The Suppressing Mechanisms Of RNA Silencing Suppressors Encoded By Tomato Yellow Leaf Curl China Virus And Its Satellite DNAβ

Tomato yellow leaf curl China virus(TYLCCNV)is a monopartite begomovirus found in China,which caused severe diseases on tobacco and tomato in Yunnan and Guangxi provinces of China in recent years.Our previous study demonstrated thatβC1.AC2 and AC4 of TYLCCNV were suppressors of RNA silencing,but molecular mechanisms underlying their suppressing activity remain unknown.In this paper,the mechanisms for RNA silencing suppression byβC1.AC2 and AC4 were studied.Co-infiltration ofβC1 with GFP in GFP transgenic Nicotiana benthamiana line 16c showed that the local GFP mRNA and GFP protein accumulations increased significantly in N.benthamiana line 16c plants at five days post co-infiltration when compared with those in plants co-infiltrated with GFP and empty vector orβC1 mutant,indicating thatβ1 was indeed a RNA silencing suppressor.The RNA binding ability of recombinantβC1 protein determined by electrophoresis mobility shift assay(EMSA)showed thatβC1 protein could bind single-stranded RNA(ssRNA) but not double-stranded RNA(dsRNA),suggesting thatβC1 protein might could not interfere with dsRNA cleaving by Dicer.Consistent with this,Northern blot also showed thatβC1 markedly diminished the accumulation of siRNAs,but could not inhibit the siRNA generation.βC1 protein could bind single-stranded siRNA but not siRNA duplex,and its ability of single-stranded siRNA binding correlated with protein concentration,demonstrating thatβC1 protein might interfere with RNA-induced silencing complex(RISC)activity,but could not inhibit the siRNA-initiated RISC assembly via binding to siRNAs.Further study revealed thatβC1 protein could inhibit GFP systemic silencing and prevent spread of silencing signal,similar to the positive control of 2b of cucumber mosaic virus(CMV). Therefore,we speculated thatβC1 might suppress RNA silencing via preventing long-distance spread of silencing signal like CMV 2b.The AC2 and AC4 genes of TYLCCNV were expressed in E.coli strain BL21 (DE3)pLys S using pET32a vector,and recombiant proteins were purified with Ni²⁺- NTA agarose affinity chromatography.The RNA binding ability of recombinant AC2 protein was determined by EMSA.The results showed that AC2 protein could bind ss/dsRNA and ss/ds siRNA,and its ability of RNA binding correlated with protein concentration,suggesting that AC2 might prevent dsRNA cleaving by Dicer or inhibit the siRNA-initiated RISC assembly via binding to siRNAs or interfere with RISC activity.The RNA binding ability of recombinant AC4 protein determined by EMSA showed that AC4 protein could not bind dsRNA and siRNA duplex,suggesting that AC4 protein might could not prevent dsRNA cleaving by Dicer and could not inhibit the siRNA-initiated RISC assembly via binding to siRNAs.But AC4 protein could bind single-stranded siRNA,and its ability of single-stranded siRNA binding correlated with protein concentration,suggesting that AC4 was likely to interfere with RISC activity.