Involvement of cathepsin B in the plant disease resistance hypersensitive response.

A diverse range of plant proteases are implicated in pathogen perception and in subsequent signalling and execution of disease resistance. We demonstrate, using protease inhibitors and virus-induced gene silencing (VIGS), that the plant papain cysteine protease cathepsin B is required for the disease resistance hypersensitive response (HR). VIGS of cathepsin B prevented programmed cell death (PCD) and compromised disease resistance induced by two distinct non-host bacterial pathogens. It also suppressed the HR triggered by transient co-expression of potato R3a and Phytophthora infestans Avr3a genes. However, VIGS of cathepsin B did not compromise HR following recognition of Cladosporium fulvum AVR4 by tomato Cf-4, indicating that plant PCD can be independent of cathepsin B. The non-host HR to Erwinia amylovora was accompanied by a transient increase in cathepsin B transcript level and enzymatic activity and induction of the HR marker gene Hsr203. VIGS of cathepsin B significantly reduced the induction of Hsr203 following E. amylovora challenge, further demonstrating a role for this protease in PCD. Whereas cathepsin B is often relocalized from the lysosome to the cytosol during animal PCD, plant cathepsin B is secreted into the apoplast, and is activated upon secretion in the absence of pathogen challenge.

Virus-induced gene silencing-based functional characterization of genes associated with powdery mildew resistance in barley.

We successfully implemented virus-induced gene silencing (VIGS) in barley (Hordeum vulgare) for the functional characterization of genes required for Mla13-mediated resistance toward the biotrophic barley pathogen Blumeria graminis f. sp. hordei. Initially, barley cultivars were screened for their ability to host the barley stripe mosaic virus (BSMV)-VIGS vector by allowing its replication and systemic movement without causing excessive symptoms. Phytoene desaturase silencing leading to photobleaching was used as a phenotypic marker alongside reverse transcription-PCR data to characterize the silencing response at the molecular level. Barley cultivar Clansman, harboring the Mla13 resistance gene, was chosen as the most suitable host for BSMV-VIGS-based functional characterization of Rar1, Sgt1, and Hsp90 in the Mla-mediated resistance toward powdery mildew. BSMV-induced gene silencing of these candidate genes, which are associated in many but not all race-specific pathways, proved to be robust and could be detected at both mRNA and protein levels for up to 21 d postinoculation. Systemic silencing was observed not only in the newly developed leaves from the main stem but also in axillary shoots. By examining fungal development from an incompatible mildew strain carrying the cognate Avr13 gene on plants BSMV silenced for Rar1, Sgt1, and Hsp90, a resistance-breaking phenotype was observed, while plants infected with BSMV control constructs remained resistant. We demonstrate that Hsp90 is a required component for Mla13-mediated race-specific resistance and that BSMV-induced VIGS is a powerful tool to characterize genes involved in pathogen resistance in barley.

Potato virus X-induced gene silencing in leaves and tubers of potato.

Virus induced gene silencing (VIGS) is increasingly used to generate transient loss-of-function assays and has potential as a powerful reverse-genetics tool in functional genomic programs as a more rapid alternative to stable transformation. A previously described potato virus X (PVX) VIGS vector has been shown to trigger silencing in the permissive host Nicotiana benthamiana. This paper demonstrates that a PVX-based VIGS vector is also effective in triggering a VIGS response in both diploid and cultivated tetraploid Solanum species. We show that systemic silencing of a phytoene desaturase gene is observed and maintained throughout the foliar tissues of potato plants and was also observed in tubers. Here we report that VIGS can be triggered and sustained on in vitro micropropagated tetraploid potato for several cycles and on in vitro generated microtubers. This approach will facilitate large-scale functional analysis of potato expressed sequence tags and provide a noninvasive reverse-genetic approach to study mechanisms involved in tuber and microtuber development.

Enhancement of virus-induced gene silencing through viral-based production of inverted-repeats.

Plant virus-based vectors carrying sequences homologous to endogenous genes trigger silencing through a homology-dependent RNA degradation mechanism. This phenomenon, called virus-induced gene silencing (VIGS), has potential as a powerful reverse-genetics tool in functional genomic programmes through transient, loss-of-function screens. Here, we describe a method to enhance the robustness of the VIGS phenotype by increasing the level of dsRNA molecule production, a critical step in the VIGS response. Incorporation of 40-60 base direct inverted-repeats into a plant viral vector generates RNA molecules that form dsRNA hairpins. A tobacco mosaic virus (TMV)-based vector carrying such inverted-repeats, homologous to a green fluorescent protein (gfp) transgene or an endogenous phytoene desaturase (pds) gene, generated a stronger and more pervasive VIGS phenotype than constructs carrying corresponding cDNA fragments in sense or antisense orientation. Real-time RT-PCR indicated that there was up to a threefold reduction in target mRNA accumulation in the tissues where VIGS was triggered by constructs carrying inverted-repeats compared to those where it was triggered by sense or antisense constructs. Moreover, an enhanced VIGS pds phenotype was observed using a different vector, based on barley stripe mosaic virus, in the monocotyledonous host barley. This demonstrates that VIGS can be significantly improved through the inclusion of small inverted-repeats in plant virus-based vectors, generating a more robust loss-of-function phenotype. This suggests that dsRNA formation can be a limiting factor in the VIGS phenomenon.