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.     

Closing the circle on the discovery of genes encoding Hrp regulon members and type III secretion system effectors in the genomes of three model Pseudomonas syringae strains

Pseudomonas syringae strains translocate large and distinct collections of effector proteins into plant cells via the type III secretion system (T3SS). Mutations in T3SS-encoding hrp genes are unable to elicit the hypersensitive response or pathogenesis in nonhost and host plants, respectively. Mutations in individual effectors lack strong phenotypes, which has impeded their discovery. P. syringae effectors are designated Hop (Hrp outer protein) or Avr (avirulence) proteins. Some Hop proteins are considered to be extracellular T3SS helpers acting at the plant-bacterium interface. Identification of complete sets of effectors and related proteins has been enabled by the application of bioinformatic and high-throughput experimental techniques to the complete genome sequences of three model strains: P. syringae pv. tomato DC3000, P. syringae pv. phaseolicola 1448A, and P. syringae pv. syringae B728a. Several recent papers, including three in this issue of Molecular Plant-Microbe Interactions, address the effector inventories of these strains. These studies establish that active effector genes in P. syringae are expressed by the HrpL alternative sigma factor and can be predicted on the basis of cis Hrp promoter sequences and N-terminal amino-acid patterns. Among the three strains analyzed, P. syringae pv. tomato DC3000 has the largest effector inventory and P. syringae pv. syringae B728a has the smallest. Each strain has several effector genes that appear inactive. Only five of the 46 effector families that are represented in these three strains have an active member in all of the strains. Web-based community resources for managing and sharing growing information on these complex effector arsenals should help future efforts to understand how effectors promote P. syringae virulence.     

Extracytoplasmic function sigma factors in Pseudomonas syringae

Genome analyses of the plant pathogens Pseudomonas syringae pv. tomato DC3000, pv. syringae B728a and pv. phaseolicola 1448A reveal fewer extracytoplasmic function (ECF) sigma factors than in related Pseudomonads with different lifestyles. We highlight the presence of a P. syringae-specific ECF sigma factor that is an interesting target for future studies because of its potential role in the adaptation of P. syringae to its specialized phytopathogenic lifestyle.     

Pseudomonas syringae exchangeable effector loci: sequence diversity in representative pathovars and virulence function in P. syringae pv. syringae B728a

Pseudomonas syringae is a plant pathogen whose pathogenicity and host specificity are thought to be determined by Hop/Avr effector proteins injected into plant cells by a type III secretion system. P. syringae pv. syringae B728a, which causes brown spot of bean, is a particularly well-studied strain. The type III secretion system in P. syringae is encoded by hrp (hypersensitive response and pathogenicity) and hrc (hrp conserved) genes, which are clustered in a pathogenicity island with a tripartite structure such that the hrp/hrc genes are flanked by a conserved effector locus and an exchangeable effector locus (EEL). The EELs of P. syringae pv. syringae B728a, P. syringae strain 61, and P. syringae pv. tomato DC3000 differ in size and effector gene composition; the EEL of P. syringae pv. syringae B728a is the largest and most complex. The three putative effector proteins encoded by the P. syringae pv. syringae B728a EEL--HopPsyC, HopPsyE, and HopPsyV--were demonstrated to be secreted in an Hrp-dependent manner in culture. Heterologous expression of hopPsyC, hopPsyE, and hopPsyV in P. syringae pv. tabaci induced the hypersensitive response in tobacco leaves, demonstrating avirulence activity in a nonhost plant. Deletion of the P. syringae pv. syringae B728a EEL strongly reduced virulence in host bean leaves. EELs from nine additional strains representing nine P. syringae pathovars were isolated and sequenced. Homologs of avrPphE (e.g., hopPsyE) and hopPsyA were particularly common. Comparative analyses of these effector genes and hrpK (which flanks the EEL) suggest that the EEL effector genes were acquired by horizontal transfer after the acquisition of the hrp/hrc gene cluster but before the divergence of modern pathovars and that some EELs underwent transpositions yielding effector exchanges or point mutations producing effector pseudogenes after their acquisition.     

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 RND-type multidrug efflux pump MexAB-OprM of the plant pathogen Pseudomonas syringae

In gram-negative bacteria, transporters belonging to the RND family are the transporters most relevant for resistance to antimicrobial compounds. In Pseudomonas aeruginosa, a clinically important pathogen, the RND-type pump MexAB-OprM has been recognized as one of the major multidrug efflux systems. Here, homologues of MexAB-OprM in the plant pathogens Pseudomonas syringae pv. phaseolicola 1448A, P. syringae pv. syringae B728a, and P. syringae pv. tomato DC3000 were identified, and mexAB-oprM-deficient mutants were generated. Determination of MICs revealed that mutation of MexAB-OprM dramatically reduced the tolerance to a broad range of antimicrobials. Moreover, the ability of the mexAB-oprM-deficient mutants to multiply in planta was reduced. RNA dot blot hybridization revealed growth-dependent regulation of the mexAB-oprM operon in P. syringae; the expression of this operon was maximal in early exponential phase and decreased gradually during further growth.     

Pseudomonas syringae pv. tomato DC3000 uses constitutive and apoplast-induced nutrient assimilation pathways to catabolize nutrients that are abundant in the tomato apoplast

The plant apoplast is the intercellular space that surrounds plant cells, in which metabolic and physiological processes relating to cell wall biosynthesis, nutrient transport, and stress responses occur. The apoplast is also the primary site of infection for hemibiotrophic pathogens such as P. syringae, which obtain nutrients directly from apoplastic fluid. We have used apoplastic fluid extracted from healthy tomato leaves as a growth medium for Pseudomonas spp. in order to investigate the role of apoplastic nutrients in plant colonization by Pseudomonas syringae. We have confirmed that apoplast extracts mimic some of the environmental and nutritional conditions that bacteria encounter during apoplast colonization by demonstrating that expression of the plant-induced type III protein secretion pathway is upregulated during bacterial growth in apoplast extracts. We used a modified phenoarray technique to show that apoplast-adapted P. syringae pv. tomato DC3000 expresses nutrient utilization pathways that allow it to use sugars, organic acids, and amino acids that are highly abundant in the tomato apoplast. Comparative analyses of the nutrient utilization profiles of the genome-sequenced strains P. syringae pv. tomato DC3000, P. syringae pv. syringae B728a, P. syringae pv. phaseolicola 1448A, and the unsequenced strain P. syringae pv. tabaci 11528 with nine other genome-sequenced strains of Pseudomonas provide further evidence that P. syringae strains are adapted to use nutrients that are abundant in the leaf apoplast. Interestingly, P. syringae pv. phaseolicola 1448A lacks many of the nutrient utilization abilities that are present in three other P. syringae strains tested, which can be directly linked to differences in the P. syringae pv. phaseolicola 1448A genome.     

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.     

The Arabidopsis thaliana JASMONATE INSENSITIVE 1 gene is required for suppression of salicylic acid-dependent defenses during infection by Pseudomonas syringae

Many plant pathogens suppress antimicrobial defenses using virulence factors that modulate endogenous host defenses. The Pseudomonas syringae phytotoxin coronatine (COR) is believed to promote virulence by acting as a jasmonate analog, because COR-insensitive 1 (coil) Arabidopsis thaliana and tomato mutants are impaired in jasmonate signaling and exhibit reduced susceptibility to P. syringae. To further investigate the role of jasmonate signaling in disease development, we analyzed several jasmonate-insensitive A. thaliana mutants for susceptibility to P. syringae pv. tomato strain DC3000 and sensitivity to COR. Jasmonate-insensitive 1 (jin1) mutants exhibit both reduced susceptibility to P. syringae pv. tomato DC3000 and reduced sensitivity to COR, whereas jasmonate-resistant 1 (jar1) plants exhibit wild-type responses to both COR and P. syringae pv. tomato DC3000. A jin1 jar1 double mutant does not exhibit enhanced jasmonate insensitivity, suggesting that JIN1 functions downstream of jasmonic acid-amino acid conjugates synthesized by JAR1. Reduced disease susceptibility in jin1 mutants is correlated with elevated expression of pathogenesis-related 1 (PR-1) and is dependent on accumulation of salicylic acid (SA). We also show that JIN1 is required for normal P. syringae pv. tomato DC3000 symptom development through an SA-independent mechanism. Thus, P. syringae pv. tomato DC3000 appears to utilize COR to manipulate JIN1-dependent jasmonate signaling both to suppress SA-mediated defenses and to promote symptom development.     

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.     

A Pseudomonas syringae pv. tomato avrE1/hopM1 mutant is severely reduced in growth and lesion formation in tomato

The model plant pathogen Pseudomonas syringae pv. tomato DC3000 grows and produces necrotic lesions in the leaves of its host, tomato. Both abilities are dependent upon the hypersensitive response and pathogenicity (Hrp) type III secretion system (TTSS), which translocates multiple effector proteins into plant cells. A previously constructed DC3000 mutant with a 9.3-kb deletion in the Hrp pathogenicity island conserved effector locus (CEL) was strongly reduced in growth and lesion formation in tomato leaves. The ACEL mutation affects three putative or known effector genes: avrE1, hopM1, and hopAA1-1. Comparison of genomic sequences of DC3000, P. syringae pv. phaseolicola 1448A, and P. syringae pv. syringae B728a revealed that these are the only effector genes present in the CEL of all three strains. AvrEl was shown to carry functional TTSS translocation signals based on the performance of a fusion of the first 315 amino acids of AvrE1 to the Cya translocation reporter. A DC3000 delta avrE1 mutant was reduced in its ability to produce lesions but not in its ability to grow in host tomato leaves. AvrE1 expressed from the 35S promoter elicited cell death in nonhost Nicotiana tabacum leaves and host tomato leaves in Agrobacterium-mediated transient expression experiments. Mutations involving combinations of avrE1, hopM1, and hopAA1-1 revealed that deletion of both avrE1 and hopM1 reproduced the strongly reduced growth and lesion phenotype of the delta CEL mutant. Furthermore, quantitative assays involving different levels of inoculum and electrolyte leakage revealed that the avrE1/hopM1 and deltaCEL mutants both were partially impaired in their ability to elicit the hypersensitive response in nonhost N. benthamiana leaves. However, the avrE1/hopM1 mutant was not impaired in its ability to deliver AvrPto1(1-100)-Cya to nonhost N. benthamiana or host tomato leaves during the first 9 h after inoculation. These data suggest that AvrE1 acts within plant cells and promotes lesion formation and that the combined action of AvrE1 and HopM1 is particularly important in promoting bacterial growth in planta.     

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.     

Yersiniabactin production by Pseudomonas syringae and Escherichia coli, and description of a second yersiniabactin locus evolutionary group

The siderophore and virulence factor yersiniabactin is produced by Pseudomonas syringae. Yersiniabactin was originally detected by high-pressure liquid chromatography (HPLC); commonly used PCR tests proved ineffective. Yersiniabactin production in P. syringae correlated with the possession of irp1 located in a predicted yersiniabactin locus. Three similarly divergent yersiniabactin locus groups were determined: the Yersinia pestis group, the P. syringae group, and the Photorhabdus luminescens group; yersiniabactin locus organization is similar in P. syringae and P. luminescens. In P. syringae pv. tomato DC3000, the locus has a high GC content (63.4% compared with 58.4% for the chromosome and 60.1% and 60.7% for adjacent regions) but it lacks high-pathogenicity-island features, such as the insertion in a tRNA locus, the integrase, and insertion sequence elements. In P. syringae pv. tomato DC3000 and pv. phaseolicola 1448A, the locus lies between homologues of Psyr_2284 and Psyr_2285 of P. syringae pv. syringae B728a, which lacks the locus. Among tested pseudomonads, a PCR test specific to two yersiniabactin locus groups detected a locus in genospecies 3, 7, and 8 of P. syringae, and DNA hybridization within P. syringae also detected a locus in the pathovars phaseolicola and glycinea. The PCR and HPLC methods enabled analysis of nonpathogenic Escherichia coli. HPLC-proven yersiniabactin-producing E. coli lacked modifications found in irp1 and irp2 in the human pathogen CFT073, and it is not clear whether CFT073 produces yersiniabactin. The study provides clues about the evolution and dispersion of yersiniabactin genes. It describes methods to detect and study yersiniabactin producers, even where genes have evolved.     

The Tat pathway of the plant pathogen Pseudomonas syringae is required for optimal virulence

Pseudomonas syringae is a gram-negative bacterium that infects a number of agriculturally important plant species. The ability of the organism to deliver virulence factors across the plant cell wall is a key to its pathogenicity. Deletion mutants in the twin arginine translocation (Tat) pathway of two pathovars of P. syringae, pvs. tomato DC3000 and maculicola ES4326, displayed a range of pleiotropic phenotypic changes, such as defects in fluorescent siderophore production, a decrease in sodium dodecyl sulfate and copper resistance, and a significant loss in fitness using Arabidopsis thaliana or tomato as plant hosts. The genome sequence of P. syringae pv. tomato DC3000 encodes a number of potential virulence factors that are predicted to be translocated via the Tat pathway, including several proteins involved in iron scavenging (two siderophore receptors, PSPTO3474 and PSPTO3294, and an aminotransferase, PSPTO2155, involved in siderophore biosynthesis). Further candidates for Tat-dependent pathogenicity determinants include the homologs of a cell wall amidase (PSPTO5528), an enzyme involved in periplasmic glucans biosynthesis (PSPTO5542), and two putative phospholipases (PSPTO3648 and PSPTOB0005). Translocation of the putative amidase, aminotransferase, glucans biosynthetic enzyme, and the two phospholipases, but not the two siderophore receptors, is shown to be dependent on the Tat pathway. Strains deleted for the genes encoding the probable aminotransferase and amidase enzymes are significantly less infectious than the wild type. We conclude that the incremental effects due to the failure to correctly localize at least two, and possibly more, Tat substrates gives rise to the attenuated fitness phenotype of the P. syringae pv. tomato DC3000 tat strain.     

Roadmap to new virulence determinants in Pseudomonas syringae: insights from comparative genomics and genome organization

Systematic comparison of the current repertoire of virulence-associated genes for three Pseudomonas syringae strains with complete genome sequences, P. syringae pv. tomato DC3,000, P. syringae pv. phaseolicola 1448A, and P. syringae pv. syringae B728a, is prompted by recent advances in virulence factor identification in P. syringae and other bacteria. Among these are genes linked to epiphytic fitness, plant- and insect-active toxins, secretion pathways, and virulence regulators, all reflected in the recently updated DC3,000 genome annotation. Distribution of virulence genes in relation to P. syringae genome organization was analyzed to distinguish patterns of conservation among genomes and association between genes and mobile genetic elements. Variable regions were identified on the basis of deviation in sequence composition and gaps in syntenic alignment among the three genomes. Mapping gene location relative to the genome structure revealed strong segregation of the HrpL regulon with variable genome regions (VR), divergent distribution patterns for toxin genes depending on association with plant or insect pathogenesis, and patterns of distribution for other virulence genes that highlight potential sources of strain-to-strain differences in host interaction. Distribution of VR among other sequenced bacterial genomes was analyzed and future plans for characterization of this potential reservoir of virulence genes are discussed.     

A bacterial cysteine protease effector protein interferes with photosynthesis to suppress plant innate immune responses

The bacterial pathogen Pseudomonas syringae pv tomato DC3000 suppresses plant innate immunity with effector proteins injected by a type III secretion system (T3SS). The cysteine protease effector HopN1, which reduces the ability of DC3000 to elicit programmed cell death in non-host tobacco, was found to also suppress the production of defence-associated reactive oxygen species (ROS) and callose when delivered by Pseudomonas fluorescens heterologously expressing a P. syringae T3SS. Purified His(6) -tagged HopN1 was used to identify tomato PsbQ, a member of the oxygen evolving complex of photosystem II (PSII), as an interacting protein. HopN1 localized to chloroplasts and both degraded PsbQ and inhibited PSII activity in chloroplast preparations, whereas a HopN1(D299A) non-catalytic mutant lost these abilities. Gene silencing of NtPsbQ in tobacco compromised ROS production and programmed cell death by DC3000. Our data reveal PsbQ as a contributor to plant immunity responses and a target for pathogen suppression.