Abstract
Polyhydroxyalkanoates (PHAs) are intracellular carbon and energy storage materials produced in various microorganisms under nutrient-limited conditions. PhaR is a regulatory protein involved in PHA synthesis. Xanthomonas oryzae pv. oryzae (Xoo) is one of the most important bacterial pathogens in rice and has PHA biosynthesis genes in its genome, but the biological function of phaR in Xoo is unknown. In this study, we investigated the effects of the mutagenesis of phaR gene in Xoo strain PXO99A. Compared to the wildtype, the PhaR gene knock-out mutant PXO99ΔphaR was hypermotile and showed decreased growth rates in both rich and limited nutrient media. PXO99ΔphaR also showed almost 75% decrease in extracellular polysaccharide (EPS) production. When inoculated in rice leaves by leaf-clipping method, PXO99ΔphaR displayed reduced virulence in terms of lesion length and bacterial multiplication compared with the wildtype strain. PXO99ΔphaR also showed enhanced hypersensitive response (HR) induction in the leaves of non-host Nicotiana benthamiana with elevated hpa1 gene expression. Introduction of a cosmid containing the phaR coding sequence restored the phenotypes of the mutant to those of the wildtype strain. These results suggest that PhaR gene is an important gene that affects multiple bacterial characteristics, including EPS production, growth rate, defense response induced harpin production and motility, related to its virulence in plant.
Highlights
Bacterial blight on rice caused by Xanthomonas oryzae pv. oryzae (Xoo) is a widely prevalent disease that causes serious rice production losses worldwide (Singh et al, 1978; Nino-Liu et al, 2006)
We demonstrated that loss of phaR in Xoo strain PXO99A alters multiple physiological and biological functions which affect bacterial growth and virulence in plant, and provided new insights into the biological function of PhaR in X. oryzae pv. oryzae
Considering phaR gene is a stress responsible gene, we selected two time points, the first at 12 h representing the early stage of growth with the equal growth rates of mutant and wildtype strains, the other at 22 h representing the later stage with differential growth rates of mutant and wildtype strains
Summary
Bacterial blight on rice caused by Xanthomonas oryzae pv. oryzae (Xoo) is a widely prevalent disease that causes serious rice production losses worldwide (Singh et al, 1978; Nino-Liu et al, 2006). Bacterial blight on rice caused by Xanthomonas oryzae pv. Regulation of PhaR in Xanthomonas and non-host, like tobacco, Xoo largely depends on Type III secretion system (T3SS) to secrete different effector proteins to cause disease or induce hypersensitive response (HR) (Wei et al, 1992; Alfano and Collmer, 2004; Song and Yang, 2010). In the model PHA-producing strain Ralstonia eutropha H16, PhaR functions as a repressor or autoregulator for the expression of PhaP and PhaR itself, both of which can tightly bind to PHB granules (York et al, 2002; Pötter et al, 2005). PHA synthesis related genes exist in phytopathogenic bacteria, including Xanthomonas, but limited research exists on the biological functions of these genes during bacterial growth and host infection. We demonstrated that loss of phaR in Xoo strain PXO99A alters multiple physiological and biological functions which affect bacterial growth and virulence in plant, and provided new insights into the biological function of PhaR in X. oryzae pv. oryzae
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