a, Gene expression of ROPGEFs during pollen germination and growth. The data are normalized expression values and standard deviation from microarray data (n = 4 for dry pollen, 30 min in vitro PT, and 4 h in vitro PT; n = 3 for semi-in-vivo PT)13 as noted in Extended Data Fig. 1b. ROPGEF8, ROPGEF9, ROPGEF11, ROPGEF12 and ROPGEF13 are expressed specifically in the dry pollen grain and pollen tube. b, BiFC assay showing the interaction between PRK6–cYFP and nYFP–GEF8, nYFP–GEF9, nYFP–GEF12, or nYFP–GEF13 (see Methods). c, A control experiment using C-terminal-deleted ROPGEF12 (ROPGEF12ΔC). The C-terminal domain is suggested to mediate the interaction with PRK2 (ref. 8). d, BiFC assay showing interaction between PRK6–cYFP and PRK6–nYFP, PRK3–nYFP, LIP1–nYFP or LIP2–nYFP. Scale bars, 50 μm. Images are representative of more than three experiments. e, Co-immunoprecipitation assay of PRK–mClover and ROPGEF12 proteins expressed in N. benthamiana leaf cells. ROPGEF12-3 × Flag protein was precipitated with full-length PRK3, PRK6 and kinase domain-deleted PRK6 (K-del), but not mClover control or cytosolic domain-deleted PRK6 (cyto-del-1). Data are representative of three experiments. For gel source data, see Supplementary Fig. 2.