Identification And Functional Analysis Of Downstream Components Co-regulated By Cyclic Di-GMP Receptor Filp And Its Interacting Protein PXO₀2715 In Xanthomonas Oryzae Pv.Oryzae

It has been shown that the cyclic diguanylate?c-di-GMP?signaling pathway is involved in regulation of bacterial virulence,extracellular polysaccharide?EPS?production,and biofilm formation,motility,etc.in the bacterial blight pathogen of rice Xanthomonas oryzae pv.oryzae?Xoo?.Several signal receptors play an important role in c-di-GMP signal recognition,reception and transmission.Filp,a c-di-GMP receptor interacts with the PilZ-domain protein PXO₀2715,both co-regulating the virulence and related gene expression in Xoo.However,the molecular mechanisms by which Filp and PXO₀2715 co-regulate the virulence expression in Xoo have not been elucidated.Therefore,identification and functional analysis of the downstream key components co-regulated by Filp/PXO₀2715 were performed in the current study,aiming to provide the scientific proof and basis for elucidating the c-di-GMP signaling pathway and its regulation of virulence in Xoo.1.Screening and identification of the Filp/PXO₀2715 co-regulated proteins.Quantitative proteomics analysis of the wildtype PXO99^A,the single gene deletion mutants?filp and?PXO₀2715,and the double gene deletion mutant?filp?PXO₀2715 was carried out by using iTRAQ technology.About 108 differentially-expressed proteins were identified to be co-regulated by Filp and PXO₀2715.Functional annotation indicated that these proteins were mostly involved in regulation of a variety of important biological functions in bacterial cells.Among them,10 Filp/PXO₀2715 co-regulated proteins were selected for the further investigation,including PXO₀0476?a two-component system regulatory protein with HD-GYP domain?,PXO₀1515?a transcriptional regulator LuxR/uhpA family?,PXO₀1585?a PhoPQ-regulated protein?,PXO₀1644 and PXO₀1883?two TonB-dependent receptors?,PXO₀3510?a prophage Lp2 protein 6?,PXO₀4157?a histidine kinase-response regulator hybrid protein?,PXO₀4752?a chemotaxis protein?,PXO₀4753?a response regulator with EAL domain?,and PXO₀5583?a phosphate-binding protein Pst S?.2.Verification of the Filp/PXO₀2715 co-regulated protein expression.Total proteins extracted from PXO99^A,?filp,?PXO₀2715,and?filp?PXO₀2715 respectively were analyzed by using Western Blot with the antibodies of ten selected proteins,while GAPDH was used as the reference protein.Results showed that expression levels of 6 proteins?PXO₀0476,PXO₀1515,PXO₀1883,PXO₀4752,PXO₀4753,and PXO₀5583?were down-regulated in each of three mutants compared with PXO99^A.In contrast,expression levels of 4 other proteins?PXO₀1585,PXO₀1644,PXO₀3510and PXO₀4157?were up-regulated in each of three mutants compared with PXO99^A.These observations of the Filp/PXO₀2715 co-regulated protein expression patterns completely verified those of the iTRAQ analysis in Part 1.3.Transcriptional analysis of the Filp/PXO₀2715 co-regulated protein genes.qRT-PCR analysis was performed to demonstrate the transcriptional patterns of the Filp/PXO₀2715 co-regulated protein genes in PXO99^A,?filp,?PXO₀2715,and?filp?PXO₀2715.Results indicated that transcript levels of 6 protein genes?PXO₀0476,PXO₀1515,PXO₀1883,PXO₀4752,PXO₀4753,and PXO₀5583?were down-regulated in each of three mutants compared with PXO99^A.In contrast,transcript levels of 4other protein genes?PXO₀1585,PXO₀1644,PXO₀3510 and PXO₀4157?were up-regulated in each of three mutants compared with PXO99^A.These gene transcription patterns were consistent with the protein expression patterns of the Filp/PXO₀2715 co-regulated proteins in Part 2.4.Functional analysis of Filp/PXO₀2715 co-regulated proteins.To reveal the biological functions of 3 co-regulated proteins?PXO₀0476,PXO₀4157 and PXO₀4752?,gene deletion mutagenesis via homologous recombination and non-marker exchange,complementation and phenotypic analysis?virulence,motility,EPS production,biofilm formation,and the extracellular enzyme activity?were performed.Results showed that PXO₀0476 and PXO₀4752 positively regulated the virulence on rice,while PXO₀4157 negatively regulated virulence.Compared with PXO99^A,?PXO₀0476,?PXO₀4157,and?PXO₀4752 displayed increased motility and EPS production.?PXO₀0476 was impaired in biofilm formation,while?PXO₀4157 and?PXO₀4752showed enhanced biofilm formation.There were no significant changes in extracellular activities of cellulase and xylanase between the wildtype and mutants.In trans expression of the full-length PXO₀0476,PXO₀4157,and PXO₀4752 in the relevant mutants restored the phenotypes to near-wildtype levels.In addition,significant increase in intracellular c-di-GMP content was found in?PXO₀0476 compared with that of PXO99^A.Therefore,PXO₀0476?a HD-GYP domain protein?might function as a phosphodiesterase?PDE?to degrade c-di-GMP,thereby regulating bacterial virulence,biofilm formation,flagellar motility,and EPS production in either positive or negative manners.In summary,through functional identification of the key components co-regulated by c-di-GMP receptor Filp and its interactor PXO₀2715,this study provides some of novel insights into c-di-GMP signaling pathway and its mediation of virulence regulation in Xoo.