CPR5 positively regulates pattern-triggered immunity via a mediator protein.

Plant cells possess a two-layered immune system consisting of pattern-triggered immunity (PTI) and effector-triggered immunity (ETI), mediated by cell surface pattern-recognition receptors and intracellular nucleotide-binding leucine-rich repeat receptors (NLRs), respectively. The CONSTITUTIVE EXPRESSION OF PR GENES 5 (CPR5) nuclear pore complex protein negatively regulates ETI, including ETI-associated hypersensitive response. Here, we show that CPR5 is essential for the activation of various PTI responses in Arabidopsis, such as resistance to the non-adapted bacterium Pseudomonas syringae pv. tomato DC3000 hrcC- . In a forward-genetic screen for suppressors of cpr5, we identified the mediator protein MED4. Mutation of MED4 in cpr5 greatly restored the defective PTI of cpr5. Our findings reveal that CPR5 plays opposite roles in regulating PTI and ETI, and genetically regulates PTI via MED4.

Transcriptomic Analysis Revealed Key Defense Genes and Signaling Pathways Mediated by the Arabidopsis thaliana Gene SAD2 in Response to Infection with Pseudomonas syringae pv. Tomato DC3000.

Nucleocytoplasmic transport receptors play key roles in the nuclear translocation of disease resistance proteins, but the associated mechanisms remain unclear. The Arabidopsis thaliana gene SAD2 encodes an importin ß-like protein. A transgenic Arabidopsis line overexpressing SAD2 (OESAD2/Col-0) showed obvious resistance to Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) compared to the wild type (Col-0), but the knockout mutant sad2-5 was susceptible. Transcriptomic analysis was then performed on Col-0, OESAD2/Col-0, and sad2-5 leaves at 0, 1, 2, and 3 days post-inoculation with Pst DC3000. A total of 1825 differentially expressed genes (DEGs) were identified as putative biotic stress defense genes regulated by SAD2, 45 of which overlapped between the SAD2 knockout and overexpression datasets. Gene Ontology (GO) analysis indicated that the DEGs were broadly involved in single-organism cellular metabolic processes and in response to stimulatory stress. Kyoto Encyclopedia of Genes and Genomes (KEGG) biochemical pathway analysis revealed that many of the DEGs were associated with the biosynthesis of flavonoids and other specialized metabolites. Transcription factor analysis showed that a large number of ERF/AP2, MYB, and bHLH transcription factors were involved in SAD2-mediated plant disease resistance. These results provide a basis for future exploration of the molecular mechanisms associated with SAD2-mediated disease resistance and establish a set of key candidate disease resistance genes.

Cold Exposure Memory Reduces Pathogen Susceptibility in Arabidopsis Based on a Functional Plastid Peroxidase System.

Chloroplasts serve as cold priming hubs modulating the transcriptional response of Arabidopsis thaliana to a second cold stimulus for several days by postcold accumulation of thylakoid ascorbate peroxidases (tAPX). In an attempt to investigate cross-priming effects of cold on plant pathogen protection, we show here that such a single 24-h cold treatment at 4°C decreased the susceptibility of Arabidopsis to virulent Pseudomonas syringae pv. tomato DC3000 but did not alter resistance against the avirulent P. syringae pv. tomato avRPM1 and P. syringae pv. tomato avrRPS4 strains or the effector-deficient P. syringae pv. tomato strain hrcC-. The effect of cold priming against P. syringae pv. tomato was active immediately after cold exposure and memorized for at least 5 days. The priming benefit was established independent of the immune regulator Enhanced Disease Susceptibility 1 (EDS1) or activation of the immune-related genes NHL10, FRK1, ICS1 and PR1 but required thylakoid-bound as well as stromal ascorbate peroxidase activities because the effect was absent or weak in corresponding knock-out-lines. Suppression of tAPX postcold regulation in a conditional-inducible tAPX-RNAi line led to increased bacterial growth numbers. This highlights that the plant immune system benefits from postcold regeneration of the protective chloroplast peroxidase system.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Pseudomonas syringae pv. actinidiae Effector HopAU1 Interacts with Calcium-Sensing Receptor to Activate Plant Immunity.

Kiwifruit canker, caused by Pseudomonas syringae pv. actinidiae (Psa), is a destructive pathogen that globally threatens the kiwifruit industry. Understanding the molecular mechanism of plant-pathogen interaction can accelerate applying resistance breeding and controlling plant diseases. All known effectors secreted by pathogens play an important role in plant-pathogen interaction. However, the effectors in Psa and their function mechanism remain largely unclear. Here, we successfully identified a T3SS effector HopAU1 which had no virulence contribution to Psa, but could, however, induce cell death and activate a series of immune responses by agroinfiltration in Nicotiana benthamiana, including elevated transcripts of immune-related genes, accumulation of reactive oxygen species (ROS), and callose deposition. We found that HopAU1 interacted with a calcium sensing receptor in N. benthamiana (NbCaS) as well as its close homologue in kiwifruit (AcCaS). More importantly, silencing CaS by RNAi in N. benthamiana greatly attenuated HopAU1-triggered cell death, suggesting CaS is a crucial component for HopAU1 detection. Further researches showed that overexpression of NbCaS in N. benthamiana significantly enhanced plant resistance against Sclerotinia sclerotiorum and Phytophthora capsici, indicating that CaS serves as a promising resistance-related gene for disease resistance breeding. We concluded that HopAU1 is an immune elicitor that targets CaS to trigger plant immunity.

Arabidopsis inositol polyphosphate kinases IPK1 and ITPK1 modulate crosstalk between SA-dependent immunity and phosphate-starvation responses.

KEY MESSAGE: Selective Arabidopsis thaliana inositol phosphate kinase functions modulate response amplitudes in innate immunity by balancing signalling adjustments with phosphate homeostasis networks. Pyrophosphorylation of InsP6 generates InsP7 and/or InsP8 containing high-energy phosphoanhydride bonds that are harnessed during energy requirements of a cell. As bona fide co-factors for several phytohormone networks, InsP7/InsP8 modulate key developmental processes. With requirements in transducing jasmonic acid (JA) and phosphate-starvation responses (PSR), InsP8 exemplifies a versatile metabolite for crosstalks between different cellular pathways during diverse stress exposures. Here we show that Arabidopsis thaliana INOSITOL PENTAKISPHOSPHATE 2-KINASE 1 (IPK1), INOSITOL 1,3,4-TRISPHOSPHATE 5/6-KINASE 1 (ITPK1), and DIPHOSPHOINOSITOL PENTAKISPHOSPHATE KINASE 2 (VIH2) implicated in InsP8 biosynthesis, suppress salicylic acid (SA)-dependent immunity. In ipk1, itpk1 or vih2 mutants, constitutive activation of defenses lead to enhanced resistance against the Pseudomonas syringae pv tomato DC3000 (PstDC3000) strain. Our data reveal that upregulated SA-signaling sectors potentiate increased expression of several phosphate-starvation inducible (PSI)-genes, previously known in these mutants. In reciprocation, upregulated PSI-genes moderate expression amplitudes of defense-associated markers. We demonstrate that SA is induced in phosphate-deprived plants, however its defense-promoting functions are likely diverted to PSR-supportive roles. Overall, our investigations reveal selective InsPs as crosstalk mediators in defense-phosphate homeostasis and in reprogramming stress-appropriate response intensities.

Bacterial type III effector-induced plant C8 volatiles elicit antibacterial immunity in heterospecific neighbouring plants via airborne signalling.

Upon sensing attack by pathogens and insect herbivores, plants release complex mixtures of volatile compounds. Here, we show that the infection of lima bean (Phaseolus lunatus L.) plants with the non-host bacterial pathogen Pseudomonas syringae pv. tomato led to the production of microbe-induced plant volatiles (MIPVs). Surprisingly, the bacterial type III secretion system, which injects effector proteins directly into the plant cytosol to subvert host functions, was found to prime both intra- and inter-specific defense responses in neighbouring wild tobacco (Nicotiana benthamiana) plants. Screening of each of 16 effectors using the Pseudomonas fluorescens effector-to-host analyser revealed that an effector, HopP1, was responsible for immune activation in receiver tobacco plants. Further study demonstrated that 1-octen-3-ol, 3-octanone and 3-octanol are novel MIPVs emitted by the lima bean plant in a HopP1-dependent manner. Exposure to synthetic 1-octen-3-ol activated immunity in tobacco plants against a virulent pathogen Pseudomonas syringae pv. tabaci. Our results show for the first time that a bacterial type III effector can trigger the emission of C8 plant volatiles that mediate defense priming via plant-plant interactions. These results provide novel insights into the role of airborne chemicals in bacterial pathogen-induced inter-specific plant-plant interactions.

Pseudomonas syringae effector HopZ3 suppresses the bacterial AvrPto1-tomato PTO immune complex via acetylation.

The plant pathogen Pseudomonas syringae secretes multiple effectors that modulate plant defenses. Some effectors trigger defenses due to specific recognition by plant immune complexes, whereas others can suppress the resulting immune responses. The HopZ3 effector of P. syringae pv. syringae B728a (PsyB728a) is an acetyltransferase that modifies not only components of plant immune complexes, but also the Psy effectors that activate these complexes. In Arabidopsis, HopZ3 acetylates the host RPM1 complex and the Psy effectors AvrRpm1 and AvrB3. This study focuses on the role of HopZ3 during tomato infection. In Psy-resistant tomato, the main immune complex includes PRF and PTO, a RIPK-family kinase that recognizes the AvrPto effector. HopZ3 acts as a virulence factor on tomato by suppressing AvrPto1Psy-triggered immunity. HopZ3 acetylates AvrPto1Psy and the host proteins PTO, SlRIPK and SlRIN4s. Biochemical reconstruction and site-directed mutagenesis experiments suggest that acetylation acts in multiple ways to suppress immune signaling in tomato. First, acetylation disrupts the critical AvrPto1Psy-PTO interaction needed to initiate the immune response. Unmodified residues at the binding interface of both proteins and at other residues needed for binding are acetylated. Second, acetylation occurs at residues important for AvrPto1Psy function but not for binding to PTO. Finally, acetylation reduces specific phosphorylations needed for promoting the immune-inducing activity of HopZ3's targets such as AvrPto1Psy and PTO. In some cases, acetylation competes with phosphorylation. HopZ3-mediated acetylation suppresses the kinase activity of SlRIPK and the phosphorylation of its SlRIN4 substrate previously implicated in PTO-signaling. Thus, HopZ3 disrupts the functions of multiple immune components and the effectors that trigger them, leading to increased susceptibility to infection. Finally, mass spectrometry used to map specific acetylated residues confirmed HopZ3's unusual capacity to modify histidine in addition to serine, threonine and lysine residues.

The small molecule Zaractin activates ZAR1-mediated immunity in Arabidopsis.

Pathogenic effector proteins use a variety of enzymatic activities to manipulate host cellular proteins and favor the infection process. However, these perturbations can be sensed by nucleotide-binding leucine-rich-repeat (NLR) proteins to activate effector-triggered immunity (ETI). Here we have identified a small molecule (Zaractin) that mimics the immune eliciting activity of the Pseudomonas syringae type III secreted effector (T3SE) HopF1r and show that both HopF1r and Zaractin activate the same NLR-mediated immune pathway in Arabidopsis Our results demonstrate that the ETI-inducing action of pathogenic effectors can be harnessed to identify synthetic activators of the eukaryotic immune system.

Enhanced Resistance of atbzip62 against Pseudomonas syringae pv. tomato Suggests Negative Regulation of Plant Basal Defense and Systemic Acquired Resistance by AtbZIP62 Transcription Factor.

The intrinsic defense mechanisms of plants toward pathogenic bacteria have been widely investigated for years and are still at the center of interest in plant biosciences research. This study investigated the role of the AtbZIP62 gene encoding a transcription factor (TF) in the basal defense and systemic acquired resistance in Arabidopsis using the reverse genetics approach. To achieve that, the atbzip62 mutant line (lacking the AtbZIP62 gene) was challenged with Pseudomonas syringae pv. tomato (Pst DC3000) inoculated by infiltration into Arabidopsis leaves at the rosette stage. The results indicated that atbzip62 plants showed an enhanced resistance phenotype toward Pst DC3000 vir over time compared to Col-0 and the susceptible disease controls, atgsnor1-3 and atsid2. In addition, the transcript accumulation of pathogenesis-related genes, AtPR1 and AtPR2, increased significantly in atbzip62 over time (0-72 h post-inoculation, hpi) compared to that of atgsnor1-3 and atsid2 (susceptible lines), with AtPR1 prevailing over AtPR2. When coupled with the recorded pathogen growth (expressed as a colony-forming unit, CFU mL-1), the induction of PR genes, associated with the salicylic acid (SA) defense signaling, in part explained the observed enhanced resistance of atbzip62 mutant plants in response to Pst DC3000 vir. Furthermore, when Pst DC3000 avrB was inoculated, the expression of AtPR1 was upregulated in the systemic leaves of Col-0, while that of AtPR2 remained at a basal level in Col-0. Moreover, the expression of AtAZI (a systemic acquired resistance -related) gene was significantly upregulated at all time points (0-24 h post-inoculation, hpi) in atbzip62 compared to Col-0 and atgsnor1-3 and atsid2. Under the same conditions, AtG3DPH exhibited a high transcript accumulation level 48 hpi in the atbzip62 background. Therefore, all data put together suggest that AtPR1 and AtPR2 coupled with AtAZI and AtG3DPH, with AtAZI prevailing over AtG3DPH, would contribute to the recorded enhanced resistance phenotype of the atbzip62 mutant line against Pst DC3000. Thus, the AtbZIP62 TF is proposed as a negative regulator of basal defense and systemic acquired resistance in plants under Pst DC3000 infection.

The Fusarium graminearum FGSG_03624 Xylanase Enhances Plant Immunity and Increases Resistance against Bacterial and Fungal Pathogens.

Fungal enzymes degrading the plant cell wall, such as xylanases, can activate plant immune responses. The Fusarium graminearum FGSG_03624 xylanase, previously shown to elicit necrosis and hydrogen peroxide accumulation in wheat, was investigated for its ability to induce disease resistance. To this aim, we transiently and constitutively expressed an enzymatically inactive form of FGSG_03624 in tobacco and Arabidopsis, respectively. The plants were challenged with Pseudomonas syringae pv. tabaci or pv. maculicola and Botrytis cinerea. Symptom reduction by the bacterium was evident, while no reduction was observed after B. cinerea inoculation. Compared to the control, the presence of the xylanase gene in transgenic Arabidopsis plants did not alter the basal expression of a set of defense-related genes, and, after the P. syringae inoculation, a prolonged PR1 expression was detected. F. graminearum inoculation experiments of durum wheat spikes exogenously treated with the FGSG_03624 xylanase highlighted a reduction of symptoms in the early phases of infection and a lower fungal biomass accumulation than in the control. Besides, callose deposition was detected in infected spikes previously treated with the xylanase and not in infected control plants. In conclusion, our results highlight the ability of FGSG_03624 to enhance plant immunity, thus decreasing disease severity.

Identification of chemoreceptor proteins for amino acids involved in host plant infection in Pseudomonas syringae pv. tabaci 6605.

Chemotaxis is crucial for Pseudomonas syringae pv. tabaci (Pta) 6605 to evoke disease in tobacco plants. Pta6605 harbors more than fifty genes for methyl-accepting chemotaxis proteins (mcp), but almost all are functionally uncharacterized. Previously we identified a dCache_1 type MCP in Pta6605 that mediates chemotaxis to γ-aminobutyric acid, called McpG. In this study, we characterized four more dCache_1 type MCPs, three of which, PscA, PscB, and PscC2, are responsible for sensing amino acids. Using a capillary chemotaxis assay, we observed that PscA, PscB, and PscC2 mutant strains had reduced chemotaxis to most amino acids, indicating that PscA and PscB mediate chemotaxis to 14 amino acids, while PscC2 has a slightly narrower ligand recognition, mediating chemotaxis to 12 amino acids. Other cellular functions were also affected in ΔpscB and ΔpscC2: swarming motility was reduced, and biofilm formation was increased. Furthermore, ΔpscB and ΔpscC2 but not ΔpscA had reduced virulence in the host tobacco plant. On the other hand, ΔpscC1 was defective in motility and did not even respond to yeast extract and was unable to cause disease. These findings supported the idea that the chemosensory pathway correlated with virulence-related phenotypes. Amino acids are abundant in tobacco apoplast; having multiple MCPs appears to support the invasion of Pta6605 into the plant.

RIN4 homologs from important crop species differentially regulate the Arabidopsis NB-LRR immune receptor, RPS2.

KEY MESSAGE: RIN4 homologs from important crop species differ in their ability to prevent ectopic activity of the nucleotide binding-leucine rich repeat resistance protein, RPS2. Pathogens deploy virulence effectors to perturb host processes. Plants utilize intracellular resistance (R) proteins to recognize pathogen effectors either by direct interaction or indirectly via effector-mediated perturbations of host components. RPM1-INTERACTING PROTEIN4 (RIN4) is a plant immune regulator that mediates the indirect activation of multiple, independently evolved R-proteins by multiple, unrelated effector proteins. One of these, RPS2 (RESISTANT TO P. SYRINGAE2), is activated upon cleavage of Arabidopsis (At)RIN4 by the Pseudomonas syringae effector AvrRpt2. To gain insight into the AvrRpt2-RIN4-RPS2 defense-activation module, we compared the function of AtRIN4 with RIN4 homologs present in a diverse range of plant species. We selected seven homologs containing conserved features of AtRIN4, including two NOI (Nitrate induced) domains, each containing a predicted cleavage site for AvrRpt2, and a C-terminal palmitoylation site predicted to mediate membrane tethering of the proteins. Palmitoylation-mediated tethering of AtRIN4 to the plasma membrane and cleavage by AvrRpt2 are required for suppression and activation of RPS2, respectively. While all seven homologs are localized at the plasma membrane, only four suppress RPS2 when transiently expressed in Nicotiana benthamiana. All seven homologs are cleaved by AvrRpt2 and, for those homologs that are able to suppress RPS2, cleavage relieves suppression of RPS2. Further, we demonstrate that the membrane-tethered, C-terminal AvrRpt2-generated cleavage fragment is sufficient for the suppression of RPS2. Lastly, we show that the membrane localization of RPS2 is unaffected by its suppression or activation status.

Genome-wide identification and analysis of Catharanthus roseus RLK1-like kinases in Nicotiana benthamiana.

BACKGROUND: The Catharanthus roseus RLK1-like kinase (CrRLK1L) is a subfamily of the RLK gene family, and members are sensors of cell wall integrity and regulators of cell polarity growth. Recent studies have also shown that members of this subfamily are involved in plant immunity. Nicotiana benthamiana is a model plant widely used in the study of plant-pathogen interactions. However, the members of the NbCrRLK1L subfamily and their response to pathogens have not been reported. RESULTS: In this study, a total of 31 CrRLK1L members were identified in the N. benthamiana genome, and these can be divided into 6 phylogenetic groups (I-VI). The members in each group have similar exon-intron structures and conserved motifs. NbCrRLK1Ls were predicted to be regulated by cis-acting elements such as STRE, TCA, ABRE, etc., and to be the target of transcription factors such as Dof and MYB. The expression profiles of the 16 selected NbCrRLK1Ls were determined by quantitative PCR. Most NbCrRLK1Ls were highly expressed in leaves but there were different and diverse expression patterns in other tissues. Inoculation with the bacterium Pseudomonas syringae or with Turnip mosaic virus significantly altered the transcript levels of the tested genes, suggesting that NbCrRLK1Ls may be involved in the response to pathogens. CONCLUSIONS: This study systematically identified the CrRLK1L members in N. benthamiana, and analyzed their tissue-specific expression and gene expression profiles in response to different pathogens and two pathogens associated molecular patterns (PAMPs). This research lays the foundation for exploring the function of NbCrRLK1Ls in plant-microbe interactions.

Altered metabolomic states elicited by Flg22 and FlgII-28 in Solanum lycopersicum: intracellular perturbations and metabolite defenses.

BACKGROUND: Surveillance of potential pathogens is a key feature of plant innate immunity. For non-self-recognition plants rely on the perception of pathogen-derived molecules. Early post-perception events activate signaling cascades, leading to the synthesis of defense-related proteins and specialized metabolites, thereby providing a broad-spectrum antimicrobial coverage. This study was concerned with tracking changes in the tomato plant metabolome following perception of the flagellum-derived elicitors (Flg22 and FlgII-28). RESULTS: Following an untargeted metabolomics workflow, the metabolic profiles of a Solanum lycopersicum cultivar were monitored over a time range of 16-32 h post-treatment. Liquid chromatography was used to resolve the complex mixture of metabolites and mass spectrometry for the detection of differences associated with the elicitor treatments. Stringent data processing and multivariate statistical tools were applied to the complex dataset to extract relevant metabolite features associated with the elicitor treatments. Following perception of Flg22 and FlgII-28, both elicitors triggered an oxidative burst, albeit with different kinetic responses. Signatory biomarkers were annotated from diverse metabolite classes which included amino acid derivatives, lipid species, steroidal glycoalkaloids, hydroxybenzoic acids, hydroxycinnamic acids and derivatives, as well as flavonoids. CONCLUSIONS: An untargeted metabolomics approach adequately captured the subtle and nuanced perturbations associated with elicitor-linked plant defense responses. The shared and unique features characterizing the metabolite profiles suggest a divergence of signal transduction events following perception of Flg22 vs. FlgII-28, leading to a differential reorganization of downstream metabolic pathways.

Bacterial rhamnolipids and their 3-hydroxyalkanoate precursors activate Arabidopsis innate immunity through two independent mechanisms.

Plant innate immunity is activated upon perception of invasion pattern molecules by plant cell-surface immune receptors. Several bacteria of the genera Pseudomonas and Burkholderia produce rhamnolipids (RLs) from l-rhamnose and (R)-3-hydroxyalkanoate precursors (HAAs). RL and HAA secretion is required to modulate bacterial surface motility, biofilm development, and thus successful colonization of hosts. Here, we show that the lipidic secretome from the opportunistic pathogen Pseudomonas aeruginosa, mainly comprising RLs and HAAs, stimulates Arabidopsis immunity. We demonstrate that HAAs are sensed by the bulb-type lectin receptor kinase LIPOOLIGOSACCHARIDE-SPECIFIC REDUCED ELICITATION/S-DOMAIN-1-29 (LORE/SD1-29), which also mediates medium-chain 3-hydroxy fatty acid (mc-3-OH-FA) perception, in the plant Arabidopsis thaliana HAA sensing induces canonical immune signaling and local resistance to plant pathogenic Pseudomonas infection. By contrast, RLs trigger an atypical immune response and resistance to Pseudomonas infection independent of LORE. Thus, the glycosyl moieties of RLs, although abolishing sensing by LORE, do not impair their ability to trigger plant defense. Moreover, our results show that the immune response triggered by RLs is affected by the sphingolipid composition of the plasma membrane. In conclusion, RLs and their precursors released by bacteria can both be perceived by plants but through distinct mechanisms.

alfaNET: A Database of Alfalfa-Bacterial Stem Blight Protein-Protein Interactions Revealing the Molecular Features of the Disease-causing Bacteria.

Alfalfa has emerged as one of the most important forage crops, owing to its wide adaptation and high biomass production worldwide. In the last decade, the emergence of bacterial stem blight (caused by Pseudomonas syringae pv. syringae ALF3) in alfalfa has caused around 50% yield losses in the United States. Studies are being conducted to decipher the roles of the key genes and pathways regulating the disease, but due to the sparse knowledge about the infection mechanisms of Pseudomonas, the development of resistant cultivars is hampered. The database alfaNET is an attempt to assist researchers by providing comprehensive Pseudomonas proteome annotations, as well as a host-pathogen interactome tool, which predicts the interactions between host and pathogen based on orthology. alfaNET is a user-friendly and efficient tool and includes other features such as subcellular localization annotations of pathogen proteins, gene ontology (GO) annotations, network visualization, and effector protein prediction. Users can also browse and search the database using particular keywords or proteins with a specific length. Additionally, the BLAST search tool enables the user to perform a homology sequence search against the alfalfa and Pseudomonas proteomes. With the successful implementation of these attributes, alfaNET will be a beneficial resource to the research community engaged in implementing molecular strategies to mitigate the disease. alfaNET is freely available for public use at http://bioinfo.usu.edu/alfanet/.

Direct Antibiotic Activity of Bacillibactin Broadens the Biocontrol Range of Bacillus amyloliquefaciens MBI600.

Bacillus amyloliquefaciens is considered the most successful biological control agent due to its ability to colonize the plant rhizosphere and phyllosphere where it outgrows plant pathogens by competition, antibiosis, and inducing plant defense. Its antimicrobial function is thought to depend on a diverse spectrum of secondary metabolites, including peptides, cyclic lipopeptides, and polyketides, which have been shown to target mostly fungal pathogens. In this study, we isolated and characterized the catecholate siderophore bacillibactin by B. amyloliquefaciens MBI600 under iron-limiting conditions and we further identified its potential antibiotic activity against plant pathogens. Our data show that bacillibactin production restrained in vitro and in planta growth of the nonsusceptible (to MBI600) pathogen Pseudomonas syringae pv. tomato. Notably, it was also related to increased antifungal activity of MBI600. In addition to bacillibactin biosynthesis, iron starvation led to upregulation of specific genes involved in microbial fitness and competition. IMPORTANCE Siderophores have mostly been studied concerning their contribution to the fitness and virulence of bacterial pathogens. In the present work, we isolated and characterized for the first time the siderophore bacillibactin from a commercial bacterial biocontrol agent. We proved that its presence in the culture broth has significant biocontrol activity against nonsusceptible bacterial and fungal phytopathogens. In addition, we suggest that its activity is due to a new mechanism of action, that of direct antibiosis, rather than by competition through iron scavenging. Furthermore, we showed that bacillibactin biosynthesis is coregulated with the transcription of antimicrobial metabolite synthases and fitness regulatory genes that maximize competition capability. Finally, this work highlights that the efficiency and range of existing bacterial biocontrol agents can be improved and broadened via the rational modification of the growth conditions of biocontrol organisms.

The truncated TNL receptor TN2-mediated immune responses require ADR1 function.

The loss of function of exocyst subunit EXO70B1 leads to autoimmunity, which is dependent on TIR-NBS2 (TN2), a truncated intracellular nucleotide-binding and leucine-rich repeat receptor (NLR). However, how TN2 triggers plant immunity and whether typical NLRs are required in TN2-activated resistance remain unclear. Through the CRISPR/Cas9 gene editing system and knockout analysis, we found that the spontaneous cell death and enhanced resistance in exo70B1-3 were independent of the full-length NLR SOC3 and its closest homolog SOC3-LIKE 1 (SOC3-L1). Additionally, knocking out SOC3-L1 or TN2 did not suppress the chilling sensitivity conferred by chilling sensitive 1-2 (chs1-2). The ACTIVATED DISEASE RESISTANCE 1 (ADR1) family and the N REQUIREMENT GENE 1 (NRG1) family have evolved as helper NLRs for many typical NLRs. Through CRISPR/Cas9 gene editing methods, we discovered that the autoimmunity of exo70B1-3 fully relied on ADR1s, but not NRG1s, and ADR1s contributed to the upregulation of TN2 transcript levels in exo70B1-3. Furthermore, overexpression of TN2 also led to ADR1-dependent autoimmune responses. Taken together, our genetic analysis highlights that the truncated TNL protein TN2-triggered immune responses require ADR1s as helper NLRs to activate downstream signaling, revealing the importance and complexity of ADR1s in plant immunity regulation.

Comparative genomic insights into the epidemiology and virulence of plant pathogenic pseudomonads from Turkey.

Pseudomonas is a highly diverse genus that includes species that cause disease in both plants and animals. Recently, pathogenic pseudomonads from the Pseudomonas syringae and Pseudomonas fluorescens species complexes have caused significant outbreaks in several agronomically important crops in Turkey, including tomato, citrus, artichoke and melon. We characterized 169 pathogenic Pseudomonas strains associated with recent outbreaks in Turkey via multilocus sequence analysis and whole-genome sequencing, then used comparative and evolutionary genomics to characterize putative virulence mechanisms. Most of the isolates are closely related to other plant pathogens distributed among the primary phylogroups of P. syringae, although there are significant numbers of P. fluorescens isolates, which is a species better known as a rhizosphere-inhabiting plant-growth promoter. We found that all 39 citrus blast pathogens cluster in P. syringae phylogroup 2, although strains isolated from the same host do not cluster monophyletically, with lemon, mandarin orange and sweet orange isolates all being intermixed throughout the phylogroup. In contrast, 20 tomato pith pathogens are found in two independent lineages: one in the P. syringae secondary phylogroups, and the other from the P. fluorescens species complex. These divergent pith necrosis strains lack characteristic virulence factors like the canonical tripartite type III secretion system, large effector repertoires and the ability to synthesize multiple bacterial phytotoxins, suggesting they have alternative molecular mechanisms to cause disease. These findings highlight the complex nature of host specificity among plant pathogenic pseudomonads.