Identification Of And Studies On Silencing Suppressors From Three Rice Viruses

RNA silencing is an evolutionally conserved antiviral mechanism in plants and some other eukaryotes. As a counter defense, most plant and some animal viruses encode one or multiple silencing suppressors. Rice is one of the most important crop plants in China. Viral disease constitutes one major threat to rice production. Rice ragged stunt virus (RRSV), which is the type species of Orayzavirus, and Southern rice black-streaked dwarf virus (SRBSDV), a newly proposed species of Fijivirus, are two rice viruses that have caused great yield losses to rice production in China in recent years.In this study, the ORFs of the non-structural proteins of RRSV and SRBSDV were cloned and ligated into the vectors pPZP212 or pEarleyGate 100 respectively. The plasmids, which were named 35S-RRSVS6 , 35S-RRSVS7, 35S-RRSVS10, 35S-SRBSDVS6 , 35S-SRBSDVS7 and 35S-SRBSDVS9 respectively, were individually transformed into the Agrobacterium tumefaciens EH105. The transformants were each mixed with a strain of Agrobacterium carrying the construct 35S-GFP and the mixtures were inoculated into leaves of Nicotiana benthamiana 16c. All innoculated leaf patches showed strong green fluorescence at 3 days post inoculation (dpi), but only in patches co-infiltrated by 35S-RRSVS6 plus 35S-GFP or 35S-SRBSDVS6 plus 35S-GFP did the fluorescence intensity maintained until 7dpi. However, the green fluorescence declined in leaf patches coexpressing GFP and mutant forms of the ORF of RRSV or SRBSDV S6 at 3 dpi and disappeared at 7dpi. In addition, in leaf patches co-infiltrated with 35S-RRSVS6, but not 35S-RRSVâ–³S6 plus 35S-GFP, the accumulation of GFP mRNA was high and that of GFP siRNA was barely detectable at 7 dpi. When cloned into PVX, pns6 of RRSV could greatly enhance the pathogenicity of the chimeric virus. We concluded from these results that the pns6 of RRSV and SRBSDV have silencing suppressor activities. Further analyses showed that RRSV pns6 could not inhibit the silencing induced by dsGFP and its silencing suppressor activities were abolished when a region involved in nucleic acid binding in this protein was deleted.Previously, we have found that the pns6 of RRSV might be a movement protein. The multi-functional nature of this protein promoted us to search possible host proteins that could interact with it. To this end, a rice cDNA library was screened using yeast two hybrid experiments with RRSV pns6 as bait. A number of positive clones were obtained and sequence analysis of the cDNA inserts resulted in the identification of 44 candidate rice proteins that might interact with RRSV pns6. These proteins were classified according to their GO annotations. The results revealed that they might be involved in a wide range of cellular processes. In addition, the cellular localization patterns of the pns6 of RRSV were observed by transiently expressing it in leaf cells of Nicotiana benthamiana. The results showed that the Pns6 of RRSV mainly accumulated in the cell periphery, forming distinct and punctate small dots there.Previous studies of this lab showed that p2 of RSV could interact with SGS3 of rice. In RSV infected rice, the expression of five ARFs that were targets of TAS3 derived tas-siRNAs was upregulated, indicating that RSV infection resulted in the alteration of the tas-siRNA pathway in rice. Further experiments were conducted in this study to explore the biological implications of the interaction between RSV p2 with OsSGS3 and the following results were obtained: 1) p2 of RSV is a silencing suppressor. It could suppress the silencing induced by sense GFP in N. benthamiana 16c and could greatly enhance the pathogenicity as well as accumulation of PVX in N. benthamiana. 2) Several auxin related genes exhibited downregulation in RSV infected rice. This indicated that the increasing in expression of auxin related genes was not a general response of rice to RSV infection, confirming the specificity of the elevated expression of the ARFs targeted by tas-siRNAs detectd by our lab previously. 3) It was shown that the region encompassing the N-terminal amino acids 1-40 is dispensable for the interaction between p2 and OsSGS3. However, deletion of the 20 amino acids located on the C terminus of p2 abolished its interaction with OsSGS3. 4) Polyclonal antibodies to OsSGS3 were prepared. 5) Transgenic rice overexpressing OsSGS3 was obtained. The inoculation experiments in this study using one tube-one seedling method indicated that the transformants showed enhanced resistance to RSV compared with wild type rice.