Some cultivars of tobacco are resistant to tobacco mosaic virus (TMV) and synthesize pathogenesis-related (PR) proteins upon infection. In a search for the signal or signals that induce resistance or PR genes, it was found that the endogenous salicylic acid levels in resistant, but not susceptible, cultivars increased at least 20-fold in infected leaves and 5-fold in uninfected leaves after TMV inoculation. Induction of PR1 genes paralleled the rise in salicylic acid levels. Since earlier work has demonstrated that treatment with exogenous salicylic acid induces PR genes and resistance, these findings suggest that salicylic acid functions as the natural transduction signal.

Summary: Leaf senescence is a complex process that is controlled by multiple developmental and environmental signals and is manifested by induced expression of a large number of different genes. In this paper we describe experiments that show, for the first time, that the salicylic acid (SA)‐signalling pathway has a role in the control of gene expression during developmental senescence. Arabidopsis plants defective in the SA‐signalling pathway (npr1 and pad4 mutants and NahG transgenic plants) were used to investigate senescence‐enhanced gene expression, and a number of genes showed altered expression patterns. Senescence‐induced expression of the cysteine protease gene SAG12, for example, was conditional on the presence of SA, together with another unidentified senescence‐specific factor. Changes in gene expression patterns were accompanied by a delayed yellowing and reduced necrosis in the mutant plants defective in SA‐signalling, suggesting a role for SA in the cell death that occurs at the final stage of senescence. We propose the presence of a minimum of three senescence‐enhanced signalling factors in senescing leaves, one of which is SA. We also suggest that a combination of signalling factors is required for the optimum expression of many genes during senescence.

Argonaute (AGO) proteins bind to small-interfering (si)RNAs and micro (mi)RNAs to target RNA silencing against viruses, transgenes and in regulation of mRNAs. Plants encode multiple AGO proteins but, in Arabidopsis, only AGO1 is known to have an antiviral role. To uncover the roles of specific AGOs in limiting virus accumulation we inoculated turnip crinkle virus (TCV) to Arabidopsis plants that were mutant for each of the ten AGO genes. The viral symptoms on most of the plants were the same as on wild type plants although the ago2 mutants were markedly hyper-susceptible to this virus. ago2 plants were also hyper-susceptible to cucumber mosaic virus (CMV), confirming that the antiviral role of AGO2 is not specific to a single virus. For both viruses, this phenotype was associated with transient increase in virus accumulation. In wild type plants the AGO2 protein was induced by TCV and CMV infection. Based on these results we propose that there are multiple layers to RNA-mediated defense and counter-defense in the interactions between plants and their viruses. AGO1 represents a first layer. With some viruses, including TCV and CMV, this layer is overcome by viral suppressors of silencing that can target AGO1 and a second layer involving AGO2 limits virus accumulation. The second layer is activated when the first layer is suppressed because AGO2 is repressed by AGO1 via miR403. The activation of the second layer is therefore a direct consequence of the loss of the first layer of defense.

The family Potyviridae encompasses ~30% of plant viruses and is responsible for significant economic losses worldwide. Recently, a small overlapping coding sequence, termed pipo , was found to be conserved in the genomes of all potyvirids. PIPO is expressed as part of a frameshift protein, P3N‐PIPO, which is essential for virus cell‐to‐cell movement. However, the frameshift expression mechanism has hitherto remained unknown. Here, we demonstrate that transcriptional slippage, specific to the viral RNA polymerase, results in a population of transcripts with an additional “A” inserted within a highly conserved GAAAAAA sequence, thus enabling expression of P3N‐PIPO. The slippage efficiency is ~2% in Turnip mosaic virus and slippage is inhibited by mutations in the GAAAAAA sequence. While utilization of transcriptional slippage is well known in negative‐sense RNA viruses such as Ebola , mumps and measles, to our knowledge this is the first report of its widespread utilization for gene expression in positive‐sense RNA viruses.

Plant viruses are difficult to control, and they decrease both the quality and yield of crops, thus threatening global food security. A new approach that uses topical application of double-stranded RNA (dsRNA) to induce antiviral RNA-interference has been shown to be effective at preventing virus infection in a range of plants following mechanical inoculation. In this study, topical application of dsRNA was effective against mechanical inoculation and aphid-mediated inoculation with the potyvirus bean common mosaic virus (BCMV). Topical application of dsRNAs targeting either the coding region of the potyviral nuclear inclusion b (NIb) protein (BCMVNIb-dsRNA) or the coat protein (CP) coding region (BCMVCP-dsRNA) protected Nicotiana benthamiana and cowpea (Vigna unguiculata) plants against mechanical inoculation with BCMV. BCMVCP-dsRNA was selected for subsequent aphid transmission experiments. BCMVCP-dsRNA was loaded onto layered double hydroxide nanoparticles to form BCMVCP-BioClay which is a more stable formulation for delivering dsRNA than naked dsRNA. BCMVCP-BioClay was shown to be successful in protecting plants against BCMV transmission by the aphid Myzus persicae. Spraying detached N. benthamiana leaves with BCMVCP-BioClay 5 days prior to exposure to viruliferous aphids protected the leaves from infection by BCMV. Importantly, spraying of intact N. benthamiana and cowpea plants with BCMVCP-BioClay 5 days prior to exposure to viruliferous aphids protected plants of both species from BCMV infection. This study demonstrates that topical application of dsRNA using BioClay protects plants from aphid-mediated virus transmission, which is an important first step toward developing practical application of this approach in crop protection.

The RNA silencing suppressor activity of the 2b protein of Cucumber mosaic virus (CMV) has been variously attributed to its nuclear targeting, its interaction with and inhibition of Argonaute 1 (AGO1), or its ability to bind small RNAs in vitro. In addition, the 2b ortholog of Tomato aspermy virus forms aggregates and binds RNAs in vitro. We have further studied the relationships between CMV 2b protein silencing suppressor activity and its subcellular distribution, protein-protein interactions in vivo, and interactions with small interfering RNAs in vitro. To do this, we tagged the protein with fluorescent markers and showed that it retained suppressor activity. We showed that the 2b protein is present in the nucleolus and that it self-interacts and interacts with AGO1 and AGO4 in vivo. Using a battery of mutants, we showed that the putative nuclear localization signals and phosphorylation motif of the 2b protein are not required for self-interaction or for interaction with AGO proteins. The occurrence of neither of these interactions or of nucleolar targeting was sufficient to provide local silencing-suppression activity. In contrast, the ability of the 2b protein to bind small RNAs appears to be indispensable for silencing suppressor function.