SAG101 forms a ternary complex with EDS1 and PAD4 and is required for resistance signaling against turnip crinkle virus.

EDS1, PAD4, and SAG101 are common regulators of plant immunity against many pathogens. EDS1 interacts with both PAD4 and SAG101 but direct interaction between PAD4 and SAG101 has not been detected, leading to the suggestion that the EDS1-PAD4 and EDS1-SAG101 complexes are distinct. We show that EDS1, PAD4, and SAG101 are present in a single complex in planta. While this complex is preferentially nuclear localized, it can be redirected to the cytoplasm in the presence of an extranuclear form of EDS1. PAD4 and SAG101 can in turn, regulate the subcellular localization of EDS1. We also show that the Arabidopsis genome encodes two functionally redundant isoforms of EDS1, either of which can form ternary complexes with PAD4 and SAG101. Simultaneous mutations in both EDS1 isoforms are essential to abrogate resistance (R) protein-mediated defense against turnip crinkle virus (TCV) as well as avrRps4 expressing Pseudomonas syringae. Interestingly, unlike its function as a PAD4 substitute in bacterial resistance, SAG101 is required for R-mediated resistance to TCV, thus implicating a role for the ternary complex in this defense response. However, only EDS1 is required for HRT-mediated HR to TCV, while only PAD4 is required for SA-dependent induction of HRT. Together, these results suggest that EDS1, PAD4 and SAG101 also perform independent functions in HRT-mediated resistance.

Enhanced disease susceptibility 1 and salicylic acid act redundantly to regulate resistance gene-mediated signaling.

Resistance (R) protein-associated pathways are well known to participate in defense against a variety of microbial pathogens. Salicylic acid (SA) and its associated proteinaceous signaling components, including enhanced disease susceptibility 1 (EDS1), non-race-specific disease resistance 1 (NDR1), phytoalexin deficient 4 (PAD4), senescence associated gene 101 (SAG101), and EDS5, have been identified as components of resistance derived from many R proteins. Here, we show that EDS1 and SA fulfill redundant functions in defense signaling mediated by R proteins, which were thought to function independent of EDS1 and/or SA. Simultaneous mutations in EDS1 and the SA-synthesizing enzyme SID2 compromised hypersensitive response and/or resistance mediated by R proteins that contain coiled coil domains at their N-terminal ends. Furthermore, the expression of R genes and the associated defense signaling induced in response to a reduction in the level of oleic acid were also suppressed by compromising SA biosynthesis in the eds1 mutant background. The functional redundancy with SA was specific to EDS1. Results presented here redefine our understanding of the roles of EDS1 and SA in plant defense.

The common metabolite glycerol-3-phosphate is a novel regulator of plant defense signaling.

Conversion of glycerol to glycerol-3-phosphate (G3P) is one of the highly conserved steps of glycerol metabolism in evolutionary diverse organisms. In plants, G3P is produced either via the glycerol kinase (GK)-mediated phosphorylation of glycerol, or via G3P dehydrogenase (G3Pdh)-mediated reduction of dihydroxyacetone phosphate (DHAP). We have recently shown that G3P levels contribute to basal resistance against the hemibiotrophic pathogen, Colletotrichum higginsianum. Since a mutation in the GLY1-encoded G3Pdh conferred more susceptibility compared to a mutation in the GLI1-encoded GK, we proposed that GLY1 is the major contributor of the total G3P pool that participates in defense against C. higginsianum.

Glycerol-3-phosphate levels are associated with basal resistance to the hemibiotrophic fungus Colletotrichum higginsianum in Arabidopsis.

Glycerol-3-phosphate (G3P) is an important component of carbohydrate and lipid metabolic processes. In this article, we provide evidence that G3P levels in plants are associated with defense to a hemibiotrophic fungal pathogen Colletotrichum higginsianum. Inoculation of Arabidopsis (Arabidopsis thaliana) with C. higginsianum was correlated with an increase in G3P levels and a concomitant decrease in glycerol levels in the host. Plants impaired in utilization of plastidial G3P (act1) accumulated elevated levels of pathogen-induced G3P and displayed enhanced resistance. Furthermore, overexpression of the host GLY1 gene, which encodes a G3P dehydrogenase (G3Pdh), conferred enhanced resistance. In contrast, the gly1 mutant accumulated reduced levels of G3P after pathogen inoculation and showed enhanced susceptibility to C. higginsianum. Unlike gly1, a mutation in a cytosolic isoform of G3Pdh did not alter basal resistance to C. higginsianum. Furthermore, act1 gly1 double-mutant plants were as susceptible as the gly1 plants. Increased resistance or susceptibility of act1 and gly1 plants to C. higginsianum, respectively, was not due to effects of these mutations on salicylic acid- or ethylene-mediated defense pathways. The act1 mutation restored a wild-type-like response in camalexin-deficient pad3 plants, which were hypersusceptible to C. higginsianum. These data suggest that G3P-associated resistance to C. higginsianum occurs independently or downstream of the camalexin pathway. Together, these results suggest a novel and specific link between G3P metabolism and plant defense.

Plastidial fatty acid levels regulate resistance gene-dependent defense signaling in Arabidopsis.

In Arabidopsis, resistance to Turnip Crinkle Virus (TCV) depends on the resistance (R) gene, HRT, and the recessive locus rrt. Resistance also depends on salicylic acid (SA), EDS1, and PAD4. Exogenous application of SA confers resistance in RRT-containing plants by increasing HRT transcript levels in a PAD4-dependent manner. Here we report that reduction of oleic acid (18:1) can also induce HRT gene expression and confer resistance to TCV. However, the 18:1-regulated pathway is independent of SA, rrt, EDS1, and PAD4. Reducing the levels of 18:1, via a mutation in the SSI2-encoded stearoyl-acyl carrier protein-desaturase, or by exogenous application of glycerol, increased transcript levels of HRT as well as several other R genes. Second-site mutations in the ACT1-encoded glycerol-3-phosphate acyltransferase or GLY1-encoded glycerol-3-phosphate dehydrogenase restored 18:1 levels in HRT ssi2 plants and reestablished a dependence on rrt. Resistance to TCV and HRT gene expression in HRT act1 plants was inducible by SA but not by glycerol, whereas that in HRT pad4 plants was inducible by glycerol but not by SA. The low 18:1-mediated induction of R gene expression was also dependent on ACT1 but independent of EDS1, PAD4, and RAR1. Intriguingly, TCV inoculation did not activate this 18:1-regulated pathway in HRT plants, but instead resulted in the induction of several genes that encode 18:1-synthesizing isozymes. These results suggest that the 18:1-regulated pathway may be specifically targeted during pathogen infection and that altering 18:1 levels may serve as a unique strategy for promoting disease resistance.

Role of salicylic acid and fatty acid desaturation pathways in ssi2-mediated signaling.

Stearoyl-acyl carrier protein desaturase-mediated conversion of stearic acid to oleic acid (18:1) is the key step that regulates the levels of unsaturated fatty acids (FAs) in cells. Our previous work with the Arabidopsis (Arabidopsis thaliana) ssi2/fab2 mutant and its suppressors demonstrated that a balance between glycerol-3-phosphate (G3P) and 18:1 levels is critical for the regulation of salicylic acid (SA)- and jasmonic acid-mediated defense signaling in the plant. In this study, we have evaluated the role of various genes that have an impact on SA, resistance gene-mediated, or FA desaturation (FAD) pathways on ssi2-mediated signaling. We show that ssi2-triggered resistance is dependent on EDS1, PAD4, EDS5, SID2, and FAD7 FAD8 genes. However, ssi2-triggered defects in the jasmonic acid pathway, morphology, and cell death phenotypes are independent of the EDS1, EDS5, PAD4, NDR1, SID2, FAD3, FAD4, FAD5, DGD1, FAD7, and FAD7 FAD8 genes. Furthermore, the act1-mediated rescue of ssi2 phenotypes is also independent of the FAD2, FAD3, FAD4, FAD5, FAD7, and DGD1 genes. Since exogenous application of glycerol converts wild-type plants into ssi2 mimics, we also studied the effect of exogenous application of glycerol on mutants impaired in resistance-gene signaling, SA, or fad pathways. Glycerol increased SA levels and induced pathogenesis-related gene expression in all but sid2, nahG, fad7, and fad7 fad8 plants. Furthermore, glycerol-induced phenotypes in various mutant lines correlate with a concomitant reduction in 18:1 levels. Inability to convert glycerol into G3P due to a mutation in the nho1-encoded glycerol kinase renders plants tolerant to glycerol and unable to induce the SA-dependent pathway. A reduction in the NHO1-derived G3P pool also results in a partial age-dependent rescue of the ssi2 morphological and cell death phenotypes in the ssi2 nho1 plants. The glycerol-mediated induction of defense was not associated with any major changes in the lipid profile and/or levels of phosphatidic acid. Taken together, our results suggest that glycerol application and the ssi2 mutation in various mutant backgrounds produce similar effects and that restoration of ssi2 phenotypes is not associated with the further desaturation of 18:1 to linoleic or linolenic acids in plastidal or extraplastidal lipids.

Oleic acid levels regulated by glycerolipid metabolism modulate defense gene expression in Arabidopsis.

Stearoyl-acyl-carrier-protein-desaturase-mediated conversion of stearic acid (18:0) to oleic acid (18:1) is a key step, which regulates levels of unsaturated fatty acids in cells. We previously showed that stearoyl-acyl-carrier-protein-desaturase mutants ssi2/fab2 carrying a loss-of-function mutation in the plastidial glycerol-3-phosphate (G3P) acyltransferase (act1) have elevated 18:1 levels and are restored in their altered defense signaling. Because G3P is required for the acylation of 18:1 by G3P acyltransferase, it was predicted that reduction of G3P levels should increase 18:1 levels and thereby revert ssi2-triggered phenotypes. Here we show that a mutation in G3P dehydrogenase restores both salicylic acid- and jasmonic acid-mediated phenotypes of ssi2 plants. The G3P dehydrogenase gene was identified by map-based cloning of the ssi2 suppressor mutant rdc8 (gly1-3) and confirmed by epistatic analysis of ssi2 with gly1-1. Restoration of ssi2-triggered phenotypes by the gly1-3 mutation was age-dependent and correlated with the levels of 18:1. Regeneration of G3P pools by glycerol application in ssi2 and ssi2 gly1-3 plants caused a marked reduction in the 18:1 levels, which rendered these plants hypersensitive to glycerol. This hypersensitivity in ssi2 was rescued by the act1 mutation. Furthermore, overexpression of the ACT1 gene resulted in enhanced sensitivity to glycerol. Glycerol application also lowered the 18:1 content in SSI2 plants and converted these into ssi2-mimics. Our results show that 18:1 levels in plastids are regulated by means of acylation with G3P, and a balance between G3P and 18:1 is critical for the regulation of salicylic acid- and jasmonic acid-mediated signaling pathways.