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

Oligonucleotide primers used in this study.

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

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> 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

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

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

Movement of the <i>X</i>. <i>fastidiosa</i> PD1671 mutant in microfluidic chambers.

<p>Twitching movement speed of wild-type <i>X</i>. <i>fastidiosa</i> Temecula 1, PD1671 mutant, and complemented PD1671 mutant (PD1671-C) cells in microfluidic flow chambers. Values shown are means and standard errors from three independent experiments. Letters above bars indicate significant differences by Kruskal-Wallis test and means were separated by the Kruskal-Wallis all pairwise comparison test (<i>P</i> = 0.02)</p

Biofilm production by the <i>X</i>. <i>fastidiosa</i> PD1671 mutant.

<p>Quantification of biofilm formation in <i>Vitis vinifera</i> xylem sap with agitation for wild-type <i>X</i>. <i>fastidiosa</i> Temecula 1, PD1671 mutant, and complemented PD1671 mutant (PD1671-C). Representative experiment shown. Different letters represent significant difference when means are compared (<i>P</i> = 0.001).</p