Biologically induced systemic acquired resistance in Arabidopsis thaliana

Local infection with a necrotizing pathogen can render plants resistant to subsequent infection by normally virulent pathogens. A system for biological induction of such systemic acquired resistance (SAR) in Arabidopsis thaliana is reported. When plants were immunized by local inoculation of a single leaf with avirulent Pseudomonas syringae pv. tomato (Pst) carrying the avrRpt2 avirulence gene, after 2 days other leaves became resistant, as measured symptomatically and by in planta bacterial growth, to challenge with a virulent Pst strain lacking this avirulence gene. Resistance was systemic and protected the plants against infection by other virulent pathogens including P. syringae pv. maculicola. Low-dose inoculation induced a strong SAR and double immunizations did not increase the level of protection indicating that the response of only a few cells to the immunizing bacteria is required. SAR was not induced by the virulent strain of Pst lacking avrRpt2. However, experiments with the Arabidopsis RPS2 disease resistance gene mutant rps2-201, which does not exhibit a local hypersensitive response to Pst carrying the corresponding avirulence gene avrRpt2, indicate that a hypersensitive response contributes to, but is not essential for, the induction of SAR. Thus, avrRpt2 activates either a branching signal pathway or separate parallel pathways for induction of localized hypersensitive resistance and SAR, with downstream potentiation of the systemic response by the local response. Using this system for the biological induction of SAR in Arabidopsis, it should be possible to dissect the molecular genetics of SAR by the isolation of mutants affected in the production, transmission, perception and transduction of the systemic signal(s).     

Two pathways act in an additive rather than obligatorily synergistic fashion to induce systemic acquired resistance and PR gene expression

Background  

Local infection with necrotizing pathogens induces whole plant immunity to secondary challenge. Pathogenesis-related genes are induced in parallel with this systemic acquired resistance response and thought to be co-regulated. The hypothesis of co-regulation has been challenged by induction of Arabidopsis PR-1 but not systemic acquired resistance in npr1 mutant plants responding to Pseudomonas syringae carrying the avirulence gene avrRpt2. However, experiments with ndr1 mutant plants have revealed major differences between avirulence genes. The ndr1-1 mutation prevents hypersensitive cell death, systemic acquired resistance and PR-1 induction elicited by bacteria carrying avrRpt2. This mutation does not prevent these responses to bacteria carrying avrB.     

Mutations in the Pseudomonas syringae avrRpt2 gene that dissociate its virulence and avirulence activities lead to decreased efficiency in AvrRpt2-induced disappearance of RIN4

The avrRpt2 gene from Pseudomonas syringae pv. tomato exhibits avirulence activity on Arabidopsis expressing the resistance gene RPS2 but promotes bacterial virulence on susceptible rps2 Arabidopsis. To understand the functional relationship between the avirulence and virulence activities of avrRpt2, we analyzed a series of six avrRpt2 mutants deficient in eliciting the RPS2-dependent hypersensitive response. We show that the mutants are also severely impaired in triggering RSP2-dependent resistance. Four of these mutants are severely impaired in their virulence activity, whereas two alleles, encoding C-terminal deletions of AvrRpt2, retain significant but slightly reduced virulence activity. Thus, the avirulence and virulence activities of avrRpt2 can be genetically uncoupled. We tested the ability of the two C-terminal deletion mutants to trigger AvrRpt2-induced elimination of the Arabidopsis RIN4 protein and show that they retain this activity but are less efficient than wild-type AvrRpt2. Thus, reduced AvrRpt2 virulence activity is correlated with reduced efficiency in the induction of RIN4 disappearance. This suggests that an alteration in kinetics of RIN4 disappearance triggered by the C-terminal deletion mutants may provide the mechanistic basis for the uncoupling of the avirulence and virulence activities of avrRpt2.     

RPS2, an Arabidopsis disease resistance locus specifying recognition of Pseudomonas syringae strains expressing the avirulence gene avrRpt2.

A molecular genetic approach was used to identify and characterize plant genes that control bacterial disease resistance in Arabidopsis. A screen for mutants with altered resistance to the bacterial pathogen Pseudomonas syringae pv. tomato (Pst) expressing the avirulence gene avrRpt2 resulted in the isolation of four susceptible rps (resistance to P. syringae) mutants. The rps mutants lost resistance specifically to bacterial strains expressing avrRpt2 as they retained resistance to Pst strains expressing the avirulence genes avrB or avrRpm1. Genetic analysis indicated that in each of the four rps mutants, susceptibility was due to a single mutation mapping to the same locus on chromosome 4. Identification of a resistance locus with specificity for a single bacterial avirulence gene suggests that this locus, designated RPS2, controls specific recognition of bacteria expressing the avirulence gene avrRpt2. Ecotype Wü-0, a naturally occurring line that is susceptible to Pst strains expressing avrRpt2, appears to lack a functional allele at RPS2, demonstrating that there is natural variation at the RPS2 locus among wild populations of Arabidopsis.     

Mutational analysis of the Arabidopsis RPS2 disease resistance gene and the corresponding pseudomonas syringae avrRpt2 avirulence gene

Plants have evolved a large number of disease resistance genes that encode proteins containing conserved structural motifs that function to recognize pathogen signals and to initiate defense responses. The Arabidopsis RPS2 gene encodes a protein representative of the nucleotide-binding site-leucine-rich repeat (NBS-LRR) class of plant resistance proteins. RPS2 specifically recognizes Pseudomonas syringae pv. tomato strains expressing the avrRpt2 gene and initiates defense responses to bacteria carrying avrRpt2, including a hypersensitive cell death response (HR). We present an in planta mutagenesis experiment that resulted in the isolation of a series of rps2 and avrRpt2 alleles that disrupt the RPS2-avrRpt2 gene-for-gene interaction. Seven novel avrRpt2 alleles incapable of eliciting an RPS2-dependent HR all encode proteins with lesions in the C-terminal portion of AvrRpt2 previously shown to be sufficient for RPS2 recognition. Ten novel rps2 alleles were characterized with mutations in the NBS and the LRR. Several of these alleles code for point mutations in motifs that are conserved among NBS-LRR resistance genes, including the third LRR, which suggests the importance of these motifs for resistance gene function.     

The Pseudomonas syringae avrRpt2 gene product promotes pathogen virulence from inside plant cells

Several bacterial avr genes have been shown to contribute to virulence on susceptible plants lacking the corresponding resistance (R) gene. The mechanisms by which avr genes promote parasitism and disease, however, are not well understood. We investigated the role of the Pseudomonas syringae pv. tomato avrRpt2 gene in pathogenesis by studying the interaction of P. syringae pv. tomato strain PstDC3000 expressing avrRpt2 with several Arabidopsis thaliana lines lacking the corresponding R gene, RPS2. We found that PstDC3000 expressing avrRpt2 grew to significantly higher levels and often resulted in the formation of more severe disease symptoms in ecotype No-0 plants carrying a mutant RPS2 allele, as well as in two Col-0 mutant lines, cpr5 rps2 and coil rps2, that exhibit enhanced resistance. We also generated transgenic A. thaliana lines expressing avrRpt2 and demonstrated, by using several different assays, that expression of avrRpt2 within the plant also promotes virulence of PstDC3000. Thus, AvrRpt2 appears to promote pathogen virulence from within the plant cell.     

Isolation of Arabidopsis genes that differentiate between resistance responses mediated by the RPS2 and RPM1 disease resistance genes.

The Arabidopsis disease resistance gene RPS2 is involved in recognition of bacterial pathogens carrying the avirulence gene avrRpt2, and the RPM1 resistance gene is involved in recognition of pathogens carrying avrRpm1 or avrB. We identified and cloned two Arabidopsis genes, AIG1 and AIG2 (for avrRpt2-induced gene), that exhibit RPS2- and avrRpt2-dependent induction early after infection with Pseudomonas syringae pv maculicola strain ES4326 carrying avrRpt2. However, ES4326 carrying avrRpm1 or avrB did not induce early expression of AIG1 and AIG2. Conversely, ES4326 carrying avrRpm1 or avrB induced early expression of the previously isolated defense-related gene ELI3, whereas ES4326 carrying avrRpt2 did not. The induction patterns of the AIG genes and ELI3 demonstrate that different resistance gene-avr gene combinations can elicit distinct defense responses. Furthermore, by examining the expression of AIG1 and ELI3 in plants infiltrated with a mixed inoculum of ES4326 carrying avrRpt2 and ES4326 carrying avrRpm1, we found that there is interference between the RPS2- and RPM1-mediated resistance responses.     

Development of a Virus-Induced Gene-Silencing System for Functional Analysis of the RPS2-Dependent Resistance Signalling Pathways in Arabidopsis

Virus-induced gene silencing (VIGS) offers a rapid and high throughput technique platform for the analysis of gene function in plants. Although routinely used in some Solanaceous species, VIGS system has not been well established in Arabidopsis thaliana (L.) Heynh. We have recently reported some factors that potentially influence tobacco rattle virus (TRV)-mediated VIGS of phytoene desaturase (PDS) and actin gene expression in Arabidopsis. In this study, we have further established that the Agrobacterium strain used for agro-inoculation significantly affects the VIGS efficiency. Strain GV3101 was highly effective; C58C1 and LBA4404 were invalid, while EHA105 was plant growth stage-dependent for TRV-induced gene silencing. Furthermore, the VIGS procedure optimised for the PDS gene was applied for the functional analysis of the disease resistance gene RPS2-mediated resistance pathway. Silencing of RPS2 led to loss of resistance to the otherwise avirulence strain of Pseudomonas syringae pv. tomato DC3000 carrying the avirulence gene AvrRpt2. Silencing of RIN4, a RPS2 repressor gene, gave rise to conversion of compatible interaction to incompatible. Silencing of NDR1, RAR1 and HSP90, known to be required for the RPS2-mediated resistance, resulted in loss of the resistance, while silencing of EDS1 and SGT1b, which are not required for the RPS2-mediated resistance, caused no change of the resistance. These results indicate that the optimised procedure for the TRV-based VIGS is a potentially powerful tool for dissecting the signal transduction pathways of disease resistance in Arabidopsis. Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at An erratum to this article is available at .     

Mutational analysis of the Arabidopsis nucleotide binding site-leucine-rich repeat resistance gene RPS2

Disease resistance proteins containing a nucleotide binding site (NBS) and a leucine-rich repeat (LRR) region compose the largest class of disease resistance proteins. These so-called NBS-LRR proteins confer resistance against a wide variety of phytopathogens. To help elucidate the mechanism by which NBS-LRR proteins recognize and transmit pathogen-derived signals, we analyzed mutant versions of the Arabidopsis NBS-LRR protein RPS2. The RPS2 gene confers resistance against Pseudomonas syringae strains carrying the avirulence gene avrRpt2. The activity of RPS2 derivatives in response to AvrRpt2 was measured by using a functional transient expression assay or by expressing the mutant proteins in transgenic plants. Directed mutagenesis revealed that the NBS and an N-terminal leucine zipper (LZ) motif were critical for RPS2 function. Mutations near the N terminus, including an LZ mutation, resulted in proteins that exhibited a dominant negative effect on wild-type RPS2. Scanning the RPS2 molecule with a small in-frame internal deletion demonstrated that RPS2 does not have a large dispensable region. Overexpression of RPS2 in the transient assay in the absence of avrRpt2 also led to an apparent resistant response, presumably a consequence of a low basal activity of RPS2. The NBS and LZ were essential for this overdose effect, whereas the entire LRR was dispensable. RPS2 interaction with a 75-kD protein (p75) required an N-terminal portion of RPS2 that is smaller than the region required for the overdose effect. These findings illuminate the pathogen recognition mechanisms common among NBS-LRR proteins.     

A key role for the Arabidopsis WIN3 protein in disease resistance triggered by Pseudomonas syringae that secrete AvrRpt2

Effector proteins injected by the pathogenic bacteria Pseudomonas syringae into plants can have profound effects on the pathogen-host interaction due to their efficient recognition by plants and the subsequent triggering of defenses. The AvrRpt2 effector triggers strong local and systemic defense (called systemic acquired resistance [SAR]) responses in Arabidopsis thaliana plants that harbor a functional RPS2 gene that encodes an R protein in the coiled-coil, nucleotide-binding domain, leucine-rich repeat class. The newly identified win3-T mutant shows greatly reduced resistance to P syringae carrying avrRpt2. In win3-T plants, RIN4 cleavage, an early AvrRpt2-induced event, is normal. However, salicylic acid accumulation is compromised, as is SAR induction and the local hypersensitive cell death response after infection by P syringae carrying avrRpt2. WIN3 encodes a member of the firefly luciferase protein superfamily. Expression of WIN3 at an infection site partially requires PAD4, a protein known to play a quantitative role in RPS2-mediated signaling. WIN3 expression in tissue distal to an infection site requires multiple salicylic acid regulatory genes. Finally, win3-T plants show modestly increased susceptibility to virulent P syringae and modestly reduced SAR in response to P. syringae carrying avrRpm1. Thus, WIN3 is a key element of the RPS2 defense response pathway and a basal and systemic defense component.     

Use of Arabidopsis thaliana and Pseudomonas syringae in the Study of Plant Disease Resistance and Tolerance

The interaction between Arabidopsis thaliana and the bacterium Pseudomonas syringae is being developed as a model experimental system for plant pathology research. Race-specific ("gene-for-gene") resistance has been demonstrated for this interaction, and pathogen genes that determine avirulence have been isolated and characterized. Because certain lines of both Arabidopsis and soybean are resistant to bacteria carrying the avirulence genes avrRpt2 and avrB, extremely similar pathogen recognition mechanisms are apparently present in these two plant species. Isogenic bacterial strains that differ by the presence of single avirulence genes are being used to analyze plant resistance. Plant resistance genes have been identified in crosses between resistant and susceptible lines. The extensive map-based cloning tools available in Arabidopsis are being used to isolate these resistance genes. In a related project, ethylene-insensitive Arabidopsis mutants are being used to examine the role of ethylene in disease development. Ethylene apparently mediates symptom formation in susceptible plants and is not required for resistance, suggesting possible strategies for enhancement of disease tolerance in crops.     

The Arabidopsis thaliana 4-coumarate:CoA ligase (4CL) gene: stress and developmentally regulated expression and nucleotide sequence of its cDNA

An Arabidopsis cDNA clone encoding 4-coumarate:CoA ligase (4CL), a key enzyme of phenylpropanoid metabolism, was identified and sequenced. The predicted amino acid sequence is similar to those of other cloned 4CL genes. Southern blot analysis indicated that 4CL is single-copy gene in Arabidopsis. Northern blots showed that 4CL expression was activated early during seedling development. The onset of 4CL expression was correlated with the onset of lignin deposition in cotyledons and roots 2–3 days after germination. The timing of the expression of a parsley 4CL1-GUS fusion in transgenic Arabidopsis seedlings was examined in parallel and was very similar to that of endogenous 4CL. In mature plants, highest 4CL expression was observed in bolting stems, where relatively large amounts of lignin accumulate. Both 4CL and 4CL1-GUS mRNA accumulation was strongly and transiently activated by wounding of mature Arabidopsis leaves. 4CL expression was specifically activated within 6 h after infiltration of Arabidopsis ecotype Columbia leaves with a Pseudomonas syringae pv. maculicola strain harboring the bacterial avirulence gene avrB, which causes in incompatible interaction. The timing of 4CL activation was identical to the previously observed activation of PAL gene expression in this interaction. No activation of 4CL expression was observed in a compatible interaction caused by a Pseudomonas syringae pv. maculicola strain without avrB.     

Expression of RPS4 in tobacco induces an AvrRps4-independent HR that requires EDS1, SGT1 and HSP90

The Arabidopsis RPS4 gene belongs to the Toll/interleukin-1 receptor/nucleotide-binding site/leucine-rich repeat (TIR-NB-LRR) class of plant resistance (R) genes. It confers resistance to Pseudomonas syringae carrying the avirulence gene avrRps4. Transient expression of genomic RPS4 driven by the 35S promoter in tobacco leaves induces an AvrRps4-independent hypersensitive response (HR). The same phenotype is seen after expression of a full-length RPS4 cDNA. This indicates that alternative splicing of RPS4 is not involved in this HR. The extent of HR is correlated with RPS4 protein levels. Deletion analyses of RPS4 domains show the TIR domain is required for the HR phenotype. Mutations in the P-loop motif of the NB domain abolish the HR. Using virus-induced gene silencing, we found that the cell death resulting from RPS4 expression is dependent on the three plant signalling components EDS1, SGT1 and HSP90. All these data suggest that heterologous expression of an R gene can result in activation of cell death even in the absence of its cognate avirulence product, and provides a system for studying the RPS4 domains required for HR.     

Defence gene expression in soybean is linked to the status of the cell death program

Soybean cell cultures (cv. Williams 82) respond to Pseudomonas syringae bacteria expressing the avirulence gene AvrA with a hypersensitive reaction, a programmed cell death (PCD) of plant cells to pathogen attack. This PCD is under control of salicylic acid (SA) via an unknown mechanism. In the presence of low concentrations of SA, the cells undergo a very rapid cell death, which needs only half of the time required for the normal hypersensitive reaction (HR). Northern blot studies for defence-related genes show that the expression of many of these genes is tightly linked to the status of the cell death program rather than to pathogen-derived elicitors. Thus the expression is much faster in the SA-accelerated PCD than in the normal hypersensitive reaction. In contrast, other pathogen-responsive genes are induced independently of the speed of PCD, indicating a divergent signalling mechanism. The production of reactive oxygen species during the oxidative burst of bacteria-inoculated soybean cells is slightly enhanced in the presence of SA but occurs at the same time as in untreated cells, suggesting that SA exhibits the control of the PCD downstream of the oxidative burst. Consistent with these findings a HR-specific marker gene is neither directly induced by H₂O₂ or SA. However, this gene shows a high expression in the regular HR and is induced much faster in the SA-accelerated PCD.     

Characterization of the Pseudomonas syringae pv. tomato AvrRpt2 protein: demonstration of secretion and processing during bacterial pathogenesis

Pseudomonas syringae pv. tomato strain DC3000 (Pst DC3000) expressing avrRpt2 is specifically recognized by plant cells expressing RPS2 activity, resulting in localized cell death and plant resistance. Furthermore, transient expression of this bacterial avrRpt2 gene in plant cells results in RPS2-dependent cell death. This indicates that the AvrRpt2 protein is recognized inside RPS2 plant cells and is sufficient for the activation of disease resistance-mediated cell death in planta. We explored the possibility that Pst DC3000 delivers AvrRpt2 protein to plant cells via the hrp (type III) secretion pathway. We now provide direct evidence that mature AvrRpt2 protein is secreted from Pst DC3000 and that secretion is hrp dependent. We also show that AvrRpt2 is N-terminally processed when Arabidopsis thaliana plants are infected with Pst DC3000 expressing avrRpt2. Similar N-terminal processing of AvrRpt2 occurred when avrRpt2 was stably expressed in A. thaliana. No cleavage of AvrRpt2 was detected in bacteria expressing avrRpt2 in culture or in the plant extracellular fluids. The N-terminus of AvrRpt2 was not required for RPS2 recognition in planta. However, this region of AvrRpt2 was essential for Pst DC3000-mediated elicitation of RPS2-dependent cell death in A. thaliana leaves.     

Ferredoxin from sweet pepper (Capsicum annuum L.) intensifying harpinpss-mediated hypersensitive response shows an enhanced production of active oxygen species (AOS)

The hypersensitive response (HR) is a form of cell death associated with plant resistance to pathogen infection. Harpin_pss, an elicitor from the bacterium Pseudomonas syringae pv. syringae, induces a HR in non-host plants. Previously, we reported an amphipathic protein from sweet pepper interfering with harpin_pss-mediated HR. In this report, we isolated and characterized a cDNA clone encoded that amphipathic protein from sweet pepper. This protein is designated as PFLP (plant ferredoxin-like protein) by virtue of its high homology with plant ferredoxin protein containing an N-terminal signal peptide responsible for chloroplast targeting and a putative 2Fe-2S domain responsible for redox activity. Recombinant PFLP obtained from Escherichia coliwas able to significantly increase active oxygen species (AOS) generation when mixed with harpin_pss in tobacco suspension cells. It also showed enhanced HR when co-infiltrated with harpin_pss in tobacco leaves. We used a transgenic tobacco suspension cells system that constitutively expresses the Pflpgene driven by the CaMV 35S promoter to study the function of PFLP in enhancing harpin_pss-mediated hypersensitive cell death in vivo. In response to harpin_pss, suspension cells derived from Pflptransgenic tobacco showed a significant increase both in the generation of AOS and in cell death as compared to the wild type. AOS inhibitors diphenylene iodonium chloride (DPI) and lanthanum chlorate (LaCl₃) were used to study the involvement of AOS in harpin_pss-induced cell death. Our results demonstrate enhanced generation of AOS is necessary to cause enhanced hypersensitive cell death in Pflp transgenic tobacco cells and it is plasma membrane-bound NADPH-oxidase-dependent. Sub-cellular localization studies showed that PFLP is present in the cytoplasm and chloroplast of Pflp transgenic tobacco cells, but only in the chloroplast, not in the cytoplasm, of wild-type tobacco cells. It is possible that PFLP can change the redox state of the cell upon harpin_pss inoculation to increase AOS generation and hypersensitive cell death. Overall, this study will provide a new insight in the functional properties of ferredoxin in hypersensitive cell death.     

Genes required for pathogenicity and hypersensitivity are conserved and interchangeable among pathovars of Pseudomonas syringae

Summary A group of pathogenicity genes was previously identified in Pseudomonas syringae pv. phaseolicola which controls the ability of the pathogen to cause disease on bean and to elicit the hypersensitive response on non-host plants. These genes, designated hrp, are located in a ca. 20 kb region which was referred to as the hrp cluster. Homologous sequences to DNA segments derived from this region were detected in several pathovars of P. syringae but not in symbiotic, saprophytic or other phytopathogenic bacteria. A Tn5-induced Hrp^- mutation was transferred from P. syringae pv. phaseolicola to P. syringae pv. tabaci and to three races of P. syringae pv. glycinea by marker exchange mutagenesis. The resulting progeny were phenotypically Hrp^-, i.e. no longer pathogenic on their respective hosts and unable to elicit the hypersensitive response on non-host plants. These mutants were restored to wild-type phenotype upon introduction of a recombinant plasmid carrying the corresponding wild-type locus from P. syringae pv. phaseolicola. The marker exchange mutants of P. syringae pv. glycinea psg0 and Psg5 which carry different avr genes for race specific avirulence did not elicit a hypersensitive response on incompatible soybean cultivars. It appears, therefore, that P. syringae pathovars possess common genes for pathogenicity which also control their interaction with non-host plants. Furthermore, the expression of race/cultivar specific incompatibility of P. syringae pv. glycinea requires a fully functional hrp region in addition to the avr genes which determine avirulence on single-gene differential cultivars of soybean.     

Suppression by ABA of salicylic acid and lignin accumulation and the expression of multiple genes,in <Emphasis Type="Italic">Arabidopsis</Emphasis> infected with <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">tomato</Emphasis>

Abscisic acid (ABA) has been implicated in determining the outcome of interactions between many plants and their pathogens. We had previously shown that increased concentrations of ABA within leaves of Arabidopsis induced susceptibility towards an avirulent strain of Pseudomonas syringae pathovar (pv.) tomato. We now show that ABA induces susceptibility via suppression of the accumulation of components crucial for a resistance response. Lignin and salicylic acid concentrations in leaves were increased during a resistant interaction but reduced when plants were treated with ABA. The reduction in lignin and salicylic acid production was independent of the development of the hypersensitive response (HR), indicating that, in this host-pathogen system, HR is not required for resistance. Genome-wide gene expression analysis using microarrays showed that treatment with ABA suppressed the expression of many defence-related genes, including those important for phenylpropanoid biosynthesis and those encoding resistance-related proteins. Together, these results show that resistance induction in Arabidopsis to an avirulent strain of P. syringae pv. tomato is regulated by ABA. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Arabidopsis DND2, a second cyclic nucleotide-gated ion channel gene for which mutation causes the "defense, no death" phenotype

A previous mutant screen identified Arabidopsis dnd1 and dnd2 "defense, no death" mutants, which exhibit loss of hypersensitive response (HR) cell death without loss of gene-for-gene resistance. The dnd1 phenotype is caused by mutation of the gene encoding cyclic nucleotide-gated (CNG) ion channel AtCNGC2. This study characterizes dnd2 plants. Even in the presence of high titers of Pseudomonas syringae expressing avrRpt2, most leaf mesophyll cells in the dnd2 mutant exhibited no HR. These plants retained strong RPS2-, RPM1-, or RPS4-mediated restriction of P. syringae pathogen growth. Mutant dnd2 plants also exhibited enhanced broad-spectrum resistance against virulent P. syringae and constitutively elevated levels of salicylic acid, and pathogenesis-related (PR) gene expression. Unlike the wild type, dnd2 plants responding to virulent and avirulent P. syringae exhibited elevated expression of both salicylate-dependent PR-1 and jasmonate and ethylene-dependent PDF1.2. Introduction of nahG+ (salicylate hydroxylase) into the dnd2 background, which removes salicylic acid and causes other defense alterations, eliminated constitutive disease resistance and PR gene expression but only weakly impacted the HR- phenotype. Map-based cloning revealed that dnd2 phenotypes are caused by mutation of a second CNG ion channel gene, AtCNGC4. Hence, loss of either of two functionally nonredundant CNG ion channels can cause dnd phenotypes. The dnd mutants provide a unique genetic background for dissection of defense signaling.