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.     

Induction of Arabidopsis defense genes by virulent and avirulent Pseudomonas syringae strains and by a cloned avirulence gene.

We developed a model system to study the signal transduction pathways leading to the activation of Arabidopsis thaliana genes involved in the defense against pathogen attack. Here we describe the identification and characterization of virulent and avirulent Pseudomonas syringae strains that elicit disease or resistance symptoms when infiltrated into Arabidopsis leaves. The virulent and avirulent strains were characterized by determining growth of the pathogen in Arabidopsis leaves and by measuring accumulation of mRNA corresponding to Arabidopsis phenylalanine ammonia-lyase (PAL), beta-1,3-glucanase (BG), and chalcone synthase (CHS) genes in infected leaves. The virulent strain, P. syringae pv maculicola ES4326, multiplied 10(5)-fold in Arabidopsis leaves and strongly elicited BG1, BG2, and BG3 mRNA accumulation but had only a modest effect on PAL mRNA accumulation. In contrast, the avirulent strain, P. syringae pv tomato MM1065, multiplied less than 10-fold in leaves and had only a minimal effect on BG1, BG2, and BG3 mRNA accumulation, but it induced PAL mRNA accumulation. No accumulation of CHS mRNA was found with either ES4326 or MM1065. We also describe the cloning of a putative avirulence (avr) gene from the avirulent strain MM1065 that caused the virulent strain ES4326 to grow less well in leaves and to strongly elicit PAL but not BG1 and BG3 mRNA accumulation. These results suggest that the Arabidopsis PAL and BG genes may be activated by distinct signal transduction pathways and show that differences in plant gene induction by virulent and avirulent strains can be attributed to a cloned presumptive avr gene.     

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.     

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.     

Bacterial non-host resistance: interactions of Arabidopsis with non-adapted Pseudomonas syringae strains

Although interactions of plants with virulent and avirulent host pathogens are under intensive study, relatively little is known about plant interactions with non-adapted pathogens and the molecular events underlying non-host resistance. Here we show that two Pseudomonas syringae strains for which Arabidopsis is a non-host plant, P. syringae pathovar (pv.) glycinea (Psg) and P. syringae pv. phaseolicola (Psp),induce salicylic acid (SA) accumulation and pathogenesis-related gene expression at inoculation sites, and that induction of these defences is largely dependent on bacterial type III secretion. The defence signalling components activated by non-adapted bacteria resemble those initiated by host pathogens, including SA, non-expressor of PR-1, non-race specific disease resistance 1, phytoalexin-deficient 4 and enhanced disease susceptibility 1. However, some differences in individual defence pathways induced by Psg and Psp exist, suggesting that for each strain, distinct sets of type III effectors are recognized by the plant. Although induction of SA-related defences occurs, it does not directly contribute to bacterial non-host resistance, because Arabidopsis mutants compromised in SA signalling and other classical defence pathways do not permit enhanced survival of Psg or Psp in leaves. The finding that numbers of non-adapted bacteria in leaf extracellular spaces rapidly decline after inoculation suggests that they fail to overcome toxic or structural defence barriers preceding SA-related responses. Consistent with this hypothesis, rapid, type III secretion system-independent upregulation of the lignin biosynthesis genes, PAL1 and BCB, which might contribute to an early induced, cell wall-based defence mechanism, occurs in response to non-adapted bacteria. Moreover, knockout of PAL1 permits increased leaf survival of non-host bacteria. In addition, different survival rates of non-adapted bacteria in leaves from Arabidopsis accessions and mutants with distinct glucosinolate composition or hydrolysis exist. Possible roles for early inducible, cell wall-based defences and the glucosinolate/myrosinase system in bacterial non-host resistance are discussed.     

Molecular characterization of avirulence gene D from Pseudomonas syringae pv. tomato

Avirulence gene D, cloned from Pseudomonas syringae pv. tomato, caused P. s. pv. glycinea to elicit a hypersensitive defense response on certain cultivars of soybean. Nucleotide sequence data for a 5.6-kb HindIII fragment containing avrD disclosed five long open-reading frames (ORFs) occurring in tandem. The phenotype conferred by avrD was expressed in P. s. pv. glycinea solely by the first of these ORFs (933 bases) that encoded a protein of 34,115 daltons. Neither a signal peptide sequence nor significant regions of hydrophobicity were present that would indicate secretion of the protein or its membrane association. Hybridization analyses revealed that some but not all P. syringae pathovars contained DNA homologous to avrD. This included weak hybridization to all tested races of P. s. pv. glycinea, although none of them express the phenotype conferred by avrD. The avrD gene occurred on an indigenous 75-kb plasmid in several P. s. pv. tomato isolates.     

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.     

Plants expressing the Pto disease resistance gene confer resistance to recombinant PVX containing the avirulence gene AvrPto

Elicitation of hypersensitive cell death and induction of plant disease resistance by Pseudomonas syringae pv. tomato (Pst) is dependent on activity of the Pst Hrp secretion system and the gene-for-gene interaction between the tomato resistance gene Pto and the bacterial avirulence gene avrPto. AvrPto was expressed transiently in resistant or susceptible plant lines via a potato virus X (PVX) vector. We found that while PVX is normally virulent on tomato, a PVX derivative expressing avrPto was only capable of infecting plants lacking a functional Pto resistance pathway. Mutations in either the Pto or Prf genes allowed systemic spread of the recombinant virus. These results indicate that recognition of AvrPto by Pto in resistant plant lines triggers a plant defense response that can confer resistance to a viral as well as a bacterial pathogen.     

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.     

Molecular characterization and nucleic acid sequence of an avirulence gene from race 6 of Pseudomonas syringae pv. glycinea.

A gene was previously cloned from Pseudomonas syringae pv. glycinea race 6, designated avirulence gene A (avrA), that controls the expression of virulence by the pathogen on specific cultivars of soybean. A 3.2-kilobase (kb) AccI subclone from the cosmid clone pPg6L3 was shown to be active when cloned into the broad-host-range vector pRK404. Transposon Tn5 mutagenesis and deletion analysis delineated a span of approximately 2.5 kb of DNA that was necessary for gene activity. The nucleotide sequence of a 3.409-kb segment of DNA which contained the avrA gene has been determined. An open reading frame of 2.721 kb of DNA, which correlates with the region of DNA defined by transposon mutagenesis and deletion analysis, was identified. The open reading frame would encode a protein of 100.866 kilodaltons, which is in good agreement with the 100-kilodalton protein expressed by Escherichia coli maxicells.     

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.     

A gene from Pseudomonas syringae pv. glycinea with homology to avirulence gene D from P. s. pv. tomato but devoid of the avirulence phenotype

A gene was cloned from Pseudomonas syringae pv. glycinea that hybridized to avirulence gene D (avrD), previously cloned from P. s. pv. tomato. Unlike avrD, the hypersensitive response (HR) was not elicited when the P. s. pv. glycinea gene was reintroduced into P. s. pv. glycinea race 4 on a broad host range plasmid and the bacteria were inoculated into soybean leaves. DNA sequence data disclosed that the P. s. pv. glycinea homologue of avrD encoded a protein containing 86% identical amino acids to avrD, with substitutions distributed throughout the protein. Two ORFs immediately downstream from the avrD homologue were more similar in P. s. pv. tomato and P. s. pv. glycinea, with 98 and 99% identical amino acids. Expression of the wildtype P. s. pv. glycinea gene and recombinant genes constructed between the P. s. pv. tomato avrD gene and its P. s. pv. glycinea homologue in both Escherichia coli and P. s. pv. glycinea indicated that the P. s. pv. glycinea gene product was formed less efficiently or was less stable than was the P. s. pv. tomato protein encoded by avrD. The data indicated that the P. s. pv. glycinea homologue represents a recessive allele of the P. s. pv. tomato avrD gene which has been modified by mutation such that it does not lead to an avirulence phenotype on the normal host plant, soybean.     

大豆细菌性斑点病菌harpin编码基因的克隆与表达

摘要：【方法、目的】利用PCR方法从丁香假单胞菌大豆致病变种（Pseudomonas syringae pv. glycinea）Psg12菌株中克隆到1026bp的hrp基因。将其定向插入到表达载体pGEX-4T-1上,并转化宿主菌BL21,IPTG诱导表达后,SDS-PAGE显示其表达产物为分子量为61 kDa的融合蛋白质。【结果】该蛋白质在性质与功能上类似于已发现的harpins,即富含甘氨酸、不含半胱氨酸,热稳定以及对蛋白酶K敏感,能够在烟草上引起典型的过敏性反应,过敏性反应还可被真核生物代谢抑制     

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.