Functional analysis of MLPK, a membrane-anchored cytosolic protein kinase, in Brassica self-incompatibility signaling

本研究的目的是评估MLPK的功能作用，MLPK是一种膜锚定的胞质蛋白激酶，被发现是自交亲和芸苔属突变体的致病因子。主要研究结果如下：1. MLPK的分子功能：我们鉴定了两种不同的MLPK转录本，MLPKf 1和MLPKf 2，它们是通过使用替代转录起始位点产生的，并且编码两种仅在N-末端不同的亚型。MLPKf 1和MLPKf 2都在乳头细胞中表达，并通过不同的分子机制定位于质膜。虽然MLPKPf 1和MLPKf 2都可以独立地互补mlpk/mlpk突变，但它们缺乏质膜定位基序的突变形式不能互补突变。此外，双分子荧光互补（BiFC）测定显示SRK和MLPK亚型在植物中的直接相互作用。这些结果表明，MLPK亚型定位于乳头细胞膜，并与SRK直接相互作用，从而参与信号转导。2. MLPK的靶分子我们通过酵母双杂交筛选与MLPK相互作用的柱头蛋白，发现了几个候选蛋白。发现一些候选物与MLPK的活性形式选择性地相互作用，并且显示出被重组MLPK在体外特异性磷酸化。拟南芥（Arabidopsis thaliana）是一种自交亲和的芸苔属植物，具有MLPK的直向同源物APK 1b。为了测试APK 1b除了SI信号转导之外的生物学功能，我们分析了APK 1b的T-DNA敲除系：然而，我们未能检测到任何表型改变，包括生育力。APK 1b在SI信号转导中的唯一功能或同源激酶的冗余可能解释APK 1b敲除突变体中缺乏异常表型。

The aim of the present study was to evaluate the functional role of MLPK, a membrane-anchored cytosolic protein kinase found as a causal factor of a self-compatible Brassica mutant. The major findings are summarized as follows.1. Molecular function of MLPK.We identified two different MLPK transcripts, MLPKf1 and MLPKf2, which are produced by using alternative transcriptional initiation sites and encode two isoforms that differ only at the N-termini. Both MLPKf1 and MLPKf2 are expressed in papilla cells and localize to the plasma membrane by different molecular mechanisms. Although both MLPKPf1 and MLPKf2 could independently complement the mlpk/mlpk mutation, their mutant forms that lack the plasma membrane localization motifs failed to complement the mutation. Furthermore, a bimolecular fluorescence complementation (BiFC) assay revealed direct interactions between SRK and the MLPK isoforms in planta. These results suggest that MLPK isoforms localize to the papilla cell membrane and interact directly with SRK to transduce SI signaling.2.Target molecule of MLPKWe searched for MLPK-interacting stigmatic proteins by yeast two-hybrid screening and found several candidates. Some candidates were found to selectively interact with active form of MLPK, and were shown to be specifically phosphorylated by the recombinant MLPK in vitro. We are now trying to elucidate the physiological function of these candidate molecules.Arabidopsis thaliana is a self-compatible Brassica plant having an MLPK ortholog, APK1b. To test for biological functions of APK1b aside from SI signal transduction, we analyzed a T-DNA knockout line for APK1b: we failed, however, to detect any phenotypic alterations including fertility. The sole function of APK1b in SI signal transduction or the redundancy with regard to homologous kinases might explain the lack of aberrant phenotype in knockout mutant of APK1b.