RIC4-mediated F-actin accumulation at the tip inhibits RLK-GFP FRAP in the apical PM. (A) Time-lapse images of RLK-GFP fluorescence in a tube overexpressing RIC4. The time series of images shows before bleaching, immediately after bleaching (0 s), and recovery of fluorescence after bleaching at the indicated time points. The bleached area is marked by a box. (B) LatB treatment reverses impairment of RLK-GFP FRAP in RIC4-overexpressing tubes. RLK-GFP was coexpressed with RIC4 in tobacco pollen tubes. 3 h after incubation, pollen tubes were treated with 5 nM LatB for 1 h and then underwent FRAP analysis. The box shows the bleached area. (C) A balance between the RIC3 and RIC4 pathways is important for vesicle fusion. RLK-GFP was coexpressed with 0.2 μg RIC3 and 0.4 μg RIC4. Transformed pollen tubes underwent FRAP 4 h after transformation. Note the recovery of pollen tube growth and vesicle fusion. Box indicates the bleached area. Bars, 10 μm. (D) Quantification of fluorescence recovery. In RIC4-overexpressing tubes, fluorescence recovery was significantly slower and greatly reduced; <10% of the original intensity was observed 3 min after photobleaching (▪). LatB treatment (▴) or RIC3 coexpression (×) induced efficient and rapid fluorescence recovery similar to what was observed in control pollen tubes. Similar results were obtained in at least three different FRAP experiments. Control, FRAP of wild-type pollen tube.