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1.
Microbiol Res ; 240: 126560, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32721820

RESUMO

Xanthomonas axonopodis pv. punicae (Xap), the bacterial blight pathogen of pomegranate, incurs substantial loss to yield and reduces export quality of this economically important fruit crop. During infection, the bacterium secretes six non-TAL (Xop) effectors into the pomegranate cells through a specialized type three secretion system (T3SS). Previously, we demonstrated the role of two key effectors, XopL and XopN in pathogenesis. Here, we investigate the role of rest effectors (XopC2, XopE1, XopQ and XopZ) on disease development. We generated null mutants for each individual effector and mutant bacterial suspension was infiltrated into pomegranate leaves. Compared to Xap wild, the mutant bacterial growth was reduced by 2.7-11.5 folds. The mutants produced lesser water-soaked lesions when infiltrated on leaves by 1.13-2.21 folds. Among the four effectors, XopC2 contributes highest for in planta bacterial growth and disease development. XopC2 efficiently suppressed the defense responses like callose deposition, reactive oxygen species (ROS) and the activation of immune responsive genes. Being a major contributor, we further characterize XopC2 for its subcellular localization, its protein structure and networking. XopC2 is localized to the plasma membrane of Nicotiana benthamiana like XopL and XopN. XopC2 is a 661 amino acids protein having 15 alpha and 17 beta helix. Our STRING and I-TASSER based analysis hinted that XopC2 interacts with multiple membrane localized plant proteins including transcription regulator of CCR4-NOT family, TTN of maintenance of chromosome family and serine/threonine-protein phosphatase 2A (PP2A) isoform. Based on the interaction it is predicted that XopC2 might involve in diverse functions like nuclear-transcribed mRNA catabolic process, maintenance of chromosome, hormone signaling and protein dephosphorylation activities and thereby suppress the plant immunity. Altogether, our study suggests that Xap largely depends on three non-TAL (Xop) effectors, including XopC2, XopL and XopN, to modulate pomegranate PTI for its unrestricted proliferation during bacterial blight development.


Assuntos
Imunidade Vegetal/fisiologia , Romã (Fruta)/imunologia , Romã (Fruta)/microbiologia , Sistemas de Secreção Tipo III/metabolismo , Xanthomonas axonopodis/fisiologia , Membrana Celular/metabolismo , Frutas , Peróxido de Hidrogênio , Desenvolvimento Vegetal , Doenças das Plantas/microbiologia , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Romã (Fruta)/genética , Espécies Reativas de Oxigênio , Tabaco , Xanthomonas axonopodis/genética
2.
Proteomics ; 20(1): e1900125, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31693783

RESUMO

Methyltransferases (MTases) are enzymes that modify specific substrates by adding a methyl group using S-adenosyl-l-methionine. Functions of MTases have been extensively studied in eukaryotic organisms and animal pathogenic bacteria. Despite their importance, mechanisms underlying MTase function in plant pathogenic bacteria have not been studied in depth, as is the case of Xanthomonas axonopodis pv. glycines (Xag) that causes bacterial pustule disease in soybean crops worldwide. Here, the association between Xag proteome alterations and three MTase-overexpressing strains, Xag(XgMT1), Xag(XgMT2), and Xag(XgMT3), compared to Xag carrying an empty vector, Xag(EV) is reported. Using label-free shotgun comparative proteomic analysis, proteins are identified in all three biological replicates of the four strains and ranged from 1004 to 1082. In comparative analyses, 124, 135, and 134 proteins are differentially changed (over twofold) by overexpression of XgMT1, XgMT2, and XgMT3, respectively. These proteins are also categorized using cluster of orthologous group (COG) analyses, allowing postulation of biological mechanisms associated with three MTases in Xag. COGs reveal that the three MTases may play distinct roles, although some functions may overlap. These results are expected to allow new insight into understanding and predicting the biological functions of MTases in plant pathogenic bacteria. Data are available via ProteomeXchange (Identifier PXD012590).


Assuntos
Proteínas de Bactérias/metabolismo , Metiltransferases/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Xanthomonas axonopodis/enzimologia , Isoenzimas/metabolismo , Doenças das Plantas/microbiologia , Soja/microbiologia , Xanthomonas axonopodis/fisiologia
3.
BMC Microbiol ; 19(1): 103, 2019 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-31113370

RESUMO

BACKGROUND: Hfq is a widely conserved bacterial RNA-binding protein which generally mediates the global regulatory activities involv ed in physiological process and virulence. The goal of this study was to characterize the biological function of hfq gene in Xanthomonas axonpodis pv. citri (Xac), the causal agent of citrus canker disease. RESULTS: An hfq mutant in Xac was generated by plasmid integration. The loss of hfq resulted in attenuation of bacterial growth, motility and biofilm formation. In addition, the hfq mutation impaired Xac resistance to H2O2 and both high and low pH environments, but did not affect the virulence to citrus. RNA-Seq analyses indicated that Hfq played roles in regulating the expression of 746 genes. In hfq mutant, gene expression related to chemotaxis, secretion system, two-component system, quorum sensing and flagellar assembly were repressed, whereas expression of ribosomal genes were significantly up-regulated. The down-regulated expression of three bacterial chemotaxis related genes and seven flagella genes, which involved in cell growth and biofilm formation, were further validated by RT-qPCR. CONCLUSIONS: The study demonstrated that hfq was involved in multiple biological processes in Xac. The results could serve as initiate points for identifying regulatory sRNAs and genes controlled by Hfq-sRNA interactions in Xac.


Assuntos
Perfilação da Expressão Gênica/métodos , Fator Proteico 1 do Hospedeiro/genética , Mutação , Xanthomonas axonopodis/fisiologia , Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Doenças das Plantas/microbiologia , Percepção de Quorum , Análise de Sequência de RNA , Xanthomonas axonopodis/crescimento & desenvolvimento
4.
Plant Cell Rep ; 37(6): 887-900, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29523964

RESUMO

KEY MESSAGE: MeCIPK23 interacts with MeCBL1/9, and they confer improved defense response, providing potential genes for further genetic breeding in cassava. Cassava (Manihot esculenta) is an important food crop in tropical area, but its production is largely affected by cassava bacterial blight. However, the information of defense-related genes in cassava is very limited. Calcium ions play essential roles in plant development and stress signaling pathways. Calcineurin B-like proteins (CBLs) and CBL-interacting protein kinases (CIPKs) are crucial components of calcium signals. In this study, systematic expression profile of 25MeCIPKs in response to Xanthomonas axonopodis pv. manihotis (Xam) infection was examined, by which seven candidate MeCIPKs were chosen for functional investigation. Through transient expression in Nicotiana benthamiana leaves, we found that six MeCIPKs (MeCIPK5, MeCIPK8, MeCIPK12, MeCIPK22, MeCIPK23 and MeCIPK24) conferred improved defense response, via regulating the transcripts of several defense-related genes. Notably, we found that MeCIPK23 interacted with MeCBL1 and MeCBL9, and overexpression of these genes conferred improved defense response. On the contrary, virus-induced gene silencing of either MeCIPK23 or MeCBL1/9 or both genes resulted in disease sensitive in cassava. To our knowledge, this is the first study identifying MeCIPK23 as well as MeCBL1 and MeCBL9 that confer enhanced defense response against Xam.


Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Manihot/imunologia , Doenças das Plantas/imunologia , Proteínas de Plantas/metabolismo , Xanthomonas axonopodis/fisiologia , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Manihot/genética , Manihot/microbiologia , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , /metabolismo , Espécies Reativas de Oxigênio/metabolismo , Tabaco/genética , Tabaco/imunologia , Tabaco/microbiologia
5.
Plant Dis ; 102(1): 67-72, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30673446

RESUMO

Several studies have shown that mixtures of plant-growth-promoting rhizobacteria (PGPR) could enhance biological control activity for multiple plant diseases through the mechanisms of induced systemic resistance or antagonism. Prior experiments showed that four individual PGPR strains-AP69 (Bacillus altitudinis), AP197 (B. velezensis), AP199 (B. velezensis), and AP298 (B. velezensis)-had broad-spectrum biocontrol activity via antagonism in growth chambers against two foliar bacterial pathogens (Xanthomonas axonopodis pv. vesicatoria and Pseudomonas syringae pv. tomato) and one of two tested soilborne fungal pathogens (Rhizoctonia solani and Pythium ultimum). Based on these findings, the overall hypothesis of this study was that a mixture of two individual PGPR strains would exhibit better overall biocontrol and plant-growth promotion than the individual PGPR strains. Two separate greenhouse experiments were conducted. In each experiment, two individual PGPR strains and their mixtures were tested for biological control of three different diseases and for plant-growth promotion in the presence of the pathogens. The results demonstrated that the two individual PGPR strains and their mixtures exhibited both biological control of multiple plant diseases and plant-growth promotion. Overall, the levels of disease suppression and growth promotion were greater with mixtures than with individual PGPR strains.


Assuntos
Bacillus/química , Agentes de Controle Biológico/química , Capsicum/microbiologia , Cucumis sativus/microbiologia , Lycopersicon esculentum/microbiologia , Controle Biológico de Vetores , Doenças das Plantas/prevenção & controle , Pseudomonas syringae/fisiologia , Pythium/fisiologia , Rhizoctonia/fisiologia , Xanthomonas axonopodis/fisiologia
6.
J Integr Plant Biol ; 60(4): 341-357, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29193868

RESUMO

Xanthomonas axonopodis pv. punicae (Xap) causing bacterial blight is an important pathogen that incurs significant losses to the exportability of pomegranate. Xap uses the Xop TTSS-effector, via the type three secretion system, to suppress pomegranate immunity. Here, we investigate the role of XopL during blight pathogenesis. We observed that XopL is essential for its in planta growth and full virulence. Leaves inoculated with Xap ΔxopL produced restricted water-soaked lesions compared to those inoculated with wild-type Xap. XopL supports Xap for its sustained multiplication in pomegranate by suppressing the plant cell death (PCD) event. We further demonstrated that XopL suppresses immune responses, such as callose deposition and production of reactive oxygen species (ROS). RT-qPCR analysis revealed that immune responsive genes were upregulated when challenged with Xap ΔxopL, whereas upregulation of such genes was compromised in the complemented strain containing the xopL gene. The transiently expressed XopL::EYFP fusion protein was localized to the plasma membrane, indicating the possible site of its action. Altogether, this study highlights that XopL is an important TTSS-effector of Xap that suppresses plant immune responses, including PCD, presumably to support the multiplication of Xap for a sufficient time-period during blight disease development.


Assuntos
Proteínas de Bactérias/metabolismo , Lythraceae/imunologia , Lythraceae/microbiologia , Imunidade Vegetal , Xanthomonas axonopodis/fisiologia , Apoptose , Membrana Celular/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Peróxido de Hidrogênio/metabolismo , Lythraceae/genética , Lythraceae/crescimento & desenvolvimento , Mutagênese , Mutação/genética , Doenças das Plantas/microbiologia , Folhas de Planta/microbiologia , Plantas Geneticamente Modificadas , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo , Frações Subcelulares/metabolismo , Tabaco/genética , Tabaco/metabolismo
7.
Mol Genet Genomics ; 290(4): 1393-402, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25675870

RESUMO

This genetic diversity study aimed to estimate the population structure and explore the use of association mapping strategies to identify linked markers for bacterial resistance, growth and fruit quality in pomegranate collections from India. In total, 88 accessions including 37 cultivated types were investigated. A total of 112 alleles were amplified by use of 44 publicly available microsatellites for estimating molecular genetic diversity and population structure. Neighbor-joining analysis, model-based population structure and principal component analysis corroborated the genetic relationships among wild-type and cultivated pomegranate collections from India. Our study placed all 88 germplasm into four clusters. We identified a cultivated clade of pomegranates in close proximity to Daru types of wild-type pomegranates that grow naturally near the foothills of the Himalayas. Admixture analysis sorted various lineages of cultivated pomegranates to their respective ancestral forms. We identified four linked markers for fruit weight, titratable acidity and bacterial blight severity. PGCT001 was found associated with both fruit weight and bacterial blight, and the association with fruit weight during both seasons analyzed was significant after Bonferroni correction. This research demonstrates effectiveness of microsatellites to resolve population structure among the wild and cultivar collection of pomegranates and future use for association mapping studies.


Assuntos
Variação Genética , Lythraceae/genética , Repetições de Microssatélites/genética , Doenças das Plantas/genética , Análise de Variância , Mapeamento Cromossômico , Análise por Conglomerados , Resistência à Doença/genética , Fluxo Gênico , Genética Populacional , Interações Hospedeiro-Patógeno , Índia , Lythraceae/classificação , Lythraceae/microbiologia , Filogenia , Doenças das Plantas/microbiologia , Análise de Componente Principal , Estações do Ano , Especificidade da Espécie , Xanthomonas axonopodis/fisiologia
8.
Appl Environ Microbiol ; 80(20): 6266-79, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25085494

RESUMO

Xanthomonas axonopodis pv. citri (Xac) is the causal agent of citrus bacterial canker (CBC) and is a serious problem worldwide. Like CBC, several important diseases in other fruits, such as mango, pomegranate, and grape, are also caused by Xanthomonas pathovars that display remarkable specificity toward their hosts. While citrus and mango diseases were documented more than 100 years ago, the pomegranate and grape diseases have been known only since the 1950s and 1970s, respectively. Interestingly, diseases caused by all these pathovars were noted first in India. Our genome-based phylogenetic studies suggest that these diverse pathogens belong to a single species and these pathovars may be just a group of rapidly evolving strains. Furthermore, the recently reported pathovars, such as those infecting grape and pomegranate, form independent clonal lineages, while the citrus and mango pathovars that have been known for a long time form one clonal lineage. Such an understanding of their phylogenomic relationship has further allowed us to understand major and unique variations in the lineages that give rise to these pathovars. Whole-genome sequencing studies including ecological relatives from their putative country of origin has allowed us to understand the evolutionary history of Xac and other pathovars that infect fruits.


Assuntos
Evolução Biológica , Doenças das Plantas/microbiologia , Xanthomonas axonopodis/genética , Xanthomonas axonopodis/patogenicidade , Anisóis , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Genes Bacterianos , Variação Genética , Genoma Bacteriano , Integrons , Lipopolissacarídeos/genética , Lythraceae/microbiologia , Mangifera/microbiologia , Dados de Sequência Molecular , Filogenia , Vitis/microbiologia , Xanthomonas axonopodis/fisiologia
9.
Plant Cell Rep ; 33(11): 1901-12, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25120000

RESUMO

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/fisiologia
10.
BMC Microbiol ; 13: 186, 2013 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-23924281

RESUMO

BACKGROUND: Xanthomonas axonopodis pv. citri (X. a. pv. citri) causes citrus canker that can result in defoliation and premature fruit drop with significant production losses worldwide. Biofilm formation is an important process in bacterial pathogens and several lines of evidence suggest that in X. a. pv. citri this process is a requirement to achieve maximal virulence since it has a major role in host interactions. In this study, proteomics was used to gain further insights into the functions of biofilms. RESULTS: In order to identify differentially expressed proteins, a comparative proteomic study using 2D difference gel electrophoresis was carried out on X. a. pv. citri mature biofilm and planktonic cells. The biofilm proteome showed major variations in the composition of outer membrane proteins and receptor or transport proteins. Among them, several porins and TonB-dependent receptor were differentially regulated in the biofilm compared to the planktonic cells, indicating that these proteins may serve in maintaining specific membrane-associated functions including signaling and cellular homeostasis. In biofilms, UDP-glucose dehydrogenase with a major role in exopolysaccharide production and the non-fimbrial adhesin YapH involved in adherence were over-expressed, while a polynucleotide phosphorylase that was demonstrated to negatively control biofilm formation in E. coli was down-regulated. In addition, several proteins involved in protein synthesis, folding and stabilization were up-regulated in biofilms. Interestingly, some proteins related to energy production, such as ATP-synthase were down-regulated in biofilms. Moreover, a number of enzymes of the tricarboxylic acid cycle were differentially expressed. In addition, X. a. pv. citri biofilms also showed down-regulation of several antioxidant enzymes. The respective gene expression patterns of several identified proteins in both X. a. pv. citri mature biofilm and planktonic cells were evaluated by quantitative real-time PCR and shown to consistently correlate with those deduced from the proteomic study. CONCLUSIONS: Differentially expressed proteins are enriched in functional categories. Firstly, proteins that are down-regulated in X. a. pv. citri biofilms are enriched for the gene ontology (GO) terms 'generation of precursor metabolites and energy' and secondly, the biofilm proteome mainly changes in 'outer membrane and receptor or transport'. We argue that the differentially expressed proteins have a critical role in maintaining a functional external structure as well as enabling appropriate flow of nutrients and signals specific to the biofilm lifestyle.


Assuntos
Proteínas de Bactérias/química , Biofilmes , Citrus/microbiologia , Doenças das Plantas/microbiologia , Proteômica , Xanthomonas axonopodis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Xanthomonas axonopodis/química , Xanthomonas axonopodis/fisiologia
11.
PLoS One ; 8(4): e62824, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23626857

RESUMO

Citrus bacterial canker caused by Xanthomonas axonopodis pv. citri is a serious disease that impacts citrus production worldwide, and X. axonopodis pv. citri is listed as a quarantine pest in certain countries. Biofilm formation is important for the successful development of a pathogenic relationship between various bacteria and their host(s). To understand the mechanisms of biofilm formation by X. axonopodis pv. citri strain XW19, the strain was subjected to transposon mutagenesis. One mutant with a mutation in a two-component response regulator gene that was deficient in biofilm formation on a polystyrene microplate was selected for further study. The protein was designated as BfdR for biofilm formation defective regulator. BfdR from strain XW19 shares 100% amino acid sequence identity with XAC1284 of X. axonopodis pv. citri strain 306 and 30-100% identity with two-component response regulators in various pathogens and environmental microorganisms. The bfdR mutant strain exhibited significantly decreased biofilm formation on the leaf surfaces of Mexican lime compared with the wild type strain. The bfdR mutant was also compromised in its ability to cause canker lesions. The wild-type phenotype was restored by providing pbfdR in trans in the bfdR mutant. Our data indicated that BfdR did not regulate the production of virulence-related extracellular enzymes including amylase, lipase, protease, and lecithinase or the expression of hrpG, rfbC, and katE; however, BfdR controlled the expression of rpfF in XVM2 medium, which mimics cytoplasmic fluids in planta. In conclusion, biofilm formation on leaf surfaces of citrus is important for canker development in X. axonopodis pv. citri XW19. The process is controlled by the two-component response regulator BfdR via regulation of rpfF, which is required for the biosynthesis of a diffusible signal factor.


Assuntos
Proteínas de Bactérias/genética , Biofilmes , Citocinas/genética , Virulência/genética , Xanthomonas axonopodis/fisiologia , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Citrus/microbiologia , Citocinas/metabolismo , Elementos de DNA Transponíveis , Regulação Bacteriana da Expressão Gênica , Ordem dos Genes , Dados de Sequência Molecular , Mutagênese Insercional , Mutação , Doenças das Plantas/microbiologia , Folhas de Planta/microbiologia , Alinhamento de Sequência , Transcrição Genética , Xanthomonas axonopodis/classificação
12.
Ann Bot ; 111(5): 925-34, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23471007

RESUMO

BACKGROUND AND AIMS: The use of vitamins including vitamin B1, B2 and K3 for the induction of systemic acquired resistance (SAR) to protect crops against plant pathogens has been evaluated previously. The use of vitamins is beneficial because it is cost effective and safe for the environment. The use of folate precursors, including ortho-aminobenzoic acid, to induce SAR against a soft-rot pathogen in tobacco has been reported previously. METHODS: In the present study, para-aminobenzoic acid (PABA, also referred to as vitamin Bx) was selected owing to its effect on the induction of SAR against Xanthomonas axonopodis pv. vesicatoria in pepper plants through greenhouse screening. KEY RESULTS: Dipping of pepper seedlings in a 1 mm PABA solution in field trials induced SAR against artificially infiltrated X. axonopodis pv. vesicatoria and naturally occurring cucumber mosaic virus. Expression of the Capsicum annuum pathogenesis-related 4 gene was primed in response to pathogen infection as assessed by quantitative real-time PCR. The accumulation of cucumber mosaic virus RNA was reduced in PABA-treated pepper plants at 40 and 105 d post-treatment. Unexpectedly, fruit yield was increased in PABA-treated plants, indicating that PABA-mediated SAR successfully protected pepper plants from infection by bacterial and viral pathogens without significant fitness allocation costs. CONCLUSIONS: The present study is the first to demonstrate the effective elicitation of SAR by a folate precursor under field conditions.


Assuntos
Ácido 4-Aminobenzoico/farmacologia , Capsicum/imunologia , Cucumovirus/fisiologia , Resistência à Doença/efeitos dos fármacos , Ácido Fólico/metabolismo , Doenças das Plantas/imunologia , Xanthomonas axonopodis/fisiologia , Ácido 4-Aminobenzoico/química , Capsicum/genética , Capsicum/microbiologia , Capsicum/virologia , Cucumovirus/efeitos dos fármacos , Resistência à Doença/genética , Resistência à Doença/imunologia , Ácido Fólico/química , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Doenças das Plantas/microbiologia , Doenças das Plantas/virologia , Transcrição Genética/efeitos dos fármacos , Xanthomonas axonopodis/efeitos dos fármacos
13.
J Bacteriol ; 194(21): 5922-31, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22942245

RESUMO

Type IV pili (T4P) are polar surface structures that play important roles in bacterial motility, biofilm formation, and pathogenicity. The protein FimX and its orthologs are known to mediate T4P formation in the human pathogen Pseudomonas aeruginosa and some other bacterial species. It was reported recently that FimX(XAC2398) from Xanthomonas axonopodis pv. citri interacts with PilZ(XAC1133) directly through the nonenzymatic EAL domain of FimX(XAC2398). Here we present experimental data to reveal that the strong interaction between FimX(XAC2398) and PilZ(XAC1133) is not conserved in P. aeruginosa and likely other Pseudomonas species. In vitro and in vivo binding experiments showed that the interaction between FimX and PilZ in P. aeruginosa is below the measurable limit. Surface plasmon resonance assays further confirmed that the interaction between the P. aeruginosa proteins is at least more than 3 orders of magnitude weaker than that between the X. axonopodis pv. citri pair. The N-terminal lobe region of FimX(XAC2398) was identified as the binding surface for PilZ(XAC1133) by amide hydrogen-deuterium exchange and site-directed mutagenesis studies. Lack of several key residues in the N-terminal lobe region of the EAL domain of FimX is likely to account for the greatly reduced binding affinity between FimX and PilZ in P. aeruginosa. All together, the results suggest that the interaction between PilZ and FimX in Xanthomonas species is not conserved in P. aeruginosa due to the evolutionary divergence among the FimX orthologs. The precise roles of FimX and PilZ in bacterial motility and T4P biogenesis are likely to vary among bacterial species.


Assuntos
Proteínas de Bactérias/metabolismo , Fímbrias Bacterianas/metabolismo , Mapeamento de Interação de Proteínas , Pseudomonas aeruginosa/fisiologia , Xanthomonas axonopodis/fisiologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Ligação Proteica , Conformação Proteica , Alinhamento de Sequência , Ressonância de Plasmônio de Superfície
14.
PLoS One ; 7(7): e42124, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22848728

RESUMO

Citrus bacterial canker caused by Xanthomonas axonopodis pv. citri is a devastating disease resulting in significant crop losses in various citrus cultivars worldwide. A biocontrol agent has not been recommended for this disease. To explore the potential of bacilli native to Taiwan to control this disease, Bacillus species with a broad spectrum of antagonistic activity against various phytopathogens were isolated from plant potting mixes, organic compost and the rhizosphere soil. Seven strains TKS1-1, OF3-16, SP4-17, HSP1, WG6-14, TLB7-7, and WP8-12 showing superior antagonistic activity were chosen for biopesticide development. The genetic identity based on 16S rDNA sequences indicated that all seven native strains were close relatives of the B. subtilis group and appeared to be discrete from the B. cereus group. DNA polymorphisms in strains WG6-14, SP4-17, TKS1-1, and WP8-12, as revealed by repetitive sequence-based PCR with the BOXA1R primers were similar to each other, but different from those of the respective Bacillus type strains. However, molecular typing of the strains using either tDNA-intergenic spacer regions or 16S-23S intergenic transcribed spacer regions was unable to differentiate the strains at the species level. Strains TKS1-1 and WG6-14 attenuated symptom development of citrus bacterial canker, which was found to be correlated with a reduction in colonization and biofilm formation by X. axonopodis pv. citri on leaf surfaces. The application of a Bacillus strain TKS1-1 endospore formulation to the leaf surfaces of citrus reduced the incidence of citrus bacterial canker and could prevent development of the disease.


Assuntos
Bacillus/fisiologia , Biofilmes/crescimento & desenvolvimento , Citrus/microbiologia , DNA Bacteriano/genética , Doenças das Plantas/microbiologia , Polimorfismo Genético , Xanthomonas axonopodis/fisiologia , Bacillus/classificação , Bacillus/genética , Agentes de Controle Biológico , Análise por Conglomerados , DNA Intergênico/genética , Dados de Sequência Molecular , Filogenia , Folhas de Planta/microbiologia , RNA Ribossômico 16S/genética , Sequências Repetitivas de Ácido Nucleico/genética , Análise de Sequência de RNA , Microbiologia do Solo
15.
Theor Appl Genet ; 125(7): 1525-37, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22798059

RESUMO

Common bacterial blight (CBB), caused by Xanthomonas axonopodis pv. phaseoli (Xap), is a major yield-limiting factor of common bean (Phaseolus vulgaris L.) production around the world. Two major CBB-resistant quantitative trait loci (QTL), linked to the sequence characterized amplified region markers BC420 and SU91, are located at chromosomes 6 and 8, respectively. Using map-based cloning approach, four bacterial artificial chromosome (BAC) clones from the BC420-QTL locus and one BAC clone containing SU91 were sequenced by Roche 454 technique and subsequently assembled using merged assemblies from three different programs. Based on the quality of the assembly, only the sequences of BAC 32H6 and 4K7 were used for candidate gene marker (CGM) development and candidate gene (CG) selection. For the BC420-QTL locus, 21 novel genes were predicted in silico by FGENESH using Medicago gene model, whereas 16 genes were identified in the SU91-QTL locus. For each putative gene, one or more primer pairs were designed and tested in the contrasting near isogenic lines. Overall, six and nine polymorphic markers were found in the SU91- and BC420-QTL loci, respectively. Afterwards, association mapping was conducted in a breeding population of 395 dry bean lines to discover marker-trait associations. Two CGMs per each locus showed better association with CBB resistance than the BC420 and SU91 markers, which include BC420-CG10B and BC420-CG14 for BC420_QTL locus, and SU91-CG10 and SU91-CG11 for SU91_QTL locus. The strong associations between CBB resistance and the CGs 10 and 14 from BC420_QTL locus and the CGs 10 and 11 from SU91_QTL locus indicate that the genes 10 and 14 from the BC420 locus are potential CGs underlying the BC420_QTL locus, whereas the genes 10 and 11 from the SU91 locus are potential CGs underlying the SU91_QTL locus. The superiority of SU91-CG11 was further validated in a recombinant inbred line population Sanilac × OAC 09-3. Thus, co-dominant CGMs, BC420-CG14 and SU91-CG11, are recommended to replace BC420 and SU91 for marker-assisted selection of common bean with resistance to CBB.


Assuntos
Resistência à Doença/genética , Estudos de Associação Genética , Phaseolus/genética , Phaseolus/microbiologia , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Xanthomonas axonopodis/fisiologia , Mapeamento Cromossômico , Cromossomos Artificiais Bacterianos/genética , Cruzamentos Genéticos , Frequência do Gene/genética , Genes de Plantas/genética , Loci Gênicos/genética , Marcadores Genéticos , Testes Genéticos , Endogamia , Modelos Lineares , Anotação de Sequência Molecular , Dados de Sequência Molecular , Fenótipo , Análise de Sequência de DNA
16.
PLoS One ; 7(6): e38226, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22675525

RESUMO

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/genética
17.
J Bacteriol ; 193(22): 6342-57, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21908674

RESUMO

Xanthomonas axonopodis pv. citrumelo is a citrus pathogen causing citrus bacterial spot disease that is geographically restricted within the state of Florida. Illumina, 454 sequencing, and optical mapping were used to obtain a complete genome sequence of X. axonopodis pv. citrumelo strain F1, 4.9 Mb in size. The strain lacks plasmids, in contrast to other citrus Xanthomonas pathogens. Phylogenetic analysis revealed that this pathogen is very close to the tomato bacterial spot pathogen X. campestris pv. vesicatoria 85-10, with a completely different host range. We also compared X. axonopodis pv. citrumelo to the genome of citrus canker pathogen X. axonopodis pv. citri 306. Comparative genomic analysis showed differences in several gene clusters, like those for type III effectors, the type IV secretion system, lipopolysaccharide synthesis, and others. In addition to pthA, effectors such as xopE3, xopAI, and hrpW were absent from X. axonopodis pv. citrumelo while present in X. axonopodis pv. citri. These effectors might be responsible for survival and the low virulence of this pathogen on citrus compared to that of X. axonopodis pv. citri. We also identified unique effectors in X. axonopodis pv. citrumelo that may be related to the different host range as compared to that of X. axonopodis pv. citri. X. axonopodis pv. citrumelo also lacks various genes, such as syrE1, syrE2, and RTX toxin family genes, which were present in X. axonopodis pv. citri. These may be associated with the distinct virulences of X. axonopodis pv. citrumelo and X. axonopodis pv. citri. Comparison of the complete genome sequence of X. axonopodis pv. citrumelo to those of X. axonopodis pv. citri and X. campestris pv. vesicatoria provides valuable insights into the mechanism of bacterial virulence and host specificity.


Assuntos
Citrus/microbiologia , Genômica , Especificidade de Hospedeiro , Doenças das Plantas/microbiologia , Xanthomonas axonopodis/genética , Xanthomonas axonopodis/patogenicidade , Proteínas de Bactérias/genética , Genoma Bacteriano , Dados de Sequência Molecular , Filogenia , Virulência , Xanthomonas/classificação , Xanthomonas/genética , Xanthomonas/patogenicidade , Xanthomonas/fisiologia , Xanthomonas axonopodis/classificação , Xanthomonas axonopodis/fisiologia
18.
PLoS One ; 6(7): e21804, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21750733

RESUMO

Xanthomonas axonopodis pv. citri (Xac) causes citrus canker disease, a major threat to citrus production worldwide. Accumulating evidence suggests that the formation of biofilms on citrus leaves plays an important role in the epiphytic survival of this pathogen prior to the development of canker disease. However, the process of Xac biofilm formation is poorly understood. Here, we report a genome-scale study of Xac biofilm formation in which we identified 92 genes, including 33 novel genes involved in biofilm formation and 7 previously characterized genes, colR, fhaB, fliC, galU, gumD, wxacO, and rbfC, known to be important for Xac biofilm formation. In addition, 52 other genes with defined or putative functions in biofilm formation were identified, even though they had not previously reported been to be associated with biofilm formation. The 92 genes were isolated from 292 biofilm-defective mutants following a screen of a transposon insertion library containing 22,000 Xac strain 306 mutants. Further analyses indicated that 16 of the novel genes are involved in the production of extracellular polysaccharide (EPS) and/or lipopolysaccharide (LPS), 7 genes are involved in signaling and regulatory pathways, and 5 genes have unknown roles in biofilm formation. Furthermore, two novel genes, XAC0482, encoding a haloacid dehalogenase-like phosphatase, and XAC0494 (designated as rbfS), encoding a two-component sensor protein, were confirmed to be biofilm-related genes through complementation assays. Our data demonstrate that the formation of mature biofilm requires EPS, LPS, both flagellum-dependent and flagellum-independent cell motility, secreted proteins and extracellular DNA. Additionally, multiple signaling pathways are involved in Xac biofilm formation. This work is the first report on a genome-wide scale of the genetic processes of biofilm formation in plant pathogenic bacteria. The report provides significant new information about the genetic determinants and regulatory mechanism of biofilm formation.


Assuntos
Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Genoma Bacteriano/genética , Xanthomonas axonopodis/genética , Proteínas de Bactérias/metabolismo , Citrus/microbiologia , Elementos de DNA Transponíveis/genética , Lipopolissacarídeos/biossíntese , Modelos Genéticos , Família Multigênica/genética , Mutagênese , Mutagênese Insercional , Doenças das Plantas/microbiologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Xanthomonas axonopodis/metabolismo , Xanthomonas axonopodis/fisiologia
19.
J Biol Chem ; 286(29): 25628-43, 2011 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-21596742

RESUMO

Xanthomonas axonopodis pv. citri (Xac) causes citrus canker, provoking defoliation and premature fruit drop with concomitant economical damage. In plant pathogenic bacteria, lipopolysaccharides are important virulence factors, and they are being increasingly recognized as major pathogen-associated molecular patterns for plants. In general, three domains are recognized in a lipopolysaccharide: the hydrophobic lipid A, the hydrophilic O-antigen polysaccharide, and the core oligosaccharide, connecting lipid A and O-antigen. In this work, we have determined the structure of purified lipopolysaccharides obtained from Xanthomonas axonopodis pv. citri wild type and a mutant of the O-antigen ABC transporter encoded by the wzt gene. High pH anion exchange chromatography and matrix-assisted laser desorption/ionization mass spectrum analysis were performed, enabling determination of the structure not only of the released oligosaccharides and lipid A moieties but also the intact lipopolysaccharides. The results demonstrate that Xac wild type and Xacwzt LPSs are composed mainly of a penta- or tetra-acylated diglucosamine backbone attached to either two pyrophosphorylethanolamine groups or to one pyrophosphorylethanolamine group and one phosphorylethanolamine group. The core region consists of a branched oligosaccharide formed by Kdo2Hex6GalA3Fuc3NAcRha4 and two phosphate groups. As expected, the presence of a rhamnose homo-oligosaccharide as O-antigen was determined only in the Xac wild type lipopolysaccharide. In addition, we have examined how lipopolysaccharides from Xac function in the pathogenesis process. We analyzed the response of the different lipopolysaccharides during the stomata aperture closure cycle, the callose deposition, the expression of defense-related genes, and reactive oxygen species production in citrus leaves, suggesting a functional role of the O-antigen from Xac lipopolysaccharides in the basal response.


Assuntos
Citrus sinensis/imunologia , Citrus sinensis/microbiologia , Imunidade Inata , Lipopolissacarídeos/química , Lipopolissacarídeos/metabolismo , Xanthomonas axonopodis/fisiologia , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sequência de Carboidratos , Citrus sinensis/anatomia & histologia , Citrus sinensis/genética , Regulação da Expressão Gênica de Plantas/imunologia , Interações Hospedeiro-Patógeno , Imunidade Inata/genética , Lipopolissacarídeos/biossíntese , Lipopolissacarídeos/isolamento & purificação , Dados de Sequência Molecular , Peróxidos/metabolismo , Folhas de Planta/genética , Folhas de Planta/imunologia , Folhas de Planta/microbiologia , Estômatos de Plantas/anatomia & histologia , Estômatos de Plantas/imunologia , Estômatos de Plantas/microbiologia , Xanthomonas axonopodis/metabolismo
20.
BMC Plant Biol ; 11: 55, 2011 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-21439092

RESUMO

BACKGROUND: Enormous work has shown that polyamines are involved in a variety of physiological processes, but information is scarce on the potential of modifying disease response through genetic transformation of a polyamine biosynthetic gene. RESULTS: In the present work, an apple spermidine synthase gene (MdSPDS1) was introduced into sweet orange (Citrus sinensis Osbeck 'Anliucheng') via Agrobacterium-mediated transformation of embryogenic calluses. Two transgenic lines (TG4 and TG9) varied in the transgene expression and cellular endogenous polyamine contents. Pinprick inoculation demonstrated that the transgenic lines were less susceptible to Xanthomonas axonopodis pv. citri (Xac), the causal agent of citrus canker, than the wild type plants (WT). In addition, our data showed that upon Xac attack TG9 had significantly higher free spermine (Spm) and polyamine oxidase (PAO) activity when compared with the WT, concurrent with an apparent hypersensitive response and the accumulation of more H2O2. Pretreatment of TG9 leaves with guazatine acetate, an inhibitor of PAO, repressed PAO activity and reduced H2O2 accumulation, leading to more conspicuous disease symptoms than the controls when both were challenged with Xac. Moreover, mRNA levels of most of the defense-related genes involved in synthesis of pathogenesis-related protein and jasmonic acid were upregulated in TG9 than in the WT regardless of Xac infection. CONCLUSION: Our results demonstrated that overexpression of the MdSPDS1 gene prominently lowered the sensitivity of the transgenic plants to canker. This may be, at least partially, correlated with the generation of more H2O2 due to increased production of polyamines and enhanced PAO-mediated catabolism, triggering hypersensitive response or activation of defense-related genes.


Assuntos
Citrus sinensis/imunologia , Peróxido de Hidrogênio/imunologia , Malus/enzimologia , Doenças das Plantas/imunologia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/imunologia , Espermidina Sintase/genética , Xanthomonas axonopodis/fisiologia , Citrus sinensis/genética , Citrus sinensis/microbiologia , Expressão Gênica , Regulação da Expressão Gênica de Plantas , Malus/genética , Doenças das Plantas/microbiologia , Proteínas de Plantas/imunologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/microbiologia , Espermidina Sintase/imunologia
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