Elucidating the suppression of root hair formation by a member of a novel, short ENTH protein family in Arabidopsis thaliana

具有EPSIN N-末端同源结构域的蛋白质通过识别磷脂酰肌醇(PIP)被招募到膜上，导致膜局部失稳，并有助于囊泡的形成和囊泡的破裂。到目前为止，所有研究的th蛋白都招募了更多的囊泡形成因子，例如在它们的长C末端存在相互作用基序的Adaptins或clathrin。拟南芥拥有7个th蛋白(Zouhar und Sauer，2014；Sauer等人)。2013)。它们中只有四个具有相互作用基序的功能C-末端。剩下的三个，这里称为EPSINOID，形成了一个系统发育亚群，具有短的、可能没有功能的C-末端，缺乏任何已知的相互作用基序。到目前为止，这个家庭还没有被描述出来。我们的初步数据显示，EPSINOID在不同的器官和组织类型中特异表达。突变分析表明，EPSINOID2是根毛生物发生中的一个负性因子。在epsinoid2中，两个独立的T-DNA等位基因具有更多、更长和异位的根毛。EPSINOID2在非毛细胞中特异表达。在亚细胞内，EPSINOID2主要是胞浆的，但在分裂细胞时与细胞板结合，这与囊泡运输的功能是一致的。然而，几乎所有已知的在根毛形成中起作用的囊泡运输因子都是积极的因子，它们的缺失会导致根毛减少或形态异常。因此，令人惊讶的是，EPSINOID2的缺失会导致更多的根毛。我们假设短长度蛋白结合了囊泡产生或运输所需的因子，但由于缺乏具有相互作用基序的长C末端，因此不能招募更多的辅助因子。这将通过非生产性的相互作用导致竞争性抑制。这种相互作用的可能候选者是Synaxins，它已被证明通过第10结构域中的特定基序与某些Long Long蛋白质相互作用。EPSINOID2也包含这样的基序，并且有几种已知的在根毛发形成中起作用的合成素，它们也在非毛细胞中表达。在非毛细胞中，合成素与EPSINOID2的非生产性相互作用可以调节其靶膜中合成素的丰度。在这个项目中，我们想用遗传、功能和生化的方法来解决以下两个主要问题：1)EPSINOID2在根毛形成的分子和遗传框架中的位置是什么？2)EPSINOID2在根毛形成中的确切作用方式是什么。我们希望这能有助于理解短长度蛋白质的一般作用的长期目标，特别是考虑潜在的竞争抑制功能。

Proteins with an EPSIN N-TERMINAL HOMOLOGY (ENTH) domain are recruited to membranes via recognition of phosphoinositides (PIPs), which leads to local membrane destabilization and contributes to vesicle formation and vesicle scission. All ENTH proteins studied so far recruit further factors of vesicle formation, such as adaptins or clathrin with interaction motifs present in their long C-termini. Arabidopsis thaliana possesses seven ENTH proteins (Zouhar und Sauer, 2014; Sauer et al. 2013). Only four of them have long, functional C-termini with interaction motifs. The three remaining ones, termed EPSINOIDs here, form a phylogenetic subgroup with short, putatively nonfunctional C-termini that lack any known interaction motifs. This family has not been characterized so far. Our preliminary data show that EPSINOIDs are specifically expressed in different organs and tissue types. Mutant analyses reveal that EPSINOID2 is a negative factor in root hair biogenesis. Two independent T-DNA alleles in epsinoid2 have more, longer and ectopic root hairs. EPSINOID2 is specifically expressed in nonhair cells. Subcellularly, EPSINOID2 is predominantly cytosolic but associates with the cell plate in dividing cells, which is in line with a function in vesicle trafficking. However, almost all known vesicle trafficking factors with roles in root hair formation act as positive factors and their loss leads to less or morphologically aberrant root hairs. Thus, it is surprising that loss of EPSINOID2 leads to more root hairs. We hypothesize that short ENTH proteins bind factors required for vesicle generation or transport but cannot recruit further cofactors due to lack of a long C-terminus with interaction motifs. This would lead to a competitive inhibition through nonproductive interactions. Possible candidates for such interactors are syntaxins, which have been shown to interact with certain long ENTH proteins via specific motifs in the ENTH domain. EPSINOID2 also contains such a motif and there are several syntaxins known with roles in root hair formation that are also expressed in nonhair cells. Nonproductive interactions of syntaxins with EPSINOID2 in nonhair cells could modulate the abundance of syntaxins in their target membranes. This would constitute a novel mechanism of action for ENTH proteins, previously not described in any organism.In this project we want to address the following two major questions with genetic, functional and biochemical methods: 1) what is the position of EPSINOID2 in the molecular and genetic framework of root hair formation? And 2) what is the exact mode of action of EPSINOID2 in root hair formation. We hope that this can contribute to the long-term goal of understanding the general role of short ENTH proteins, with special consideration of a potential competitive inhibitory function.