Multiple suppressors of RNA silencing encoded by both genomic RNAs of the crinivirus, Tomato chlorosis virus

Abstract

Viruses express proteins with silencing suppression activity to counteract the RNA silencing-mediated defense response of the host. In the family Closteroviridae, examples of multiple-component RNA silencing suppression systems have been reported. To ascertain if this is a general strategy in this group of viruses, we have explored the bipartite genome of Tomato chlorosis virus (ToCV, genus Crinivirus). We have identified the RNA1-encoded p22 protein as an effective silencing suppressor by using a Agrobacterium co-infiltration assay. p22 suppressed local RNA silencing induced either by sense RNA or dsRNA very efficiently, but did not interfere with short or long-distance systemic spread of silencing. We have also demonstrated by using the heterologous vector PVX the silencing suppression activity of the RNA-2 encoded coat protein (CP) and minor coat protein (CPm). In this study, we demonstrate an even greater complexity of silencing suppressor activity for a plant virus, and for the first time we show the presence of RNA silencing suppressor genes encoded by both genomic RNA molecules of a bipartite genome in the complex family Closteroviridae.

The Road to RNA Silencing is Paved with Plant-Virus Interactions

“…The closterovirus citrus tristeza virus contains three VSR, with different specificities: the P23 inhibits intracellular silencing, the CP inhibits long-distance signaling and the P20 does both (Lu et al, 2004). The crinivirus tomato chlorosis virus also contains three VSR: the P22 encoded by RNA 1 interfered with local RNA silencing but not with the spread of the silencing signal, while both the major and minor capsid proteins, encoded by RNA 2, also showed local RNA silencing suppression activity (Cañizares et al, 2008). On the other hand, the crinivirus sweet potato chlorotic stunt virus expresses a small dsRNase that cleaves siRNAs such that they cannot function in RNA silencing and works in concert with the P22 protein of this virus (Cuellar et al, 2006;Kreuze et al, 2005).…”

P22 of tomato chlorosis virus, an RNA silencing suppressor, is naturally expressed in the infected plant

“…The proteins are not well studied and their putative functions are associated with virus replication, encapsidation, movement, and whitefly transmission (11)(12)(13). The p22 protein encoded in the RNA1, as well as the major (CP) and minor (CPm) coat proteins encoded in the RNA2 of ToCV are implicated as RNA silencing suppressors (RSS) (14). Until now, the CP is the only viral protein being detected in ToCV-infected tomato plants (15).…”

“…As an RNA silencing suppressor (RSS), p22 is important for ToCV infection and it might also be involved in viral pathogenicity (14). The RSS property of p22 has been further supported by its preferential binding to long dsRNAs in vitro and protecting these long RNAs from cleavage by dicer (16).…”

Characterization and full genome sequence analysis of a Chinese isolate of tomato chlorosis virus

“…ORF 1b encoded a 59 kDa RNA-dependent RNA polymerase (RdRp) that was conserved among members of the genus Crinivirus (11). ORF 2 (nt 7662-8243) encoded a 22 kDa protein (p22), which was identified to be an effective silencing suppressor (22). ORF 3 (nt 8263-8418) encoded a 5 kDa protein (p5) possessing a central transmembrane domain, but its function remains unknown (11).…”

“…KC887999), possessing 238 nt and 213 nt 5´-and 3´-UTRs, respectively, and encoding nine putative proteins, p4 (ORF 1, nt 239 to 340), Hsp70h (ORF 2, nt 733 to 2397), p8 (ORF 3, nt 2407 to 2610), p59 (ORF 4, nt 2562 to 4115), p9 (ORF 5, nt 4097 to 4333), CP (ORF 6, nt 4333 to 5106), CPm (ORF 7, nt 5112 to 7121), p27 (ORF 8, nt 7125 to 7823) and p7 (ORF 9, nt 7832 to 8029). Hsp70h, p59, CP and CPm may participate in viral movement, encapsidation, vector-mediated transmission and silencing suppression (11,12,14,22). P4 and p7 were transmembrane proteins, but their functions remain unknown, and p7 was a novel protein that had no equivalent in genomes of other criniviruses (12).…”