Cloning Of Xanthomonas Tale Genes And Base-Editing Techniques Established For Rice And Bacterial Gene Editing

Bacterial blight of rice(BB)is one of the most ruinous bacterial diseases globally,which causes large rice yield and quality losses every year.Xanthomonas oryzae pv oryzae(Xoo)is the causal agent of BB,which contains a class of effector proteins with specific structure and function,namely TALE(Transcriptional Activator-Like Effector).TAL effectors play a vital role in the virulence process of Xanthomonas.And the importance of TAL effectors is mainly reflected after secretion into host cells.TAL effectors directly interact with the host susceptibility and resistance gene in the nucleus.In this study,the Xoo-rice research model system was used to conduct research,many new TAL effectors were cloned,we analyzed the interaction between these effectors and sweet susceptible gene and Xa1 resistant gene in host rice systematically.According to the mechanism of TAL effectors causing disease,a new material of rice bacterial blight was created by using single base gene-editing technology,a single base editing system for base plant pathogenic bacteria was successfully constructed.The results Includes the following five parts.1.The establishment of an efficient TALe genes cloning systemIn the first part of this study,Gibson Assembly,a cloning method widely used in molecular biology laboratories,was used according to the characteristics of conserved nucleotide sequences at the N and C terminals of the TALe gene family.pZW-Gib and p HM1-Gib were successfully developed,which can quickly clone the TALe gene from the genome of Xanthomonas,successfully constructed by molecular design and modification.pZW-Gib can efficiently clone the TALe gene fragment between Sph I restriction enzyme sites from the genome.The high efficiency of this system was confirmed by cloning and functional verification of the effector Avr Xa7 with a known function.The second upgraded cloning system,p HM1-Gib,can efficiently fish TALe gene fragments between Bam HI restriction enzyme sites.After plasmid molecular modification,the high copy replication origin was introduced,and the p HM1 vector was given high copy characteristics in E.coli.The background elimination marker gene ccdb was added for reducing the process of cloning and screening.TAL effector cloning and intermediate vector construction for functional verification were simplified.Hence,the efficiency of TALe gene cloning and functional research was improved.The function of the cloned framework p HM1-Gib was verified by using the newly cloned virulence factor Pth Xo2 B and then successfully applied to two African strains,CFBP7321 and CFBP7325.All the TAL effectors were cloned.The known virulence effectors Tal C and Tal F can effectively restore the virulence of the virulencedeficient strain ME2,indicating the high efficiency and universality of the p HM1-Gib cloning system.Through NCBI database sequence alignment and restriction site analysis,the cloning system can be widely used to clone and study the function of TALe genes from different Xanthomonas.2.The function study of TAL effector Pth Xo2 B from PXO61The TAL effector is at the core of the interaction between bacteria and host plants,and it plays an essential role in the pathogenesis of host plants.In the second part of this study,the whole genome of the Philippine Xoo strain PXO61 was sequenced.We analyzed the distribution of the TAL effector around the genome,the cloning system pZW-Gib was applied,and 17 TALe genes were successfully cloned.The toxicity function of the cloned TALe genes was analyzed,which confirmed the function of the known toxicity factor Pth Xo3.Meanwhile,a new toxicity factor Pth Xo2 B was found in PXO61,while other TAL effectors showed no obvious pathogenicity.According to the RVD composition of the intermediate repeat region of Pth Xo2 B,the target of Pth Xo2 B in the host rice was predicted.Meanwhile,the difference in toxicity of Pth Xo2 B in different rice varieties was systematically analyzed,and it was found that Pth Xo2 B has a similar pathogenic mechanism to Pth Xo2.In other words,the expression of Os SWEET13 was induced to promote the pathogen’s infection,which was confirmed by the loss of pathogenicity of Pth Xo2 B on the Os SWEET13 mutant plants.Further analysis of the characteristics of the promoter EBE sequence of Os SWEET13,a target gene of Pth Xo2 and Pth Xo2 B,in different rice cultivar backgrounds showed a coevolutionary phenomenon RVD composition of the TAL effectors repeat region represented by Pth Xo2 s and the promoter region EBE of Os SWEET13.3.Analysis of interaction between TAL effector and resistance gene Xa1Previous studies have shown that rice bacterial blight resistance gene Xa1 can recognize full-length TAL effectors to induce HR response and has a broad spectrum of disease resistance potential.At the same time,it can suppress immune function by a class of Cterminal truncated TAL effectors(i TALEs or trunc TALEs).Here,we applied the highly efficient TALe-cloning system developed previously to express the TALe gene libraries of different strains in the PH strain and analyze the ability of different TAL effectors to induce Xa1 immunity systematically.We observed the performance of different TAL effectors to induce the HR of immune response in rice containing Xa1.Some full-length TAL effectors were unable to induce Xa1 mediated immune response HR,among which Pth Xo2 B and Pth Xo3 in strain PXO61 could bypass Xa1 immunity to play a normal toxic role.It was found that the diversity of TAL effectors from African strain AXO1947 also existed,in which the Tal C virulence factor could not activate Xa1 immunity.Still,it did not restore the toxicity of the PH strain.Further,through domain replacement,we found that the differences in the intermediate repeat regions influence whether Xa1-mediated immunity could be induced or not;Through random deletion mutations in the intermediate repeat region of Pth Xo2 B and Avr Xa7,We found that the combination of the intermediate repeat region determined activation of Xa1-mediated immunity.The African strains could not cause disease on the rice containing Xa1,indicating that the African strain did not contain the inhibitory factor of Xa1-mediated immunity HR;later,this speculation was confirmed by the ability of i TALE to give the African strain the ability to break through Xa1 immunity.The Blp I-Aat II domain of Tal C and Pth Xo3 was replaced with i Tal3 a,and the results showed that the inhibition of Xa1 immunity by the i TAL effector was also related to the composition of the intermediate repeat region.4.Single-base editing technology creates new materials with resistance to bacterial blightAt present,it is considered that the most important strategy to control BB is to breed new varieties resistant to this disease.The fourth part of this research Considered the BB epidemic and control situation in Africa,the urgency of research and breeding based on TAL effector pathogenesis.We applied single-base editing techniques recently highly regarded CBE and ABE,through the target genes’ leading RNA designing and building dual-source expression vector,using the method of agrobacterium mediated genetic transformation to infect rice callus,The EBE region of Os SWEET14,a vital rice susceptible gene,was edited in model rice variety Kitaake;Single base gene editing lines were successfully obtained through tissue culture,resistance screening and genotype identification of T0,T1 and T2 generation lines;After the progeny isolation and genotype screening,the plants without transgenic marker were obtained.After pathogen resistance detection and a series of molecular experiments,the strains resistant to the virulent TAL effector Tal C widely distributed in African Xoo strains were successfully obtained.On-line sequence analysis software was used to detect potential off-target sites,and no off-target phenomenon was found;The important agronomic traits of the resistant strain were evaluated,and the yield and quality of the resistant strain were not significantly different from that of the wild type;We further analyzed the relationship between the genotype and phenotype by the edited strains,it was found that cytosine-edited plants showed resistance phenotype to Tal C.In contrast,adenine-edited plants showed no-resistance phenotype to Tal C,revealing the difference of resistance to effectors of different single base loci mutations;The RVD binding amino acid sequence differences of TAL effectors were analyzed and predicted according to the crystal structure characteristics.The obtained Tal C resistant rice plants showed moderate resistance to wild-type African strains,which provided materials for further study on the pathogenic mechanism of African strains and the prevention and control of African BB.5.The application of Single-base editing in plant pathogenWe found that large amounts of antibiotics during the transgenic process were not economical or ecologically safe.Thereinto,Agrobacterium tumefacien strain LBA4404 is widely used in plant genetic transformation.In the fifth part of this study,a novel Agrobacterium tumefacien single-base editing system was used to edit three genes(Thy A/GuaA/Rec A)in the LBA4404 strain.The target gene translation was terminated in advance,and the nutrition-deficient strain with a mutation at four sites of three genes was created.A single gene mutation strain growth experiment results found the GuaA gene mutation is not enough to inhibit the growth of strains completely.Thy A single locus of single-base mutation is not enough to restrain the growth of the strain for a long time.The risk of bacterial mutation reversed homologous recombination key Rec A gene mutation can improve the stability of the auxotroph strain genome.After several rounds of editing,mutant strains with two site mutations of Thy A,single-site mutation of GuaA and single-site mutation of Rec A were created.Nicotiana benthamiana transfection experiment proved that the nutrition-deficient strain could be effectively applied to plant infection and transgene.It could be predicted that the use of antibiotics would be significantly reduced in the stable transformation of plants,which had high ecological value and economic value.At the same time,to further expand the application range of the pathogen bacterial single-base editing system,the single-base editing system of Xanthomonas was successfully constructed in this study through molecular modification,which could realize the editing of up to four sites at the same time;This system can also be effectively applied to the gene-editing of Pseudomonas syringae and Erwinia.In summary,this study started with the cloning of TAL effectors in the pathogen,investigated the mechanism of TAL effectors causing bacterial blight of rice through interaction between the pathogen and rice,we found a new virulence factor Pth Xo2 B,and analyzed the interaction between TAL effector and Xa1 systematically;Finally,we combined the theory and application,and the rice single base editing technology was applied to create new resistant materials.At the end,the single base gene editing system for broad-spectrum application in plant pathogenic bacteria was developed.