Isolation Of Tal Genes From Four Xanthomonas Species And Identification Of Tale Targets In Host Plants

Bacterial blight of cotton(BLB),soybean bacterial spot disease,phaseolus vulgaris and cowpea bacterial blight are important and catastrophic bacterial diseases in crop ecosystem,respectively caused by Xanthomonas campestris pv.malvacearum(Xcm),Xanthomonas axonopodis pv.glycines(Xag),Xanthomonas campestris pv.phaseoli(Xcp)and Xanthomonas axonopodis pv.vigniacola(Xav).Like other Xanthomonas pathogens,Xcm,Xag,Xcp and Xav secret large amount of T3SS-secreted effectors(T3SEs)into plants via a type-III secretion system(T3SS)which is highly conserved in Gram-negative plant pathogenic bacteria,to trigger host resistance or susceptibility in plants.T3SEs include transcription activator-like(tal)effectors(TALEs)and Non-TALE effectors.However,of Xcm,Xag,Xav and Xcp,the types,the number and the diversity of TALEs are seldom reported.Also how the TALEs play their roles in bacterial pathogenicity and their target genes in host plants are rarely reported.In this paper,we tried to isolate tal genes from these four bacterial species and identify their target genes in their host plants.Major results are as the following.1.Construction of genetic manipulation system of 4 kind of XanthomonasIn order to find out whether genetic manipulation such as knock-out and knock-in can be operated in tal genes,four representative strains of Xanthomonas were selected,that is ATCC43911 strain of Xav,ATCC11648 strain of Xav,ATCC49119 strain of Xcp,and Xss-V₂-18strain of Xcm as original strains.Based on highly conserved tal genes in sequence,p B-C8B::Tn5,a plasmid with homolog-interchangeable fragments of a tal gene flanking EZ-Tn5 tansposon,was constructed.Then,the plasmid was electroporated into the four receptor bacteria cells.Km resistance screening found that the plasmid can enter into the four kinds of Xanthomonas.Given that C8B is a tal gene derived from X.oryzae,homologous recombination of the tal genes was presumed to occur in clones resistant to km.Further,genomes with km resistance of colonies of Xag ATCC43911 strain were extracted and the probe was constructed by using the Sph I fragment of the middle repeat region of a tal gene.Southern blot results demonstrated that the wild-type ATCC43911contained 6 tal genes.The location of tal genes changed and more tal bands were showed in Tn5-inserted mutants because the Tn5 transposon was inserted in a single or double copies into tal genes of the tested strain.These results suggested that the Tn5 transposon was inserted into the tal genes successfully.Construction of Tn5-inserted mutants of Xag in tal genes will provide fundamental basis to understand roles of these tal genes in bacterial pathogenicity in soybean.2.Deletion of tal genes in X.campestris pv.malvacearum and pathogenicity detection of tal mutants in cottonSince mutation technology based on the Tn5 transposon can only achieve the insertion mutants of Xanthomonas in tal gene.In order to obtain the knockout mutants of the tal gene,the carrier p KMSA1 and p KMSA2,which fused with the left and right homologous arms out of the repeat domain of tal gene,were constructed on the suicide plasmid p KMS1 vector.PKMSA1 was transformed into Xss-V₂-18 cells by electrotransformation,and Km resisitance and sucrose medium were screened to obtain mutants which can grow on NA but sensitive to Km.After screening of colony-PCR,candidate mutants containing tal gene fusion fragment were obtained.The genomes of candidate mutants were extracted and digested with Bam HI.Southern Blot was operated with probe of Sph I fragment of a tal gene.It showed that M1,M2,M3 and M4mutants were obtained based on spectrum of tal genes band.Compared with tal gene bands of wild-type strain,M1 lacked tal3,M2 lacked tal2,M3 lacked tal2 and tal4,M4 lacked tal2,tal4,tal5 and tal6.Using M4 as the original strain,p KMSA2 was used to knock out the remaining tal genes.According to the above screening steps,M5 and M6 mutants were obtained by Southern Blot.The M5 mutant lacked tal3 and all 6 tal genes were deleted in M6.The mutants were inoculated in cotton cotyledons,compared with wild type,the pathogenicity of M2,M3,M5 and M6 on cotton decreased significantly,while no significant changes in the pathogenicity of M1 and M4 mutants on cotton.It is speculated that tal2and tal1 play an important role in determining symptoms of BLB.Correspondingly,measurement of bacterial growth on cotton cotyledons showed that growth of tal-deleted mutants was significantly lower than that of the wild type on the fourth day after inoculation,but there was no significant difference between the mutants.According to the same strategy,the tal genes of Xcp and Xav could be knocked out and knockout mutants of partial tal genes were obtained.The successful knockout of Xcm in tal genes has laid a good material system for further revealing the mechanism of each tal gene in the pathogenicity of BLB.3.Tal genes isolation from four Xanthomonas species and the detection of tal gene roles in bacterial pathogenicity in plantsIn order to find out that the tal genes are present in Xanthomonas chromosome or plasmid,genomic DNA and plasmid DNA were obtained according to two extraction methods.DNA were digested with Bam HI and southern blot was operated with probe of Sph I fragment of a tal gene.And it was found:Xcp has 2 tal genes,Xag has 4 tal genes,Xav has 2 tal genes,while Xcm has 6 tal genes.The tal genes of the first 3 pathogens were located on the chromosomes,while the 6 tal genes of Xcm were located on the plasmid.According to the location of the hybridization signal bands of tal genes,the corresponding tal bands were recovered,and the tal genes library was constructed on the p B vector.The Sph I fragment of tal gene was used as a probe for colony hybridization in situ.The positive clones were identified by digestion with Sph I according to the size and type of the bands,and positive clones with target tal genes were obtained.The candidate positive clones with tal gene were confirmed by Southern blot.6tal genes of Xcm,4 tal genes of Xav,1 tal gene of Xcp and 3 tal genes of Xag were obtained.In view of the 102 bp central repeat unit in tal genes,in order to get the sequence,Tn5 transposon was inserted to sequence the 6 tal genes obtained from Xcm.According to the result of digestion of restriction endonuclease,colonies which Tn5 was inserted in the middle position of the tal gene were sequenced.Sequencing results showed that the 6 tal genes of Xcm were not the same of tal genes in the database.There were27.5 repeat units in tal1,25.5 in ta2,21.5 in tal3,18.5 in tal4,15.5 in tal5and 13.5 in tal6.In order to evaluate the contribution of tal genes in the pathogenicity of Xcm,each of 6 tal genes were cloned in an expression vector p HM1.Electrophoresis and Western blotting displayed that the product of each tal genes were expressed in the tal-free strain M6 when the tal genes were transferred.Pathogenicity assays demonstrated that tal1 and tal2,but not other four tal genes,made M6 to cause mesophyll cell necrosis in the infiltrated areas of cotton leaves 7 days after inoculation.This necrosis was similar to that caused by the wild-type strain of Xcm,indicating that tal1 and tal2 play an important role in bacterial pathogenicity of Xcm in cotton.Using the same strategy above,some of tal genes were isolated from other three Xanthomonas species.4.Identification of TALE targets in cottonIn order to identify TALE2 targets in cotton to reveal pathogenicity mechanism,the wild type strain Xss-V₂-18,the tal2-containing strain M6-TALE2 and the tal-free strain M6 were infiltrated into cotton leaves by using needleless syringes.The infiltrated areas of cotton leaves were collected for RNA extraction 72 hours after the inoculation.The extracted RNAs were run by RNA-seq technology and the differentially expressed genes in presence of TALE2 were analized and compared with those caused by the tal-free strain M6.The predicted EBEs targeted by TALE2in the promoters of the up-regulated genes triggered by TALE2 were collected in a library manner.Totally,eleven EBEs in 6 gene promoter regions were used to detect whether they were bound by TALE2 protein or not by EMSA methodology.EMSA test confirmed that gene LOC107894299 and LOC107911681 may be target genes of TALE2.To confirm this point,specific DNA sequence was selected in the promoter region of LOC107894299 and LOC107911681 to synthesize d TALE,namely d TALE1681 and d TALE4299 respectively,acting on these two gene promoters.The synthesized d TALE genes were constructed on p HM1 vector and transformed into tal-free strain M6.M6-d TALE1681and M6-d TALE4299 strains were obtained and inoculated into cotton cotyledons.Five days later,it was found that M6-d TALE1681 and M6-d TALE4299 strains,like M6-TAL2,stimulated cotton to produce water soaking.The results of RT-PCR detection showed that,under the action of d TALE1681 and TALE2,the expression of LOC107911681 was significantly higher than that of the wild type,Xss-V₂-18,M2 and M6strains.However,the changes in the expression of LOC107894299 were not obvious.This suggests that the LOC107911681 gene may be a target of TALE2.Whether it is or not,it needs to be tested and verified further.