Abstract
Cyclic diguanylate monophosphate (c-di-GMP) is a secondary messenger present in bacteria. The GGDEF-domain proteins can participate in the synthesis of c-di-GMP as diguanylate cyclase (DGC) or bind with c-di-GMP to function as a c-di-GMP receptor. In the genome of Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial blight of rice, there are 11 genes that encode single GGDEF domain proteins. The GGDEF domain protein, PXO_02019 (here GdpX6 [GGDEF-domain protein of Xoo 6]) was characterized in the present study. Firstly, the DGC and c-di-GMP binding activity of GdpX6 was confirmed in vitro. Mutation of the crucial residues D403 residue of the I site in GGDEF motif and E411 residue of A site in GGDEF motif of GdpX6 abolished c-di-GMP binding activity and DGC activity of GdpX6, respectively. Additionally, deletion of gdpX6 significantly increased the virulence, swimming motility, and decreased sliding motility and biofilm formation. In contrast, overexpression of GdpX6 in wild-type PXO99A strain decreased the virulence and swimming motility, and increased sliding motility and biofilm formation. Mutation of the E411 residue but not D403 residue of the GGDEF domain in GdpX6 abolished its biological functions, indicating the DGC activity to be imperative for its biological functions. Furthermore, GdpX6 exhibited multiple subcellular localization in bacterial cells, and D403 or E411 did not contribute to the localization of GdpX6. Thus, we concluded that GdpX6 exhibits DGC activity to control the virulence, swimming and sliding motility, and biofilm formation in Xoo.
Highlights
The phytopathogen Xanthomonas oryzae pv. oryzae (Xoo) causes the bacterial leaf blight in rice, one of the most devastating bacterial diseases of rice [1]
diguanylate cyclase (DGC) indicated that GGE411 EF residues of active site (A-site), RXXD403 residues of I-site, Mg2+, and GTP binding sites were conserved in GdpX6 (Figure 1b)
The results showed that expression of point mutation protein GdpX6D403A -GFP in ∆gdpX6 restored the virulence to near-wild-type levels, while expression of GdpX6E411A -GFP in ∆gdpX6 showed similar virulence to that of the mutant ∆gdpX6 strain (Figure 4a,b)
Summary
The phytopathogen Xanthomonas oryzae pv. oryzae (Xoo) causes the bacterial leaf blight in rice, one of the most devastating bacterial diseases of rice [1]. Oryzae (Xoo) causes the bacterial leaf blight in rice, one of the most devastating bacterial diseases of rice [1]. Xoo mainly invades the rice through the wound or water pores, colonizes the xylem vessels of rice leaves [1]. The bacterial leaf blight disease causes different degrees of reduction rice yield with 70% yield reduction in the most severe cases [2,3]. Multiple virulence factors like exopolysaccharide (EPS), biofilm, extracellular enzymes, and adhesins contribute to the virulence of Xoo [4]. These virulence factors have been found to be regulated by several signaling systems, including the two-component systems, 4.0/)
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