Spreading of RNA targeting and DNA methylation in RNA silencing requires transcription of the target gene and a putative RNA-dependent RNA polymerase

RNA silencing is a sequence-specific RNA degradation process that follows the recognition of double-stranded RNA. Here, we show that virus vectors carrying parts of a green fluorescent protein (GFP) transgene targeted RNA silencing in Nicotiana benthamiana and Arabidopsis against the entire GFP RNA. These results indicate that there was spreading of RNA targeting from the initiator region into the adjacent 5'and 3' regions of the target gene. Spreading was accompanied by methylation of the corresponding GFP DNA. It also was dependent on transcription of the transgene and on the putative RNA-dependent RNA polymerase, SDE1/SGS2. These findings indicate that SDE1/SGS2 produces doublestranded RNA using the target RNA as a template. RNA silencing of ribulose-1,5-bisphosphate carboxylase/oxygenase and phytoene desaturase was not associated with the spreading of RNA targeting or DNA methylation, indicating that these endogenous RNAs are not templates for SDE1/SGS2

Agrobacterium transient expression system as a tool for the isolation of disease resistance genes: application to the Rx2 locus in potato

33 ref.International audienceRx2 confers resistance against potato virus X (PVX). To clone Rx2, we developed a system based on Agrobacterium-mediated transient expression of candidate R genes in transgenic tobacco leaves expressing the PVX coat protein elicitor of Rx2-mediated resistance. Using this system, a potato gene eliciting HR specifically in the presence of the elicitor was identified. Based on genetical and functional analysis, it is concluded that the cloned gene is Rx2. The transient expression system is potentially adaptable to cloning of any other resistance gene. The Rx2 locus is on chromosome V of potato and the encoded protein is highly similar to the products of Rx1 and Rxh1 encoded on potato chromosome XII. Rxh1 has been shown elsewhere to encode a potato cyst nematode resistance gene Gpa2. All three proteins are in the leucine zipper-nucleotide binding site-leucine rich repeat class of resistance gene products. Rx1 and Rx2 are functionally identical and are almost identical in the C terminal region consistent with a role of the leucine rich repeats in recognition of the PVX coat protein. In the N terminal, half there are some regions where the Rx1 and Rx2 proteins are more similar to each other than to the Rxh1 protein. However, in other regions these proteins are more similar to Rxh1 than to each other. Based on this mosaic pattern of sequence similarity, we conclude that sequence exchange occurs repeatedly between genetically unlinked disease resistance genes through a process of gene conversion

Stepwise artificial evolution of a plant disease resistance gene

Genes encoding plant nucleotide-binding leucine-rich repeat (NB-LRR) proteins confer dominant resistance to diverse pathogens. The wild-type potato NB-LRR protein Rx confers resistance against a single strain of potato virus X (PVX), whereas LRR mutants protect against both a second PVX strain and the distantly related poplar mosaic virus (PopMV). In one of the Rx mutants there was a cost to the broad-spectrum resistance because the response to PopMV was transformed from a mild disease on plants carrying wild-type Rx to a trailing necrosis that killed the plant. To explore the use of secondary mutagenesis to eliminate this cost of broad-spectrum resistance, we performed random mutagenesis of the N-terminal domains of this broad-recognition version of Rx and isolated four mutants with a stronger response against the PopMV coat protein due to enhanced activation sensitivity. These mutations are located close to the nucleotide-binding pocket, a highly conserved structure that likely controls the “switch” between active and inactive NB-LRR conformations. Stable transgenic plants expressing one of these versions of Rx are resistant to the strains of PVX and the PopMV that previously caused trailing necrosis. We conclude from this work that artificial evolution of NB-LRR disease resistance genes in crops can be enhanced by modification of both activation and recognition phases, to both accentuate the positive and eliminate the negative aspects of disease resistance

An RNA-dependent RNA Polymerase prevents meristem invasion by potato virus X and is required for the activity but not the production of a systemic silencing signal

One of the functions of RNA silencing in plants is antiviral defense. A hallmark of RNA silencing is spreading of the silenced state through the plant. Little is known about the nature of the systemic silencing signal and the proteins required for its production, transport, and reception in plant tissues. Here, we show that the RNA-dependent RNA polymerase RDR6 in Nicotiana benthamiana is involved in defense against potato virus X at the level of systemic spreading and in exclusion of the virus from the apical growing point. It has no effect on primary replication and cell-to-cell movement of the virus and does not contribute significantly to the formation of virus-derived small interfering (si) RNA in a fully established potato virus X infection. In grafting experiments, the RDR6 homolog was required for the ability of a cell to respond to, but not to produce or translocate, the systemic silencing signal. Taking these findings together, we suggest a model of virus defense in which RDR6 uses incoming silencing signal to generate double-stranded RNA precursors of secondary siRNA. According to this idea, the secondary siRNAs mediate RNA silencing as an immediate response that slows down the systemic spreading of the virus into the growing point and newly emerging leaves