Roxana Andrea Roeschlin

The Xanthomonas axonopodis pv. citri flagellum is required for mature biofilm and canker development.

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.

Novel demonstration of RNAi in citrus reveals importance of citrus callose synthase in defence against Xanthomonas citri subsp. citri

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.

Characterization of a variant of Xanthomonas citri subsp. citri that triggers a host-specific defense response.

Citrus is an economically important fruit crop that is severely afflicted by Asiatic citrus bacterial canker (CBC), a disease caused by the phytopathogen Xanthomonas citri subsp. citri (X. citri). To gain insight into the molecular epidemiology of CBC, 42 Xanthomonas isolates were collected from a range of Citrus spp. across 17 different orchards in Tucumán, Argentina and subjected to molecular, biochemical, and pathogenicity tests. Analysis of genome-specific X. citri markers and DNA polymorphisms based on repetitive elements-based polymerase chain reaction showed that all 42 isolates belonged to X. citri. Interestingly, pathogenicity tests showed that one isolate, which shares >90% genetic similarity to the reference strain X. citri T, has host range specificity. This new variant of X. citri subsp. citri, named X. citri A(T), which is deficient in xanthan production, induces an atypical, noncankerous chlorotic phenotype in Citrus limon and C. paradisi and weak cankerous lesions in C. aurantifolia and C. clementina leaves. In C. limon, suppression of canker development is concomitant with an oxidative burst; xanthan is not implicated in the phenotype induced by this interaction, suggesting that other bacterial factors would be involved in triggering the defense response.

Xanthan Pyruvilation Is Essential for the Virulence of Xanthomonas campestris pv. campestris

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.

Resistance to citrus canker induced by a variant of Xanthomonas citri ssp. citri is associated with a hypersensitive cell death response involving autophagy-associated vacuolar processes.

Xanthomonas citri ssp. citri (X. citri) is the causal agent of Asiatic citrus canker, a disease that seriously affects most commercially important Citrus species worldwide. We have identified previously a natural variant, X. citri AT , that triggers a host-specific defence response in Citrus limon. However, the mechanisms involved in this canker disease resistance are unknown. In this work, the defence response induced by X. citri AT was assessed by transcriptomic, physiological and ultrastructural analyses, and the effects on bacterial biofilm formation were monitored in parallel. We show that X. citri AT triggers a hypersensitive response associated with the interference of biofilm development and arrest of bacterial growth in C. limon. This plant response involves an extensive transcriptional reprogramming, setting in motion cell wall reinforcement, the oxidative burst and the accumulation of salicylic acid (SA) and phenolic compounds. Ultrastructural analyses revealed subcellular changes involving the activation of autophagy-associated vacuolar processes. Our findings show the activation of SA-dependent defence in response to X. citri AT and suggest a coordinated regulation between the SA and flavonoid pathways, which is associated with autophagy mechanisms that control pathogen invasion in C. limon. Furthermore, this defence response protects C. limon plants from disease on subsequent challenges by pathogenic X. citri. This knowledge will allow the rational exploitation of the plant immune system as a biotechnological approach for the management of the disease.