Three distinct suppressors of RNA silencing encoded by a 20-kb viral RNA genome

Abstract

Viral infection in both plant and invertebrate hosts requires a virus-encoded function to block the RNA silencing antiviral defense. Here, we report the identification and characterization of three distinct suppressors of RNA silencing encoded by the Ϸ20-kb plus-strand RNA genome of citrus tristeza virus (CTV). When introduced by genetic crosses into plants carrying a silencing transgene, both p20 and p23, but not coat protein (CP), restored expression of the transgene. Although none of the CTV proteins prevented DNA methylation of the transgene, export of the silencing signal (capable of mediating intercellular silencing spread) was detected only from the F1 plants expressing p23 and not from the CP-or p20-expressing F1 plants, demonstrating suppression of intercellular silencing by CP and p20 but not by p23. Thus, intracellular and intercellular silencing are each targeted by a CTV protein, whereas the third, p20, inhibits silencing at both levels. Notably, CP suppresses intercellular silencing without interfering with intracellular silencing. The novel property of CP suggests a mechanism distinct to p20 and all of the other viral suppressors known to interfere with intercellular silencing and that this class of viral suppressors may not be consistently identified by Agrobacterium coinfiltration because it also induces RNA silencing against the infiltrated suppressor transgene. Our analyses reveal a sophisticated viral counter-defense strategy that targets the silencing antiviral pathway at multiple steps and may be essential for protecting CTV with such a large RNA genome from antiviral silencing in the perennial tree host.RNA interference ͉ citrus tristeza virus ͉ virus synergy ͉ antiviral immunity T he discoveries of viral suppressors of RNA silencing play an important role in establishing RNA silencing as a natural antiviral response in both plant and invertebrate hosts (1). Many plant viral proteins have been identified as suppressors of RNA silencing since the initial reports in late 1998 (2-4). RNA silencing suppressors from different virus taxons are structurally diverse. However, they are typically required for long-distance virus spread and influence virulence and accumulation levels in infected plants (5-7). Progress is being made toward understanding the molecular mechanisms involved in viral suppression of RNA silencing (8-11). In RNA silencing assays, plant viral suppressors differ by their ability to suppress intracellular and͞or intercellular silencing (12-16). Intercellular silencing is mediated by the non-cell-autonomous silencing signal that can spread to destroy homologous RNAs in neighboring or distant tissues that do not contain the initial trigger, such as a silencing transgene or a replicating virus (17-19). The differential silencing suppression is best illustrated by the potyviral helper component proteinase (HC-Pro) and the cucumber mosaic virus (CMV) 2b protein. When introduced by genetic crosses into the Nicotiana tabacum line 6b5 carrying a silencing ␤-glucuronidase (GUS) transgen...

Suppressors of RNA silencing encoded by plant viruses and their role in viral infections

“…It was recently established that three different proteins, p20, p23 and the CP of this complex virus have silencing suppression activities [12]. Both p20 and p23, but not CP, suppressed RNA silencing in the agro-infiltration assay, and were able to reverse transgene silencing.…”

“…An exhaustive review of the methodology for suppressor identification has recently been compiled by Moissiard and Voinnet [11]. In the interests of not being duplicative, we have chosen to highlight features of four procedures that have been used most frequently, alone and in combination, to verify the silencing suppression activity of a given viral gene, or to identify multiple suppressors encoded by a single virus [12].…”

“…Interestingly, p20 and CP, but not p23 prevented intercellular signaling of RNA silencing. It was suggested that suppression of RNA silencing at multiple steps of the silencing pathway may be essential for viruses with large RNA genomes such as CTV [12].…”

Citrus tristeza virus infection induces the accumulation of viral small RNAs (21–24-nt) mapping preferentially at the 3′-terminal region of the genomic RNA and affects the host small RNA profile

“…Three of these proteins (p25, p20 and p23) are silencing suppressors in N icotiana spp. (Lu et al 2004). Besid es, p23, an RN A-bind ing protein (López et al 2000) that controls the asym m etrical accum ulation of (+) and (-) CTV RN As (Satyanarayana et al 2002), is a pathogenicity d eterm inant w hen expressed ectopically in several Citrus spp.…”

Gene silencing: concepts, applications, and perspectives in woody plants

“…CTV, for instance, is known to contain at least three distinct suppressors of RNAi. Proteins p20, p23, and the coat protein act in different stages of RNAi, affecting both intercellular (p20 and coat protein) and intracellular (p20 and p23) silencing (Lu et al, 2004). RNAi suppressor proteins can alter the pattern of gene expression in virus hosts, since they affect both miRNA and siRNA pathways, leading to developmental abnormalities and possibly viral symptoms (Voinnet, 2005).…”