Xylella fastidiosa pil-chp operon is involved in regulating key structural genes of both type I and IV pili

Xylella fastidiosa is the causal agent of Pierce's disease (PD) in grapevines. It has type I and type IV pili, which are both virulence factors involved in the PD-associated processes of motility, aggregation, and biofilm formation. Many questions remain as to how the two pili are regulated. We previously identified a X. fastidiosa pil-chp chemosensory-like cluster as an operon composed of genes pilG-I-J-L-chpB-C. In this study, we deleted pilG (resulting in a ∆pilG-I strain) and pilJ and discovered that both mutants (∆pilG-I and ∆pilJ) had reduced virulence after 24 weeks post-inoculation, whereas ∆chpB and ∆chpC did not. Both ∆pilG-I and ∆pilJ lost motility and were impaired in biofilm formation in rich artificial media and xylem sap. Gene expression was significantly downregulated for representative fimbrial adhesin and motility genes in ∆pilG-I, and to a lesser extent in ∆pilJ. Our data suggest that Pil, but not Chp, proteins are virulence factors, and pilG-I-J are involved in transcriptional regulation of type I and IV pili virulence genes and therefore motility and biofilm formation. To our knowledge, this is the first report of a chemotaxis-like operon involved in the regulation of key structural genes of both type I and type IV pili

In vitro inhibition of pathogenic Verticillium dahliae, causal agent of potato wilt disease in China by Trichoderma isolates

Twenty (20) of Verticillium dahliae were isolated from wilted potato specimens collected from six districts in Guizhou, China. All the isolates were evaluated for pathogenicity on two potato cultivars, Favorita (susceptible) and Hui-2 (resistant) using the root dip inoculation (RDI) and microsclerotia inoculation (MI). All of the V. dahliae isolates appeared to be pathogenic on both cultivars but VGZ-HZ-4 isolate gave the highest wilt incidence comparing to the others, seconded by VGZ-SC-1 and VGZ-XW-1. Combined analysis of wilt incidence resulting from using two inoculation methods for VGZ-HZ-4 and VGZ-XW-1 isolates on the two potato cultivars showed that the MI gave a higher wilt incidence than that of the RDI and cultivar Favorita had a higher wilt incidence than that of Hui-2. These two V. dahliae isolates were further used as representative isolates for mycelial inhibition (MyI) test with 33 Trichoderma isolates under a dual culture condition on potato dextrose agar plate. The 33 Trichoderma isolates consisting of 21 isolates isolated from potato soils from seven districts of Guizhou, 11 isolates from single spore isolates of the TGZ-150 isolate preserved at Guizhou Institute of Plant Protection (GZIPP) and one isolate TGZ-OLD-81 also preserved at the GZIPP. Most of the single spore isolates and TGZ-SC-4 were found to have higher MyI efficiency than that of the rest. The results indicate that the Trichoderma isolates in this study have initial modes of action of biological control to protect potato crop against V. dahlia.Keywords: Trichoderma, potato wilt disease, growth inhibition, Verticillium dahliae, antagonistic fungi.African Journal of Biotechnology, Vol 13(31) 3402-341

Identification of an Operon, Pil-Chp, That Controls Twitching Motility and Virulence in Xylella fastidiosa

Xylella fastidiosa is an important phytopathogenic bacterium that causes many serious plant diseases, including Pierce`s disease of grapevines. Disease manifestation by X. fastidiosa is associated with the expression of several factors, including the type IV pili that are required for twitching motility. We provide evidence that an operon, named Pil-Chp, with genes homologous to those found in chemotaxis systems, regulates twitching motility. Transposon insertion into the pilL gene of the operon resulted in loss of twitching motility (pilL is homologous to cheA genes encoding kinases). The X. fastidiosa mutant maintained the type IV pili, indicating that the disrupted pilL or downstream operon genes are involved in pili function, and not biogenesis. The mutated X. fastidiosa produced less biofilm than wild-type cells, indicating that the operon contributes to biofilm formation. Finally, in planta the mutant produced delayed and less severe disease, indicating that the Pil-Chp operon contributes to the virulence of X. fastidiosa, presumably through its role in twitching motility.United States Department of Agriculture Cooperative State Research, EducationUnited States Department of Agriculture Cooperative State Research, EducationUniversity of CaliforniaUniversity of CaliforniaNanobiotechnology CenterNanobiotechnology CenterNational Science Foundation (NSF)National Science Foundation (NSF)[ECS-9876771

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

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

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

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> 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