bacLIFE: a user-friendly computational workflow for genome analysis and prediction of lifestyle-associated genes in bacteria.

Bacteria have an extensive adaptive ability to live in close association with eukaryotic hosts, exhibiting detrimental, neutral or beneficial effects on host growth and health. However, the genes involved in niche adaptation are mostly unknown and their functions poorly characterized. Here, we present bacLIFE ( https://github.com/Carrion-lab/bacLIFE ) a streamlined computational workflow for genome annotation, large-scale comparative genomics, and prediction of lifestyle-associated genes (LAGs). As a proof of concept, we analyzed 16,846 genomes from the Burkholderia/Paraburkholderia and Pseudomonas genera, which led to the identification of hundreds of genes potentially associated with a plant pathogenic lifestyle. Site-directed mutagenesis of 14 of these predicted LAGs of unknown function, followed by plant bioassays, showed that 6 predicted LAGs are indeed involved in the phytopathogenic lifestyle of Burkholderia plantarii and Pseudomonas syringae pv. phaseolicola. These 6 LAGs encompassed a glycosyltransferase, extracellular binding proteins, homoserine dehydrogenases and hypothetical proteins. Collectively, our results highlight bacLIFE as an effective computational tool for prediction of LAGs and the generation of hypotheses for a better understanding of bacteria-host interactions.

An atypical 3-ketoacyl ACP synthase III required for acyl homoserine lactone synthesis in Pseudomonas syringae pv. syringae B728a.

The last step of the initiation phase of fatty acid biosynthesis in most bacteria is catalyzed by the 3-ketoacyl-acyl carrier protein (ACP) synthase III (FabH). Pseudomonas syringae pv. syringae strain B728a encodes two FabH homologs, Psyr_3467 and Psyr_3830, which we designated PssFabH1 and PssFabH2, respectively. Here, we explored the roles of these two 3-ketoacyl-ACP synthase (KAS) III proteins. We found that PssFabH1 is similar to the Escherichia coli FabH in using acetyl-acetyl-coenzyme A (CoA ) as a substrate in vitro, whereas PssFabH2 uses acyl-CoAs (C4-C10) or acyl-ACPs (C6-C10). Mutant analysis showed that neither KAS III protein is essential for the de novo fatty acid synthesis and cell growth. Loss of PssFabH1 reduced the production of an acyl homoserine lactone (AHL) quorum-sensing signal, and this production was partially restored by overexpressing FabH homologs from other bacteria. AHL production was also restored by inhibiting fatty acid elongation and providing exogenous butyric acid. Deletion of PssFabH1 supports the redirection of acyl-ACP toward biosurfactant synthesis, which in turn enhances swarming motility. Our study revealed that PssFabH1 is an atypical KAS III protein that represents a new KAS III clade that functions in providing a critical fatty acid precursor, butyryl-ACP, for AHL synthesis.IMPORTANCEAcyl homoserine lactones (AHLs) are important quorum-sensing compounds in Gram-negative bacteria. Although their formation requires acylated acyl carrier proteins (ACPs), how the acylated intermediate is shunted from cellular fatty acid synthesis to AHL synthesis is not known. Here, we provide in vivo evidence that Pseudomonas syringae strain B728a uses the enzyme PssFabH1 to provide the critical fatty acid precursor butyryl-ACP for AHL synthesis. Loss of PssFabH1 reduces the diversion of butyryl-ACP to AHL, enabling the accumulation of acyl-ACP for synthesis of biosurfactants that contribute to bacterial swarming motility. We report that PssFabH1 and PssFabH2 each encode a 3-ketoacyl-acyl carrier protein synthase (KAS) III in P. syringae B728a. Whereas PssFabH2 is able to function in redirecting intermediates from ß-oxidation to fatty acid synthesis, PssFabH1 is an atypical KAS III protein that represents a new KAS III clade based on its sequence, non-involvement in cell growth, and novel role in AHL synthesis.

Current methods for monitoring Pseudomonas syringae biofilm development.

This work reviews biofilm investigation techniques and highlights the benefits and drawbacks of each approach focusing especially on Pseudomonas syringae and may serve as a comprehensive guide for any early-career researchers starting with the topic of biofilm. Each approach with applications of individual microscopy and spectroscopy techniques is summarized together with characterization of Pseudomonas syringae and its role in pathogenesis.

Naïve Bayes Classifiers and accompanying dataset for Pseudomonas syringae isolate characterization.

The Pseudomonas syringae species complex (PSSC) is a diverse group of plant pathogens with a collective host range encompassing almost every food crop grown today. As a threat to global food security, rapid detection and characterization of epidemic and emerging pathogenic lineages is essential. However, phylogenetic identification is often complicated by an unclarified and ever-changing taxonomy, making practical use of available databases and the proper training of classifiers difficult. As such, while amplicon sequencing is a common method for routine identification of PSSC isolates, there is no efficient method for accurate classification based on this data. Here we present a suite of five Naïve bayes classifiers for PCR primer sets widely used for PSSC identification, trained on in-silico amplicon data from 2,161 published PSSC genomes using the life identification number (LIN) hierarchical clustering algorithm in place of traditional Linnaean taxonomy. Additionally, we include a dataset for translating classification results back into traditional taxonomic nomenclature (i.e. species, phylogroup, pathovar), and for predicting virulence factor repertoires.

Genomic characterization of Pseudomonas syringae pv. syringae from Callery pear and the efficiency of associated phages in disease protection.

The Pseudomonas syringae species complex is a heterogeneous group of plant pathogenic bacteria associated with a wide distribution of plant species. Advances in genomics are revealing the complex evolutionary history of this species complex and the wide array of genetic adaptations underpinning their diverse lifestyles. Here, we genomically characterize two P. syringae isolates collected from diseased Callery pears (Pyrus calleryana) in Berkeley, California in 2019 and 2022. We also isolated a lytic bacteriophage, which we characterized and evaluated for biocontrol efficiency. Using a multilocus sequence analysis and core genome alignment, we classified the P. syringae isolates as members of phylogroup 2, related to other strains previously isolated from Pyrus and Prunus. An analysis of effector proteins demonstrated an evolutionary conservation of effectoromes across isolates classified in PG2 and yet uncovered unique effector profiles for each, including the two newly identified isolates. Whole-genome sequencing of the associated phage uncovered a novel phage genus related to Pseudomonas syringae pv. actinidiae phage PHB09 and the Flaumdravirus genus. Finally, using in planta infection assays, we demonstrate that the phage was equally useful in symptom mitigation of immature pear fruit regardless of the Pss strain tested. Overall, this study demonstrates the diversity of P. syringae and their viruses associated with ornamental pear trees, posing spill-over risks to commercial pear trees and the possibility of using phages as biocontrol agents to reduce the impact of disease.IMPORTANCEGlobal change exacerbates the spread and impact of pathogens, especially in agricultural settings. There is a clear need to better monitor the spread and diversity of plant pathogens, including in potential spillover hosts, and for the development of novel and sustainable control strategies. In this study, we characterize the first described strains of Pseudomonas syringae pv. syringae isolated from Callery pear in Berkeley, California from diseased tissues in an urban environment. We show that these strains have divergent virulence profiles from previously described strains and that they can cause disease in commercial pears. Additionally, we describe a novel bacteriophage that is associated with these strains and explore its potential to act as a biocontrol agent. Together, the data presented here demonstrate that ornamental pear trees harbor novel P. syringae pv. syringae isolates that potentially pose a risk to local fruit production, or vice versa-but also provide us with novel associated phages, effective in disease mitigation.

Molecular Characterization and Genome Mechanical Features of Two Newly Isolated Polyvalent Bacteriophages Infecting Pseudomonas syringae pv. garcae.

Coffee plants have been targeted by a devastating bacterial disease, a condition known as bacterial blight, caused by the phytopathogen Pseudomonas syringae pv. garcae (Psg). Conventional treatments of coffee plantations affected by the disease involve frequent spraying with copper- and kasugamycin-derived compounds, but they are both highly toxic to the environment and stimulate the appearance of bacterial resistance. Herein, we report the molecular characterization and mechanical features of the genome of two newly isolated (putative polyvalent) lytic phages for Psg. The isolated phages belong to class Caudoviricetes and present a myovirus-like morphotype belonging to the genuses Tequatrovirus (PsgM02F) and Phapecoctavirus (PsgM04F) of the subfamilies Straboviridae (PsgM02F) and Stephanstirmvirinae (PsgM04F), according to recent bacterial viruses' taxonomy, based on their complete genome sequences. The 165,282 bp (PsgM02F) and 151,205 bp (PsgM04F) genomes do not feature any lysogenic-related (integrase) genes and, hence, can safely be assumed to follow a lytic lifestyle. While phage PsgM02F produced a morphogenesis yield of 124 virions per host cell, phage PsgM04F produced only 12 virions per host cell, indicating that they replicate well in Psg with a 50 min latency period. Genome mechanical analyses established a relationship between genome bendability and virion morphogenesis yield within infected host cells.

In Vivo Production of dsRNA Using Bacteriophage ϕ6 in Pseudomonas syringae Cit7 Cells.

RNA interference (RNAi), also known as post-transcriptional gene silencing (PTGS), is one of the emerging genetic engineering techniques to effectively silence or inhibit the expression of target genes. This chapter describes a method for in vivo production of dsRNA in non-pathogenic Pseudomonas syringae strains using phage ϕ6 RNA-dependent RNA polymerase, extraction and purification of dsRNA from bacterial solution, and the use of dsRNA to induce silencing of green fluorescent protein (GFP) in transgenic Nicotiana benthamiana.

Role of DEAD-box RNA helicases in low-temperature adapted growth of Antarctic Pseudomonas syringae Lz4W.

IMPORTANCE: RNA metabolism is important as RNA acts as a link between genomic information and functional biomolecules, thereby playing a critical role in cellular response to environment. We investigated the role of DEAD-box RNA helicases in low-temperature adapted growth of P. syringae, as this group of enzymes play an essential role in modulation of RNA secondary structures. This is the first report on the assessment of all major DEAD-box RNA helicases in any Antarctic bacterium. Of the five RNA helicases, three (srmB, csdA, and dbpA) are important for the growth of the Antarctic P. syringae at low temperature. However, the requisite role of dbpA and the indispensable requirement of csdA for low-temperature adapted growth are a novel finding of this study. Growth analysis of combinatorial deletion strains was performed to understand the functional interaction among helicase genes. Similarly, genetic complementation of RNA helicase mutants was conducted for identification of gene redundancy in P. syringae.

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.

Diversity and ice nucleation activity of Pseudomonas syringae in drone-based water samples from eight lakes in Austria.

Bacteria from the Pseudomonas syringae complex (comprised of at least 15 recognized species and more than 60 different pathovars of P. syringae sensu stricto) have been cultured from clouds, rain, snow, streams, rivers, and lakes. Some strains of P. syringae express an ice nucleation protein (hereafter referred to as ice+) that catalyzes the heterogeneous freezing of water. Though P. syringae has been sampled intensively from freshwater sources in the U.S. and France, little is known about the genetic diversity and ice nucleation activity of P. syringae in other parts of the world. We investigated the haplotype diversity and ice nucleation activity at -8 °C (ice+) of strains of P. syringae from water samples collected with drones in eight freshwater lakes in Austria. A phylogenetic analysis of citrate synthase (cts) sequences from 271 strains of bacteria isolated from a semi-selective medium for Pseudomonas revealed that 69% (188/271) belonged to the P. syringae complex and represented 32 haplotypes in phylogroups 1, 2, 7, 9, 10, 13, 14 and 15. Strains within the P. syringae complex were identified in all eight lakes, and seven lakes contained ice+ strains. Partial 16S rDNA sequences were analyzed from a total of 492 pure cultures of bacteria isolated from non-selective medium. Nearly half (43.5%; 214/492) were associated with the genus Pseudomonas. Five of the lakes (ALT, GRU, GOS, GOL, and WOR) were all distinguished by high levels of Pseudomanas (p ≤ 0.001). HIN, the highest elevation lake, had the highest percentage of ice+ strains. Our work highlights the potential for uncovering new haplotypes of P. syringae in aquatic habitats, and the use of robotic technologies to sample and characterize microbial life in remote settings.

A computational model of Pseudomonas syringae metabolism unveils a role for branched-chain amino acids in Arabidopsis leaf colonization.

Bacterial pathogens adapt their metabolism to the plant environment to successfully colonize their hosts. In our efforts to uncover the metabolic pathways that contribute to the colonization of Arabidopsis thaliana leaves by Pseudomonas syringae pv tomato DC3000 (Pst DC3000), we created iPst19, an ensemble of 100 genome-scale network reconstructions of Pst DC3000 metabolism. We developed a novel approach for gene essentiality screens, leveraging the predictive power of iPst19 to identify core and ancillary condition-specific essential genes. Constraining the metabolic flux of iPst19 with Pst DC3000 gene expression data obtained from naïve-infected or pre-immunized-infected plants, revealed changes in bacterial metabolism imposed by plant immunity. Machine learning analysis revealed that among other amino acids, branched-chain amino acids (BCAAs) metabolism significantly contributed to the overall metabolic status of each gene-expression-contextualized iPst19 simulation. These predictions were tested and confirmed experimentally. Pst DC3000 growth and gene expression analysis showed that BCAAs suppress virulence gene expression in vitro without affecting bacterial growth. In planta, however, an excess of BCAAs suppress the expression of virulence genes at the early stages of infection and significantly impair the colonization of Arabidopsis leaves. Our findings suggesting that BCAAs catabolism is necessary to express virulence and colonize the host. Overall, this study provides valuable insights into how plant immunity impacts Pst DC3000 metabolism, and how bacterial metabolism impacts the expression of virulence.

Exoribonuclease RNase R protects Antarctic Pseudomonas syringae Lz4W from DNA damage and oxidative stress.

IMPORTANCE: Bacterial exoribonucleases play a crucial role in RNA maturation, degradation, quality control, and turnover. In this study, we have uncovered a previously unknown role of 3'-5' exoribonuclease RNase R of Pseudomonas syringae Lz4W in DNA damage and oxidative stress response. Here, we show that neither the exoribonuclease function of RNase R nor its association with the RNA degradosome complex is essential for this function. Interestingly, in P. syringae Lz4W, hydrolytic RNase R exhibits physiological roles similar to phosphorolytic 3'-5' exoribonuclease PNPase of E. coli. Our data suggest that during the course of evolution, mesophilic E. coli and psychrotrophic P. syringae have apparently swapped these exoribonucleases to adapt to their respective environmental growth conditions.

Pseudomonas syringae isolated in lichens for the first time: Unveiling Peltigera genus as the exclusive host.

Pseudomonas syringae is a bacterial complex that is widespread through a range of environments, typically associated with plants where it can be pathogenic, but also found in non-plant environments such as clouds, precipitation, and surface waters. Understanding its distribution within the environment, and the habitats it occupies, is important for examining its evolution and understanding behaviours. After a recent study found P. syringae living among a range of vascular plant species in Iceland, we questioned whether lichens could harbour P. syringae. Sixteen different species of lichens were sampled all over Iceland, but only one lichen genus, Peltigera, was found to consistently harbour P. syringae. Phylogenetic analyses of P. syringae from 10 sampling points where lichen, tracheophyte, and/or moss were simultaneously collected showed significant differences between sampling points, but not between different plants and lichens from the same point. Furthermore, while there were similarities in the P. syringae population in tracheophytes and Peltigera, the densities in Peltigera thalli were lower than in moss and tracheophyte samples. This discovery suggests P. syringae strains can localize and survive in organisms beyond higher plants, and thus reveals opportunities for studying their influence on P. syringae evolution.

Diversity and characterization of antagonistic bacteria against Pseudomonas syringae pv. actinidiae isolated from kiwifruit rhizosphere.

Kiwifruit bacterial canker caused by Pseudomonas syringae pv. actinidiae (Psa) is a severe global disease. However, effective biological control agents for controlling Psa are currently unavailable. This study aimed to screen potential biological control agents against Psa from the kiwifruit rhizosphere. In this study, a total of 722 isolates of bacteria were isolated from the rhizosphere of kiwifruit orchards in five regions of China. A total of 82 strains of rhizosphere bacteria showed antagonistic effects against Psa on plates. Based on amplified ribosomal DNA restriction analysis (ARDRA), these antagonistic rhizosphere bacteria were grouped into 17 clusters. BLAST analyses based on 16S rRNA gene sequence revealed 95.44%-100% sequence identity to recognized species. The isolated strains belonged to genus Acinetobacter, Bacillus, Chryseobacterium, Flavobacterium, Glutamicibacter, Lysinibacillus, Lysobacter, Pseudomonas, Pseudarthrobacter, and Streptomyces, respectively. A total of four representative strains were selected to determine their extracellular metabolites and cell-free supernatant activity against Psa in vitro. They all produce protease and none of them produce glucanase. One strain of Pseudomonas sp. produces siderophore. Strains of Bacillus spp. and Flavobacteria sp. produce cellulase, and Flavobacteria sp. also produce chitinase. Our results suggested that the kiwifruit rhizosphere soils contain a variety of antagonistic bacteria that effectively inhibit the growth of Psa.

Machine learning uncovers the Pseudomonas syringae transcriptome in microbial communities and during infection.

IMPORTANCE: Pseudomonas syringae pv. tomato DC3000 is a model plant pathogen that infects tomatoes and Arabidopsis thaliana. The current understanding of global transcriptional regulation in the pathogen is limited. Here, we applied iModulon analysis to a compendium of RNA-seq data to unravel its transcriptional regulatory network. We characterize each co-regulated gene set, revealing the activity of major regulators across diverse conditions. We provide new insights on the transcriptional dynamics in interactions with the plant immune system and with other bacterial species, such as AlgU-dependent regulation of flagellar genes during plant infection and downregulation of siderophore production in the presence of a siderophore cheater. This study demonstrates the novel application of iModulons in studying temporal dynamics during host-pathogen and microbe-microbe interactions, and reveals specific insights of interest.

Diversity and characteristics of plant immunity-activating bacteria from Brassicaceae plants.

BACKGROUND: Microorganisms that activate plant immune responses are useful for application as biocontrol agents in agriculture to minimize crop losses. The present study was conducted to identify and characterize plant immunity-activating microorganisms in Brassicaceae plants. RESULTS: A total of 25 bacterial strains were isolated from the interior of a Brassicaceae plant, Raphanus sativus var. hortensis. Ten different genera of bacteria were identified: Pseudomonas, Leclercia, Enterobacter, Xanthomonas, Rhizobium, Agrobacterium, Pantoea, Rhodococcus, Microbacterium, and Plantibacter. The isolated strains were analyzed using a method to detect plant immunity-activating microorganisms that involves incubation of the microorganism with tobacco BY-2 cells, followed by treatment with cryptogein, a proteinaceous elicitor of tobacco immune responses. In this method, cryptogein-induced production of reactive oxygen species (ROS) in BY-2 cells serves as a marker of immune activation. Among the 25 strains examined, 6 strains markedly enhanced cryptogein-induced ROS production in BY-2 cells. These 6 strains colonized the interior of Arabidopsis plants, and Pseudomonas sp. RS3R-1 and Rhodococcus sp. RS1R-6 selectively enhanced plant resistance to the bacterial pathogens Pseudomonas syringae pv. tomato DC3000 and Pectobacterium carotovorum subsp. carotovorum NBRC 14082, respectively. In addition, Pseudomonas sp. RS1P-1 effectively enhanced resistance to both pathogens. We also comprehensively investigated the localization (i.e., cellular or extracellular) of the plant immunity-activating components produced by the bacteria derived from R. sativus var. hortensis and the components produced by previously isolated bacteria derived from another Brassicaceae plant species, Brassica rapa var. perviridis. Most gram-negative strains enhanced cryptogein-induced ROS production in BY-2 cells via the presence of cells themselves rather than via extracellular components, whereas many gram-positive strains enhanced ROS production via extracellular components. Comparative genomic analyses supported the hypothesis that the structure of lipopolysaccharides in the outer cell envelope plays an important role in the ROS-enhancing activity of gram-negative Pseudomonas strains. CONCLUSIONS: The assay method described here based on elicitor-induced ROS production in cultured plant cells enabled the discovery of novel plant immunity-activating bacteria from R. sativus var. hortensis. The results in this study also suggest that components involved in the ROS-enhancing activity of the bacteria may differ depending largely on genus and species.

Phyllosphere bacterial strains Rhizobium b1 and Bacillus subtilis b2 control tomato leaf diseases caused by Pseudomonas syringae pv. tomato and Alternaria solani.

AIMS: Show that tomato leaf phyllosphere bacteria are candidates for biocontrol of tomato leaf diseases. METHODS AND RESULTS: Seven bacterial isolates from surface-sterilized Moneymaker tomato plants were tested for growth inhibition of 14 tomato pathogens on potato dextrose agar. Biocontrol assays were conducted with tomato leaf pathogens, Pseudomonas syringae pv. tomato (Pto) and Alternaria solani (A. solani). Two potential isolates showing the greatest inhibition were identified by 16S rDNA sequencing as Rhizobium sp. (isolate b1) and Bacillus subtilis (isolate b2), both produce protease and isolate b2 cellulase. Both reduced tomato leaf infections by Pto and A. solani in detached leaf bioassays. Both bacteria b1 and b2 reduced pathogen development in a tomato growth trial. Bacteria b2 also induced the tomato plant salicylic acid (SA) immune response pathway. Disease suppression in biocontrol assays with b1 and b2 varied between five commercial tomato varieties. CONCLUSIONS: Tomato phyllosphere bacteria when used as phyllosphere inoculants, inhibited tomato diseases caused by Pto and A. solani.

Methylome Response to Proteasome Inhibition by Pseudomonas syringae Virulence Factor Syringolin A.

DNA methylation is an important epigenetic mark required for proper gene expression and silencing of transposable elements. DNA methylation patterns can be modified by environmental factors such as pathogen infection, in which modification of DNA methylation can be associated with plant resistance. To counter the plant defense pathways, pathogens produce effector molecules, several of which act as proteasome inhibitors. Here, we investigated the effect of proteasome inhibition by the bacterial virulence factor syringolin A (SylA) on genome-wide DNA methylation. We show that SylA treatment results in an increase of DNA methylation at centromeric and pericentromeric regions of Arabidopsis chromosomes. We identify several CHH differentially methylated regions (DMRs) that are enriched in the proximity of transcriptional start sites. SylA treatment does not result in significant changes in small RNA composition. However, significant changes in genome transcriptional activity can be observed, including a strong upregulation of resistance genes that are located on chromosomal arms. We hypothesize that DNA methylation changes could be linked to the upregulation of some atypical members of the de novo DNA methylation pathway, namely AGO3, AGO9, and DRM1. Our data suggests that modification of genome-wide DNA methylation resulting from an inhibition of the proteasome by bacterial effectors could be part of an epi-genomic arms race against pathogens. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Biophysical and proteomic analyses of Pseudomonas syringae pv. tomato DC3000 extracellular vesicles suggest adaptive functions during plant infection.

Vesiculation is a process employed by Gram-negative bacteria to release extracellular vesicles (EVs) into the environment. EVs from pathogenic bacteria play functions in host immune modulation, elimination of host defenses, and acquisition of nutrients from the host. Here, we observed EV production of the bacterial speck disease causal agent, Pseudomonas syringae pv. tomato (Pto) DC3000, as outer membrane vesicle release. Mass spectrometry identified 369 proteins enriched in Pto DC3000 EVs. The EV samples contained known immunomodulatory proteins and could induce plant immune responses mediated by bacterial flagellin. Having identified two biomarkers for EV detection, we provide evidence for Pto DC3000 releasing EVs during plant infection. Bioinformatic analysis of the EV-enriched proteins suggests a role for EVs in antibiotic defense and iron acquisition. Thus, our data provide insights into the strategies this pathogen may use to develop in a plant environment. IMPORTANCE The release of extracellular vesicles (EVs) into the environment is ubiquitous among bacteria. Vesiculation has been recognized as an important mechanism of bacterial pathogenesis and human disease but is poorly understood in phytopathogenic bacteria. Our research addresses the role of bacterial EVs in plant infection. In this work, we show that the causal agent of bacterial speck disease, Pseudomonas syringae pv. tomato, produces EVs during plant infection. Our data suggest that EVs may help the bacteria to adapt to environments, e.g., when iron could be limiting such as the plant apoplast, laying the foundation for studying the factors that phytopathogenic bacteria use to thrive in the plant environment.

The Ralstonia pseudosolanacearum effector RipE1 is recognized at the plasma membrane by NbPtr1, the Nicotiana benthamiana homologue of Pseudomonas tomato race 1.

The bacterial wilt disease caused by soilborne bacteria of the Ralstonia solanacearum species complex (RSSC) threatens important crops worldwide. Only a few immune receptors conferring resistance to this devastating disease are known so far. Individual RSSC strains deliver around 70 different type III secretion system effectors into host cells to manipulate the plant physiology. RipE1 is an effector conserved across the RSSC and triggers immune responses in the model solanaceous plant Nicotiana benthamiana. Here, we used multiplexed virus-induced gene silencing of the nucleotide-binding and leucine-rich repeat receptor family to identify the genetic basis of RipE1 recognition. Specific silencing of the N. benthamiana homologue of Solanum lycopersicoides Ptr1 (confers resistance to Pseudomonas syringae pv. tomato race 1) gene (NbPtr1) completely abolished RipE1-induced hypersensitive response and immunity to Ralstonia pseudosolanacearum. The expression of the native NbPtr1 coding sequence was sufficient to restore RipE1 recognition in Nb-ptr1 knockout plants. Interestingly, RipE1 association with the host cell plasma membrane was necessary for NbPtr1-dependent recognition. Furthermore, NbPtr1-dependent recognition of RipE1 natural variants is polymorphic, providing additional evidence for the indirect mode of activation of NbPtr1. Altogether, this work supports NbPtr1 relevance for resistance to bacterial wilt disease in Solanaceae.