| Brassinosteroids(BRs)are polyhydroxylated sterol phytohormones that distributed widely in plants and play lots of vital roles in regulating plant growth and development.BAK1,also named SERK3 for its role in regulating somatic development,which could form a complex with BRI1 as a co-receptor to perceive BRs and initiate early BR signaling,interact with pattern recognition receptor(PRR)involved in regulating plant immunity signal,and interact with other RKs to regulate cell death.To further elucidate the biological function of BAK1 in regulating plant growth and development,BAK1-ITERACTING RECEPTOR 3(AtBIR3)was identified in AtBAK1-overexpressing Arabidopsis by immunoprecipitation and MS assays.The protein structure,gene expression patterns in tissue,subcelluar localization,and kinase activity of AtBIR3 were analyzed.The interaction between SERKs and AtBIR3,and the molecular mechanism of affection of AtBIR3 on BR signaling and regulating plant growth and development were investigated.The homologue gene of AtBIR3 in tomato named SlBIR3 were identified through homology cloning technique and the protein structure,subcellular localization,kinase activity and the interaction with SlBAK1 of SlBIR3 were analyzed.The sensitivity of SlBIR3-overexpressing seedling to flg22 was investigated.The function of SlBIR3 in resistance to PstDC3000 and Botrytis cinerea and regulating spontaneous cell death were explored.The main results are as follows.1.BAK1-ITERACTING RECEPTOR 3(AtBIR3)was identified in AtBAK1-overexpressing Arabidopsis by immunoprecipitation and MS assays.AtBIR3 is 1806 bp in length,encoding 601 amino acids which has a typical structure like other LRR-RK including leucine-rich repeat regions in extracellular domain,transmembrane domain,Ser/Thr kinase region in cytoplasmic domain.AtBIR3 lacks conserved RD motif in VIb subdomain which is necessary for the kinase activity.The subcellular localization and kinase activity assays proved that AtBIR3 mainly distributes on plasma membrane and has no autophosphorylation and transphosphprylation activity.2.AtBIR3-overexpressing in Col-0 could reduce the plant height,fertility and the sensitivity to exogenous BR treatment.The Y2 H,MBP pull down,and co-immunoprecipitation(Co-IP)assays proved that AtBIR3 could interact with SERKs,and AtBIR3 could be phosphorylated by BAK1.The Co-IP assay results showed that BIR3 could disturb the interaction of BAK1 and BRI1 resulting in the interruption of BR signaling and finally leading to the extremely dwarfed phenotype of the transgenic plants.3.T-DNA mutant bir3-2 showed a slightly decreased BR signaling according to the phenotype and the sensitivity to BR of bir3-2 mutant.This result was further confirmed by the phenotype of bir3-2bri1-301.This may due to the functional redundancy between AtBIR2,AtBIR4,and AtBIR3 genes and the degradation of BAK1 in bir3-2.According to the sensitivity of bak1-4:BAK1-GFP and bir3-2bak1-4:BAK1-GFP to BR treatment,we conclude that knockout of BIR3 could enhance BR signaling under the condition of having enough BAK1 proteins.4.The homologue gene of AtBIR3 in tomato,named SlBIR3(Solyc05g006570.1.1),was cloned through homology cloning technique which has a 63.59% similarity to AtBIR3 in amino acids and fall in to a same clade with AtBIR3 in phylogenetic tree.SlBIR3 is 1833 bp in length,encoding 610 amino acids which has a similar structure like AtBIR3 and lacks conserved RD motif.5.The subcellular localization and kinase activity assays showed that SlBIR3 is a membrane-localized non-RD kinase.A series of experiments proved that SlBIR3 could interact with SlBAK1 and AtBAK1 and could be phosphorylated by AtBAK1.Through Transcriptome RT-PCR analysis,we found SlBIR3 transcript was present in all tissues and could response to pathogen or PAMP treatment.6.According to the phenotype and the expression level of BR signaling marker genes SlCPD and SlDWARF in Sl BIR3-overexpressing lines,we conclude that SlBIR3 has a weak effect on BR signaling pathway and BR-mediated plant growth and development in tomato.Similar results were obtained when overexpression of SlBIR3 in Arabidopsis.The results of seedling growth inhibition assay and the expression level of FRK1 in SlBIR3-overexpressing Arabidopsis induced by flg22 showed that overexpression of SlBIR3 could block PTI signaling.The resistance of SlBIR3-overexpressing plants to PstDC3000 has no change,but the resistance to Botrytis cinerea is weakened.Co-silenceing SlBIR3 with SlSERK3 A or SlSERK3 B together could induce spontaneous cell death with the up-regulated of SlPR1b1 and SlPR2 indicating SlBIR3 cooperates with SlSERK3 genes to regulate cell death.In conclusion,our study revealed that BIR3 negatively regulates plant growth and development through BR signaling and also involved in the negative regulation PTI signaling and spontaneous cell death. |
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