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

具有胰蛋白酶N端同源（ENTH）结构域的蛋白质通过识别磷酸肌醇（PIP）被募集到膜上，这导致局部膜不稳定并有助于囊泡形成和囊泡断裂。迄今为止研究的所有ENTH蛋白招募囊泡形成的进一步因素，如适应素或网格蛋白，其长C-末端存在相互作用基序。拟南芥具有七种ENTH蛋白（Zouhar und Sauer，2014; Sauer et al. 2013）。其中只有四个具有长的、功能性的C-末端和相互作用基序。剩下的三个，在这里被称为EPSINOIDS，形成了一个系统发育亚组，具有短的，pupelin-nonfunctional 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。我们的初步数据表明，EPSINOIDS在不同的器官和组织类型中特异性表达。突变体分析表明EPSINOID 2在根毛生物发生中是一个负因子。epsinoid 2中两个独立的T-DNA等位基因具有更多、更长和异位的根毛。EPSINOID 2在非毛细胞中特异性表达。在亚细胞内，EPSINOID 2主要存在于胞质中，但在分裂细胞中与细胞板结合，这与囊泡运输功能一致。然而，几乎所有已知的囊泡运输因子与根毛形成的作用作为积极的因素和他们的损失导致较少或形态异常根毛。因此，令人惊讶的是，EPSINOID 2的缺失导致更多的根毛。我们假设，短ENTH蛋白结合囊泡生成或运输所需的因子，但由于缺乏长C-末端与相互作用基序，不能招募进一步的辅因子。这将导致通过非生产性相互作用的竞争性抑制。这种相互作用物的可能候选物是突触融合蛋白，其已被证明通过ENTH结构域中的特定基序与某些长ENTH蛋白相互作用。EPSINOID 2也含有这样的基序，并且已知有几种在根毛形成中起作用的突触融合蛋白也在非毛细胞中表达。在非毛细胞中，突触融合蛋白与EPSINOID 2的非生产性相互作用可以调节其靶细胞膜中突触融合蛋白的丰度。这将构成ENTH蛋白的一种新的作用机制，以前没有在任何生物中描述过。在这个项目中，我们想用遗传学、功能学和生物化学的方法来解决以下两个主要问题：1）EPSINOID 2在根毛形成的分子和遗传框架中的位置是什么？和2）EPSINOID 2在根毛形成中的确切作用方式是什么。我们希望这可以有助于理解短ENTH蛋白的一般作用的长期目标，特别考虑潜在的竞争性抑制功能。

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.