| Legumes form rhizomes by establishing a symbiotic relationship with rhizobia,which in turn converts nitrogen from the air into urea nitrogen to be used by the plant.Soybean is an important legume crop and is one of the main raw materials for edible oil and protein.However,soybean production in China does not take full advantage of the symbiotic nitrogen fixation properties of soybeans,which not only results in a large waste of nitrogen fertilizer in soybean production,but also causes great pollution to the farmland environment.In the process of establishing a symbiotic relationship between rhizobia and soybean,the type III effector of rhizobia plays an important role in the establishment and regulation of the symbiotic relationship.However,the soybean signaling pathways in which the type III effectors of rhizobia are involved are not clear.In this thesis,we conducted an in-depth study on the effect of soybean signaling network involved in type III effector Nop T of Rhizobium on symbiotic nodulation.Firstly,the effect on nodulation after Nop T mutant was determined by performing nodulation phenotype analysis on different soybean germplasm resources.Based on the chromosome segments substituted lines(CSSL)and the high generation recombinant inbred line(RIL)population constructed by the group.QTL localization of the responsive Nop T gene in soybean was performed to determine the location of the candidate gene on the chromosome.Protein precipitation(IP)was then used to screen soybean genes that could directly interact with Nop T at the protein level,and genes with high potential for interaction were screened for direct interaction analysis in combination with QTL localization results.The genes with direct interaction with Nop T(Gm TIP,Nop T Interact Protein)were identified by Bi FC(bimolecular fluorescence complementation technique)and Pull-Down protein interaction analysis methods.We also used overexpressed transgenic hairy roots to determine the effect of the Gm TIP gene on Nop T and wild-type rhizobia-induced nodulation phenotypes in the presence of overexpression.The differential genes induced by Nop T were mined using RNA-seq sequencing,and the network relationship between genes was analyzed to further mine the downstream reciprocal genes of Gm TIP in soybean,and the reciprocal relationship was verified using Bi FC and Pull-Down,and overexpressed transgenic hairy roots were used for nodulation phenotype analysis.Finally,by constructing a double mutant of another type III effector Nop P and Nop T in rhizobia,the signaling association between Nop P and Nop T in regulating soybean symbiotic nodulation was analyzed,and the soybean gene network that they jointly influence was resolved based on the results of RNA-seq induced by the double mutant and single mutation in rhizobia,and a signaling network transduction model was established.The main results obtained in this study were as follows:(1)Construction of Nop T mutants and determination of the effect of Nop T on the nodulation phenotype of soybean.Using a tri-parental matting approach,an antibiotic-encoding gene sequence was inserted 20 bp downstream of the start codon of the type III effector Nop T-encoding gene in the genome of Rhizobium HH103,which prevented Nop T protein synthesis while making Rhizobium carry the corresponding antibiotic resistance,completing the construction of the Nop T mutant.Thirty cultivated soybean,wild soybean,and landrace soybean with different representativeness were selected from northeastern soybean germplasm resources for nodulation phenotype analysis to determine the effect of Nop T mutants on the nodulation phenotypes of different soybean germplasm resources,and it was found that the Nop T mutations had positive and negative regulatory effects on the nodulation phenotypes of different soybean germplasm resources.These results indicate that Nop T plays an important regulatory role in soybean nodulation and also suggest the existence of Nop T-responsive genes in soybean.(2)Localization of Nop T-responsive QTLs in soybean.In the nodulation analysis,it was found that parents of CSSL(Suinong 14 and ZYD00006)and the high-generation recombinant self-compatible parents(Charleston and Dongnong 594)produced relatively obvious nodulation phenotypic changes after inoculation with the Nop T mutant,and this result supports the possibility of using the CSSL and high-generation RIL populations to locate Nop T-responsive genes.QTL localization of nodulation-related genes using both populations showed that a total of 19 QTL were localized in the high-generation RIL when inoculated with wild-type Sinorhizobium fredii HH103(here after HH103)in the parental and genetic populations,and a total of 11 QTLs were identified when inoculated with Nop T mutants.A total of 10 QTLs were localized in the case of wild-type Rhizobium HH103 inoculation in the introgression line population and 6 QTLs in the case of Nop T mutant inoculation.There were five QTLs with similar and overlapping QTLs among these QTLs,which were located on chromosomes 2,10,17,15,18,and 19.The contribution rate of QTL ranged from 2.7%-20.35%.These QTLs provided a reference for further mining of Nop T direct reciprocal genes.(3)Identification of Nop T direct reciprocal protein Gm PBS1.The screening of Nop T direct reciprocal protein was performed using protein precipitation method,and the obtained protein bands were analyzed by mass spectrometry to obtain a total of 12 valid protein spots,and the gene coding sequences of the 12 protein spots were identified by comparison in Phytozome database.Combined with the results of QTL localization,a serine protein kinase(Gm PBS1)located on chromosome 19 was selected as a candidate protein for direct interaction with Nop T for in-depth study.The Pull-Down protein interaction assay also further confirmed that Nop T and Gm PBS1 can directly interact with each other in the presence of in vitro protein expression.Nodulation experiments using Gm PBS1 overexpressing soybean hairy roots revealed that the nodulation phenotype induced by the Nop T mutant in the presence of Gm PBS1 overexpression was consistent with that induced by wild-type HH103 in wild-type soybean.This result supports the involvement of Gm PBS1 in the signaling pathway of Nop T-regulated soybean nodulation.(4)Analysis of Nop T-influenced differential genes and mining of Gm PBS1-interacting genes.Gene expression changes in soybean roots after inoculation with wild-type rhizobia and Nop T mutants were analyzed using RNA-seq sequencing,and the results showed that 2789 and1472 differentially expressed genes were identified at 0.5 h and 6 h after inoculation with rhizobia(wild-type and Nop T mutant),respectively.Among these differentially expressed genes there was a similar co-expression pattern between Gm PBS1 and Gm RIN4.q RT-PCR results indicated that Gm RIN4 b was specifically expressed in soybean roots,and Bi FC and Pull-Down experiments confirmed the reciprocal relationship between Gm PBS1 and Gm RIN4 b.The nodulation phenotype of Gm RIN4 b overexpressing hairy roots also supported the existence of a reciprocal relationship with Nop T and Nop T are mutually exclusive.(5)Signaling crosstalk between Nop T and Nop P for soybean nodules.The Nop T and Nop P double mutants HH103ΩNop T&Nop P were constructed,and the nodulation phenotype analysis revealed that the number of root nodules induced by the Nop T and Nop P double mutations was significantly reduced compared with those induced by inoculation with wild-type rhizobia,Nop T single mutant and Nop P single mutant,while the number of root nodules induced by the Nop T single mutant was significantly more than that induced by wild-type HH103.The number of nodules induced by Nop T single mutant was significantly more than that induced by wild-type rhizobia,and the number of nodules induced by Nop P single mutant was significantly less than that induced by wild-type rhizobia.The number of nodules induced by single mutants did not follow the same trend as that induced by double mutants,which counteracted the effect of Nop T and Nop P single mutants on nodule phenotype,suggesting a signaling association between Nop T and Nop P.Comparing the characteristics of differentially expressed genes induced by the HH103ΩNop T&Nop P double mutant with different single mutants and wild-type rhizobia in the RNA-seq sequencing results,we found that the expression change of Gm PBS1 was also induced by Nop P,and identified seven candidate genes Glyma.16g212300(Gm Rj2),Glyma.17 g217000(Gm CHIT5),Glyma.07g095800(Gm PR5),Glyma.02g138800(Gm MAPK2),Glyma.03g236300(Gm RBOHB),Glyma.02g247600(Gm BIK1),Glyma.16G090700(Gm RIN4b)are involved in signaling networks involving both Nop P and NopT. |
