Vapd In Xylella Fastidiosa Is A Thermostable Protein With Ribonuclease Activity.

Xylella fastidiosa strain 9a5c is a gram-negative phytopathogen that is the causal agent of citrus variegated chlorosis (CVC), a disease that is responsible for economic losses in Brazilian agriculture. The most well-known mechanism of pathogenicity for this bacterial pathogen is xylem vessel occlusion, which results from bacterial movement and the formation of biofilms. The molecular mechanisms underlying the virulence caused by biofilm formation are unknown. Here, we provide evidence showing that virulence-associated protein D in X. fastidiosa (Xf-VapD) is a thermostable protein with ribonuclease activity. Moreover, protein expression analyses in two X. fastidiosa strains, including virulent (Xf9a5c) and nonpathogenic (XfJ1a12) strains, showed that Xf-VapD was expressed during all phases of development in both strains and that increased expression was observed in Xf9a5c during biofilm growth. This study is an important step toward characterizing and improving our understanding of the biological significance of Xf-VapD and its potential functions in the CVC pathosystem.10e014576

Broad Substrate-Specific Phosphorylation Events Are Associated With the Initial Stage of Plant Cell Wall Recognition in Neurospora crassa.

Fungal plant cell wall degradation processes are governed by complex regulatory mechanisms, allowing the organisms to adapt their metabolic program with high specificity to the available substrates. While the uptake of representative plant cell wall mono- and disaccharides is known to induce specific transcriptional and translational responses, the processes related to early signal reception and transduction remain largely unknown. A fast and reversible way of signal transmission are post-translational protein modifications, such as phosphorylations, which could initiate rapid adaptations of the fungal metabolism to a new condition. To elucidate how changes in the initial substrate recognition phase of Neurospora crassa affect the global phosphorylation pattern, phospho-proteomics was performed after a short (2 min) induction period with several plant cell wall-related mono- and disaccharides. The MS/MS-based peptide analysis revealed large-scale substrate-specific protein phosphorylation and de-phosphorylations. Using the proteins identified by MS/MS, a protein-protein-interaction (PPI) network was constructed. The variance in phosphorylation of a large number of kinases, phosphatases and transcription factors indicate the participation of many known signaling pathways, including circadian responses, two-component regulatory systems, MAP kinases as well as the cAMP-dependent and heterotrimeric G-protein pathways. Adenylate cyclase, a key component of the cAMP pathway, was identified as a potential hub for carbon source-specific differential protein interactions. In addition, four phosphorylated F-Box proteins were identified, two of which, Fbx-19 and Fbx-22, were found to be involved in carbon catabolite repression responses. Overall, these results provide unprecedented and detailed insights into a so far less well known stage of the fungal response to environmental cues and allow to better elucidate the molecular mechanisms of sensory perception and signal transduction during plant cell wall degradation

Structural and phylogenetic analysis of Xf-VapD.

<p>(A) The prediction of the 3D structure of Xf-VapD was performed using Phyre V 2.0 (red cartoon). A model was obtained with 66% of the Xf-VapD sequence and 100% confidence using the single highest scoring template. The structure was edited using PyMol. On the right, the amino acid sequence is shown with the respective secondary structure and confidence. The amino acid residues highlighted in red correspond to the area in the red cartoon shown in the 3D structure. (B) The neighbor-joining consensus tree inferred for the amino acid sequences of VapD. The values above the branches indicate the Bayesian posterior probabilities for the amino acids (PPaa) and nucleotides (PPnt). The values below the branches indicate the results of the neighbor-joining method for the amino acids (BSaa) and nucleotides (BSnt) following 1,000 bootstrap replicates. The minus symbol (‒) indicates that no support was reached for this node. The groups are the same as those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145765#pone.0145765.g001" target="_blank">Fig 1</a>.</p

Purification of recombinant Xf-VapD.

<p>A chromatogram showing the two steps used in the purification of Xf-VapD: Xf-VapD fused with a trx-tag was obtained first, and then trypsin cleavage was used to remove the trx-tag. On the right, a 12.5% SDS-PAGE is shown for the Xf-VapD purification process. The lanes are as follows: M, protein marker; 1, Xf-VapD fused to trx-tag (31 kDa); 2, Xf-VapD and trx-tag after cleavage with trypsin; 3, totally purified Xf-VapD (16.5 kDa); and 4, trx-tag (~13 kDa). The gel was stained using Coomassie brilliant blue.</p

Western blot analyses of Xf-VapD expression in <i>X</i>. <i>fastidiosa</i> cells.

<p>(A) Strain 9a5c in planktonic and biofilm growth. (B) Strain J1a12 in planktonic growth. The band values were obtained using ImageJ software. The values above the bars indicate the average of three biological replicates. The error bars indicate the standard errors of the means. The asterisk (*) indicates a significant difference compared to the days of growth indicated in the brackets (Student’s <i>t</i>-test, <i>p</i><0.05).</p

Multiple sequence alignments to VapD proteins.

<p>Group 1: The <i>X</i>. <i>fastidiosa</i> strains 9a5c and Ann-1 (Xf9a5c and XfAnn-1, respectively), <i>G</i>. <i>anatis</i> (Ga), <i>A</i>. <i>actinomycetemcomitans</i> (Aa), and <i>N</i>. <i>meningitides</i> (Nm). Group 2: <i>E</i>. <i>coli</i> (Ec), <i>X</i>. <i>campestris</i> (Xc), and <i>H</i>. <i>influenza</i> (Hi). Group 3: <i>H</i>. <i>pylori</i> (Hp) and <i>R</i>. <i>equi</i> (Re). The identity values for Xf-VapD are shown in parentheses at the end of each sequence.</p

Secondary structure and thermo stability assay of Xf-VapD.

<p>(A) Circular dichroism spectrum (200 nm–260 nm) of the recombinant purified Xf-VapD. The secondary structure of the protein was found to be 69% α-helices, 19% β-sheets, and 12% random coils. (B) Thermal unfolding/refolding of Xf-VapD. At 83°C during the heating phase, 50% of the proteins were unfolded (red marker). The conformational structure of the protein was not restored during the cooling phase.</p