Functional Characterization Of Pxo₀3877 And PXO₀1021 Which Putatively Encode C-di-GMP-metabolizing Enzymes In Xanthomonas Oryzae Pv. Oryzae

Rice bacterial leaf blight is a serious bacterial disease caused by Xanthomonas oryzae pv. oryzae（Xoo）. Xoo-rice interaction is one of the important model systems to study the pathogen-host plant interactions. C-di-GMP is a ubiquitous second messenger existing in bacteria, regulating the pathogenicity, biof ilm formation, EPS production, and many other biological functions. Diguanylate cyclases（DGC） and phosphodiesterase（PDE） control the synthesis and degradation of c-di-GMP, respectively. In recent years, our laboratory has been continuously studing the c-di-GMP signaling pathways and its regulatory mechanism in Xoo to reveal its role in virulence regulat ion. Previous studies have identif ied PXO₀3877 and PXO₀1021 genes of Xoo. They encode EAL single-domain protein and PAS/GGDEF/ EAL hybr id protein respectively, based on which we speculated that they might perform PDE and DGC activity, respectively. In this study, we firstly carried out comparative analys is of c-di-GMP-metabolism related genes in Xoo by bioinformatics tools, and then thoroughly analyzed the biological functions of PXO₀3877 and PXO₀1021 gene focusing on essential sites and each domain. The main results are listed as follows.1. Comparative analysis of the GGDEF, EAL and HD-GYP domain proteins in Xoo genome. At genom ic level, bioinformatics analysis was carried out on c-di-GMP-metabolism related genes in Xoo strains PXO99 A, MAFF311018 and KACC10331. A variety of different signal input domains（REC, PAS, GAF, HAMP, etc.） are encoded by these genes, suggesting they are able to perceive environment stimuli and correspondingly regulate the activity of downstream output domains. By analyzing sequence characteristics of the output structure domains（GGDEF, EAL and HD-GYP）, we found some proteins may possess DGC or PDE activity, while others might function asc-di-GMP receptors or binding proteins. Although these 77 genes are scattered in the genomes, there are some hot spots with gene aggregation. Most homologous genes in the three strains share similar sequences, while several of them only exist in one or two strains.2. Functional analysis of PXO₀3877 point mutant and its complementary strains. We constructed complemented strains for the PXO₀3877 mutant containing either the full length of PXO₀3877, or the point-mutation version（?PXO₀3877:: E153 A and ?PXO₀3877::E176A） to test their virulence-related phenotypes. The results showed that ?PXO₀3877 and ?PXO₀3877:: E153 A resulted in significantly decreased virulence, biofilm formation and EPS production. ?PXO₀3877::E176A has no significant effect on phenotypes. Especially, ?PXO₀3877:: E153 A is much less EPS production than ?PXO₀3877. The motility of all strains has no obvious difference. The results indicated that E153, but not E176 is essential for the activity of PXO₀3877.3. Functional analysis of PXO₀1021 complementary and overexpression strains. We constructed the complemented strain of PXO₀1021 mutant with the full- length gene, and over-expression strains containing the full- length PXO₀1021, or its GGDEF or EAL domains to test their virulence-related phenotypes. The results showed that the PXO₀1021 mutant had increased virulence, while overexpression strain of full- length PXO₀1021 had decreased virulence. Meanwhile, overexpression strains containing GGDEF or EAL domains of PXO₀1021 did not show signif icant changes. All strains had no significant effect on biofilm formation, EPS production, and motility. As GGDEF and EAL domains might interfere with each other’s function, that’s probably why the single domain of PXO₀1021 was not sufficient for theactivity and only the full length displayed complementary activity.In summary, we used bioinformatics tools to analyze the types, numbers and sequence characteristics of c-di-GMP-metabolism related genes in Xoo. In addition, we focused on the function of two genes PXO₀3877 and PXO₀1021, and confirmed that they played the opposite roles in regulat ing the pathogenicity of Xoo. The relationship between pathogenicity and extracellular polysaccharides or biofilm formation was further studied. These results will help us elucidate the c-di-GMP signaling pathway and its regulatory mechanisms.