BackgroundArgonaute (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 FindingsTo 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/SignificanceBased 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 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.

BackgroundVirus-induced deterrence to aphid feeding is believed to promote plant virus transmission by encouraging migration of virus-bearing insects away from infected plants. We investigated the effects of infection by an aphid-transmitted virus, cucumber mosaic virus (CMV), on the interaction of Arabidopsis thaliana, one of the natural hosts for CMV, with Myzus persicae (common names: ‘peach-potato aphid’, ‘green peach aphid’). Methodology/Principal FindingsInfection of Arabidopsis (ecotype Col-0) with CMV strain Fny (Fny-CMV) induced biosynthesis of the aphid feeding-deterrent 4-methoxy-indol-3-yl-methylglucosinolate (4MI3M). 4MI3M inhibited phloem ingestion by aphids and consequently discouraged aphid settling. The CMV 2b protein is a suppressor of antiviral RNA silencing, which has previously been implicated in altering plant-aphid interactions. Its presence in infected hosts enhances the accumulation of CMV and the other four viral proteins. Another viral gene product, the 2a protein (an RNA-dependent RNA polymerase), triggers defensive signaling, leading to increased 4MI3M accumulation. The 2b protein can inhibit ARGONAUTE1 (AGO1), a host factor that both positively-regulates 4MI3M biosynthesis and negatively-regulates accumulation of substance(s) toxic to aphids. However, the 1a replicase protein moderated 2b-mediated inhibition of AGO1, ensuring that aphids were deterred from feeding but not poisoned. The LS strain of CMV did not induce feeding deterrence in Arabidopsis ecotype Col-0. Conclusions/SignificanceInhibition of AGO1 by the 2b protein could act as a booby trap since this will trigger antibiosis against aphids. However, for Fny-CMV the interplay of three viral proteins (1a, 2a and 2b) appears to balance the need of the virus to inhibit antiviral silencing, while inducing a mild resistance (antixenosis) that is thought to promote transmission. The strain-specific effects of CMV on Arabidopsis-aphid interactions, and differences between the effects of Fny-CMV on this plant and those seen previously in tobacco (inhibition of resistance to aphids) may have important epidemiological consequences.

The cucumber mosaic virus (CMV) 2b protein not only inhibits anti-viral RNA silencing but also quenches transcriptional responses of plant genes to jasmonic acid, a key signalling molecule in defence against insects. This suggested that it might affect interactions between infected plants and aphids, insects that transmit CMV. We found that infection of tobacco with a 2b gene deletion mutant (CMVΔ2b) induced strong resistance to aphids (Myzus persicae) while CMV infection fostered aphid survival. Using electrical penetration graph methodology we found that higher proportions of aphids showed sustained phloem ingestion on CMV-infected plants than on CMVΔ2b-infected or mock-inoculated plants although this did not increase the rate of growth of individual aphids. This indicates that while CMV infection or certain viral gene products might elicit aphid resistance, the 2b protein normally counteracts this during a wild-type CMV infection. Our findings suggest that the 2b protein could indirectly affect aphid-mediated virus transmission.

Plant volatiles play important roles in attraction of certain pollinators and in host location by herbivorous insects. Virus infection induces changes in plant volatile emission profiles, and this can make plants more attractive to insect herbivores, such as aphids, that act as viral vectors. However, it is unknown if virus-induced alterations in volatile production affect plant-pollinator interactions. We found that volatiles emitted by cucumber mosaic virus (CMV)-infected tomato (Solanum lycopersicum) and Arabidopsis thaliana plants altered the foraging behaviour of bumblebees (Bombus terrestris). Virus-induced quantitative and qualitative changes in blends of volatile organic compounds emitted by tomato plants were identified by gas chromatography-coupled mass spectrometry. Experiments with a CMV mutant unable to express the 2b RNA silencing suppressor protein and with Arabidopsis silencing mutants implicate microRNAs in regulating emission of pollinator-perceivable volatiles. In tomato, CMV infection made plants emit volatiles attractive to bumblebees. Bumblebees pollinate tomato by ‘buzzing’ (sonicating) the flowers, which releases pollen and enhances self-fertilization and seed production as well as pollen export. Without buzz-pollination, CMV infection decreased seed yield, but when flowers of mock-inoculated and CMV-infected plants were buzz-pollinated, the increased seed yield for CMV-infected plants was similar to that for mock-inoculated plants. Increased pollinator preference can potentially increase plant reproductive success in two ways: i) as female parents, by increasing the probability that ovules are fertilized; ii) as male parents, by increasing pollen export. Mathematical modeling suggested that over a wide range of conditions in the wild, these increases to the number of offspring of infected susceptible plants resulting from increased pollinator preference could outweigh underlying strong selection pressures favoring pathogen resistance, allowing genes for disease susceptibility to persist in plant populations. We speculate that enhanced pollinator service for infected individuals in wild plant populations might provide mutual benefits to the virus and its susceptible hosts.