John P. Carr

Salicylic acid: a likely endogenous signal in the resistance response of tobacco to viral infection

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 PRl 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.

An Antiviral Defense Role of AGO2 in Plants

Background 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. Methodology/Principal Findings 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. Conclusions/Significance 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.

Salicylic Acid Interferes with Tobacco Mosaic Virus Replication via a Novel Salicylhydroxamic Acid-Sensitive Mechanism.

Salicylic acid (SA) induces resistance to all plant pathogens, including bacteria, fungi, and viruses, but the mechanism by which SA engenders resistance to viruses is not known. Pretreatment of tobacco mosaic virus (TMV)-susceptible (nn genotype) tobacco tissue with SA reduced the levels of viral RNAs and viral coat protein accumulating after inoculation with TMV. Viral RNAs were not affected equally, suggesting that SA treatment interferes with TMV replication. Salicylhydroxamic acid (SHAM), an inhibitor of the mitochondrial alternative oxidase, antagonized both SA-induced resistance to TMV in nn genotype plants and SA-induced acquired resistance in resistant (NN genotype) tobacco. SHAM did not inhibit induction of the PR-1 pathogenesis-related protein or induction of resistance to Erwinia carotovora or Botrytis cinerea by SA. This indicates that SA induces resistance to TMV via a novel SHAM-sensitive signal transduction pathway (potentially involving alternative oxidase), which is distinct from that leading to resistance to bacteria and fungi.

Cyanide Restores N Gene–Mediated Resistance to Tobacco Mosaic Virus in Transgenic Tobacco Expressing Salicylic Acid Hydroxylase

Salicylhydroxamic acid (SHAM), an inhibitor of alternative oxidase (AOX), blocks salicylic acid–induced resistance to tobacco mosaic virus (TMV) but does not inhibit pathogenesis-related PR-1 protein synthesis or resistance to fungal and bacterial pathogens. We found that the synthetic resistance-inducing chemical 2,6-dichloroisonicotinic acid also induced Aox transcript accumulation and SHAM-sensitive resistance to TMV. The respiratory inhibitors antimycin A and KCN also induced Aox transcript accumulation and resistance to TMV but did not induce PR-1 accumulation. Tobacco plants of the TMV-resistant cultivar Samsun NN transformed with the salicylic acid hydroxylase (nahG) gene could no longer restrict virus to the inoculation site, resulting in spreading necrosis instead of discrete necrotic lesions. Treatment with KCN restored TMV localization and normal lesion morphology. SHAM antagonized this effect, allowing virus escape and spreading necrosis to resume. The results demonstrate the importance of the SHAM-sensitive (potentially AOX-dependent) signal transduction pathway in mediating virus localization early in the hypersensitive response.

Salicylic acid-induced resistance to viruses and other pathogens: a parting of the ways?

Resistance genes allow plants to recognize specific pathogens. Recognition results in the activation of a variety of defence responses, including localized programmed cell death (the hypersensitive response), synthesis of pathogenesis-related proteins and induction of systemic acquired resistance. These responses are co-ordinated by a branching signal transduction pathway. In tobacco, one branch activates virus resistance, and might require the mitochondrial alternative oxidase to operate. Here we discuss the evidence for this virus-specific branch of the transduction pathway and assess what must be done to further understand virus resistance and the role of the alternative oxidase in its induction.

Salicylic Acid Can Induce Resistance to Plant Virus Movement

Salicylic acid (SA) treatment has recently been reported to inhibit replication of tobacco mosaic virus (TMV) in inoculated tissue. Furthermore, resistance is induced via a novel defensive signal transduction pathway sensitive to inhibition by salicylhydroxamic acid (SHAM; S. Chivasa, A. M. Murphy, M. Naylor, and J. P. Carr, Plant Cell 9: 547–557, 1997). The goals of this study were to determine if replication of viruses other than TMV could be inhibited by SA and, if so, whether the resistance to other viruses could also be prevented by SHAM. Potato virus X (PVX) RNA accumulation in inoculated tobacco leaf tissue was reduced by SA treatment and resistance was dependent on the SHAM-sensitive signaling pathway. However, although symptoms of cucumber mosaic virus (CMV) infection were delayed in SA-treated tobacco plants, this was not due to inhibition of replication but rather to inhibition of systemic movement of the virus. 14CO2-feeding experiments indicated that SA-induced interference with long-distance...

Transcriptional and post-transcriptional regulation of ribulose 1,5-bisphosphate carboxylase gene expression in light- and dark-grown amaranth cotyledons.

The regulation of expression of the genes encoding the large subunit (LSU) and small subunit (SSU) of ribulose 1,5-bisphosphate carboxylase (RuBPCase) was examined in 1- through 8-day-old, dark-grown (etiolated) and light-grown amaranth cotyledons. RuBPCase specific activity in light-grown cotyledons increased during this 8-day period to a level 15-fold higher than in dark-grown cotyledons. Under both growth conditions, the accumulation of the LSU and SSU polypeptides was not coordinated. Initial detection of the SSU occurred 1 and 2 days after the appearance of the LSU in light- and dark-grown cotyledons, respectively. Furthermore, although the levels of the LSU were similar in both light- and dark-grown seedlings, the amount of the SSU followed clearly the changes in enzyme activity. Synthesis of these two polypeptides was dramatically different in etiolated versus light-grown cotyledons. In light the synthesis of both subunits was first observed on day 2 and continued throughout the growth of the cotyledons. In darkness the rate of synthesis of both subunits was much lower than in light and occurred only as a burst between days 2 and 5 after planting. However, mRNAs for both subunits were present in etiolated cotyledons at similar levels on days 4 through 7 (by Northern analysis) and were functional in vitro, despite their apparent inactivity in vivo after day 5. In addition, since both LSU and SSU mRNA levels were lower in dark- than in light-grown seedlings, our results indicate that both transcriptional and post-transcriptional controls modulate RuBPCase production in developing amaranth cotyledons.

Virulence and Differential Local and Systemic Spread of Cucumber mosaic virus in Tobacco are Affected by the CMV 2b Protein

A mutant of the Cucumber mosaic virus subgroup IA strain Fny (Fny-CMV) lacking the gene encoding the 2b protein (Fny-CMVdelta2b) induced a symptomless systemic infection in tobacco. Both the accumulation of Fny-CMVdelta2b in inoculated tissue and the systemic movement of the virus appeared to proceed more slowly than for wild-type Fny-CMV. The influence of the 2b protein on virus movement in the inoculated leaf was examined using viral constructs derived from Fny-CMV and Fny-CMVdelta2b expressing the green fluorescent protein. Laser scanning confocal microscopy was used to visualize the movement of these viruses. Whereas the wild-type virus spread between the epidermal cells as well as the mesophyll cells, the mutant virus spread less efficiently through the epidermal layer and moved preferentially through the mesophyll. Thus, the 2b protein of Fny-CMV influences the dynamics of movement of the virus both within the inoculated leaf and through the whole plant. We propose that this altered movement profile of Fny-CMVdelta2b results in the absence of disease symptoms in tobacco.

Disruption of Two Defensive Signaling Pathways by a Viral RNA Silencing Suppressor

The Cucumber mosaic virus (CMV) 2b counter-defense protein disrupts plant antiviral mechanisms mediated by RNA silencing and salicylic acid (SA). We used microarrays to investigate defensive gene expression in 2b-transgenic Arabidopsis thaliana plants. Surprisingly, 2b inhibited expression of few SA-regulated genes and, in some instances, enhanced the effect of SA on certain genes. Strikingly, the 2b protein inhibited changes in the expression of 90% of genes regulated by jasmonic acid (JA). Consistent with this, infection of plants with CMV, but not the 2b gene-deletion mutant CMVDelta2b, strongly inhibited JA-inducible gene expression. JA levels were unaffected by infection with either CMV or CMVDelta2b. Although the CMV-Arabidopsis interaction is a compatible one, SA accumulation, usually considered to be an indicator of plant resistance, was increased in CMV-infected plants but not in CMVDelta2b-infected plants. Thus, the 2b protein inhibits JA signaling at a step downstream of JA biosynthesis but it primes induction of SA biosynthesis by another CMV gene product or by the process of infection itself. Like many plant viruses, CMV is aphid transmitted. JA is important in plant defense against insects. This raises the possibility that disruption of JA-mediated gene expression by the 2b protein may influence CMV transmission by aphids.

Activation of multiple antiviral defence mechanisms by salicylic acid

SUMMARY The plant signal molecule salicylic acid (SA) can induce resistance to a wide range of pathogen types. In the case of viruses, SA can stimulate the inhibition of all three main stages in virus infection: replication, cell-to-cell movement and long-distance movement. Induction of resistance by SA appears to depend, in part, on downstream signalling via the mitochondrion. However, evidence has recently emerged that SA may stimulate a separate downstream pathway, leading to the induction of an additional mechanism of resistance based on RNA interference. In this review our aims are to document these recent advances and to suggest possible future avenues of research on SA-induced resistance to viruses.