Identification And Functional Characterization Of The Phosphodiesterase Pde R-interacting Proteins In Xanthomonas Oryzae Pv. Oryzae

Xanthomonas oryzae pv. oryzae（Xoo） causes bacterial leaf blight of rice, which is one of the most important bacterial diseases in worldwild rice production. Xoo enters xylem tissues of rice leaves through water pores or wounds. Occurrence of the disease mainly depedents on the virulence factors including type III system and its effectors, exopolysaccrides（EPS） and extracellular enzymes. c-di-GMP is a second messenger wildly existing in bacteria, regulating a variety of biological process, such as biofilm formation, motility, virulence and cell cycle. The diguanylate cyclases（DGCs） containing GGDEF domain and phosphodiesterases（PDEs） containing EAL or HD-GYP domians are responsible for c-di-GMP synthesis and degradation, respectively. Previous work in our lab showed that Pde R is a response regulator containing GGDEF, EAL and REC domains, which harbors PDE activity to degrade c-di-GMP. Pde R, together with histidine kinase Pde K forms a two-componet system to regulate EPS production and bacterial virulence. This study is to further understand the molecular mechanism of Pde R-mediated virulence regulation in Xoo, by searching Pde R’s interacting proteins and analyzing the functions of these interactors. The goal is to clarify the functional mechanism of Pde R and provide more scientific basis for fully understanding the c-di-GMP signaling pathway and bacterial pathogenesis. The main results are listed as following: 1. Screening and identification of Pde R-interacting proteins in XooFirst, we constructed the c DNA library of PXO99 A, then performed yeast two-hybrid（Y2H） screening using Pde R as bait to obatin Pde R-interacting proteins. After Y2 H and GST pull-down experiments, we confirmed two interacting proteins: PXO₀4421（renamed as Tri P） and PXO₀0987. The interaction between Tri P and Pde R was inhibited by high concentration of c-di-GMP. Tri P is a predicted Omp R/Pho B family transcriptional regulator, containing an N-terminal REC domain and a C-terminal helix-turn-helix（HTH） DNA binding domain, which is supposed to regulate downstream genes transcription. PXO₀0987 is one of the acetyltransferases in Xoo, which is located in the flagellar regulon of Xoo and might be involved in regulating flagellar motility. 2. Functional analysis of transcriptional regulator Tri PWe firstly generated a tri P in-frame deletion mutant using homologous recombination method, and measured its effect on phenotypes and bacterial virulence. Compared with wild-type strain PXO99 A, the tri P mutant showed decreased EPS production and attenuated Xoo virulence, while flagellar-dependent swimming motility was not influenced. These phenotypes were consistence with those of the pde R mutant, suggesting that they might be in the same signaling pathway. We further analyzed the differentially expressed genes in tri P and pde R mutants by RNA-seq technology. The results indicated that mutation of tri P tends to stimulate genes expression, while mutation of pde R tends to inhibit genes expression besides inactivating some genes expression, suggesting that Tri P and Pde R have common, but also distinct regulatory pathways.3. Functional analysis of acetyltransferase PXO₀0987We constructed an PXO₀0987 in-frame deletion mutant using homologous recombination method, and studied its effect on phenotypes and bacterial virulence. Compared with wild-type strain, PXO₀0987 deletion mutant resulted in decreased EPS production, increased flagellar-dependent motility, decreased biofilm formation and attenuated Xoo virulence. Thus we speculated that the attenuated virulence might be due to reduced level of virulence factors. Furthermore, we thought PXO₀0987 might be involved in flagellin posttranslational modification since it is located in the predicted glycosylation island of Xoo. We extracted flagellin proteins from wild-type and PXO₀0987 mutant strains to test their glycosylation. Result showed that mutation of PXO₀0987 abolished the glycosylation of wild-type flagellins, indicating that PXO₀0987 is involved in flagellin glycosylation modification. 4. Subcellular localization analysis of Pde RIn this study, we constructed a Pde R-GFP fusion expression vector to observe Pde R’s subcellular localization in Xoo, and found that Pde R mainly located at two poles of the cell. We then analyzed the factors which might be responsible for its pole-localization status. Firstly, we studied whether Pde R’s three functional domains affect its localization. The results showed that GGDEF and REC domians are required for Pde R’s pole localization. Secondly, we studied whether Pde R’s interacting proteins Pde K and Tri P affect its subcellular localization. The result showed that Tri P influenc Pde R’s pole localization. Therefore, the subcellular localization of Pde R was determined by its interacting protein Tri P, and t he REC and GGDEF domains.In summary, through functional analysis of Pde R’s interacting proteins, we demonstrated that Tri P was invovled in the c-di-GMP signaling pathway mediated by Pde R. PXO₀0987 had its own regulatory functions and invovled in flagellin glycosylation, but whether it was also invovled in the Pde R-mediated signaling pathway remains unclear. Furthermore, we found that Pde R was mainly located at the poles of bacterial cells, which might be related to its virulence regulation. Therefore, thi s study has further revealed the regulatory mechanism of Pde R and provide more scientific evidence for understanding the c-di-GMP signaling mechanism in Xoo.