Effect Of Gene Deletion Of Clpx And SpeE On Pathogenicity In Xanthomonas Oryzae Pv.oryzicola

Rice bacterial leaf streak(BLS),caused by with Xanthomonas oryzae pv.oryzicola(Xoc),is one of the most important diseases of rice.The disease has become another major rice disease after rice blast,bacterial leaf blight and rice sheath blight,causing serious damage to rice in China and Southeast Asia.At present,there is a lack of comprehensive and in-depth research on the pathogenic mechanism of bacteria Xoc.Identifying pathogenic-related genes and analyzing the functions of different genes are great significance for revealing the pathogenic mechanism of Xoc.In this study,transposon Tn5 was used to mutagenize X.oryzae pv.oryzicola Xoc RS105,and a mutant library containing 1500 strains was constructed.After in vivo screening,two mutants with altered pathogenicity were obtained from 1500 transposon insertion mutants,which are numbered respectively R-160 and R-168.The flanking sequences of the transposon Tn5 insertion site in the two mutants were amplified by inverse PCR.By sequence alignment,it was found that the transposon Tn5 in the mutants R-160 and R-168 was inserted into ATP-dependent Clp protease ATP-binding subunit encoding gene clp X and spermidine synthase encoding gene spe E respectively.Based on the above results,gene deletion strains ?clp X and ?spe E were constructed by knocking out the clp X and spe E genes in framework from Xoc RS105 by homologous recombination.The pathogenicity of the above two genes deletion strains to rice was determined,and the results showed that the strains ?clp X and ?spe E were less pathogenic than the wild-type strain Xoc RS105,which were found respectively the pathogenic ability of the mutants R-160 and R-168 is similar.The results indicated that the genes clp X and spe E belonged to the pathogenic related genes of rice pathogen Xoc RS105.ClpX belongs to the Hsp100 protein family and is a component of the intracellular protein quality cont-rol system,which plays roles in recognizing and degrading abnormal proteins in various biological cells to maintain homeostasis in the cell.In order to reveal the possible role of ClpX in pathogenicity of Xoc,the complementary strain ?clp X::clp X of the clp X gene knockout strain and the heterologous complementary strain ?clp X::clp XEc of the clp X gene from Escherichia coli were constructed.The pathogenicity-related phenotypes of wild-type Xoc RS105 and its derivatives were determined,and the results showed that afterclp X deletion,the pathogenicity was reduced.Other traits such as motility,production of extracellular proteases and polysaccharides,growth,sensitive to heavy metal ion and antibiotics stress,changes under test conditions.the exercise ability became weak;the ability to produce extracellular proteases decreased by 25%;and the ability to produce extracellular polysaccharides decreased by 20%,which indicates that the clp X gene has a certain degree of resistance to stress in Xoc;the colonization ability of ?clp X in rice leaves decreased in the colonization experiment.By the heterologous complementation of the clp X gene of bacteria and the clp X gene from E.coli,the phenotype can be restored,indicating that the clp X gene plays an important role in Xanthomonas oryzae in environmental adaptation.In order to analyze the function of spe E gene and its role in pathogenicity,the content of spermidine in cells of spe E knockout strain ?spe E and its wild-type Xoc RS105 in the growth phase of stable phase was determined by high performance liquid chromatography HPLC.The content of spermidine in the cells of?spe E was significantly lower than that in the wild-type Xoc RS105,which indicating that the gene spe E is involved in the synthesis of spermidine.On this basis,the complementary strain ?spe E::spe E of the spe E deletion strain was constructed and determined.The pathogenicity-related phenotypes of wild-type Xoc RS105 and the above-mentioned strains were determined,the results showed that after spe E was deleted,the pathogenicity was reduced;the motility was enhanced;the resistance to heat was weakened;it is sensitive to Erythromycin,Streptomycin,Vancomycin,vitamin K3,Triton X-100,which shows that spe E gene has a certain effect on antibiotics,metal ion,oxidative stress and other stress in Xoc;the colonization ability of ?spe E in rice leaves decreased in the colonization experiment,and the above phenotypes can be restored after spe E gene complementation.Spermidine is ubiquitous in eukaryotic and prokaryotic cells and regulates the process of biological adaptation to adversity.This study suggests that the spe E gene in Xoc bacteria is involved in the synthesis of spermidine to regulate the environment adaptation and pathogenicity of the bacteria.