Variability in the level of RNA silencing suppression caused by triple gene block protein 1 (TGBp1) from various potexviruses during infection

Abstract

RNA silencing is an important defence mechanism against virus infection, and many plant viruses encode RNA silencing suppressors as a counter defence. In this study, we analysed the RNA silencing suppression ability of multiple virus species of the genus Potexvirus. Nicotiana benthamiana plants exhibiting RNA silencing of a green fluorescent protein (GFP) transgene showed reversal of GFP fluorescence when systemically infected with potexviruses. However, the degree of GFP fluorescence varied among potexviruses. Agrobacterium-mediated transient expression assay in N. benthamiana leaves demonstrated that the triple gene block protein 1 (TGBp1) encoded by these potexviruses has drastically different levels of silencing suppressor activity, and these differences were directly related to variations in the silencing suppression ability during virus infection. These results suggest that suppressor activities differ even among homologous proteins encoded by viruses of the same genus, and that TGBp1 contributes to the variation in the level of RNA silencing suppression by potexviruses. Moreover, we investigated the effect of TGBp1 encoded by Plantago asiatica mosaic virus (PlAMV), which exhibited a strong suppressor activity, on the accumulation of microRNA, virus genomic RNA and virus-derived small interfering RNAs. INTRODUCTIONRNA silencing is an RNA-guided gene regulatory mechanism that operates in a wide variety of eukaryotic organisms. RNA silencing begins with processing of an RNA trigger into small RNAs (~21-30 nt) by the RNase III-type enzyme Dicer (Bernstein et al., 2001). In plants, small RNAs are divided into two classes: small interfering RNAs (siRNAs) and microRNAs (miRNAs) (Mallory & Vaucheret, 2006;Brodersen & Voinnet, 2006). siRNAs are generated from double-stranded RNAs (dsRNAs) which are usually derived from transgenes, viruses or endogenous non-coding RNA genes. On the other hand, miRNAs are generated from genome-encoded precursor RNAs with imperfect stem-loop structures. These small RNAs are then incorporated into multicomponent RNA-induced silencing complexes (RISC), which contain an Argonaute (AGO) family protein and lead to homologous RNA cleavage or translational repression, and (or) DNA/chromatin methylation (Hammond, 2005;Xie et al., 2004;Chapman & Carrington, 2007).One of the important roles of RNA silencing in plants is antiviral defence (Ratcliff et al., 1997;Vance & Vaucheret, 2001;Baulcombe, 2004;Ding & Voinnet, 2007). During virus infection, dsRNAs derived from viral replication intermediates or highly structured viral genomic RNAs trigger RNA silencing directed against the viruses. Consequently, initially symptomatic plants recover from infection and become resistant to secondary infection with homologous viruses. To counteract RNA silencing, viruses have evolved RNA silencing suppressors. More than 40 RNA silencing suppressors have been identified in plant, animal, insect and fungal viruses (Li & Ding, 2006). These suppressors do not have obvious sequence similarity to one another, and t...

In Planta Recognition of a Double-Stranded RNA Synthesis Protein Complex by a Potexviral RNA Silencing Suppressor

“…TGBp1 suppresses S-RNAi but not IR-RNAi (Senshu et al, 2009). This is consistent with the finding that TGBp1 inactivates SGS3/RDR6-mediated dsRNA synthesis because SGS3 and RDR6 are only involved in S-RNAi, but AGO1 and DCL4 are involved in both S-RNAi and IR-RNAi.…”

“…Indeed, in the TGBp1 transgenic plants, the levels of tasiRNA were significantly reduced ( Figure 1C; Supplemental Table 2), and the levels of tasiRNA target mRNA increased ( Figure 1D). Although previous reports have indicated that PVX-TGBp1 transformants do not exhibit any visible developmental abnormalities (Dunoyer et al, 2004), this may be due to the low level of suppressor activity of PVX TGBp1 (Senshu et al, 2009).…”

“…RNA isolation and RNA gel blot analysis of sRNAs and mRNAs were performed as described previously (Senshu et al, 2009). Digoxigenin (DIG)-labeled RNA probes corresponding to nucleotides 180 to 767 of TAS2 (At2g39680) and nucleotides 58 to 863 of TAS3 (At3g17185) were used to detect TAS2 tasiRNA and TAS3 tasiRNA, respectively.…”

“…pCAMBIA1301-PIAMV-TGBp1, a binary vector expressing PlAMV-TGBp1, was described previously (Senshu et al, 2009). To generate binary vectors expressing TGBp1 mutants, we introduced mutations into pCAMBIA1301-PlAMV-TGBp1 by performing recombinant PCR using sets of two partially complementary primers: PlTGBp1AKT-F and PlTGBp1AKT-R, PlTGBp1E92A-F and PlTGBp1E92A-R, PlTGBp1P110L-F and PlTGBp1P110L-R, and PlTGBp1T192A-F and PlTGBp1T192A-R for CAMBIA1301-PlAMV-TGBp1 AKT , -TGBp1 E82A , -TGBp1 P110L , and -TGBp1 T192A , respectively.…”

“…Nevertheless, subsequent studies showed that PVX TGBp1 suppresses RNA silencing of the green fluorescent protein (GFP) gene in agroinfiltrated leaves (local silencing) and spread of the silencing into upper noninoculated tissues in GFP transgenic plants (systemic silencing) (Voinnet et al, 2000). In a previous study, we showed that the levels of RNA silencing suppressor activity differ depending on the virus species and that of PVX is relatively low among potexviruses (Senshu et al, 2009).…”