Establishment Of The Inducing Expression Detection System Of Xanthomonas Oryzae Pv.oryzicola T3SS Genes

Xanthomonas oryzae pv.oryzicola(Xanthomonas oryzae pv.oryzicola,Xoc)causes bacterial leaf streak(Bacterial leaf streak,BLS)in host plant rice,causing a threatening damage to rice-planting area in Asia.Xoc is mainly dependent on the type III secretion system(T3SS)which is encoded by hrp cluster(T3SS gene)for secreting the type III effector protein into rice cells,which leading to disease resistance or susceptibility in rice.As the key virulence associated factor of Xoc,T3 SS is the necessary component of the Xoc virulence network,however there is still a lack of an efficient expression detection system to analyze the Xoc virulence network.In order to analazy the T3 SS genes regulating expression efficiently,in this study we established the inducing-expression detection system of Xoc T3 SS genes containing both the transcriptional expression level and protein expression level.In order to establish the transcriptional-expression detection system,we constructed the T3 SS genes promoter-probe-vectors pHG2-hrpGxoc,pHG2-hrpXoc,pHG1-hrcCxoc.The result of promoter GUS activity assays and qRT-PCR showed that this transcriptional system is premilinary worked in XOM3 induction system and can efficiently analyze the gene transcriptional expression.The pHG2-hrpGxoc,pHG2-hrpXoc,pHG1-hrcCxoc were introduced into Xoc mutants of the virulence regulator genes trh,lrpX and zur,and the results of GUS activity assay and qRT-PCR indicated that this expression detection system can be effectively applied in analyzing the T3 SS gene transcriptional expression.Introduced pHG2-hrpXoc into RS105 and R?hrpG and inoculate into the leaf of host rice and non-host tobacco,and the results of GUS activity and bacterial growth indicated that this system can efficiently applied in regulating expression analisis in live plant tissue,which is an innovation in the interaction research technology.In order to analyze the T3 SS gene protein expression efficiently,in this study we used the synthetic biology to construct protein expression vectors pHM1-hrpGxoc::3myc ? pH1-hrpGxoc::3myc ?pH3-hrpGxoc::flag ? pHM1-hrpXoc::flag ? pH1-hrpXoc::flag ?pH3-hrcCxoc::flag.Introduced pHM1-hrpGxoc::3myc ?pH1-hrpGxoc::3myc?pHM1-hrpXoc::flag?pH1-hrpXoc::flag into RS105 and R?hrpG and the result of western blot showed that this system worked.Introduced the pH3-hrpGxoc::flag ? pH1-hrpXoc::flag ?pH3-hrcCxoc::flag into R?trh?R?zur?R?lrpX and the western blot interacting with XOM3 showed that the hrpG?hrpX and hrcC genes protein expression and transcriptional expression is consistent,indicating that this system can efficiently analyze the T3 SS protein expression.Introduced the plasmid of pHM1-hrpGxoc::3myc ?pH1-hrpGxoc::3myc into RS105 and R?hrpG and inoculated to the leaves of rice IR24,and the result of water soaking showed that these plasmids can restores the pathogenicity of R?hrpG on host rice,indicating that these protein expression vectors can also be effectively applied to functional complementation analysis.The results above indicated that the Xoc T3 SS gene inducing expression detection system established in this study can be effectively applied in gene expression analysis both in Xoc-XOM3,Xoc-host and non-host tissue interaction systems,and help to further analyze Xoc virulence regulation network and identificate key pathogenic virulence genes,which is help to develop pesticides against the pathogenic target genes and disease-resistant rice varieties.