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Plant pathogens can cause serious diseases that impact global agriculture. The plant innate immunity, when fully activated, can halt pathogen growth in plants. Despite extensive studies into the molecular and genetic bases of plant immunity against pathogens, the influence of plant immunity in global pathogen metabolism to restrict pathogen growth is poorly understood. Here, we developed RNA sequencing pipelines for analyzing bacterial transcriptomes in planta and determined high-resolution transcriptome patterns of the foliar bacterial pathogen Pseudomonas syringae in Arabidopsis thaliana with a total of 27 combinations of plant immunity mutants and bacterial strains. Bacterial transcriptomes were analyzed at 6 h post infection to capture early effects of plant immunity on bacterial processes and to avoid secondary effects caused by different bacterial population densities in planta. We identified specific “immune-responsive” bacterial genes and processes, including those that are activated in susceptible plants and suppressed by plant immune activation. Expression patterns of immune-responsive bacterial genes at the early time point were tightly linked to later bacterial growth levels in different host genotypes. Moreover, we found that a bacterial iron acquisition pathway is commonly suppressed by multiple plant immune-signaling pathways. Overexpression of a P. syringae sigma factor gene involved in iron regulation and other processes partially countered bacterial growth restriction during the plant immune response triggered by AvrRpt2. Collectively, this study defines the effects of plant immunity on the transcriptome of a bacterial pathogen and sheds light on the enigmatic mechanisms of bacterial growth inhibition during the plant immune response.

High humidity has a strong influence on the development of numerous diseases affecting the above-ground parts of plants (the phyllosphere) in crop fields and natural ecosystems, but the molecular basis of this humidity effect is not understood. Previous studies have emphasized immune suppression as a key step in bacterial pathogenesis. Here we show that humidity-dependent, pathogen-driven establishment of an aqueous intercellular space (apoplast) is another important step in bacterial infection of the phyllosphere. Bacterial effectors, such as Pseudomonas syringae HopM1, induce establishment of the aqueous apoplast and are sufficient to transform non-pathogenic P. syringae strains into virulent pathogens in immunodeficient Arabidopsis thaliana under high humidity. Arabidopsis quadruple mutants simultaneously defective in a host target (AtMIN7) of HopM1 and in pattern-triggered immunity could not only be used to reconstitute the basic features of bacterial infection, but also exhibited humidity-dependent dyshomeostasis of the endophytic commensal bacterial community in the phyllosphere. These results highlight a new conceptual framework for understanding diverse phyllosphere–bacterial interactions. A combination of high humidity and bacterial effectors, such as Pseudomonas syringae HopM1, creates an aqueous environment in the apoplast of immunodeficient Arabidopsis thaliana that allows non-pathogenic P. syringae strains to become virulent pathogens. Bacterial pathogenesis in plants High humidity has a profound influence on the development of numerous plant diseases in crop fields and natural ecosystems, but the molecular basis of this humidity effect is not understood. Sheng Yang He and colleagues show that plant pathogens such as Pseudomonas syringae actively establish an aqueous leaf apoplast—that is, a space between the cells and the cell walls—in a humidity-dependent manner through the secretion of conserved bacterial effectors. The effectors also cause alterations in the leaf-associated microbiota. This is a crucial step in plant infection by bacteria and the effectors involved are sufficient to transform non-pathogenic strains into virulent pathogens only under high humidity. Through elegant genetics work, the authors define immune suppression and aqueous apoplast formation as the minimal set of host processes required for bacterial pathogenesis in plant leaves.

Environmental conditions profoundly affect plant disease development; however, the underlying molecular bases are not well understood. Here we show that elevated temperature significantly increases the susceptibility of Arabidopsis to Pseudomonas syringae pv. tomato ( Pst ) DC3000 independently of the phyB/PIF thermosensing pathway. Instead, elevated temperature promotes translocation of bacterial effector proteins into plant cells and causes a loss of ICS1-mediated salicylic acid (SA) biosynthesis. Global transcriptome analysis reveals a major temperature-sensitive node of SA signalling, impacting ~60% of benzothiadiazole (BTH)-regulated genes, including ICS1 and the canonical SA marker gene, PR1 . Remarkably, BTH can effectively protect Arabidopsis against Pst DC3000 infection at elevated temperature despite the lack of ICS1 and PR1 expression. Our results highlight the broad impact of a major climate condition on the enigmatic molecular interplay between temperature, SA defence and function of a central bacterial virulence system in the context of a widely studied susceptible plant–pathogen interaction. Temperature is known to influence plant disease development. Here Huot et al. show that elevated temperature can enhance Pseudomonas syringae effector delivery into plant cells and suppress SA biosynthesis while also finding a temperature-sensitive branch of the SA signaling pathway in Arabidopsis .

Plants and animals are in constant association with a variety of microbes. Although much is known about how pathogenic and symbiotic microbes interact with plants, less is known about the population dynamics, adaptive traits, and transcriptional features of the vast number of microbes that make up the bulk of the plant microbiota. The majority of microbiota taxa are either commensal, natural mutants of pathogens, or pathogens that encounter strong immune responses due to plant recognition of pathogen effectors. How these “nonpathogenic” microbes interact with plants is poorly understood, especially during long-term, steady-state interactions, which are more reflective of plant–microbiota interactions in nature. In this study, we embarked upon long-term population and in planta transcriptomic studies of commensal endophytic bacteria and compared them to nonpathogenic or effector-triggered immunity-inducing strains of the bacterial pathogen Pseudomonas syringae. Our results led to the discovery of multiplication–death equilibrium as a common basis for the shared long-term static population densities of these bacteria. A comprehensive in planta transcriptomic analysis using multiple time points after inoculation revealed a striking similarity between the transcriptomic features of nonpathogenic P. syringae to that of bacteria in stationary phase in vitro, a metabolically active physiological state in which the production of adaptive secondary metabolites and stress responses are induced. We propose that the long-term population and transcriptomic features of nonpathogenic bacteria captured in this study likely reflect the physiological steady state encountered by the bulk of endophytic microbiota—excluding virulent pathogens—in their life-long interactions with plants in nature.

Plants are continuously threatened by pathogen attack and, as such, they have evolved mechanisms to evade, escape and defend themselves against pathogens. However, it is not known what types of defense mechanisms a plant would already possess to defend against a potential pathogen that has not co-evolved with the plant. We addressed this important question in a comprehensive manner by studying the responses of 1041 accessions of Arabidopsis thaliana to the foliar pathogen Pseudomonas syringae pv. tomato (Pst) DC3000. We characterized the interaction using a variety of established methods, including different inoculation techniques, bacterial mutant strains, and assays for the hypersensitive response, salicylic acid (SA) accumulation and reactive oxygen species production. Fourteen accessions showed resistance to infection by Pst DC3000. Of these, two accessions had a surface-based mechanism of resistance, six showed a hypersensitive-like response while three had elevated SA levels. Interestingly, A. thaliana was discovered to have a recognition system for the effector AvrPto, and HopAM1 was found to modulate Pst DC3000 resistance in two accessions. Our comprehensive study has significant implications for the understanding of natural disease resistance mechanisms at the species level and for the ecology and evolution of plant–pathogen interactions.

The gamma-proteobacterial plant pathogen Pseudomonas syringae pv. tomato DC3000 uses the type III secretion system to inject ca. 28 Avr/Hop effector proteins into plants, which enables the bacterium to grow from low inoculum levels to produce bacterial speck symptoms in tomato, Arabidopsis thaliana, and (when lacking hopQ1-1) Nicotiana benthamiana. The effectors are collectively essential but individually dispensable for the ability of the bacteria to defeat defenses, grow, and produce symptoms in plants. Eighteen of the effector genes are clustered in six genomic islands/islets. Combinatorial deletions involving these clusters and two of the remaining effector genes revealed a redundancy-based structure in the effector repertoire, such that some deletions diminished growth in N. benthamiana only in combination with other deletions. Much of the ability of DC3000 to grow in N. benthamiana was found to be due to five effectors in two redundant-effector groups (REGs), which appear to separately target two high-level processes in plant defense: perception of external pathogen signals (AvrPto and AvrPtoB) and deployment of antimicrobial factors (AvrE, HopM1, HopR1). Further support for the membership of HopR1 in the same REG as AvrE was gained through bioinformatic analysis, revealing the existence of an AvrE/DspA/E/HopR effector superfamily, which has representatives in virtually all groups of proteobacterial plant pathogens that deploy type III effectors.

Understanding the molecular determinants underlying the interaction between the leaf and human pathogenic bacteria is key to provide the foundation to develop science-based strategies to prevent or decrease the pathogen contamination of leafy greens. In this study, we conducted a dual RNA-sequencing analysis to simultaneously define changes in the transcriptomic profiles of the plant and the bacterium when they come in contact. We used an economically relevant vegetable crop, lettuce (Lactuca sativa L. cultivar Salinas), and a model plant, Arabidopsis thaliana Col-0, as well as two pathogenic bacterial strains that cause disease outbreaks associated with fresh produce, Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium 14028s (STm 14028s). We observed commonalities and specificities in the modulation of biological processes between Arabidopsis and lettuce and between O157:H7 and STm 14028s during early stages of the interaction. We detected a larger alteration of gene expression at the whole transcriptome level in lettuce and Arabidopsis at 24 h post inoculation with STm 14028s compared to that with O157:H7. In addition, bacterial transcriptomic adjustments were substantially larger in Arabidopsis than in lettuce. Bacterial transcriptome was affected at a larger extent in the first 4 h compared to the subsequent 20 h after inoculation. Overall, we gained valuable knowledge about the responses and counter-responses of both bacterial pathogen and plant host when these bacteria are residing in the leaf intercellular space. These findings and the public genomic resources generated in this study are valuable for additional data mining.

Background The ability to target and manipulate protein-based cellular processes would accelerate plant research; yet, the technology to specifically and selectively target plant-expressed proteins is still in its infancy. Leucine-rich repeats (LRRs) are ubiquitously present protein domains involved in mediating protein–protein interactions. LRRs confer the binding specificity to the highly diverse variable lymphocyte receptor (VLR) antibodies (including VLRA, VLRB and VLRC types) that jawless vertebrates make as the functional equivalents of jawed vertebrate immunoglobulin-based antibodies. Results In this study, VLRBs targeting an effector protein from a plant pathogen, HopM1, were developed by immunizing lampreys and using yeast surface display to select for high-affinity VLRBs. HopM1-specific VLRBs (VLR M1 ) were expressed in planta in the cytosol, the trans -Golgi network, and the apoplast. Expression of VLR M1 was higher when the protein localized to an oxidizing environment that would favor disulfide bridge formation (when VLR M1 was not localized to the cytoplasm), as disulfide bonds are necessary for proper VLR folding. VLR M1 specifically interacted in planta with HopM1 but not with an unrelated bacterial effector protein while HopM1 failed to interact with a non-specific VLRB. Conclusions In the future, VLRs may be used as flexible modules to bind proteins or carbohydrates of interest in planta , with broad possibilities for their use by binding directly to their targets and inhibiting their action, or by creating chimeric proteins with new specificities in which endogenous LRR domains are replaced by those present in VLRs.

The ability to target and manipulate protein-based cellular processes would accelerate plant research; yet, the technology to specifically and selectively target plant-expressed proteins is still in its infancy. Leucine-rich repeats (LRRs) are ubiquitously present protein domains involved in mediating protein–protein interactions. LRRs confer the binding specificity to the highly diverse variable lymphocyte receptor (VLR) antibodies (including VLRA, VLRB and VLRC types) that jawless vertebrates make as the functional equivalents of jawed vertebrate immunoglobulin-based antibodies. Here, VLRBs targeting an effector protein from a plant pathogen, HopM1, were developed by immunizing lampreys and using yeast surface display to select for high-affinity VLRBs. HopM1-specific VLRBs (VLRM1) were expressed in planta in the cytosol, the trans-Golgi network, and the apoplast. Expression of VLRM1 was higher when the protein localized to an oxidizing environment that would favor disulfide bridge formation (when VLRM1 was not localized to the cytoplasm), as disulfide bonds are necessary for proper VLR folding. VLRM1 specifically interacted in planta with HopM1 but not with an unrelated bacterial effector protein while HopM1 failed to interact with a non-specific VLRB. Later, VLRs may be used as flexible modules to bind proteins or carbohydrates of interest in planta, with broad possibilities for their use by binding directly to their targets and inhibiting their action, or by creating chimeric proteins with new specificities in which endogenous LRR domains are replaced by those present in VLRs.

The γ-proteobacterial plant pathogen Pseudomonas syringae pv. tomato DC3000 uses the type III secretion system to inject ca. 28 Avr/Hop effector proteins into plants, which enables the bacterium to grow from low inoculum levels to produce bacterial speck symptoms in tomato, Arabidopsis thaliana, and (when lacking hopQ1-1) Nicotiana benthamiana. The effectors are collectively essential but individually dispensable for the ability of the bacteria to defeat defenses, grow, and produce symptoms in plants. Eighteen of the effector genes are clustered in six genomic islands/islets. Combinatorial deletions involving these clusters and two of the remaining effector genes revealed a redundancy-based structure in the effector repertoire, such that some deletions diminished growth in N. benthamiana only in combination with other deletions. Much of the ability of DC3000 to grow in N. benthamiana was found to be due to five effectors in two redundant-effector groups (REGs), which appear to separately target two high-level processes in plant defense: perception of external pathogen signals (AvrPto and AvrPtoB) and deployment of antimicrobial factors (AvrE, HopM1, HopR1). Further support for the membership of HopR1 in the same REG as AvrE was gained through bioinformatic analysis, revealing the existence of an AvrE/DspA/E/HopR effector superfamily, which has representatives in virtually all groups of proteobacterial plant pathogens that deploy type III effectors.