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Xanthomonas axonopodis pv. glycines ( Xag ) is a phytopathogenic bacterium causing bacterial pustule disease in soybean. Functions of DNA methyltransferases have been characterized in animal pathogenic bacteria, but are poorly understood in plant pathogens. Here, we report that functions of a putative DNA methyltransferase, EadM, in Xag . An EadM-overexpressing strain, Xag (EadM), was less virulent than the wild-type carrying an empty vector, Xag (EV). Interestingly, the viable cell numbers of Xag (EadM) were much lower (10-fold) than those of Xag (EV) at the same optical density. Comparative proteomic analysis revealed that proteins involved in cell wall/membrane/envelope and iron-transport were more abundant. Based on proteomic analysis we carried out diverse phenotypic assays. Scanning electron microscopy revealed abnormal bacterial envelopes in Xag (EadM). Additionally, Xag (EadM) showed decreased stress tolerance against ciprofloxacin and sorbitol, but enhanced resistance to desiccation. Exopolysaccharide production in Xag (EadM) was also decreased. Production of siderophores, which are iron-chelators, was much higher in Xag (EadM). As in Xag , Escherichia coli expressing EadM showed significantly reduced (1000-fold) viable cell numbers at the same optical density. Thus, EadM is associated with virulence, envelope biogenesis, stress tolerance, exopolysaccharide production, and siderophore production. Our results provide valuable and fundamental information regarding DNA methyltransferase functions and their related cellular mechanisms in plant pathogenic bacteria.

Background: Xanthomonas axonopodis pv. citri (Xac) is an obligate aerobic phytopathogen constantly exposed to hydrogen peroxide produced by normal aerobic respiration and by the plant defense response during plant-pathogen interactions. Four putative catalase genes have been identified in silico in the Xac genome, designated as katE, catB, srpA (monofunctional catalases) and katG (bifunctional catalase). Methodology/Principal Findings: Xac catalase activity was analyzed using native gel electrophoresis and semi-quantitative RT-PCR. We demonstrated that the catalase activity pattern was regulated in different growth stages displaying the highest levels during the stationary phase. KatE was the most active catalase in this phase of growth. At this stage cells were more resistant to hydrogen peroxide as was determined by the analysis of CFU after the exposition to different H2O2 concentrations. In addition, Xac exhibited an adaptive response to hydrogen peroxide, displaying higher levels of catalase activity and H2O2 resistance after treatment with sub-lethal concentrations of the oxidant. In the plant-like medium XVM2 the expression of KatE was strongly induced and in this medium Xac was more resistant to H2O2. A XackatE mutant strain was constructed by insertional mutagenesis. We observed that catalase induction in stationary phase was lost meanwhile the adaptive response to peroxide was maintained in this mutant. Finally, the XackatE strain was assayed in planta during host plant interaction rendering a less aggressive phenotype with a minor canker formation. Conclusions: Our results confirmed that in contrast to other Xanthomonas species, Xac catalase-specific activity is induced during the stationary phase of growth in parallel with the bacterial resistance to peroxide challenge. Moreover, Xac catalases expression pattern is modified in response to any stimuli associated with the plant or the microenvironment it provides. The catalase KatE has been shown to have an important function for the colonization and survival of the bacterium in the citrus plant during the pathogenic process. Our work provides the first genetic evidence to support a monofunctional catalase as a virulence factor in Xac.

Bacterial blight caused by Xanthomonas axonopodis pv. punicae is a major disease of pomegranate. Bacterial blight drastically reduces the yield and quality of fruits, which are critical for pomegranate production. Precise and early diagnosis of bacterial blight is crucial for active surveillance and effective management of the disease. Symptoms based disease diagnostic methods are labor-intensive, time-consuming and may not detect disease on asymptomatic plants. DNA-based disease diagnostics using polymerase chain reaction (PCR) are reliable, precise, accurate and quick. PCR coupled with agarose gel electrophoresis (PCR-AGE), PCR coupled with capillary electrophoresis (PCR-CE) and real-time PCR (qPCR) were applied for the early and accurate diagnosis of bacterial blight in pomegranate. PCR-CE and qPCR were capable of diagnosing bacterial blight 6 to 10 days before symptom appearance, with detection limits of 100 fg and 10 fg of bacterial DNA respectively. However, conventional PCR-AGE detected pathogen at the onset of disease symptoms with a detection limit of 10 pg of bacterial DNA. qPCR detected bacterial blight in orchards that did not show any disease symptoms. Our data demonstrate that qPCR is more sensitive than other PCR methods along with being reliable for early diagnosis.

Key message The soybean Rxp gene, encoding a bHLH transcription factor and an ACT-like domain, has an rxp allele producing a truncated protein that confers resistance to pustule-causing Xanthomonas axonopodis pv. glycines . In soybean, bacterial pustules caused by Xanthomonas axonopodis pv. glycines lead to premature defoliation and decreased yield in warm, wet climates. In the USA, approximately 70 years ago, bacterial pustules were eliminated by introducing a recessive resistance allele, rxp , of the Rxp gene, representing the first example of successful soybean breeding for durable disease resistance in North America. In this study, we isolated this historical Rxp gene from resistant soybean varieties using positional cloning. The 1.06 Mb region where Rxp was reported to reside was narrowed down to an 11.1 kb region containing a single gene, Glyma.17g090500 . The resistance allele, rxp , contains a T insertion. A complementation test of the Rxp allele in resistant plants confirmed the identification of the Rxp gene. The product of the susceptible wild-type allele, Rxp , is presumed to be a basic helix–loop–helix (bHLH) transcription factor with an aspartate kinase, chorismate mutase, and TyrA (ACT)–like domain. This gene was mainly expressed in extended leaves, and its homologs were identified to be distributed in angiosperms. A total of six alleles were obtained: four from spontaneous variation, including the wild-type and three mutant alleles that encoded truncated proteins, and two from ethyl methanesulfonate mutants, including an allele that encoded a truncated protein and a missense allele. By evaluating the resistance of these six alleles, we found that the loss of function of RXP decreased the bacterial pustule lesions. This study provides important insights into the soybean rxp allele, which confers durable resistance to bacterial pustules.

Cassava bacterial blight (CBB), incited by Xanthomonas axonopodis pv. manihotis (Xam), is the most important bacterial disease of cassava, a staple food source for millions of people in developing countries. Here we present a widely applicable strategy for elucidating the virulence components of a pathogen population. We report Illumina-based draft genomes for 65 Xam strains and deduce the phylogenetic relatedness of Xam across the areas where cassava is grown. Using an extensive database of effector proteins from animal and plant pathogens, we identify the effector repertoire for each sequenced strain and use a comparative sequence analysis to deduce the least polymorphic of the conserved effectors. These highly conserved effectors have been maintained over 11 countries, three continents, and 70 y of evolution and as such represent ideal targets for developing resistance strategies.

Understanding the evolutionary history and potential of bacterial pathogens is critical to prevent the emergence of new infectious bacterial diseases. Xanthomonas axonopodis subsp. citri (Xac) (synonym X. citri subsp. citri ), which causes citrus canker, is one of the hardest-fought plant bacterial pathogens in US history. Here, we sequenced 21 Xac strains (14 XacA, 3 XacA* and 4 XacA w ) with different host ranges from North America and Asia and conducted comparative genomic and evolutionary analyses. Our analyses suggest that acquisition of beneficial genes and loss of detrimental genes most likely allowed XacA to infect a broader range of hosts as compared with XacA w and XacA*. Recombination was found to have occurred frequently on the relative ancient branches, but rarely on the young branches of the clonal genealogy. The ratio of recombination/mutation ρ/θ was 0.0790±0.0005, implying that the Xac population was clonal in structure. Positive selection has affected 14% (395 out of 2822) of core genes of the citrus canker-causing Xanthomonas . The genes affected are enriched in ‘carbohydrate transport and metabolism’ and ‘DNA replication, recombination and repair’ genes ( P <0.05). Many genes related to virulence, especially genes involved in the type III secretion system and effectors, are affected by positive selection, further highlighting the contribution of positive selection to the evolution of citrus canker-causing Xanthomonas . Our results suggest that both metabolism and virulence genes provide advantages to endow XacA with higher virulence and a wider host range. Our analysis advances our understanding of the genomic basis of specialization by positive selection in bacterial evolution.

The nuclear factor Y (NF-Y) transcription factor complex is important in plant growth, development and stress response. Information regarding this transcription factor complex is limited in cassava (Manihot esculenta). In this study, 15 MeNF-YAs, 21 MeNF-YBs and 15 MeNF-YCs were comprehensively characterized during plant defence. Gene expression in MeNF-Ys was examined during interaction with the bacterial pathogen Xanthomonas axonopodis pv. manihotis (Xam). The yeast two-hybrid system was employed to investigate protein-protein interactions in the heterotrimeric NF-Y transcription factor complex. The in vivo roles of MeNF-Ys were revealed by virus-induced gene silencing (VIGS) in cassava. The regulation of MeNF-Ys in response to Xam indicated their possible roles in response to cassava bacterial blight. Protein-protein interaction assays identified the heterotrimeric NF-Y transcription factor complex (MeNF-YA1/3, MeNF-YB11/16 and MeNF-YC11/12). Moreover, the members of the heterotrimeric NF-Y transcription factor complex were located in the cell nucleus and conferred transcriptional activation activity to the CCAAT motif. Notably, the heterotrimeric NF-Y transcription factor complex positively regulated plant disease resistance to Xam, confirmed by a disease phenotype in overexpressing plants in Nicotiana benthamiana and VIGS in cassava. Consistently, the heterotrimeric NF-Y transcription factor complex positively regulated the expression of pathogenesis-related genes (MePRs). The NF-Y transcription factor complex (MeNF-YA1/3, MeNF-YB11/16 and MeNF-YC11/12) characterized here was shown to play a role in transcriptional activation of MePR promoters, contributing to the plant defence response in cassava.

Xanthomonas axonopodis pv. citri (Xac) belongs to the Gram-negative species, causing citrus canker that seriously affects the fruit yield and quality of many rutaceae plants. Herein, we found that compound 2-(butyldisulfanyl) quinazolin-4(3H)-one exhibited remarkable anti-Xac activity in vitro with a half effective concentration (EC50) of 2.6 μg/mL, while the positive controls thiodiazole-copper with 57 μg/mL and bismerthiazol with 68 μg/mL and this compound showed great anti-citrus canker activity in vivo. This active compound also was confirmed to reduce biofilm formation, increase the level of reactive oxygen species, damage the morphological structure of the bacteria, and cause bacterial death. Proteomics and RT-qPCR analysis results indicated that this compound down-regulated the expression of enzymes in the MEP (2-methyl-D-erythritol 4-phosphate) pathway and might achieve destructive ability of Xac. Overall, this study indicates that such derivatives could be a promising scaffold to develop novel bactericides to control citrus canker.

Recent studies have demonstrated that an appropriate light environment is required for the establishment of efficient vegetal resistance responses in several plant-pathogen interactions. The photoreceptors implicated in such responses are mainly those belonging to the phytochrome family. Data obtained from bacterial genome sequences revealed the presence of photosensory proteins of the BLUF (Blue Light sensing Using FAD), LOV (Light, Oxygen, Voltage) and phytochrome families with no known functions. Xanthomonas axonopodis pv. citri is a Gram-negative bacterium responsible for citrus canker. The in silico analysis of the X. axonopodis pv. citri genome sequence revealed the presence of a gene encoding a putative LOV photoreceptor, in addition to two genes encoding BLUF proteins. This suggests that blue light sensing could play a role in X. axonopodis pv. citri physiology. We obtained the recombinant Xac-LOV protein by expression in Escherichia coli and performed a spectroscopic analysis of the purified protein, which demonstrated that it has a canonical LOV photochemistry. We also constructed a mutant strain of X. axonopodis pv. citri lacking the LOV protein and found that the loss of this protein altered bacterial motility, exopolysaccharide production and biofilm formation. Moreover, we observed that the adhesion of the mutant strain to abiotic and biotic surfaces was significantly diminished compared to the wild-type. Finally, inoculation of orange (Citrus sinensis) leaves with the mutant strain of X. axonopodis pv. citri resulted in marked differences in the development of symptoms in plant tissues relative to the wild-type, suggesting a role for the Xac-LOV protein in the pathogenic process. Altogether, these results suggest the novel involvement of a photosensory system in the regulation of physiological attributes of a phytopathogenic bacterium. A functional blue light receptor in Xanthomonas spp. has been described for the first time, showing an important role in virulence during citrus canker disease.

Background R genes are a key component of genetic interactions between plants and biotrophic bacteria and are known to regulate resistance against bacterial invasion. The most common R proteins contain a nucleotide-binding site and a leucine-rich repeat (NBS-LRR) domain. Some NBS-LRR genes in the soybean genome have also been reported to function in disease resistance. In this study, the number of NBS-LRR genes was found to correlate with the number of disease resistance quantitative trait loci (QTL) that flank these genes in each chromosome. NBS-LRR genes co-localized with disease resistance QTL. The study also addressed the functional redundancy of disease resistance on recently duplicated regions that harbor NBS-LRR genes and NBS-LRR gene expression in the bacterial leaf pustule (BLP)-induced soybean transcriptome. Results A total of 319 genes were determined to be putative NBS-LRR genes in the soybean genome. The number of NBS-LRR genes on each chromosome was highly correlated with the number of disease resistance QTL in the 2-Mb flanking regions of NBS-LRR genes. In addition, the recently duplicated regions contained duplicated NBS-LRR genes and duplicated disease resistance QTL, and possessed either an uneven or even number of NBS-LRR genes on each side. The significant difference in NBS-LRR gene expression between a resistant near-isogenic line (NIL) and a susceptible NIL after inoculation of Xanthomonas axonopodis pv. glycines supports the conjecture that NBS-LRR genes have disease resistance functions in the soybean genome. Conclusions The number of NBS-LRR genes and disease resistance QTL in the 2-Mb flanking regions of each chromosome was significantly correlated, and several recently duplicated regions that contain NBS-LRR genes harbored disease resistance QTL for both sides. In addition, NBS-LRR gene expression was significantly different between the BLP-resistant NIL and the BLP-susceptible NIL in response to bacterial infection. From these observations, NBS-LRR genes are suggested to contribute to disease resistance in soybean. Moreover, we propose models for how NBS-LRR genes were duplicated, and apply Ks values for each NBS-LRR gene cluster.