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.     

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.     

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.     

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.     

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.     

Pseudomonas syringae effector avrB confers soybean cultivar-specific avirulence on Soybean mosaic virus adapted for transgene expression but effector avrPto does not

Soybean mosaic virus (SMV) was adapted for transgene expression in soybean and used to examine the function of avirulence genes avrB and avrPto of Pseudomonas syringae pvs. glycinea and tomato, respectively. A cloning site was introduced between the P1 and HC-Pro genes in 35S-driven infectious cDNAs of strains SMV-N and SMV-G7. Insertion of the uidA gene or the green fluorescent protein gene into either modified cDNA and bombardment into primary leaves resulted in systemic expression that reflected the pattern of viral movement into uninoculated leaves. Insertion of avrB blocked symptom development and detectable viral movement in cv. Harosoy, which carries the Rpg1-b resistance gene corresponding to avrB, but not in cvs. Keburi or Hurrelbrink, which lack Rpg1-b. In Keburi and Hurrelbrink, symptoms caused by SMV carrying avrB appeared more quickly and were more severe than those caused by the virus without avrB. Insertion of avrPto enhanced symptoms in Harosoy, Hurrelbrink, and Keburi. This result was unexpected because avrPto was reported to confer avirulence on P. syringae pv. glycinea inoculated to Harosoy. We inoculated Harosoy with P syringae pv. glycinea expressing avrPto, but observed no hypersensitive reaction, avrPto-dependent induction of pathogenesis-related protein la, or limitation of bacterial population growth. In Hurrelbrink, avrPto enhanced bacterial multiplication and exacerbated symptoms. Our results establish SMV as an expression vector for soybean. They demonstrate that resistance triggered by avrB is effective against SMV, and that avrB and avrPto have general virulence effects in soybean. The results also led to a reevaluation of the reported avirulence activity of avrPto in this plant.     

Genetic and physical localization of the soybean Rpg1-b disease resistance gene reveals a complex locus containing several tightly linked families of NBS-LRR genes

Alleles or tightly linked genes at the soybean (Glycine max L. Merr.) Rpg1 locus confer resistance to strains of Pseudomonas syringae pv. glycinea that express the avirulence genes avrB or avrRpm1. We have previously mapped Rpg1-b (the gene specific for avrB) to a cluster of resistance genes (R genes) with diverse specificities in molecular linkage group F. Here, we describe the high-resolution physical and genetic mapping of Rpg1-b to a 0.16-cM interval encompassed by two overlapping BAC clones spanning approximately 270 kilobases. Rpg1-b is part of a complex locus containing numerous genes related to previously characterized coiled coil-nucleotide binding site-leucine rich repeat (CC-NBS-LRR)-type R genes that are spread throughout this region. Phylogenetic and Southern blot analyses group these genes into four distinct subgroups, some of which are conserved in the common bean, Phaseolus vulgaris, indicating that this R gene cluster may predate the divergence of Phaseolus and Glycine. Members from different subgroups are physically intermixed and display a high level of polymorphism between soybean cultivars, suggesting that this region is rearranging at a high frequency. At least five CC-NBS-LRR-type genes cosegregate with Rpg1-b in our large mapping populations.     

AvrB mutants lose both virulence and avirulence activities on soybean and Arabidopsis

The Pseudomonas syringae pv. glycinea effector protein AvrB induces resistance responses in soybean varieties that contain the resistance gene Rpg1-b and Arabidopsis varieties that carry RPM1. In addition to this avirulence activity, AvrB also enhances bacterial virulence on soybean plants that lack Rpg1-b and induces a chlorotic phenotype on Arabidopsis plants that lack RPM1. We screened a library of avrB mutants for loss of avirulence on soybean and Arabidopsis, and assayed selected avirulence mutants for loss of virulence on both plants. All mutants screened were recognized similarly on both plant species. Nine single-site avrB mutations that affected avirulence localized to a solvent-accessible pocket in the protein structure. Seven of these mutated residues are absolutely conserved between AvrB and its nine homologues. Avirulence mutants generally lost virulence enhancement on susceptible soybean varieties and lost the ability to induce a chlorotic response on the rpm1 null Arabidopsis variety Mt-0. Three of four avirulence mutants tested failed to interact with RIN4, an Arabidopsis protein previously shown to be required for RPM1 function. Our results suggest that soybean and Arabidopsis recognize AvrB in the same manner, and that AvrB enzymatic activity is required for its function as an avirulence and virulence effector on two different plant species.     

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.     

Sequence domains required for the activity of avirulence genes avrB and avrC from Pseudomonas syringae pv. glycinea.

avrB and avrC from Pseudomonas syringae pv. glycinea share significant amino acid homology but interact with different soybean resistance genes to elicit the hypersensitive defense reaction. Recombinant genes constructed between avrB and avrC revealed that the central regions were required for avirulence gene activity but the 5' and 3' termini were interchangeable. Recombinants involving the central regions did not yield any detectable avirulence gene activity, and no new avirulence phenotypes were observed from any of the chimeric genes. These results suggest that the protein products of avrB and avrC possess catalytic properties that are required for the avirulence phenotypes.     

Functional homologs of the Arabidopsis RPM1 disease resistance gene in bean and pea.

We showed that a bacterial avirulence (avr) gene function, avrPpiA1, from the pea pathogen Pseudomonas syringae pv pisi, is recognized by some, but not all, genotypes of Arabidopsis. Thus, an avr gene functionally defined on a crop species is also an avr gene on Arabidopsis. The activity of avrPpiA1 on a series of Arabidopsis genotypes is identical to that of the avrRpm1 gene from P.s. pv maculicola previously defined using Arabidopsis. The two avr genes are homologous and encode nearly identical predicted products. Moreover, this conserved avr function is also recognized by some bean and pea cultivars in what has been shown to be a gene-for-gene manner. We further demonstrated that the Arabidopsis disease resistance locus, RPM1, conditioning resistance to avrRpm1, also conditions resistance to bacterial strains carrying avrPpiA1. Therefore, bean, pea, and conceivably other crop species contain functional and potentially molecular homologs of RPM1.     

The RPM1 plant disease resistance gene facilitates a rapid and sustained increase in cytosolic calcium that is necessary for the oxidative burst and hypersensitive cell death

Early events occurring during the hypersensitive resistance response (HR) were examined using the avrRpm1/RPM1 gene-for-gene interaction in Arabidopsis challenged by Pseudomonas syringae pv. tomato. Increases in cytosolic Ca2+ were measured in whole leaves using aequorin-mediated bioluminescence. During the HR a sustained increase in Ca2+ was observed which was dependent on the presence of both a functional RPM1 gene product and delivery of the cognate avirulence gene product AvrRpm1. The sequence-unrelated avirulence gene avrB, which also interacts with RPM1, generated a significantly later but similarly prolonged increase in cytosolic Ca2+. Accumulation of H2O2 at reaction sites, as revealed by electron microscopy, occurred within the same time frame as the changes in cytosolic Ca2+. The NADPH oxidase inhibitor diphenylene iodonium chloride did not affect the calcium signature, but did block H2O2 accumulation and the HR. By contrast, the calcium-channel blocker LaCl3 suppressed the increase in cytosolic Ca2+ as well as H2O2 accumulation and the HR, placing calcium elevation upstream of the oxidative burst.     

Molecular characterization of cloned avirulence genes from race 0 and race 1 of Pseudomonas syringae pv. glycinea.

A wide-host-range cosmid cloning vector, pLAFR3, was constructed and used to make cosmid libraries of partially digested Sau3A DNA from race 0 and race 1 of Pseudomonas syringae pv. glycinea. Two avirulence genes, avrB0 and avrC, cloned from race 0, elicited the hypersensitivity reaction (HR) on specific cultivars of soybean. Race 4 transconjugants containing avrB0 induced a dark brown necrotic HR within 24 h on the soybean cultivars Harosoy and Norchief, whereas race 4 transconjugants containing avrC induced a light brown necrotic HR within 48 h on the soybean cultivars Acme, Peking, Norchief, and Flambeau. An additional avirulence gene, avrB1, cloned from race 1, appeared to be identical to avrB0 from race 0. The avrB0 and avrC genes from race 0 were characterized by restriction enzyme mapping, Southern blot analysis, Tn5 transposon mutagenesis, and site-directed gene replacements. The effects of these three genes on the in planta bacterial growth of race 4 transconjugants have also been examined. The identification and cloning of avrB1 provides genetic evidence for a gene-for-gene interaction in the bacterial blight disease of soybean, as avrB1 from race 1 interacts with the soybean disease resistance locus, Rpg1.     

Disease development in ethylene-insensitive Arabidopsis thaliana infected with virulent and avirulent Pseudomonas and Xanthomonas pathogens.

The plant hormone ethylene has been hypothesized to play roles both in disease resistance and in disease susceptibility. These processes were examined by using isogenic virulent and avirulent bacterial pathogens and mutants of Arabidopsis thaliana that were altered in ethylene physiology. Ethylene-insensitive ein1 and ein2 mutants of Arabidopsis were resistant to Pseudomonas syringae pv. tomato made avirulent by the addition of the cloned avirulence genes avrRpt2, avrRpm1, or avrB; this suggests that ethylene is not required for active resistance against avirulent bacteria. In a second set of experiments, susceptibility was monitored with virulent P. s. pv. tomato, P. s. pv. maculicola, or Xanthomonas campestris pv. campestris strains. Wild-type Arabidopsis and ein1 mutants were susceptible to these strains, but ein2 mutants developed only minimal disease symptoms. Despite these reduced symptoms, virulent P. s. pv. tomato grew extensively within ein2 leaves. The Pseudomonas phytotoxin coronatine induces ethylene biosynthesis and diseaselike symptoms on many plant species, but the reduced symptomology of ein2 mutants could not be attributed to insensitivity to coronatine. The enhanced disease tolerance of ein2 plants suggests that ethylene may mediate pathogen-induced damage, but the absence of tolerance in ein1 mutants has yet to be explained.     

Characterization and expression of two avirulence genes cloned from Pseudomonas syringae pv. glycinea.

Two avirulence genes, avrB and avrC, from race 0 of Pseudomonas syringae pv. glycinea, were sequenced and found to encode single protein products of 36 and 39 kilodaltons, respectively. The proteins had neither recognizable signal peptide sequences nor significant stretches of hydrophobic amino acids that might indicate membrane association. Both avrB and avrC had relatively low position 3 and overall G+C contents, which suggests that they may have been recently introduced into P. syringae pv. glycinea. The deduced amino acid sequences of the proteins encoded by avrB and avrC shared 42% identical amino acids. However, when introduced into race 4 of P. syringae pv. glycinea, each gene directed a unique pattern of hypersensitive reactions on several differential soybean cultivars. The avrC protein was overproduced in Escherichia coli cells and deposited as insoluble inclusion bodies in the cell cytoplasm. The avrC protein could be solubilized with urea-octyl glucoside treatment, but neither the solubilized protein nor the intact inclusion bodies elicited a hypersensitive reaction in soybean leaves.     

Identification of Pseudomonas syringae pathogens of Arabidopsis and a bacterial locus determining avirulence on both Arabidopsis and soybean.

To develop a model system for molecular genetic analysis of plant-pathogen interactions, we studied the interaction between Arabidopsis thaliana and the bacterial pathogen Pseudomonas syringae pv tomato (Pst). Pst strains were found to be virulent or avirulent on specific Arabidopsis ecotypes, and single ecotypes were resistant to some Pst strains and susceptible to others. In many plant-pathogen interactions, disease resistance is controlled by the simultaneous presence of single plant resistance genes and single pathogen avirulence genes. Therefore, we tested whether avirulence genes in Pst controlled induction of resistance in Arabidopsis. Cosmids that determine avirulence were isolated from Pst genomic libraries, and the Pst avirulence locus avrRpt2 was defined. This allowed us to construct pathogens that differed only by the presence or absence of a single putative avirulence gene. We found that Arabidopsis ecotype Col-0 was susceptible to Pst strain DC3000 but resistant to the same strain carrying avrRpt2, suggesting that a single locus in Col-0 determines resistance. As a first step toward genetically mapping the postulated resistance locus, an ecotype susceptible to infection by DC3000 carrying avrRpt2 was identified. The avrRpt2 locus from Pst was also moved into virulent strains of the soybean pathogen P. syringae pv glycinea to test whether this locus could determine avirulence on soybean. The resulting strains induced a resistant response in a cultivar-specific manner, suggesting that similar resistance mechanisms may function in Arabidopsis and soybean.     

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.     

Identification and molecular mapping of a single Arabidopsis thaliana locus determining resistance to a phytopathogenic Pseudomonas syringae isolate

We present a model pathosystem to dissect genetically the disease resistance response of plants against phytopathogenic bacteria. The interaction between Pseudomonas syringae pathovar maculicola (Psm) and Arabidopsis thaliana displays phenotypic varia-ion which depends on the genotype of both partners. Compatible interactions are defined by sustained in-planta bacterial growth and are normally accompanied of their appearance. For compatible interactions, resistance is defined by limited in-planta bacterial growth accompanied by a typical 'hypersensitive response' (HR). We show that at least parts of this system fit the paradigms of Flor's 'gene-for-gene' hypothesis. We identify functionally a putative bacterial avirulence gene (avrRpm 1) from a Psm isolate which conditions the HR on A. thaliana ecotypes Oy-0 abd Col- 0, but not Nd-0. We also demonstrate that resistance to the Psm strain from which avrRpm1 was isolated segregates as a single trait in the crosses Col-o x Nd-0 and Nd-0 x Oy-0. Furthermore, we map this locus (RPM1) molecularly in the Col-0 x Nd-0 cross to a relatively small interval defined by two RFLP markers on A. thliana chromosome 3. Resistance in the second cross also maps to this locus and co-segregates with resistance to avrRpm1.