<i>X</i>. <i>fastidiosa</i> biofilm formation in different grape saps under continuous flow in microfluidic chambers.

<p><i>X</i>. <i>fastidiosa</i> biofilm formation in different grape saps under continuous flow in microfluidic chambers.</p

<i>Xylella fastidiosa</i> biofilm formation and aggregation in saps from PD susceptible and resistant <i>Vitis</i> spp. in microfluidic chambers.

<p><i>X</i>. <i>fastidiosa</i> was cultured in xylem saps from different susceptible and resistant <i>Vitis</i> spp. for a period of ten days. <i>V</i>. <i>vinifera</i> was from California. Black arrow points to biofilm mound. Scale bar is 50 μm.</p

<i>Xylella fastidiosa</i> populations in different saps in renewed culture.

<p>Saps were <i>Vitis vinifera</i> (squares), <i>V</i>. <i>aestivalis</i> (diamonds), and <i>V</i>. <i>champinii</i> (triangles). Shown are mean log units of populations with standard deviation error bars.</p

Biofilm formation by <i>Xylella fastidiosa</i> in microfluidic chambers.

<p>(A) Sap was pumped in through the inlets on the left, cells through the inlets on top and bottom, and outflow through the outlets on the right. The chamber model used has two 1 mm wide channels running in parallel in which <i>X</i>. <i>fastidiosa</i> was cultured in sap from a susceptible and resistant hosts (top and bottom channels, respectively). The insert shows scanning electron micrograph of the mature biofilm structure obtained following disassembly of the microfluidic chamber after 10 days. The wall dividing the channels is 50 μm wide. (B) Time-lapse of aggregation and biofilm development of <i>X</i>. <i>fastidiosa</i> cultured for 10 days in xylem sap from Pierce’s disease-susceptible <i>Vitis vinifera</i> (left) and Pierce’s disease-resistant <i>V</i>. <i>champinii</i> (right). White arrows point to star-shaped aggregates and black arrows shows mounds. Scale bar is 50 μm. (C) Z-scan of pillars and mound structures in mature biofilm of <i>X</i>. <i>fastidiosa</i> formed in <i>V</i>. <i>vinifera</i> sap. Series of images captured after ten days of growth under continuous flow of Pierce’s disease-susceptible <i>V</i>. <i>vinifera</i> xylem sap in a microfluidic chamber. Images obtained in a central region of the chamber and through Z-scan from the lower to upper chamber surface, which is a distance of 160 μm. Note the central region is devoid of cells and is surrounded by three major mounds (one on the left and two on the right). Scale bar is 100 μm. Diagram on bottom right represents three biofilm mounds in the Z-scan with each plane, shown as a box outlined in light grey, representing the position of the captured images.</p

Characterization of the <i>Xylella fastidiosa</i> PD1671 Gene Encoding Degenerate c-di-GMP GGDEF/EAL Domains, and Its Role in the Development of Pierce’s Disease

<div><p><i>Xylella fastidiosa</i> is an important phytopathogenic bacterium that causes many serious plant diseases including Pierce’s disease of grapevines. <i>X</i>. <i>fastidiosa</i> is thought to induce disease by colonizing and clogging xylem vessels through the formation of cell aggregates and bacterial biofilms. Here we examine the role in <i>X</i>. <i>fastidiosa</i> virulence of an uncharacterized gene, PD1671, annotated as a two-component response regulator with potential GGDEF and EAL domains. GGDEF domains are found in c-di-GMP diguanylate cyclases while EAL domains are found in phosphodiesterases, and these domains are for c-di-GMP production and turnover, respectively. Functional analysis of the PD1671 gene revealed that it affected multiple <i>X</i>. <i>fastidiosa</i> virulence-related phenotypes. A Tn5 PD1671 mutant had a hypervirulent phenotype in grapevines presumably due to enhanced expression of <i>gum</i> genes leading to increased exopolysaccharide levels that resulted in elevated biofilm formation. Interestingly, the PD1671 mutant also had decreased motility <i>in vitro</i> but did not show a reduced distribution in grapevines following inoculation. Given these responses, the putative PD1671 protein may be a negative regulator of <i>X</i>. <i>fastidiosa</i> virulence.</p></div

Putative PD1671 domains.

<p><b>A</b>) Boxes represent the three PD1671 domains with domain names above the boxes and amino acid numbers below the boxes. Bacterial diguanylate cyclase and phosphodiesterase consensus sequences listed in boxes (X is any amino acid), and PD1671 aligned sequences listed below the boxes at their approximate locations. Arrow head denotes Tn5 insertion point. <b>B</b>) REC domain alignment. <i>Xylella fastidiosa</i> PD1671 REC domain alignment with functional REC protein and <i>X</i>. <i>fastidiosa</i> predicted c-di-GMP protein containing REC domain. Grey boxed/bold amino acids are the phosphorylation site, grey boxed/non-bold amino acids are the intermolecular recognition site, and bold/underlined amino acids are the dimerization interface. <b>C</b>) GGDEF domain. Top sequence group is hybrid GGDEF-EAL domain-containing proteins enzymatic in both domains, middle sequence group is non-enzymatic hybrid GGDEF-EAL domain-containing proteins, and bottom sequence group is <i>X</i>. <i>fastidiosa</i> predicted GGDEF domain proteins. Underlined amino acids are the allosteric I site, RxxD, and grey boxed/bold amino acids are the GGDEF sequences. Underlined/bold PD1671 residues denote a potential RxxD site. <b>D</b>) EAL alignment. Top sequence group is hybrid GGDEF-EAL containing proteins enzymatic in both subunits, middle sequence group is non-enzymatic hybrid GGDEF-EAL domain proteins, and bottom sequence group is <i>X</i>. <i>fastidiosa</i> predicted EAL proteins. Grey boxed/bold amino acids are signature EAL sequence and underlined/bold residues are DDFGTG sequences. Alignment comparison sequences: Ec = <i>Escherichia coli</i>, Lp = <i>Legionella pneumophilia</i>, Ms = <i>Mycobacterium smegmatis</i>, Mt = <i>Mycobacterium tuberculosis</i>, Pa = <i>Pseudomonas aeruginosa</i>, Pf = <i>Pseudomonas fluorescens</i>, Pp = <i>Pseudomonas putida</i>, Rs = <i>Rhodobacter sphaeroides</i>, Vp = <i>Vibrio parahaemolyticus</i>, Xf = <i>Xylella fastidiosa</i>, Xo = <i>Xanthomonas oryzae</i>.</p

Relative <i>X</i>. <i>fastidiosa</i> exopolysaccharide (EPS) production.

<p>^a Average dry weight of EPS (mg mL^-1). The experiments were performed three times with five replicates each. The standard deviations are shown.</p><p>^b Statistically significant compared to wild-type (<i>P</i><0.0001).</p><p>Relative <i>X</i>. <i>fastidiosa</i> exopolysaccharide (EPS) production.</p

Relative <i>X</i>. <i>fastidiosa</i> exoenzyme activity.

<p>^a Extracellular enzyme activities were estimated from the diameter (mm) of the halo zones of supernatant enzymatic activity surrounding each well. All assays were performed three times, with five replicate plates each. The standard deviations of the means for each enzyme are shown.</p><p>Relative <i>X</i>. <i>fastidiosa</i> exoenzyme activity.</p

Oligonucleotide primers used in this study.

<p>^a RT-PCR—reverse transcriptase-polymerase chain reaction.</p><p>Oligonucleotide primers used in this study.</p

Relative <i>X</i>. <i>fastidiosa</i> RNA levels.

<p>^a RT-PCR (reverse transcriptase-polymerase chain reaction) experiments performed in <i>Vitis vinifera</i> xylem sap (three to six independent experiments with three replicates each). The standard deviations of the normalized means are shown. Expression of the gene regions was normalized to <i>dnaQ</i> gene expression [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121851#pone.0121851.ref042" target="_blank">42</a>]. Gene segments amplified: PD1671-REC domain (115 to 357bp), PD1671-GGDEF domain (601 to 858bp), PD1671-EAL domain (1325 to 1621bp), <i>gumD</i> (628–847bp), and <i>gumJ</i> (4–229bp).</p><p>^b Statistically significant compared to wild-type (<i>P</i><0.01).</p><p>Relative <i>X</i>. <i>fastidiosa</i> RNA levels.</p