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In plants there are several classes of 21–24-nt short RNAs that regulate gene expression. The most conserved class is the microRNAs (miRNAs), although some miRNAs are found only in specific species. We used high-throughput pyrosequencing to identify conserved and nonconserved miRNAs and other short RNAs in tomato fruit and leaf. Several conserved miRNAs showed tissue-specific expression, which, combined with target gene validation results, suggests that miRNAs may play a role in fleshy fruit development. We also identified four new nonconserved miRNAs. One of the validated targets of a novel miRNA is a member of the CTR family involved in fruit ripening. However, 62 predicted targets showing near perfect complementarity to potential new miRNAs did not validate experimentally. This suggests that target prediction of plant short RNAs could have a high false-positive rate and must therefore be validated experimentally. We also found short RNAs from a Solanaceae-specific foldback transposon, which showed a miRNA/miRNA*-like distribution, suggesting that this element may function as a miRNA gene progenitor. The other Solanaceae-specific class of short RNA was derived from an endogenous pararetrovirus sequence inserted into the tomato chromosomes. This study opens a new avenue in the field of fleshy fruit biology by raising the possibility that fruit development and ripening may be under miRNA regulation.

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RNA silencing is an evolutionarily conserved surveillance system that occurs in a broad range of eukaryotic organisms. In plants, RNA silencing acts as an antiviral system; thus, successful virus infection requires suppression of gene silencing. A number of viral suppressors have been identified so far; however, the molecular bases of silencing suppression are still poorly understood. Here we show that p19 of Cymbidium ringspot virus (CymRSV) inhibits RNA silencing via its small RNA‐binding activity in vivo . Small RNAs bound by p19 in planta are bona fide double‐stranded siRNAs and they are silencing competent in the in vitro RNA‐silencing system. p19 also suppresses RNA silencing in the heterologous Drosophila in vitro system by preventing siRNA incorporation into RISC. During CymRSV infection, p19 markedly diminishes the amount of free siRNA in cells by forming p19–siRNA complexes, thus making siRNAs inaccessible for effector complexes of RNA‐silencing machinery. Furthermore, the obtained results also suggest that the p19‐mediated sequestration of siRNAs in virus‐infected cells blocks the spread of the mobile, systemic signal of RNA silencing.

Background: High-throughput sequencing technology is capable to identify novel short RNAs in plant species. We used Solexa sequencing to find new microRNAs in one of the model legume species, barrel medic (Medicago truncatula).Results: 3,948,871 reads were obtained from two separate short RNA libraries generated from total RNA extracted fromM. truncatulaleaves, representing 1,563,959 distinct sequences. 2,168,937 reads were mapped to the availableM. truncatulagenome corresponding to 619,175 distinct sequences. 174,504 reads representing 25 conserved miRNA families showed perfect matches to known miRNAs. We also identified 26 novel miRNA candidates that were potentially generated from 32 loci. Nine of these loci produced eight distinct sequences, for which the miRNA* sequences were also sequenced. These sequences were not described in other plant species and accumulation of these eight novel miRNAs was confirmed by Northern blot analysis. Potential target genes were predicted for most conserved and novel miRNAs.Conclusion: Deep sequencing of short RNAs fromM. truncatulaleaves identified eight new miRNAs indicating that specific miRNAs exist in legume species.