| Two model bacterial pathogens in rice,Xanthomonas oryzae pv.oryzicola(Xoc)and X.oryzae pv.oryzae(Xoo),belonging to Xanthomonas oryzae species,cause bacterial leaf streak(BLS)and bacterial blight(BB)in rice,respectively.The diseases are two of the most severe diseases in rice and constrain rice production in China and the world.These two pathogens possess hrp genes as other gram-negative plant pathogenic bacteria.The hrp genes confer the pathogenic bacteria to the ability to trigger hypersensitive response(HR)on nonhost plants and to be pathogenic on susceptible host plants.The clustered hrp genes also encode conserved proteins composing of a type Ⅲ secretion system(T3SS)that delivers T3SS effectors into host cells to cause disease or to trigger defense in plants.However,it is unclear what roles of Xanthomonas oryzae hrpD6 gene plays in HR in tobacco and in pathogenicity in rice.In this study,we constructed a deletion mutant of hrpD6 gene by using marker-exchange method.PCR and Southern blot analysis demonstrated that the hrpD6 gene was knocked out successfully.in planta assays indicated the hrpD6 mutant lost the ability to induce HR in tobacco,to trigger water-soaked symptoms in seedlings rice and to cause BB or BLS in adult rice.Importantly,the bacterial growth in rice tissues was tremendously reduced.Complementation assays confirmed that hrpD6 gene could restore HR induction in tobacco,pathogenicity and bacterial growth in rice to the hrpD6 mutant.Reverse transcriptional polymerase chain reaction(RT-PCR)revealed that the expression of hrpD6 was not only induced by rice cells,but also controlled by hrpG and hrpX.Intriguingly,the expression of hpal,encoding a harpin protein,was dependent on hrpD6,implying that hrpD6 regulates the expression of hpal.the yeast two blot showed that HrpD6 interacted with hpal,hpa2,and hrpD5.Western bolt and secretion assay with avrXa10 as a reporter demonstrated that HrpD6 protein was secreted through T3SS apparatus into rice cells.Our results provide molecular clues to understand whether hrpD6 is involved in the formation of T3S apparatus and in regulation of other hpa-hrp-hrc gene expression or not for HR induction in tobacco and pathogenicity in riceScreening the rice genes which interact with T3SS effectors is the basis to understand molecular interactions between host plants and pathogens.The yeast two-hybrid system(Y2H)is one of the most effectively molecular and genetic methods to isolate unknown proteins with known baits.In order to isolate rice gene which may interact with Hrp proteins(some maybe T3SS effectors)of Xooc,eight hrp genes in the hrp cluster were constructed into a vector pGBKT-7,respectively,as the baits to prey their interacting proteins from a rice cDNA library that was obtained previously in our lab.Totally,we got eight groups rice genes which were preyed by hrcN、hrcU、hrcQ、hrpB4、hrpB5、hrpD5、hrpD6 and hpaF,respectively.This suggested that these Hrp proteins are not only involved in T3SS formation,but in interaction with host cells.There are no interacting proteins in rice with the corn T3SS components.To investigate how HrpD6 interacts with its interacting protein OSHRPD6,we first sequenced OshrpD6 gene.The sequence showed that OshrpD6 encods a S-adenosyl-L-methionine synthetase(SAMS).SAMS is a important enzyme for plant metabolism which catalyzes biosynthesis of SAM from Met and ATP.Comparison of OSHRPD6 with its homologous in other plants indicated that OSHRPD6 is also existence in corn、barlay and wheat plant.Transcriptional expression assays displayed the expression of OshrpD6 was constutitive in rice,but increased after 8 hours when rice was infected by the pathogen.Northern blot results confirmed that the expression of OshrpD6 was decreased when rice was inoculated with the hrpD6 or hrpX mutants.This strongly suggest that OSHRPD6 is a target of HrpD6.The HrpD6-OSHRPD6 interaction provides a clue for further understanding how OSHRPD6 involves in X.oryzae-rice pathosystem. |
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