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.     

The Erwinia amylovora avrRpt2EA gene contributes to virulence on pear and AvrRpt2EA is recognized by Arabidopsis RPS2 when expressed in pseudomonas syringae

The enterobacterium Erwinia amylovora is a devastating plant pathogen causing necrotrophic fire blight disease of apple, pear, and other rosaceous plants. In an attempt to identify genes induced during infection of host plants, we identified and cloned a putative effector gene, avrRpt2EA. The deduced amino-acid sequence of the translated AvrRpt2EA protein is homologous to the effector protein AvrRpt2 previously reported in Pseudomonas syringae pv. tomato. These two proteins share 58% identity (70% similarity) in the functional domain; however, the secretion and translocation signal domain varied. The avrRpt2EA promoter region contains a typical 'hrp box,' which suggests that avrRpt2EA is regulated by the alternative sigma factor, HrpL. avrRpt2EA was detected in all E. amylovora strains tested but not in other closely related Erwinia species. An avrRpt2EA deletion mutant was reduced in its ability to cause systemic infection on immature pear fruits as compared with the wild-type strain, indicating that avrRpt2EA acts as a virulence factor on its native host. Growth of P. syringae pv. tomato DC3000 expressing avrRpt2EA was 10-fold higher than that of P. syringae pv. tomato DC3000 in an Arabidopsis rps2 mutant, indicating that avrRpt2EA promotes virulence of P. syringae pv. tomato DC3000 on Arabidopsis similar to P. syringae pv. tomato avrRpt2. When avrRpt2EA was expressed in P. syringae pv. tomato DC3000 in its native form, a weak hypersensitive response (HR) was induced in Arabidopsis; however, a hybrid protein containing the P. syringae pv. tomato avrRpt2 signal sequence, when expressed from the P syringae pv. tomato avrRpt2 promoter, caused a strong HR. Thus, the signal sequence and promoter of avrRpt2EA may affect its expression, secretion, or translocation, singly or in combination, in P. syringae pv. tomato DC3000. These results indicated that avrRpt2EA is genetically recognized by the RPS2 disease resistance gene in Arabidopsis when expressed in P. syringae pv. tomato DC3000. The results also suggested that although distinct pathogens such as E. amylovora and P. syringae may contain similar effector genes, expression and secretion of these effectors can be under specific regulation by the native pathogen.     

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.     

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.     

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.     

The cloned avirulence gene avrPto induces disease resistance in tomato cultivars containing the Pto resistance gene.

Resistance of tomato plants to the bacterial pathogen Pseudomonas syringae pv. tomato race 0 is controlled by the locus Pto. A bacterial avirulence gene was cloned by constructing a cosmid library from an avirulent P. syringae pv. tomato race, conjugating the recombinants into a strain of P. syringae pv. maculicola virulent on a tomato cultivar containing Pto, and screening for those clones that converted the normally virulent phenotype to avirulence. The cloned gene, designated avrPto, reduced multiplication of P. syringae pv. tomato transconjugants specifically on Pto tomato lines, as demonstrated by bacterial growth curve analyses. Analysis of F2 populations revealed cosegregation of resistance to P. syringae pv. tomato transconjugants carrying avrPto with resistance to P. syringae pv. tomato race 0. Surprisingly, mutation of avrPto in P. syringae pv. tomato race 0 does not eliminate the avirulent phenotype of race 0, suggesting that additional, as yet uncharacterized, avirulence genes and/or resistance genes may contribute to specificity in the avrPto-Pto interaction. Genetic analysis indicates that this resistance gene(s) would be tightly linked to Pto. Interestingly, P. syringae pv. glycinea transconjugants carrying avrPto elicit a typical hypersensitive resistant response in the soybean cultivar Centennial, suggesting conservation of Pto function between two crop plants, tomato and soybean.     

Pseudomonas syringae pv. syringae B728a hydrolyses indole-3-acetonitrile to the plant hormone indole-3-acetic acid

Nitrilase enzymes catalyse the hydrolysis of nitrile compounds to the corresponding carboxylic acid and ammonia, and have been identified in plants, bacteria and fungi. There is mounting evidence to support a role for nitrilases in plant–microbe interactions, but the activity of these enzymes in plant pathogenic bacteria remains unexplored. The genomes of the plant pathogenic bacteria Pseudomonas syringae pv. syringae B728a and Pseudomonas syringae pv. tomato DC3000 contain nitrilase genes with high similarity to characterized bacterial arylacetonitrilases. In this study, we show that the nitrilase of P. syringae pv. syringae B728a is an arylacetonitrilase, which is capable of hydrolysing indole-3-acetonitrile to the plant hormone indole-3-acetic acid, and allows P. syringae pv. syringae B728a to use indole-3-acetonitrile as a nitrogen source. This enzyme may represent an additional mechanism for indole-3-acetic acid biosynthesis by P. syringae pv. syringae B728a, or may be used to degrade and assimilate aldoximes and nitriles produced during plant secondary metabolism. Nitrilase activity was not detected in P. syringae pv. tomato DC3000, despite the presence of a homologous nitrilase gene. This raises the interesting question of why nitrilase activity has been retained in P. syringae pv. syringae B728a and not in P. syringae pv. tomato DC3000.     

In silico analysis reveals multiple putative type VI secretion systems and effector proteins in Pseudomonas syringae pathovars

Type VI secretion systems (T6SS) of Gram-negative bacteria form injectisomes that have the potential to translocate effector proteins into eukaryotic host cells. In silico analysis of the genomes in six Pseudomonas syringae pathovars revealed that P. syringae pv. tomato DC3000, pv. tabaci ATCC 11528, pv. tomato T1 and pv. oryzae 1-6 each carry two putative T6SS gene clusters (HSI-I and HSI-II; HSI: Hcp secretion island), whereas pv. phaseolicola 1448A and pv. syringae B728 each carry one. The pv. tomato DC3000 HSI-I and pv. tomato T1 HSI-II possess a highly similar organization and nucleotide sequence, whereas the pv. tomato DC3000, pv. oryzae 1-6 and pv. tabaci 11528 HSI-II are more divergent. Putative effector orthologues vary in number among the strains examined. The Clp-ATPases and IcmF orthologues form distinct phylogenetic groups: the proteins from pv. tomato DC3000, pv. tomato T1, pv. oryzae and pv. tabaci 11528 from HSI-II group together with most orthologues from other fluorescent pseudomonads, whereas those from pv. phaseolicola, pv. syringae, pv. tabaci, pv. tomato T1 and pv. oryzae from HSI-I group closer to the Ralstonia solanacearum and Xanthomonas orthologues. Our analysis suggests multiple independent acquisitions and possible gene attrition/loss of putative T6SS genes by members of P. syringae.     

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.     

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.     

Natural variation in RPS2-mediated resistance among Arabidopsis accessions: correlation between gene expression profiles and phenotypic responses

Natural variation in gene expression (expression traits or e-traits) is increasingly used for the discovery of genes controlling traits. An important question is whether a particular e-trait is correlated with a phenotypic trait. Here, we examined the correlations between phenotypic traits and e-traits among 10 Arabidopsis thaliana accessions. We studied defense against Pseudomonas syringae pv tomato DC3000 (Pst), with a focus on resistance gene-mediated resistance triggered by the type III effector protein AvrRpt2. As phenotypic traits, we measured growth of the bacteria and extent of the hypersensitive response (HR) as measured by electrolyte leakage. Genetic variation among accessions affected growth of Pst both with (Pst avrRpt2) and without (Pst) the AvrRpt2 effector. Variation in HR was not correlated with variation in bacterial growth. We also collected gene expression profiles 6 h after mock and Pst avrRpt2 inoculation using a custom microarray. Clusters of genes whose expression levels are correlated with bacterial growth or electrolyte leakage were identified. Thus, we demonstrated that variation in gene expression profiles of Arabidopsis accessions collected at one time point under one experimental condition has the power to explain variation in phenotypic responses to pathogen attack.     

Natural variation in the Arabidopsis response to the avirulence gene hopPsyA uncouples the hypersensitive response from disease resistance

The plant hypersensitive response (HR) is tightly associated with gene-for-gene resistance and has been proposed to function in containing pathogens at the invasion site. This tight association has made it difficult to unequivocally evaluate the importance of HR for plant disease resistance. Here, hopPsyA from Pseudomonas syringae pv. syringae 61 is identified as a new avirulence gene for Arabidopsis that triggers resistance in the absence of macroscopic HR. Resistance to P. syringae pv. tomato DC3000 expressing hopPsyA was EDS1-dependent and NDR1-independent. Intriguingly, several Arabidopsis accessions were resistant to DC3000(hopPsyA) in the absence of HR. This is comparable to the Arabidopsis response to avrRps4, but it is shown that hopPsyA does not signal through RPS4. In a cross between two hopPsyA-resistant accessions that differ in their HR response, the HR segregated as a recessive phenotype regulated by a single locus. This locus, HED1 (HR regulator in EDS1 pathway), is proposed to encode a protein whose activity can cause suppression of the EDS1-dependent HR signaling pathway. HED1-regulated symptomless gene-for-gene resistance responses may explain some cases of Arabidopsis resistance to bacteria that are classified as nonhost resistance.     

A Pseudomonas syringae pv. tomato DC3000 mutant lacking the type III effector HopQ1-1 is able to cause disease in the model plant Nicotiana benthamiana

The model pathogen Pseudomonas syringae pv. tomato DC3000 causes bacterial speck in tomato and Arabidopsis, but Nicotiana benthamiana, an important model plant, is considered to be a non-host. Strain DC3000 injects approximately 28 effector proteins into plant cells via the type III secretion system (T3SS). These proteins were individually delivered into N. benthamiana leaf cells via T3SS-proficient Pseudomonas fluorescens, and eight, including HopQ1-1, showed some capacity to cause cell death in this test. Four gene clusters encoding 13 effectors were deleted from DC3000: cluster II (hopH1, hopC1), IV (hopD1, hopQ1-1, hopR1), IX (hopAA1-2, hopV1, hopAO1, hopG1), and native plasmid pDC3000A (hopAM1-2, hopX1, hopO1-1, hopT1-1). DC3000 mutants deleted for cluster IV or just hopQ1-1 acquired the ability to grow to high levels and produce bacterial speck lesions in N. benthamiana. HopQ1-1 showed other hallmarks of an avirulence determinant in N. benthamiana: expression in the tobacco wildfire pathogen P. syringae pv. tabaci 11528 rendered this strain avirulent in N. benthamiana, and elicitation of the hypersensitive response in N. benthamiana by HopQ1-1 was dependent on SGT1. DC3000 polymutants involving other effector gene clusters in a hopQ1-1-deficient background revealed that clusters II and IX contributed to the severity of lesion symptoms in N. benthamiana, as well as in Arabidopsis and tomato. The results support the hypothesis that the host ranges of P. syringae pathovars are limited by the complex interactions of effector repertoires with plant anti-effector surveillance systems, and they demonstrate that N. benthamiana can be a useful model host for DC3000.     

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.