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...

Tomato bushy stunt virus (TBSV) and other tombusviruses encode a p19 protein (P19), which is a suppressor of RNAi. Wild-type TBSV or p19-defective mutants initially show a similar infection course in Nicotiana benthamiana, but the absence of an active P19 results in viral RNA degradation followed by recovery from infection. P19 homodimers sequester 21-nt virus-derived duplex siRNAs, and it is thought that this prevents the programming of an antiviral RNAinduced silencing complex to avoid viral RNA degradation. Here we report on chromatographic fractionation (gel filtration, ion exchange, and hydroxyapatite) of extracts from healthy or infected Nicotiana benthamiana plants in combination with in vitro assays for ribonuclease activity and detection of TBSV-derived siRNAs. Only extracts of plants infected with p19 mutants provided a source of sequence-nonspecific but ssRNA-targeted in vitro ribonuclease activity that coeluted with components of a wide molecular weight range. In addition, we isolated a discrete Ϸ500-kDa protein complex that contained Ϸ21-nt TBSV-derived siRNAs and that exhibited ribonuclease activity that was TBSV sequencepreferential, ssRNA-specific, divalent cation-dependent, and insensitive to a ribonuclease inhibitor. We believe that this study provides biochemical evidence for a virus-host system that infection in the absence of a fully active RNAi suppressor induces ssRNA-specific ribonuclease activity, including that conferred by a RNA-induced silencing complex, which is likely the cause for the recovery of plants from infection.suppression ͉ silencing ͉ RNA-induced silencing complex

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Cultivating crops under saline conditions is of high importance due to global fresh water shortage for irrigation. Crithmum maritimum is a halophytic plant that has a long history of human consumption and was suggested as a cash crop for biosaline agriculture. Our results highlight variations existing among Crithmum maritimum genotypes from different geographic origins regarding salt-induced changes in plant growth, flowering behavior and leaf metabolites with nutritional value. Our results indicate that genotypic characteristics should be taken into account when evaluating wild plant species for future crop cultivation.

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