Mechanistic Studies Of Suppression Of Post-transcriptional Gene Silencing By The Tomato Yellow Leaf Curl China Virus-associated DNA Satellite

Betasatellites represent novel DNA molecules that are associated with some monopartite begomoviruses in the Geminiviridae family including Tomato yellow leaf curl China virus (TYLCCNV). Although these monopartite begomoviruses are able to infect their hosts alone, they often are incapable of inducing typical symptoms, and require betasatellites for accumulation to high titers and for elicitation of disease symptoms. The betasatellite contains a single open reading frame (βCl) that encodes a symptom determinant. Earlier studies have shown that βCl proteins are potent post-transcriptional gene silencing (PTGS) suppressors, but the underlying mechanism has not been elucidated. To gain more insights into the mode of action of βCl in PTGS suppression and the functions of the key proteins involved in RNA silencing against geminivirus infection, the following studies were carried out:1The suppression of βCl on PTGS:whole genome tiling microarrays were used to probe the changes of transcriptome profile in response to TYLCCNB or βCl. The most pronounced of the differentially expressed gene, was a Nicotiana benthamiana ortholog of tobacco calmodulin-like protein, which has been previously identified as an endogenous regulator of gene silencing Calmodulin(rgs-CaM). In other Solanaceae hosts, TYLCCNV infections containing the betasatellite also exhibited up-regulated expression of the rgs-CaM orthologs in tobacco(Ntrgs-CaM) and tomato (Slrgs-CaM). Transgenic plants over-expressing Nbrgs-CaM displayed developmental abnormities reminiscent of βCl-associated morphological alterations. Nbrgs-CaM suppressed RNA silencing in an Agrobacterium infiltration assay and, when over-expressed, blocked TYLCCNV-induced gene silencing. Genetic evidence obtained with transgenic RNAi lines of Nbrgs-CaM showed that Nbrgs-CaM mediated the βCl functions in PTGS suppression and symptom modulation, and was required for efficient virus infection. Moreover, the tobacco and tomato orthologs of Nbrgs-CaM also possessed endogenous gene silencing suppressor (ESR) activity and were induced by betasatellite to promote virus infection in these Solanaceae hosts.2RDR6-mediated RNA silencing against geminivirus infection:βCl-induced Nbrgs-CaM suppressed sense RNA-induced PTGS (S-PTGS) but not hairpin RNA-induced PTGS, and inhibited the production of secondary small interfering RNAs (siRNAs). Further studies showed that βC1and Nbrgs-CaM repress RNA-dependent RNA polymerase6(RDR6) expression, and Nbrgs-CaM is genetically required for βC1to repress NbRDR6expression. RDR6-deficient N. benthamiana plants were defective in mounting an effective RNA silencing response and thus were hypersensitive to TYLCCNV infection. More significantly, TYLCCNV could overcome host range restrictions to infect Arabidopsis thaliana when the plants carried a RDR6mutation.3SGS3-mediated RNA silencing against geminivirus infection:In Arabidopsis thaliana, efficient RNA silencing requires RDR6and its double-stranded RNA (dsRNA)-binding partner, Suppressor of Gene Silencing3(SGS3) to amplify secondary siRNAs that allow plants to mount effective defense response against redundant endogenous mRNA or virus infection. Therefore, to better understand the role of Nbrgs-CaM in S-PTGS pathway and the potential effect of Nbrgs-CaM on the function of NbSGS3, we cloned NbSGS3gene from N. benthamiana. We found NbSGS3was required for producing S-PTGS, establishing and maintaining GFP systemic silencing. Moreover, NbSGS3plays an essential role in triggering antiviral gene silencing response upon TYLCCNV-mediated gene silencing and TYLCCNV infection. Using a yeast two-hybrid interaction assay and bimolecular fluorescence complementation analysis, we revealed that Nbrgs-CaM interacted with NbSGS3. Subsequently, the overexpression of Nbrgs-CaM altered the subcellular localization of NbSGS3from the’siRNA-bodies’to the cell membrane, and decreased the accumulation of the NbSGS3. Those data illustrated that Nbrgs-CaM could suppress RNA silencing possibly by changing the localization of NbSGS3in the ’siRNA-bodies’and degrading the NbSGS3protein.4The mechanism of the upregulation of Nbrgs-CaM induced by βC1:βC1has also been shown to be a suppressor in transcriptional gene silencing (TGS) and when ectopically expressed, βC1causes global reductions in host genome cytosine methylation. Using a chemical inhibitor of DNA methylation, we found the transcription of Nbrgs-CaM was silenced by a DNA methylation-related mechanism under normal conditions, but was induced in the presence of TYLCCNB or βCl. A mutant βCl protein (βCl3A) with three lysine residues in the nuclear localization signal (49KKK51) being replaced by alanine residues, which could not suppress TGS, was neither able to up-regulate the expression of Nbrgs-CaM nor suppress PTGS. This study showed the PTGS suppressor activity of βCl is dependent on its TGS suppression.Overall, our findings demonstrate a distinct mechanism of viral suppressors of RNA silencing (VSRs) for suppressing PTGS through usurpation of a host ESR, and highlight an essential role for RDR6and SGS3in RNA silencing defense response against geminivirus infection, and explain the relationship between βCl-mediated TGS and PTGS suppression.