Reduction in vertical transmission rate of bean common mosaic virus in bee-pollinated common bean plants.

Vertical transmission, the transfer of pathogens across generations, is a critical mechanism for the persistence of plant viruses. The transmission mechanisms are diverse, involving direct invasion through the suspensor and virus entry into developing gametes before achieving symplastic isolation. Despite the progress in understanding vertical virus transmission, the environmental factors influencing this process remain largely unexplored. We investigated the complex interplay between vertical transmission of plant viruses and pollination dynamics, focusing on common bean (Phaseolus vulgaris). The intricate relationship between plants and pollinators, especially bees, is essential for global ecosystems and crop productivity. We explored the impact of virus infection on seed transmission rates, with a particular emphasis on bean common mosaic virus (BCMV), bean common mosaic necrosis virus (BCMNV), and cucumber mosaic virus (CMV). Under controlled growth conditions, BCMNV exhibited the highest seed transmission rate, followed by BCMV and CMV. Notably, in the field, bee-pollinated BCMV-infected plants showed a reduced transmission rate compared to self-pollinated plants. This highlights the influence of pollinators on virus transmission dynamics. The findings demonstrate the virus-specific nature of seed transmission and underscore the importance of considering environmental factors, such as pollination, in understanding and managing plant virus spread.

Investigating the interactions of endornaviruses with each other and with other viruses in common bean, Phaseolus vulgaris.

BACKGROUND: Plant viruses of the genus Alphaendornavirus are transmitted solely via seed and pollen and generally cause no apparent disease. It has been conjectured that certain plant endornaviruses may confer advantages on their hosts through improved performance (e.g., seed yield) or resilience to abiotic or biotic insult. We recently characterised nine common bean (Phaseolus vulgaris L.) varieties that harboured either Phaseolus vulgaris endornavirus (PvEV1) alone, or PvEV1 in combination with PvEV2 or PvEV1 in combination with PvEV2 and PvEV3. Here, we investigated the interactions of these endornaviruses with each other, and with three infectious pathogenic viruses: cucumber mosaic virus (CMV), bean common mosaic virus (BCMV), and bean common mosaic necrosis virus (BCMNV). RESULTS: In lines harbouring PvEV1, PvEV1 and PvEV2, or PvEV1, PvEV2 plus PvEV3, the levels of PvEV1 and PvEV3 RNA were very similar between lines, although there were variations in PvEV2 RNA accumulation. In plants inoculated with infectious viruses, CMV, BCMV and BCMNV levels varied between lines, but this was most likely due to host genotype differences rather than to the presence or absence of endornaviruses. We tested the effects of endornaviruses on seed production and seedborne transmission of infectious pathogenic viruses but found no consistent relationship between the presence of endornaviruses and seed yield or protection from seedborne transmission of infectious pathogenic viruses. CONCLUSIONS: It was concluded that endornaviruses do not interfere with each other's accumulation. There appears to be no direct synergy or competition between infectious pathogenic viruses and endornaviruses, however, the effects of host genotype may obscure interactions between endornaviruses and infectious viruses. There is no consistent effect of endornaviruses on seed yield or susceptibility to seedborne transmission of other viruses.

Identification and characterization of Phaseolus vulgaris endornavirus 1, 2 and 3 in common bean cultivars of East Africa.

Persistent viruses include members of the family Endornavirus that cause no apparent disease and are transmitted exclusively via seed or pollen. It is speculated that these RNA viruses may be mutualists that enhance plant resilience to biotic and abiotic stresses. Using reverse transcription coupled polymerase chain reactions, we investigated if common bean (Phaseolus vulgaris L.) varieties popular in east Africa were hosts for Phaseolus vulgaris endornavirus (PvEV) 1, 2 or 3. Out of 26 bean varieties examined, four were infected with PvEV1, three were infected with both PvEV1 and PvEV2 and three had infections of all three (PvEV) 1, 2 and 3. Notably, this was the first identification of PvEV3 in common bean from Africa. Using high-throughput sequencing of two east African bean varieties (KK022 and KK072), we confirmed the presence of these viruses and generated their genomes. Intra- and inter-species sequence comparisons of these genomes with comparator sequences from GenBank revealed clear species demarcation. In addition, phylogenetic analyses based on sequences generated from the helicase domains showed that geographical distribution does not correlate to genetic relatedness or the occurrence of endornaviruses. These findings are an important first step towards future investigations to determine if these viruses engender positive effects in common bean, a vital crop in east Africa.

The cucumber mosaic virus 1a protein regulates interactions between the 2b protein and ARGONAUTE 1 while maintaining the silencing suppressor activity of the 2b protein.

The cucumber mosaic virus (CMV) 2b viral suppressor of RNA silencing (VSR) is a potent counter-defense and pathogenicity factor that inhibits antiviral silencing by titration of short double-stranded RNAs. It also disrupts microRNA-mediated regulation of host gene expression by binding ARGONAUTE 1 (AGO1). But in Arabidopsis thaliana complete inhibition of AGO1 is counterproductive to CMV since this triggers another layer of antiviral silencing mediated by AGO2, de-represses strong resistance against aphids (the insect vectors of CMV), and exacerbates symptoms. Using confocal laser scanning microscopy, bimolecular fluorescence complementation, and co-immunoprecipitation assays we found that the CMV 1a protein, a component of the viral replicase complex, regulates the 2b-AGO1 interaction. By binding 2b protein molecules and sequestering them in P-bodies, the 1a protein limits the proportion of 2b protein molecules available to bind AGO1, which ameliorates 2b-induced disease symptoms, and moderates induction of resistance to CMV and to its aphid vector. However, the 1a protein-2b protein interaction does not inhibit the ability of the 2b protein to inhibit silencing of reporter gene expression in agroinfiltration assays. The interaction between the CMV 1a and 2b proteins represents a novel regulatory system in which specific functions of a VSR are selectively modulated by another viral protein. The finding also provides a mechanism that explains how CMV, and possibly other viruses, modulates symptom induction and manipulates host-vector interactions.

Viral Perturbation of Alternative Splicing of a Host Transcript Benefits Infection.

Pathogens disturb alternative splicing patterns of infected eukaryotic hosts. However, in plants it is unknown if this is incidental to infection or represents a pathogen-induced remodeling of host gene expression needed to support infection. Here, we compared changes in transcription and protein accumulation with changes in transcript splicing patterns in maize (Zea mays) infected with the globally important pathogen sugarcane mosaic virus (SCMV). Our results suggested that changes in alternative splicing play a major role in determining virus-induced proteomic changes. Focusing on maize phytoene synthase1 (ZmPSY1), which encodes the key regulatory enzyme in carotenoid biosynthesis, we found that although SCMV infection decreases total ZmPSY1 transcript accumulation, the proportion of splice variant T001 increases by later infection stages so that ZmPSY1 protein levels are maintained. We determined that ZmPSY1 has two leaf-specific transcripts, T001 and T003, distinguished by differences between the respective 3'-untranslated regions (UTRs). The shorter 3'-UTR of T001 makes it the more efficient mRNA. Nonsense ZmPSY1 mutants or virus-induced silencing of ZmPSY1 expression suppressed SCMV accumulation, attenuated symptoms, and decreased chloroplast damage. Thus, ZmPSY1 acts as a proviral host factor that is required for virus accumulation and pathogenesis. Taken together, our findings reveal that SCMV infection-modulated alternative splicing ensures that ZmPSY1 synthesis is sustained during infection, which supports efficient virus infection.

Virus Infection of Plants Alters Pollinator Preference: A Payback for Susceptible Hosts?

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.

The biochemical properties of the two Arabidopsis thaliana isochorismate synthases.

The important plant hormone salicylic acid (SA; 2-hydroxybenzoic acid) regulates several key plant responses including, most notably, defence against pathogens. A key enzyme for SA biosynthesis is isochorismate synthase (ICS), which converts chorismate into isochorismate, and for which there are two genes in Arabidopsis thaliana One (AtICS1) has been shown to be required for increased SA biosynthesis in response to pathogens and its expression can be stimulated throughout the leaf by virus infection and exogenous SA. The other (AtICS2) appears to be expressed constitutively, predominantly in the plant vasculature. Here, we characterise the enzymatic activity of both isozymes expressed as hexahistidine fusion proteins in Escherichia coli. We show for the first time that recombinant AtICS2 is enzymatically active. Both isozymes are Mg2+-dependent with similar temperature optima (ca. 33°C) and similar Km values for chorismate of 34.3 ± 3.7 and 28.8 ± 6.9 µM for ICS1 and ICS2, respectively, but reaction rates were greater for ICS1 than for ICS2, with respective values for Vmax of 63.5 ± 2.4 and 28.3 ± 2.0 nM s-1 and for kcat of 38.1 ± 1.5 and 17.0 ± 1.2 min-1 However, neither enzyme displayed isochorismate pyruvate lyase (IPL) activity, which would enable these proteins to act as bifunctional SA synthases, i.e. to convert chorismate into SA. These results show that although Arabidopsis has two functional ICS enzymes, it must possess one or more IPL enzymes to complete biosynthesis of SA starting from chorismate.

Cucumber mosaic virus and its 2b protein alter emission of host volatile organic compounds but not aphid vector settling in tobacco.

BACKGROUND: Aphids, including the generalist herbivore Myzus persicae, transmit cucumber mosaic virus (CMV). CMV (strain Fny) infection affects M. persicae feeding behavior and performance on tobacco (Nicotiana tabacum), Arabidopsis thaliana and cucurbits in varying ways. In Arabidopsis and cucurbits, CMV decreases host quality and inhibits prolonged feeding by aphids, which may enhance virus transmission rates. CMV-infected cucurbits also emit deceptive, aphid-attracting volatiles, which may favor virus acquisition. In contrast, aphids on CMV-infected tobacco (cv. Xanthi) exhibit increased survival and reproduction. This may not increase transmission but might increase virus and vector persistence within plant communities. The CMV 2b counter-defense protein diminishes resistance to aphid infestation in CMV-infected tobacco plants. We hypothesised that in tobacco CMV and its 2b protein might also alter the emission of volatile organic compounds that would influence aphid behavior. RESULTS: Analysis of headspace volatiles emitted from tobacco plants showed that CMV infection both increased the total quantity and altered the blend produced. Furthermore, experiments with a CMV 2b gene deletion mutant (CMV∆2b) showed that the 2b counter-defense protein influences volatile emission. Free choice bioassays were conducted where wingless M. persicae could choose to settle on infected or mock-inoculated plants under a normal day/night regime or in continual darkness. Settling was recorded at 15 min, 1 h and 24 h post-release. Statistical analysis indicated that aphids showed no marked preference to settle on mock-inoculated versus infected plants, except for a marginally greater settlement of aphids on mock-inoculated over CMV-infected plants under normal illumination. CONCLUSIONS: CMV infection of tobacco plants induced quantitative and qualitative changes in host volatile emission and these changes depended in part on the activity of the 2b counter-defense protein. However, CMV-induced alterations in tobacco plant volatile emission did not have marked effects on the settling of aphids on infected versus mock-inoculated plants even though CMV-infected plants are higher quality hosts for M. persicae.

Salicylic acid treatment and expression of an RNA-dependent RNA polymerase 1 transgene inhibit lethal symptoms and meristem invasion during tobacco mosaic virus infection in Nicotiana benthamiana.

BACKGROUND: Host RNA-dependent RNA polymerases (RDRs) 1 and 6 contribute to antiviral RNA silencing in plants. RDR6 is constitutively expressed and was previously shown to limit invasion of Nicotiana benthamiana meristem tissue by potato virus X and thereby inhibit disease development. RDR1 is inducible by salicylic acid (SA) and several other phytohormones. But although it contributes to basal resistance to tobacco mosaic virus (TMV) it is dispensable for SA-induced resistance in inoculated leaves. The laboratory accession of N. benthamiana is a natural rdr1 mutant and highly susceptible to TMV. However, TMV-induced symptoms are ameliorated in transgenic plants expressing Medicago truncatula RDR1. RESULTS: In MtRDR1-transgenic N. benthamiana plants the spread of TMV expressing the green fluorescent protein (TMV.GFP) into upper, non-inoculated, leaves was not inhibited. However, in these plants exclusion of TMV.GFP from the apical meristem and adjacent stem tissue was greater than in control plants and this exclusion effect was enhanced by SA. TMV normally kills N. benthamiana plants but although MtRDR1-transgenic plants initially displayed virus-induced necrosis they subsequently recovered. Recovery from disease was markedly enhanced by SA treatment in MtRDR1-transgenic plants whereas in control plants SA delayed but did not prevent systemic necrosis and death. Following SA treatment of MtRDR1-transgenic plants, extractable RDR enzyme activity was increased and Western blot analysis of RDR extracts revealed a band cross-reacting with an antibody raised against MtRDR1. Expression of MtRDR1 in the transgenic N. benthamiana plants was driven by a constitutive 35S promoter derived from cauliflower mosaic virus, confirmed to be non-responsive to SA. This suggests that the effects of SA on MtRDR1 are exerted at a post-transcriptional level. CONCLUSIONS: MtRDR1 inhibits severe symptom development by limiting spread of virus into the growing tips of infected plants. Thus, RDR1 may act in a similar fashion to RDR6. MtRDR1 and SA acted additively to further promote recovery from disease symptoms in MtRDR1-transgenic plants. Thus it is possible that SA promotes MtRDR1 activity and/or stability through post-transcriptional effects.

Abnormal glycosphingolipid mannosylation triggers salicylic acid-mediated responses in Arabidopsis.

The Arabidopsis thaliana protein GOLGI-LOCALIZED NUCLEOTIDE SUGAR TRANSPORTER (GONST1) has been previously identified as a GDP-d-mannose transporter. It has been hypothesized that GONST1 provides precursors for the synthesis of cell wall polysaccharides, such as glucomannan. Here, we show that in vitro GONST1 can transport all four plant GDP-sugars. However, gonst1 mutants have no reduction in glucomannan quantity and show no detectable alterations in other cell wall polysaccharides. By contrast, we show that a class of glycosylated sphingolipids (glycosylinositol phosphoceramides [GIPCs]) contains Man and that this mannosylation is affected in gonst1. GONST1 therefore is a Golgi GDP-sugar transporter that specifically supplies GDP-Man to the Golgi lumen for GIPC synthesis. gonst1 plants have a dwarfed phenotype and a constitutive hypersensitive response with elevated salicylic acid levels. This suggests an unexpected role for GIPC sugar decorations in sphingolipid function and plant defense signaling. Additionally, we discuss these data in the context of substrate channeling within the Golgi.

Domains of the cucumber mosaic virus 2b silencing suppressor protein affecting inhibition of salicylic acid-induced resistance and priming of salicylic acid accumulation during infection.

The cucumber mosaic virus (CMV) 2b silencing suppressor protein allows the virus to overcome resistance to replication and local movement in inoculated leaves of plants treated with salicylic acid (SA), a resistance-inducing plant hormone. In Arabidopsis thaliana plants systemically infected with CMV, the 2b protein also primes the induction of SA biosynthesis during this compatible interaction. We found that CMV infection of susceptible tobacco (Nicotiana tabacum) also induced SA accumulation. Utilization of mutant 2b proteins expressed during infection of tobacco showed that the N- and C-terminal domains, which had previously been implicated in regulation of symptom induction, were both required for subversion of SA-induced resistance, while all mutants tested except those affecting the putative phosphorylation domain had lost the ability to prime SA accumulation and expression of the SA-induced marker gene PR-1.

Interference with jasmonic acid-regulated gene expression is a general property of viral suppressors of RNA silencing but only partly explains virus-induced changes in plant-aphid interactions.

The cucumber mosaic virus (CMV) 2b viral suppressor of RNA silencing (VSR) inhibits host responses to jasmonic acid (JA), a chemical signal regulating resistance to insects. Previous experiments with a CMV subgroup IA strain and its 2b gene deletion mutant suggested that VSRs might neutralize aphid (Myzus persicae) resistance by inhibiting JA-regulated gene expression. To further investigate this, we examined JA-regulated gene expression and aphid performance in Nicotiana benthamiana infected with Potato virus X, Potato virus Y, Tobacco mosaic virus and a subgroup II CMV strain, as well as in transgenic plants expressing corresponding VSRs (p25, HC-Pro, 126 kDa and 2b). All the viruses or their VSRs inhibited JA-induced gene expression. However, this did not always correlate with enhanced aphid performance. Thus, VSRs are not the sole viral determinants of virus-induced changes in host-aphid interactions and interference with JA-regulated gene expression cannot completely explain enhanced aphid performance on virus-infected plants.

Investigating the Interactions of the Cucumber Mosaic Virus 2b Protein with the Viral 1a Replicase Component and the Cellular RNA Silencing Factor Argonaute 1.

The cucumber mosaic virus (CMV) 2b protein is a suppressor of plant defenses and a pathogenicity determinant. Amongst the 2b protein's host targets is the RNA silencing factor Argonaute 1 (AGO1), which it binds to and inhibits. In Arabidopsis thaliana, if 2b-induced inhibition of AGO1 is too efficient, it induces reinforcement of antiviral silencing by AGO2 and triggers increased resistance against aphids, CMV's insect vectors. These effects would be deleterious to CMV replication and transmission, respectively, but are moderated by the CMV 1a protein, which sequesters sufficient 2b protein molecules into P-bodies to prevent excessive inhibition of AGO1. Mutant 2b protein variants were generated, and red and green fluorescent protein fusions were used to investigate subcellular colocalization with AGO1 and the 1a protein. The effects of mutations on complex formation with the 1a protein and AGO1 were investigated using bimolecular fluorescence complementation and co-immunoprecipitation assays. Although we found that residues 56-60 influenced the 2b protein's interactions with the 1a protein and AGO1, it appears unlikely that any single residue or sequence domain is solely responsible. In silico predictions of intrinsic disorder within the 2b protein secondary structure were supported by circular dichroism (CD) but not by nuclear magnetic resonance (NMR) spectroscopy. Intrinsic disorder provides a plausible model to explain the 2b protein's ability to interact with AGO1, the 1a protein, and other factors. However, the reasons for the conflicting conclusions provided by CD and NMR must first be resolved.

Induction of aphid resistance in tobacco by the cucumber mosaic virus CMV∆2b mutant is jasmonate-dependent.

Cucumber mosaic virus (CMV) is vectored by aphids, including Myzus persicae. Tobacco (Nicotiana tabacum 'Xanthi') plants infected with a mutant of the Fny strain of CMV (Fny-CMVΔ2b, which cannot express the CMV 2b protein) exhibit strong resistance against M. persicae, which is manifested by decreased survival and reproduction of aphids confined on the plants. Previously, we found that the Fny-CMV 1a replication protein elicits aphid resistance in plants infected with Fny-CMVΔ2b, whereas in plants infected with wild-type Fny-CMV this is counteracted by the CMV 2b protein, a counterdefence protein that, among other things, inhibits jasmonic acid (JA)-dependent immune signalling. We noted that in nontransformed cv. Petit Havana SR1 tobacco plants aphid resistance was not induced by Fny-CMVΔ2b, suggesting that not all tobacco varieties possess the factor(s) with which the 1a protein interacts. To determine if 1a protein-induced aphid resistance is JA-dependent in Xanthi tobacco, transgenic plants were made that expressed an RNA silencing construct to diminish expression of the JA co-receptor CORONATINE-INSENSITIVE 1. Fny-CMVΔ2b did not induce resistance to M. persicae in these transgenic plants. Thus, aphid resistance induction by the 1a protein requires JA-dependent defensive signalling, which is countered by the CMV 2b protein.

Biased pollen transfer by bumblebees favors the paternity of virus-infected plants in cross-pollination.

We used a green fluorescent protein marker gene for paternity analysis to determine if virus infection affected male reproductive success of tomato in bumblebee-mediated cross-pollination under glasshouse conditions. We found that bumblebees that visited flowers of infected plants showed a strong preference to subsequently visit flowers of non-infected plants. The behavior of the bumblebees to move toward non-infected plants after pollinating virus-infected plants appears to explain the paternity data, which demonstrate a statistically significant ∼10-fold bias for fertilization of non-infected plants with pollen from infected parents. Thus, in the presence of bumblebee pollinators, CMV-infected plants exhibit enhanced male reproductive success.

The Effects of Cucumber Mosaic Virus and Its 2a and 2b Proteins on Interactions of Tomato Plants with the Aphid Vectors Myzus persicae and Macrosiphum euphorbiae.

Cucumber mosaic virus (CMV), a major tomato pathogen, is aphid-vectored in the non-persistent manner. We investigated if CMV-induced volatile organic compounds (VOCs) or other virus-induced cues alter aphid-tomato interactions. Y-tube olfactometry showed that VOCs emitted by plants infected with CMV (strain Fny) attracted generalist (Myzus persicae) and Solanaceae specialist (Macrosiphum euphorbiae) aphids. Myzus persicae preferred settling on infected plants (3 days post-inoculation: dpi) at 1h post-release, but at 9 and 21 dpi, aphids preferentially settled on mock-inoculated plants. Macrosiphum euphorbiae showed no strong preference for mock-inoculated versus infected plants at 3 dpi but settled preferentially on mock-inoculated plants at 9 and 21 dpi. In darkness aphids showed no settling or migration bias towards either mock-inoculated or infected plants. However, tomato VOC blends differed in light and darkness, suggesting aphids respond to a complex mix of olfactory, visual, and other cues influenced by infection. The LS-CMV strain induced no changes in aphid-plant interactions. Experiments using inter-strain recombinant and pseudorecombinant viruses showed that the Fny-CMV 2a and 2b proteins modified tomato interactions with Macrosiphum euphorbiae and Myzus persicae, respectively. The defence signal salicylic acid prevents excessive CMV-induced damage to tomato plants but is not involved in CMV-induced changes in aphid-plant interactions.

Infection of Arabidopsis by cucumber mosaic virus triggers jasmonate-dependent resistance to aphids that relies partly on the pattern-triggered immunity factor BAK1.

Many aphid-vectored viruses are transmitted nonpersistently via transient attachment of virus particles to aphid mouthparts and are most effectively acquired or transmitted during brief stylet punctures of epidermal cells. In Arabidopsis thaliana, the aphid-transmitted virus cucumber mosaic virus (CMV) induces feeding deterrence against the polyphagous aphid Myzus persicae. This form of resistance inhibits prolonged phloem feeding but promotes virus acquisition by aphids because it encourages probing of plant epidermal cells. When aphids are confined on CMV-infected plants, feeding deterrence reduces their growth and reproduction. We found that CMV-induced inhibition of growth as well as CMV-induced inhibition of reproduction of M. persicae are dependent upon jasmonate-mediated signalling. BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1 (BAK1) is a co-receptor enabling detection of microbe-associated molecular patterns and induction of pattern-triggered immunity (PTI). In plants carrying the mutant bak1-5 allele, CMV induced inhibition of M. persicae reproduction but not inhibition of aphid growth. We conclude that in wildtype plants CMV induces two mechanisms that diminish performance of M. persicae: a jasmonate-dependent and PTI-dependent mechanism that inhibits aphid growth, and a jasmonate-dependent, PTI-independent mechanism that inhibits reproduction. The growth of two crucifer specialist aphids, Lipaphis erysimi and Brevicoryne brassicae, was not affected when confined on CMV-infected A. thaliana. However, B. brassicae reproduction was inhibited on CMV-infected plants. This suggests that in A. thaliana CMV-induced resistance to aphids, which is thought to incentivize virus vectoring, has greater effects on polyphagous than on crucifer specialist aphids.

The effects of extracellular adenosine 5'-triphosphate on the tobacco proteome.

Extracellular adenosine 5'-triphosphate (eATP) is emerging as an important plant signalling compound capable of mobilising intracellular second messengers such as Ca(2+), nitric oxide, and reactive oxygen species. However, the downstream molecular targets and the spectrum of physiological processes that eATP regulates are largely unknown. We used exogenous ATP and a non-hydrolysable analogue as probes to identify the molecular and physiological effects of eATP-mediated signalling in tobacco. 2-DE coupled with MS/MS analysis revealed differential protein expression in response to perturbation of eATP signalling. These proteins are in several functional classes that included photosynthesis, mitochondrial ATP synthesis, and defence against oxidative stress, but the biggest response was in the pathogen defence-related proteins. Consistent with this, impairment of eATP signalling induced resistance against the bacterial pathogen Erwinia carotovora subsp. carotovora. In addition, disease resistance activated by a fungal pathogen elicitor (xylanase from Trichoderma viride) was concomitant with eATP depletion. These results reveal several previously unknown putative molecular targets of eATP signalling, which pinpoint eATP as an important hub at which regulatory signals of some major primary metabolic pathways and defence responses are integrated.

Extracellular ATP is a regulator of pathogen defence in plants.

In healthy plants extracellular ATP (eATP) regulates the balance between cell viability and death. Here we show an unexpected critical regulatory role of eATP in disease resistance and defensive signalling. In tobacco, enzymatic depletion of eATP or competition with non-hydrolysable ATP analogues induced pathogenesis-related (PR) gene expression and enhanced resistance to tobacco mosaic virus and Pseudomonas syringae pv. tabaci. Artificially increasing eATP concentrations triggered a drop in levels of the important defensive signal chemical salicylic acid (SA) and compromised basal resistance to viral and bacterial infection. Inoculating tobacco leaf tissues with bacterial pathogens capable of activating PR gene expression triggered a rapid decline in eATP. Conversely, inoculations with mutant bacteria unable to induce defence gene expression failed to deplete eATP. Furthermore, a collapse in eATP concentration immediately preceded PR gene induction by SA. Our study reveals a previously unsuspected role for eATP as a negative regulator of defensive signal transduction and demonstrates its importance as a key signal integrating defence and cell viability in plants.

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.