Final Report Summary - GAMETEFUSION (Molecular and functional characterization of sperm-expressed proteins with potential fusogenic roles during double fertilization in Arabidopsis thaliana) Sexual reproduction is a key developmental process in the plant life cycle. In angiosperms, seeds are the direct products of fusion of the two male with two female gametes (double fertilization). Despite the relevance of double fertilization and its obvious economic and agronomical interest, our understanding of the molecular and signaling mechanisms controlling this crucial biological process are still rather limited.This project aimed to offer a holistic approach to the signalling and molecular mechanisms controlling double fertilization in Angiosperms. The initial strategy consisted in using a combination of bioinformatics tools and comparative transcriptomic analysis to select sperm‐enriched candidates sharing structural similarities or homology to known fusogenic proteins. The functional characterization of these candidates was expected to reveal insights into cellular events occurring transiently during double fertilization, such as gamete recognition, adhesion or fusion. For the majority of the candidates an initial functional characterization through the analysis of single T-DNA insertion lines failed to reveal any associated fertility defects expected for proteins with potential functions in gamete interactions. These results strongly suggest that genes expressed in male gametes might be endowed with a robust genetic redundancy. We focused our attention in two sperm-enriched tetraspanins, AtTET11 and AtTET12, due to their physiological importance as signaling mediators of cell-cell interactions in diverse biological processes in animal systems, namely adhesion and gamete fusion. However, knowledge about the biological function of tetraspanins in plants was still very scarce. The detailed expression analysis of all 17 gene family members of Arabidopsis thaliana confirmed TETs’ localization as membrane associated proteins preferentially expressed in specific reproductive tissues or cells, including male and female gametes. TETs showed distinct, sometimes overlapping expression patterns and a post-pollination developmental regulation, consistent with functions in diverse cellular events occurring during plant sexual reproduction. We could also show that plant tetraspanins were able to assemble in homo- and heterodimeric complexes in vivo. Similarly to their animal counterparts, TETs’ functions in plants may depend on the assembly of multi-molecular membrane complexes that rely on patterns of co-expression and regulation of potential binding partners. The unique punctate and polarized localization pattern found for TET11/TET12 in the interface of both sperm cells suggests the existence of a membrane scaffold that could be involved in mediating sperm-sperm cell adhesion or communication. This specific sperm cell microdomain may fulfill different possible functions: (1) polarized localization of fusogenic peptides; (2) establishment of a sperm-sperm cytoplasmic continuity (cell-cell communication) or (3) promoting sperm-sperm adhesion and Male Germ Unit (MGU) stability. Addressing the functional redundancy of plant TETs in general and specifically for TET11/TET12 was hindered by the lack of available T-DNA insertions and gene silencing strategies failed to address the desired function of this molecular complex in sperm cells. As an alternative approach we decided to identify potential TET11/TET12 binding partners in sperm cells. Using the publically available “Arabidopsis Membrane Interactome Database” we identified two potential TET12 sperm-enriched binding partners. The functional analysis of a double knockout in these genes showed defects in seed set, with predominant incidence of single fertilization events. Morphological changes in MGU assembly associated to sperm-sperm and sperm-VN connection seem to underlie the fertility defects, indicating that functional interactions within the MGU are necessary for the correct differentiation, maturation or acquisition of sperm cell fusogenic competence. The identification of novel gamete enriched proteins like those identified within this project is of significant relevance to understand cellular processes and signalling mechanisms underlying gamete differentiation and double fertilization, contributing to disclose possible conserved/divergent evolutionary mechanisms in cell-cell signaling and fusion in plants and animals, a topic of interest to the general scientific community.The identification of partner molecules in female gametes or sperm cells will be instrumental for a comprehensive view of the signalling and genetic pathways operating during double fertilization. Following this research topic we explored our laboratory’s expertise in order to obtain enough FACS purified Arabidopsis sperm cells to generate a sperm cell-specific membrane based-split ubiquitin (MbSUS) yeast two hybrid cDNA library. This unique experimental tool will be vital to boost the discovery of new molecular interactions underlying cellular events transiently occurring during double fertilization that otherwise would be extremely difficult to detect in planta. Probing proteins of interest or other male or female gamete bait candidates previously identified (e.g. EC1, HAP2, GEX2) in a cell type specific manner will undoubtedly minimize possible false positives arising from unspecific interactions with homologous genes. Several evidences reported in the literature and results obtained within the course of this project seem to support that transcripts present in sperm cells may have different cellular origins or be stored in a translational repressive state until delivery into the female gametes. Using our FACS methods we isolated the different cellular components of pollen in order to obtain a better resolution of transcriptional activity of the different cellular components. Candidates are being currently tested using a dual fluorescent labeling technique allowing targeting of specific mRNAs to explore their cellular dynamics and localized translation within cells of the male gametophyte. If successful, this approach will represent the first application of an in vivo system in imaging mRNA movement and localization in plant gametes and possibly to track delivery of mRNA transcripts during double fertilization. In the course of this project we have developed a set of experimental conditions and unique molecular tools that are either already available or will be made available to the scientific community in the near future. Present and future research outcomes are expected to bring new insights into this complex but vital cellular process in plants and foster the development of new research areas and new scientific collaborations. Ultimately the results arising from these studies are expected to promote the development of novel strategies in the manipulation of the fertilization process and for particular applications in economic interesting species in breeding programs.