Plant growth-promoting rhizobacteria systemically protect Arabidopsis thaliana against Cucumber mosaic virus by a salicylic acid and NPR1-independent and jasmonic acid-dependent signaling pathway

Arabidopsis thaliana ecotype Columbia plants (Col-0) treated with plant growth-promoting rhizobacteria (PGPR) Serattia marcescens strain 90-166 and Bacillus pumilus strain SE34 had significantly reduced symptom severity by Cucumber mosaic virus (CMV). In some cases, CMV accumulation was also significantly reduced in systemically infected leaves. The signal transduction pathway(s) associated with induced resistance against CMV by strain 90-166 was determined using mutant strains and transgenic and mutant Arabidopsis lines. NahG plants treated with strains 90-166 and SE34 had reduced symptom severity indicating that the resistance did not require salicylic acid (SA). Strain 90-166 naturally produces SA under iron-limited conditions. Col-0 and NahG plants treated with the SA-deficient mutant, 90-166-1441, had significantly reduced CMV symptom severity with reduced virus accumulation in Col-0 plants. Another PGPR mutant, 90-166-2882, caused reduced disease severity in Col-0 and NahG plants. In a time course study, strain 90-166 reduced virus accumulation at 7 but not at 14 and 21 days post-inoculation (dpi) on the non-inoculated leaves of Col-0 plants. NahG and npr1-1 plants treated with strain 90-166 had reduced amounts of virus at 7 and 14 dpi but not at 21 dpi. In contrast, no decrease in CMV accumulation occurred in strain 90-166-treated fad3-2 fad7-2 fad8 plants. These data indicate that the protection of Arabidopsis against CMV by strain 90-166 follows a signaling pathway for virus protection that is independent of SA and NPR1, but dependent on jasmonic acid.     

NbALD1 mediates resistance to turnip mosaic virus by regulating the accumulation of salicylic acid and the ethylene pathway in Nicotiana benthamiana

AGD2-LIKE DEFENCE RESPONSE PROTEIN 1 (ALD1) triggers plant defence against bacterial and fungal pathogens by regulating the salicylic acid (SA) pathway and an unknown SA-independent pathway. We now show that Nicotiana benthamiana ALD1 is involved in defence against a virus and that the ethylene pathway also participates in ALD1-mediated resistance. NbALD1 was up-regulated in plants infected with turnip mosaic virus (TuMV). Silencing of NbALD1 facilitated TuMV infection, while overexpression of NbALD1 or exogenous application of pipecolic acid (Pip), the downstream product of ALD1, enhanced resistance to TuMV. The SA content was lower in NbALD1-silenced plants and higher where NbALD1 was overexpressed or following Pip treatments. SA mediated resistance to TuMV and was required for NbALD1-mediated resistance. However, on NahG plants (in which SA cannot accumulate), Pip treatment still alleviated susceptibility to TuMV, further demonstrating the presence of an SA-independent resistance pathway. The ethylene precursor, 1-aminocyclopropanecarboxylic acid (ACC), accumulated in NbALD1-silenced plants but was reduced in plants overexpressing NbALD1 or treated with Pip. Silencing of ACS1, a key gene in the ethylene pathway, alleviated the susceptibility of NbALD1-silenced plants to TuMV, while exogenous application of ACC compromised the resistance of Pip-treated or NbALD1 transgenic plants. The results indicate that NbALD1 mediates resistance to TuMV by positively regulating the resistant SA pathway and negatively regulating the susceptible ethylene pathway.     

Priming for enhanced ARGONAUTE2 activation accompanies induced resistance to cucumber mosaic virus in Arabidopsis thaliana

Systemic acquired resistance (SAR) is a broad-spectrum disease resistance response that can be induced upon infection from pathogens or by chemical treatment, such as with benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH). SAR involves priming for more robust activation of defence genes upon pathogen attack. Whether priming for SAR would involve components of RNA silencing remained unknown. Here, we show that upon leaf infiltration of water, BTH-primed Arabidopsis thaliana plants accumulate higher amounts of mRNA of ARGONAUTE (AGO)2 and AGO3, key components of RNA silencing. The enhanced AGO2 expression is associated with prior-to-activation trimethylation of lysine 4 in histone H3 and acetylation of histone H3 in the AGO2 promoter and with induced resistance to the yellow strain of cucumber mosaic virus (CMV[Y]). The results suggest that priming A. thaliana for enhanced defence involves modification of histones in the AGO2 promoter that condition AGO2 for enhanced activation, associated with resistance to CMV(Y). Consistently, the fold-reduction in CMV(Y) coat protein accumulation by BTH pretreatment was lower in ago2 than in wild type, pointing to reduced capacity of ago2 to activate BTH-induced CMV(Y) resistance. A role of AGO2 in pathogen-induced SAR is suggested by the enhanced activation of AGO2 after infiltrating systemic leaves of plants expressing a localized hypersensitive response upon CMV(Y) infection. In addition, local inoculation of SAR-inducing Pseudomonas syringae pv. maculicola causes systemic priming for enhanced AGO2 expression. Together our results indicate that defence priming targets the AGO2 component of RNA silencing whose enhanced expression is likely to contribute to SAR.     

Nicotiana benthamiana asparagine synthetase associates with IP-L and confers resistance against tobacco mosaic virus via the asparagine-induced salicylic acid signalling pathway

Asparagine synthetase is a key enzyme that catalyses the conversion of amide groups from glutamine or ammonium to aspartate, which leads to the generation of asparagine. However, the role of asparagine synthetase in plant immunity remains largely unknown. Here, we identified a Nicotiana benthamiana asparagine synthetase B (NbAS-B) that associates with tomato mosaic virus coat protein-interacting protein L (IP-L) using the yeast two-hybrid assay and examined its role in tobacco mosaic virus (TMV) resistance. The association of IP-L with NbAS-B was further confirmed by in vivo co-immunoprecipitation, luciferase complementation imaging, and bimolecular fluorescence complementation assays. IP-L and NbAS-B interact in the nucleus and cytosol and IP-L apparently stabilizes NbAS-B, thus enhancing its accumulation. The expressions of IP-L and NbAS-B are continuously induced on TMV-green fluorescent protein (GFP) infection. Co-silencing of IP-L and NbAS-B facilitates TMV-GFP infection. Overexpression of NbAS-B in tobacco reduces TMV-GFP infection by significantly improving the synthesis of asparagine. Furthermore, the external application of asparagine significantly inhibits the infection of TMV-GFP by activating the salicylic acid signalling pathway. These findings hold the potential for the future application of asparagine in the control of TMV.     

Signaling requirements and role of salicylic acid in HRT- and rrt-mediated resistance to turnip crinkle virus in Arabidopsis

Inoculation of turnip crinkle virus (TCV) on the resistant Arabidopsis ecotype Di-17 elicits a hypersensitive response (HR), which is accompanied by increased expression of pathogenesis-related (PR) genes. Previous genetic analyses revealed that the HR to TCV is conferred by HRT, which encodes a coiled-coil (CC), nucleotide-binding site (NBS) and leucine-rich repeat (LRR) class resistance (R) protein. In contrast to the HR, resistance to TCV requires both HRT and a recessive allele at a second locus designated rrt. Here, we demonstrate that unlike most CC-NBS-LRR R genes, HRT/rrt-mediated resistance is dependent on EDS1 and independent of NDR1. Resistance is also independent of RAR1 and SGT1. HRT/rrt-mediated resistance is compromised in plants with reduced salicylic acid (SA) content as a consequence of mutations eds5, pad4, or sid2. By contrast, HR is not affected by mutations in eds1, eds5, pad4, sid2, ndr1, rar1, or sgt1b. Resistance to TCV is restored in both SA-deficient Di-17 plants expressing the nahG transgene and mutants containing the eds1, eds5, or sid2 mutations by exogenous application of SA or the SA analog benzo(1,2,3)thiadiazole-7-carbothioic acid (BTH). In contrast, SA/BTH treatment failed to enhance resistance in HRT pad4, Col-0, or hrt homozygous progeny of a cross between Di-17 and Col-0. Thus, HRT and PAD4 are required for SA-induced resistance. Exogenously supplied SA or high endogenous levels of SA, due to the ssi2 mutation, overcame the suppressive effects of RRT and enhanced resistance to TCV, provided the HRT allele was present. High levels of SA upregulate HRT expression via a PAD4-dependent pathway. As Col-0 transgenic lines expressing high levels of HRT were resistant to TCV, but lines expressing moderate to low levels of HRT were not, we conclude that SA enhances resistance in the RRT background by upregulating HRT expression. These data suggest that the HRT-TCV interaction is unable to generate sufficient amounts of SA required for a stable resistance phenotype, and the presence of rrt possibly corrects this deficiency.     

Glutathione contributes to resistance responses to TMV through a differential modulation of salicylic acid and reactive oxygen species

Systemic acquired resistance (SAR) is induced by pathogens and confers protection against a broad range of pathogens. Several SAR signals have been characterized, but the nature of the other unknown signalling by small metabolites in SAR remains unclear. Glutathione (GSH) has long been implicated in the defence reaction against biotic stress. However, the mechanism that GSH increases plant tolerance against virus infection is not entirely known. Here, a combination of a chemical, virus-induced gene-silencing-based genetics approach, and transgenic technology was undertaken to investigate the role of GSH in plant viral resistance in Nicotiana benthamiana. Tobacco mosaic virus (TMV) infection results in increasing the expression of GSH biosynthesis genes NbECS and NbGS, and GSH content. Silencing of NbECS or NbGS accelerated oxidative damage, increased accumulation of reactive oxygen species (ROS), compromised plant resistance to TMV, and suppressed the salicylic acid (SA)-mediated signalling pathway. Application of GSH or l -2-oxothiazolidine-4-carboxylic acid (a GSH activator) alleviated oxidative damage, decreased accumulation of ROS, elevated plant local and systemic resistance, enhanced the SA-mediated signalling pathway, and increased the expression of ROS scavenging-related genes. However, treatment with buthionine sulfoximine (a GSH inhibitor) accelerated oxidative damage, elevated ROS accumulation, compromised plant systemic resistance, suppressed the SA-mediated signalling pathway, and reduced the expression of ROS-regulating genes. Overexpression of NbECS reduced oxidative damage, decreased accumulation of ROS, increased resistance to TMV, activated the SA-mediated signalling pathway, and increased the expression of the ROS scavenging-related genes. We present molecular evidence suggesting GSH is essential for both local and systemic resistance of N. benthamiana to TMV through a differential modulation of SA and ROS.     

Four sequence positions of the movement protein of Cucumber mosaic virus determine the virulence against cmv1‐mediated resistance in melon

The resistance to a set of strains of Cucumber mosaic virus (CMV) in the melon accession PI 161375, cultivar ‘Songwhan Charmi’, is dependent on one recessive gene, cmv1, which confers total resistance, whereas a second set of strains is able to overcome it. We tested 11 strains of CMV subgroups I and II in the melon line SC12‐1‐99, which carries the gene cmv1, and showed that this gene confers resistance to strains of subgroup II only and that restriction is not related to either viral replication or cell‐to‐cell movement. This is the first time that a resistant trait has been correlated with CMV subgroups. Using infectious clones of the CMV strains LS (subgroup II) and FNY (subgroup I), we generated rearrangements and viral chimaeras between both strains and established that the determinant of virulence against the gene cmv1 resides in the first 209 amino acids of the movement protein, as this region from FNY is sufficient to confer virulence to the LS clone in the line SC12‐1‐99. A comparison of the sequences of the strains of both subgroups in this region shows that there are five main positions shared by all strains of subgroup II, which are different from those of subgroup I. Site‐directed mutagenesis of the CMV‐LS clone to substitute these residues for those of CMV‐FNY revealed that a combination of four of these changes [the group 64–68 (SNNLL to HGRIA), and the point mutations R81C, G171T and A195I] was required for a complete gain of function of the LS MP in the resistant melon plant.     

Coat protein-mediated resistance against an Indian isolate of the Cucumber mosaic virus subgroup IB in Nicotiana benthamiana

Coat protein (CP) -mediated resistance against an Indian isolate of the Cucumber mosaic virus (CMV) subgroup IB was demonstrated in transgenic lines of Nicotiana benthamiana through Agrobacterium tumefaciens-mediated transformation. Out of the fourteen independently transformed lines developed, two lines were tested for resistance against CMV by challenge inoculations. The transgenic lines exhibiting complete resistance remained symptomless throughout life and showed reduced or no virus accumulation in their systemic leaves after virus challenge. These lines also showed virus resistance against two closely related strains of CMV. This is the first report of CP-mediated transgenic resistance against a CMV subgroup IB member isolated from India.     

Tobacco Rar1, EDS1 and NPR1/NIM1 like genes are required for N-mediated resistance to tobacco mosaic virus

The tobacco N gene confers resistance to tobacco mosaic virus (TMV) and encodes a Toll-interleukin-1 receptor/nucleotide binding site/leucine-rich repeat (TIR-NBS-LRR) class protein. We have developed and used a tobacco rattle virus (TRV) based virus induced gene silencing (VIGS) system to investigate the role of tobacco candidate genes in the N-mediated signalling pathway. To accomplish this we generated transgenic Nicotiana benthamiana containing the tobacco N gene. The transgenic lines exhibit hypersensitive response (HR) to TMV and restrict virus spread to the inoculated site. This demonstrates that the tobacco N gene can confer resistance to TMV in heterologous N. benthamiana. We have used this line to study the role of tobacco Rar1-, EDS1-, and NPR1/NIM1- like genes in N-mediated resistance to TMV using a TRV based VIGS approach. Our VIGS analysis suggests that these genes are required for N function. EDS1-like gene requirement for the N function suggests that EDS1 could be a common component of bacterial, fungal and viral resistance signalling mediated by the TIR-NBS-LRR class of resistance proteins. Requirement of Rar1- like gene for N-mediated resistance to TMV and some powdery mildew resistance genes in barley provide the first example of converging points in the disease resistance signalling pathways mediated by TIR-NBS-LRR and CC-NBS-LRR proteins. The TRV based VIGS approach as described here to study N-mediated resistance signalling will be useful for the analysis of not only disease resistance signalling pathways but also of other signalling pathways in genetically intractable plant systems.     

Propyl gallate,a free radical scavenger,counteracts the benefits of exogenously applied salicylic acid and aggravates the deleterious effects of the southern bean mosaic virus in Rhizobium-nodulated Phaseolus vulgaris plants

Infection of Rhizobium-nodulated Phaseolus vulgaris by the southern bean mosaic virus (SBMV) markedly decreased the growth and nodulation of plants. Exogenous applications of salicylic acid (SA) at concentrations ≥10 μM further decreased growth and nodulation of virus-infected (V) plants. Only SA concentration of 5 μM in the solution improved the growth, nodulation, chlorophyll concentration and the catabolism of ureide in leaves of V plants. The spray of leaves with 2 mM propyl gallate (+Pg) decreased growth, nodulation as well as the chlorophyll and leaf ureide concentrations in V plants, regardless of the concentration of SA at which plants were grown. Ultrastructural damages in leaf cells caused by SBMV were also enhanced in V+Pg plants. The massive proliferation of virus particles and the presence of virus crystalline arrays within symbiosomes of nodules in V+Pg plants were attributed to proliferation of branched plasmodesmata in leaf vascular-parenchyma cell walls that facilitated virus movement. Virus particles were never observed in leaf and nodule tissues of V plants not sprayed with Pg. Exogenous applications of SA hindered while Pg increased the symbiotic performance of H plants, pointing out the complexity to be addressed in breeding for virus resistance in Rhizobium-nodulated beans.     

Characterization of an Arabidopsis–Phytophthora Pathosystem: resistance requires a functional PAD2 gene and is independent of salicylic acid, ethylene and jasmonic acid signalling

Arabidopsis accessions were screened with isolates of Phytophthora porri originally isolated from other crucifer species. The described Arabidopsis-Phytophthora pathosystem shows the characteristics of a facultative biotrophic interaction similar to that seen in agronomically important diseases caused by Phytophthora species. In susceptible accessions, extensive colonization of the host tissue occurred and sexual and asexual spores were formed. In incompatible combinations, the plants reacted with a hypersensitive response (HR) and the formation of papillae at the sites of attempted penetration. Defence pathway mutants such as jar1 (jasmonic acid-insensitive), etr1 (ethylene receptor mutant) and ein2 (ethylene-insensitive) remained resistant towards P. porri. However, pad2, a mutant with reduced production of the phytoalexin camalexin, was hyper-susceptible. The accumulation of salicylic acid (SA) and PR1 protein was strongly reduced in pad2. Surprisingly, this lack of SA accumulation does not appear to be the cause of the hyper-susceptibility because interference with SA signalling in nahG plants or sid2 or npr1 mutants had only a minor effect on resistance. In addition, the functional SA analogue benzothiadiazol (BTH) did not induce resistance in susceptible plants including pad2. Similarly, the complete blockage of camalexin biosynthesis in pad3 did not cause susceptibility. Resistance of Arabidopsis against P. porri appears to depend on unknown defence mechanisms that are under the control of PAD2.     

The hypersensitive induced reaction 3 (HIR3) gene contributes to plant basal resistance via an EDS1 and salicylic acid‐dependent pathway

The hypersensitive‐induced reaction (HIR) gene family is associated with the hypersensitive response (HR) that is a part of the plant defense system against bacterial and fungal pathogens. The involvement of HIR genes in response to viral pathogens has not yet been studied. We now report that the HIR3 genes of Nicotiana benthamiana and Oryza sativa (rice) were upregulated following rice stripe virus (RSV) infection. Silencing of HIR3s in N. benthamiana resulted in an increased accumulation of RSV RNAs, whereas overexpression of HIR3s in N. benthamiana or rice reduced the expression of RSV RNAs and decreased symptom severity, while also conferring resistance to Turnip mosaic virus, Potato virus X, and the bacterial pathogens Pseudomonas syringae and Xanthomonas oryzae. Silencing of HIR3 genes in N. benthamiana reduced the content of salicylic acid (SA) and was accompanied by the downregulated expression of genes in the SA pathway. Transient expression of the two HIR3 gene homologs from N. benthamiana or the rice HIR3 gene in N. benthamiana leaves caused cell death and an accumulation of SA, but did not do so in EDS1‐silenced plants or in plants expressing NahG. The results indicate that HIR3 contributes to plant basal resistance via an EDS1‐ and SA‐dependent pathway.     

Arabidopsis GH3-LIKE DEFENSE GENE 1 is required for accumulation of salicylic acid, activation of defense responses and resistance to Pseudomonas syringae

In Arabidopsis, the GH3-like gene family consists of 19 members, several of which have been shown to adenylate the plant hormones jasmonic acid, indole acetic acid and salicylic acid (SA). In some cases, this adenylation has been shown to catalyze hormone conjugation to amino acids. Here we report molecular characterization of the GH3-LIKE DEFENSE GENE 1 (GDG1), a member of the GH3-like gene family, and show that GDG1 is an important component of SA-mediated defense against the bacterial pathogen Pseudomonas syringae. Expression of GDG1 is induced earlier and to a higher level in response to avirulent pathogens compared to virulent pathogens. gdg1 null mutants are compromised in several pathogen defense responses, including activation of defense genes and resistance against virulent and avirulent bacterial pathogens. Accumulation of free and glucoside-conjugated SA (SAG) in response to pathogen infection is compromised in gdg1 mutants. All defense-related phenotypes of gdg1 can be rescued by external application of SA, suggesting that gdg1 mutants are defective in the SA-mediated defense pathway(s) and that GDG1 functions upstream of SA. Our results suggest that GDG1 contributes to both basal and resistance gene-mediated inducible defenses against P. syringae (and possibly other pathogens) by playing a critical role in regulating the levels of pathogen-inducible SA. GDG1 is allelic to the PBS3 (avrPphB susceptible) gene.     

Arabidopsis thaliana GLUTATHIONE‐S‐TRANSFERASE THETA 2 interacts with RSI1/FLD to activate systemic acquired resistance

A partly infected plant develops systemic acquired resistance (SAR) and shows heightened resistance during subsequent infections. The infected parts generate certain mobile signals that travel to the distal tissues and help to activate SAR. SAR is associated with epigenetic modifications of several defence‐related genes. However, the mechanisms by which mobile signals contribute to epigenetic changes are little known. Previously, we have shown that the Arabidopsis REDUCED SYSTEMIC IMMUNITY 1 (RSI1, alias FLOWERING LOCUS D; FLD), which codes for a putative histone demethylase, is required for the activation of SAR. Here, we report the identification of GLUTATHIONE‐S‐TRANSFERASE THETA 2 (GSTT2) as an interacting factor of FLD. GSTT2 expression increases in pathogen‐inoculated as well as pathogen‐free distal tissues. The loss‐of‐function mutant of GSTT2 is compromised for SAR, but activates normal local resistance. Complementation lines of GSTT2 support its role in SAR activation. The distal tissues of gstt2 mutant plants accumulate significantly less salicylic acid (SA) and express a reduced level of the SA biosynthetic gene PAL1. In agreement with the established histone modification activity of FLD, gstt2 mutant plants accumulate an enhanced level of methylated and acetylated histones in the promoters of WRKY6 and WRKY29 genes. Together, these results demonstrate that GSTT2 is an interactor of FLD, which is required for SAR and SAR‐associated epigenetic modifications.