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Curr Microbiol ; 76(7): 879-887, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31089795


The goal of this study was to elucidate the role of the outer membrane protein A (ompA) gene of Xanthomonas axonopodis pv. glycines in bacterial pustule pathogenesis of soybean. An ompA mutant of X. axonopodis pv. glycines KU-P-SW005 was shown to significantly decrease cellulase, pectate lyase, and polysaccharide production. The production of these proteins in the ompA mutant was approximately five times lower than that of the wildtype. The ompA mutant also exhibited modified biofilm development. More importantly, the mutant reduced disease severity to the soybean. Ten days after inoculation, the virulence rating of the susceptible soybean cv. SJ4 inoculated with the ompA mutant was 11.23%, compared with 87.98% for the complemented ompA mutant. Production of cellulase, pectate lyase, polysaccharide was restored, biofilm, and pustule numbers were restored in the complemented ompA mutant that did not differ from the wild type. Taken together, these data suggest that OmpA-mediated invasion plays an important role in protein secretion during pathogenesis to soybean.

Proteínas da Membrana Bacteriana Externa/metabolismo , Doenças das Plantas/microbiologia , Soja/microbiologia , Xanthomonas axonopodis/genética , Xanthomonas axonopodis/patogenicidade , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Celulase/metabolismo , Teste de Complementação Genética , Mutação , Folhas de Planta/microbiologia , Polissacarídeo-Liase/metabolismo , Polissacarídeos Bacterianos/metabolismo , Virulência/genética
BMC Res Notes ; 8: 155, 2015 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-25880211


BACKGROUND: The Gram-negative xylem-limited bacterium, Xylella fastidiosa, is an important plant pathogen that infects a number of high value crops. The Temecula 1 strain infects grapevines and induces Pierce's disease, which causes symptoms such as scorching on leaves, cluster collapse, and eventual plant death. In order to understand the pathogenesis of X. fastidiosa, researchers routinely perform gene deletion studies and select mutants via antibiotic markers. METHODS: Site-directed pilJ mutant of X. fastidiosa were generated and selected on antibiotic media. Mutant cultures were assessed by PCR to determine if they were composed of purely transformant cells or included mixtures of non-transformants cells. Then pure pilJ mutant and wildtype cells were mixed in PD2 medium and following incubation and exposure to kanamycin were assessed by PCR for presence of mutant and wildtype populations. RESULTS: We have discovered that when creating clones of targeted mutants of X. fastidiosa Temecula 1 with selection on antibiotic plates, X. fastidiosa lacking the gene deletion often persist in association with targeted mutant cells. We believe this phenomenon is due to spontaneous antibiotic resistance and/or X. fastidiosa characteristically forming aggregates that can be comprised of transformed and non-transformed cells. A combined population was confirmed by PCR, which showed that targeted mutant clones were mixed with non-transformed cells. After repeated transfer and storage the non-transformed cells became the dominant clone present. CONCLUSIONS: We have discovered that special precautions are warranted when developing a targeted gene mutation in X. fastidiosa because colonies that arise following transformation and selection are often comprised of transformed and non-transformed cells. Following transfer and storage the cells can consist primarily of the non-transformed strain. As a result, careful monitoring of targeted mutant strains must be performed to avoid mixed populations and confounding results.

Deleção de Genes , Genes Bacterianos/genética , Doenças das Plantas/microbiologia , Vitis/microbiologia , Xylella/genética , Mutação/genética , Xylella/patogenicidade
PLoS One ; 10(3): e0121851, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25811864


Xylella fastidiosa is an important phytopathogenic bacterium that causes many serious plant diseases including Pierce's disease of grapevines. X. fastidiosa is thought to induce disease by colonizing and clogging xylem vessels through the formation of cell aggregates and bacterial biofilms. Here we examine the role in X. fastidiosa virulence of an uncharacterized gene, PD1671, annotated as a two-component response regulator with potential GGDEF and EAL domains. GGDEF domains are found in c-di-GMP diguanylate cyclases while EAL domains are found in phosphodiesterases, and these domains are for c-di-GMP production and turnover, respectively. Functional analysis of the PD1671 gene revealed that it affected multiple X. fastidiosa virulence-related phenotypes. A Tn5 PD1671 mutant had a hypervirulent phenotype in grapevines presumably due to enhanced expression of gum genes leading to increased exopolysaccharide levels that resulted in elevated biofilm formation. Interestingly, the PD1671 mutant also had decreased motility in vitro but did not show a reduced distribution in grapevines following inoculation. Given these responses, the putative PD1671 protein may be a negative regulator of X. fastidiosa virulence.

Proteínas de Bactérias/genética , Doenças das Plantas/microbiologia , Domínios e Motivos de Interação entre Proteínas/genética , Xylella/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Biofilmes , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Espaço Extracelular/enzimologia , Espaço Extracelular/metabolismo , Regulação Bacteriana da Expressão Gênica , Dados de Sequência Molecular , Mutação , Polímeros/metabolismo , Alinhamento de Sequência , Virulência/genética , Xylella/metabolismo , Xylella/patogenicidade
Mol Plant Microbe Interact ; 24(10): 1198-206, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21692637


Xylella fastidiosa is an important phytopathogenic bacterium that causes many serious plant diseases, including Pierce's disease of grapevines. Disease manifestation by X. fastidiosa is associated with the expression of several factors, including the type IV pili that are required for twitching motility. We provide evidence that an operon, named Pil-Chp, with genes homologous to those found in chemotaxis systems, regulates twitching motility. Transposon insertion into the pilL gene of the operon resulted in loss of twitching motility (pilL is homologous to cheA genes encoding kinases). The X. fastidiosa mutant maintained the type IV pili, indicating that the disrupted pilL or downstream operon genes are involved in pili function, and not biogenesis. The mutated X. fastidiosa produced less biofilm than wild-type cells, indicating that the operon contributes to biofilm formation. Finally, in planta the mutant produced delayed and less severe disease, indicating that the Pil-Chp operon contributes to the virulence of X. fastidiosa, presumably through its role in twitching motility.

Doenças das Plantas/microbiologia , Xylella/genética , Xylella/patogenicidade , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Sequência de Bases , Biofilmes/crescimento & desenvolvimento , DNA Bacteriano/genética , Fímbrias Bacterianas/genética , Fímbrias Bacterianas/fisiologia , Fímbrias Bacterianas/ultraestrutura , Genes Bacterianos , Interações Hospedeiro-Patógeno/genética , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Movimento/fisiologia , Mutação , Óperon , Homologia de Sequência de Aminoácidos , Virulência/genética , Vitis/microbiologia , Xylella/fisiologia , Xylella/ultraestrutura
Phytopathology ; 99(8): 996-1004, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19594319


Three races of Xanthomonas axonopodis pv. glycines were identified on pustule disease resistant and susceptible soybean cultivars based on virulence phenotype. For race 3, an avrBs3 homolog, avrXg1 was identified that conferred resistance expressed as a hypersensitive response on resistant cultivar Williams 82. Mutations in two predicted functional domains of avrXg1 resulted in gained virulence on Williams 82 and an increase in bacterial population number on susceptible cultivars. Expression of avrXg1 in race 1, that is predicted to confer a nonspecific HR, led to virulence on susceptible cultivars Spencer and PI 520733. Expression of avrXg1 in race 2, that is predicted of carrying avrBs3-like genes, resulted in gained virulence and fitness of pathogen on both resistant and susceptible cultivars. The results demonstrate multifunctions for avrXg1 dependent on pathogen and plant genetic backgrounds.

Proteínas de Bactérias/genética , Soja/microbiologia , Xanthomonas/patogenicidade , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Genoma Bacteriano , Dados de Sequência Molecular , Isoformas de Proteínas , Especificidade da Espécie , Virulência