Cloning And Functional Analysis Of XopX Gene And AvrBs3/PthA Gene Of Xanthomonas Oryzae

The gram-negative plant-pathogenic bacteria Xanthomonas oryzae pv.oryzae(Xoo) and Xanthomonas oryzicola pv.oryzicola(Xooc),which are the most important bacterial pathogens in the rice,cause bacterial blight and bacteria leaf streak in rice,respectively. These pathogen possess hrp genes and avirulence(avr) or virulence(vir) as other gram-negative plant pathogenic bacteria.The hrp genes confer the pathotgenic bacteria to the ability to trigger hypersensitive response(HR) on nonhost plants and to be pathogenic on susceptible host plants.The avirulence(avr) or virulence(vir) genes determine host specificity via gene-for-gene interactions.XopX effector protein from Xanthomonas campestris pv.vesicatoria(Xcv) is proposed to targets basic innate immunity in plants,resulting in enhanced plant disease susceptibility. However,the function of the homolog with xopX revealed by the genome sequence of X.oryzae pv.oryzae(Xoo) and X.oryzicola pv.oryzicola(Xooc) is uncertain.A XopXoo gene of Xoo and A xopXoc of Xooc were cloned and mutated.Both of the xopX mutants induced hypersensitive response in tobacco and maintained water soaking symptoms in rice seedlings,but it was greatly reduced in causing disease symptoms and multipling in rice leaves.Complementation of the xopX mutants with xopXoo and xopXoc,respectively, recovered the bacterial growth and the ability to cause the lesion length to the wild type,suggested that xopXoo and xopXooc involves in pathogenicity of Xoo and Xooc in rice. Interestingly,xopXoc,2800bp,contains a 2271 bp open reading frame with the ATG start codon,but no PIP box in the promoter region.The gene product is putatively a 756 amino acid protein and weights 79.5 kDa.And the C region of XopXoc has a CDK (cyclin-dependent kinase)-activating kinase(AAK97227) homolous peptide of the serine/threonine-specific kinase subfamily,which is an essential virulence determinant.Hpal and xopXoc genes were deleted and a double hap1-xopX mutant was obtained.These mutants all maintained water soaking symptoms in rice seedlings,but extremely reduced in causing disease symptoms and bacterial population in rice.The double hap1-xopX mutant was the lowest in bacterial population,and the hpa1 mutant was lower than the xopXoc.Interestingly,double hap1-xopX mutant did not trigger HR in tobacco, while the single mutation either in hpal or xopX locus kept the ability to elicit HR.Reverse transcriptional analysis indicated that the marker genes in the signaling pathways in HR caused by the hpal and xopX mutants,respectively,were trasncripted and expressed differently.Hpal and xopX genes induce HR in plant through various ddefence signal pathways,such as salicylic acid-mediated SAR(system acquired resistance),including the marker gene PR-1a(pathogenesis-related protein),and MAPK signaling cascades,including the MAPK(mitogen-activated protein kinase),WIPK(wound-induced protein kinase) and HMGR2(3-hydroxy-3-methylglutaryl CoA reductase),xopXoc mutant didn't induce the transcription of HMGR2 before 4 hours after syringe infiltration in tobacco.Hpa1 may inhibite the transcription of HMGR2.The interactions between Xanthomonas oryzae pv.oryzae and rice are controlled in a gene-for-gene manner.However,the basis of the gene-for-gene specificity is not well understood.In this lab,a DNA fragment(359 bp) of the avrXa3 gene containing three nuclear localization signal(NLS) motifs present in all members of avrBs3/pthA family was used as a probe to screen the genomic library of C8 strrain of Xoo.We found that C8 strain particularly possesses about 6.0Kb size of diverse members of the family existed in the pathogen genome.So,C8 genome was digested with BamHI and 5.5-6.5 kb fragment were collected,forming a partial genomic library.2 positive clones,avr/pthC8a and avr/pthC8b, were obtained through colony in situ hybridization.Compared with other avr/pth genes, Partial sequence of avr/pthC8a shows avr/pthC8a dose not have a BamHI enzyme site which is conserved in the family.The positive avr gene clones were transferred into the compatible recipient PXO99~A.It is found that in DV85 and Asominiori warter soaking disappeared and it was not apparently increased in bacterial population.Pathogenicity tests in near isogenic lines of rice confirmed that the lesion length was rifely declined.It showed avr/pthC8a and avr/pthC8b were,might be directively,involved in avirulence of the pathogen and the formation of warter soaking symptom.