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

具有EPSIN n端同源结构域（ENTH）的蛋白质通过磷酸肌苷（PIPs）的识别被招募到膜上，这导致局部膜不稳定，并有助于囊泡的形成和囊泡的分裂。迄今为止研究的所有ENTH蛋白都招募了囊泡形成的进一步因子，如在其长c端存在相互作用基序的适应性蛋白或网格蛋白。拟南芥具有7个ENTH蛋白（Zouhar und Sauer, 2014; Sauer et al. 2013）。其中只有四种具有长而功能性的c末端和相互作用基序。剩下的三个，在这里被称为epsinoid，形成一个系统发育亚群，具有短的，假定无功能的c端，缺乏任何已知的相互作用基序。到目前为止，这个家族还没有被确定。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EPSINOID2在根毛生物发生中是一个负性因子。epsinoid2的两个独立的T-DNA等位基因具有更多、更长和异位的根毛。EPSINOID2在非毛细胞中特异性表达。在细胞下，EPSINOID2主要是细胞质，但在细胞分裂时与细胞板结合，这与囊泡运输的功能一致。然而，几乎所有已知的与根毛形成有关的囊泡运输因子都是积极因子，它们的缺失导致根毛减少或形态异常。因此，令人惊讶的是，EPSINOID2的缺失导致了更多的根毛。我们假设短ENTH蛋白结合囊泡生成或运输所需的因子，但由于缺乏具有相互作用基序的长c端，因此无法招募进一步的辅因子。这将通过非生产性互动导致竞争性抑制。这种相互作用的可能候选物是句法素，它们已被证明通过ENTH结构域的特定基序与某些长ENTH蛋白相互作用。EPSINOID2也含有这样的基序，并且有几种已知的在根毛形成中起作用的合成素也在非毛细胞中表达。合成素与EPSINOID2在非毛细胞中的非生产性相互作用可以调节合成素在其靶膜中的丰度。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体中描述过。在本项目中，我们希望通过遗传学、功能性和生化方法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处于什么位置？2) EPSINOID2在根毛形成中的确切作用模式是什么？我们希望这有助于理解短ENTH蛋白的一般作用，并特别考虑潜在的竞争性抑制功能的长期目标。

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.