Molecular Mechanisms Of Symbiosis Receptor Kinase Regulating Symbiosis Nitrogen Fixation In Lotus Japonicus

Specific interactions between legume and rhizobia result in the formation of a highly specialized plant organ-the root nodule,and this process is also called Root Nodule Symbiosis（RNS）.In the nodules,the host cells provide organic carbon com-pounds to the rhizobia and guarantee a stable living environment,in return,rhizobia reduce the free nitrogen（N₂）in the atmosphere to ammonia by nitrogen fixation to promote plant growth,thus,both the plant host and bacterial symbiont appear to derive benefit.In the past 20 years,nearly 200 genes were identified involved in RNS through forward genetics and reverse genetics in the model legumes Lotus japonicus and Medi-cago truncatula.The symbiosis receptor-like kinase（Sym RK）is a key regulator for early signal recognition between rhizobia and legumes,but the regulatory mechanism is unclear.In this study,the functions of Sym RK and its interacting proteins in the early stage of symbiosis of root nodules were explored using protein interaction,CRISPR knockout,plant genetic transformation and other techniques in Lotus japonicus,and the results are list below:1. Both M.loti MAFF30309 and flg22 trigger immune responses in the roots of wild-type L.japonicus,but pretreatment with M.loti significantly suppresses host de-fense responses triggered by flg22;2. To assess genes involved in suppression of flg22-triggered host defense responses,loss-of-function mutants of Nod Factor Receptor-like Kinase 1 and 5（nfr1,nfr5）and Symbiosis Receptor-like Kinase（ems61,symrk-409）were pretreated with M.loti,followed by treatment with flg22.Suppression of flg22-triggered defense re-sponses were observed in nfr1 and nfr5 but not symrk mutant plants which indicates Sym RK,but not NFR1 or NFR5,is required for suppression of host defense responses during symbiosis.3. To reveal the mechanism of Sym RK involved suppression of flg22-triggered defense responses by pretreatment with M.loti.The kinase domain of Lj Sym RK was used as a bait to screen a yeast two-hybrid library for Lj Sym RK-interacting proteins.Among those candidates identified was a protein that shared high homology with the Arabidopsis leucine-rich repeat receptor kinase At BAK1.We cloned the full-length gene from L.japonicas referred to it as Lj BAK1.Ectopic expression of Lj BAK1 could partially rescue the defective phenotypes of bri1-5,and bak1-4,indicates that Lj BAK1is an ortholog of Arabidopsis BAK1 with similar biological functions regarding induc-tion of innate immunity and plant development.4. To confirm the interaction between Lj Sym RK and Lj BAK1,yeast two-hybrid and co-immunoprecipitation were carried out.Lj BAK1 specific interacts with Sym RK but not NFR1 or NFR5 in yeast cells,and this interaction were conserved among leg-umes and nonlegumes.5. To study the kinase activity of Sym RK and Lj BAK1,we expressed and purified recombinant proteins of Lj BAK1（His-Lj BAK1-CD）and Sym RK（MBP-Sym RK-CD）in E.coli.An in vitro kinase assay showed that both Lj Sym RK and Lj BAK1 had auto-phosphorylation and transphosphorylation activity and can phosphorylated each other.At the same time,Sym RK inhibits the kinase activity of Lj BAK1,and the kinase activ-ity of Sym RK is required for inhibitory effects on Lj BAK1 in vitro.To dissect the cor-relation between the kinase activity of Lj BAK1,two stable transgenic lines expressing Lj BAK1 with（G₄S）₂-FLAG tag were carried out in wild-type Lotus japonicus.Trans-genic plants were treated with flg22 followed by immunoprecipitation with FLAG an-tibody,the output was analysis by western-blot using anti-FLAG and anti-p Ser/Thr/Tyr antibodies.The results showed that flg22 induced phosphorylation of Lj BAK1 was sup-pressed by pretreatment with M.loti.6. We generated two independent Ljbak1 mutant lines using CRISPR-Cas9 editing with two guide RNAs targeting Lj BAK1,both Ljbak1 lines make truncated proteins with a frameshift mutation within the Lj BAK1 gene.Compared with wild-type MG20,Ljbak1 mutant plants showed a semi-dwarfed phenotype and insensitive to flg22,and the densities of Ips and Its in Ljbak1 mutant plants significantly increased.These results indicate Lj BAK1 negative regulates symbiosis process between legumes and rhizobia,and provide a partial mechanism of suppression of defense response for the action of Sym RK during the symbiosis.7. Using Y2H and Bi Fc protein interaction methods,we found Sym RK interacting E3 ligase（SIE3,a RING[Really Interesting New Gene]-containing E3 ligase）and Sym RK interacting protein 1（SIP1,an ARID-type DNA-binding protein）interact with each other.Moreover,SIE3 associated with itself to form a homodimer.8. The cysteine 266 residue（Cys266）in the CRA domain was found to be essential for SIE3 dimerization,and substitution Cys266 with Ser（C266S）abolished the di-meration and biological function of SIE3,these results suggested SIE3 may functions as a homodimer.