Construction And Characteristic Analysis For The HrpX/hrpG Gene Knock-out Mutant Of Xanthomonas Oryzae Pv. Oryzae

The gram-negative plant-pathogenic bacteria Xanthomonas oryzae pv.oryzae(Xoo) is the most important bacterial pathogens in the rice, which causes rice bacterial leaf blight. Like other Xanthomonas species, Xoo possesses an hrp gene cluster, which is essential for type III secretion systems(T3SS). Type III secretion system mediates the translocation of effector proteins across the bacterial membrane into the host cells, which are involved in induction of the hypersensitive response (HR) in resistant host or nonhost plants and pathogenicity in susceptible host plants. So, identification of the genes encoding T3SS effectors is a key step to understanding the function of the T3SS in plant pathogens. In Xanthomonas species, expression of the structural genes of the T3SS and some effector genes are mediated by the hrpG and hrpX gene products, both of which were present in Xoo as well as in Xac and Xcc. HrpX can mediate hrpB-hrpF region, several genes regulated in a HrpX-dependent manner possess the consensus nucleotide sequence TTCGC…N15…TTCGC (PIP box). The PIP box is a useful tool for identifying candidate genes in the HrpX regulon. hrpX gene and hrpA operon expressing is mediated by hrpG gene product. In Xoo, hrpX and hrpG genes are detached from hrp gene cluster, locating in tandem, and their transcriptional orientations are opposite. Between hrpX and hrpG codon sequences there are some noncoding sequences including their respective promoter sequences.In this study hrpX/hrpG genes of Xoo were cloned into a suicide mutant vector pKNG101, formed a mutant vector of pKNG-△hrpX/△hrpG. The Xoo mutant for hrpX/hrpG genes is acquired by kanamycin resistance selection through homologous recombination. The Xoo mutant for hrpX/hrpG genes was greatly reduced in causing disease symptoms. The result showed that hrpX and hrpG genes play an important role in pathogenicity of Xoo. Electron microscope observation discovered surface of mutant had more or less protuberances. Then five gene microarraies were made in CapitalBio corporatione, analyzing the gene expression profiles of Xoo JxoI,Xoc RS63,Xv5, mutant and water during different stages of interaction with rice. Many up-regulated and down-regulated genes were found. Comparing mutant and water, up-regulated genes were 1,061 and down-regulated genes were 1,620 from 57,259 detected genes. Meanwhile comparing JxoI and mutant, up-regulated genes were 356 and down-regulated genes were 382 from 57,259 detected genes. In this study, some gene expression dynamics were obtained by analyzing the gene expression profiles of during the different stages of interactions with rice. Some of the new candidate bacterial genes related to pathogenesis were identified. To understand the mechanism of pathogenic bacteria is crucial for developing strategies to prevent plant disease.