GEX3 interacts with GEX2 to function in gamete attachment and plasma membrane fusion in Arabidopsis

Male-female gamete interactions lead to double fertilization in angiosperms, but the molecular mechanisms that govern these interactions are poorly understood. Through EMS mutagenesis, we identified two Arabidopsis mutants defective in sperm-egg fusion, both of which harbored mutations in GAMETE INTERACTIONPROTEIN 1 (GIP1)/GAMETE EXPRESSED 3 (GEX3). GIP1/GEX3 encodes a sperm-specific plasma membrane protein whose loss leads to severe fertility defects due to impaired double fertilization. Using a gip1-cr1 cdka;1 double mutant that produces pollen with a single sperm-like cell, we demonstrated that GIP1/GEX3 is required for preferential fertilization of the egg cell. Further analysis revealed that GIP1/GEX3 functions at two stages: gamete attachment and subsequent plasma membrane fusion. Importantly, GIP1/GEX3 physically interacts with the sperm membrane protein GEX2 to regulate these two critical steps. Our work thus reveals a key regulatory module that mediates gamete attachment and plasma membrane fusion, providing new insight into the mechanisms that control double fertilization in flowering plants.