Functional Identification Of C-di-GMP Signaling Components Regulated By Filp/PXO₀2715 In Xanthomonas Oryzae Pv. Oryzae

Bacterial blight of rice caused by Xanthomonas oryzae pv.oryzae?Xoo?is one of the most important bacterial diseases,threatening the safe production of rice in the world,especially in the subtropical regions and in many countries in Africa.Xoo is both an important plant pathogen and one of the bacterial models for studying the molecular mechanism of interaction with plants.Cyclic guanosine phosphate?c-di-GMP?signaling pathways are involved in regulation of virulence expression in Xoo.Under c-di-GMP signaling pathways regulations,Xoo can use a variety of virulence factors to regulate virulence in rice.Diguanylate cyclase?DGC?with a GGDEF domain synthesizes c-di-GMP,while phosphodiesterase?PDE?harboring the EAL or HD-GYP domains degrades c-di-GMP.Filp?PXO₀0403?is a FimX-like protein with degenerate EAL/GGDEF domains that loses the DGC and PDE enzymatic activity,but acts as a c-di-GMP receptor in Xoo.Filp specifically interacts with the degenerate PilZ domain protein PXO₀2715,thereby co-regulating bacterial virulence.In our lab early study,by isobaric quantitative labeling?iTRAQ?assay,about 150 differentially-expressed proteins?DEPs?co-regulated by Filp and PXO₀2715 were identified from the comparative analysis of wild type strain PXO99^A,single gene deletion mutant?filp and?PXO₀2715,and double gene deletion mutant?filp?PXO₀2715.In this study,functional characterization of 6 DEPs were performed,including two c-di-GMP metabolism-related proteins PXO₀0476 and PXO₀4753,two TCS-related proteins PXO₀4752 and PXO₀4157,one PhoPQ-regulated protein PXO₀1585,and one Ton B receptor PXO₀1883.Based on 6 previously-constructed gene deletion mutants of six DEPs,the construction of the relevant complementary strains was carried out in this study.The wild type,mutant and complimentary strains were comparatively assayed for their phenotypes,such as virulence,motility,extracellular polysaccharide?EPS?production,biofilm formation,and growth in vitro or in vivo.In addition,enzymatic activities for c-di-GMP turnover,subcellular localization in the cell,and protein interactions with Filp and PXO₀2715 of 6 proteins were also analyzed.The results showed that PXO₀0476,PXO₀4752 and PXO₀1883 positively regulated virulence of Xoo,in contrast,PXO₀1585 and PXO₀4157 negatively regulated bacterial virulence,while PXO₀4753 had no significant effect on virulence.Enhanced sliding motility in?PXO₀0476,?PXO₀4157,?PXO₀1585,and?PXO₀1883 and decreased one in?PXO₀4752 and?PXO₀4753 were observed compared to that in wild type.No significant changes in swimming motility in all of mutants were found.Compared with wild type,?PXO₀0476,?PXO₀4157,?PXO₀4752,and?PXO₀4753 showed increased EPS production,and?PXO₀0476,?PXO₀1883,and?PXO₀4157 displayed enhanced biofilm formation,and other mutants did not change significantly.The PDE activity of PXO₀0476 and PXO₀0476^D53A,PXO₀4753 and PXO₀4753^E192A was examined by Ultra-performance liquid chromatography?UPLC?analysis,demonstrating that PXO₀0476had PDE activity,and PXO₀4753 had relatively weak PDE activity.But,PXO₀0476^D53A and PXO₀4753^E192A did not degrade the c-di-GMP as compared to original proteins.GFP fusion expression and cell localization analysis showed that PXO₀0476,PXO₀1585,and PXO₀4157 showed the same non-polar localization in Xoo cells as Filp and PXO₀2715 did,while PXO₀4752 and PXO₀4753showed unipolar localization.No direct interactions between each of six proteins with either Filp or PXO₀2715 were observed by Y2H analysis.It is suggested that Filp/PXO₀2715 might indirectly regulating the expression of these proteins that ultimately affect c-di-GMP level and Xoo virulence.