Choi Jae-Han, Kim Ji-Woo, Oh Man-Ho
Department of Biological Sciences, College of Biological Sciences and Biotechnology, Chungnam National University, Daejeon, 34134, South Korea.
Genes Genomics. 2022 Dec;44(12):1477-1485. doi: 10.1007/s13258-022-01292-3. Epub 2022 Sep 2.
Plant growth and development are complex processes modulated by numerous genes, transcription factors, hormones, and peptides. Several reports implicate the membrane-localized Catharanthus roseus receptor-like kinase1 (CrRLK1L) protein, FERONIA (FER), involved in plant development. However, protein targets of FER remain poorly characterized.
FER recombinant proteins were analyzed, and FER-interacting proteins were identified, to better understand the function of the Arabidopsis thaliana FER (AtFER) gene in plant development.
AtFER-interacting proteins were identified through Yeast-Two Hybrid (Y2H) and validated by bimolecular fluorescence complementation (BiFC). Autophosphorylation activity was evaluated in AtFER site-directed and deletion mutants.
AtFER cytoplasmic kinase domain (Flag-FER-CD) is autophosphorylated at the Thr residue (s), with T559 and T664 as important sites for AtFER kinase activity. In addition, the carboxy terminal region is essential for AtFER kinase activity. Y2H identified an Armadillo (ARM)-repeat protein (At4g16490) with tandem copies of a degenerate protein sequence motif, a U-BOX 9 (PUB9, At3g07360), IQ-DOMAIN 7 (IQD7, At1g17480), and heteroglycan glucosidase 1 (HGL1, At3g23640) as AtFER-interacting proteins. BiFC confirmed the in vivo interactions between these four proteins and AtFER in tobacco (Nicotiana benthamiana) leaf transient expression assays. The RAPID ALKALINIZATION FACTOR1 (RALF1) peptide, which is a FER ligand, induced the expression of genes encoding the four AtFER-interacting proteins.
The AtFER-interacting proteins identified in this study are likely involved in FER-mediated intracellular signaling pathways that are essential in plant growth and development, and possibly plant immunity.
植物的生长和发育是由众多基因、转录因子、激素和肽调节的复杂过程。一些报道表明，膜定位的长春花类受体激酶1（CrRLK1L）蛋白FERONIA（FER）参与植物发育。然而，FER的蛋白质靶点仍未得到很好的表征。
分析FER重组蛋白并鉴定与FER相互作用的蛋白，以更好地了解拟南芥FER（AtFER）基因在植物发育中的功能。
通过酵母双杂交（Y2H）鉴定与AtFER相互作用的蛋白，并通过双分子荧光互补（BiFC）进行验证。在AtFER定点和缺失突变体中评估自磷酸化活性。
AtFER细胞质激酶结构域（Flag-FER-CD）在苏氨酸残基处发生自磷酸化，其中T559和T664是AtFER激酶活性的重要位点。此外，羧基末端区域对AtFER激酶活性至关重要。Y2H鉴定出一种犰狳（ARM）重复蛋白（At4g16490），其具有简并蛋白序列基序的串联拷贝、一个U盒9（PUB9，At3g07360）、IQ结构域7（IQD7，At1g17480）和杂聚糖葡糖苷酶1（HGL1，At3g23640）作为与AtFER相互作用的蛋白。BiFC在烟草（本氏烟草）叶片瞬时表达试验中证实了这四种蛋白与AtFER在体内的相互作用。FER配体快速碱化因子1（RALF1）肽诱导了编码这四种与AtFER相互作用蛋白的基因的表达。
本研究中鉴定出的与AtFER相互作用的蛋白可能参与FER介导的细胞内信号通路，这些信号通路在植物生长发育以及可能的植物免疫中至关重要。
