The Functional Domain of GCS1-Based Gamete Fusion Resides in the Amino Terminus in Plant and Parasite Species

Toshiyuki Mori,Tsuneyoshi Kuroiwa,Makoto Hirai,Shin-Ya Miyagishima,Frederic Berger

doi:10.1371/journal.pone.0015957

Toshiyuki Mori, Tsuneyoshi Kuroiwa + Show 3 more

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https://doi.org/10.1371/journal.pone.0015957

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Abstract

Fertilization is one of the most important processes in all organisms utilizing sexual reproduction. In a previous study, we succeeded in identifying a novel male gametic transmembrane protein GCS1 (GENERATIVE CELL SPECIFIC 1), also called HAP2 (HAPLESS 2) in the male-sterile Arabidopsis thaliana mutants, as a factor critical to gamete fusion in flowering plants. Interestingly, GCS1 is highly conserved among various eukaryotes covering plants, protists and invertebrates. Of these organisms, Chlamydomonas (green alga) and Plasmodium (malaria parasite) GCS1s similarly show male gametic expression and gamete fusion function. Since it is generally believed that protein factors controlling gamete fusion have rapidly evolved and different organisms utilize species-specific gamete fusion factors, GCS1 may be an ancient fertilization factor derived from the common ancestor of those organisms above. And therefore, its molecular structure and function are important to understanding the common molecular mechanics of eukaryotic fertilization. In this study, we tried to detect the central functional domain(s) of GCS1, using complementation assay of ArabidopsisGCS1 mutant lines expressing modified GCS1. As a result, the positively-charged C-terminal sequence of this protein is dispensable for gamete fusion, while the highly conserved N-terminal domain is critical to GCS1 function. In addition, in vitro fertilization assay of Plasmodium berghei (mouse malaria parasite) knock-in lines expressing partly truncated GCS1 showed similar results. Those findings above indicate that the extracellular N-terminus alone is sufficient for GCS1-based gamete fusion.

Highlights

Angiosperm fertilization is comprised of certain processes, from pollination to gamete fusion [1]
Construction of Modified AtGCS1s As previously described, AtGCS1 is composed of an N-terminus signal sequence (SS), which probably leads to its cell membrane localization, a long body sequence containing the HAP2-GCS1 domain that is highly conserved among GCS1-possessing organisms, a hydrophobic transmembrane (TM) domain and a highly basic C-terminal histidine-rich (HR) sequence (Figure 1A) [2,3,11]
In which long sequences of AtGCS1 were deleted or exchanged mainly [11], we used a different strategy that partly disrupts characteristic AtGCS1 domains with a fluorescence tag insert so that we could see the importance of each domain, and normal expression of the modified AtGCS1s as well

Summary

Introduction

Angiosperm fertilization is comprised of certain processes, from pollination to gamete fusion [1]. Namely egg and central cells, exist close by in an embryo sac and fuse with these sperm cells to produce an embryo and an endosperm, respectively (double fertilization). We succeeded in identifying the novel protein GCS1 in male generative cells isolated from Lilium longiflorum pollen [2]. Lilium and Arabidopsis GCS1s were demonstrated to be expressed exclusively in male gametes (generative and sperm cells) and localized to the cell surface [2,3]. Arabidopsis GCS1 mutant pollen exhibits serious male sterility in which none of the sperm cells are able to fuse with female gametes, suggesting that GCS1 is an indispensable factor for gamete fusion [2,3]

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