GAMETEFUSION · Molecular and functional characterization of sperm-expressed proteins with potential fusogenic roles during double fertilization in Arabidopsis thaliana

Sexual reproduction is a fundamental biological process common among eukaryotes. In angiosperms, fertilization although associated with pollination, is actually a fairly late phenomenon in the process of reproduction and involves a double fusion event between two male and two female gametes. In contrast to animal fertilization, the molecular mechanisms underlying this process in flowering plants are poorly understood and the molecular players unknown. With this project we intend to explore recent advances in the field, namely the availability of expression profiles of plant gametes to identify the molecular players which mediate sperm-egg cell/central cell fusion in Arabidopsis thaliana. We used bioinformatics approaches to identify plasma membrane proteins with expression in sperm cells and sharing structural similarities to known fusogenic peptides. The first candidates are two sperm-specific proteins which belong to the conserved family of tetraspanins. Functional analysis of the candidate proteins will be performed in knockout, amiRNA and overexpression transgenic lines. Interacting proteins in sperm cells and in the female partner cells will be identified using yeast-two hybrid system and validated by functional analysis. Candidate proteins will be assayed for specific functions during double fertilization, such as preferential gamete targeting, fusogenic properties, polyspermy block and fertilization barriers using manipulation of gene expression and proteomic approaches coupled to in vivo and in vitro assays. We also intend to develop an in vivo fluorescent based method to detect and track specific sperm-expressed mRNAs, in order to understand the importance of intercellular communication during double fertilization and initiation of embryogenesis. This project is addressing fundamental questions involving one of the most complex biological processes in nature and might provide novel tools for biotechnology applications and crop improvement.