Extended Data Fig. 6: The microtubule cage is essential for HUG-cage assembly and male germ-unit organization.

(a) GFP–MAP4–MBD around the sperm nuclei in pollen tubes. Arrow, the sperm projection into the invagination of the vegetative nucleus. Scale bar, 10 μm. (c) Co-expression of GFP–MAP4–MBD and HUG2–RFP showing the microtubule cage is inside the HUG2–GFP cage. Scale bar, 5 μm. (b) Relative fluorescence intensity along the white line in (a). (c, d) Confocal imaging of GFP–MBD- and HUG1–GFP-expressing mature pollen treated with oryzalin at 25 °C and 4 °C. Scale bar, 5 μm. (e, f) Treatment of mature pollen grains with 5 μM oryzalin overnight partially disturbs HUG1–GFP localization outside the peri-germ cell membrane, causing pollen-tube branching or bulging, but does not affect the integrity of MGU. Scale bar, 10 μm. (g) Colchicine treatment of HUG1–GFP-expressing pollen. Arrow, VN-connecting filamentous structures labeled by HUG1–GFP. Scale bar, 2 μm. The mock-treated pollen is the same as that in (c, d). (h) Treatment of mature pollen grains with 1 mM colchicine disrupts HUG1–GFP localization outside the peri-germ cell membrane but does not disassemble the MGU. Asterisks indicate sperm nuclei. Scale bar, 10 μm. (i) Frequency of pollen developmental stages after supplementation to inflorescence with oryzalin for 3 d. (j) Pollen-germination rate after treatment in (i). n = 500 for each sample. Data are the mean ± S.D. Two-sided Students’ t-test, P = 0.0668 for Oryzalin-treated WT, P = 0.0585 for GFP-MAP4-MBD and P = 0.0712 for HUG1-GFP. n.s. non-significant.