Identification Of Two Interacting Proteins Of Cyclic Di-GMP Receptors Filp And PXO₀2374in Xanthomonas Oryzae Pv. Oryzae

A series of signal transduction pathways involved in the regulation of virulence and infectionprogress during interaction with host plant rice. C-di-GMP is a ubiquitous second messenger present ina wide variety of bacteria signaling cascade, regulating virulence, biofilm formation,exopolysaccharides, motility, and other processes. C-di-GMP receptors as signal recognition, receivingand delivery units can play a regulatory function in gene transcription, translation and post translationlevel. Previous study shows that PXO99Astrain encodes26c-di-GMP related proteins, the changes ofc-di-GMP can influence production of EPS and pathogenicity of Xoo, indicate that c-di-GMP signalingsystem is very important in regulation of Xoo pathogenicity. It has been proved that Filp which encodesdegenerated GGDEF and EAL domains and PXO₀2374protein which has PilZ domain function ascyclic di-GMP receptors to regulate virulence of Xanthomonas oryzae pv. oryzae, however, regulationand mechanism of its downstream is unclear. In this study, further reseaech on screening thedownstream proteins will help us reveal c-di-GMP signaling mechanism in Xoo. Results are as follows.1. Identify that Filp can interact with PilZ domain protein PXO₀2715. Constructed yeastexpression plasmid pGBKFilp, confirmed bait doesn’t autonomously activate the reporter genes inY2HGold, and not toxic when expressed in yeast. Constructed yease expression vectors of three PilZdomain proteins in Xoo, pGAD2715, pGAD2374, pGAD49, results show Filp can interact withPXO₀2715, not PXO₀2374nor PXO₀0049by Y2H. Expressed and purified Filp protein with GSTtag and PXO₀2715protein with His6tag, proved that Filp can interact with PXO₀2715by GSTpull-down assay.2. Identify interacting domains of Filp with PXO₀2715. Constructed expression vectors with lackof different domian or sigle domain of Filp, analysed interaction model between different domains ofthe two proteins. Results show that lack of EAL domain of Filp cannot interact with PXO₀2715, lackof REC, PAS and GGDEF domain do not affect interaction between Filp and PXO₀2715. More over,GGDEF or EAL sigle domain of Filp can interact with PXO₀2715.3. Identify PXO₀2374interactive proteins using Y2H. Screening the cDNA library of Xoo byY2H system using PXO₀2374as bait protein, we got107positive clones. Through PCR andsequencing analysis, we got14candidate genes. Finally, we successfully screened two PXO₀2374specific interaction proteins PXO₀3510and PXO₀5583by Y2H and GST Pull-Down assay.In summary, we prove that Filp can interact with PilZ domain protein, and the EAL domain of Filpis necessary, also single domain GGDEF of Filp can also interact with PXO₀2715. PilZ domainprotein PXO₀2374can interact with PXO₀3510and PXO₀5583. Identification of Filp andPXO₀2374interactive protein can help us reveal the c-di-GMP signaling mechanisms in Xoo.