Extended Data Fig. 4: ECS1 and ECS2 are almost quantitatively secreted from an apical network of the mature egg cell to the extracellular space only after gamete fusion.

a, b, ECS1-mCitrine (mCit) at the apical domain forming a network before fertilization. c, d, ECS2–mCit accumulate at the apical domain forming a network before fertilization. b, d, Enlargement of apical domains of egg cells shown in a, b. e–h, ECS1–mCit (e, f) and ECS2–mCit (g, h) are secreted from the egg cell to the extracellular space. Synergids are largely degenerated as indicated by the lack or diminished signal of the synergid marker. Volume projections of z-stacks from ECS1/2–mCit (yellow), an egg cell expressed Golgi–mScarlet (mScar; red) and a synergid expressed endoplasmic reticulum marker tagged to mTurquoise2 (mTur; cyan) are shown. i, FM4-64 staining showing that the cortical network containing ECS2–mCit is located at the plasma membrane. j, Signal intensity plot along the arrow shown in i indicates that ECS2–Cit is located at or just below the plasma membrane. k, Single optical section through the cortical network shows weak ECS2–Cit signals outside the network. l, Overexposure of the same optical section shown in k illustrates ECS2–Cit signals throughout the egg cytoplasm. m, Overexposed image showing ECS2–GFP accumulating in the apical egg cell domain and the endoplasmic reticulum maker EC1.2::erRFP marking the boundaries of the egg cell. A pollen tube expressing DsRed driven by the Lat52 promoter was used to monitor pollen tube perception. During pollen tube arrival ECS2–GFP was not yet released. n, Intensity plot profile showing relative fluorescence signal intensities of ECS2–GFP (green line) and erRFP (red line) along a dashed line drawn across the egg cells (indicated in the left image) confirming the microscopic observation. o, Sperm cells defective in gamete fusion (gcs1 mutant) did not trigger ECS2–GFP release. p, Intensity plot profile as in n showing that egg cell-localized ECS2–GFP and synergid cell-localized erRFP signals do not overlap. Ec, egg cell; pt, pollen tube; sp., sperm cells; sy, synbergid cells. Scale bars, 10 μm.