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

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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.