Syringae Pv Maculicola Research Articles

Chloride salt enhances plant resistance to biotic stresses.

Biotic stresses caused by bacterial and fungal pathogens damage crops; identifying treatments that enhance disease resistance provides important information for understanding plant defenses and sustainable agriculture. Salt stress affects crop yields worldwide; however, studies have focused on the toxic sodium ion, leaving the effects of the chloride ion unclear. In this study, we found that irrigation with a combination of chloride salts (MgCl2, CaCl2, and KCl) suppressed the cell death phenotype of the ceramide kinase mutant acd5. Chloride salt pre-irrigation also significantly limited the cell death caused by Pseudomonas syringae pv maculicola infection and inhibited the multiplication of this bacterial pathogen in a mechanism partially dependent on the salicylic acid pathway. Moreover, chloride salt pre-irrigation improved plant defenses against the fungal pathogen challenge, confining the lesion area caused by Botrytis cinerea infection. Furthermore, the growth of herbivorous larvae of Spodoptera exigua was retarded by feeding on chloride salt irrigated plants. Thus, our data suggest that treatment with Cl- increases broad spectrum resistance to biotic challenges.

Direct acetylation of a conserved threonine of RIN4 by the bacterial effector HopZ5 or AvrBsT activates RPM1-dependent immunity in Arabidopsis

Plant pathogenic bacteria deliver effectors into plant cells to suppress immunity and promote pathogen survival; however, these effectors can be recognized by plant disease resistance proteins to activate innate immunity. The bacterial acetyltransferase effectors HopZ5 and AvrBsT trigger immunity in Arabidopsis thaliana genotypes lacking SUPPRESSOR OF AVRBST-ELICITED RESISTANCE 1 (SOBER1). Using an Arabidopsis accession, Tscha-1, that naturally lacks functional SOBER1 but is unable to recognize HopZ5, we demonstrated that RESISTANCE TO P. SYRINGAE PV MACULICOLA 1 (RPM1) and RPM1-INTERACTING PROTEIN 4 (RIN4) are indispensable for HopZ5- or AvrBsT-triggered immunity. Remarkably, T166 of RIN4, the phosphorylation of which is induced by AvrB and AvrRpm1, is directly acetylated by HopZ5 and AvrBsT. Furthermore, we demonstrated that the acetylation of RIN4 T166 is required and sufficient for HopZ5- or AvrBsT-triggered RPM1-dependent defense activation. Finally, we showed that SOBER1 interferes with HopZ5- or AvrBsT-triggered immunity by deacetylating RIN4 T166. Collectively, our study elucidates detailed molecular mechanisms underlying the activation and suppression of plant innate immunity triggered by two bacterial acetyltransferases, HopZ5 and AvrBsT, from different bacterial pathogens.

MEDEA-interacting protein LONG-CHAIN BASE KINASE 1 promotes pattern-triggered immunity in Arabidopsis thaliana.

Arabidopsis LONG-CHAIN BASE KINASE 1 (LCBK1) interacts with MEDEA, a component of PCR2 complex that negatively regulates immunity. LCBK1 phosphorylates phytosphingosine and thereby promotes stomatal immunity against bacterial pathogens. Arabidopsis polycomb-group repressor complex2 (PRC2) protein MEDEA (MEA) suppresses both pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). MEA represses the expression of RPS2 and thereby attenuates AvrRpt2 effector-mediated ETI. However, the mechanism of MEA-mediated PTI diminution was not known. By screening the Arabidopsis cDNA library using yeast-2-hybrid interaction, we identified LONG-CHAIN BASE KINASE1 (LCBK1) as an MEA-interacting protein. We found that lcbk1 mutants are susceptible to virulent bacterial pathogens, such as Pseudomonas syringae pv maculicola (Psm) and P. syringae pv tomato (Pst) but not the avirulent strain of Pst that carries AvrRpt2 effector. Pathogen inoculation induces LCBK1 expression, especially in guard cells. We found that LCBK1 has a positive regulatory role in stomatal closure after pathogen inoculation. WT plants close stomata within an hour of Pst inoculation or flg22 (a 22 amino acid peptide from bacterial flagellin protein that activates PTI) treatment, but not lcbk1 mutants. LCBK1 phosphorylates phytosphingosine (PHS). Exogenous application of phosphorylated PHS (PHS-P) induces stomatal closure and rescues loss-of-PTI phenotype of lcbk1 mutant plants. MEA overexpressing (MEA-Oex) plants are defective, whereas loss-of-function mea-6 mutants are hyperactive in PTI-induced stomatal closure. Exogenous application of PHS-P rescues loss-of-PTI in MEA-Oex plants. Results altogether demonstrate that LCBK1 is an interactor of MEA that positively regulates PTI-induced stomatal closure in Arabidopsis.

A MPK3/6-WRKY33-ALD1-Pipecolic Acid Regulatory Loop Contributes to Systemic Acquired Resistance.

Plants induce systemic acquired resistance (SAR) upon localized exposure to pathogens. Pipecolic acid (Pip) production via AGD2-LIKE DEFENSE RESPONSE PROTEIN1 (ALD1) is key for SAR establishment. Here, we report a positive feedback loop important for SAR induction in Arabidopsis thaliana We showed that local activation of the MAP kinases MPK3 and MPK6 is sufficient to trigger Pip production and mount SAR. Consistent with this, mutations in MPK3 or MPK6 led to compromised Pip accumulation upon inoculation with the bacterial pathogen Pseudomonas syringae pv tomato DC3000 (Pto) AvrRpt2, which triggers strong sustained MAPK activation. By contrast, P. syringae pv maculicola and Pto, which induce transient MAPK activation, trigger Pip biosynthesis and SAR independently of MPK3/6. ALD1 expression, Pip accumulation, and SAR were compromised in mutants defective in the MPK3/6-regulated transcription factor WRKY33. Chromatin immunoprecipitation showed that WRKY33 binds to the ALD1 promoter. We found that Pip triggers activation of MPK3 and MPK6 and that MAPK activation after Pto AvrRpt2 inoculation is compromised in wrky33 and ald1 mutants. Collectively, our results reveal a positive regulatory loop consisting of MPK3/MPK6, WRKY33, ALD1, and Pip in SAR induction and suggest the existence of distinct SAR activation pathways that converge at the level of Pip biosynthesis.

Different Modes of Negative Regulation of Plant Immunity by Calmodulin-Related Genes.

Plant immune responses activated through the perception of microbe-associated molecular patterns, leading to pattern-triggered immunity, are tightly regulated. This results in low immune responses in the absence of pathogens and a rapid return to the resting state following an activation event. Here, we show that two CALMODULIN-LIKE genes, CML46 and CML47, negatively regulate salicylic acid accumulation and immunity in Arabidopsis (Arabidopsis thaliana). The double mutant cml46 cml47 is highly resistant to the pathogen Pseudomonas syringae pv maculicola (Pma). The effects of cml46 cml47 on Pma growth are genetically additive to that of cbp60a, a known negative regulator in the CALMODULIN-BINDING PROTEIN60 (CBP60) family. Transcriptome profiling revealed the effects of cbp60a and cml46 cml47 on both common and separate sets of genes, with the majorities of these differentially expressed genes being Pma responsive. CBP60g, a positive regulator of immunity in the CBP60 family, was found to be transcriptionally regulated by CBP60a, CML46, and CML47 Analysis of the flg22-induced mRNA levels of CBP60g in cbp60a and cml46 cml47 revealed that cml46 cml47 plants have higher induced expression while cbp60a plants retain elevated levels longer than wild-type plants. Assays for the effect of flg22 treatment on Pma growth showed that the effect is stronger in cml46 cml47 plants and lasts longer in cbp60a plants. Thus, the expression pattern of CBP60g is reflected in flg22-induced resistance to Pma.

The Proteasome Acts as a Hub for Plant Immunity and Is Targeted by Pseudomonas Type III Effectors.

Recent evidence suggests that the ubiquitin-proteasome system is involved in several aspects of plant immunity and that a range of plant pathogens subvert the ubiquitin-proteasome system to enhance their virulence. Here, we show that proteasome activity is strongly induced during basal defense in Arabidopsis (Arabidopsis thaliana). Mutant lines of the proteasome subunits RPT2a and RPN12a support increased bacterial growth of virulent Pseudomonas syringae pv tomato DC3000 (Pst) and Pseudomonas syringae pv maculicola ES4326. Both proteasome subunits are required for pathogen-associated molecular pattern-triggered immunity responses. Analysis of bacterial growth after a secondary infection of systemic leaves revealed that the establishment of systemic acquired resistance (SAR) is impaired in proteasome mutants, suggesting that the proteasome also plays an important role in defense priming and SAR In addition, we show that Pst inhibits proteasome activity in a type III secretion-dependent manner. A screen for type III effector proteins from Pst for their ability to interfere with proteasome activity revealed HopM1, HopAO1, HopA1, and HopG1 as putative proteasome inhibitors. Biochemical characterization of HopM1 by mass spectrometry indicates that HopM1 interacts with several E3 ubiquitin ligases and proteasome subunits. This supports the hypothesis that HopM1 associates with the proteasome, leading to its inhibition. Thus, the proteasome is an essential component of pathogen-associated molecular pattern-triggered immunity and SAR, which is targeted by multiple bacterial effectors.

Pectin Biosynthesis Is Critical for Cell Wall Integrity and Immunity in Arabidopsis thaliana.

Plant cell walls are important barriers against microbial pathogens. Cell walls of Arabidopsis thaliana leaves contain three major types of polysaccharides: cellulose, various hemicelluloses, and pectins. UDP-D-galacturonic acid, the key building block of pectins, is produced from the precursor UDP-D-glucuronic acid by the action of glucuronate 4-epimerases (GAEs). Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) repressed expression of GAE1 and GAE6 in Arabidopsis, and immunity to Pma ES4326 was compromised in gae6 and gae1 gae6 mutant plants. These plants had brittle leaves and cell walls of leaves had less galacturonic acid. Resistance to specific Botrytis cinerea isolates was also compromised in gae1 gae6 double mutant plants. Although oligogalacturonide (OG)-induced immune signaling was unaltered in gae1 gae6 mutant plants, immune signaling induced by a commercial pectinase, macerozyme, was reduced. Macerozyme treatment or infection with B. cinerea released less soluble uronic acid, likely reflecting fewer OGs, from gae1 gae6 cell walls than from wild-type Col-0. Although both OGs and macerozyme-induced immunity to B. cinerea in Col-0, only OGs also induced immunity in gae1 gae6. Pectin is thus an important contributor to plant immunity, and this is due at least in part to the induction of immune responses by soluble pectin, likely OGs, that are released during plant-pathogen interactions.

Two N-terminal acetyltransferases antagonistically regulate the stability of a nod-like receptor in Arabidopsis.

Nod-like receptors (NLRs) serve as immune receptors in plants and animals. The stability of NLRs is tightly regulated, though its mechanism is not well understood. Here, we show the crucial impact of N-terminal acetylation on the turnover of one plant NLR, Suppressor of NPR1, Constitutive 1 (SNC1), in Arabidopsis thaliana. Genetic and biochemical analyses of SNC1 uncovered its multilayered regulation by different N-terminal acetyltransferase (Nat) complexes. SNC1 exhibits a few distinct N-terminal isoforms generated through alternative initiation and N-terminal acetylation. Its first Met is acetylated by N-terminal acetyltransferase complex A (NatA), while the second Met is acetylated by N-terminal acetyltransferase complex B (NatB). Unexpectedly, the NatA-mediated acetylation serves as a degradation signal, while NatB-mediated acetylation stabilizes the NLR protein, thus revealing antagonistic N-terminal acetylation of a single protein substrate. Moreover, NatA also contributes to the turnover of another NLR, RESISTANCE TO P. syringae pv maculicola 1. The intricate regulation of protein stability by Nats is speculated to provide flexibility for the target protein in maintaining its homeostasis.

Arabidopsis PECTIN METHYLESTERASEs Contribute to Immunity against Pseudomonas syringae

Pectins, major components of dicot cell walls, are synthesized in a heavily methylesterified form in the Golgi and are partially deesterified by pectin methylesterases (PMEs) upon export to the cell wall. PME activity is important for the virulence of the necrotrophic fungal pathogen Botrytis cinerea. Here, the roles of Arabidopsis PMEs in pattern-triggered immunity and immune responses to the necrotrophic fungus Alternaria brassicicola and the bacterial hemibiotroph Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) were studied. Plant PME activity increased during pattern-triggered immunity and after inoculation with either pathogen. The increase of PME activity in response to pathogen treatment was concomitant with a decrease in pectin methylesterification. The pathogen-induced PME activity did not require salicylic acid or ethylene signaling, but was dependent on jasmonic acid signaling. In the case of induction by A. brassicicola, the ethylene response factor, but not the MYC2 branch of jasmonic acid signaling, contributed to induction of PME activity, whereas in the case of induction by Pma ES4326, both branches contributed. There are 66 PME genes in Arabidopsis, suggesting extensive genetic redundancy. Nevertheless, selected pme single, double, triple and quadruple mutants allowed significantly more growth of Pma ES4326 than wild-type plants, indicating a role of PMEs in resistance to this pathogen. No decreases in total PME activity were detected in these pme mutants, suggesting that the determinant of immunity is not total PME activity; rather, it is some specific effect of PMEs such as changes in the pattern of pectin methylesterification.

Pipecolic acid enhances resistance to bacterial infection and primes salicylic acid and nicotine accumulation in tobacco

Distinct amino acid metabolic pathways constitute integral parts of the plant immune system. We have recently identified pipecolic acid (Pip), a lysine-derived non-protein amino acid, as a critical regulator of systemic acquired resistance (SAR) and basal immunity to bacterial infection in Arabidopsis thaliana. In Arabidopsis, Pip acts as an endogenous mediator of defense amplification and priming. For instance, Pip conditions plants for effective biosynthesis of the phenolic defense signal salicylic acid (SA), accumulation of the phytoalexin camalexin, and expression of defense-related genes. Here, we show that tobacco plants respond to leaf infection by the compatible bacterial pathogen Pseudomonas syringae pv tabaci (Pstb) with a significant accumulation of several amino acids, including Lys, branched-chain, aromatic, and amide group amino acids. Moreover, Pstb strongly triggers, alongside the biosynthesis of SA and increases in the defensive alkaloid nicotine, the production of the Lys catabolites Pip and α-aminoadipic acid. Exogenous application of Pip to tobacco plants provides significant protection to infection by adapted Pstb or by non-adapted, hypersensitive cell death-inducing P. syringae pv maculicola. Pip thereby primes tobacco for rapid and strong accumulation of SA and nicotine following bacterial infection. Thus, our study indicates that the role of Pip as an amplifier of immune responses is conserved between members of the rosid and asterid groups of eudicot plants and suggests a broad practical applicability for Pip as a natural enhancer of plant disease resistance.

The CALMODULIN-BINDING PROTEIN60 Family Includes Both Negative and Positive Regulators of Plant Immunity

Two members of the eight-member CALMODULIN-BINDING PROTEIN60 (CBP60) gene family, CBP60g and SYSTEMIC ACQUIRED RESISTANCE DEFICIENT1 (SARD1), encode positive regulators of plant immunity that promote the production of salicylic acid (SA) and affect the expression of SA-dependent and SA-independent defense genes. Here, we investigated the other six family members in Arabidopsis (Arabidopsis thaliana). Only cbp60a mutations affected growth of the bacterial pathogen Pseudomonas syringae pv maculicola ES4326. In contrast to cbp60g and sard1 mutations, cbp60a mutations reduced pathogen growth, indicating that CBP60a is a negative regulator of immunity. Bacterial growth was increased by cbp60g only in the presence of CBP60a, while the increase in growth due to sard1 was independent of CBP60a, suggesting that the primary function of CBP60g may be to counter the repressive effect of CBP60a. In the absence of pathogen, levels of SA as well as of several SA-dependent and SA-independent pathogen-inducible genes were higher in cbp60a plants than in the wild type, suggesting that the enhanced resistance of cbp60a plants may result from the activation of immune responses prior to pathogen attack. CBP60a bound calmodulin, and the calmodulin-binding domain was defined at the C-terminal end of the protein. Transgenes encoding mutant versions of CBP60a lacking the ability to bind calmodulin failed to complement null cbp60a mutations, indicating that calmodulin-binding ability is required for the immunity-repressing function of CBP60a. Regulation at the CBP60 node involves negative regulation by CBP60a as well as positive regulation by CBP60g and SARD1, providing multiple levels of control over the activation of immune responses.

A Putative Nucleoporin 96 Is Required for Both Basal Defense and Constitutive Resistance Responses Mediated bysuppressor of npr1-1,constitutive 1

The Arabidopsis thaliana suppressor of npr1-1, constitutive 1 (snc1) mutant contains a gain-of-function mutation in a Toll Interleukin1 receptor-nucleotide binding-Leu-rich repeat-type resistance gene (R-gene), which leads to constitutive activation of disease resistance response against pathogens. In a screen for suppressors of snc1, a recessive mutation, designated mos3 (for modifier of snc1,3), was found to suppress the constitutive pathogenesis-related gene expression and resistance to virulent Pseudomonas syringae maculicola ES4326 and Peronospora parasitica Noco2 in snc1. In addition, mos3 is also compromised in resistance mediated by Resistance to Peronospora parasitica 4 (RPP4), Resistance to Pseudomonas syringae pv maculicola (RPM1), and Resistance to Pseudomonas syringae 4 (RPS4). Single mutant mos3 plants exhibited enhanced disease susceptibility to P. s. pv maculicola ES4326, suggesting that MOS3 is required for basal resistance to pathogens as well. mos3-1 was identified by map-based cloning, and it encodes a protein with high sequence similarity to human nucleoporin 96. Localization of the MOS3-green fluorescent protein fusion to the nuclear envelope further indicates that MOS3 may encode a nucleoporin, suggesting that nuclear and cytoplasmic trafficking plays an important role in both R-gene-mediated and basal disease resistance.

Arabidopsis RIN4 negatively regulates disease resistance mediated by RPS2 and RPM1 downstream or independent of the NDR1 signal modulator and is not required for the virulence functions of bacterial type III effectors AvrRpt2 or AvrRpm1.

Bacterial pathogens deliver type III effector proteins into the plant cell during infection. On susceptible (r) hosts, type III effectors can contribute to virulence. Some trigger the action of specific disease resistance (R) gene products. The activation of R proteins can occur indirectly via modification of a host target. Thus, at least some type III effectors are recognized at site(s) where they may act as virulence factors. These data indicate that a type III effector's host target might be required for both initiation of R function in resistant plants and pathogen virulence in susceptible plants. In Arabidopsis thaliana, RPM1-interacting protein 4 (RIN4) associates with both the Resistance to Pseudomonas syringae pv maculicola 1 (RPM1) and Resistance to P. syringae 2 (RPS2) disease resistance proteins. RIN4 is posttranslationally modified after delivery of the P. syringae type III effectors AvrRpm1, AvrB, or AvrRpt2 to plant cells. Thus, RIN4 may be a target for virulence functions of these type III effectors. We demonstrate that RIN4 is not the only host target for AvrRpm1 and AvrRpt2 in susceptible plants because its elimination does not diminish their virulence functions. In fact, RIN4 negatively regulates AvrRpt2 virulence function. RIN4 also negatively regulates inappropriate activation of both RPM1 and RPS2. Inappropriate activation of RPS2 is nonspecific disease resistance 1 (NDR1) independent, in contrast with the established requirement for NDR1 during AvrRpt2-dependent RPS2 activation. Thus, RIN4 acts either cooperatively, downstream, or independently of NDR1 to negatively regulate RPS2 in the absence of pathogen. We propose that many P. syringae type III effectors have more than one target in the host cell. We suggest that a limited set of these targets, perhaps only one, are associated with R proteins. Thus, whereas any pathogen virulence factor may have multiple targets, the perturbation of only one is necessary and sufficient for R activation.

The pattern of systemic acquired resistance induction within the Arabidopsis rosette in relation to the pattern of translocation.

Local leaf infections by a necrogenic pathogen can lead to systemic acquired resistance (SAR) in untreated leaves. We reasoned that, whatever the nature of the long-distance signal, if it is transported in the phloem, the pattern of SAR induced within the plant by treatment of a single leaf should match the pattern of translocation out of that leaf. The source-sink relationships (orthostichies) in the Arabidopsis rosette were established with [14C]Suc or phloem-mobile 3-aminotriazole at herbicidal concentrations. SAR was activated by infiltrating a single Columbia leaf with Pseudomonas syringae pv maculicola DC3000/avrRPM1, which causes a hypersensitive response. The pattern of SAR in the rosette was monitored by assessing the growth of wild-type DC3000 and by measuring the SAR markers salicylic acid and PR1 transcripts. Although the orthostichy of a single leaf was clearly limited to a row of vertically aligned leaves, SAR and SAR markers were also found outside the orthostichy. This indicates that, whatever the nature of the long-distance signal from the treated leaf to the upper responding leaves, its transport is either not limited exclusively to the phloem or the minor proportion of translocate that is not confined to the orthostichy contains enough of the SAR systemic signal to set in motion events leading to the establishment of the SAR state in the upper leaves.

Age-related resistance in Arabidopsis is a developmentally regulated defense response to Pseudomonas syringae.

Age-related resistance (ARR) has been observed in a number of plant species; however, little is known about the biochemical or molecular mechanisms involved in this response. Arabidopsis becomes more resistant, or less susceptible, to virulent Pseudomonas syringae (pv tomato or maculicola) as plants mature (in planta bacterial growth reduction of 10- to 100-fold). An ARR-like response also was observed in response to certain environmental conditions that accelerate Arabidopsis development. ARR occurs in the Arabidopsis mutants pad3-1, eds7-1, npr1-1, and etr1-4, suggesting that ARR is a distinct defense response, unlike the induced systemic resistance or systemic acquired resistance responses. However, three salicylic acid (SA) accumulation-deficient plant lines, NahG, sid1, and sid2, did not exhibit ARR. A heat-stable antibacterial activity was detected in intercellular washing fluids in response to Pst inoculation in wild-type ARR-competent plants but not in NAHG: These data suggest that the ability to accumulate SA is necessary for the ARR response and that SA may act as a signal for the production of the ARR-associated antimicrobial compound(s) and/or it may possess direct antibacterial activity against P. syringae.

Arabidopsis dth9 mutation identifies a gene involved in regulating disease susceptibility without affecting salicylic acid-dependent responses.

To determine which components of the plant defense response make important contributions to limiting pathogen attack, an M(2) mutagenized population of a transgenic Arabidopsis line was screened for mutants showing constitutive expression of beta-glucuronidase activity driven by the promoter region of the CEVI-1 gene. The CEVI-1 gene originally was isolated from tomato plants and has been shown to be induced in susceptible varieties of tomato plants by virus infection in a salicylic acid-independent manner. We report here the characterization of a recessive mutant, detachment9 (dth9). This mutant is more susceptible to both virulent and avirulent forms of the oomycete Peronospora and also exhibits increased susceptibility to the moderately virulent bacterial pathogen Pseudomonas syringae pv maculicola ES4326. However, this mutant is not affected in salicylic acid metabolism and shows normal expression of pathogenesis-related (PR) genes after pathogen attack. Furthermore, after inoculation with avirulent pathogens, the dth9 mutant shows a compromised systemic acquired resistance response that cannot be complemented by exogenous application of salicylic acid, although this molecule is able to promote normal activation of PR genes. Therefore, the dth9 mutation defines a regulator of disease susceptibility that operates upstream or independently of salicylic acid. Pleiotropy is also evident in the dth9 mutant in the sense that the shoots of dth9 plants are insensitive to the exogenously applied auxin analog 2,4-dichlorophenoxyacetic acid.

PAD4 Functions Upstream from Salicylic Acid to Control Defense Responses in Arabidopsis

The Arabidopsis PAD4 gene was previously shown to be required for synthesis of camalexin in response to infection by the virulent bacterial pathogen Pseudomonas syringae pv maculicola ES4326 but not in response to challenge by the non-host fungal pathogen Cochliobolus carbonum. In this study, we show that pad4 mutants exhibit defects in defense responses, including camalexin synthesis and pathogenesis-related PR-1 gene expression, when infected by P. s. maculicola ES4 326. No such defects were observed in response to infection by an isogenic avirulent strain carrying the avirulence gene avrRpt2. In P. s. maculicola ES4 326-infected pad4 plants, synthesis of salicylic acid (SA) was found to be reduced and delayed when compared with SA synthesis in wild-type plants. Moreover, treatment of pad4 plants with SA partially reversed the camalexin deficiency and PR-1 gene expression phenotypes of P. s. maculicola ES4 326-infected pad4 plants. These findings support the hypothesis that PAD4 acts upstream from SA accumulation in regulating defense response expression in plants infected with P. s. maculicola ES4 326. A working model of the role of PAD4 in governing expression of defense responses is presented.

The cpr5 mutant of Arabidopsis expresses both NPR1-dependent and NPR1-independent resistance.

The cpr5 mutant was identified from a screen for constitutive expression of systemic acquired resistance (SAR). This single recessive mutation also leads to spontaneous expression of chlorotic lesions and reduced trichome development. The cpr5 plants were found to be constitutively resistant to two virulent pathogens, Pseudomonas syringae pv maculicola ES4326 and Peronospora parasitica Noco2; to have endogenous expression of the pathogenesis-related gene 1 (PR-1); and to have an elevated level of salicylic acid (SA). Lines homozygous for cpr5 and either the SA-degrading bacterial gene nahG or the SA-insensitive mutation npr1 do not express PR-1 or exhibit resistance to P. s. maculicola ES4326. Therefore, we conclude that cpr5 acts upstream of SA in inducing SAR. However, the cpr5 npr1 plants retained heightened resistance to P. parasitica Noco2 and elevated expression of the defensin gene PDF1.2, implying that NPR1-independent resistance signaling also occurs. We conclude that the cpr5 mutation leads to constitutive expression of both an NPR1-dependent and an NPR1-independent SAR pathway. Identification of this mutation indicates that these pathways are connected in early signal transduction steps and that they have overlapping functions in providing resistance.

Arabidopsis enhanced disease susceptibility mutants exhibit enhanced susceptibility to several bacterial pathogens and alterations in PR-1 gene expression.

To identify plant defense responses that limit pathogen attack, Arabidopsis eds mutants that exhibit enhanced disease susceptibility to the virulent bacterial pathogen Pseudomonas syringae pv maculicola ES4326 were previously identified. In this study, we show that each of four eds mutants (eds5-1, eds6-1, eds7-1, and eds9-1) has a distinguishable phenotype with respect to the degree of susceptibility to a panel of bacterial phytopathogens and the ability to activate pathogenesis-related PR-1 gene expression after pathogen attack. None of the four eds mutants exhibited observable defects in mounting a hypersensitive response. Although all four eds mutants were also capable of mounting a systemic acquired resistance response, enhanced growth of P. s. maculicola ES4326 was still apparent in the secondarily infected leaves of three of the eds mutants. These data indicate that eds genes define a diverse set of previously unknown defense responses that affect resistance to virulent pathogens.

A mutation in Arabidopsis that leads to constitutive expression of systemic acquired resistance.

Systemic acquired resistance (SAR) is a nonspecific defense response in plants that is associated with an increase in the endogenous level of salicylic acid (SA) and elevated expression of pathogenesis-related (PR) genes. To identify mutants involved in the regulation of PR genes and the onset of SAR, we transformed Arabidopsis with a reporter gene containing the promoter of a beta-1,3-glucanase-encoding PR gene (BGL2) and the coding region of beta-glucuronidase (GUS). The resulting transgenic line (BGL2-GUS) was mutagenized, and the M2 progeny were scored for constitutive GUS activity. We report the characterization of one mutant, cpr1 (constitutive expressor of PR genes), that was identified in this screen and shown by RNA gel blot analysis also to have elevated expression of the endogenous PR genes BGL2, PR-1, and PR-5. Genetic analyses indicated that the phenotype conferred by cpr1 is caused by a single, recessive nuclear mutation and is suppressed in plants producing a bacterial salicylate hydroxylase, which inactivates SA. Furthermore, biochemical analysis showed that the endogenous level of SA is elevated in the mutant. Finally, the cpr1 plants were found to be resistant to the fungal pathogen Peronospora parasitica NOCO2 and the bacterial pathogen Pseudomonas syringae pv maculicola ES4326, which are virulent in wild-type BGL2-GUS plants. Because the cpr1 mutation is recessive and associated with an elevated endogenous level of SA, we propose that the CPR1 gene product acts upstream of SA as a negative regulator of SAR.