Functional Analysis Of CIK Receptor-like Kinases In Arabidopsis Thaliana

As well studied receptor kinases,SERKs(SOMATIC EMBRYOGENESIS RECEPTOR KINASEs)have been shown to be involved in multiple biological processes in plant growth,development and stress adaptation as key co-receptors.Previous work in our lab demonstrated that CIK(CLAVATA3 INSENSITIVE RECEPTOR KINASE)receptor-like kinases act as co-receptors of CLV1(CLAVATA1),CLV2/CRN(CORYNE)and RPK2(RECEPTOR-LIKE PROTEIN KINASE 2)to perceive the CLV3 signal to repress the expression of the downstream transcription factor WUS(WUSCHEL)and then maintain the homeostasis of shoot apical meristem.They also act as co-receptors of BAM1(BARELY ANY MERISTEM1)/BAM2 and RPK2 to regulate early anther development in Arabidopsis.Both CIKs and SERKs belong to LRR-RLK II subfamily and they share similar protein structures.Therefore,it is speculated that CIKs may be a new group of co-receptor kinases involved in the sensing of multiple signals,and then affecting various biological processes in Arabidopsis.However,the research on CIKs has just begun.It is far to well characterize the functional diversity of CIKs.Therefore,it is necessary to systematically explore the functions of CIKs.This project mainly explores the functions of CIKs from three aspects.Frist,in order to figure out any biological processes in which CIKs may be involved in,cik multiple mutants were constructed by genetic cross and treated with a series of abiotic stresses and phytohormones.Second,to find receptors that interact with CIKs and the downstream components of CIK-mediated signaling pathways,we used the extracellular and cytoplasmic domains of CIK2 as baits to screen a yeast two-hybrid library.Third,receptor kinases that interact with CIKs were identified by a mass spectrometry,and the functions of the receptor complexesAfter treat with mannitol,high concentrations of sucrose,NaCl,KNO₃,IAA,ABA and JA,cik mutants and wild type showed no significant difference in root length.But in the presence of BL,the roots of ciks were more curly than the wild type.This result indicates that ciks are hypersensitive to BL.In addition,qRT-PCR experiments have further confirmed that CIKs are negative regulators of BR signaling.BiFC analyses showed that CIKs interacted with BR receptor BRI1.These results suggest that CIKs are not involved in osmotic stress,high concentration of sucrose stress,and salt stresses such as NaCl and KNO₃,and are not involved in the response to plant hormones,including IAA,ABA and JA.But CIKs may involve in the regulation of BR signaling pathway as a negative regulator,and the detailed molecular mechanisms require further investigation.We identified a total of 77 proteins that interact with the extracellular domain of CIK2 by screening a yeast-two hybrid library.Then 18 proteins were selected to confirm the interaction.Among them,12 proteins showed interactions with CIK2.A total of 151 proteins have been identified when CIK2 cytoplasmic domain was used as the bait,then 16 proteins were selected to confirm the interaction and 14 of them showed confirmed interactions.Further analysis revealed that these proteins may be involved in plant growth,development and pathogenic defense responses.In addition,a CIK-interacting protein MIK2(MDIS1-INTERACTING RECEPTOR LIKE KINASE 2)was identified by a mass spectrometry.To further explore the biological processes that MIK2 may be involved in,we found its homologous gene AT1G35710 by phylogenetic analysis and named it MIK3.The result of BiFC confirmed the interaction between MIK2,MIK3 and CIKs,suggesting that MIK2 and MIK3 may be receptor proteins of CIKs.A double mutant mik2-2 mik3cr114#was constructed with the help of CRISPR/Cas9 technology.We treated the double knockout mutant with abiotic stresses and phytohormones.Similar root length between mik2-2 mik3cr 114#and wild type was observed in the presence of mannitol,high concentrations of sucrose,NaCl,KNO₃,IAA,ABA,JA,and BL,indicating that MIKs do not respond to osmotic stresses such as mannitol and sucrose treatment,and do not respond to salt stresses such as NaCl and KNO₃ treatment,and they are not involved in signaling pathways of hormones such as IAA,ABA,JA and BL.Therefore,what kind of signal pathways mediated by the complexes of CIKs/MIKs,and what ligands perceived by them require further research in the future.Based on above results,our study found that CIKs are not involved in osmotic stress,high concentration of sucrose stress,salt stress of NaCl and KNO₃,and responses to plant hormones IAA,ABA and JA.CIKs may be involved in BR signaling pathway as a negative regulator of BR.CIKs may be involved in plant growth,development and pathogenic defense responses.MIKs,receptor kinases interacting with CIKs,do not respond to stresses and phytohormones described above.These efforts have made a meaningful attempt to reveal the biological functions of CIKs and laid the foundation for future work.