Biological Relevance of a Stable Biochemical Interaction between the Tombusvirus-Encoded P19 and Short Interfering RNAs

Abstract

The Tomato bushy stunt virus (TBSV)-encoded p19 protein (P19) is widely used as a robust tool to suppress RNA interference (RNAi) in various model organisms. P19 dimers appropriate 21-nucleotide (nt) duplex short interfering RNAs (siRNAs) generated by Dicer presumably to prevent programming of the RNA-induced silencing complex (RISC). In the context of virus infection, this model predicts that P19 mutants compromised for siRNA binding cannot prevent RISC-mediated degradation of TBSV RNA and thus reduce viral pathogenicity. To test this, we used P19/43 (R3W), which is less pathogenic than wild-type P19 (wtP19), and P19/75-78 (RR3GG), with pathogenicity properties (i.e., viral spread and symptom induction) comparable to those of a P19-null mutant. We demonstrate that P19/43 still suppresses RNAi-mediated viral RNA degradation in infected Nicotiana benthamiana, while P19/75-78 is unable to prevent this clearance of viral RNA, leading to an irreversible recovery phenotype. Gel filtration and immunoprecipitation assays show that at the onset of the infection, wtP19, P19/43, and P19/75-78 readily accumulate, and they form dimers. The wtP19 is stably associated with duplex ϳ21-nt TBSV siRNAs, while P19/75-78 does not bind these molecules, and the electrostatic interaction of P19/43 with siRNAs is perturbed for ϳ21-nt duplexes but not for longer siRNAs. This is the first clear demonstration of a direct correlation between a novel structurally orchestrated siRNA binding of an RNAi suppressor and its roles in viral pathogenesis. The findings should be particularly valuable for the RNAi field in general because the P19 mutants enable precise determination of siRNA appropriation effects.RNA interference (RNAi) is a posttranscriptional RNA silencing process that is evolutionary conserved across eukaryotic kingdoms to regulate the levels of specific RNAs during development. RNAi also functions as a host defense system against virus infections, as was first shown for plant viruses (1). As a countermeasure, viruses have evolved suppressors of gene silencing (15,26,27,32,38). One of the biochemically best characterized suppressors is a protein of 19 kDa (P19) encoded by Tomato bushy stunt virus (TBSV) and related tombusviruses that is functional as a suppressor in several model systems (4,12,13,40,41).During TBSV replication in plants, abundant levels of highly structured genomic single-stranded RNA (ssRNA) and doublestranded RNAs (dsRNAs) accumulate that potentially form excellent substrates for one of the first steps in RNAi: Dicer-mediated cleavage of dsRNAs into duplex short interfering RNAs (siRNAs) (2,19,20,43). In a subsequent step of RNAi, these ds-siRNAs unwind to donate one of the siRNA strands to the RNA-induced silencing complex (RISC). This forms the catalytic entity for RNA degradation that uses the incorporated siRNA to specifically target complementary RNAs (7-9, 14, 16, 17). However, suppressors of RNAi, like P19, can interfere with this process (10, 33).The X-ray crystallographic structure of the P19-siRNA com...

RNAi-associated ssRNA-specific ribonucleases in Tombusvirus P19 mutant-infected plants and evidence for a discrete siRNA-containing effector complex

“…1C). Previously, it was shown that, in plants infected with wt Tombusvirus, viral-derived siRNAs exclusively associated with P19 (12,14,29). These findings, along with those reported here, agree with the model that, during infection, wtP19 dimers sequester Dicer-generated Ϸ21-nt TBSV duplex siRNAs to avoid their programming of a high-MW antiviral RISC-like complex and thus prevent the onset of RNAi-mediated viral RNA degradation (2).…”

“…The progression of infection and symptom development on N. benthamiana plants inoculated with a TBSV p19-mutant devoid of P19 expression (TBSV-dP19) is initially very similar to that observed for wt TBSV (9,14,15). However, instead of eventually succumbing to a lethal necrosis, plants infected with tombusviruses devoid of P19 expression develop a recovery phenotype associated with viral RNA clearance (2).…”

“…One of the best characterized suppressors is the Tombusvirusencoded p19 protein (P19); it interferes with RNA silencing in plants and RNAi in other model systems (2,3,8). In the context of Tombusvirus infections, P19 prevents RNAi-mediated viral RNA degradation, which correlates with a dramatic exacerbation of the disease phenotype compared with infections with virus mutants not expressing P19 (9)(10)(11)(12)(13)(14)(15)(16).…”

MicroRNAs in tomato plants

“…These results demonstrated that the tomato miRNA pathway is markedly compromised by P19, in particular when this protein is relatively abundant, as occurs during natural infection [52]. P19 is a high-affinity short (19-21 nt) double-stranded RNAbinding protein and that dimers of P19 bind siRNA duplexes in a 1 : 1 stoichiometric ratio [53][54][55][56]. This binding prohibits bound duplexes from unwinding and programming the RISC [57].…”

“…This binding prohibits bound duplexes from unwinding and programming the RISC [57]. As a consequence, RISC-mediated degradation of cognate viral RNAs is repressed [53].…”