Unraveling Plant Responses to Bacterial Pathogens through Proteomics

Plant pathogenic bacteria cause diseases in important crops and seriously and negatively impact agricultural production. Therefore, an understanding of the mechanisms by which plants resist bacterial infection at the stage of the basal immune response or mount a successful specific R-dependent defense response is crucial since a better understanding of the biochemical and cellular mechanisms underlying these interactions will enable molecular and transgenic approaches to crops with increased biotic resistance. In recent years, proteomics has been used to gain in-depth understanding of many aspects of the host defense against pathogens and has allowed monitoring differences in abundance of proteins as well as posttranscriptional and posttranslational processes, protein activation/inactivation, and turnover. Proteomics also offers a window to study protein trafficking and routes of communication between organelles. Here, we summarize and discuss current progress in proteomics of the basal and specific host defense responses elicited by bacterial pathogens

A plant natriuretic peptide-like molecule of the pathogen Xanthomonas axonopodis pv. citri causes rapid changes in the proteome of its citrus host

<p>Abstract</p> <p>Background</p> <p>Plant natriuretic peptides (PNPs) belong to a novel class of peptidic signaling molecules that share some structural similarity to the N-terminal domain of expansins and affect physiological processes such as water and ion homeostasis at nano-molar concentrations. The citrus pathogen Xanthomonas axonopodis pv. citri possesses a PNP-like peptide (XacPNP) uniquely present in this bacteria. Previously we observed that the expression of <it>XacPNP </it>is induced upon infection and that lesions produced in leaves infected with a XacPNP deletion mutant were more necrotic and lead to earlier bacterial cell death, suggesting that the plant-like bacterial PNP enables the plant pathogen to modify host responses in order to create conditions favorable to its own survival.</p> <p>Results</p> <p>Here we measured chlorophyll fluorescence parameters and water potential of citrus leaves infiltrated with recombinant purified XacPNP and demonstrate that the peptide improves the physiological conditions of the tissue. Importantly, the proteomic analysis revealed that these responses are mirrored by rapid changes in the host proteome that include the up-regulation of Rubisco activase, ATP synthase CF1 α subunit, maturase K, and α- and β-tubulin.</p> <p>Conclusions</p> <p>We demonstrate that XacPNP induces changes in host photosynthesis at the level of protein expression and in photosynthetic efficiency in particular. Our findings suggest that the biotrophic pathogen can use the plant-like hormone to modulate the host cellular environment and in particular host metabolism and that such modulations weaken host defence.</p

A Filamentous Hemagglutinin-Like Protein of Xanthomonas axonopodis pv. citri, the Phytopathogen Responsible for Citrus Canker, Is Involved in Bacterial Virulence

Xanthomonas axonopodis pv. citri, the phytopathogen responsible for citrus canker has a number of protein secretion systems and among them, at least one type V protein secretion system belonging to the two-partner secretion pathway. This system is mainly associated to the translocation of large proteins such as adhesins to the outer membrane of several pathogens. Xanthomonas axonopodis pv. citri possess a filamentous hemagglutinin-like protein in close vicinity to its putative transporter protein, XacFhaB and XacFhaC, respectively. Expression analysis indicated that XacFhaB was induced in planta during plant-pathogen interaction. By mutation analysis of XacFhaB and XacFhaC genes we determined that XacFhaB is involved in virulence both in epiphytic and wound inoculations, displaying more dispersed and fewer canker lesions. Unexpectedly, the XacFhaC mutant in the transporter protein produced an intermediate virulence phenotype resembling wild type infection, suggesting that XacFhaB could be secreted by another partner different from XacFhaC. Moreover, XacFhaB mutants showed a general lack of adhesion and were affected in leaf surface attachment and biofilm formation. In agreement with the in planta phenotype, adhesin lacking cells moved faster in swarming plates. Since no hyperflagellation phenotype was observed in this bacteria, the faster movement may be attributed to the lack of cell-to-cell aggregation. Moreover, XacFhaB mutants secreted more exopolysaccharide that in turn may facilitate its motility. Our results suggest that this hemagglutinin-like protein is required for tissue colonization being mainly involved in surface attachment and biofilm formation, and that plant tissue attachment and cell-to-cell aggregation are dependent on the coordinated action of adhesin molecules and exopolysaccharides

Expression Analysis of Small Heat Shock Proteins During Compatible and Incompatible Plant-Pathogen Interactions

Abstract The study of small heat shock proteins in plant-pathogen interactions is a subject that has to be further investigated. While Hsp70 and Hsp90 participate in the defense response, the role of this small Hsps remains elusive. In this work we analyzed the expression of small Hsps in citrus canker and the bacterial spot of pepper as well as in the incompatible interactions of the bacterial pathogens that cause these diseases, Xanthomonas axonopodis pv. citri and Xanthomonas campestris pv. vesicatoria, with non-host plants. Our results show that although with different expression profiles, small Hsps are induced during both compatible and incompatible interactions giving light to the idea that these chaperones are components of the basal immune plant response

3-methylcrotonyl Coenzyme A (CoA) carboxylase complex is involved in the Xanthomonas citri subsp. citri lifestyle during citrus infection.

Citrus canker is a disease caused by the phytopathogen Xanthomonas citri subsp. citri (Xcc), bacterium which is unable to survive out of the host for extended periods of time. Once established inside the plant, the pathogen must compete for resources and evade the defenses of the host cell. However, a number of aspects of Xcc metabolic and nutritional state, during the epiphytic stage and at different phases of infection, are poorly characterized. The 3-methylcrotonyl-CoA carboxylase complex (MCC) is an essential enzyme for the catabolism of the branched-chain amino acid leucine, which prevents the accumulation of toxic intermediaries, facilitates the generation of branched chain fatty acids and/or provides energy to the cell. The MCC complexes belong to a group of acyl-CoA carboxylases (ACCase) enzymes dependent of biotin. In this work, we have identified two ORFs (XAC0263 and XAC0264) encoding for the α and β subunits of an acyl-CoA carboxylase complex from Xanthomonas and demonstrated that this enzyme has MCC activity both in vitro and in vivo. We also found that this MCC complex is conserved in a group of pathogenic gram negative bacteria. The generation and analysis of an Xcc mutant strain deficient in MCC showed less canker lesions in the interaction with the host plant, suggesting that the expression of these proteins is necessary for Xcc fitness during infection

Swarming motility of <i>X. axonopodis</i> pv. <i>citri</i> wild type and Δ<i>XacFhaB</i>, Δ<i>XacFhaC</i> and Δ<i>gumD</i> strains.

<p>(A) The different strains were centrally inoculated on SB plates fortified with 0.7% agar. After 6 days incubation at 28°C the plates were photographed on a light transiluminator to denote the different swarming phenotype. (B) The motility zone of swarming colonies grown as in A was measured at different times of incubation during a period of 10 days. Each data point showed in the figure is an average of 4 independent experiments, error bars indicate the standard error. The results are representative of four independent experiments.</p

Analysis of the factors involved in the swarming motility of <i>X. axonopodis</i> pv. <i>citri</i> wild type and Δ<i>XacFhaB</i> and Δ<i>XacFhaC</i> strains.

<p>(A) For flagellar proteins immunodetection, whole-cell extracts from the XacWT, Δ<i>XacFhaB</i> and Δ<i>XacFhaC</i> strains isolated from the center (C) or the border (B) of the swarming colony grown as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004358#pone-0004358-g005" target="_blank">Figure 5A</a> were analyzed by Western blotting developed with anti-flagellin polyclonal antibodies. Samples were standardized as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004358#s4" target="_blank">Materials and Methods</a>. (B) Cells of XacWT, Δ<i>XacFhaB</i> and Δ<i>XacFhaC</i> strains isolated from the center or the border of the advancing swarm were stained for flagellar structures (indicated by arrows) and observed under light microscopy at 100× magnification. The photographs are representative of three experiments in which several fields of view were observed. (C) Xanthan production in XOL medium of XacWT, Δ<i>XacFhaB</i> and Δ<i>XacFhaC</i> mutants strains. Each data point is the mean of three experiments, error bars indicate the standard error. (D) Total RNA was extracted from XacWT and Δ<i>XacFhaB</i> mutant grown in SB for 48 h at 28°C and <i>gumD</i> expression was analyzed by RT-PCR using specific primers. 16S rRNA was used as a constitutive control.</p