Pseudomonas syringae coffee blight is associated with the horizontal transfer of plasmid-encoded type III effectors.

The emergence of new pathogens is an ongoing threat to human health and agriculture. While zoonotic spillovers received considerable attention, the emergence of crop diseases is less well studied. Here, we identify genomic factors associated with the emergence of Pseudomonas syringae bacterial blight of coffee. Fifty-three P. syringae strains from diseased Brazilian coffee plants were sequenced. Comparative and evolutionary analyses were used to identify loci associated with coffee blight. Growth and symptomology assays were performed to validate the findings. Coffee isolates clustered in three lineages, including primary phylogroups PG3 and PG4, and secondary phylogroup PG11. Genome-wide association study of the primary PG strains identified 37 loci, including five effectors, most of which were encoded on a plasmid unique to the PG3 and PG4 coffee strains. Evolutionary analyses support the emergence of coffee blight in PG4 when the coffee-associated plasmid and associated effectors derived from a divergent plasmid carried by strains associated with other hosts. This plasmid was only recently transferred into PG3. Natural diversity and CRISPR-Cas9 plasmid curing were used to show that strains with the coffee-associated plasmid grow to higher densities and cause more severe disease symptoms in coffee. This work identifies possible evolutionary mechanisms underlying the emergence of a new lineage of coffee pathogens.

Host-specific signal perception by PsaR2 LuxR solo induces Pseudomonas syringae pv. actinidiae virulence traits.

Plant-associated bacteria, including pathogens, recognise host-derived signals to activate specific responses. The genome of Pseudomonas syringae pv. actinidiae (Psa), the aetiological agent of bacterial canker of kiwifruit, encodes for three putative LuxR-like receptors. Proteins of this family are usually involved in the quorum sensing system, through the perception of autoinducers (AHLs) produced by a cognate LuxI. However, Psa does not produce AHLs according to the lack of LuxI-encoding gene. It has been proposed that the so-called LuxR solos may be involved in the perception of environmental stimuli. We thus hypothesised that Psa LuxR-like receptors could be involved in host-derived signal sensing. Psa virulence traits, i.e., biofilm formation, motility and endophytic colonisation, were stimulated by growing the pathogen in host plant extracts, but not in non-host plant extracts or rich medium. Moreover, the phenotypic analyses of Psa mutant strains lacking the LuxR solo-encoding genes, demonstrated that PsaR2 plays a major role in host recognition and induction of virulence responses. The heterologous expression of PsaR2, followed by affinity chromatography and fraction activity assessment, confirmed the specific recognition of plant-derived components by this sensor. Overall, these data provide a deeper understanding of the regulation of Psa virulence through interkingdom communication, which represents a interesting target for the development of tolerant/resistant genotypes or innovative control strategies.

Genomic analyses of Pseudomonas syringae pv. actinidiae isolated in Korea suggest the transfer of the bacterial pathogen via kiwifruit pollen.

Introduction. The bacterial pathogen, Pseudomonas syringae pv. actinidiae (Psa), has emerged as a major threat to kiwifruit cultivation throughout the world. One pandemic strain (from the Psa3 group) has occurred in various geographical regions. It is important to understand how this pathogen is being transmitted.Aim. Although Psa has been found in Korea since 1992, the isolates were until recently of a distinct type (Psa2). Recently, the more virulent Psa3 type has been detected. The purpose of this study was to describe the variety of Psa3 now found in Korea.Methodology. Strains were isolated from kiwifruit plants in Korea and from pollen imported into Korea from New Zealand. The genomes of 10 isolates were sequenced using the Illumina platform and compared to the completely assembled genomes of pandemic Psa3 strains from New Zealand and China. Comparisons were also made with pandemic strains from Chile and non-pandemic Psa3 isolates from China.Results. Six of the 10 Psa3 isolates from Korea show a clear relationship with New Zealand isolates. Two isolates show a distinct relationship to isolates from Chile; one further isolate has a sequence that is highly similar to that of M228, a strain previously isolated in China; and the last isolate belongs to the Psa3 group, but is not a member of the pandemic lineage.Conclusion. This analysis establishes that there have been multiple routes of transmission of the Psa3 pandemic strain into Korea. One route has involved the importation of pollen from New Zealand. A second route probably involves importation from Chile.

Pseudomonas bacteriocin syringacin M released upon desiccation suppresses the growth of sensitive bacteria in plant necrotic lesions.

Bacteriocins are regarded as important factors mediating microbial interactions, but their exact role in community ecology largely remains to be elucidated. Here, we report the characterization of a mutant strain, derived from Pseudomonas syringae pv. tomato DC3000 (Pst), that was incapable of growing in plant extracts and causing disease. Results showed that deficiency in a previously unannotated gene saxE led to the sensitivity of the mutant to Ca2+ in leaf extracts. Transposon insertions in the bacteriocin gene syrM, adjacent to saxE, fully rescued the bacterial virulence and growth of the ΔsaxE mutant in plant extracts, indicating that syrM-saxE encode a pair of bacteriocin immunity proteins in Pst. To investigate whether the syrM-saxE system conferred any advantage to Pst in competition with other SyrM-sensitive pathovars, we compared the growth of a SyrM-sensitive strain co-inoculated with Pst strains with or without the syrM gene and observed a significant syrM-dependent growth reduction of the sensitive bacteria on plate and in lesion tissues upon desiccation-rehydration treatment. These findings reveal an important biological role of SyrM-like bacteriocins and help to understand the complex strategies used by P. syringae in adaptation to the phyllosphere niche in the context of plant disease.

Transgenic expression of plant-specific insert of potato aspartic proteases (StAP-PSI) confers enhanced resistance to Botrytis cinerea in Arabidopsis thaliana.

The plant-specific insert of Solanum tuberosum aspartic proteases (StAP-PSI) has high structural similarity with NK-lysin and granulysin, two saposin-like proteins (SAPLIPs) with antimicrobial activity. Recombinant StAP-PSI and some SAPLIPs show antimicrobial activity against pathogens that affect human and plants. In this work, we transformed Arabidopsis thaliana plants with StAP-PSI encoding sequence with its corresponding signal peptide under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Results obtained show that StAP-PSI significantly enhances Arabidopsis resistance against Botrytis cinerea infection. StAP-PSI is secreted into the leaf apoplast and acts directly against pathogens; thereby complementing plant innate immune responses. Data obtained from real-time PCR assays show that the constitutive expression of StAP-PSI induces the expression of genes that regulate jasmonic acid signalling pathway, such as PDF1.2, in response to infection due to necrotrophic pathogens. On the other hand, according to the data described for other antimicrobial peptides, the presence of the StAP-PSI protein in the apoplast of A. thaliana leaves is responsible for the expression of salicylic acid-associated genes, such as PR-1, irrespective of infection with B. cinerea. These results indicate that the increased resistance demonstrated by A. thaliana plants that constitutively express StAP-PSI owing to B. cinerea infection compared to the wild-type plants is a consequence of two factors, i.e., the antifungal activity of StAP-PSI and the overexpression of A. thaliana defense genes induced by the constitutive expression of StAP-PSI. We suggest that the use of this protein would help in minimizing the ecological and health risks that arise from the use of pesticides. We suggest that the use of this protein would help in minimizing the ecological and health risks that arise from the spreading of resistance of agriculturally important pathogens.

Efficient Surface Display of Diisopropylfluorophosphatase (DFPase) in E. coli for Biodegradation of Toxic Organophosphorus Compounds (DFP and Cp).

Compounds including organophosphorus pesticides (OPs) and chemical nerve agents are toxic compounds synthesized recently which disrupt the mechanisms of neural transmission. Therefore, a critical requirement is the development of a bio-refining technology to facilitate the biodegradation of organophosphorus pollutants. The diisopropylfluorophosphatase (DFPase, EC 3.1.8.2) from the ganglion and brain of Loligo vulgaris acts on P-F bonds present in some OPs. Intracellular production of OPs-degrading enzymes or the use of native bacteria and fungi leads to a low degradation rate of OPs due to a mass transfer issue which reduces the overall catalytic efficiency. To overcome this challenge, we expressed DFPase on the surface of E. coli for the first time by employing the N-terminal domain of the ice nucleation protein (InaV-N) as an anchoring motif. Tracking the recombinant protein confirmed that DFPase is successfully located on the outer membrane. Further studies on its activity to degrade diisopropylfluorophosphate (DFP) showed its significant ability for the biodegradation of diisopropylfluorophosphate (DFP) with a specific activity of 500 U/mg of wet cell weight. Recombinant cells could also degrade chlorpyrifos (Cp) with an activity equivalent to a maximum value of 381.44 U/ml with a specific activity of 476.75 U/mg of cell, analyzed using HPLC technique. The optimum activity of purified DFPase was found at 30 °C. A more increased activity was also obtained in the presence of glucose-mineral-salt (GMS) supplemented with tryptone and 100 mg/L Co(2+) ion. These results highlight the high potential of the InaV-N anchoring domain to produce an engineered bacterium that can be used in the bioremediation of pesticide-contaminated environments.

Antimicrobial activity of essential oils of Thymus vulgaris and Origanum vulgare on phytopathogenic strains isolated from soybean.

The aim of this work was to study the antimicrobial activity of essential oils obtained from Thymus vulgaris (thyme) and Origanum vulgare (oregano) on phytopathogenic Pseudomonas species isolated from soybean. Strains with characteristics of P. syringae were isolated from leaves of soybean plants with blight symptoms. Ten of these could be identified in Group Ia of LOPAT as P. syringae. Six of these were confirmed as P. syringae using 16S rRNA, indicating the presence of these phytopathogenic bacteria in east and central Argentina. All the phytopathogenic bacteria were re-isolated and identified from the infected plants. MIC values for thyme were 11.5 and 5.7 mg·ml(-1) on P. syringae strains, while oregano showed variability in the inhibitory activity. Both essential oils inhibited all P. syringae strains, with better inhibitory activity than the antibiotic streptomycin. The oils were not bactericidal for all pseudomonads. Both oils contained high carvacrol (29.5% and 19.7%, respectively) and low thymol (1.5%). Natural products obtained from aromatic plants represent potential sources of molecules with biological activity that could be used as new alternatives for the treatment of phytopathogenic bacteria infections.

The Pseudomonas viridiflava phylogroups in the P. syringae species complex are characterized by genetic variability and phenotypic plasticity of pathogenicity-related traits.

As a species complex, Pseudomonas syringae exists in both agriculture and natural aquatic habitats. P.viridiflava, a member of this complex, has been reported to be phenotypically largely homogenous. We characterized strains from different habitats, selected based on their genetic similarity to previously described P.viridiflava strains. We revealed two distinct phylogroups and two different kinds of variability in phenotypic traits and genomic content. The strains exhibited phase variation in phenotypes including pathogenicity and soft rot on potato. We showed that the presence of two configurations of the Type III Secretion System [single (S-PAI) and tripartite (T-PAI) pathogenicity islands] are not correlated with pathogenicity or with the capacity to induce soft rot in contrast to previous reports. The presence/absence of the avrE effector gene was the only trait we found to be correlated with pathogenicity of P.viridiflava. Other Type III secretion effector genes were not correlated with pathogenicity. A genomic region resembling an exchangeable effector locus (EEL) was found in S-PAI strains, and a probable recombination between the two PAIs is described. The ensemble of the variability observed in these phylogroups of P.syringae likely contributes to their adaptability to alternating opportunities for pathogenicity or saprophytic survival.

In planta expression or delivery of potato aphid Macrosiphum euphorbiae effectors Me10 and Me23 enhances aphid fecundity.

The interactions between aphids and their host plants seem to be analogous to those of plant-microbial pathogens. Unlike microbial pathogen effectors, little is known about aphid effectors and their ability to interfere with host immunity. To date, only three functional aphid effectors have been reported. To identify potato aphid (Macrosiphum euphorbiae) effectors, we developed a salivary gland transcriptome using Illumina technology. We generated 85 million Illumina reads from salivary glands and assembled them into 646 contigs. Ab initio sequence analysis predicted secretion signal peptides in 24% of these sequences, suggesting that they might be secreted into the plant during aphid feeding. Eight of these candidate effectors with secretion signal peptides were functionally characterized using Agrobacterium tumefaciens-mediated transient overexpression in Nicotiana benthamiana. Two candidate effectors, Me10 and Me23, increased aphid fecundity, suggesting their ability to suppress N. benthamiana defenses. Five of these candidate effectors, including Me10 and Me23, were also analyzed in tomato by delivering them through the Pseudomonas syringae type three secretion system. In tomato, only Me10 increased aphid fecundity. This work identified two additional aphid effectors with ability to manipulate the host for their advantage.

Pseudomonas syringae pv. tomato DC3000 CmaL (PSPTO4723), a DUF1330 family member, is needed to produce L-allo-isoleucine, a precursor for the phytotoxin coronatine.

Pseudomonas syringae pv. tomato DC3000 produces the phytotoxin coronatine, a major determinant of the leaf chlorosis associated with DC3000 pathogenesis. The DC3000 PSPTO4723 (cmaL) gene is located in a genomic region encoding type III effectors; however, it promotes chlorosis in the model plant Nicotiana benthamiana in a manner independent of type III secretion. Coronatine is produced by the ligation of two moieties, coronafacic acid (CFA) and coronamic acid (CMA), which are produced by biosynthetic pathways encoded in separate operons. Cross-feeding experiments, performed in N. benthamiana with cfa, cma, and cmaL mutants, implicate CmaL in CMA production. Furthermore, analysis of bacterial supernatants under coronatine-inducing conditions revealed that mutants lacking either the cma operon or cmaL accumulate CFA rather than coronatine, supporting a role for CmaL in the regulation or biosynthesis of CMA. CmaL does not appear to regulate CMA production, since the expression of proteins with known roles in CMA production is unaltered in cmaL mutants. Rather, CmaL is needed for the first step in CMA synthesis, as evidenced by the fact that wild-type levels of coronatine production are restored to a ΔcmaL mutant when it is supplemented with 50 µg/ml l-allo-isoleucine, the starting unit for CMA production. cmaL is found in all other sequenced P. syringae strains with coronatine biosynthesis genes. This characterization of CmaL identifies a critical missing factor in coronatine production and provides a foundation for further investigation of a member of the widespread DUF1330 protein family.

Silencing and heterologous expression of ppo-2 indicate a specific function of a single polyphenol oxidase isoform in resistance of dandelion (Taraxacum officinale) against Pseudomonas syringae pv. tomato.

Dandelion (Taraxacum officinale) possesses an unusually high degree of disease resistance. As this plant exhibits high polyphenol oxidase (PPO) activity and PPO have been implicated in resistance against pests and pathogens, we analyzed the potential involvement of five PPO isoenzymes in the resistance of dandelion against Botrytis cinerea and Pseudomonas syringae pv. tomato. Only one PPO (ppo-2) was induced during infection, and ppo-2 promoter and ß-glucuronidase marker gene fusions revealed strong induction of the gene surrounding lesions induced by B. cinerea. Specific RNAi silencing reduced ppo-2 expression only, and concomitantly increased plant susceptibility to P. syringae pv. tomato. At 4 days postinoculation, P. syringae pv. tomato populations were strongly increased in the ppo-2 RNAi lines compared with wild-type plants. When the dandelion ppo-2 gene was expressed in Arabidopsis thaliana, a plant having no PPO gene, active protein was formed and protein extracts of the transgenic plants exhibited substrate-dependent antimicrobial activity against P. syringae pv. tomato. These results clearly indicate a strong contribution of a specific, single PPO isoform to disease resistance. Therefore, we propose that specific PPO isoenzymes be included in a new family of pathogenesis-related (PR) proteins.

Pseudomonas sax genes overcome aliphatic isothiocyanate-mediated non-host resistance in Arabidopsis.

Most plant-microbe interactions do not result in disease; natural products restrict non-host pathogens. We found that sulforaphane (4-methylsulfinylbutyl isothiocyanate), a natural product derived from aliphatic glucosinolates, inhibits growth in Arabidopsis of non-host Pseudomonas bacteria in planta. Multiple sax genes (saxCAB/F/D/G) were identified in Pseudomonas species virulent on Arabidopsis. These sax genes are required to overwhelm isothiocyanate-based defenses and facilitate a disease outcome, especially in the young leaves critical for plant survival. Introduction of saxCAB genes into non-host strains enabled them to overcome these Arabidopsis defenses. Our study shows that aliphatic isothiocyanates, previously shown to limit damage by herbivores, are also crucial, robust, and developmentally regulated defenses that underpin non-host resistance in the Arabidopsis-Pseudomonas pathosystem.

The phytopathogen Pseudomonas syringae pv. tomato DC3000 has three high-affinity iron-scavenging systems functional under iron limitation conditions but dispensable for pathogenesis.

High-affinity iron scavenging through the use of siderophores is a well-established virulence determinant in mammalian pathogenesis. However, few examples have been reported for plant pathogens. Here, we use a genetic approach to investigate the role of siderophores in Pseudomonas syringae pv. tomato DC3000 (DC3000) virulence in tomato. DC3000, an agronomically important pathogen, has two known siderophores for high-affinity iron scavenging, yersiniabactin and pyoverdin, and we uncover a third siderophore, citrate, required for growth when iron is limiting. Though growth of a DC3000 triple mutant unable to either synthesize or import these siderophores is severely restricted in iron-limited culture, it is fully pathogenic. One explanation for this phenotype is that the DC3000 triple mutant is able to directly pirate plant iron compounds such as heme/hemin or iron-nicotianamine, and our data indicate that DC3000 can import iron-nicotianamine with high affinity. However, an alternative explanation, supported by data from others, is that the pathogenic environment of DC3000 (i.e., leaf apoplast) is not iron limited but is iron replete, with available iron of >1 µM. Growth of the triple mutant in culture is restored to wild-type levels by supplementation with a variety of iron chelates at >1 µM, including iron(III) dicitrate, a dominant chelate of the leaf apoplast. This suggests that lower-affinity iron import would be sufficient for DC3000 iron nutrition in planta and is in sharp contrast to the high-affinity iron-scavenging mechanisms required in mammalian pathogenesis.

The hrpZ gene of Pseudomonas syringae pv. phaseolicola enhances resistance to rhizomania disease in transgenic Nicotiana benthamiana and sugar beet.

To explore possible sources of transgenic resistance to the rhizomania-causing Beet necrotic yellow vein virus (BNYVV), Nicotiana benthamiana plants were constructed to express the harpin of Pseudomonas syringae pv. phaseolicola (HrpZ(Psph)). The HrpZ protein was expressed as an N-terminal fusion to the PR1 signal peptide (SP/HrpZ) to direct harpin accumulation to the plant apoplast. Transgene integration was verified by mPCR in all primary transformants (T0), while immunoblot analysis confirmed that the protein HrpZ(Psph) was produced and the signal peptide was properly processed. Neither T0 plants nor selfed progeny (T1) showed macroscopically visible necrosis or any other macroscopic phenotypes. However, plants expressing the SP/HrpZ(Psph) showed increased vigor and grew faster in comparison with non-transgenic control plants. Transgenic resistance was assessed after challenge inoculation with BNYVV on T1 progeny by scoring of disease symptoms and by DAS-ELISA at 20 and 30 dpi. Transgenic and control lines showed significant differences in terms of the number of plants that became infected, the timing of infection and the disease symptoms displayed. Plants expressing the SP/HrpZ(Psph) developed localized leaf necrosis in the infection area and had enhanced resistance upon challenge with BNYVV. In order to evaluate the SP/HrpZ-based resistance in the sugar beet host, A. rhizogenes-mediated root transformation was exploited as a transgene expression platform. Upon BNYVV inoculation, transgenic sugar beet hairy roots showed high level of BNYVV resistance. In contrast, the aerial non-transgenic parts of the same seedlings had virus titers that were comparable to those of the seedlings that were untransformed or transformed with wild type R1000 cells. These findings indicate that the transgenically expressed SP/HrpZ protein results in enhanced rhizomania resistance both in a model plant and sugar beet, the natural host of BNYVV. Possible molecular mechanisms underlying the enhanced resistance and plant growth phenotypes observed in SP/HrpZ transgenic plants are discussed.

Mutations in γ-aminobutyric acid (GABA) transaminase genes in plants or Pseudomonas syringae reduce bacterial virulence.

Pseudomonas syringae pv. tomato DC3000 is a bacterial pathogen of Arabidopsis and tomato that grows in the apoplast. The non-protein amino acid γ-amino butyric acid (GABA) is produced by Arabidopsis and tomato and is the most abundant amino acid in the apoplastic fluid of tomato. The DC3000 genome harbors three genes annotated as gabT GABA transaminases. A DC3000 mutant lacking all three gabT genes was constructed and found to be unable to utilize GABA as a sole carbon and nitrogen source. In complete minimal media supplemented with GABA, the mutant grew less well than wild-type DC3000 and showed strongly reduced expression of hrpL and avrPto, which encode an alternative sigma factor and effector, respectively, associated with the type III secretion system. The growth of the gabT triple mutant was weakly reduced in Arabidopsis ecotype Landberg erecta (Ler) and strongly reduced in the Ler pop2-1 GABA transaminase-deficient mutant that accumulates higher levels of GABA. Much of the ability to grow on GABA-amended minimal media or in Arabidopsis pop2-1 leaves could be restored to the gabT triple mutant by expression in trans of just gabT2. The ability of DC3000 to elicit the hypersensitive response (HR) in tobacco leaves is dependent upon deployment of the type III secretion system, and the gabT triple mutant was less able than wild-type DC3000 to elicit this HR when bacteria were infiltrated along with GABA at levels of 1 mm or more. GABA may have multiple effects on P. syringae-plant interactions, with elevated levels increasing disease resistance.

Interference of quorum sensing in Pseudomonas syringae by bacterial epiphytes that limit iron availability.

Leaf surfaces harbour bacterial epiphytes that are capable of influencing the quorum sensing (QS) system, density determination through detection of diffusible signal molecules, of the plant-pathogen Pseudomonas syringae pv. syringae (Pss) which controls expression of extracellular polysaccharide production, motility and other factors contributing to virulence to plants. Approximately 11% of the bacterial epiphytes recovered from a variety of plants produced a diffusible factor capable of inhibiting the QS system of Pss as indicated by suppression of ahlI. Blockage of QS by these interfering strains correlated strongly with their ability to limit iron availability to Pss. A direct relationship between the ability of isogenic Escherichia coli strains to sequester iron via their production of different siderophores and their ability to suppress QS in Pss was also observed. Quorum sensing induction was inversely related to iron availability in culture media supplemented with iron chelators or with FeCl(3). Co-inoculation of interfering strains with Pss onto leaves increased the number of resultant disease lesions over twofold compared with that on plants inoculated with Pss alone. Transposon-generated mutants of interfering strains in which QS inhibition was blocked did not increase disease when co-inoculated with Pss. Increased disease incidence was also not observed when a non-motile mutant of Pss was co-inoculated onto plants with QS interfering bacteria suggesting that these strains enhanced the motility of Pss in an iron-dependent manner, leading to an apparent increase in virulence of this pathogen. Considerable cross-talk mediated by iron scavenging apparently occurs on plants, thereby altering the behaviour of bacteria such as Pss that exhibit important QS-dependent traits in this habitat.

Transcriptional profile of Pseudomonas syringae pv. phaseolicola NPS3121 in response to tissue extracts from a susceptible Phaseolus vulgaris L. cultivar.

BACKGROUND: Pseudomonas syringae pv. phaseolicola is a Gram-negative plant-pathogenic bacterium that causes "halo blight" disease of beans (Phaseolus vulgaris L.). This disease affects both foliage and pods, and is a major problem in temperate areas of the world. Although several bacterial genes have been determined as participants in pathogenesis, the overall process still remains poorly understood, mainly because the identity and function of many of the genes are largely unknown. In this work, a genomic library of P. syringae pv. phaseolicola NPS3121 was constructed and PCR amplification of individual fragments was carried out in order to print a DNA microarray. This microarray was used to identify genes that are differentially expressed when bean leaf extracts, pod extracts or apoplastic fluid were added to the growth medium. RESULTS: Transcription profiles show that 224 genes were differentially expressed, the majority under the effect of bean leaf extract and apoplastic fluid. Some of the induced genes were previously known to be involved in the first stages of the bacterial-plant interaction and virulence. These include genes encoding type III secretion system proteins and genes involved in cell-wall degradation, phaseolotoxin synthesis and aerobic metabolism. On the other hand, most repressed genes were found to be involved in the uptake and metabolism of iron. CONCLUSION: This study furthers the understanding of the mechanisms involved, responses and the metabolic adaptation that occurs during the interaction of P. syringae pv. phaseolicola with a susceptible host plant.

The life history of the plant pathogen Pseudomonas syringae is linked to the water cycle.

Pseudomonas syringae is a plant pathogen well known for its capacity to grow epiphytically on diverse plants and for its ice-nucleation activity. The ensemble of its known biology and ecology led us to postulate that this bacterium is also present in non-agricultural habitats, particularly those associated with water. Here, we report the abundance of P. syringae in rain, snow, alpine streams and lakes and in wild plants, in addition to the previously reported abundance in epilithic biofilms. Each of these substrates harbored strains that corresponded to P. syringae in terms of biochemical traits, pathogenicity and pathogenicity-related factors and that were ice-nucleation active. Phylogenetic comparisons of sequences of four housekeeping genes of the non-agricultural strains with strains of P. syringae from disease epidemics confirmed their identity as P. syringae. Moreover, strains belonging to the same clonal lineage were isolated from snow, irrigation water and a diseased crop plant. Our data suggest that the different substrates harboring P. syringae modify the structure of the associated populations. Here, we propose a comprehensive life cycle for P. syringae--in agricultural and non-agricultural habitats--driven by the environmental cycle of water. This cycle opens the opportunity to evaluate the importance of non-agricultural habitats in the evolution of a plant pathogen and the emergence of virulence. The ice-nucleation activity of all strains from snow, unlike from other substrates, strongly suggests that P. syringae plays an active role in the water cycle as an ice nucleus in clouds.

A translocation signal for delivery of oomycete effector proteins into host plant cells.

Bacterial, oomycete and fungal plant pathogens establish disease by translocation of effector proteins into host cells, where they may directly manipulate host innate immunity. In bacteria, translocation is through the type III secretion system, but analogous processes for effector delivery are uncharacterized in fungi and oomycetes. Here we report functional analyses of two motifs, RXLR and EER, present in translocated oomycete effectors. We use the Phytophthora infestans RXLR-EER-containing protein Avr3a as a reporter for translocation because it triggers RXLR-EER-independent hypersensitive cell death following recognition within plant cells that contain the R3a resistance protein. We show that Avr3a, with or without RXLR-EER motifs, is secreted from P. infestans biotrophic structures called haustoria, demonstrating that these motifs are not required for targeting to haustoria or for secretion. However, following replacement of Avr3a RXLR-EER motifs with alanine residues, singly or in combination, or with residues KMIK-DDK--representing a change that conserves physicochemical properties of the protein--P. infestans fails to deliver Avr3a or an Avr3a-GUS fusion protein into plant cells, demonstrating that these motifs are required for translocation. We show that RXLR-EER-encoding genes are transcriptionally upregulated during infection. Bioinformatic analysis identifies 425 potential genes encoding secreted RXLR-EER class proteins in the P. infestans genome. Identification of this class of proteins provides unparalleled opportunities to determine how oomycetes manipulate hosts to establish infection.

Global virulence regulation networks in phytopathogenic bacteria.

Phytopathogens coordinate multifaceted life histories and deploy stratified virulence determinants via complex, global regulation networks. We dissect the global regulation of four distantly related model phytopathogens to evaluate large-scale events and mechanisms that determine successful pathogenesis. Overarching themes include dependence on centralized cell-to-cell communication systems, pervasive two-component signal-transduction systems, post-transcriptional regulation systems, AraC-like regulators and sigma factors. Although these common regulatory systems control virulence, each functions in different capacities, and to differing ends, in the diverse species. Hence, the virulence regulation network of each species determines its survival and success in various life histories and niches.