DRD1-Pol V-dependent self-silencing of an exogenous silencer restricts the non-cell autonomous silencing of an endogenous target gene.

In plants, the exogenous transgene transcribing inverted-repeat (exo-IR) sequences produces double-stranded RNAs that are processed by DCL4. The 21-nt small interfering RNAs generated function as mobile signals to trigger non-cell autonomous silencing of target endogenes in the neighboring 10-15 cells. The potential involvement of nuclear silencing pathway components in signal spreading or sensing in target cells is not clear. Here, we demonstrate that the exo-IR silencer (exo-Pdsi) is negatively autoregulated through methylation spreading, which acts in cis to reinforce the self-silencing of the silencer. Mutations affecting nuclear proteins DRD1 and Pol V (NRPE1 or NRPD2) relieved exo-Pdsi self-silencing, resulting in higher levels of Pdsi transcripts, which increased the non-cell autonomous silencing of endo-PDS. Our results suggest that in an experimental silencing pathway, methylation spreading on a silencer transgene may not have a direct endogenous plant counterpart when the protein-encoding gene is the target. DRD1-Pol V-dependent de novo methylation, by acting in cis to reinforce self-silencing of exo-IR, may play a role in restraining the inappropriate silencing of active protein-coding genes in plants.

RNA-dependent RNA polymerase 1 from Nicotiana tabacum suppresses RNA silencing and enhances viral infection in Nicotiana benthamiana.

Endogenous eukaryotic RNA-dependent RNA polymerases (RDRs) produce double-stranded RNA intermediates in diverse processes of small RNA synthesis in RNA silencing pathways. RDR6 is required in plants for posttranscriptional gene silencing induced by sense transgenes (S-PTGS) and has an important role in amplification of antiviral silencing. Whereas RDR1 is also involved in antiviral defense in plants, this does not necessarily proceed through triggering silencing. In this study, we show that Nicotiana benthamiana transformed with RDR1 from Nicotiana tabacum (Nt-RDR1 plants) exhibits hypersusceptibility to Plum pox potyvirus and other viruses, resembling RDR6-silenced (RDR6i) N. benthamiana. Analysis of transient induction of RNA silencing in N. benthamiana Nt-RDR1 and RDR6i plants revealed that Nt-RDR1 possesses silencing suppression activity. We found that Nt-RDR1 does not interfere with RDR6-dependent siRNA accumulation but turns out to suppress RDR6-dependent S-PTGS. Our results, together with previously published data, suggest that RDR1 might have a dual role, contributing, on one hand, to salicylic acid-mediated antiviral defense, and suppressing, on the other hand, the RDR6-mediated antiviral RNA silencing. We propose a scenario in which the natural loss-of-function variant of RDR1 in N. benthamiana may be the outcome of selective pressure to maintain a high RDR6-dependent antiviral defense, which would be required to face the hypersensitivity of this plant to a large number of viruses.

Identification of a movement protein of rice yellow stunt rhabdovirus.

Rice yellow stunt rhabdovirus (RYSV) encodes seven genes in its negative-sense RNA genome in the order 3'-N-P-3-M-G-6-L-5'. The existence of gene 3 in the RYSV genome and an analogous gene(s) of other plant rhabdoviruses positioned between the P and M genes constitutes a unique feature for plant rhabdoviruses that is distinct from animal-infecting rhabdoviruses in which the P and M genes are directly linked. However, little is known about the function of these extra plant rhabdovirus genes. Here we provide evidence showing that the protein product encoded by gene 3 of RYSV, P3, possesses several properties related to a viral cell-to-cell movement protein (MP). Analyses of the primary and secondary protein structures suggested that RYSV P3 is a member of the "30K" superfamily of viral MPs. Biolistic bombardment transcomplementation experiments demonstrated that RYSV P3 can support the intercellular movement of a movement-deficient potexvirus mutant in Nicotiana benthamiana leaves. In addition, Northwestern blot analysis indicated that the RYSV P3 protein can bind single-stranded RNA in vitro, a common feature of viral MPs. Finally, glutathione S- transferase pull-down assays revealed a specific interaction between the RYSV P3 protein and the N protein which is a main component of the ribonucleocapsid, a subviral structure believed to be involved in the intercellular movement of plant rhabdoviruses. Together, these data suggest that RYSV P3 is likely a MP of RYSV, thus representing the first example of characterized MPs for plant rhabdoviruses.