Identification Of The Transcriptional Regulator HpaR1 Interacting Protein In Xanthomonas Campestris Pv.Campestris

Xanthomonas campestris pv.campestris(Xcc)is an important model strain for studying the interaction between plant pathogens and plants.In our previous work,we have identified HpaR1 as an important global transcriptional regulator in Xcc,which can regulate a variety of cellular processes,and found that HpaR1 can co-regulate a series of cellular processes through interaction with another global transcriptional regulator Clp.Therefore,whether HpaR1 interacts with other proteins in the genome is a question that needs to be answered in this project.In this study,co-immunoprecipitation(Co-IP)was used to screen proteins that can interact with HpaR1.We first constructed an Xcc strain capable of expressing a 3×Flag tag HpaR1(HpaR1::3×Flag)protein,then performed a Co-IP experiment.The proteins captured by immunoprecipitation was analyzed and identified by mass spectrometry,and a series of candidate protein that interacts with HpaR1 were obtained.Through Pull-Down and bacterial two-hybrid experiments,it was confirmed that HpaR1 interacts with the two proteins numbered XC?1355 and XC?3597 in the Xcc genome.XC?1355 is annotated as a DNA-binding protein of the HU family,and we first conduct genetic analysis of its function.By constructing deletion mutants and performing phenotype testing,it was found that the extracellular polysaccharides and extracellular enzymes of the XC?1355 gene deletion mutants had no significant changes compared with the wild-type strain,but the pathogenicity was significantly reduced.In order to understand the functional relationship between hpaR1 and XC?1355,we further constructed a cross-complementary strain of hpaR1 and XC?1355 genes and double deletion mutants of these two genes in this study,and test a series of phenotype analysis.The results showed that the hpaR1 gene can improve the pathogenicity of the XC?1355 deletion mutant,indicating that the hpaR1 gene may have partial functional redundancy or functional complementarity with the XC?1355 gene.In order to explore whether the XC?1355 protein regulates the expression of the hpaR1 gene,we overexpressed and purified the XC?1355 protein,and performed EMSA experiments on the obtained protein and hpaR1 promoter DNA fragments,and found that XC?1355can bind to the promoter region of the hpaR1 gene.This study identified a new HpaR1 interaction protein XC?1355,and analyzed the function of XC?1355,which confirmed that XC?1355 is a DNA binding protein that regulates the pathogenicity of Xcc.This study provides a solid foundation for further research on the regulation mechanism of HpaR1 and the pathogenic mechanism of Xcc.