A disease resistance gene in Arabidopsis with specificity for two different pathogen avirulence genes.

The RPS3 and RPM1 disease resistance loci of Arabidopsis confer resistance to Pseudomonas syringae strains that carry the avirulence genes avrB and avrRpm1, respectively. We have previously shown that RPS3 and RPM1 are closely linked genetically. Here, we show that RPS3 and RPM1 are in fact the same gene. We screened a mutagenized Arabidopsis population with a P. syringae strain carrying avrB and found 12 susceptible mutants. All 12 mutants were also susceptible to an isogenic strain carrying avrRpm1, indicating a loss of both RPS3 and RPM1 functions. No mutants were recovered that lost only RPS3 function. Genetic analysis of four independent mutants revealed that the lesions were in RPS3. Thus, a single gene in Arabidopsis confers resistance that is specific to two distinct pathogen avirulence genes--a gene-for-genes interaction. This observation suggests that the RPS3/RPM1 gene product can bind multiple pathogen ligands, or alternatively, that it does not function as a receptor.     

Lignification induced by pseudomonads harboring avirulent genes on Arabidopsis

The responses of Arabidopsis thaliana ecotypes to the bacterial pathogen Pseudomonas syringae pv. maculicola 4326 (Psm4326) harboring cloned avirulence genes avrB and avrRpt2 from P. syringae pv. glycinea were examined. Psm4326 containing avirulent genes, avrB and avrRpt2 induced lignification and peroxidase activities in the bacteria infiltrated leaves of Col-O only and not in Mt-O, Bla-2 and Po-1. However, Arabidopsis ecotypes infiltrated with Psm4326 harboring with and without avirulent genes all showed differential induction of mRNA for peroxidase gene and lignin accumulation up to 24 h after infiltration. Only avrB gene in Col-O showed strong corelationship between peroxidase mRNA expression as well as lignification gradually up to 36 h after infiltration. These results extend previous observations that avirulence genes from pathogens of one host plant can be recognized by non-host plants and provide the genetic framework for analysis of the plant-specific response to the bacterial avirulent gene products in A. thaliana.     

Signals involved in Arabidopsis resistance to Trichoplusia ni caterpillars induced by virulent and avirulent strains of the phytopathogen Pseudomonas syringae

Plants have evolved different but interconnected strategies to defend themselves against herbivorous insects and microbial pathogens. We used an Arabidopsis/Pseudomonas syringae pathosystem to investigate the impact of pathogen-induced defense responses on cabbage looper (Trichoplusia ni) larval feeding. Arabidopsis mutants [npr1, pad4, eds5, and sid2(eds16)] or transgenic plants (nahG) that are more susceptible to microbial pathogens and are compromised in salicylic acid (SA)-dependent defense responses exhibited reduced levels of feeding by T. ni compared with wild-type plants. Consistent with these results, Arabidopsis mutants that are more resistant to microbial pathogens and have elevated levels of SA (cpr1 and cpr6) exhibited enhanced levels of T. ni feeding. These experiments suggested an inverse relationship between an active SA defense pathway and insect feeding. In contrast to these results, there was increased resistance to T. ni in wild-type Arabidopsis ecotype Columbia plants that were infected with P. syringae pv. maculicola strain ES4326 (Psm ES4326) expressing the avirulence genes avrRpt2 or avrB, which elicit a hypersensitive response, high levels of SA accumulation, and systemic acquired resistance to bacterial infection. Similar results were obtained with other ecotypes, including Landsberg erecta, Cape Verdi Islands, and Shakdara. When infected with Psm ES4326(avrRpt2) or Psm ES4326(avrB), nahG transgenic and npr1 mutant plants (which are more susceptible to virulent and avirulent P. syringae strains) failed to show the increased insect resistance exhibited by wild-type plants. It was surprising that wild-type plants, as well as nahG and npr1 plants, infected with Psm ES4326 not expressing avrRpt2 or avrB, which elicits disease, became more susceptible to T. ni. Our results suggest two potentially novel systemic signaling pathways: a systemic response elicited by HR that leads to enhanced T. ni resistance and overrides the SA-mediated increase in T. ni susceptibility, and a SA-independent systemic response induced by virulent pathogens that leads to enhanced susceptibility to T. ni.     

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.     

A resistance gene product of the nucleotide binding site -- leucine rich repeats class can form a complex with bacterial avirulence proteins in vivo

Resistance (R) genes in plants mediate gene-for-gene disease resistance. The ligand-receptor model, which explains the gene-for-gene specificity, predicts a physical interaction between an elicitor, which is directly or indirectly encoded by an avirulence (avr) gene in the pathogen, and the corresponding R gene product. The nucleotide binding site (NBS) - leucine rich repeats (LRR) class of R genes is the largest known class of R genes. Here we report that an NBS-LRR R protein and its cognate Avr protein form a complex together in the plant cell. The Arabidopsis thaliana R genes RPS2 and RPM1 confer gene-for-gene disease resistance to strains of the phytopathogenic bacterium Pseudomonas syringae carrying the avr genes avrRpt2 and avrB, respectively. Using transient expression of these genes in Arabidopsis leaf mesophyll protoplasts, we first demonstrated that the protoplast system is appropriate for the investigation of the gene-for-gene recognition mechanism. Formation of an in vivo complex containing the RPS2 and AvrRpt2 proteins was demonstrated by co-immunoprecipitation of the proteins following expression of the genes in protoplasts. This complex contained at least one additional plant protein of approximately 75 kDa. Unexpectedly, RPS2 also formed a complex with AvrB. We speculate that complex formation between AvrRpt2 and RPS2 is productive and leads to the elicitation of the resistance response, whilst complex formation between AvrB and RPS2 is unproductive and possibly competes with complex formation between AvrRpt2 and RPS2.     

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.     

Identification of a disease resistance locus in Arabidopsis that is functionally homologous to the RPG1 locus of soybean

A new disease resistance locus in Arabidopsis, RPS3 , was identified using a previously cloned avirulence gene from a non- Arabidopsis pathogen. The avrB avirulence gene from the soybean pathogen Pseudomonas syringae pv. glycinea was transferred into a P. syringae pv. tomato strain that is virulent on Arabidopsis , and conversion to avirulence was assayed on Arabidopsis plants. The avrB gene had avirulence activity on most, but not all, Arabidopsis ecotypes. Of 53 ecotypes examined, 45 were resistant to a P. syringae pv. tomato strain carrying avrB , and eight were susceptible. The inheritance of this resistance was examined using crosses between the resistant ecotype Col-0 and the susceptible ecotype Bla-2. In F₂ plants from this cross, the ratio of resistant:susceptible plants was approximately 3:1, indicating that resistance to P. syringae expressing avrB is determined by a single dominant locus in ecotype Col-0, which we have designated RPS3 . Using RFLP analysis, RPS3 was mapped to chromosome 3, adjacent to markers M583 and G4523, and ≤ 1 cM from another disease resistance locus, RPM1 . In soybean, resistance to P. syringae strains that carry avrB is controlled by the locus RPG1 . Thus, RPG1 and RPS3 both confer avrB -specific disease resistance, suggesting that these genes may be homologs.     

Large-scale structure-function analysis of the Arabidopsis RPM1 disease resistance protein

The Arabidopsis RPM1 gene confers resistance against Pseudomonas syringae expressing either the AvrRpm1 or the AvrB type III effector protein. We present an exhaustive genetic screen for mutants that no longer recognize avrRpm1. Using an inducible avrRpm1 expression system, we identified 110 independent mutations. These mutations represent six complementation groups. None discriminates between avrRpm1 and avrB recognition. We identified 95 rpm1 alleles and present a detailed structure--function analysis of the RPM1 protein. Several rpm1 mutants retain partial function, and we deduce that their residual activity is dependent on the level of avrRpm1 signal. In these mutants, the hypersensitive response remains activated if the signal goes above a certain threshold. Missense mutations in rpm1 are highly enriched in the nucleotide binding domain, suggesting that this region plays a key role either in the hypersensitive response associated with RPM1 activation or in RPM1 stability. Cluster analysis of rpm1 alleles defines functionally important residues that are highly conserved between nucleotide binding site leucine-rich repeat R proteins and those that are unique to RPM1. Regions of RPM1 to which no loss-of-function alleles map may represent domains in which variation is tolerated and may contribute to the evolution of new R gene specificities.     

Soybean Resistance Genes Specific for Different Pseudomonas Syringae Avirulence Genes Are Allelic, or Closely Linked, at the Rpg1 Locus

RPG1 and RPM1 are disease resistance genes in soybean and Arabidopsis, respectively, that confer resistance to Pseudomonas syringae strains expressing the avirulence gene avrB. RPM1 has recently been demonstrated to have a second specificity, also conferring resistance to P. syringae strains expressing avrRpm1. Here we show that alleles, or closely linked genes, exist at the RPG1 locus in soybean that are specific for either avrB or avrRpm1 and thus can distinguish between these two avirulence genes.     

A putative nucleoporin 96 Is required for both basal defense and constitutive resistance responses mediated by suppressor 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.     

Overexpression of the plasma membrane-localized NDR1 protein results in enhanced bacterial disease resistance in Arabidopsis thaliana

Previous studies have established that mutations in the NDR1 gene in Arabidopsis thaliana suppress the resistance response of three resistance proteins, RPS2, RPM1, and RPS5, to Pseudomonas syringae pv. tomato (Pst) strain DC3000 containing the cognate effector genes, avrRpt2, avrRpm1, and avrpPhB, respectively. NDR1 is a plasma membrane (PM)-localized protein, and undergoes several post-translational modifications including carboxy-terminal processing and N-linked glycosylation. Expression of NDR1 under the NDR1 native promoter complements the ndr1-1 mutation, while overexpression of NDR1 results in enhanced resistance to virulent Pst. Sequence analysis and mass spectrometry suggest that NDR1 is localized to the PM via a C-terminal glycosylphosphatidyl-inositol (GPI) anchor. GPI modification would potentially place NDR1 on the outer surface of the PM, perhaps allowing NDR1 to act as a transducer of pathogen signals and/or interact directly with the pathogen.     

Virulence of the phytopathogen Pseudomonas syringae pv. maculicola is rpoN dependent

We cloned the rpoN (ntrA and glnF) gene encoding sigma(54) from the phytopathogen Pseudomonas syringae pv. maculicola strain ES4326. The P. syringae ES4326 rpoN gene complemented Pseudomonas aeruginosa, Escherichia coli, and Klebsiella aerogenes rpoN mutants for a variety of rpoN mutant phenotypes, including the inability to utilize nitrate as sole nitrogen source. DNA sequence analysis of the P. syringae ES4326 rpoN gene revealed that the deduced amino acid sequence was most similar (86% identity; 95% similarity) to the sigma(54) protein encoded by the Pseudomonas putida rpoN gene. A marker exchange protocol was used to construct an ES4326 rpoN insertional mutation, rpoN::Km(r). In contrast to wild-type ES4326, ES4326 rpoN::Km(r) was nonmotile and could not utilize nitrate, urea, C(4)-dicarboxylic acids, several amino acids, or concentrations of ammonia below 2 mM as nitrogen sources. rpoN was essential for production of the phytotoxin coronatine and for expression of the structural genes encoding coronamic acid. In addition, ES4326 rpoN::Km(r) did not multiply or elicit disease symptoms when infiltrated into Arabidopsis thaliana leaves, did not elicit the accumulation of several Arabidopsis defense-related mRNAs, and did not elicit a hypersensitive response (HR) when infiltrated into tobacco (Nicotiana tabacum) leaves. Furthermore, whereas P. syringae ES4326 carrying the avirulence gene avrRpt2 elicited an HR when infiltrated into Arabidopsis ecotype Columbia leaves, ES4326 rpoN::Km(r) carrying avrRpt2 elicited no response. Constitutive expression of ES4326 hrpL in ES4326 rpoN::Km(r) partially restored defense-related mRNA accumulation, showing a direct role for the hrp cluster in host defense gene induction in a compatible host-pathogen interaction. However, constitutive expression of hrpL in ES4326 rpoN::Km(r) did not restore coronatine production, showing that coronatine biosynthesis requires factors other than hrpL.     

Arabidopsis thaliana EDS4 contributes to salicylic acid (SA)-dependent expression of defense responses: evidence for inhibition of jasmonic acid signaling by SA

The Arabidopsis enhanced disease susceptibility 4 (eds4) mutation causes enhanced susceptibility to infection by the bacterial pathogen Pseudomonas syringae pv. maculicola ES4326 (Psm ES4326). Gene-for-gene resistance to bacteria carrying the avirulence gene avrRpt2 is not significantly affected by eds4. Plants homozygous for eds4 exhibit reduced expression of the pathogenesis-related gene PR-1 after infection by Psm ES4326, weakened responses to treatment with the signal molecule salicylic acid (SA), impairment of the systemic acquired resistance response, and reduced accumulation of SA after infection with Psm ES4326. These phenotypes indicate that EDS4 plays a role in SA-dependent signaling. SA has been shown to have a negative effect on activation of gene expression by the signal molecule jasmonic acid (JA). Two mutations that cause reduced SA levels, eds4 and pad4, cause heightened responses to inducers of JA-dependent gene expression, providing genetic evidence to support the idea that SA interferes with JA-dependent signaling. Two possible working models of the role of EDS4 in governing activation of defense responses are presented.     

Identification of three putative signal transduction genes involved in R gene-specified disease resistance in Arabidopsis

The RPS5 disease resistance gene of Arabidopsis mediates recognition of Pseudomonas syringae strains that possess the avirulence gene avrPphB. By screening for loss of RPS5-specified resistance, we identified five pbs (avrPphB susceptible) mutants that represent three different genes. Mutations in PBS1 completely blocked RPS5-mediated resistance, but had little to no effect on resistance specified by other disease resistance genes, suggesting that PBS1 facilitates recognition of the avrPphB protein. The pbs2 mutation dramatically reduced resistance mediated by the RPS5 and RPM1 resistance genes, but had no detectable effect on resistance mediated by RPS4 and had an intermediate effect on RPS2-mediated resistance. The pbs2 mutation also had varying effects on resistance mediated by seven different RPP (recognition of Peronospora parasitica) genes. These data indicate that the PBS2 protein functions in a pathway that is important only to a subset of disease-resistance genes. The pbs3 mutation partially suppressed all four P. syringae-resistance genes (RPS5, RPM1, RPS2, and RPS4), and it had weak-to-intermediate effects on the RPP genes. In addition, the pbs3 mutant allowed higher bacterial growth in response to a virulent strain of P. syringae, indicating that the PBS3 gene product functions in a pathway involved in restricting the spread of both virulent and avirulent pathogens. The pbs mutations are recessive and have been mapped to chromosomes I (pbs2) and V (pbs1 and pbs3).     

Expression of the Pseudomonas syringae avirulence protein AvrB in plant cells alleviates its dependence on the hypersensitive response and pathogenicity (Hrp) secretion system in eliciting genotype-specific hypersensitive cell death.

The nonpathogenic bacteria Pseudomonas fluorescens and Escherichia coli can elicit a genotype-specific hypersensitive response (HR) in plants if they express both the HR and pathogenesis (Hrp) protein secretion system and the HrpZ harpin from P. syringae pv syringae 61 and a P. syringae avirulence (avr) gene whose presence is recognized by a corresponding disease resistance gene in the plant. We have found that the recognition event appears to require transfer of the Avr protein into the plant cell. Elicitation of a genotype-specific HR was observed with avrB+ P. fluorescens in soybean and Arabidopsis plants carrying resistance genes RPG1 and RPM1, respectively, and with avrPto+ E. coll in tomato plants carrying resistance gene PTO, but only if the Hrp secretion system, HrpZ, and the appropriate Avr proteins were produced in the same bacterial cell. The failure of avrB hyperexpression and exogenous AvrB or HrpZ to alleviate these requirements in soybean and Arabidopsis suggests that the site of AvrB action is not in the bacterial cell or plant apoplast. An Arabidopsis rps3 (rpm1) glabrous1 mutant was transformed with constructs expressing avrB and was crossed with an Arabidopsis ecotype Columbia (RPM1 GLABROUS1) plant. F1 seedlings (identified by their kanamycin-resistant, pubescent phenotype) exhibited extensive necrosis on cotyledon leaves 10 days postgermination. Ecotype Columbia and rps3-1 leaves biolistically cobombarded with plasmids expressing the beta-glucuronidase (GUS) gene and avrB failed to produce GUS activity (indicative of cell death) only when RPM1 and avrB were present in the leaf. Thus, both stable and transient expression of avrB in Arabidopsis resulted in RPM1-dependent necrosis, and the only demonstrable site of action for AvrB was inside plant cells.     

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.     

Arabidopsis NHO1 is required for general resistance against Pseudomonas bacteria

Nonhost interactions are prevalent between plants and specialized phytopathogens. Although it has great potential for providing crop plants with durable resistance, nonhost resistance is poorly understood. Here, we show that nonhost resistance is controlled, at least in part, by general resistance. Arabidopsis plants are resistant to the nonhost pathogen Pseudomonas syringae pv phaseolicola NPS3121 and completely arrest bacterial multiplication in the plant. Ten Arabidopsis mutants were isolated that were compromised in nonhost (nho) resistance to P. s. phaseolicola. Among these, nho1 is caused by a single recessive mutation that defines a novel gene. nho1 is defective in nonspecific resistance to Pseudomonas bacteria, because it also supported the growth of P. s. tabaci and P. fluorescens bacteria, both of which are nonpathogenic on Arabidopsis. In addition, the nho1 mutation also compromised resistance mediated by RPS2, RPS4, RPS5, and RPM1. Interestingly, the nho1 mutation had no effect on the growth of the virulent bacteria P. s. maculicola ES4326 and P. s. tomato DC3000, but it partially restored the in planta growth of the DC3000 hrpS(-) mutant bacteria. Thus, the virulent bacteria appear to evade or suppress NHO1-mediated resistance by means of an Hrp-dependent virulence mechanism.