Investigating the Effects of Temperature on Pathogen Propagation in Arabidopsis.

Temperature is one of the most prominent environmental factors that influence plant immunity. Depending on the plant-pathogen system, increased temperature may inhibit or enhance disease resistance or immunity in plants. Measuring the effect of temperature on plant immunity is the first step toward revealing climate effects on plant-pathogen interactions and molecular regulators of temperature sensitivity of plant immunity. Quantification of plant disease resistance or susceptibility under different temperatures can be accomplished by assessing pathogen growth over time in infected plants or tissues. Here, we present a protocol for quantifying pathogen growth in the most studied system of Arabidopsis thaliana and Pseudomonas syringae pathovar tomato (Pst) DC3000. We discuss important factors to consider for assaying pathogen growth in plants under different temperatures. This protocol can be used to assess temperature sensitivity of resistance in different plant genotypes and to various pathovars.

Marker and readout genes for defense priming in Pseudomonas cannabina pv. alisalensis interaction aid understanding systemic immunity in Arabidopsis.

Following localized infection, the entire plant foliage becomes primed for enhanced defense. However, specific genes induced during defense priming (priming-marker genes) and those showing increased expression in defense-primed plants upon rechallenge (priming-readout genes) remain largely unknown. In our Arabidopsis thaliana study, genes AT1G76960 (function unknown), CAX3 (encoding a vacuolar Ca2+/H+ antiporter), and CRK4 (encoding a cysteine-rich receptor-like protein kinase) were strongly expressed during Pseudomonas cannabina pv. alisalensis-induced defense priming, uniquely marking the primed state for enhanced defense. Conversely, PR1 (encoding a pathogenesis-related protein), RLP23 and RLP41 (both encoding receptor-like proteins) were similarly activated in defense-primed plants before and after rechallenge, suggesting they are additional marker genes for defense priming. In contrast, CASPL4D1 (encoding Casparian strip domain-like protein 4D1), FRK1 (encoding flg22-induced receptor-like kinase), and AT3G28510 (encoding a P loop-containing nucleoside triphosphate hydrolases superfamily protein) showed minimal activation in uninfected, defense-primed, or rechallenged plants, but intensified in defense-primed plants after rechallenge. Notably, mutation in only priming-readout gene NHL25 (encoding NDR1/HIN1-like protein 25) impaired both defense priming and systemic acquired resistance, highlighting its previously undiscovered pivotal role in systemic plant immunity.

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.

Pseudomonas effector AvrB is a glycosyltransferase that rhamnosylates plant guardee protein RIN4.

The plant pathogen Pseudomonas syringae encodes a type III secretion system avirulence effector protein, AvrB, that induces a form of programmed cell death called the hypersensitive response in plants as a defense mechanism against systemic infection. Despite the well-documented catalytic activities observed in other Fido (Fic, Doc, and AvrB) proteins, the enzymatic activity and target substrates of AvrB have remained elusive. Here, we show that AvrB is an unprecedented glycosyltransferase that transfers rhamnose from UDP-rhamnose to a threonine residue of the Arabidopsis guardee protein RIN4. We report structures of various enzymatic states of the AvrB-catalyzed rhamnosylation reaction of RIN4, which reveal the structural and mechanistic basis for rhamnosylation by a Fido protein. Collectively, our results uncover an unexpected reaction performed by a prototypical member of the Fido superfamily while providing important insights into the plant hypersensitive response pathway and foreshadowing more diverse chemistry used by Fido proteins and their substrates.

Using an RNA Aptamer to Inhibit the Action of Effector Proteins of Plant Pathogens.

In previous work, we experimentally demonstrated the possibility of using RNA aptamers to inhibit endogenous protein expression and their function within plant cells In the current work, we show that our proposed method is suitable for inhibiting the functions of exogenous, foreign proteins delivered into the plant via various mechanisms, including pathogen proteins. Stringent experimentation produced robust RNA aptamers that are able to bind to the recombinant HopU1 effector protein of P. syringae bacteria. This research uses genetic engineering methods to constitutively express/transcribe HopU1 RNA aptamers in transgenic A. thaliana. Our findings support the hypothesis that HopU1 aptamers can actively interfere with the function of the HopU1 protein and thereby increase resistance to phytopathogens of the genus P. syringae pv. tomato DC 3000.

Mutation of a highly conserved amino acid in RPM1 causes leaf yellowing and premature senescence in wheat.

KEY MESSAGE: A point mutation of RPM1 triggers persistent immune response that induces leaf premature senescence in wheat, providing novel information of immune responses and leaf senescence. Leaf premature senescence in wheat (Triticum aestivum L.) is one of the most common factors affecting the plant's development and yield. In this study, we identified a novel wheat mutant, yellow leaf and premature senescence (ylp), which exhibits yellow leaves and premature senescence at the heading and flowering stages. Consistent with the yellow leaves phenotype, ylp had damaged and collapsed chloroplasts. Map-based cloning revealed that the phenotype of ylp was caused by a point mutation from Arg to His at amino acid 790 in a plasma membrane-localized protein resistance to Pseudomonas syringae pv. maculicola 1 (RPM1). The point mutation triggered excessive immune responses and the upregulation of senescence- and autophagy-associated genes. This work provided the information for understanding the molecular regulatory mechanism of leaf senescence, and the results would be important to analyze which mutations of RPM1 could enable plants to obtain immune activation without negative effects on plant growth.

The structure of MucD from Pseudomonas syringae revealed N-terminal loop-mediated trimerization of HtrA-like serine protease.

Protein quality control mechanisms are essential for maintaining cellular integrity, and the HtrA family of serine proteases plays a crucial role in handling folding stress in prokaryotic periplasm. Escherichia coli harbors three HtrA members, namely, DegS, DegP, and DegQ, which share a common domain structure. MucD, a putative HtrA family member that resembles DegP, is involved in alginate biosynthesis regulation and the stress response. Pseudomonas syringae causes plant diseases and opportunistic infections in humans. This study presents the high-resolution structure of MucD from Pseudomonas syringae (psMucD), revealing its composition as a typical HtrA family serine protease with protease and PDZ domains. Its findings suggest that psMucD containing one PDZ domain is a trimer in solution, and psMucD trimerization is mediated by its N-terminal loop. Sequence and structural analyses revealed similarities and differences with other HtrA family members. Additionally, this study provides a model of psMucD's catalytic process, comparing it with other members of the HtrA family of serine proteases.

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.

The genetic and physiological basis of Arabidopsis thaliana tolerance to Pseudomonas viridiflava.

The opportunistic pathogen Pseudomonas viridiflava colonizes > 50 agricultural crop species and is the most common Pseudomonas in the phyllosphere of European Arabidopsis thaliana populations. Belonging to the P. syringae complex, it is genetically and phenotypically distinct from well-characterized P. syringae sensu stricto. Despite its prevalence, we lack knowledge of how A. thaliana responds to its native isolates at the molecular level. Here, we characterize the host response in an A. thaliana - P. viridiflava pathosystem. We measured host and pathogen growth in axenic infections and used immune mutants, transcriptomics, and metabolomics to determine defense pathways influencing susceptibility to P. viridiflava infection. Infection with P. viridiflava increased jasmonic acid (JA) levels and the expression of ethylene defense pathway marker genes. The immune response in a susceptible host accession was delayed compared with a tolerant one. Mechanical injury rescued susceptibility, consistent with an involvement of JA. The JA/ethylene pathway is important for suppression of P. viridiflava, yet suppression capacity varies between accessions. Our results shed light on how A. thaliana can suppress the ever-present P. viridiflava, but further studies are needed to understand how P. viridiflava evades this suppression to spread broadly across A. thaliana populations.

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.

Ptr1 and ZAR1 immune receptors confer overlapping and distinct bacterial pathogen effector specificities.

Some nucleotide-binding and leucine-rich repeat receptors (NLRs) indirectly detect pathogen effectors by monitoring their host targets. In Arabidopsis thaliana, RIN4 is targeted by multiple sequence-unrelated effectors and activates immune responses mediated by RPM1 and RPS2. These effectors trigger cell death in Nicotiana benthamiana, but the corresponding NLRs have yet not been identified. To identify N. benthamiana NLRs (NbNLRs) that recognize Arabidopsis RIN4-targeting effectors, we conducted a rapid reverse genetic screen using an NbNLR VIGS library. We identified that the N. benthamiana homolog of Ptr1 (Pseudomonas tomato race 1) recognizes the Pseudomonas effectors AvrRpt2, AvrRpm1, and AvrB. We demonstrated that recognition of the Xanthomonas effector AvrBsT and the Pseudomonas effector HopZ5 is conferred independently by the N. benthamiana homolog of Ptr1 and ZAR1. Interestingly, the recognition of HopZ5 and AvrBsT is contributed unequally by Ptr1 and ZAR1 in N. benthamiana and Capsicum annuum. In addition, we showed that the RLCK XII family protein JIM2 is required for the NbZAR1-dependent recognition of AvrBsT and HopZ5. The recognition of sequence-unrelated effectors by NbPtr1 and NbZAR1 provides an additional example of convergently evolved effector recognition. Identification of key components involved in Ptr1 and ZAR1-mediated immunity could reveal unique mechanisms of expanded effector recognition.

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.

Single-cell profiling of Arabidopsis leaves to Pseudomonas syringae infection.

Plant response to pathogen infection varies within a leaf, yet this heterogeneity is not well resolved. We expose Arabidopsis to Pseudomonas syringae or mock treatment and profile >11,000 individual cells using single-cell RNA sequencing. Integrative analysis of cell populations from both treatments identifies distinct pathogen-responsive cell clusters exhibiting transcriptional responses ranging from immunity to susceptibility. Pseudotime analyses through pathogen infection reveals a continuum of disease progression from an immune to a susceptible state. Confocal imaging of promoter-reporter lines for transcripts enriched in immune cell clusters shows expression surrounding substomatal cavities colonized or in close proximity to bacterial colonies, suggesting that cells within immune clusters represent sites of early pathogen invasion. Susceptibility clusters exhibit more general localization and are highly induced at later stages of infection. Overall, our work shows cellular heterogeneity within an infected leaf and provides insight into plant differential response to infection at a single-cell level.

The Arabidopsis thaliana onset of leaf death 12 mutation in the lectin receptor kinase P2K2 results in an autoimmune phenotype.

BACKGROUND: Plant immunity relies on the perception of immunogenic signals by cell-surface and intracellular receptors and subsequent activation of defense responses like programmed cell death. Under certain circumstances, the fine-tuned innate immune system of plants results in the activation of autoimmune responses that cause constitutive defense responses and spontaneous cell death in the absence of pathogens. RESULTS: Here, we characterized the onset of leaf death 12 (old12) mutant that was identified in the Arabidopsis accession Landsberg erecta. The old12 mutant is characterized by a growth defect, spontaneous cell death, plant-defense gene activation, and early senescence. In addition, the old12 phenotype is temperature reversible, thereby exhibiting all characteristics of an autoimmune mutant. Mapping the mutated locus revealed that the old12 phenotype is caused by a mutation in the Lectin Receptor Kinase P2-TYPE PURINERGIC RECEPTOR 2 (P2K2) gene. Interestingly, the P2K2 allele from Landsberg erecta is conserved among Brassicaceae. P2K2 has been implicated in pathogen tolerance and sensing extracellular ATP. The constitutive activation of defense responses in old12 results in improved resistance against Pseudomonas syringae pv. tomato DC3000. CONCLUSION: We demonstrate that old12 is an auto-immune mutant and that allelic variation of P2K2 contributes to diversity in Arabidopsis immune responses.

Characterization of Disease Resistance Induced by a Pyrazolecarboxylic Acid Derivative in Arabidopsis thaliana.

Systemic acquired resistance (SAR) is a potent innate immunity system in plants that is induced through the salicylic acid (SA)-mediated signaling pathway. Here, we characterized 3-chloro-1-methyl-1H-pyrazole-5-carboxylic acid (CMPA) as an effective SAR inducer in Arabidopsis. The soil drench application of CMPA enhanced a broad range of disease resistance against the bacterial pathogen Pseudomonas syringae and fungal pathogens Colletotrichum higginsianum and Botrytis cinerea in Arabidopsis, whereas CMPA did not show antibacterial activity. Foliar spraying with CMPA induced the expression of SA-responsible genes such as PR1, PR2 and PR5. The effects of CMPA on resistance against the bacterial pathogen and the expression of PR genes were observed in the SA biosynthesis mutant, however, while they were not observed in the SA-receptor-deficient npr1 mutant. Thus, these findings indicate that CMPA induces SAR by triggering the downstream signaling of SA biosynthesis in the SA-mediated signaling pathway.

Plant extracellular self-DNA inhibits growth and induces immunity via the jasmonate signaling pathway.

Plants have evolved sophisticated mechanisms to detect various forms of danger. Damage-associated molecular patterns (DAMPs) are endogenous danger molecules that are released from damaged cells and activate the innate immunity. Recent evidence suggests that plant extracellular self-DNA (esDNA) can serve as a DAMP molecule. However, the mechanisms by which esDNA functions are largely unknown. In this study, we confirmed that esDNA inhibits root growth and triggers reactive oxygen species (ROS) production in a concentration- and species-specific manner in Arabidopsis (Arabidopsis thaliana) and tomato (Solanum lycopersicum L.). Furthermore, by combining RNA sequencing, hormone measurement, and genetic analysis, we found that esDNA-mediated growth inhibition and ROS production are achieved through the jasmonic acid (JA) signaling pathway. Specifically, esDNA induces JA production and the expression of JA-responsive genes. The esDNA-mediated growth inhibition, ROS production, and gene expression are impaired in the JA-related mutants. Finally, we found that the JA signaling pathway is required for the esDNA-elicited resistance against the pathogens Botrytis cinerea and Pseudomonas syringae pv. tomato DC3000. This finding highlights the importance of JA signaling in esDNA-mediated biological effects, thereby providing insight into how esDNA functions as a DAMP.

Pseudomonas syringae Type III Secretion Protein HrpP Manipulates Plant Immunity To Promote Infection.

The bacterial plant pathogen Pseudomonas syringae deploys a type III secretion system (T3SS) to deliver effector proteins into plant cells to facilitate infection, for which many effectors have been characterized for their interactions. However, few T3SS Hrp (hypersensitive response and pathogenicity) proteins from the T3SS secretion apparatus have been studied for their direct interactions with plants. Here, we show that the P. syringae pv. tomato DC3000 T3SS protein HrpP induces host cell death, suppresses pattern-triggered immunity (PTI), and restores the effector translocation ability of the hrpP mutant. The hrpP-transgenic Arabidopsis lines exhibited decreased PTI responses to flg22 and elf18 and enhanced disease susceptibility to P. syringae pv. tomato DC3000. Transcriptome analysis reveals that HrpP sensing activates salicylic acid (SA) signaling while suppressing jasmonic acid (JA) signaling, which correlates with increased SA accumulation and decreased JA biosynthesis. Both yeast two-hybrid and bimolecular fluorescence complementation assays show that HrpP interacts with mitogen-activated protein kinase kinase 2 (MKK2) on the plant membrane and in the nucleus. The HrpP truncation HrpP1-119, rather than HrpP1-101, retains the ability to interact with MKK2 and suppress PTI in plants. In contrast, HrpP1-101 continues to cause cell death and electrolyte leakage. MKK2 silencing compromises SA signaling but has no effect on cell death caused by HrpP. Overall, our work highlights that the P. syringae T3SS protein HrpP facilitates effector translocation and manipulates plant immunity to facilitate bacterial infection. IMPORTANCE The T3SS is required for the virulence of many Gram-negative bacterial pathogens of plants and animals. This study focuses on the sensing and function of the T3SS protein HrpP during plant interactions. Our findings show that HrpP and its N-terminal truncation HrpP1-119 can interact with MKK2, promote effector translocation, and manipulate plant immunity to facilitate bacterial infection, highlighting the P. syringae T3SS component involved in the fine-tuning of plant immunity.

Polyamine-mediated mechanisms contribute to oxidative stress tolerance in Pseudomonas syringae.

Bacterial phytopathogens living on the surface or within plant tissues may experience oxidative stress because of the triggered plant defense responses. Although it has been suggested that polyamines can defend bacteria from this stress, the mechanism behind this action is not entirely understood. In this study, we investigated the effects of oxidative stress on the polyamine homeostasis of the plant pathogen Pseudomonas syringae and the functions of these compounds in bacterial stress tolerance. We demonstrated that bacteria respond to H2O2 by increasing the external levels of the polyamine putrescine while maintaining the inner concentrations of this compound as well as the analogue amine spermidine. In line with this, adding exogenous putrescine to media increased bacterial tolerance to H2O2. Deletion of arginine decarboxylase (speA) and ornithine decarboxylate (speC), prevented the synthesis of putrescine and augmented susceptibility to H2O2, whereas targeting spermidine synthesis alone through deletion of spermidine synthase (speE) increased the level of extracellular putrescine and enhanced H2O2 tolerance. Further research demonstrated that the increased tolerance of the ΔspeE mutant correlated with higher expression of H2O2-degrading catalases and enhanced outer cell membrane stability. Thus, this work demonstrates previously unrecognized connections between bacterial defense mechanisms against oxidative stress and the polyamine metabolism.

Distinct function of SPL genes in age-related resistance in Arabidopsis.

In plants, age-related resistance (ARR) refers to a gain of disease resistance during shoot or organ maturation. ARR associated with vegetative phase change, a transition from juvenile to adult stage, is a widespread agronomic trait affecting resistance against multiple pathogens. How innate immunity in a plant is differentially regulated during successive stages of shoot maturation is unclear. In this work, we found that Arabidopsis thaliana showed ARR against its bacterial pathogen Pseudomonas syringae pv. tomato DC3000 during vegetative phase change. The timing of the ARR activation was associated with a temporal drop of miR156 level. The microRNA miR156 maintains juvenile phase by inhibiting the accumulation and translation of SPL transcripts. A systematic inspection of the loss- and gain-of-function mutants of 11 SPL genes revealed that a subset of SPL genes, notably SPL2, SPL10, and SPL11, activated ARR in adult stage. The immune function of SPL10 was independent of its role in morphogenesis. Furthermore, the SPL10 mediated an age-dependent augmentation of the salicylic acid (SA) pathway partially by direct activation of PAD4. Disrupting SA biosynthesis or signaling abolished the ARR against Pto DC3000. Our work demonstrated that the miR156-SPL10 module in Arabidopsis is deployed to operate immune outputs over developmental timing.

Pangenomic analysis reveals plant NAD+ manipulation as an important virulence activity of bacterial pathogen effectors.

Nicotinamide adenine dinucleotide (NAD+) has emerged as a key component in prokaryotic and eukaryotic immune systems. The recent discovery that Toll/interleukin-1 receptor (TIR) proteins function as NAD+ hydrolases (NADase) links NAD+-derived small molecules with immune signaling. We investigated pathogen manipulation of host NAD+ metabolism as a virulence strategy. Using the pangenome of the model bacterial pathogen Pseudomonas syringae, we conducted a structure-based similarity search from 35,000 orthogroups for type III effectors (T3Es) with potential NADase activity. Thirteen T3Es, including five newly identified candidates, were identified that possess domain(s) characteristic of seven NAD+-hydrolyzing enzyme families. Most Pseudomonas syringae strains that depend on the type III secretion system to cause disease, encode at least one NAD+-manipulating T3E, and many have several. We experimentally confirmed the type III-dependent secretion of a novel T3E, named HopBY, which shows structural similarity to both TIR and adenosine diphosphate ribose (ADPR) cyclase. Homologs of HopBY were predicted to be type VI effectors in diverse bacterial species, indicating potential recruitment of this activity by microbial proteins secreted during various interspecies interactions. HopBY efficiently hydrolyzes NAD+ and specifically produces 2'cADPR, which can also be produced by TIR immune receptors of plants and by other bacteria. Intriguingly, this effector promoted bacterial virulence, indicating that 2'cADPR may not be the signaling molecule that directly initiates immunity. This study highlights a host-pathogen battleground centered around NAD+ metabolism and provides insight into the NAD+-derived molecules involved in plant immunity.