RESUMO
Red/far-red light-sensing bacteriophytochrome photoreceptor (BphP) pathways play key roles in bacterial physiology and ecology. These bilin-binding proteins photoswitch between two states, Pr (red absorbing) and Pfr (far-red absorbing). The isomerization of the chromophore and the downstream structural changes result in the light signal transduction. The agricultural pathogen Xanthomonas campestris pv. campestris (Xcc) code for a single bathy-like type BphP (XccBphP), previously shown to negatively regulate several light-mediated biological processes involved in virulence. Here, we generated three different full-length variants with single amino acid changes within its GAF domain that affect the XccBphP photocycle favouring its Pr state: L193Q, L193N and D199A. While D199A recombinant protein locks XccBphP in a Pr-like state, L193Q and L193N exhibit a significant enrichment of the Pr form in thermal equilibrium. The X-ray crystal structures of the three variants were solved, resembling the wild-type protein in the Pr state. Finally, we studied the effects of altering the XccBphP photocycle on the exopolysaccharide xanthan production and stomatal aperture assays as readouts of its bacterial signalling pathway. Null-mutant complementation assays show that the photoactive Pr-favoured XccBphP variants L193Q and L193N tend to negatively regulate xanthan production in vivo. In addition, our results indicate that strains expressing these variants also promote stomatal apertures in challenged plant epidermal peels, compared to wild-type Xcc. The findings presented in this work provide new evidence on the Pr state of XccBphP as a negative regulator of the virulence-associated mechanisms by light in Xcc.
Assuntos
Arabidopsis/microbiologia , Pigmentos Biliares/metabolismo , Fitocromo/química , Fitocromo/genética , Doenças das Plantas/microbiologia , Virulência , Xanthomonas campestris/fisiologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Luz , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Fitocromo/metabolismoRESUMO
Xanthan, the main exopolysaccharide (EPS) synthesized by Xanthomonas spp., contributes to bacterial stress tolerance and enhances attachment to plant surfaces by helping in biofilm formation. Therefore, xanthan is essential for successful colonization and growth in planta and has also been proposed to be involved in the promotion of pathogenesis by calcium ion chelation and, hence, in the suppression of the plant defense responses in which this cation acts as a signal. The aim of this work was to study the relationship between xanthan structure and its role as a virulence factor. We analyzed four Xanthomonas campestris pv. campestris mutants that synthesize structural variants of xanthan. We found that the lack of acetyl groups that decorate the internal mannose residues, ketal-pyruvate groups, and external mannose residues affects bacterial adhesion and biofilm architecture. In addition, the mutants that synthesized EPS without pyruvilation or without the external mannose residues did not develop disease symptoms in Arabidopsis thaliana. We also observed that the presence of the external mannose residues and, hence, pyruvilation is required for xanthan to suppress callose deposition as well as to interfere with stomatal defense. In conclusion, pyruvilation of xanthan seems to be essential for Xanthomonas campestris pv. campestris virulence.
Assuntos
Arabidopsis/microbiologia , Biofilmes/crescimento & desenvolvimento , Glucanos/metabolismo , Doenças das Plantas/microbiologia , Polissacarídeos Bacterianos/química , Xanthomonas campestris/patogenicidade , Interações Hospedeiro-Patógeno , Mutação , Folhas de Planta/microbiologia , Estômatos de Plantas/microbiologia , Polissacarídeos Bacterianos/genética , Polissacarídeos Bacterianos/metabolismo , Ácido Pirúvico/química , Virulência , Fatores de Virulência/química , Fatores de Virulência/genética , Fatores de Virulência/metabolismo , Xanthomonas campestris/genética , Xanthomonas campestris/crescimento & desenvolvimento , Xanthomonas campestris/fisiologiaRESUMO
UNLABELLED: Cruciferous plants are important edible vegetables widely consumed around the world, including cabbage, cauli-flower and broccoli. The main disease that affects crucifer plants is black rot, caused by Xanthomonas campestris pv. campestris (Xcc). In order to better understand this specific plant-pathogen interaction, proteins responsive to Xcc infection in resistant (União) and susceptible (Kenzan) Brassica oleracea cultivars were investigated by 2-DE followed by mass spectrometry. A total of 47 variable spots were identified and revealed that in the susceptible interaction there is a clear reduction in the abundance of proteins involved in energetic metabolism and defense. It was interesting to observe that in the resistant interaction, these proteins showed an opposite behavior. Based on our results, we conclude that resistance is correlated with the ability of the plant to keep sufficient photosynthesis metabolism activity to provide energy supplies necessary for an active defense. As a follow-up study, qRT-PCR analysis of selected genes was performed and revealed that most genes showed an up-regulation trend from 5 to 15days after inoculation (DAI), showing highest transcript levels at 15DAI. These results revealed the gradual accumulation of transcripts providing a more detailed view of the changes occurring during different stages of the plant-pathogen interaction. BIOLOGICAL SIGNIFICANCE: In this study we have compared cultivars of Brassica oleracea (cabbage), susceptible and resistant to black rot, by using the classical 2-DE approach. We have found that resistance is correlated with the ability of the plant to keep sufficient photosynthesis metabolism activity to provide energy supplies necessary for an active defense.
Assuntos
Brassica/microbiologia , Interações Hospedeiro-Patógeno/imunologia , Xanthomonas campestris/fisiologia , Brassica/química , Brassica/imunologia , Brassica/metabolismo , Eletroforese em Gel Bidimensional , Metabolismo Energético , Espectrometria de Massas , Fotossíntese , Proteômica/métodos , Regulação para Cima , Xanthomonas campestris/patogenicidadeRESUMO
Plants, when exposed to certain pathogens, may display a form of genotype-independent resistance, known as non-host response. In this study, the response of Citrus sinensis (sweet orange) leaves to Xanthomonas campestris pv. vesicatoria (Xcv), a pepper and tomato pathogenic bacterium, was analyzed through biochemical assays and cDNA microarray hybridization and compared with Asiatic citrus canker infection caused by Xanthomonas citri subsp. citri. Citrus leaves exposed to the non-host bacterium Xcv showed hypersensitive response (HR) symptoms (cell death), a defense mechanism common in plants but poorly understood in citrus. The HR response was accompanied by differentially expressed genes that are associated with biotic stress and cell death. Moreover, 58 transcription factors (TFs) were differentially regulated by Xcv in citrus leaves, including 26 TFs from the stress-associated families AP2-EREBP, bZip, Myb and WRKY. Remarkably, in silico analysis of the distribution of expressed sequence tags revealed that 10 of the 58 TFs, belonging to C2C2-GATA, C2H2, CCAAT, HSF, NAC and WRKY gene families, were specifically over-represented in citrus stress cDNA libraries. This study identified candidate TF genes for the regulation of key steps during the citrus non-host HR. Furthermore, these TFs might be useful in future strategies of molecular breeding for citrus disease resistance.
Assuntos
Citrus sinensis/metabolismo , Citrus sinensis/microbiologia , Interações Hospedeiro-Patógeno , Proteínas de Plantas/metabolismo , Fatores de Transcrição/metabolismo , Xanthomonas campestris/fisiologia , Alelos , Morte Celular , Citrus sinensis/citologia , Citrus sinensis/genética , Etiquetas de Sequências Expressas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Interações Hospedeiro-Patógeno/genética , Folhas de Planta/citologia , Folhas de Planta/genética , Folhas de Planta/microbiologia , Proteínas de Plantas/genética , Estresse Fisiológico/genéticaRESUMO
NAC proteins, which are plant-specific transcription factors, have been identified to play important roles in plant response to stresses and in plant development. The full-length cDNAs that encode 2 putative NAC proteins, designated as MmATAF1 and MmNAP, respectively, were cloned from Mikania micrantha by rapid amplification of cDNA ends. The full-length cDNAs of MmATAF1 and MmNAP were 1329 and 1072 bp, respectively, and they encoded deduced proteins of 260- and 278-amino acid residues, respectively. The proteins MmATAF1 and MmNAP had a calculated molecular mass of 29.81 and 32.55 kDa and a theoretical isoelectric point of 7.08 and 9.00, respectively. Nucleotide sequence data indicated that both MmATAF1 and MmNAP contained 2 introns and 3 exons and that they shared a conserved genomic organization. Multiple sequence alignments showed that MmATAF1 showed high sequence identity with ATAF1 of Arabidopsis thaliana (61%) and that MmNAP showed high sequence identity with NAP of A. thaliana (67%) and CitNAC of Citrus sinensis Osbeck (62%). Phylogenetic analysis showed that the predicted MmATAF1 and MmNAP proteins were classified into the ATAF and NAP subgroups, respectively. Transient expression analysis of onion epidermal cells indicated nuclear localization of both MmATAF1-GFP and MmNAP-GFP fusion proteins. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis indicated that MmATAF1 was expressed in all the tissues tested, but in varying abundance, while MmNAP was specifically expressed in stems, petioles, shoots, and leaves, but not in roots. The transcript levels of MmATAF1 and MmNAP in shoots and in infected stems were induced and strengthened by wounding, exogenous ZnSO(4), abscisic acid, salicylic acid, and Cuscuta campestris infection on the basis of semi-quantitative RT-PCR and real-time PCR analyses, respectively. Collectively, these results indicated that MmATAF1 and MmNAP, besides having roles in M. micrantha adaptation to C. campestris infection and abiotic stresses, also integrated signals derived from both C. campestris infection and abiotic stresses.
Assuntos
Mikania/genética , Proteínas de Plantas/genética , Caules de Planta/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Sequência de Bases , Núcleo Celular/metabolismo , Clonagem Molecular , Expressão Gênica , Regulação da Expressão Gênica de Plantas , Interações Hospedeiro-Patógeno , Mikania/metabolismo , Mikania/microbiologia , Dados de Sequência Molecular , Especificidade de Órgãos , Filogenia , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , Caules de Planta/metabolismo , Caules de Planta/microbiologia , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Estresse Fisiológico , Fatores de Transcrição/metabolismo , Xanthomonas campestris/fisiologiaRESUMO
Attempted infection of plants by pathogens elicits a complex defensive response. In many non-host and incompatible host interactions it includes the induction of defence-associated genes and a form of localized cell death (LCD), purportedly designed to restrict pathogen advance, collectively known as the hypersensitive response (HR). It is preceded by an oxidative burst, generating reactive oxygen species (ROS) that are proposed to cue subsequent deployment of the HR, although neither the origin nor the precise role played by ROS in the execution of this response are completely understood. We used tobacco plants expressing cyanobacterial flavodoxin to address these questions. Flavodoxin is an electron shuttle present in prokaryotes and algae that, when expressed in chloroplasts, specifically prevents ROS formation in plastids during abiotic stress episodes. Infiltration of tobacco wild-type leaves with high titres of Xanthomonas campestris pv. vesicatoria (Xcv), a non-host pathogen, resulted in ROS accumulation in chloroplasts, followed by the appearance of localized lesions typical of the HR. In contrast, chloroplast ROS build-up and LCD were significantly reduced in Xcv-inoculated plants expressing plastid-targeted flavodoxin. Metabolic routes normally inhibited by pathogens were protected in the transformants, whereas other aspects of the HR, including the induction of defence-associated genes and synthesis of salicylic and jasmonic acid, proceeded as in inoculated wild-type plants. Therefore, ROS generated in chloroplasts during this non-host interaction are essential for the progress of LCD, but do not contribute to the induction of pathogenesis-related genes or other signalling components of the response.
Assuntos
Morte Celular , Cloroplastos/metabolismo , Nicotiana/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Xanthomonas campestris/fisiologia , Ciclopentanos/metabolismo , Flavodoxina/metabolismo , Regulação da Expressão Gênica de Plantas , Oxilipinas/metabolismo , Doenças das Plantas , Folhas de Planta/genética , Folhas de Planta/metabolismo , Folhas de Planta/microbiologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/microbiologia , RNA de Plantas/genética , Ácido Salicílico/metabolismo , Nicotiana/genética , Nicotiana/microbiologiaRESUMO
Black rot of cruciferous plants, caused by Xanthomonas campestris pv. campestris, causes severe losses in agriculture around the world. This disease affects several cultures, including cabbage and broccoli, among others. Proteome studies of this bacterium have been reported; however, most of them were performed using the bacterium grown under culture media conditions. Recently, we have analyzed the proteome of X. campestris pv. campestris during the interaction with the susceptible cultivar of Brassica oleracea and several proteins were identified. The objective of the present study was to analyze the expressed proteins of X. campestris pv. campestris during the interaction with the resistant cultivar of B. oleracea. The bacterium was infiltrated in the leaves of the resistant plant and recovered for protein extraction and two-dimensional electrophoresis. The protein profile was compared with that of the bacterium isolated from the susceptible host and the results obtained revealed a group of proteins exclusive to the resistant interaction. Among the proteins identified in this study were plant and bacterium proteins, some of which were exclusively expressed during the resistant interaction.
Assuntos
Proteínas de Bactérias/análise , Brassica/química , Interações Hospedeiro-Patógeno , Proteínas de Plantas/análise , Proteoma/análise , Xanthomonas campestris/química , Brassica/microbiologia , Eletroforese em Gel Bidimensional , Xanthomonas campestris/fisiologiaRESUMO
Pathogen-induced stomatal closure is part of the plant innate immune response. Phytopathogens using stomata as a way of entry into the leaf must avoid the stomatal response of the host. In this article, we describe a factor secreted by the bacterial phytopathogen Xanthomonas campestris pv campestris (Xcc) capable of interfering with stomatal closure induced by bacteria or abscisic acid (ABA). We found that living Xcc, as well as ethyl acetate extracts from Xcc culture supernatants, are capable of reverting stomatal closure induced by bacteria, lipopolysaccharide, or ABA. Xcc ethyl acetate extracts also complemented the infectivity of Pseudomonas syringae pv tomato (Pst) mutants deficient in the production of the coronatine toxin, which is required to overcome stomatal defense. By contrast, the rpfF and rpfC mutant strains of Xcc, which are unable to respectively synthesize or perceive a diffusible molecule involved in bacterial cell-to-cell signaling, were incapable of reverting stomatal closure, indicating that suppression of stomatal response by Xcc requires an intact rpf/diffusible signal factor system. In addition, we found that guard cell-specific Arabidopsis (Arabidopsis thaliana) Mitogen-Activated Protein Kinase3 (MPK3) antisense mutants were unresponsive to bacteria or lipopolysaccharide in promotion of stomatal closure, and also more sensitive to Pst coronatine-deficient mutants, showing that MPK3 is required for stomatal immune response. Additionally, we found that, unlike in wild-type Arabidopsis, ABA-induced stomatal closure in MPK3 antisense mutants is not affected by Xcc or by extracts from Xcc culture supernatants, suggesting that the Xcc factor might target some signaling component in the same pathway as MPK3.
Assuntos
Arabidopsis/imunologia , Estômatos de Plantas/imunologia , Transdução de Sinais/imunologia , Fatores de Virulência/imunologia , Xanthomonas campestris/fisiologia , Xanthomonas campestris/patogenicidade , Arabidopsis/enzimologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/imunologia , Imunidade Inata , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/imunologia , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Pseudomonas syringae/genética , Pseudomonas syringae/imunologiaRESUMO
Virulence of the black rot pathogen Xanthomonas campestris pv. campestris (Xcc) is regulated by cell-cell signalling involving the diffusible signal factor DSF. Synthesis and perception of DSF require products of genes within the rpf cluster (for regulation of pathogenicity factors). RpfF directs DSF synthesis whereas RpfC and RpfG are involved in DSF perception. Here we have examined the role of the rpf/DSF system in biofilm formation in minimal medium using confocal laser-scanning microscopy of GFP-labelled bacteria. Wild-type Xcc formed microcolonies that developed into a structured biofilm. In contrast, an rpfF mutant (DSF-minus) and an rpfC mutant (DSF overproducer) formed only unstructured arrangements of bacteria. A gumB mutant, defective in xanthan biosynthesis, was also unable to develop the typical wild-type biofilm. Mixed cultures of gumB and rpfF mutants formed a typical biofilm in vitro. In contrast, in mixed cultures the rpfC mutant prevented the formation of the structured biofilm by the wild-type and did not restore wild-type biofilm phenotypes to gumB or rpfF mutants. These effects on structured biofilm formation were correlated with growth and disease development by Xcc strains in Nicotiana benthamiana leaves. These findings suggest that DSF signalling is finely balanced during both biofilm formation and virulence.
Assuntos
Proteínas de Bactérias/metabolismo , Nicotiana/microbiologia , Doenças das Plantas/microbiologia , Xanthomonas campestris/fisiologia , Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Microscopia Confocal , Mutação , Folhas de Planta/microbiologia , Percepção de Quorum , Virulência , Xanthomonas campestris/genética , Xanthomonas campestris/patogenicidadeRESUMO
The genus Xanthomonas is a diverse and economically important group of bacterial phytopathogens, belonging to the gamma-subdivision of the Proteobacteria. Xanthomonas axonopodis pv. citri (Xac) causes citrus canker, which affects most commercial citrus cultivars, resulting in significant losses worldwide. Symptoms include canker lesions, leading to abscission of fruit and leaves and general tree decline. Xanthomonas campestris pv. campestris (Xcc) causes black rot, which affects crucifers such as Brassica and Arabidopsis. Symptoms include marginal leaf chlorosis and darkening of vascular tissue, accompanied by extensive wilting and necrosis. Xanthomonas campestris pv. campestris is grown commercially to produce the exopolysaccharide xanthan gum, which is used as a viscosifying and stabilizing agent in many industries. Here we report and compare the complete genome sequences of Xac and Xcc. Their distinct disease phenotypes and host ranges belie a high degree of similarity at the genomic level. More than 80% of genes are shared, and gene order is conserved along most of their respective chromosomes. We identified several groups of strain-specific genes, and on the basis of these groups we propose mechanisms that may explain the differing host specificities and pathogenic processes.
Assuntos
Genoma Bacteriano , Plantas/microbiologia , Xanthomonas/genética , Xanthomonas/fisiologia , Ordem dos Genes/genética , Interações Hospedeiro-Parasita , Dados de Sequência Molecular , Filogenia , Regulon/genética , Origem de Replicação/genética , Especificidade da Espécie , Virulência/genética , Xanthomonas/classificação , Xanthomonas/patogenicidade , Xanthomonas campestris/genética , Xanthomonas campestris/patogenicidade , Xanthomonas campestris/fisiologiaRESUMO
Xanthomonas campestris pv. campestris is a pathogen of cruciferous plants. We studied the survival of the wild type strain and mutant derivatives which are deficient in exopolysaccharide (EPS) or in extracellular protease synthesis in soil microcosms in order to test the hypothesis that, in this environment, adherence to soil particles and scavenging of nutrients are very important strategies for bacterial survival. In sterile soil microcosms, differences in survival were only observed between the EPS producer and its mutant. In non-sterile soil experiments, survival of Prt- mutant was similar to EPS- mutant, suggesting that both characteristics have a strong influence in survival in the presence of the natural bacterial community. Bacterial decrease represented by the slope of regression lines was higher in non-sterile soil microcosms due to the influence of biotic interactions.
Assuntos
Microbiologia do Solo , Xanthomonas campestris/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Ecologia , Endopeptidases/deficiência , Endopeptidases/genética , Endopeptidases/fisiologia , Polissacarídeos Bacterianos/genética , Polissacarídeos Bacterianos/fisiologia , Xanthomonas campestris/genéticaRESUMO
Endochitinases are widely distributed among higher plants, including a number of important crop species. They are generally considered to be involved in plant defence against potential pathogens. We have cloned a class IV chitinase gene (AtchitIV) from Arabidopsis thaliana. Southern blot analysis allowed the detection of two cross-hybridising genes in the A. thaliana genome. AtchitIV transcripts are detected in seedpods, but not in roots, inflorescence stems, leaves and flowers of healthy plants. The transcripts accumulated very rapidly in leaves after inoculation with Xanthomonas campestris. Maximum mRNA accumulation was reached one hour after infection and decreased to very low levels 72 hours after induction. This result suggests an involvement of AtchitIV in the initial events of the hypersensitive reaction. Nevertheless, A. thaliana plants transformed with the gus gene under the control of a class IV chitinase bean promoter, showed GUS activity in seed embryos. These data, together with the constitutive expression of the endogenous gene in the seedpods, points to additional physiological roles for this protein.