No abstract

Arabidopsis thaliana encodes four Dicer-like (DCL) proteins and five dsRNA-binding (DRB) proteins. We have previously demonstrated that DCL4 specifically interacts with DRB4 in vitro. Here we describe the interaction between DCL4 and DRB4 in vivo. The phenotype of a mutant with a defect in DCL4 (dcl4-2) was similar to that of a mutant with a defect in DRB4 (drb4-1): both mutant plants had elongated and downwardly curled rosette leaves and over-accumulated anthocyanin. In immunoprecipitation experiments with either anti-DCL4 or anti-DRB4 antibody and crude extracts of wild-type Arabidopsis plants, co-immunoprecipitation of DCL4 and DRB4 was detected, indicating that DCL4 interacts with DRB4 in vivo. This interaction was confirmed by immunoprecipitation experiments using extracts from dcl4-2, drb4-1, or transgenic plants expressing the hemagglutinin-tagged version of DCL4 or DRB4. The results of immunoprecipitation experiments also suggest that most DCL4 is associated with DRB4, but that some DRB4 is free or associated with other proteins. Reduced accumulation of the TAS1 and TAS3 trans-acting siRNA (ta-siRNA) and over accumulation of their target mRNAs (At5g18040 and auxin response factors ARF3 and ARF4) were detected in both drb4-1 and dcl4-2 mutants. These results indicate that DRB4, together with DCL4, functions in the ta-siRNA biogenesis.

In Arabidopsis thaliana, Dicer-like 3 (DCL3) and Dicer-like 4 (DCL4) cleave long, perfect double-stranded RNAs (dsRNAs) into 24 and 21 nucleotides (nt) small interfering RNAs, respectively, which in turn function in RNA-directed DNA methylation and RNA interference, respectively. To reveal how DCL3 and DCL4 individually recognize long perfect dsRNAs as substrates, we biochemically characterized DCL3 and DCL4 and compared their enzymatic properties. DCL3 preferentially cleaves short dsRNAs with 5′ phosphorylated adenosine or uridine and a 1 nt 3′ overhang, whereas DCL4 cleaves long dsRNAs with blunt ends or with a 1 or 2 nt 3′ overhang with similar efficiency. DCL3 produces 24 nt RNA duplexes with 2 nt 3′ overhangs by the 5′ counting rule. Inorganic phosphate, NaCl and KCl enhance DCL3 activity but inhibit DCL4 activity. These results indicate that plants use DCLs with distinct catalytic profiles to ensure each dsRNA substrate generates only a specific length of siRNAs that trigger a unique siRNA-mediated response.

Bell peppers (Capsicum annuum) harbour a large dsRNA virus. The linear genome (14.7 kbp) of two isolates from Japanese and USA bell pepper cultivars were completely sequenced and compared. They shared extensive sequence identity and contained a single, long ORF encoding a 4815 aa protein. This polyprotein contained conserved motifs of putative viral methyltransferase (MTR), helicase 1 (Hel-1), UDP-glycosyltransferase and RNA-dependent RNA polymerase. This unique arrangement of conserved domains has not been reported in any of the known endornaviruses. Hence this virus, for which the name Bell pepper endornavirus (BPEV) is proposed, is a distinct species in the genus Endornavirus (family Endornaviridae). The BPEVencoded polyprotein contains a cysteine-rich region between the MTR and Hel-1 domains, with conserved CXCC motifs shared among several endornaviruses, suggesting an additional functional domain. In agreement with general endornavirus features, BPEV contains a nick in the positive-strand RNA molecule. The virus was detected in all bell pepper cultivars tested and transmitted through seed but not by graft inoculations. Analysis of dsRNA patterns and RT-PCR using degenerate primers revealed putative variants of BPEV, or closely related species, infecting other C. annuum genotypes and three other Capsicum species (C. baccatum, C. chinense and C. frutescens).