RESUMO
This paper introduces cotton fibers impregnated with biogenic silver nanoparticles (AgNPs), synthesized from a Fusarium oxysporum fungal filtrate (FF) solution, and open up the possibility for their use in medical environment and agriculture clothing as means to avoid microbial spreading. After thorough AgNPs characterization, regarding their physical, chemical and biochemical properties, Minimum Inhibitory Concentrations (MIC) against some human and orange tree pathogens were determined. We report the strong AgNPs activity against Candida parapsilosis and Xanthomonas axonopodis pv. citri (Xac) that was morphologically characterized, pointing to strong AgNPs effects on microorganisms' membranes. Cotton fibers were then impregnated with AgNPs suspension and these maintained strong antimicrobial activity even after repeated mechanical washing cycles (up to 10). Reported data might point to an application for biogenic AgNPs as potent agrochemicals, as well as, to their application in textiles for antiseptic clothing for medical and agronomic applications.
Assuntos
Antibacterianos/química , Candida/crescimento & desenvolvimento , Nanopartículas Metálicas/química , Prata/química , Têxteis , Xanthomonas axonopodis/crescimento & desenvolvimentoRESUMO
KEY MESSAGE: An RNAseq-based analysis of the cassava plants inoculated with Xam allowed the identification of transcriptional upregulation of genes involved in jasmonate metabolism, phenylpropanoid biosynthesis and putative targets for a TALE. Cassava bacterial blight, a disease caused by the gram-negative bacterium Xanthomonas axonopodis pv. manihotis (Xam), is a major limitation to cassava production worldwide and especially in developing countries. The molecular mechanisms underlying cassava susceptibility to Xam are currently unknown. To identify host genes and pathways leading to plant susceptibility, we analyzed the transcriptomic responses occurring in cassava plants challenged with either the non-pathogenic Xam strain ORST4, or strain ORST4(TALE1 Xam ) which is pathogenic due to the major virulence transcription activator like effector TALE1 Xam . Both strains triggered similar responses, i.e., induction of genes related to photosynthesis and phenylpropanoid biosynthesis, and repression of genes related to jasmonic acid signaling. Finally, to search for TALE1 Xam virulence targets, we scanned the list of cassava genes induced upon inoculation of ORST4(TALE1 Xam ) for candidates harboring a predicted TALE1 Xam effector binding element in their promoter. Among the six genes identified as potential candidate targets of TALE1 Xam a gene coding for a heat shock transcription factor stands out as the best candidate based on their induction in presence of TALE1 Xam and contain a sequence putatively recognized by TALE1 Xam .
Assuntos
Perfilação da Expressão Gênica , Manihot/genética , Doenças das Plantas/genética , Xanthomonas axonopodis/crescimento & desenvolvimento , Álcoois Benzílicos/metabolismo , Análise por Conglomerados , Genes de Plantas/genética , Interações Hospedeiro-Patógeno , Manihot/microbiologia , Análise de Sequência com Séries de Oligonucleotídeos , Fotossíntese/genética , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Análise de Componente Principal , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Virulência , Xanthomonas axonopodis/patogenicidade , Xanthomonas axonopodis/fisiologiaRESUMO
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.
Assuntos
Proteínas de Bactérias/metabolismo , Citrus sinensis/microbiologia , Interações Hospedeiro-Patógeno/fisiologia , Xanthomonas axonopodis/crescimento & desenvolvimento , Xanthomonas axonopodis/fisiologia , Sequência de Aminoácidos , Aderência Bacteriana , Proteínas de Bactérias/química , Biofilmes , Contagem de Colônia Microbiana , Biologia Computacional , Deleção de Genes , Genes Bacterianos/genética , Histidina Quinase , Dados de Sequência Molecular , Movimento/fisiologia , Processos Fotoquímicos , Doenças das Plantas/microbiologia , Folhas de Planta/microbiologia , Polissacarídeos Bacterianos/biossíntese , Proteínas Quinases/metabolismo , Proteínas Recombinantes/metabolismo , Xanthomonas axonopodis/enzimologia , Xanthomonas axonopodis/genéticaRESUMO
Xanthomonas axonopodis pv. citri (Xac) is the causative agent of citrus canker. This bacterium develops a characteristic biofilm on both biotic and abiotic surfaces. To evaluate the participation of the single flagellum of Xac in biofilm formation, mutants in the fliC (flagellin) and the flgE (hook) genes were generated. Swimming motility, assessed on 0.25â% agar plates, was markedly reduced in fliC and flgE mutants. However, the fliC and flgE mutants exhibited a flagellar-independent surface translocation on 0.5â% agar plates. Mutation of either the rpfF or the rpfC gene, which both encode proteins involved in cell-cell signalling mediated by diffusible signal factor (DSF), led to a reduction in both flagellar-dependent and flagellar-independent surface translocation, indicating a regulatory role for DSF in both types of motility. Confocal laser scanning microscopy of biofilms produced in static culture demonstrated that the flagellum is also involved in the formation of mushroom-shaped structures and water channels, and in the dispersion of biofilms. The presence of the flagellum was required for mature biofilm development on lemon leaf surfaces. The absence of flagellin produced a slight reduction in Xac pathogenicity and this reduction was more severe when the complete flagellum structure was absent.
Assuntos
Biofilmes/crescimento & desenvolvimento , Citrus/microbiologia , Flagelos/metabolismo , Doenças das Plantas/microbiologia , Folhas de Planta/microbiologia , Xanthomonas axonopodis/patogenicidade , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Flagelos/fisiologia , Flagelina/genética , Flagelina/metabolismo , Mutação , Xanthomonas axonopodis/crescimento & desenvolvimento , Xanthomonas axonopodis/fisiologiaRESUMO
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 H(2)O(2) concentrations. In addition, Xac exhibited an adaptive response to hydrogen peroxide, displaying higher levels of catalase activity and H(2)O(2) 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 H(2)O(2). 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.
Assuntos
Catalase/metabolismo , Citrus/microbiologia , Xanthomonas axonopodis/enzimologia , Xanthomonas axonopodis/patogenicidade , Adaptação Fisiológica/efeitos dos fármacos , Catalase/biossíntese , Catalase/genética , Meios de Cultura , Farmacorresistência Bacteriana/efeitos dos fármacos , Indução Enzimática/efeitos dos fármacos , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/microbiologia , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Isoenzimas/biossíntese , Isoenzimas/genética , Isoenzimas/metabolismo , Mutação/genética , Virulência/efeitos dos fármacos , Xanthomonas axonopodis/efeitos dos fármacos , Xanthomonas axonopodis/crescimento & desenvolvimentoRESUMO
Xanthomonas axonopodis pv. citri (Xac) causes citrus canker and the completion of the Xac genome sequence has opened up the possibility of investigating basic cellular mechanisms at the genomic level. Copper compounds have been extensively used in agriculture to control plant diseases. The copA and copB genes, identified by annotation of the Xac genome, encode homologues of proteins involved in copper resistance. A gene expression assay by Northern blotting revealed that copA and copB are expressed as a unique transcript specifically induced by copper. Synthesis of the gene products was also induced by copper, reaching a maximum level at 4 h after addition of copper to the culture medium. CopA was a cytosolic protein and CopB was detected in the cytoplasmic membrane. The gene encoding CopA was disrupted by the insertion of a transposon, leading to mutant strains that were unable to grow in culture medium containing copper, even at the lowest CuSO(4) concentration tested (0.25 mM), whereas the wild-type strain was able to grow in the presence of 1 mM copper. Cell suspensions of the wild-type and mutant strains in different copper concentrations were inoculated in lemon leaves to analyse their ability to induce citrus canker symptoms. Cells of mutant strains showed higher sensitivity than the wild-type strain in the presence of copper, i.e. they were not able to induce citrus canker symptoms at high copper concentrations and exhibited a more retarded growth in planta.