Characterization of the Xylella fastidiosa PD1671 gene encoding degenerate c-di-GMP GGDEF/EAL domains, and its role in the development of Pierce's disease.

Luciana Cursino,Thomas J Burr,Cheryl D Galvani,Leonardo De La Fuente,Kelly R Patel,Yaxin Li,Dusit Athinuwat,Harvey C Hoch,Patricia Mowery,Paulo A Zaini,Leo Eberl

doi:10.1371/journal.pone.0121851

Abstract

Xylella fastidiosa is an important phytopathogenic bacterium that causes many serious plant diseases including Pierce’s disease of grapevines. X. fastidiosa 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 X. fastidiosa 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 X. fastidiosa virulence-related phenotypes. A Tn5 PD1671 mutant had a hypervirulent phenotype in grapevines presumably due to enhanced expression of gum genes leading to increased exopolysaccharide levels that resulted in elevated biofilm formation. Interestingly, the PD1671 mutant also had decreased motility in vitro but did not show a reduced distribution in grapevines following inoculation. Given these responses, the putative PD1671 protein may be a negative regulator of X. fastidiosa virulence.

Highlights

Xylella fastidiosa is a motile, xylem-limited bacterium transmitted to plants by xylem sap-feeding insects [1]
We report examination of the X. fastidiosa PD1671 protein, and provide evidence that its sequence is degenerative in both the GGDEF and EAL domains; it is involved in biofilm formation, presumably through regulation of gum gene expression and extracellular polymeric substance (EPS) production, and subsequent Pierce’s disease development
Gene segments amplified: PD1671-REC domain (115 to 357bp), PD1671-GGDEF domain (601 to 858bp), PD1671-EAL domain (1325 to 1621bp), gumD (628–847bp), and gumJ (4–229bp). b Statistically significant compared to wild-type (P

Summary

Introduction

Xylella fastidiosa is a motile, xylem-limited bacterium transmitted to plants by xylem sap-feeding insects [1]. Numerous molecular regulators are proposed to facilitate switching from motility to biofilm formation, such as the quorum sensing molecule diffusible signal factor (DSF) [13]. Hybrid proteins have been identified that contain both GGDEF and EAL domains [14,15,16,17]. The Eal protein contains an EAL domain and was recently shown to be involved in antibiotic resistance and biofilm formation [20]. RpfG has an HD-GYP domain and is a response regulator involved in quorum sensing and biofilm formation [13]. We report examination of the X. fastidiosa PD1671 protein, and provide evidence that its sequence is degenerative in both the GGDEF and EAL domains; it is involved in biofilm formation, presumably through regulation of gum gene expression and EPS production, and subsequent Pierce’s disease development

Materials and Methods

Results

Discussion