Virulence Analysis And Detection Of T3SS-secreted Effectors Of Xanthomonas

X.oryzae pv.oryzae,a pathovar of X.oryzae,causes bacterial leaf blight(BLB)on rice(Oryzae sativa).BLB is one of the most serious diseases in rice and makes great loss in rice production.A type ? secretion system(T3SS)in the pathogen plays a key role in determining BLB disease development in rice by injecting many T3SEs(T3SS-secreted effectors)into plant cells.Two kinds of T3SEs were classified into transcriptional activator-like effectors(TAL effectors)and Non-TAL effectors.Normally the T3SS device is highly conserved in Xanthomonas species.To better understand the differences of the T3SS and T3SEs and what roles of T3SEs take in pathogenicity of Xanthomonas species,in the first part of this study,sixty Xoo strains from nine rice growing provinces of China were collected.Using the central Sph I-fragment of pthXo1 gene as a probe,Southern blotting assay was run to investigate the numbers and sizes of TAL effector genes in those isolates.Based on the sizes and numbers of tal genes,these strains were classified into 16 different tal genotypes(designated 1 to 16).To determine the pathogenicity of these tal genotypes in near isogenic lines(NILs)of rice which contain different resistant(R)genes,sixteen tal genotype strains were inoculated in nine NILs by leaf clipping method.The pathogenicity assay results indicated that GD0612and JS137-1 strains were much stronger virulent in the rice lines,whereas KS1-20,GZ29,GD36 and OS14 genotypes weak.The clade ? of the SWEET gene family,consisting of OsSWEET11,OsSWEET12,OsSWEET13,OsSWEET14 and OsSWEET15,has been proved to be major BLB susceptibility genes(S)in rice.To determine whether the expression of these genes are activated or not,these 16genotype strains were inoculated in a genome-sequenced rice line(Nipponbare).qRT-PCR results demonstrated that most strains had the ability to activate the expression of one or two SWEET genes,particularly OsSWEET13 and OsSWEET14,while only LN4 strain did not,suggesting that there is(are)unknown susceptible genes in rice targeted by unknown tal gene(s)of LN4.Non-TAL effectors have been proved to suppress PAMP-triggered immunity(PTI)in plants.To know the virulence roles of six Non-TAL effector genes in rice,avrBs2(inhibiting PTI in plants),hpa1(triggering hypersensitive response(HR)in tobacco and other four genes(xopP,xopY,xopAA and xopL,causing HR in tobacco)were selected.Thus,in the second part of this study,a tal-free mutant of PXO99~A,designed PH,was used as a recipient for deletion of these six gene gradually by a marker free knock-out strategy.Then,all the deletion of these six genes resulted in a mutant PH?PAAY21L.Interestingly,PH?PAAY21L still had ability to induce HR in tobacco,implying that there is(are)still other unknown HR elicitor(s)in the mutant.After introducing PthXo1(a major virulent effector for BLB development in rice)into sets of the mutant regarding these six genes,the pathogenicity test in rice indicated that AvrBs2 and Hpa1 played more important role in contribution to bacterial virulence than other four.Most species of Xanthomonas genus in?-Proteobacteria are plant pathogenic bacteria,causing HR on non-host plants and pathogenicity on susceptible host plants.However,it remains unclear which Xanthomonas species lacking of T3SS and T3SEs that can be the targets to design specific probes for quarantine detection.Here,seven Xanthomonas species were collected and detected.PCR and Southern blot results showed that X.campestris pv.musacearum ICMP287 and ATCC49084 strains,X.axonopodis pv.vasculorum ATCC13901 strain,and X.axonopodis pv.allii LMG576 and LMG578 strains lacked the tal genes which encode TAL effectors,of these three species X.axonopodis pv.vasculorum ATCC13901possesses neither hpa1 nor xopQ and hrcC genes for the T3SS;and X.campestris pv.phaseoli ATCC49119 strain possesses xopQ and hrcC genes but hpa1.HR assays on tobacco demonstrated that only X.axonopodis pv.glycines ATCC43911 strain did not trigger HR,despite that hpa1,xopQ,hrcC and tal genes were detected in this strain.These results provided fundamental basis for further understanding the differences of pathogenic determinants and designing specific quarantine probes among them.