Background

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.

Objective

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.

Methods

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).

Results

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.

Conclusion

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.

中文翻译：

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BIK1 的磷酸化对于与下游信号成分的相互作用至关重要

背景

Botrytis 诱导的激酶 1 (BIK1) 是一种受体样细胞质激酶 (RLCK)，参与高等植物的防御、生长和发育。它与各种受体样激酶 (RLK) 相互作用，例如油菜素类固醇不敏感 1 (BRI1)、鞭毛蛋白敏感 2 (FLS2) 和拟南芥危险信号肽 1 (PEPR1) 的感知，但对 BIK1 下游的信号传导知之甚少。

客观的

在本研究中，我们旨在鉴定拟南芥BIK1 (AtBIK1) 和芸苔BIK1 (BrBIK1) 相互作用蛋白，它们是拟南芥中的下游信号成分。此外，还检查了 BIK1 磷酸化对其相互作用的影响。

方法

对于酵母二杂种 (Y2H) 筛选，使用了青菜cDNA 激活域 (AD) 文库和拟南芥cDNA 文库。在 pDONR207 或 pDONR/zeo 中对适当的开放阅读框（AtBIK1、BrBIK1、AtRGP2、AtPATL2、AtPP7）进行逆反应（LR）重组，使用分离的 YFP 目标载体 pDEST-GWVYNE 和 pDEST-GWVYCE 生成 N-或C 端分别与 N 端和 C 端黄色荧光蛋白 (YFP) 部分融合。将重组载体转化到农杆菌菌株 GV3101 中。所描述的 GST-AtBIK1、Flag-AtBIK1 和 Flag-BrBIK1 构建体被用作使用 QuikChange XL 定点诱变试剂盒 (Stratagene) 进行定点诱变的模板。

结果

结果表明，拟南芥BIK1 (AtBIK1) 在酪氨酸和苏氨酸残基上表现出很强的自磷酸化激酶活性，而拟南芥BIK1 (BrBIK1)在体外不表现出自磷酸化激酶活性。在这里，我们证明了在 Y2H 系统中，四种蛋白质（RGP2、PATL2、PP7 和 SULTR4.1）与 BrBIK1 相互作用，但不与 AtBIK1 相互作用。确认 BIK1 与本氏烟草中蛋白质候选物之间的相互作用, 进行了 BiFC 分析，发现只有 BrBIK1 结合了所测试的三种蛋白质。三个磷酸化位点 T90、T362 和 T368，基于 AtBIK1 和 BrBIK1 之间的氨基酸序列比对，并对 AtBIK1 和 BrBIK 进行定点诱变 (SDM)。BrBIK1 中的 S90T、P362T 和 A369T 突变恢复了与 AtBIK1 相当的苏氨酸残基上的自磷酸化激酶活性。然而，与野生型 AtBIK1 相比，AtBIK1 中的 T90A、T362P 和 T368A 突变并未改变苏氨酸残基上的自磷酸化激酶活性。BiFC 结果表明，BIK1 突变恢复了激酶活性，导致与 RGP2、PATL2 或 PP7 蛋白的结合活性丧失。

结论

Phospho-BIK1 可能参与植物先天免疫，而非磷酸化 BIK1 可能通过与 RGP2、PATL2 和 PP7 相互作用调节植物生长发育。