The phytopathogen Xanthomonas campestris senses and effluxes salicylic acid via a sensor HepR and an RND family efflux pump to promote virulence in host plants

Salicylic acid(SA)plays an essential role in plant defense against biotrophic and semi-biotrophic pathogens.Following pathogen recognition,SA biosynthesis dramatically increases at the infection site of the host plant.The manner in which pathogens sense and tolerate the onslaught of SA stress to survive in the plant following infection remains to be understood.The objective of this work was to determine how the model phytopathogen Xanthomonas campestris pv.campestris(Xcc)senses and effluxes SA during infection inside host plants.First,RNA-Seq analysis identified an SA-responsive operon Xcc4167-Xcc4171,encoding a MarR family transcription factor HepR and an RND(resistance-nodulation-cell division)family efflux pump HepABCD in Xcc.Electrophoretic mobility shift assays and DNase I footprint analysis revealed that HepR neg-atively regulated hepABCD expression by specifically binding to an AT-rich region of the promoter of the hepRABCD operon,Phep.Second,isothermal titration calorimetry and further genetic analysis suggest that HepR is a novel SA sensor.SA binding released HepR from its cognate promoter Pnep and then induced the expression of hepABCD.Third,the RND family efflux pump HepABCD was responsible for SA efflux.The hepRABCD cluster was also involved in the regulation of culture pH and quorum sensing signal diffusible signaling factor turnover.Finally,the hepRABCD cluster was transcribed during the XC1 infection of Chinese radish and was required for the full virulence of Xcc in Chinese radish and cabbage.These findings suggest that the ability of Xcc to co-opt the plant defense signal SA to activate the multidrug efflux pump may have evolved to ensure Xcc survival and virulence in susceptible host plants.

Using Moringa oleifera Extracts to Control Blackrot （Xanthomonas campestris）in Field Grown Rape （Brassica napus）

The leaf, bark and seed extracts ofMoringa oleifera were evaluated for their efficacy under field conditions in suppressing Xanthomonas campestris pv. campestris in rape （Brassica napus. L.）. Xanthomonas campestris pv. campestris is an important bacterial pathogen of agricultural importance causing devastating black rot disease of Brassicas. Three extracts concentrations of 60, 100 and 140% were sprayed as foliar applications weekly and the antibacterial activity was evaluated by recording number of totally defoliated plants. The three extracts showed significant effect against the test pathogen （p 〉 0.05）. The antibacterial activity of seed extract demonstrated higher activity against the Xanthomonas campestris pv. campestris as evidenced by lower mean leaf defoliation of 1.59 cm followed by bark （2.58 cm） and lastly leaf extracts （2.96 cm） （p 〈 0.05）. There were no significant differences based on the concentration levels used. Observations revealed that 100% and 140% levels were not significantly different from each other on enhancing growth of the stem diameter. Moringa seed at 60% concentration level can be used to enhance growth of rape. The conclusion is that Moringa seed extracts can be effectively implemented to suppress Xanthomonas campestris pv. campestris pathogen in field grown rape in an integrated disease control program.

Isolation and Characterization of a Phytotoxin from Xanthomonas campestris pv. retroflexus

A phytotoxin from Xanthomonas campestris pv. retroflexus was isolated using a chromatographer and HPLC, and the components were identified to be a mixture of minor molecular compounds including organic acids and cyclo-（proline-phenylalanine）. The greenhouse cultivation test was used to determine the influence of the isolated fractions on the growth of target weed redroot pigweed （Amaranthus retroflexus L）. The experimental results demonstrated that the cyclo-（Pro-Phe） had the weed inhibit activity obviously on dicotyledonous weed and the mixture with six organic acids showed stronger bioactivity. Further, greenhouse and field test were processed, and the test showed that the use of the toxin appeared to have the potential to be developed further as a bioherbicide system to control weedy grasses.

iTRAQ proteomics reveals the regulatory response to Xanthomonas campestris pv.vesicatoria in resistant vs.susceptible pepper genotypes

Bacterial spot(BS)is a severe bacterial disease induced by Xanthomonas campestris pv.vesicatoria(Xcv),a pathogen that causes serious damage to pepper growth and yield.It is therefore important to study the mechanisms of pepper resistance to Xcv and to breed and promote Xcvresistant pepper varieties.However,studies of the responses to Xcv infection in peppers at the protein level are limited.Here,we examined Xcv-induced proteomic changes in leaves of the BS susceptible bell pepper ECW and the resistant bell pepper VI037601 using the isobaric tags for relative and absolute quantitation(iTRAQ)-based protein labeling technology.A total of 6,120 distinct proteins were identified,and there were 1,289 significantly differentially accumulated proteins(DAPs)in ECW and VI037601 leaves after Xcv inoculation.Among these,339(250up-and 89 down-regulated)and 479(300 up-and 179 down-regulated)DAPs were specifically identified in ECW and VI037601,respectively,with 459(364 up-and 95 down-regulated)similarly expressed DAPs being shared by ECW and VI037601.Based on bioinformatics analysis,many defense-associated proteins were identified as up-regulated in ECW and VI037601,especially the proteins involved in plant-pathogen interaction,phenylpropanoid biosynthesis,protein processing in the endoplasmic reticulum,and MAPK signaling pathway-plant.Moreover,we evaluated transcript levels of six differentially expressed genes from the iTRAQ results by q RT-PCR.The analysis revealed transcriptional changes that were consistent with the changes at the protein level.This study will provide a valuable resource for understanding the molecular basis of pepper resistance to Xcv infection and for improving the disease resistance of pepper cultivars.

Characterization of Chinese Isolates of Mango Bacterial Canker Xanthomonas campestris pv. mangiferaeindicae

Mango bacterial canker is caused by Xanthomonas campestris pv. mangiferaeindicae. During 2009 and 2013,leaves,twigs and fruits of mango were collected from commercial and experimental mango fields with typical canker symptoms in Hainan,Guangxi,Guangdong and Szechwan Provinces of China. The causal agent was identified as X. campestris pv. mangiferaeindicae through KC semi-selective medium isolation,pathogenicity tests,and sequencing of the gyrB gene.

PCR-based Assay for the Detection of Xanthomonas campestris pv. mangiferaeindicae Causing Bacterial Black Spot in Mango

[Objective] This study aimed to develop a PCR assay for detecting Xanthomonas campestris pv. mangiferaeindicae(Xcm) in culture and in planta. [Method] Primers(Xcm HF and Xcm HR) were designed based on the partial sequence of hrp B gene from xanthomonads to develop a PCR assay for Xcm. Furthermore, specificity and sensitivity of the primer pairs were analyzed in detection of genomic DNA and cell from Xcm. [Result] Amplication was positive only with genomic DNA from positive control ATCC11637 and 12 Xcm strains; no PCR products were amplified with genomic DNA from ten other xanthomonads and seven other bacterial species. The sensitivity of detection was 2.4 pg/μl genomic DNA, and 1.8 × 104CFU/ml cells. The primers also worked well for pathogen detection in direct PCR assays of Xcm colonies grown on liquid medium and in PCR assays of total DNA from leaf, branch and fruit lesions. [Conclusion] A PCR assay was successfully established for rapid detection of Xcm in culture and in planta.

Genetic and Proteomic Analyses of a Xanthomonas campestris pv.campestris purC Mutant Deficient in Purine Biosynthesis and Virulence

Bacterial proliferation in hosts requires activation of a number of housekeeping pathways, including purine de novo biosynthesis. Although inactivation of purine biosynthesis genes can attenuate virulence, it is unclear which biochemical or virulence factors are associated with the purine biosynthesis pathway in vivo. We report that inactivation of purC, a gene encoding phosphoribosylaminoimidazole-succinocarboxamide synthase, caused complete loss of virulence in Xanthomonas campestris pv. cam- pestris, the causal agent of black rot disease of cruciferous plants. The purC mutant was a purine auxotroph; it could not grow on minimal medium, whereas addition of purine derivatives, such as hypoxanthine or adenine plus guanine, restored growth of the mutant. The purC mutation also significantly enhanced the production of an unknown purine synthesis associated pigment and extracellular polysaccharides by the bacterium. In addition, comparative proteomic analyses of bacteria grown on rich and minimal media revealed that the purC mutation affected the expression levels of diverse proteins involved in purine and pyrimidine synthesis, carbon and energy metabolisms, iron uptake, proteolysis, protein secretion, and signal transduction. These results provided clues to understanding the contributions of purine synthesis to bacterial virulence and interactions with host immune systems.

Investigation of the Metabolic Flux Analysis Method for Overdetermined System

With the xanthan synthesis in Xanthomoaas campestris as an example, two methods for metabolic flux analysis of overdetermined system, the experimental data error minimization method and the equation error minimization method, are compared from their mathematical basis, rationality of the results and the easiness of computation. The results show that the experimental data error minimization method is appropriate in metabolic flux analysis of overdetermined system.

New insights on molecular regulation of biofilm formation in plant-associated bacteria

Biofilms are complex bacterial assemblages with a defined three-dimensional architecture, attached to solid surfaces, and surrounded by a self-produced matrix generally composed of exopolysaccharides, proteins, lipids and extracellular DNA. Biofilm formation has evolved as an adaptive strategy of bacteria to cope with harsh environmental conditions as well as to establish antagonistic or beneficial interactions with their host. Plant-associated bacteria attach and form biofilms on different tissues including leaves, stems,vasculature, seeds and roots. In this review, we examine the formation of biofilms from the plant-associated bacterial perspective and detail the recently-described mechanisms of genetic regulation used by these organisms to orchestrate biofilm formation on plant surfaces. In addition, we describe plant host signals that bacterial pathogens recognize to activate the transition from a planktonic lifestyle to multicellular behavior.