Genetic analysis of acd6-1 reveals complex defense networks and leads to identification of novel defense genes in Arabidopsis

Pathogen infection leads to the activation of defense signaling networks in plants. To study these networks and the relationships between their components, we introduced various defense mutations into acd6-1 , a constitutive gain-of-function Arabidopsis mutant that is highly disease resistant. acd6-1 plants show spontaneous cell death, reduced stature, and accumulate high levels of camalexin (an anti-fungal compound) and salicylic acid (SA; a signaling molecule). Disruption of several defense genes revealed that in acd6-1 , SA levels/signaling were positively correlated with the degree of disease resistance and defense gene expression. Salicylic acid also modulates the severity of cell death. However, accumulation of camalexin in acd6-1 is largely unaffected by reducing the level of SA. In addition, acd6-1 shows ethylene- and jasmonic acid-mediated signaling that is antagonized and therefore masked by the presence of SA. Mutant analysis revealed a new relationship between the signaling components NPR1 and PAD4 and also indicated that multiple defense pathways were required for phenotypes conferred by acd6-1 . In addition, our data confirmed that the size of acd6-1 was inversely correlated with SA levels/signaling. We exploited this unique feature of acd6-1 to identify two genes disrupted in acd6-1 suppressor ( sup ) mutants: one encodes a known SA biosynthetic component (SID2) and the other encodes an uncharacterized putative metalloprotease (At5g20660). Taken together, acd6-1 is a powerful tool not only for dissecting defense regulatory networks but also for discovering novel defense genes.     

The gain-of-function Arabidopsis acd6 mutant reveals novel regulation and function of the salicylic acid signaling pathway in controlling cell death, defenses, and cell growth.

We isolated a dominant gain-of-function Arabidopsis mutant, accelerated cell death 6 (acd6), with elevated defenses, patches of dead and enlarged cells, reduced stature, and increased resistance to Pseudomonas syringae. The acd6-conferred phenotypes are suppressed by removing a key signaling molecule, salicylic acid (SA), by using the nahG transgene, which encodes SA hydroxylase. This suppression includes phenotypes that are not induced by application of SA to wild-type plants, indicating that SA acts with a second signal to cause many acd6-conferred phenotypes. acd6-nahG plants show hyperactivation of all acd6-conferred phenotypes after treatment with a synthetic inducer of the SA pathway, benzo(1,2, 3)thiadiazole-7-carbothioic acid (BTH), suggesting that SA acts with and also modulates the levels and/or activity of the second defense signal. acd6 acts partially through a NONEXPRESSOR OF PR 1 (NPR1) gene-independent pathway that activates defenses and confers resistance to P. syringae. Surprisingly, BTH-treated acd6-nahG plants develop many tumor-like abnormal growths, indicating a possible role for SA in modulating cell growth.     

The immune components ENHANCED DISEASE SUSCEPTIBILITY 1 and PHYTOALEXIN DEFICIENT 4 are required for cell death caused by overaccumulation of ceramides in Arabidopsis

Sphingolipids have key functions in plant membrane structure and signaling. Perturbations of plant sphingolipid metabolism often induce cell death and salicylic acid (SA) accumulation; SA accumulation, in turn, promotes sphingolipid metabolism and further cell death. However, the underlying molecular mechanisms remain unclear. Here, we show that the Arabidopsis thaliana lipase-like protein ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) and its partner PHYTOALEXIN DEFICIENT 4 (PAD4) participate in sphingolipid metabolism and associated cell death. The accelerated cell death 5 (acd5) mutants accumulate ceramides due to a defect in ceramide kinase and show spontaneous cell death. Loss of function of EDS1, PAD4 or SALICYLIC ACID INDUCTION DEFICIENT 2 (SID2) in the acd5 background suppressed the acd5 cell death phenotype and prevented ceramide accumulation. Treatment with the SA analogue benzothiadiazole partially restored sphingolipid accumulation in the acd5 pad4 and acd5 eds1 double mutants, showing that the inhibitory effect of the pad4-1 and eds1-2 mutations on acd5-conferred sphingolipid accumulation partly depends on SA. Moreover, the pad4-1 and eds1-2 mutations substantially rescued the susceptibility of the acd5 mutant to Botrytis cinerea. Consistent with this, B. cinerea-induced ceramide accumulation requires PAD4 or EDS1. Finally, examination of plants overexpressing the ceramide synthase gene LAG1 HOMOLOGUE2 suggested that EDS1, PAD4 and SA are involved in long-chain ceramide metabolism and ceramide-associated cell death. Collectively, our observations reveal that EDS1 and PAD4 mediate ceramide (especially long-chain ceramide) metabolism and associated cell death, by SA-dependent and SA-independent pathways.     

Salicylic acid-mediated cell death in the Arabidopsis len3 mutant

The Arabidopsis lesion initiation 3 (len3) mutant develops lesions on leaves without pathogen attack. len3 plants exhibit stunted growth, constitutively express pathogenesis-related (PR) genes, PR-1, PR-2, and PR-5, and accumulate elevated levels of salicylic acid (SA). Furthermore, len3 is a semidominant, male gametophytic lethal mutation with partial defects in female gametophytic development. To determine the signaling pathway activated in len3 plants, we crossed the len3 plants with nahG, npr1-1, and pad4-1 plants and analyzed the phenotypes of the double mutants. The len3-conferred phenotypes, including cell death and PR-1 expressions, were suppressed in the double mutants. Thus SA, NPR1, and PAD4 are required for the phenotypes. However, none of these double mutants could completely suppress the len3-conferred stunted growth. This result suggests that an SA-, NPR1-, and PAD4-independent pathway is also involved in the phenotype. Treatment with BTH (benzo(1,2,3)thiadiazole-7-carbothioic acid), an SA analog, induced cell death in len3 nahG plants but not in len3 npr1 or len3 pad4 plants, suggesting the involvement of the PAD4-dependent but SA-independent second signal pathway in cell death in len3 plants.     

Salicylic acid-independent ENHANCED DISEASE SUSCEPTIBILITY1 signaling in Arabidopsis immunity and cell death is regulated by the monooxygenase FMO1 and the Nudix hydrolase NUDT7

Arabidopsis thaliana ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) controls defense activation and programmed cell death conditioned by intracellular Toll-related immune receptors that recognize specific pathogen effectors. EDS1 is also needed for basal resistance to invasive pathogens by restricting the progression of disease. In both responses, EDS1, assisted by its interacting partner, PHYTOALEXIN-DEFICIENT4 (PAD4), regulates accumulation of the phenolic defense molecule salicylic acid (SA) and other as yet unidentified signal intermediates. An Arabidopsis whole genome microarray experiment was designed to identify genes whose expression depends on EDS1 and PAD4, irrespective of local SA accumulation, and potential candidates of an SA-independent branch of EDS1 defense were found. We define two new immune regulators through analysis of corresponding Arabidopsis loss-of-function insertion mutants. FLAVIN-DEPENDENT MONOOXYGENASE1 (FMO1) positively regulates the EDS1 pathway, and one member (NUDT7) of a family of cytosolic Nudix hydrolases exerts negative control of EDS1 signaling. Analysis of fmo1 and nudt7 mutants alone or in combination with sid2-1, a mutation that severely depletes pathogen-induced SA production, points to SA-independent functions of FMO1 and NUDT7 in EDS1-conditioned disease resistance and cell death. We find instead that SA antagonizes initiation of cell death and stunting of growth in nudt7 mutants.     

The role of salicylic acid in the induction of cell death in Arabidopsis acd11

Salicylic acid (SA) is implicated in the induction of programmed cell death (PCD) associated with pathogen defense responses because SA levels increase in response to PCD-inducing infections, and PCD development can be inhibited by expression of salicylate hydroxylase encoded by the bacterial nahG gene. The acd11 mutant of Arabidopsis (Arabidopsis thaliana L. Heynh.) activates PCD and defense responses that are fully suppressed by nahG. To further study the role of SA in PCD induction, we compared phenotypes of acd11/nahG with those of acd11/eds5-1 and acd11/sid2-2 mutants deficient in a putative transporter and isochorismate synthase required for SA biosynthesis. We show that sid2-2 fully suppresses SA accumulation and cell death in acd11, although growth inhibition and premature leaf chlorosis still occur. In addition, application of exogenous SA to acd11/sid2-2 is insufficient to restore cell death. This indicates that isochorismate-derived compounds other than SA are required for induction of PCD in acd11 and that some acd11 phenotypes require NahG-degradable compounds not synthesized via isochorismate.     

Structure-function analysis of the plasma membrane- localized Arabidopsis defense component ACD6

The ACCELERATED CELL DEATH 6 (ACD6) protein, composed of an ankyrin-repeat domain and a predicted transmembrane region, is a necessary positive regulator of Arabidopsis defenses. ACD6 overexpression confers enhanced disease resistance by priming stronger and quicker defense responses during pathogen infection, plant development or treatment with an agonist of the key defense regulator salicylic acid (SA). Modulation of ACD6 affects both SA-dependent and SA-independent defenses. ACD6 localizes to the plasma membrane and is an integral membrane protein with a cytoplasmic ankyrin domain. An activated version of ACD6 with a predicted transmembrane helix mutation called ACD6-1 has the same localization and overall topology as the wild-type protein. A genetic screen for mutants that suppress acd6-1-conferred phenotypes identified 17 intragenic mutations of ACD6. The majority of these mutations reside in the ankyrin domain and in predicted transmembrane helices, suggesting that both ankyrin and transmembrane domains are important for ACD6 function. One mutation (S638F) also identified a key residue in a putative loop between two transmembrane helices. This mutation did not alter the stability or localization of ACD6, suggesting that S635 is a critical residue for ACD6 function. Based on structural modeling, two ankyrin domain mutations are predicted to be in surface-accessible residues. As ankyrin repeats are protein interaction modules, these mutations may disrupt protein-protein interactions. A plausible scenario is that information exchange between the ankyrin and transmembrane domains is involved in activating defense signaling.     

Arabidopsis local resistance to Botrytis cinerea involves salicylic acid and camalexin and requires EDS4 and PAD2, but not SID2, EDS5 or PAD4

Salicylic acid (SA) is an important regulator of plant defense responses, and a variety of Arabidopsis mutants impaired in resistance against bacterial and fungal pathogens show defects in SA accumulation, perception, or signal transduction. Nevertheless, the role of SA-dependent defense responses against necrotrophic fungi is currently unclear. We determined the susceptibility of a set of previously identified Arabidopsis mutants impaired in defense responses to the necrotrophic fungal pathogen Botrytis cinerea. The rate of development of B. cinerea disease symptoms on primary infected leaves was affected by responses mediated by the genes EIN2, JAR1, EDS4, PAD2, and PAD3, but was largely independent of EDS5, SID2/ICS1, and PAD4. Furthermore, plants expressing a nahG transgene or treated with a phenylalanine ammonia lyase (PAL) inhibitor showed enhanced symptoms, suggesting that SA synthesized via PAL, and not via isochorismate synthase (ICS), mediates lesion development. In addition, the degree of lesion development did not correlate with defensin or PR1 expression, although it was partially dependent upon camalexin accumulation. Although npr1 mutant leaves were normally susceptible to B. cinerea infection, a double ein2 npr1 mutant was significantly more susceptible than ein2 plants, and exogenous application of SA decreased B. cinerea lesion size through an NPR1-dependent mechanism that could be mimicked by the cpr1 mutation. These data indicate that local resistance to B. cinerea requires ethylene-, jasmonate-, and SA-mediated signaling, that the SA affecting this resistance does not require ICS1 and is likely synthesized via PAL, and that camalexin limits lesion development.     

A recessive mutation in the Arabidopsis SSI2 gene confers SA- and NPR1-independent expression of PR genes and resistance against bacterial and oomycete pathogens

The Arabidopsis thaliana NPR1 gene is required for salicylic acid (SA)-induced expression of pathogenesis-related (PR) genes and systemic acquired resistance. However, loss-of-function mutations in NPR1 do not confer complete loss of PR gene expression or disease resistance. Thus these responses also can be activated via an NPR1-independent pathway that currently remain to be elucidated. The ssi2-1 mutant, identified in a genetic screen for suppressors of npr1-5, affects signaling through the NPR1-independent defense pathway(s). In comparison with the wild-type (SSI2 NPR1) plants and the npr1-5 mutant (SSI2 npr1-5), the ssi2-1 npr1-5 double mutant and the ssi2-1 NPR1 single mutant constitutively express PR genes [PR-1, BGL2 (PR-2) and PR-5]; accumulate elevated levels of SA; spontaneously develop lesions; and possess enhanced resistance to a virulent strain of Peronospora parasitica. The ssi2-1 mutation also confers enhanced resistance to Pseudomonas syringae pv. tomato (Pst); however, this is accomplished primarily via an NPR1-dependent pathway. Analysis of ssi2-1 NPR1 nahG and ssi2-1 npr1-5 nahG plants revealed that elevated SA levels were not essential for the ssi2-1-conferred phenotypes. However, expression of the nahG transgene did reduce the intensity of some ssi2-1-conferred phenotypes, including PR-1 expression, and disease resistance. Based on these results, SSI2 or an SSI2-generated signal appears to modulate signaling of an SA-dependent, NPR1-independent defense pathway, or an SA- and NPR1-independent defense pathway.     

ACD6, a novel ankyrin protein, is a regulator and an effector of salicylic acid signaling in the Arabidopsis defense response

The previously reported Arabidopsis dominant gain-of-function mutant accelerated cell death6-1 (acd6-1) shows spontaneous cell death and increased disease resistance. acd6-1 also confers increased responsiveness to the major defense signal salicylic acid (SA). To further explore the role of ACD6 in the defense response, we cloned and characterized the gene. ACD6 encodes a novel protein with putative ankyrin and transmembrane regions. It is a member of one of the largest uncharacterized gene families in higher plants. Steady state basal expression of ACD6 mRNA required light, SA, and an intact SA signaling pathway. Additionally, ACD6 mRNA levels were increased in the systemic, uninfected tissue of Pseudomonas syringae-infected plants as well as in plants treated with the SA agonist benzothiazole (BTH). A newly isolated ACD6 loss-of-function mutant was less responsive to BTH and upon P. syringae infection had reduced SA levels and increased susceptibility. Conversely, plants overexpressing ACD6 showed modestly increased SA levels, increased resistance to P. syringae, and BTH-inducible and/or a low level of spontaneous cell death. Thus, ACD6 is a necessary and dose-dependent activator of the defense response against virulent bacteria and can activate SA-dependent cell death.     

Uncoupling salicylic acid-dependent cell death and defense-related responses from disease resistance in the Arabidopsis mutant acd5

Salicylic acid (SA) is required for resistance to many diseases in higher plants. SA-dependent cell death and defense-related responses have been correlated with disease resistance. The accelerated cell death 5 mutant of Arabidopsis provides additional genetic evidence that SA regulates cell death and defense-related responses. However, in acd5, these events are uncoupled from disease resistance. acd5 plants are more susceptible to Pseudomonas syringae early in development and show spontaneous SA accumulation, cell death, and defense-related markers later in development. In acd5 plants, cell death and defense-related responses are SA dependent but they do not confer disease resistance. Double mutants with acd5 and nonexpressor of PR1, in which SA signaling is partially blocked, show greatly attenuated cell death, indicating a role for NPR1 in controlling cell death. The hormone ethylene potentiates the effects of SA and is important for disease symptom development in Arabidopsis. Double mutants of acd5 and ethylene insensitive 2, in which ethylene signaling is blocked, show decreased cell death, supporting a role for ethylene in cell death control. We propose that acd5 plants mimic P. syringae-infected wild-type plants and that both SA and ethylene are normally involved in regulating cell death during some susceptible pathogen infections.     

Suppression and Restoration of Lesion Formation in Arabidopsis lsd Mutants

Systemic acquired resistance (SAR) is a broad-spectrum, systemic defense response that is activated in many plant species after pathogen infection. We have previously described Arabidopsis mutants that constitutively express SAR and concomitantly develop lesions simulating disease (lsd). Here, we describe two new mutants, lsd6 and lsd7, that develop spontaneous necrotic lesions and possess elevated levels of salicylic acid (SA) as well as heightened disease resistance, similar to the previously characterized lsd and accelerated cell death (acd2) mutants. Genetic analysis of lsd6 and lsd7 showed that the mutant phenotypes segregated as simple dominant traits. When crossed with transgenic Arabidopsis plants containing the SA-degrading enzyme salicylate hydroxylase, the F1 progeny showed suppression of both SAR gene expression and resistance. In addition, salicylate hydroxylase suppressed lesion formation in the F1 progeny, suggesting that SA or some SA-dependent process may have a role in pathogen-associated cell death. Surprisingly, lesions were restored in the lsd6 F1 progeny after the application of either 2,6-dichloroisonicotinic acid or SA. Lesions were not restored by treatment with either compound in the lsd7 F1 plants. Our findings demonstrate that steps early in the signal transduction pathway leading to SAR and disease resistance are potentiated by later events, suggesting feedback control of lesion formation.     

The disease resistance signaling components EDS1 and PAD4 are essential regulators of the cell death pathway controlled by LSD1 in Arabidopsis

Specific recognition of pathogens is mediated by plant disease resistance (R) genes and translated into a successful defense response. The extent of associated hypersensitive cell death varies from none to an area encompassing cells surrounding an infection site, depending on the R gene activated. We constructed double mutants in Arabidopsis between positive regulators of R function and a negative regulator of cell death, LSD1, to address whether genes required for normal R function also regulate the runaway cell death observed in lsd1 mutants. We report here that EDS1 and PAD4, two signaling genes that mediate some but not all R responses, also are required for runaway cell death in the lsd1 mutant. Importantly, this novel function of EDS1 and PAD4 is operative when runaway cell death in lsd1 is initiated through an R gene that does not require EDS1 or PAD4 for disease resistance. NDR1, another component of R signaling, also contributes to the control of plant cell death. The roles of EDS1 and PAD4 in regulating lsd1 runaway cell death are related to the interpretation of reactive oxygen intermediate-derived signals at infection sites. We further demonstrate that the fate of superoxide at infection sites is different from that observed at the leading margins of runaway cell death lesions in lsd1 mutants.     

Plant immunity: the EDS1 regulatory node

ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) and its interacting partner, PHYTOALEXIN DEFICIENT 4 (PAD4), constitute a regulatory hub that is essential for basal resistance to invasive biotrophic and hemi-biotrophic pathogens. EDS1 and PAD4 are also recruited by Toll-Interleukin-1 receptor (TIR)-type nucleotide binding-leucine rich repeat (NB-LRR) proteins to signal isolate-specific pathogen recognition. Recent work points to a fundamental role of EDS1 and PAD4 in transducing redox signals in response to certain biotic and abiotic stresses. These intracellular proteins are important activators of salicylic acid (SA) signaling and also mediate antagonism between the jasmonic acid (JA) and ethylene (ET) defense response pathways. EDS1 forms several molecularly and spatially distinct complexes with PAD4 and a newly discovered in vivo signaling partner, SENESCENCE ASSOCIATED GENE 101 (SAG101). Together, EDS1, PAD4 and SAG101 provide a major barrier to infection by both host-adapted and non-host pathogens.     

Arabidopsis sfd mutants affect plastidic lipid composition and suppress dwarfing, cell death, and the enhanced disease resistance phenotypes resulting from the deficiency of a fatty acid desaturase

A loss-of-function mutation in the Arabidopsis SSI2/FAB2 gene, which encodes a plastidic stearoyl-acyl-carrier protein desaturase, has pleiotropic effects. The ssi2 mutant plant is dwarf, spontaneously develops lesions containing dead cells, accumulates increased salicylic acid (SA) levels, and constitutively expresses SA-mediated, NPR1-dependent and -independent defense responses. In parallel, jasmonic acid-regulated signaling is compromised in the ssi2 mutant. In an effort to discern the involvement of lipids in the ssi2-conferred developmental and defense phenotypes, we identified suppressors of fatty acid (stearoyl) desaturase deficiency (sfd) mutants. The sfd1, sfd2, and sfd4 mutant alleles suppress the ssi2-conferred dwarfing and lesion development, the NPR1-independent expression of the PATHOGENESIS-RELATED1 (PR1) gene, and resistance to Pseudomonas syringae pv maculicola. The sfd1 and sfd4 mutant alleles also depress ssi2-conferred PR1 expression in NPR1-containing sfd1 ssi2 and sfd4 ssi2 plants. By contrast, the sfd2 ssi2 plant retains the ssi2-conferred high-level expression of PR1. In parallel with the loss of ssi2-conferred constitutive SA signaling, the ability of jasmonic acid to activate PDF1.2 expression is reinstated in the sfd1 ssi2 npr1 plant. sfd4 is a mutation in the FAD6 gene that encodes a plastidic omega6-desaturase that is involved in the synthesis of polyunsaturated fatty acid-containing lipids. Because the levels of plastid complex lipid species containing hexadecatrienoic acid are depressed in all of the sfd ssi2 npr1 plants, we propose that these lipids are involved in the manifestation of the ssi2-conferred phenotypes.     

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.     

Ethylene and jasmonic acid signaling affect the NPR1-independent expression of defense genes without impacting resistance to Pseudomonas syringae and Peronospora parasitica in the Arabidopsis ssi1 mutant

Salicylic acid (SA), ethylene, and jasmonic acid (JA) are important signaling molecules in plant defense to biotic stress. An intricate signaling network involving SA, ethylene, and JA fine tunes plant defense responses. SA-dependent defense responses in Arabidopsis thaliana are mediated through NPR1-dependent and -independent mechanisms. We have previously shown that activation of an NPR1-independent defense mechanism confers enhanced disease resistance and constitutive expression of the pathogenesis-related (PR) genes in the Arabidopsis ssi1 mutant. In addition, the ssi1 mutant constitutively expresses the defensin gene PDF1.2. Moreover, SA is required for the ssi1-conferred constitutive expression of PDF1.2 in addition to PR genes. Hence, the ssi1 mutant appears to target a step common to SA- and ethylene- or JA-regulated defense pathways. In the present study, we show that, in addition to SA, ethylene and JA signaling also are required for the ssi1-conferred constitutive expression of PDF1.2 and the NPR1-independent expression of PR-1. Furthermore, the ethylene-insensitive ein2 and JA-insensitive jar1 mutants enhance susceptibility of ssi1 plants to the necrotrophic fungus Botrytis cinerea. However, defects in either the ethylene- or JA-signaling pathways do not compromise ssi1-conferred resistance to the bacterial pathogen Pseudomonas synringae pv. maculicola and the oomycete pathogen Peronospora parasitica. Interestingly, ssi1 exhibits a marginal increase in the levels of ethylene and JA, suggesting that low endogenous levels of these phytohormones are sufficient to activate expression of defense genes. Taken together, our results indicate that although cross talk in ssi1 renders expression of ethylene- or JA-responsive defense genes sensitive to SA and vice versa, it does not affect downstream signaling leading to resistance.     

Multiple roles of WIN3 in regulating disease resistance, cell death, and flowering time in Arabidopsis

The salicylic acid (SA) regulatory gene HOPW1-1-INTERACTING3 (WIN3) was previously shown to confer resistance to the biotrophic pathogen Pseudomonas syringae. Here, we report that WIN3 controls broad-spectrum disease resistance to the necrotrophic pathogen Botrytis cinerea and contributes to basal defense induced by flg22, a 22-amino acid peptide derived from the conserved region of bacterial flagellin proteins. Genetic analysis indicates that WIN3 acts additively with several known SA regulators, including PHYTOALEXIN DEFICIENT4, NONEXPRESSOR OF PR GENES1 (NPR1), and SA INDUCTION-DEFICIENT2, in regulating SA accumulation, cell death, and/or disease resistance in the Arabidopsis (Arabidopsis thaliana) mutant acd6-1. Interestingly, expression of WIN3 is also dependent on these SA regulators and can be activated by cell death, suggesting that WIN3-mediated signaling is interconnected with those derived from other SA regulators and cell death. Surprisingly, we found that WIN3 and NPR1 synergistically affect flowering time via influencing the expression of flowering regulatory genes FLOWERING LOCUS C and FLOWERING LOCUS T. Taken together, our data reveal that WIN3 represents a novel node in the SA signaling networks to regulate plant defense and flowering time. They also highlight that plant innate immunity and development are closely connected processes, precise regulation of which should be important for the fitness of plants.     

Constitutive salicylic acid-dependent signaling in cpr1 and cpr6 mutants requires PAD4

Salicylic acid (SA)-dependent signaling controls activation of a set of plant defense mechanisms that are important for resistance to a variety of microbial pathogens. Many Arabidopsis mutants that display altered SA-dependent signaling have been isolated. We used double mutant analysis to determine the relative positions of the pad4, cpr1, cpr5, cpr6, dnd1 and dnd2 mutations in the signal transduction network leading to SA-dependent activation of defense gene expression and disease resistance. The pad4 mutation causes failure of SA accumulation in response to infection by certain pathogens, while the other mutations cause constitutively high levels of SA, defense gene expression and resistance. The cpr1 pad4, cpr5 pad4, cpr6 pad4, dnd1 pad4 and dnd2 pad4 double mutants were constructed and assayed for stature, presence of spontaneous lesions, resistance to Pseudomonas syringae and Peronospora parasitica, SA levels, expression of PAD4, PR-1 and PDF1.2, and accumulation of camalexin. We found that the effects of the cpr1 and cpr6 mutations on SA-dependent gene expression are completely dependent on PAD4 function. In contrast, SA accumulation in the lesion-mimic mutant cpr5 is partially PAD4-independent, while in dnd1 and dnd2 mutants it is completely PAD4-independent. A model describing a possible arrangement of activities in the signal transduction network is presented.