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是一种膜锚定的胞浆蛋白激酶，被发现是甘蓝自交亲和突变体的致病因子。主要研究结果总结如下：1.研究结果。我们鉴定了两种不同的MLPK转录本，MLPKf1和MLPKf2，它们是通过使用不同的转录起始点而产生的，它们编码的两种异构体只在N末端不同。MLPKf1和MLPKf2均在乳头细胞中表达，并通过不同的分子机制定位于质膜。虽然MLPKPf1和MLPKf2都可以独立地补充mlpk/mlpk突变，但它们缺乏质膜定位基序的突变形式无法补充突变。此外，双分子荧光互补(BIFC)分析揭示了SRK与植物中MLPK亚型之间的直接相互作用。这些结果表明MLPK亚型定位于乳头细胞膜，并直接与SRK相互作用传递SI信号。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，找到了几个候选蛋白。一些候选者被发现选择性地与MLPK的活性形式相互作用，并且在体外被重组的MLPK特异性地磷酸化。我们现在正试图阐明这些候选分子的生理功能。拟南芥是一种具有MLPK同源基因APK1b的自交亲和植物。为了测试APK1b除SI信号转导外的生物学功能，我们分析了APK1b的T-DNA基因敲除系：然而，我们未能检测到包括生育在内的任何表型变化。APK1b在SI信号转导中的单一功能或与同源激酶相关的冗余可能解释了APK1b基因敲除突变体缺乏异常表型的原因。

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.