Nematode ascaroside enhances resistance in a broad spectrum of plant–pathogen systems

Recognition of specific molecule signatures of microbes, including pathogens, induces innate immune responses in plants, as well as in animals. Analogously, a nematode pheromone, the ascaroside ascr#18, induces hallmark plant defences including activation of (a) mitogen‐activated protein kinases, (b) salicylic acid‐ and jasmonic acid‐mediated defence signalling pathways and (c) defence gene expression and provides protection to a broad spectrum of pathogens. Ascr#18 is a member of an evolutionarily conserved family of nematode signalling molecules and is the major ascaroside secreted by plant–parasitic nematodes. Here, we report the effects of ascr#18 on resistance in four of the major economically important crops: maize, rice, wheat and soybean to some of their associated pathogens. Treatment with low nanomolar to low micromolar concentrations of ascr#18 provided from partial to strong protection in seven of eight plant–pathogen systems tested with viruses, bacteria, fungi, oomycetes and nematodes. This research may have potential to improve agricultural sustainability by reducing use of potentially harmful agrochemicals and enhance food security worldwide.     

Restoration of defective cross talk in ssi2 mutants: role of salicylic acid, jasmonic acid, and fatty acids in SSI2-mediated signaling

The Arabidopsis mutants ssi2 and fab2 are defective in stearoyl ACP desaturase, which causes altered salicylic acid (SA)- and jasmonic acid (JA)-mediated defense signaling. Both ssi2 and fab2 plants show spontaneous cell death, express PR genes constitutively, accumulate high levels of SA, and exhibit enhanced resistance to bacterial and oomycete pathogens. In contrast to constitutive activation of the SA pathway, ssi2 and fab2 plants are repressed in JA-mediated induction of the PDF1.2 gene, which suggests that the SSI2-mediated signaling pathway modulates cross talk between the SA and JA pathways. In this study, we have characterized two recessive nonallelic mutants in the ssi2 background, designated as rdc (restorer of defective cross talk) 2 and rdc8. Both ssi2 rdc mutants are suppressed in constitutive SA signaling, show basal level expression of PR-1 gene, and induce high levels of PDF1.2 in response to exogenous application of JA. Interestingly, while the rdc8 mutation completely abolishes spontaneous cell death in ssi2 rdc8 plants, the ssi2 rdc2 plants continue to show some albeit reduced cell death. Fatty acid (FA) analysis showed a reduction in 16:3 levels in ssi2 rdc8 plants, which suggests that this mutation may limit the flux of FAs into the prokaryotic pathway of glycerolipid biosynthesis. Both rdc2 and rdc8 continue to accumulate high levels of 18:0, which suggests that 18:0 levels were responsible for neither constitutive SA signaling nor repression of JA-induced expression of the PDF1.2 gene in ssi2 plants. We also analyzed SA and JA responses of the fab2-derived shs1 mutant, which accumulates levels of 18:0 over 50% lower than those in the fab2 plants. Even though fab2 shs1 plants were morphologically bigger than fab2 plants, they expressed PR genes constitutively, showed HR-like cell death, and accumulated elevated levels of SA. However, unlike the ssi2 rdc plants, fab2 shs1 plants were unable to induce high levels of PDF1.2 expression in response to exogenous application of JA. Together, these results show that defective cross talk in ssi2 can be restored by second site mutations and is independent of morphological size of the plants, cell death, and elevated levels of 18:0.     

The glabra1 Mutation Affects Cuticle Formation and Plant Responses to Microbes

Systemic acquired resistance (SAR) is a form of defense that provides resistance against a broad spectrum of pathogens in plants. Previous work indicates a role for plastidial glycerolipid biosynthesis in SAR. Specifically, mutations in FATTY ACID DESATURASE7 (FAD7), which lead to reduced trienoic fatty acid levels and compromised plastidial lipid biosynthesis, have been associated with defective SAR. We show that the defective SAR in Arabidopsis (Arabidopsis thaliana) fad7-1 plants is not associated with a mutation in FAD7 but rather with a second-site mutation in GLABRA1 (GL1), a gene well known for its role in trichome formation. The compromised SAR in gl1 plants is associated with impairment in their cuticles. Furthermore, mutations in two other components of trichome development, GL3 and TRANSPARENT TESTA GLABRA1, also impaired cuticle development and SAR. This suggests an overlap in the biochemical pathways leading to cuticle and trichome development. Interestingly, exogenous application of gibberellic acid (GA) not only enhanced SAR in wild-type plants but also restored SAR in gl1 plants. In contrast to GA, the defense phytohoromes salicylic acid and jasmonic acid were unable to restore SAR in gl1 plants. GA application increased levels of cuticular components but not trichome formation on gl1 plants, thus implicating cuticle, but not trichomes, as an important component of SAR. Our findings question the prudence of using mutant backgrounds for genetic screens and underscore a need to reevaluate phenotypes previously studied in the gl1 background.Plants have evolved a large array of defense mechanisms to resist infection by pathogens. Upon recognition, the host plant initiates one or more signal transduction pathways that activate various plant defenses and thereby prevent pathogen colonization. In many cases, resistance is associated with increased expression of defense genes, including the pathogenesis-related (PR) genes and the accumulation of salicylic acid (SA) in the inoculated leaf. Induction of these responses is accompanied by localized cell death at the site of pathogen entry, which can often restrict the spread of pathogen to cells within and immediately surrounding the lesions. This phenomenon, known as the hypersensitive response, is one of the earliest visible manifestations of induced defense responses and resembles programmed cell death in animals (Dangl et al., 1996; Gray, 2002; Glazebrook, 2005; Kachroo and Kachroo, 2006). Concurrent with hypersensitive response development, defense reactions are triggered in sites both local and distal from the primary infection. This phenomenon, known as systemic acquired resistance (SAR), is accompanied by a local and systemic increase in SA and jasmonic acid (JA) and a concomitant up-regulation of a large set of defense genes (Durrant and Dong, 2004; Truman et al., 2007; Vlot et al., 2009).SAR involves the generation of a mobile signal in the primary leaves that, upon translocation to the distal tissues, activates defense responses resulting in broad-spectrum resistance. The production of the mobile signal takes places within 3 to 6 h of avirulent pathogen inoculation in the primary leaves (Smith-Becker et al., 1998), and the inoculated leaf must remain attached for at least 4 h after inoculation for immunity to be induced in the systemic tissues (Rasmussen et al., 1991). Mutations compromising SA synthesis or impairing SA, JA, or auxin signaling abolish SAR (Durrant and Dong, 2004; Truman et al., 2007, 2010). SAR is also dependent on the SALICYLIC ACID-BINDING PROTEIN2 (SABP2)-catalyzed conversion of methyl SA to SA in the distal tissues (Kumar and Klessig, 2003). Recent studies have suggested that methyl SA is the mobile signal required to initiate SAR in distal tissues in tobacco (Nicotiana tabacum; Park et al., 2007) and Arabidopsis (Arabidopsis thaliana; Liu et al., 2010), although another group reported a disparity in their findings related to the role of methyl SA in Arabidopsis (Attaran et al., 2009). Notably, the time point of requirement of SABP2 activity (between 48 and 72 h post inoculation; Park et al., 2009) does not coincide with the early generation and/or translocation of the mobile signal into distal tissues (within 6 h post inoculation).The mutations acyl carrier protein4 (acp4), long-chain acyl-CoA synthetase2 (lacs2), and lacs9, which are impaired in fatty acid (FA)/lipid flux (Schnurr et al., 2004; Xia et al., 2009), also compromise SAR (Xia et al., 2009). Detailed characterization has shown that the SAR defect in acp4, lacs2, and lacs9 mutants correlates with their defective cuticles. Analysis of the SAR response in acp4 plants has shown that these plants can generate the mobile signal required for inducing SAR but are unable to respond to it. It is likely that the defective cuticle in these plants impairs their ability to perceive the SAR signal, because mechanical abrasion of cuticles disrupts SAR in wild-type plants (Xia et al., 2009). This SAR-disruptive effect of cuticle abrasion is highly specific, because it does not alter local defenses and hinders SAR only during the time frame during which the mobile signal is translocated to distal tissues.SAR is also compromised in plants that contain a mutation in glycerol-3-phosphate dehydrogenase (Nandi et al., 2004). The glycerol-3-phosphate dehydrogenase (GLY1) reduces dihydroxyacetone phosphate to generate glycerol-3-phosphate, an obligatory component and precursor for the biosynthesis of all plant glycerolipids. Consequently, a mutation in GLY1 results in reduced carbon flux through the prokaryotic pathway of lipid biosynthesis, which leads to a reduction in the hexadecatrienoic (16:3) FAs (Miquel et al., 1998; Kachroo et al., 2004). Carbon flux and SAR are also impaired in plants containing mutations in FATTY ACID DESATURASE7 (FAD7; Chaturvedi et al., 2008). The FAD7 enzyme desaturates 16:2 and 18:2 FA species present on plastidial lipids to 16:3 and 18:3, respectively. Consequently, the fad7 mutant plants accumulate significantly reduced levels of trienoic FAs (16:3 and 18:3). Compromised SAR in mutants affected in certain plastidial FA/lipid pathways has prompted the suggestion that plastidial FA/lipids participate in SAR (Chaturvedi et al., 2008). Such a tempting conclusion is also favored by the fact that SAR requires the DIR1-encoded nonspecific lipid transfer protein, which is required for the generation and/or translocation of the mobile signal (Maldonado et al., 2002). In addition, azelaic acid, a dicarboxylic acid, was recently shown to prime SA biosynthesis and thereby SAR (Jung et al., 2009). The fact that azelaic acid is derived from oleic acid, a FA well known for its role in defense (Kachroo et al., 2003, 2004, 2005, 2007, 2008; Chandra-Shekara et al., 2007; Jiang et al., 2009; Venugopal et al., 2009; Xia et al., 2009), further suggests that FA/lipids might participate in SAR.This study was undertaken to reexamine the role of the FA/lipid pathways in SAR and to determine the nature of the FA/lipid species mediating SAR in fad7-1 plants. Our results show that impaired FA/lipid flux is not associated with compromised SAR in fad7-1 plants but, rather, with an abnormal cuticle, which is the result of a nonallelic mutation in the GLABRA1 (GL1) gene. Besides GL1, other mutations affecting trichome formation also compromised cuticle and thereby SAR. A compensatory effect of exogenous GA on gl1 plants suggests that GA might participate in resistance to bacterial pathogens by restoring cuticle formation.     

RPG1-B-Derived Resistance to AvrB-Expressing Pseudomonas syringae Requires RIN4-Like Proteins in Soybean

Soybean (Glycine max) RPG1-B (for resistance to Pseudomonas syringae pv glycinea) mediates species-specific resistance to P. syringae expressing the avirulence protein AvrB, similar to the nonorthologous RPM1 in Arabidopsis (Arabidopsis thaliana). RPM1-derived signaling is presumably induced upon AvrB-derived modification of the RPM1-interacting protein, RIN4 (for RPM1-interacting 4). We show that, similar to RPM1, RPG1-B does not directly interact with AvrB but associates with RIN4-like proteins from soybean. Unlike Arabidopsis, soybean contains at least four RIN4-like proteins (GmRIN4a to GmRIN4d). GmRIN4b, but not GmRIN4a, complements the Arabidopsis rin4 mutation. Both GmRIN4a and GmRIN4b bind AvrB, but only GmRIN4b binds RPG1-B. Silencing either GmRIN4a or GmRIN4b abrogates RPG1-B-derived resistance to P. syringae expressing AvrB. Binding studies show that GmRIN4b interacts with GmRIN4a as well as with two other AvrB/RPG1-B-interacting isoforms, GmRIN4c and GmRIN4d. The lack of functional redundancy among GmRIN4a and GmRIN4b and their abilities to interact with each other suggest that the two proteins might function as a heteromeric complex in mediating RPG1-B-derived resistance. Silencing GmRIN4a or GmRIN4b in rpg1-b plants enhances basal resistance to virulent strains of P. syringae and the oomycete Phytophthora sojae. Interestingly, GmRIN4a- or GmRIN4b-silenced rpg1-b plants respond differently to AvrB-expressing bacteria. Although both GmRIN4a and GmRIN4b function to monitor AvrB in the presence of RPG1-B, GmRIN4a, but not GmRIN4b, negatively regulates AvrB virulence activity in the absence of RPG1-B.One of the myriad plant defense responses activated upon pathogen invasion is signaling induced via the activation of resistance (R) proteins. R gene-mediated resistance is generally activated in response to race-specific pathogen effectors, termed avirulence proteins (Avr), and often results in the development of a hypersensitive reaction at the site of pathogen entry (Dangl et al., 1996). The hypersensitive reaction is a form of programmed cell death that results in the formation of necrotic lesions around the site of pathogen entry and is thought to help prevent pathogen spread by confining it to the dead cells.A majority of the known R proteins contain conserved structural domains, including N-terminal coiled coil (CC) or Toll-interleukin 1 receptor (TIR)-like domains, central nucleotide-binding site (NBS), and C-terminal Leu-rich repeat (LRR) domains (Martin et al., 2003). While some R proteins “perceive” pathogen presence via direct physical interactions with the cognate Avr proteins (Scofield et al., 1996; Jia et al., 2000; Leister and Katagiri, 2000; Deslandes et al., 2003), several others likely do so indirectly. This led to the suggestion that R proteins monitor the presence of Avr proteins by “guarding” other host proteins targeted by the pathogen effector (Van der Biezen and Jones, 1998; Innes, 2004; Jones and Dangl, 2006). Avr proteins enhance pathogen virulence in genetic backgrounds lacking cognate R proteins by targeting components of the host basal defense machinery, including “guardee” proteins (Chang et al., 2000; Guttman and Greenberg, 2001; Chen et al., 2004, Kim et al., 2005b; Ong and Innes, 2006; van Esse et al., 2007; Shan et al., 2008; Xiang et al., 2008). However, some Avr proteins were found to also target host proteins that do not contribute to the virulence function of the effector (Shang et al., 2006; Shabab et al., 2008; Zhou and Chai, 2008; Zipfel and Rathjen, 2008). This led to the proposition that plants express “decoy” proteins that mimic Avr-guardee recognition in the presence of the R protein. This decoy model suggests that, unlike guardees, decoy proteins do not directly contribute to host basal immunity, such that Avr-derived alterations of decoys do not enhance pathogen virulence in plants lacking the R protein (van der Hoorn and Kamoun, 2008).A well-studied example of an indirect mode of effector recognition is that of the Arabidopsis (Arabidopsis thaliana) R protein, RPM1 (for resistance to Pseudomonas syringae pv maculicola 1). RPM1 mediates resistance against bacteria expressing two different Avr proteins, AvrRpm1 (AvrRpm1_PmaM6) and AvrB (AvrB1_Pgyrace4). Although RPM1 does not directly interact with either AvrRpm1 or AvrB, it does associate with RIN4 (for RPM1-interacting 4), which interacts with AvrRpm1 and AvrB. RIN4 is required for RPM1-induced resistance to AvrRpm1/AvrB-expressing P. syringae (Mackey et al., 2002). Both AvrRpm1 and AvrB induce the phosphorylation of RIN4, which is thought to induce RPM1-mediated resistance signaling. RIN4 also associates with a second Arabidopsis R protein, RPS2 (for resistance to P. syringae), which mediates resistance against P. syringae expressing AvrRpt2. RPS2-mediated signaling is activated when AvrRpt2 (AvrRpt2_PtoJL1065), a Cys protease, cleaves RIN4 (Axtell and Staskawicz, 2003; Mackey et al., 2003; Kim et al., 2005a). The AvrRpt2-triggered loss of RIN4 compromises RPM1-mediated resistance, because RIN4 is not available for phosphorylation (Ritter and Dangl, 1996; Axtell and Staskawicz, 2003; Mackey et al., 2003).The avirulence effector AvrB was first isolated from a P. syringae strain colonizing soybean (Glycine max) and used to identify the cognate resistance locus RPG1 in soybean (Staskawicz et al., 1987; Keen and Buzzell, 1991). This locus contains the RPG1-B (for resistance to P. syringae pv glycinea) gene, which encodes a CC-NBS-LRR protein conferring resistance to AvrB-expressing P. syringae in soybean (Bisgrove et al., 1994; Ashfield et al., 2004). Unlike RPM1, RPG1-B does not confer specificity to AvrRpm1 (Ashfield et al., 1995). However, as in Arabidopsis, the soybean RPG1-B-derived hypersensitive reaction to AvrB-expressing bacteria is inhibited by the presence of AvrRpt2-expressing bacteria (Axtell and Staskawicz, 2003, Mackey et al., 2003; Ashfield et al., 2004). This suggests that RPG1-B and RPM1 might utilize common signaling components even though they share very limited sequence identity. Therefore, we investigated the possible involvement of RIN4-like proteins in RPG1-B-mediated resistance signaling. In addition to Arabidopsis, RIN4-like proteins have also been identified in tomato (Solanum lycopersicum) and lettuce (Lactuca sativa; Jeuken et al., 2009; Luo et al., 2009). In tomato, the NBS-LRR protein, Prf (for Pseudomonas resistance and fenthion sensitivity), and its interacting protein kinase, Pto, mediate resistance to the AvrPto (AvrPto1_PtoJL1065)-expressing strain of P. syringae (Scofield et al., 1996; Tang et al., 1996; Kim et al., 2002; Mucyn et al., 2006). AvrPto binds RIN4 proteins from both Arabidopsis (AtRIN4) and tomato (SlRIN4). Similar to AvrRpt2, AvrPto induces the proteolysis of RIN4, albeit only in the presence of Pto and Prf (Luo et al., 2009). However, in the case of AvrPto, degradation of RIN4 is the result of induced proteolytic activity in the plant, rather than that of AvrPto itself. In Lactuca (lettuce) species, the L. saligna RIN4 allele was recently shown to be essential for resistance to an avirulent strain of the downy mildew pathogen, Bremia lactucae (Jeuken et al., 2009).Here, we report that two functionally nonredundant isoforms of soybean RIN4 (GmRIN4) function in RPG1-B-derived resistance as well as in the virulence activity of AvrB in the absence of RPG1-B.     

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.     

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.     

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.     

Glycerol-3-phosphate and systemic immunity

Glycerol-3-phosphate (G3P), a conserved three-carbon sugar, is an obligatory component of energy-producing reactions including glycolysis and glycerolipid biosynthesis. G3P can be derived via the glycerol kinase-mediated phosphorylation of glycerol or G3P dehydrogenase (G3Pdh)-mediated reduction of dihydroxyacetone phosphate. Previously, we showed G3P levels contribute to basal resistance against the hemibiotrophic pathogen, Colletotrichum higginsianum. Inoculation of Arabidopsis with C. higginsianum correlated with an increase in G3P levels and a concomitant decrease in glycerol levels in the host. Plants impaired in GLY1 encoded G3Pdh accumulated reduced levels of G3P after pathogen inoculation and showed enhanced susceptibility to C. higginsianum. Recently, we showed that G3P is also a potent inducer of systemic acquired resistance (SAR) in plants. SAR is initiated after a localized infection and confers whole-plant immunity to secondary infections. SAR involves generation of a signal at the site of primary infection, which travels throughout the plants and alerts the un-infected distal portions of the plant against secondary infections. Plants unable to synthesize G3P are defective in SAR and exogenous G3P complements this defect. Exogenous G3P also induces SAR in the absence of a primary pathogen. Radioactive tracer experiments show that a G3P derivative is translocated to distal tissues and this requires the lipid transfer protein, DIR1. Conversely, G3P is required for the translocation of DIR1 to distal tissues. Together, these observations suggest that the cooperative interaction of DIR1 and G3P mediates the induction of SAR in plants.Glycerol-3-phosphate (G3P) is an obligatory component of energy-producing reactions including glycolysis and glycerolipid biosynthesis.^1,2 G3P levels in the plant are regulated by enzymes directly/indirectly involved in G3P biosynthesis, as well as those involved in G3P catabolism. G3P is synthesized via the glycerol kinase (GK)-mediated phosphorylation of glycerol,³ or the G3P dehydrogenase (G3Pdh)-mediated reduction of dihydroxyacetone phosphate (DHAP)⁴ (Fig. 1). DHAP is derived from glycolysis via triosephosphate isomerase activity on glyceraldehyde-3-phosphate, or from the conversion of glycerol to dihydroxacetone (DHA) by glycerol dehydrogenase (Glydh) followed by phosphorylation of DHA to DHAP by DHA kinase (DHAK). G3P is catabolized either upon its conversion to glycerol by glycerol-3-phoshatase (GPP) or its utilization in glycerolipid/triacylglycerol biosynthesis. In Arabidopsis, the total G3P pool is derived from the activities of five G3Pdh isoforms and one GK isoform present in three cellular locations^5-9; GK and two of the G3Pdh isoforms are present in the cytoplasm, two other G3Pdh isoforms localize to plastids, and one to the mitochondria. One of the plastid localized G3Pdh isoforms, designated GLY1, was previously shown to be required for glycerolipid biosynthesis; a mutation in GLY1 compromised lipids synthesized via the plastidal pathway of lipid biosynthesis. The fact that exogenous application of glycerol to gly1 plants normalizes plastidal lipid levels¹⁰ and that GLY1 encodes a G3Pdh⁴ suggests that the G3P pool generated via the GLY1 catalyzed reaction is required for the biosynthesis of plastidal lipids. Intriguingly, unlike GLY1, neither the chloroplastic, nor the two cytosolic isoforms of G3Pdh, contribute to plastidal and/or extraplastidal lipid biosynthesis.⁹Open in a separate windowFigure 1.A condensed scheme of glycerol-3-phosphate metabolism in plants. Glycerol is phosphorylated to glycerol-3-phosphate (G3P) by glycerol kinase (GK; GLI1). G3P can also be generated by G3P dehydrogenase (G3Pdh) via the reduction of dihydroxyacetone phosphate (DHAP). DHAP is derived from glycolysis via triosephosphate isomerase (TPI) activity on glyceraldehyde-3-phosphate (Gld-3-P), or from the conversion of glycerol to dihydroxacetone (DHA) by glycerol dehydrogenase (Glydh) followed by phosphorylation of DHA to DHAP by DHA kinase (DHAK). G3Pdh isoforms are present in both the cytosol and the plastids (represented by the oval). GLY1 is one of the two plastidial G3Pdh isoforms that plays an important role in plastidial glycerolipid biosynthesis. In the plastids, G3P is acylated with oleic acid (18:1) by the ACT1-encoded G3P acyltransferase. This ACT1-utilized 18:1 is derived from the stearoyl-acyl carrier protein (ACP)-desaturase (SACPD)-catalyzed desaturation of stearic acid (18:0). The 18:1-ACP generated by SACPD either enters the prokaryotic lipid biosynthetic pathway through acylation of G3P or is exported out (dotted line) of the plastids as a coenzyme A (CoA)-thioester to enter the eukaryotic lipid biosynthetic pathway. Membranous fatty acid desaturases (FAD) catalyze desaturation of FAs present on membranous glycerolipids. Other abbreviations used are: GL, glycerolipid; FAS, fatty acid synthase; ACC, acetyl-CoA carboxylase; Lyso-PA, acyl-G3P; PA, phosphatidic acid; PG, phosphatidylglycerol; MGDG, monogalactosyldiacylglycerol; DGDG, digalactosyldiacylglycerol; SL, sulfolipid; DAG, diacylglycerol.For glycerolipid biosynthesis, G3P is first acylated with the fatty acid (FA) oleic acid (18:1), to form lyso-phosphatidic acid (lyso-PA) via the activity of the soluble G3P acyltransferase (GPAT) encoded by the ACT1 gene in Arabidopsis¹¹ (Fig. 1). 18:1 in turn is derived from the saturated FA, stearic acid (18:0), via the activity of soluble stearoyl-acyl carrier protein desaturases (SACPD),¹² which introduce a single cis double bond in 18:0. The 18:1 generated via this reaction is either exported out of the plastids or acylated at the sn-1 position of G3P. Previously, we have shown that 18:1 levels are important regulators of plant defense signaling. In Arabidopsis, 18:1 is synthesized via the SSI2/FAB2-encoded SACPD,¹² which uses 18:0 as a substrate. A mutation in SSI2 results in the accumulation of 18:0 and a reduction in 18:1 levels. The mutant plants show stunting, spontaneous lesion formation, constitutive PR gene expression, and enhanced resistance to bacterial and oomycete pathogens.^4,12-17 Characterization of ssi2 suppressor mutants has shown that the altered defense-related phenotypes are the result of the reduction in the levels of the unsaturated FA, 18:1, which causes induction of several resistance (R) genes.^4,14,18,19 Restoration of 18:1 levels, via mutations in ACT1,¹⁴ GLY1⁴ or ACP4,¹⁸ normalizes R gene expression in ssi2 plants. The low 18:1-mediated induction of R gene expression and the associated defense signaling can also be suppressed by simultaneous mutations in EDS1 and the genes governing salicylic acid (SA) biosynthesis (SID2, EDS5).¹⁹ Furthermore, the functional redundancy between EDS1 and SA likely masks the requirement for EDS1 by several coiled coil (CC)- nucleotide binding site (NBS)- leucine rich repeat (LRR) proteins,¹⁹ previously thought to function independent of EDS1.²⁰ Thus, the reliance on EDS1 for signaling mediated by CC-NBS-LRR proteins becomes evident only in the absence of SA.The plastidal 18:1 levels are also regulated via the chloroplastic G3P pool and vice-versa. However, 18:1 and G3P appear to function distinctly in defense signaling. For example, G3P levels are important for basal defense against the hemibiotrophic fungus, Colletotrichum higginsianum.^21,22 Genetic mutations affecting G3P synthesis in Arabidopsis enhance susceptibility to C. higginsianum. Conversely, plants accumulating increased G3P show enhanced resistance. More recently, we demonstrated roles for G3P in R-mediated defense leading to systemic acquired resistance (SAR).⁹ R-mediated defense against the avirulent bacterial pathogen P. syringae is associated with a rapid increase in G3P levels; G3P levels peak within 6 h of inoculation with avirulent bacteria (avrRpt2), in resistant plants expressing the R gene RPS2. Strikingly, accumulation of G3P, in the infected and systemic tissues, precedes the accumulation of other metabolites known to be essential for SAR; SA,^23,24 jasmonic acid (JA)²⁵ and azelaic acid (AA)²⁶ accumulated at least 24 h post pathogen inoculation. Furthermore, mutants defective in G3P synthesis are compromised in SAR but accumulated normal levels of SA, AA, and JA. Compromised SAR in G3P deficient mutants was restored by exogenous application of G3P, thus arguing a role for G3P in SAR. This was further supported by the fact that exogenous G3P induced SAR in the absence of the primary pathogen in both Arabidopsis and soybean.⁹ That fact that G3P is a conserved metabolite common to prokaryotes, plants, and humans further corroborates the conserved nature of SAR signaling. Interestingly, although exogenous G3P did not induce SA biosynthesis, SAR conferred by exogenous G3P was dependent on SA. These results suggest that the onset and/or establishment of SAR likely requires basal, but not induced levels of SA, in the distal tissues. It is possible that the relatively small increase in SA observed in the systemic tissues during SAR is an indirect response that contributes to generalized resistance, rather than SAR itself. Interestingly, both G3P conferred SAR, and the systemic movement of G3P were dependent on the lipid transfer protein, DIR1, a well-known positive regulator of SAR.²⁷ Conversely, systemic movement of DIR1 required G3P. These findings did not correlate with the fact that G3P is cytosolic while DIR1 was a predicted apoplastic protein. To resolve this issue, we studied the localization of DIR1, and found that it is in fact a symplastic protein. The symplastic location of DIR1 was further corroborated when GFP fused to the signal peptide from DIR1 localized to the endoplasmic reticulum, rather than the typical cytoplasmic and nuclear location of GFP (Fig. 2). These results suggested that the symplastic movement of DIR1 is likely critical for SAR, and supported the facts that G3P and DIR1 are interdependent for translocation to systemic tissues. However, these findings could not explain how a lipid transfer-like protein might associate with the phosphorylated sugar G3P, to move systemically. Analysis of G3P in the leaf extracts showed that it was derivatized into an unknown compound before/during translocation. It is likely that the G3P derivative has a lipid moiety via which it associates with DIR1 for transfer. In summary, we showed that DIR1 together with a G3P-derived compound are sufficient for the induction of SAR in wild type plants. Our findings provide strong evidence in support of a direct defense-signaling role for G3P and warranty further analysis of its metabolic pathway(s) for their role(s) in various modes of plant defense.Open in a separate windowFigure 2.Confocal micrograph showing localization of GFP fused to DIR1 transit peptide (TP) or GFP alone in Nicotiana benthamiana plants expressing RFP-tagged nuclear histone protein H2B. Arrow indicates nucleus, arrowhead indicates endoplasmic reticulum.     

Silencing genes encoding omega-3 fatty acid desaturase alters seed size and accumulation of Bean pod mottle virus in soybean

Omega-3 fatty acid desaturase (FAD3)-catalyzed conversion of linoleic acid to linolenic acid (18:3) is an important step for the biosynthesis of fatty acids as well as the phytohormone jasmonic acid (JA) in plants. We report that silencing three microsomal isoforms of GmFAD3 enhanced the accumulation of Bean pod mottle virus (BPMV) in soybean. The GmFAD3-silenced plants also accumulated higher levels of JA, even though they contained slightly reduced levels of 18:3. Consequently, the GmFAD3-silenced plants expressed JA-responsive pathogenesis-related genes constitutively and exhibited enhanced susceptibility to virulent Pseudomonas syringae. Increased accumulation of BPMV in GmFAD3-silenced plants was likely associated with their JA levels, because exogenous JA application also increased BPMV accumulation. The JA-derived increase in BPMV levels was likely not due to repression of salicylic acid (SA)-derived signaling because the GmFAD3-silenced plants were enhanced in SA-dependent defenses. Furthermore, neither exogenous SA application nor silencing the SA-synthesizing phenylalanine ammonia lyase gene altered BPMV levels in soybean. In addition to the altered defense responses, the GmFAD3-silenced plants also produced significantly larger and heavier seed. Our results indicate that loss of GmFAD3 enhances JA accumulation and, thereby, susceptibility to BPMV in soybean.