Translocation Of Effector PthXol From Bacterial Blight Pathogen Into Rice Cells And Identification Of Rice Exportin For PthXol Target Gene

The Cya reporting system is the most widely applied in studying the effector secretion and transport of plant pathogenic bacteria.The first BlaM(beta lactamase)report system designed for the study of the pathogenic bacteria of animals,the transformation material is cell,not tissue or biological whole,the technology is convenient and easy to do.Based on the BlaM reporting system,the author explored and established a set of real-time detection techniques for the interaction of pathogenic bacteria and rice protoplasts to mediate the transport of BlaM marker effector.Xanthomonas oryzae pv.Oryzae(Xoo)and X.oryzae pv.Oryzicola(Xoc)are major bacterial pathogens in rice,and rmain species of plant pathogenic xanthomonds.The pathogenic bacteria plays a pathogenetic role through a variety of effector(transcription activator-like effectors,TALEs).In the coevolution between pathogenic bacteria and their host plants,different TALE effect proteins play a role as the virulence or avirulence factor on different cultivars.In the long-term game between Xanthomonas and its host rice,Xanthomonas can retain the TALE effector protein,beneficial to its infection and reproduction.These TALE effector proteins,such as PthXo1 from Xoo strain,usually play the role as virulence factors.Similar to transcriptional activation,these effectors have specific target genes in rice.When rice is attacked by Xanthomonas,these virulence factor can be transported to t rice cells by type ? secretion system,changing the expression of specific target genes in host plants.Previous studies have found that the TALE effector protein PthXo1 of Xoo can induce the expression of its target gene OsSWEETll,thereby affecting the pathogenicity of bacteria or the defense response of host.However,little is know about the process after the up-regulation of target gene.We speculated that a class of nuclear export proteins might assist them to export from the nucleus,which is also poorly understood.In the present study,author aim to identify the nuclear export protein that can assist OsSWEET11 in transporting PthXo1.We selected PthXol TALE effect in Xoo,and used the constructed knockout and covering strains in our lab to inoculate the susceptible cultivar Nipponbare.Based on that,author analysed the relative expression levels of 6 Exportin gene by RT-qPCR assay.Author found that 6h and 12h after inoculation,the expression of ExportinT,Exportin4 and Exportin7 were up-regulated;while 24h and 48h after inoculation,PthXo1 can induce the expression of Exportin7.These results suggest that Exportin7 can assist OsSWEET11 to export from the nucleus.To prove our hypothesis,Exportin7 was selected as the candidate gene to carry out the follow-up experiment.A plant expression vector pCAMBI1300 was then constructed by ligating the cloned fragment to OsExportin7 gene,used for ChIP(chromatin immunoprecipitation assay)experiment.Author found that OsExportin7 could bind to the OsSWEET11 promoter region and CDS region,confirming that OsExportin7 can assist OsSWEET11 in transporting PthXol from the nucleus.To test the point further and specify the location of OsExportin7 in palisade cells,a plant expression vector pCAMBI1300:YFP was then constructed by ligating the cloned fragment to OsExportin7 gene,used for the Agrobacterium-mediated transient expression analysis.The results showed that OsExportin7 mainly located in nucleus.This was further proof about the findings in the ChIP-PCR results.Then author used the yeast two-hybrid system to investigate the interaction of the TALE effector PthXo1 and OsExportin7,and there was no significant interaction.In order to determine the pathogenicity of PthXol on non host plant tobacco,author observed the possible eliciting hypersensitive response in tobacco.The results showed that the loss of PthXo1 could decrease the pathogenicity of Xanthomonas on non host plant tobacco.Author further analysed the relative expression levels of disease-susceptibility genes and disease-resistance genes(Xa1,xa5,Xa13,xa13,Xa3/26)by RT-qPCR assay.The results showed that PthXol can induce the expression of disease-susceptibility genesXa13,Xa3/26,while it can inhibit the expression of xa13 gene.Of these,SWEET11(Xa13)is the target gene of the PthXol,which is consistent with the conclusion of previous research.