Housekeeping gene sequencing and multilocus variable-number tandem-repeat analysis to identify subpopulations within Pseudomonas syringae pv. maculicola and Pseudomonas syringae pv. tomato that correlate with host specificity

Pseudomonas syringae pv. maculicola causes bacterial spot on Brassicaceae worldwide, and for the last 10 years severe outbreaks have been reported in the Loire Valley, France. P. syringae pv. maculicola resembles P. syringae pv. tomato in that it is also pathogenic for tomato and causes the same types of symptoms. We used a collection of 106 strains of P. syringae to characterize the relationships between P. syringae pv. maculicola and related pathovars, paying special attention to P. syringae pv. tomato. Phylogenetic analysis of gyrB and rpoD gene sequences showed that P. syringae pv. maculicola, which causes diseases in Brassicaceae, forms six genetic lineages within genomospecies 3 of P. syringae strains as defined by L. Gardan et al. (Int. J. Syst. Bacteriol. 49[Pt 2]:469-478, 1999), whereas P. syringae pv. tomato forms two distinct genetic lineages. A multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) conducted with eight minisatellite loci confirmed the genetic structure obtained with rpoD and gyrB sequence analyses. These results provide promising tools for fine-scale epidemiological studies on diseases caused by P. syringae pv. maculicola and P. syringae pv. tomato. The two pathovars had distinct host ranges; only P. syringae pv. maculicola strains were pathogenic for Brassicaceae. A subpopulation of P. syringae pv. maculicola strains that are pathogenic for Pto-expressing tomato plants were shown to lack avrPto1 and avrPtoB or to contain a disrupted avrPtoB homolog. Taking phylogenetic and pathological features into account, our data suggest that the DC3000 strain belongs to P. syringae pv. maculicola. This study shows that P. syringae pv. maculicola and P. syringae pv. tomato appear multiclonal, as they did not diverge from a single common ancestral group within the ancestral P. syringae genomospecies 3, and suggests that pathovar specificity within P. syringae may be due to independent genetic events.     

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.     

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.     

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.     

Arabidopsis enhanced disease susceptibility mutants exhibit enhanced susceptibility to several bacterial pathogens and alterations in PR-1 gene expression.

To identify plant defense responses that limit pathogen attack, Arabidopsis eds mutants that exhibit enhanced disease susceptibility to the virulent bacterial pathogen Pseudomonas syringae pv maculicola ES4326 were previously identified. In this study, we show that each of four eds mutants (eds5-1, eds6-1, eds7-1, and eds9-1) has a distinguishable phenotype with respect to the degree of susceptibility to a panel of bacterial phytopathogens and the ability to activate pathogenesis-related PR-1 gene expression after pathogen attack. None of the four eds mutants exhibited observable defects in mounting a hypersensitive response. Although all four eds mutants were also capable of mounting a systemic acquired resistance response, enhanced growth of P. s. maculicola ES4326 was still apparent in the secondarily infected leaves of three of the eds mutants. These data indicate that eds genes define a diverse set of previously unknown defense responses that affect resistance to virulent pathogens.     

Comparison of Randomly Amplified Polymorphic DNA with Amplified Fragment Length Polymorphism To Assess Genetic Diversity and Genetic Relatedness within Genospecies III of Pseudomonas syringae

Recently, DNA pairing analyses showed that Pseudomonas syringae pv. tomato and related pathovars, including P. syringae pv. maculicola, form a genomic species (Pseudomonas tomato) (L. Gardan, H. L. Shafik, and P. A. D. Grimont, p. 445-448, in K. Rudolph, T. J. Burr, J. W. Mansfield, D. Stead, A. Vivian, and J. von Kietzell, ed., Pseudomonas syringae Pathovars and Related Pathogens, 1997). The genetic diversity of 23 strains belonging to this genomic species and 4 outgroup strains was analyzed with randomly amplified polymorphic DNA (RAPD) and amplified fragment length polymorphic (AFLP) techniques. Simple boiling of P. syringae cells was suitable for subsequent DNA amplification to obtain reliable patterns in RAPD and AFLP analyses. In general, the grouping of P. syringae strains by both analysis techniques corresponded well with the classification obtained from an RFLP analysis of ribosomal DNA operons, DNA pairing studies, and an analysis of pathogenicity data. However, two strains of P. syringae pv. maculicola produced distinct DNA patterns compared to the DNA patterns of other P. syringae pv. maculicola strains; these patterns led us to assume that horizontal transfer of DNA could occur between bacterial populations. Both techniques used in this study have high discriminating power because strains of P. syringae pv. tomato and P. syringae pv. maculicola which were indistinguishable by other techniques, including pathogenicity tests on tomato, were separated into two groups by both RAPD and AFLP analyses. In addition, data analysis showed that the AFLP method was more efficient for assessing intrapathovar diversity than RAPD analysis and allowed clear delineation between intraspecific and interspecific genetic distances, suggesting that it could be an alternative to DNA pairing studies. However, it was not possible to distinguish the two races of P. syringae pv. tomato on the basis of an analysis of the data provided by either the AFLP or RAPD technique.     

A putative nucleoporin 96 Is required for both basal defense and constitutive resistance responses mediated by suppressor of npr1-1,constitutive 1

The Arabidopsis thaliana suppressor of npr1-1, constitutive 1 (snc1) mutant contains a gain-of-function mutation in a Toll Interleukin1 receptor-nucleotide binding-Leu-rich repeat-type resistance gene (R-gene), which leads to constitutive activation of disease resistance response against pathogens. In a screen for suppressors of snc1, a recessive mutation, designated mos3 (for modifier of snc1,3), was found to suppress the constitutive pathogenesis-related gene expression and resistance to virulent Pseudomonas syringae maculicola ES4326 and Peronospora parasitica Noco2 in snc1. In addition, mos3 is also compromised in resistance mediated by Resistance to Peronospora parasitica 4 (RPP4), Resistance to Pseudomonas syringae pv maculicola (RPM1), and Resistance to Pseudomonas syringae 4 (RPS4). Single mutant mos3 plants exhibited enhanced disease susceptibility to P. s. pv maculicola ES4326, suggesting that MOS3 is required for basal resistance to pathogens as well. mos3-1 was identified by map-based cloning, and it encodes a protein with high sequence similarity to human nucleoporin 96. Localization of the MOS3-green fluorescent protein fusion to the nuclear envelope further indicates that MOS3 may encode a nucleoporin, suggesting that nuclear and cytoplasmic trafficking plays an important role in both R-gene-mediated and basal disease resistance.     

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.     

Two Arabidopsis srfr (suppressor of rps4-RLD) mutants exhibit avrRps4-specific disease resistance independent of RPS4

RPS4 specifies the Arabidopsis disease resistance response to Pseudomonas syringae pv. tomato expressing avrRps4 and was cloned based on the identification of RLD as a naturally occurring susceptible accession. To dissect the molecular and genetic basis of disease resistance, we used a genetic approach to identify suppressor mutations that reactivate the avrRps4-triggered defense response in RLD. In this report, we describe two non-allelic srfr (suppressor of rps4-RLD) mutants, srfr1 and srfr3, that were susceptible to virulent P. syringae pv. tomato strain DC3000, but resistant to DC3000 expressing avrRps4. In quantitative bacterial growth assays, growth of DC3000 was similar in wild-type control and both mutant lines, indicating that basal resistance was not enhanced in srfr1 and srfr3. Growth of DC3000 (avrRps4) was approximately 30-fold lower in srfr1 and srfr3 than in RLD, but intermediate compared with fully resistant Col-0 and transgenic RLD containing RPS4-Col. The srfr1 and srfr3 mutants did not develop spontaneous lesions prior to inoculation or constitutively express the pathogenesis-related gene PR-1. Therefore, srfr1 and srfr3 constitute novel avr-specific mutants that differ from previously described Arabidopsis mutants with elevated disease resistance. The srfr1 and srfr3 mutations were recessive, and both mapped to the bottom of chromosome IV. Genetic analysis indicated that resistance in srfr1 and srfr3 was independent of the rps4-RLD allele, but dependent on a second gene in RLD. We propose that SRFR1 and SRFR3 are negative regulators of avrRps4-triggered gene-for-gene disease resistance.     

Age-related resistance in Arabidopsis is a developmentally regulated defense response to Pseudomonas syringae

Age-related resistance (ARR) has been observed in a number of plant species; however, little is known about the biochemical or molecular mechanisms involved in this response. Arabidopsis becomes more resistant, or less susceptible, to virulent Pseudomonas syringae (pv tomato or maculicola) as plants mature (in planta bacterial growth reduction of 10- to 100-fold). An ARR-like response also was observed in response to certain environmental conditions that accelerate Arabidopsis development. ARR occurs in the Arabidopsis mutants pad3-1, eds7-1, npr1-1, and etr1-4, suggesting that ARR is a distinct defense response, unlike the induced systemic resistance or systemic acquired resistance responses. However, three salicylic acid (SA) accumulation-deficient plant lines, NahG, sid1, and sid2, did not exhibit ARR. A heat-stable antibacterial activity was detected in intercellular washing fluids in response to Pst inoculation in wild-type ARR-competent plants but not in NAHG: These data suggest that the ability to accumulate SA is necessary for the ARR response and that SA may act as a signal for the production of the ARR-associated antimicrobial compound(s) and/or it may possess direct antibacterial activity against P. syringae.     

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.     

Arabidopsis displays centromeric DNA hypomethylation and cytological alterations of heterochromatin upon attack by pseudomonas syringae

Plant tissues display major alterations upon the perception of microbial pathogens. Changes of cytoplasmic and apoplastic components that sense and transduce plant defenses have been extensively characterized. In contrast, less information is available about modifications affecting the plant nuclear genome under these circumstances. Here, we investigated whether the Arabidopsis thaliana DNA methylation status is altered in tissues responding to the attack of Pseudomonas syringae pv. tomato DC3000. We applied amplified fragment length polymorphism analysis to monitor cytosine methylation at anonymous 5'-CCGG-3' and 5'-GATC-3' sites in naive and infected samples. Plant genomic fragments reducing methylation upon infection, including peri/centromeric repeats such as the 180-bp unit, Athila retrotansposon, and a portion of the nuclear insertion of mitochondrial DNA, were isolated and characterized. P. syringae pv. tomato-induced hypomethylation was detected by high-performance liquid chromatography assays and at the molecular level it did not seem to equally affect all 5-methyl cytosine (5-mC) residues. Nuclei from challenged tissues displayed structural chromatin alterations, including loosening of chromocenters, which also were stimulated by avirulent P. syringae pv. tomato, but not by the P. syringae pv. tomato hrpL- mutant. Finally, P. syringae pv. tomato-induced hypomethylation was found to occur in the absence of DNA replication, suggesting that it involves an active demethylation mechanism. All these responses occurred at 1 day postinfection, largely preceding massive plant cell death generated by pathogen attack.     

Constitutive activation of jasmonate signaling in an Arabidopsis mutant correlates with enhanced resistance to Erysiphe cichoracearum,Pseudomonas syringae,and Myzus persicae

In Arabidopsis spp., the jasmonate (JA) response pathway generally is required for defenses against necrotrophic pathogens and chewing insects, while the salicylic acid (SA) response pathway is generally required for specific, resistance (R) gene-mediated defenses against both biotrophic and necrotrophic pathogens. For example, SA-dependent defenses are required for resistance to the biotrophic fungal pathogen Erysiphe cichoracearum UCSC1 and the bacterial pathogen Pseudomonas syringae pv. maculicola, and also are expressed during response to the green peach aphid Myzus persicae. However, recent evidence indicates that the expression of JA-dependent defenses also may confer resistance to E. cichoracearum. To confirm and to extend this observation, we have compared the disease and pest resistance of wild-type Arabidopsis plants with that of the mutants coil, which is insensitive to JA, and cev1, which has constitutive JA signaling. Measurements of the colonization of these plants by E. cichoracearum, P. syringae pv. maculicola, and M. persicae indicated that activation of the JA signal pathway enhanced resistance, and was associated with the activation of JA-dependent defense genes and the suppression of SA-dependent defense genes. We conclude that JA and SA induce alternative defense pathways that can confer resistance to the same pathogens and pests.     

PAD4 functions upstream from salicylic acid to control defense responses in Arabidopsis.

The Arabidopsis PAD4 gene was previously shown to be required for synthesis of camalexin in response to infection by the virulent bacterial pathogen Pseudomonas syringae pv maculicola ES4326 but not in response to challenge by the non-host fungal pathogen Cochliobolus carbonum. In this study, we show that pad4 mutants exhibit defects in defense responses, including camalexin synthesis and pathogenesis-related PR-1 gene expression, when infected by P. s. maculicola ES4 326. No such defects were observed in response to infection by an isogenic avirulent strain carrying the avirulence gene avrRpt2. In P. s. maculicola ES4 326-infected pad4 plants, synthesis of salicylic acid (SA) was found to be reduced and delayed when compared with SA synthesis in wild-type plants. Moreover, treatment of pad4 plants with SA partially reversed the camalexin deficiency and PR-1 gene expression phenotypes of P. s. maculicola ES4 326-infected pad4 plants. These findings support the hypothesis that PAD4 acts upstream from SA accumulation in regulating defense response expression in plants infected with P. s. maculicola ES4 326. A working model of the role of PAD4 in governing expression of defense responses is presented.     

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.     

A High-Throughput Forward Genetic Screen Identifies Genes Required for Virulence of Pseudomonas syringae pv. maculicola ES4326 on Arabidopsis

Successful pathogenesis requires a number of coordinated processes whose genetic bases remain to be fully characterized. We utilized a high-throughput, liquid media-based assay to screen transposon disruptants of the phytopathogen Pseudomonas syringae pv. maculicola ES4326 to identify genes required for virulence on Arabidopsis. Many genes identified through this screen were involved in processes such as type III secretion, periplasmic glucan biosynthesis, flagellar motility, and amino acid biosynthesis. A small set of genes did not fall into any of these functional groups, and their disruption resulted in context-specific effects on in planta bacterial growth.