR were performed to check the expression levels of NbHDA6mRNA using total RNAs from TYLCV or PVX-V2or mock-inoculated N. benthamiana plants. The results suggested that TYLCV or PVX-V2infection significantly attenuated the accumulation of NbHDA6mRNA. We next used RNAi technology to inhibit NbHDA6expression in N. benthamiana by using silencing vector TRV. The symptoms on NbHDA6-silenced plants seemed to be more severe as compared with WT plants after agroinoculated with TYLCV. Southern blot analysis further confirmed that the accumulation levels of viral DNAs were higher in the NbHDA6-silenced plants. Furthermore, results of bisulfite sequencing and methylation-sensitive restriction endonuclease-PCR showed that the cytosine methylation levels of TYLCV in NbHDA6-silenced plants were reduced as compared with WT plants.These results suggest that the expression level of NbHDA6impacts the infection efficiency of TYLCV and NbHDA6is necessary for host plants to defense against viral infection by TGS.To further understand the mechanism of V2-NbHDA6interaction, the full-length V2as well as the full-length NbHDA6were fused to GST, expressed and purified from Escherichia coli cells, respectively, then used in detecting the histone deacetylase activity. NbHDA6itself has a weak histone deacetylase activity, but this activity was not influenced by V2. We then fused a truncated NbMET1(containing the first Bromo-adjacent homology domain) to MBP, fusion protein was purified from E. coli cells and used in in vitro competitive pull down assay. We found that the amounts of MBP-NbMET1pulled down by GST-NbHDA6were reduced with increasing amounts of V2in the mixture. These results provide evidence that V2competes with NbMETl for directly binding to NbHDA6. Our findings suggest that V2interacts with host HDA6and interferes the recruitment of MET1by HDA6resulting in decrease of DNA methylation on DNA virus by plant TGS and increase of host susceptibility to TYLCV infection.