Department of Biological Sciences, College of Biological Sciences and Biotechnology, Chungnam National University, Daejeon, 34134, Republic of Korea.
Genes Genomics. 2021 Nov;43(11):1269-1276. doi: 10.1007/s13258-021-01148-2. Epub 2021 Aug 27.
Botrytis-induced Kinase 1 (BIK1) is a receptor-like cytoplasmic kinase (RLCK) involved in the defense, growth, and development of higher plants. It interacts with various receptor-like kinases (RLKs) such as Brassinosteroid Insensitive 1 (BRI1), Flagellin Sensitive 2 (FLS2), and Perception of the Arabidopsis Danger Signal Peptide 1 (PEPR1), but little is known about signaling downstream of BIK1.
In this study, we aimed to identify Arabidopsis thaliana BIK1 (AtBIK1) and Brassica rapa BIK1 (BrBIK1) interacting proteins, which is downstream signaling components in Arabidopsis. In addition, the effect of BIK1 phosphorylation on their interaction were examined.
For yeast two hybrid (Y2H) screening, a B. rapa cDNA activation domain (AD) library and an A. thaliana cDNA library were used. Reverse reaction (LR) recombinations of appropriate open reading frames (AtBIK1, BrBIK1, AtRGP2, AtPATL2, AtPP7) in either pDONR207 or pDONR/zeo were performed with the split-YFP destination vectors pDEST-GWVYNE and pDEST-GWVYCE to generate N- or C-terminal fusions with the N- and C-terminal yellow fluorescent protein (YFP) moieties, respectively. Recombined vectors were transformed into Agrobacterium strain GV3101. The described GST-AtBIK1, Flag-AtBIK1, and Flag-BrBIK1 constructs were used as templates for site-directed mutagenesis with a QuikChange XL Site-Directed Mutagenesis Kit (Stratagene).
In results, A. thaliana BIK1 (AtBIK1) displays strong autophosphorylation kinase activity on tyrosine and threonine residues, whereas B. rapa BIK1 (BrBIK1) does not exhibit autophosphorylation kinase activity in vitro. Herein, we demonstrated that four proteins (RGP2, PATL2, PP7, and SULTR4.1) interact with BrBIK1 but not AtBIK1 in a Y2H system. To confirm interactions between BIK1 and protein candidates in Nicotiana benthamiana, BiFC analysis was performed and it was found that only BrBIK1 bound the three proteins tested. Three phosphosites, T90, T362, and T368, based on amino acid sequence alignment between AtBIK1 and BrBIK1, and performed site-directed mutagenesis (SDM) on AtBIK1 and BrBIK. S90T, P362T, and A369T mutations in BrBIK1 restored autophosphorylation kinase activity on threonine residues comparable to AtBIK1. However, T90A, T362P, and T368A mutations in AtBIK1 did not alter autophosphorylation kinase activity on threonine residues compared with wild-type AtBIK1. BiFC results showed that BIK1 mutations restored kinase activity led to the loss of the binding activity to RGP2, PATL2, or PP7 proteins.
Phospho-BIK1 might be involved in plant innate immunity, while non-phospho BIK1 may regulate plant growth and development through interactions with RGP2, PATL2, and PP7.
博来霉素诱导激酶 1（BIK1）是一种参与高等植物防御、生长和发育的受体样胞质激酶（RLCK）。它与各种受体样激酶（RLKs）相互作用，如油菜素内酯不敏感 1（BRI1）、鞭毛敏感 2（FLS2）和拟南芥危险信号肽 1 感知（PEPR1），但对 BIK1 下游信号的了解甚少。
本研究旨在鉴定拟南芥 BIK1（AtBIK1）和芸薹属 BIK1（BrBIK1）相互作用蛋白，作为拟南芥中 BIK1 的下游信号成分。此外，还研究了 BIK1 磷酸化对其相互作用的影响。
为了进行酵母双杂交（Y2H）筛选，使用了芸薹属 cDNA 激活结构域（AD）文库和拟南芥 cDNA 文库。通过反向反应（LR）重组适当的开放阅读框（AtBIK1、BrBIK1、AtRGP2、AtPATL2、AtPP7）在 pDONR207 或 pDONR/zeo 中，与分裂-YFP 目的载体 pDEST-GWVYNE 和 pDEST-GWVYCE 分别进行 N-或 C-末端融合，分别与 N-和 C-末端黄色荧光蛋白（YFP）片段融合。将重组载体转化为农杆菌菌株 GV3101。用 GST-AtBIK1、Flag-AtBIK1 和 Flag-BrBIK1 构建体作为模板，用 QuikChange XL 定点诱变试剂盒（Stratagene）进行定点突变。
在结果中，拟南芥 BIK1（AtBIK1）在酪氨酸和苏氨酸残基上显示出强烈的自身磷酸化激酶活性，而芸薹属 BIK1（BrBIK1）在体外没有自身磷酸化激酶活性。在此，我们证明了四个蛋白（RGP2、PATL2、PP7 和 SULTR4.1）在 Y2H 系统中与 BrBIK1 相互作用，但不与 AtBIK1 相互作用。为了确认 BIK1 与烟草原生质体中的蛋白候选物之间的相互作用，进行了 BiFC 分析，结果发现只有 BrBIK1 与测试的三种蛋白结合。根据 AtBIK1 和 BrBIK1 之间的氨基酸序列比对，有三个磷酸化位点 T90、T362 和 T368，进行了定点突变（SDM）AtBIK1 和 BrBIK。BrBIK1 中的 S90T、P362T 和 A369T 突变恢复了与 AtBIK1 相当的 Thr 残基的自身磷酸化激酶活性。然而，AtBIK1 中的 T90A、T362P 和 T368A 突变与野生型 AtBIK1 相比，并未改变 Thr 残基的自身磷酸化激酶活性。BiFC 结果表明，激酶活性恢复的 BIK1 突变导致与 RGP2、PATL2 或 PP7 蛋白的结合活性丧失。
磷酸化的 BIK1 可能参与植物先天免疫，而非磷酸化的 BIK1 可能通过与 RGP2、PATL2 和 PP7 相互作用来调节植物的生长和发育。
