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

In angiosperms, male-female gamete interaction leads to double fertilization, yet the molecular mechanisms governing gamete interaction remain poorly understood. Through EMS mutagenesis, we identified two Arabidopsis mutants defective in sperm-egg fusion, both harboring mutation site in GAMETE INTERACTION PROTEIN 1 (GIP1)/GAMETE EXPRESSED 3 (GEX3). GIP1/GEX3 encodes a sperm-specific plasma membrane protein and its 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 the preferential fertilization of the egg cell. Further analysis revealed that GIP1/GEX3 functions at two distinct stages: gamete attachment and subsequent plasma membrane fusion. Importantly, we show that GIP1/GEX3 physically interacts with the sperm membrane protein GEX2 to regulate these two critical steps. Our work thus uncovers a key regulatory module mediating gamete attachment and plasma membrane fusion, providing new mechanistic insight into the control of double fertilization in flowering plants.