A Research Of The Biological Function Of Cyclic Diguanylate Receptor N5MapZ In Citrus Cankers

The Xantho,monas axonopodis pv.citri(Xac)invades the leaves,branches and fruits of the host,causing the citrus canker disease,which is one of the most important bacterial diseases that seriously harm citrus production worldwide.Cyclic diguanylate(c-di-GMP)is an important second messenger molecule widely existing in bacteria discovered in recent years,regulating a variety of biological processes of cell differentiation,motility,and the production of various virulence,including many biological functions of the three types of secretory system and effector factors,extracellular polysaccharides(EPS),extracellular enzymes and so on,which is a key regulator of bacterial pathogenicity.GGDEF domain proteins usually have the diguanylate cyclase(DGCs)activity and are responsible for c-di-GMP synthesis,while EAL or HD-GYP domain proteins typically have phosphodiesterase(PDEs)activity and are responsible for c-di-GMP degradation.Cyclic diguanylate regulates multiple physiological functions through binding to its receptors.At present,41 c-di-GMP receptors have been found in Xacjx-6,of which four are PilZ protein receptors,XAC1133,N5MapZ,XAC1971,and XAC3518,respectively.The study found that the PilZ domain protein XAC1133 interacts with the EAL domain protein FimX(XAC2398)and regulates the function of the type IV secretion system;it was first discovered that the PilZ domain protein XCC6012 can exist as a tetramer,while XAC1971 and XCC6012 are homologous proteins;XAC3518 is related to the synthesis of cellulose;N5MapZ has not been reported as an important receptor protein of c-di-GMP of PilZ domain.Previous studies have shown that PilZ proteins regulate physiological functions through protein and protein interactions.In this study,the yeast two-hybrid library screening method was used to identify the interaction proteins of N5MapZ,preliminary identification of prmC as the interaction candidate protein of N5MapZ,further biological function analysis of the mutants of the two genes,the results suggest that the pair of proteins Interaction between may be real.This study may reveal novel mechanisms of action of c-di-GMP receptors,the results of the study are as follows.1.Screening and Identification of N5MapZ Interaction Proteins.First,we constructed a genomic expression library of Xacjx-6 strain,using yeast two-hybrid(Y2H)method,and N5MapZ protein as the bait protein,and found two interacting candidate proteins which are prmC and XAC3490 respectively,Subsequently,the interaction between prmC and target protein N5MapZ was mainly verified by bacterial two-hybrid and Co-IP.The results showed that there was a strong interaction between the prmC and N5MapZ proteins in the yeast system;while in the bacterial system,the interaction of the two proteins was not as strong as the yeast system but there was still a weak interaction.However when it was tested in vitro using Co-IP,no interaction between these two proteins was detected.Therefore,whether there is an interaction between these two proteins still needs to be further explored.2.Functional studies of N5MapZ and prmC.We first constructed a mutant strain of N5MapZ and prmC,and a double mutant strain of N5MapZ and prmC,respectively,by using the method of homologous recombination.Then the phenotypes of the mutants were determined.A complementary strain of A prmC was constructed and a functional complementation experiment was performed.The study found that the phenotype traits regulated by the prmC mutant strain were significantly more than the phenotypic traits regulated by the N5MapZ mutant strain.Compared with Xacjx-6,the growth rate of ?AprmC decreased,the extracellular polysaccharide yield decreased,the extracellular enzyme activity decreased,the motility was almost immobile,the ability to form biofilm increased,and there was precipitation when it was grown in the liquid medium of NYG.And C-AprmC can basically restore the trait of AprmC to the level of wild type strain;the growth rate of ?N5MapZ;the formation of extracellular polysaccharide,the activity of enzyme and the motility have no obvious changes compared to that of wild type strain;but the overexpression strain of N5MapZ had a slight decrease in its motility and increased biofilm production.