Type Ⅲ Translocators In Xanthomonas Oryzae Pv.oryzae And Their Function In Suppressing Rice Defense Responses

Type Ⅲ(T3)proteic effectors occupy most of the virulence determinants in eukaryote-pathogenic Gram-negative bacteria.During infection,bacteria may deploy a nanomachinery called translocon to deliver T3 effectors into host cells,wherein the effectors fulfil their pathological functions.T3 translocon is hypothetically assembled by bacterial translocators,which have been identified as one hydrophilic and two hydrophobic proteins in animal-pathogenic bacteria but remain unclear in plant pathogens.This study show that Hpa2,HrpF and XopN have a transport effect on the PthXo1 effector in PXO99A strains,are translocator components of Xoo,and they can enter plant cells,but only XopN can inhibit host plant defense responses Xanthomonas outer proteins(Xops)are virulent effectors or virulence-assistant components secreted by the type Ⅲ secretion system in plant-pathogenic bacteria that belong to the Xanthomonas genus,including the rice bacterial blight pathogen.However,there is little knowledge about Xop effectors,different Xops functional mechanisms and their pathological responses to different rice varieties,and the degree of specialization of specific Xop toxic effects.1.Identification of the type Ⅲ translocon composition in Xanthomonas oryzae pv.oryzaeBy the Ji and Wang research of our laboratory,Experiment result shows Hpa1 protein can transport the two T3 effectors of bacteria PthXol and AvrXa10 into the host Nipponbare and IRBB10,respectively.Hpal was identified as a translocator.On this basis,we selected Hpa2,HrpF and XopN of PXO99A strains to continue research.Hpa2,HrpF and XopN were found to be indispensable virulence components of PXO99A by constructing related mutants for inoculation experiment.They had different effects on the virulence of PXO99A bacteria in susceptible rice varieties,and had no effect on the secretion of PthXol-Cya.The results showed that Hpa2,HrpF and XopN mediating from-bacteria-into-rice-cell translocation of PthXo1 by measuring the bacterial content and the transport of PthXo1-Cya,and the transport of PthXo1 will activate the expression of OsSWEET11,and its virulence caused the symptom of bacterial blight in susceptible rice varieties.And then,By constructing hpa2,hrpF and xopN single,double and triple gene knockout mutants of PXO99A strains,and comparing the translocation levels of PthXo1 by inoculation of Nipponbare,we found that the three translocators of Hpa2,HrpF and XopN cooperate to mediate PthXo1 transport.Finally,through the analysis of the secretion and transport,and the study of bacterial virulence,it was confirmed that Hpa2-Cya,HrpF-Cya and XopN-Cya were secreted through the T3 pathway of bacteria.XopN could inhibit the defense response of susceptible rice varieties,but Hpa2 and HrpF had no similar function.It is suggested that the different components of T3 translocation not only mediate the common function of bacterial effector transport,but also have unique virulence mechanism.2.Effector XopN of Xanthomonas oryzae pv.oryzae plays a Virulent Role in Rice Varieties That Possess OsSWEET11 HomologsXopN of Xanthomouas oryzae pv.oryzae PXO99A is a toxic effector that makes rice sick by affecting the host’s immune response.In our laboratory,Hu Li conducted a preliminary experimental exploration on whether XopN caused differences in pathogenic bacteria toxicity due to different rice varieties,and found that XopN of PXO99A strain has different pathogenicity on different rice varieties and will affect the HR response of tobacco.On the basis of this study,we conduct more in-depth research,and also verify the previous results.By using the double crossover homologous-recombination technique,the XopN gene-knockout mutant ΔxopN and genetically complementing strain ΔxopN/xopN were generated under background of the bacterial wild-type strain PXO99A.By comparing both recombinant strains with PXO99A in terms of bacterial multiplication in nutrition broth,It was found that XopN affects the reproduction speed of bacteria.Based on bacterial populations of PXO99A,ΔxopN and ΔxopN/xopN multiplied in rice leaves,as well as severities of subsequently developed leaf blight symptoms,the 14 rice varieties fall into two categories.The results showed that the effect of xopN gene knockout on Xoo toxicity was related to rice varieties,and showed great differences because of different rice varieties,XopN only affects the pathological responses of the four rice varieties IRBB13,IRBB208,Asominori,and Nipponbare;PXO99A has a lower virulence effect on IRBB13,but XopN has a greater effect on the virulence of bacteria;while PXO99A has a strong toxicity on IRBB208,Asominori,and Nipponbare rice varieties,but XopN has a relatively small effect on virulence.Through inoculation experiments and RT-qPCR analysis,it was found that XopN gene can promote the expression of dominant gene OsSWEET11 and inhibit the expression of recessive gene OsSWEET11.Through tobacco HR experiments,the knockout of xopN gene will slow down the HR response.Therefore,XopN is a pluripotent effector and plays a virulence role in rice varieties that possess OsSWEET11 or its homologs.XopN also is necessary to the bacterial multiplication and has an additive contribution to the induction of hypersensitive response by the bacteria in a non-host plant.