RESUMO

Huanglongbing (HLB), the current major threat for Citrus species, is caused by intracellular alphaproteobacteria of the genus Candidatus Liberibacter (CaL), with CaL asiaticus (CLas) being the most prevalent species. This bacterium inhabits phloem cells and is transmitted by the psyllid Diaphorina citri. A gene encoding a putative serralysin-like metalloprotease (CLIBASIA_01345) was identified in the CLas genome. The expression levels of this gene were found to be higher in citrus leaves than in psyllids, suggesting a function for this protease in adaptation to the plant environment. Here, we study the putative role of CLas-serralysin (Las1345) as virulence factor. We first assayed whether Las1345 could be secreted by two different surrogate bacteria, Rhizobium leguminosarum bv. viciae A34 (A34) and Serratia marcescens. The protein was detected only in the cellular fraction of A34 and S. marcescens expressing Las1345, and increased protease activity of those bacteria by 2.55 and 4.25-fold, respectively. In contrast, Las1345 expressed in Nicotiana benthamiana leaves did not show protease activity nor alterations in the cell membrane, suggesting that Las1345 do not function as a protease in the plant cell. Las1345 expression negatively regulated cell motility, exopolysaccharide production, and biofilm formation in Xanthomonas campestris pv. campestris (Xcc). This bacterial phenotype was correlated with reduced growth and survival on leaf surfaces as well as reduced disease symptoms in N. benthamiana and Arabidopsis. These results support a model where Las1345 could modify extracellular components to adapt bacterial shape and appendages to the phloem environment, thus contributing to virulence.

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Transcription activator-like effectors (TALEs) are important effectors of Xanthomonas spp. that manipulate the transcriptome of the host plant, conferring susceptibility or resistance to bacterial infection. Xanthomonas citri ssp. citri variant AT (X. citri AT ) triggers a host-specific hypersensitive response (HR) that suppresses citrus canker development. However, the bacterial effector that elicits this process is unknown. In this study, we show that a 7.5-repeat TALE is responsible for triggering the HR. PthA4AT was identified within the pthA repertoire of X. citri AT followed by assay of the effects on different hosts. The mode of action of PthA4AT was characterized using protein-binding microarrays and testing the effects of deletion of the nuclear localization signals and activation domain on plant responses. PthA4AT is able to bind DNA and activate transcription in an effector binding element-dependent manner. Moreover, HR requires PthA4AT nuclear localization, suggesting the activation of executor resistance (R) genes in host and non-host plants. This is the first case where a TALE of unusually short length performs a biological function by means of its repeat domain, indicating that the action of these effectors to reprogramme the host transcriptome following nuclear localization is not limited to 'classical' TALEs.

Assuntos

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Transgenic expression of the pepper Bs2 gene confers resistance to Xanthomonas campestris pv. vesicatoria (Xcv) pathogenic strains which contain the avrBs2 avirulence gene in susceptible pepper and tomato varieties. The avrBs2 gene is highly conserved among members of the Xanthomonas genus, and the avrBs2 of Xcv shares 96% homology with the avrBs2 of Xanthomonas citri subsp. citri (Xcc), the causal agent of citrus canker disease. A previous study showed that the transient expression of pepper Bs2 in lemon leaves reduced canker formation and induced plant defence mechanisms. In this work, the effect of the stable expression of Bs2 gene on citrus canker resistance was evaluated in transgenic plants of Citrus sinensis cv. Pineapple. Interestingly, Agrobacterium-mediated transformation of epicotyls was unsuccessful when a constitutive promoter (2× CaMV 35S) was used in the plasmid construction, but seven transgenic lines were obtained with a genetic construction harbouring Bs2 under the control of a pathogen-inducible promoter, from glutathione S-transferase gene from potato. A reduction of disease symptoms of up to 70% was observed in transgenic lines expressing Bs2 with respect to non-transformed control plants. This reduction was directly dependent on the Xcc avrBs2 gene since no effect was observed when a mutant strain of Xcc with a disruption in avrBs2 gene was used for inoculations. Additionally, a canker symptom reduction was correlated with levels of the Bs2 expression in transgenic plants, as assessed by real-time qPCR, and accompanied by the production of reactive oxygen species. These results indicate that the pepper Bs2 resistance gene is also functional in a family other than the Solanaceae, and could be considered for canker control.

Assuntos

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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

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Huanglongbing (HLB), a devastating citrus disease caused by the bacterium Candidatus Liberibacter spp., is now responsible for significant economic losses worldwide. Yet, no effective disease control has been found, and the non-cultivability of the bacterium has severely hampered studies on the pathogen. The 16S rDNA gene is a well-characterized sequence, essential for cell survival, and is used for bacterial identification or assignment of close relationships at the genus and species levels. Quantitative Real-Time PCR (qPCR) assays based on 16S rDNA genes are widely used in the detection of Ca. Liberibacter spp. in multiplex reactions. We have developed for the first time a set of qPCR primers based on the conserved 16S rDNA gene, which specifically and simultaneously detects in a singleplex reaction, all three bacterial species associated with HLB, and can differentiateCa.Liberibacter asiaticus or africanus from americanus by their characteristic melting curves. The assay is very sensitive, and it was possible to amplify expected DNA fragments with an efficiency of 98 % using the Syber Green system and a Ct value lower than tested methods for HLB diagnosis. The application of this fast, simple and efficient detection methodology could also be important in the detection of all species of HLB-associated Liberibacters and could contribute to early pathogen detection, a crucial step in the development of preventive strategies aimed at avoiding the dissemination of this devastating disease in HLB-free areas.(AU)

Assuntos

RESUMO

Huanglongbing (HLB), a devastating citrus disease caused by the bacterium Candidatus Liberibacter spp., is now responsible for significant economic losses worldwide. Yet, no effective disease control has been found, and the non-cultivability of the bacterium has severely hampered studies on the pathogen. The 16S rDNA gene is a well-characterized sequence, essential for cell survival, and is used for bacterial identification or assignment of close relationships at the genus and species levels. Quantitative Real-Time PCR (qPCR) assays based on 16S rDNA genes are widely used in the detection of Ca. Liberibacter spp. in multiplex reactions. We have developed for the first time a set of qPCR primers based on the conserved 16S rDNA gene, which specifically and simultaneously detects in a singleplex reaction, all three bacterial species associated with HLB, and can differentiateCa.Liberibacter asiaticus or africanus from americanus by their characteristic melting curves. The assay is very sensitive, and it was possible to amplify expected DNA fragments with an efficiency of 98 % using the Syber Green system and a Ct value lower than tested methods for HLB diagnosis. The application of this fast, simple and efficient detection methodology could also be important in the detection of all species of HLB-associated Liberibacters and could contribute to early pathogen detection, a crucial step in the development of preventive strategies aimed at avoiding the dissemination of this devastating disease in HLB-free areas.

Assuntos

RESUMO

Xanthomonas citri subsp. citri (Xcc) develops a biofilm structure both in vitro and in vivo. Despite all the progress achieved by studies regarding biofilm formation, many of its mechanisms remain poorly understood. This work focuses on the identification of new genes involved in biofilm formation and how they are related to motility, virulence and chemotaxis in Xcc. A Tn5 library of approximately 6000 Xcc (strain 306) mutants was generated and screened to search for biofilm formation defective strains. We identified 23 genes not previously associated with biofilm formation. The analysis of the 23 mutants not only revealed the involvement of new genes in biofilm formation, but also reinforced the importance of exopolysaccharide production, motility and cell surface structures in this process. This collection of biofilm-defective mutants underscores the multifactorial genetic programme underlying the establishment of biofilm in Xcc.

Assuntos

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Xanthomonas citri ssp. citri (Xcc) is the causal agent of citrus canker. This bacterium develops a characteristic biofilm on both biotic and abiotic surfaces. A biofilm-deficient mutant was identified in a screening of a transposon mutagenesis library of the Xcc 306 strain constructed using the commercial Tn5 transposon EZ-Tn5 Tnp Transposome (Epicentre). Sequence analysis of a mutant obtained in the screening revealed that a single copy of the EZ-Tn5 was inserted at position 446 of hrpM, a gene encoding a putative enzyme involved in glucan synthesis. We demonstrate for the first time that the product encoded by the hrpM gene is involved in ß-1,2-glucan synthesis in Xcc. A mutation in hrpM resulted in no disease symptoms after 4 weeks of inoculation in lemon and grapefruit plants. The mutant also showed reduced ability to swim in soft agar and decreased resistance to H(2)O(2) in comparison with the wild-type strain. All defective phenotypes were restored to wild-type levels by complementation with the plasmid pBBR1-MCS containing an intact copy of the hrpM gene and its promoter. These results indicate that the hrpM gene contributes to Xcc growth and adaptation in its host plant.

Assuntos

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Citrus is an economically important fruit crop that is severely afflicted by citrus canker, a disease caused by the bacterial phytopathogen, Xanthomonas citri subsp. citri (Xcc). GenBank houses a large collection of Expressed Sequence Tags (ESTs) enriched with transcripts generated during the defence response against this pathogen; however, there are currently no strategies in citrus to assess the function of candidate genes. This has greatly limited research as defence signalling genes are often involved in multiple pathways. In this study, we demonstrate the efficacy of RNA interference (RNAi) as a functional genomics tool to assess the function of candidate genes involved in the defence response of Citrus limon against the citrus canker pathogen. Double-stranded RNA expression vectors, encoding hairpin RNAs for citrus host genes, were delivered to lemon leaves by transient infiltration with transformed Agrobacterium. As proof of principle, we have established silencing of citrus phytoene desaturase (PDS) and callose synthase (CalS1) genes. Phenotypic and molecular analyses showed that silencing vectors were functional not only in lemon plants but also in other species of the Rutaceae family. Using silencing of CalS1, we have demonstrated that plant cell wall-associated defence is the principal initial barrier against Xanthomonas infection in citrus plants. Additionally, we present here results that suggest that H2O2 accumulation, which is suppressed by xanthan from Xcc during pathogenesis, contributes to inhibition of xanthan-deficient Xcc mutant growth either in wild-type or CalS1-silenced plants. With this work, we have demonstrated that high-throughput reverse genetic analysis is feasible in citrus.

Assuntos

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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 0.25 % agar plates, was markedly reduced in fliC and flgE mutants. However, the fliC and flgE mutants exhibited a flagellar-independent surface translocation on 0.5 % 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

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In this review, we summarise the current knowledge on three pathogens that exhibit distinct tissue specificity and modes of pathogenesis in citrus plants. Xanthomonas axonopodis pv. citri causes canker disease and invades the host leaf mesophyll tissue through natural openings and can also survive as an epiphyte. Xylella fastidiosa and Candidatus Liberibacter are vectored by insects and proliferate in the vascular system of the host, either in the phloem (Candidatus Liberibacter) or xylem (X. fastidiosa) causing variegated chlorosis and huanglongbing diseases, respectively. Candidatus Liberibacter can be found within host cells and is thus unique as an intracellular phytopathogenic bacterium. Genome sequence comparisons have identified groups of species-specific genes that may be associated with the particular lifestyle, mode of transmission or symptoms produced by each phytopathogen. In addition, components that are conserved amongst bacteria may have diverse regulatory actions underpinning the different bacterial lifestyles; one example is the divergent role of the Rpf/DSF cell-cell signalling system in X. citri and X. fastidiosa. Biofilm plays a key role in epiphytic fitness and canker development in X. citri and in the symptoms produced by X. fastidiosa. Bacterial aggregation may be associated with vascular occlusion of the xylem vessels and symptomatology of variegated chlorosis.

Assuntos

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To evaluate the role of salicylic acid (SA) in Nb-mediated hypersensitive resistance to Potato virus X (PVX) avirulent strain ROTH1 in Solanum tuberosum, we have constructed SA-deficient transgenic potato plant lines by overexpressing the bacterial enzyme salicylate hydroxylase (NahG), which degrades SA. Evaluation of these transgenic lines revealed hydrogen peroxide accumulation and spontaneous lesion formation in an age- and light-dependent manner. In concordance, NahG potato plants were more sensitive to treatment with methyl viologen, a reactive oxygen species-generating compound. In addition, when challenged with PVX ROTH1, NahG transgenic lines showed a decreased disease-resistance response to infection and were unable to induce systemic acquired resistance. However, the avirulent viral effector, the PVX 25-kDa protein, does induce expression of the pathogenesis-related gene PR-1a in NahG potato plants. Taken together, our data indicate that SA is involved in local and systemic defense responses mediated by the Nb gene in Solanum tuberosum. This is the first report to show that basal levels of SA correlate with hypersensitive resistance to PVX.

Assuntos

RESUMO

The phytopathogenic bacterium Xanthomonas axonopodis pv. citri is responsible for the canker disease affecting citrus plants throughout the world. Here, we have evaluated the role of bacterial attachment and biofilm formation in leaf colonization during canker development on lemon leaves. Crystal violet staining and confocal laser scanning microscopy analysis of X. axonopodis pv. citri strains expressing the green fluorescent protein were used to evaluate attachment and biofilm formation on abiotic and biotic (leaf) surfaces. Wild-type X. axonopodis pv. citri attached to and formed a complex, structured biofilm on glass in minimal medium containing glucose. Similar attachment and structured biofilm formation also were seen on lemon leaves. An X. axonopodis pv. citri gumB mutant strain, defective in production of the extracellular polysaccharide xanthan, did not form a structured biofilm on either abiotic or biotic surfaces. In addition, the X. axonopodis pv. citri gumB showed reduced growth and survival on leaf surfaces and reduced disease symptoms. These findings suggest an important role for formation of biofilms in the epiphytic survival of X. axonopodis pv. citri prior to development of canker disease.

Assuntos

RESUMO

Although cyclic glucans have been shown to be important for a number of symbiotic and pathogenic bacterium-plant interactions, their precise roles are unclear. Here, we examined the role of cyclic beta-(1,2)-glucan in the virulence of the black rot pathogen Xanthomonas campestris pv campestris (Xcc). Disruption of the Xcc nodule development B (ndvB) gene, which encodes a glycosyltransferase required for cyclic glucan synthesis, generated a mutant that failed to synthesize extracellular cyclic beta-(1,2)-glucan and was compromised in virulence in the model plants Arabidopsis thaliana and Nicotiana benthamiana. Infection of the mutant bacterium in N. benthamiana was associated with enhanced callose deposition and earlier expression of the PATHOGENESIS-RELATED1 (PR-1) gene. Application of purified cyclic beta-(1,2)-glucan prior to inoculation of the ndvB mutant suppressed the accumulation of callose deposition and the expression of PR-1 in N. benthamiana and restored virulence in both N. benthamiana and Arabidopsis plants. These effects were seen when cyclic glucan and bacteria were applied either to the same or to different leaves. Cyclic beta-(1,2)-glucan-induced systemic suppression was associated with the transport of the molecule throughout the plant. Systemic suppression is a novel counterdefensive strategy that may facilitate pathogen spread in plants and may have important implications for the understanding of plant-pathogen coevolution and for the development of phytoprotection measures.

Assuntos

RESUMO

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

RESUMO

Xanthomonas axonopodis pathovar citri (Xac) causes bacterial citrus canker, a serious disease of most citrus species. Xanthomonas campestris pv. campestris (Xcc) is the causal agent of black rot disease in cruciferous plants. In Xcc, cell-cell signaling is mediated by diffusible signal factor (DSF). Synthesis of DSF depends on RpfB and RpfF. DSF perception and signal transduction have been suggested to involve a two-component system comprising RpfC and RpfG. It has been proposed that these proteins participate in a signal transduction system linking changes in the environment to the synthesis of DSF and the expression of virulence genes. Although the cluster of the rpf genes in Xac has synteny with the corresponding cluster in Xcc, two genes (rpfH and rpfI) are absent in Xac. To investigate DSF-mediated regulation during Xac-Citrus limon interaction, we constructed two strains of Xac, one with a mutation in the rpfF gene, leading to an inability to produce DSF, and one with a mutation in the rpfC gene leading to an overproduction of DSF. These mutants also show decreased levels of extracellular cyclic â-(1,2)-glucans and decreased production of endoglucanase and protease extracellular enzymes. The Xac DSF-deficient rpfF and the DSF-hyper producing rpfC mutants are both severely compromised in their ability to cause canker symptoms in lemon leaves compared to the wild-type. Here we provide evidence that rpf genes in Xac are involved in controlling virulence factors mediated by DSF.

RESUMO

Xanthan is the major exopolysaccharide secreted by Xanthomonas spp. Despite its diverse roles in bacterial pathogenesis of plants, little is known about the real implication of this molecule in Xanthomonas pathogenesis. In this study we show that in contrast to Xanthomonas campestris pv campestris strain 8004 (wild type), the xanthan minus mutant (strain 8397) and the mutant strain 8396, which is producing truncated xanthan, fail to cause disease in both Nicotiana benthamiana and Arabidopsis (Arabidopsis thaliana) plants. In contrast to wild type, 8397 and 8396 strains induce callose deposition in N. benthamiana and Arabidopsis plants. Interestingly, treatment with xanthan but not truncated xanthan, suppresses the accumulation of callose and enhances the susceptibility of both N. benthamiana and Arabidopsis plants to 8397 and 8396 mutant strains. Finally, in concordance, we also show that treatment with an inhibitor of callose deposition previous to infection induces susceptibility to 8397 and 8396 strains. Thus, xanthan suppression effect on callose deposition seems to be important for Xanthomonas infectivity.

Assuntos