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Phenylalanine ammonia-lyase (PAL) has a crucial role in secondary phenylpropanoid metabolism and is one of the most extensively studied enzymes with respect to plant responses to biotic and abiotic stress. Here, we identified the pepper (Capsicum annuum) PAL (CaPAL1) gene, which was induced in pepper leaves by avirulent Xanthomonas campestris pv. vesicatoria (Xcv) infection. CaPAL1-silenced pepper plants exhibited increased susceptibility to virulent and avirulent Xcv infection. Reactive oxygen species (ROS), hypersensitive cell death, expression of the salicylic acid (SA)-dependent marker gene CaPR1, SA accumulation, and induction of PAL activity were significantly compromised in the CaPAL1-silenced pepper plants during Xcv infection. Overexpression (OX) of CaPAL1 in Arabidopsis conferred increased resistance to Pseudomonas syringae pv. tomato (Pst) and Hyaloperonospora arabidopsidis infection. CaPAL1-OX leaves exhibited restricted Pst growth, increased ROS burst and cell death, and induction of PR1 expression and SA accumulation. The increase in PAL activity in healthy and Pst-infected leaves was higher in CaPAL1-OX plants than in wild-type Arabidopsis. Taken together, these results suggest that CaPAL1 acts as a positive regulator of SA-dependent defence signalling to combat microbial pathogens via its enzymatic activity in the phenylpropanoid pathway.

Bacterial type VI secretion systems (T6SSs) are molecular weapons designed to deliver toxic effectors into prey cells. These nanomachines have an important role in inter-bacterial competition and provide advantages to T6SS active strains in polymicrobial environments. Here we analyze the genome of the biocontrol agent Pseudomonas putida KT2440 and identify three T6SS gene clusters (K1-, K2- and K3-T6SS). Besides, 10 T6SS effector–immunity pairs were found, including putative nucleases and pore-forming colicins. We show that the K1-T6SS is a potent antibacterial device, which secretes a toxic Rhs-type effector Tke2. Remarkably, P. putida eradicates a broad range of bacteria in a K1-T6SS-dependent manner, including resilient phytopathogens, which demonstrates that the T6SS is instrumental to empower P. putida to fight against competitors. Furthermore, we observed a drastically reduced necrosis on the leaves of Nicotiana benthamiana during co-infection with P. putida and Xanthomonas campestris . Such protection is dependent on the activity of the P. putida T6SS. Many routes have been explored to develop biocontrol agents capable of manipulating the microbial composition of the rhizosphere and phyllosphere. Here we unveil a novel mechanism for plant biocontrol, which needs to be considered for the selection of plant wardens whose mission is to prevent phytopathogen infections.

As well as their importance to nutrition, fatty acids (FA) represent a unique group of quorum sensing chemicals that modulate the behavior of bacterial population in virulence. However, the way in which full-length, membrane-bound receptors biochemically detect FA remains unclear. Here, we provide genetic, enzymological and biophysical evidences to demonstrate that in the phytopathogenic bacterium Xanthomonas campestris pv. campestris, a medium-chain FA diffusible signal factor (DSF) binds directly to the N-terminal, 22 amino acid-length sensor region of a receptor histidine kinase (HK), RpfC. The binding event remarkably activates RpfC autokinase activity by causing an allosteric change associated with the dimerization and histidine phosphotransfer (DHp) and catalytic ATP-binding (CA) domains. Six residues were found essential for sensing DSF, especially those located in the region adjoining to the inner membrane of cells. Disrupting direct DSF-RpfC interaction caused deficiency in bacterial virulence and biofilm development. In addition, two amino acids within the juxtamembrane domain of RpfC, Leu172 and Ala178, are involved in the autoinhibition of the RpfC kinase activity. Replacements of them caused constitutive activation of RpfC-mediated signaling regardless of DSF stimulation. Therefore, our results revealed a biochemical mechanism whereby FA activates bacterial HK in an allosteric manner, which will assist in future studies on the specificity of FA-HK recognition during bacterial virulence regulation and cell-cell communication.

The genus Xanthomonas is a diverse and economically important group of bacterial phytopathogens, belonging to the gamma-subdivision of the Proteobacteria. Xanthomonas axonopodis pv. citri (Xac) causes citrus canker, which affects most commercial citrus cultivars, resulting in significant losses worldwide. Symptoms include canker lesions, leading to abscission of fruit and leaves and general tree decline. Xanthomonas campestris pv. campestris (Xcc) causes black rot, which affects crucifers such as Brassica and Arabidopsis. Symptoms include marginal leaf chlorosis and darkening of vascular tissue, accompanied by extensive wilting and necrosis. Xanthomonas campestris pv. campestris is grown commercially to produce the exopolysaccharide xanthan gum, which is used as a viscosifying and stabilizing agent in many industries. Here we report and compare the complete genome sequences of Xac and Xcc. Their distinct disease phenotypes and host ranges belie a high degree of similarity at the genomic level. More than 80% of genes are shared, and gene order is conserved along most of their respective chromosomes. We identified several groups of strain-specific genes, and on the basis of these groups we propose mechanisms that may explain the differing host specificities and pathogenic processes.

It has long been known that phytopathogenic bacteria react to plant-specific stimuli or environmental factors. However, how bacterial cells sense these environmental cues remains incompletely studied. Recently, three kinds of histidine kinases (HKs) were identified as receptors to perceive plant-associated or quorum-sensing signals. Among these kinases, HK VgrS detects iron depletion by binding to ferric iron via an ExxE motif, RpfC binds diffusible signal factor (DSF) by its N-terminal peptide and activates its autokinase activity through relaxation of autoinhibition, and PcrK specifically senses plant hormone–cytokinin and elicits bacterial responses to oxidative stress. These HKs are critical sensors that regulate the virulence of a Gram-negative bacterium, Xanthomonas campestris pv. campestris . Research progress on the signal perception of phytopathogenic bacterial HKs suggests that inter-kingdom signalling between host plants and pathogens controls pathogenesis and can be used as a potential molecular target to protect plants from bacterial diseases. This article is part of the theme issue ‘Biotic signalling sheds light on smart pest management’.

Black rot, caused by Xanthomonas campestris pv. campestris ( Xcc ) significantly affects the production of cabbage and other cruciferous vegetables. Plant antioxidant system plays an important role in pathogen invasion and is one of the main mechanisms underlying resistance to biological stress. Therefore, it is important to study the resistance mechanisms of the cabbage antioxidant system during the early stages of Xcc . In this study, 10 8 CFU/mL (OD 600  = 0.1) Xcc race1 was inoculated on “zhonggan 11” cabbage using the spraying method. The effects of Xcc infection on the antioxidant system before and after Xcc inoculation (0, 1, 3, and 5 d) were studied by physiological indexes determination, transcriptome and metabolome analyses. We concluded that early Xcc infection can destroy the balance of the active oxygen metabolism system, increase the generation of free radicals, and decrease the scavenging ability, leading to membrane lipid peroxidation, resulting in the destruction of the biofilm system and metabolic disorders. In response to Xcc infection, cabbage clears a series of reactive oxygen species (ROS) produced during Xcc infection via various antioxidant pathways. The activities of antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) increased after Xcc infection, and the ROS scavenging rate increased. The biosynthesis of non-obligate antioxidants, such as ascorbic acid (AsA) and glutathione (GSH), is also enhanced after Xcc infection. Moreover, the alkaloid and vitamin contents increased significantly after Xcc infection. We concluded that cabbage could resist Xcc invasion by maintaining the stability of the cell membrane system and improving the biosynthesis of antioxidant substances and enzymes after infection by Xcc . Our results provide theoretical basis and data support for subsequent research on the cruciferous vegetables resistance mechanism and breeding to Xcc .

Plant‐pathogenic bacteria colonise their hosts using various strategies, exploiting both natural openings and wounds in leaves and roots. The vascular pathogen Xanthomonas campestris pv. campestris (Xcc) enters its host through hydathodes, organs at the leaf margin involved in guttation. Subsequently, Xcc breaches the hydathode–xylem barrier and progresses into the xylem vessels causing systemic disease. To elucidate the mechanisms that underpin the different stages of an Xcc infection, a need exists to image bacterial progression in planta in a non‐invasive manner. Here, we describe a phenotyping setup and Python image analysis pipeline for capturing 16 independent Xcc infections in Arabidopsis thaliana plants in parallel over time. The setup combines an RGB camera for imaging disease symptoms and an ultrasensitive CCD camera for monitoring bacterial progression inside leaves using bioluminescence. The method reliably quantified bacterial growth in planta for two bacterial species, that is, vascular Xcc and the mesophyll pathogen Pseudomonas syringae pv. tomato (Pst). The camera resolution allowed Xcc imaging already in the hydathodes, yielding reproducible data for the first stages prior to the systemic infection. Data obtained through the image analysis pipeline was robust and validated findings from other bioluminescence imaging methods, while requiring fewer samples. Moreover, bioluminescence was reliably detected within 5 min, offering a significant time advantage over our previously reported method with light‐sensitive films. Thus, this method is suitable to quantify the resistance level of a large number of Arabidopsis thaliana accessions and mutant lines to different bacterial strains in a non‐invasive manner for phenotypic screenings. Development of a non‐invasive high‐throughput phenotyping pipeline to visualise and quantify progression of bioluminescent bacterial pathogens in Arabidopsis rosettes over time.

Background Xanthomonas campestris pv. campestris ( Xcc ) is a Gram-negative bacterium that can cause black rot disease in crucifers. The lipoprotein outer membrane localization (Lol) system is involved in the lipoprotein sorting to the outer membrane. Although Xcc has a set of annotated lol genes, there is still little known about the physiological role in this phytopathogen. In this study, we aimed to characterize the role of LolB of Xcc in bacterial attachment, stress tolerance, and virulence. Results To characterize the role of LolB, lolB mutant was constructed and phenotypic evaluation was performed. The lolB mutant revealed reductions in bacterial attachment, extracellular enzyme production, and virulence. Mutation of lolB also resulted in reduced tolerance to a myriad of stresses, including heat and a range of membrane-perturbing agents. Trans-complementation of lolB mutant with intact lolB gene reverted these altered phenotypes to the wild-type levels. From subsequent reporter assay and reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) analysis, the expression of genes that encode the major extracellular enzymes and the stress-related proteins was reduced after lolB mutation. Conclusions The results in this work contribute to the functional understanding of lolB in Xanthomonas for the first time, and provide new insights into the function of lolB in bacteria.

Most Gram-negative phytopathogenic bacteria inject type III effector (T3E) proteins into plant cells to manipulate signaling pathways to the pathogen’s benefit. In resistant plants, specialized immune receptors recognize single T3Es or their biochemical activities, thus halting pathogen ingress. However, molecular function and mode of recognition for most T3Es remains elusive. Here, we show that the Xanthomonas T3E XopH possesses phytase activity, i.e., dephosphorylates phytate ( myo -inositol- hexakis phosphate, InsP 6 ), the major phosphate storage compound in plants, which is also involved in pathogen defense. A combination of biochemical approaches, including a new NMR-based method to discriminate inositol polyphosphate enantiomers, identifies XopH as a naturally occurring 1-phytase that dephosphorylates InsP 6 at C1. Infection of Nicotiana benthamiana and pepper by Xanthomonas results in a XopH-dependent conversion of InsP 6 to InsP 5. 1-phytase activity is required for XopH-mediated immunity of plants carrying the Bs7 resistance gene, and for induction of jasmonate- and ethylene-responsive genes in N. benthamiana . Plant pathogens translocate type III effector (T3E) proteins that may be recognized by plants to trigger immunity. Here, the authors show that the Xanthomonas T3E XopH possesses a novel 1-phytase activity that is required for XopH-mediated immunity of plants carrying the Bs7 resistance gene.

RpfG is a paradigm for a class of widespread bacterial two-component regulators with a CheY-like receiver domain attached to a histidine-aspartic acid-glycine-tyrosine-proline (HD-GYP) cyclic di-GMP phosphodiesterase domain. In the plant pathogen Xanthomonas campestris pv. campestris (Xcc), a two-component system comprising RpfG and the complex sensor kinase RpfC is implicated in sensing and responding to the diffusible signaling factor (DSF), which is essential for cell—cell signaling. RpfF is involved in synthesizing DSF, and mutations of rpfF, rpfG, or rpfC lead to a coordinate reduction in the synthesis of virulence factors such as extracellular enzymes, biofilm structure, and motility. Using yeast two-hybrid analysis and fluorescence resonance energy transfer experiments in Xcc, we show that the physical interaction of RpfG with two proteins with diguanylate cyclase (GGDEF) domains controls a subset of RpfG-regulated virulence functions. RpfG interactions were abolished by alanine substitutions of the three residues of the conserved GYP motif in the HD-GYP domain. Changing the GYP motif or deletion of the two GGDEF-domain proteins reduced Xcc motility but not the synthesis of extracellular enzymes or bio-film formation. RpfG-GGDEF interactions are dynamic and depend on DSF signaling, being reduced in the rpfF mutant but restored by DSF addition. The results are consistent with a model in which DSF signal transduction controlling motility depends on a highly regulated, dynamic interaction of proteins that influence the localized expression of cyclic di-GMP.