The Establishment Of Genetic Operation System And The Functional Analysis Of Hrp Genes And Members AvrBs3/pthA Gene Family Of Xanthomonas Oryzae In Rice

Xanthomonas oryzae pv.oryzae(Xoo) and Xanthomonas oryzae pv.oryzicola(Xooc) cause bacterial blight(BB) and bacterial leaf streak(BLS) in rice(Oryza sativa), respectively.BB is one of the most serious diseases in rice,and BLS is emerging in importance in rice.Xoo and Xooc are considered as the model pathogens of rice,which is taken as the model cereal crop.A high throughput and reliable strategy is required to study the function,interregulation,and interplays among repertoire genes in pathogenicity,such as the hrp genes which determine the ability of the bacterium to trigger hypersensitive response(HR) in non-host plants and pathogenicity in host rice.Here we reported a novel target inserted mutagenesis in Xoo and Xooc through single exchange by a vector pK18mobGâ…¡and knock-out mutagenesis for single and multiple genes in Xooc through two homologous crossover events mediated by a suicide vector pKMS1 containing a sacB gene.Successfully obtain three hrpF and four hrcV inserted mutant with different sites of the strain PXo99~A of Xoo and four hrpF and hrcV inserted mutant with different sites of the strain RS105 of Xooc;and 5 single(hrcV,hrpF,hrpE,hpaB,and hpal),4 double(hpaland hpa2,hpa1 and hrpF,hrpE and hrpF,and hpaB and hrpF),and 2 triple(hpaB,hpa1 and hrpF,and hpa2,hpa1 and hrpF) hrp knock-out mutants of Xooc have been constructed in this report.The results suggest that the established mutagenesis for the target inserted and nonmarker knock-out of single and multiple genes in Xooc mediated by the suicide vector pK18mobGâ…¡and pKMS1 provides a fundamental approach to the functional genomics of Xanthomonas oryzae and will facilitate the understanding of pathogenicity mechanisms of Xanthomonas oryzae in rice. Evidence indicates that each race of Xanthomonas oryzae contain more than 15 members of avrBs3/pthA family genes.They have almost identical the 5 'and 3' terminals. The difference is the repeat number of the 102 bp repeat unit among them.In this study,we use the 359 bp DNA fragment of 3' terminal of the avrXa3 gene,a member of avrBs3/pthA family genes containing nuclear localization signals and acidic transcription activation domain,as the probe for genomic DNA Southern blot analysis of the Chinese Xoo and Xooc strains.The copies of avrBs3/pthA gene are different in races of these two pathogens and found some bands in all races and some of the bands are specific in certain races. Weaker virulent races contain fewer members of avrBs3/pthA family genes,and high virulent races keep more member of avrBs3/pthA genes with more repeat numbers of the 102 bp repeat unit.Using the same 359 bp fragment as the probe for colony in situ hybridization,we obtained the 35 positive clones from genomic library of Xooc strains RS105,then classified as 28 avrBs3/pthA genes after digestion and Southern blotting analysis.34 positive clones were introduced into PXO99~A strain of Xoo individually.The conjugants of PXO99A with individual avrBs3/pthA genes were inoculated into leaves of rice seedlings and adult rice, respectively,by needless syringes and leaf-clipping methods.We found that PXO99~A containing p6,p8,p9,p10 and p14 positive clones could trigger a specific HR (Hypersensitive response) in the rice eultivar IRBB10 containing a resistance gene Xa10. Sequencing showed that the genes in those positive clones were the members of the avrBs3/pthA family,thus named as avr/pth6,avr/pth8,avr/pth9,avr/pth10 and avr/pth14, respectively.Protein sequence comparison revealed that these five proteins at C-terminals lacked SGSVGGTI residues which are indispensable in AvrBs3/PthA members of Xoo.The flanking sequences comparison revealed that a specific GTTTCTG sequence existed in the promoter region of these avrBs3/pthA genes of Xooc but in that of Xoo.Interestingly,a direct and repeat sequence of a transferable transposon existed in the promoter and terminator regions in the avr/pth14.The BamHI fragment of the avr/pth6 and avr/pth8 genes replacing the corresponding BamHI fragment of the avrXa3 gene resulted in two new fusion genes,avrBre6 and avrBre8.These two fusion genes could retain avirulence to PXO99~A on IRBB10,respectively,suggested that avrBre6 and avrBre8 were the avirulence genes matching Xa10 in rice.This is the first report on avirulence genes in Xooc.The results in this study revealed that Xoo strain exist members of avrBs3/pthA genes,and probably some particular virulence factors inhibit avirulence activities of specific avrBs3/pthA genes in rice.We postulated that might be the reason that rice lacks major resistance gene against bacterial leaf streak.The hrcV gene in plant and animal pathogenic bacteria are highly conservative and the product of hrcV gene is the component protein of typeâ…¢secretion system(T3SS), suggesting to form a ring structure in the inner membrane of bacteria.In this study we obtained inserted mutants,PXV268,PXV917,PXV106 and PXV452,in PXO99~A strain and RSV268,RSV917,RSV106 and RSV452 in RS105 strain,respectively,through single homologous exchange method,and a deleted mutant RS105â–³hrcV through double homologous exchange method.PXV268 and RSV268 mutants still triggered HR on nonhost and retained pathogenicity on rice.The rest of hrcV mutant,PXV917,PXV106, PXV452,RSV917,RSV106,RSV452,lost pathogenicity on Rice and the ability of triggering HR response on nonhost tobacco.The yeast two-hybrid results demonstrated that the HrcV protein interacted with T3SS component proteins,HrcN,HrcJ and HrcR,and T3SS effector,Avr/pth28,AvrXa3 and Avr/pth13,respectively.Truncated HrcV assays displayed that the N-terminal of the HrcV protein interacted with HrcJ and HrcR proteins and the C-terminal did with HrcN.We proposed that the C-terminal deletion in the HrcV protein might affect the secretion of AvrBs3/pthA proteins into rice cells.T3SS effctors were secreted through the typeâ…¢secretion system(T3SS) coding by the hrp genes of Xooc and then passed through the Hrp pilus and translocated into plant cells via HrpF translocon encoded by the hrpF gene,leading to resistance or susceptibility in rice.The hpal gene encodes a Harpin-like protein which elicits HR on non-host tobacco and the hpa2 gene is highly conserved in various Xanthomonas species and codes a protein similar to lysozyme.In this study we found that the single mutant of hrpF and hpa1 gene as well as the double mutant of hpa1 and hpa2 still triggered HR on tobacco and caused water soaking lesions on rice leaves but water soaking symptoms did not developed.The double mutant of the hrpF and hpa1 genes and the the triple mutant of the hrpF,hpa1 and hpa2 genes did not trigger HR on tobacco and form water soaking symptoms on rice.We also found that the hrpF mutant did not cause bacterial leaf streak symptom and the single mutant of the hpa1 gene and the double mutant of the hpa1 and hpa2 gene formed shorter leaf streak symptoms after inoculation by spraying the bacteria into the surface of rice leaves.Scanning electron microscope(SEM) revealed that the single mutant of the hpa1 or the hrpF gene infected rice through stomata,but the double mutant of the hpa1 and hrpF genes and the triple mutant of the hrpF,hpa1 and hpa2 genes lost the ability to infect rice through stomata and to multiply around stomata.The SEM also showed that the single mutant of the hrpF or hpalgene,the doubl mutant of the hpa1 and hpa2 genes interacted with rice callus cells,but the double mutant of the hrpF and hpa1 genes and the triple mutant of the hrpF,hpa1 and hpa2 genes lost interacting capabilities with callus cells.The results above suggested that the synergies function of the hpa1 and hrpF gene is the reason for formation of water soaking symptoms caused by Xooc,and possibly HrpF and Hpa1 are the two major translocators for release of plant liquids out of cells.We postulated that the hrpF gene is the key in determining the expansion of water soakin symptoms in rice and the hrpF and hpa1 genes control the bacteria to infect rice through stomata,but mutations in thoses genes would affect interactions of other T3SS effects with rice leading to reduction in development of bacterial leaf streak symptoms and nutrition parasitic relationships between Xooc and rice.Therefore,the elucidation of the function of the hrpF and hpa1 genes surely facilitate our understanding molecular mechanisms of formation of water-soaking symptoms in rice by the pathogen.The hpaB and hrpE genes are necessary for pathogenicity on rice and HR induction on tobacco.Double mutant of the hpaB and hrpF genes retained HR induction on tobacco but lost pathogenicity on Rice.The triple mutant of the hrpF,hpaB and hpa1 genes lost the pathogenicity on rice and HR induction on tobacco.The interactions between those mutants and rice callus showed:the single mutant of the hpaB gene and the double mutant of the hrpF and hpaB gene interacted with rice callus cells,but the triple mutant of the hrpF, hpaB and hpa1 genes and the mutant of the hrpE gene lost the ability to interact with rice callus cells.Scanning electronic microscope results showed that the hrpE mutation made the pathogen lose pathogenicity and not be capable to form Hrp pilus apparatus.Putatively, the HpaB protein specifically controlled the secretion of effectors which cause water soaking symptoms in rice.Some T3SS effectors,inhibiting the HR induction on tobacco by the pathogen,might be secreted in HpaB-independent manner,or translocated in HrpF-dependent manner into plant cells.The Hpa1(Harpin) protein might not be secreted into plant cells through the HrpF translocon.Three hrpF mutant,PXF322.PXF410 and PXF744,of Xoo strain PXO99~A were constructed in this study.The mutants still kept the ability to cause water soaking lesions in rice after they were inoculated into rice seedlings by an injection method and to elicit HR response on tobacco,but lost pathogenicity in adult rice when they were inoculated in rice by leaf-clip method.The hrpF gene could restore the ability of foaming bacterial blight symtoms in rice to the hrpF mutant.These results confirmed that the hrpF gene of Xoo is a critial gene in pathogenicity of the pathogen in rice.However,the mutation in the hrpF gene does not completely block the secretion of T3SS effectors into plant cells.