Extended Data Figure 1. BIK1-GFP is functional in plants and undergoes endocytosis.

a. BIK1-GFP is functional as confirmed by BIK1 phosphorylation in 35S::BIK1-GFP expressing Col-0 cotyledons after 1 μM flg22 treatment. MPK6 is a loading control and the black stippled line indicates discontinuous segments from the same gel. b. BIK1-GFP restored ROS production in bik1 upon flg22 treatment. Leaf discs from WT, bik1 and BIK1-GFP complementation (Line 1 and 2) were treated with 100 nM flg22 for ROS measurement using a luminometer over 50 min. Data are shown as means ± SEM (WT, bik1: n = 42; BIK1-GFP/bik1 n =45). c. Time-lapse SDCM shows that BIK1-GFP endosomal puncta are highly mobile with puncta that disappear (red circle), appear (yellow circle), and rapidly move in and out of the plane of view (white circle). Scale bar, 5 μm. d-k. BIK1-GFP localizes to endosomal puncta and PM in cross-sectional images of epidermal cells. The abaxial epidermal cells of cotyledons expressing BIK1-GFP were imaged with SDCM with a Z-step of 0.3 μm. A subset of the cross-sectional images is shown at the indicated depth (3, 6, 9, 12, 15, 18 and 21 μm) along with the maximum-intensity projection of all 67 images through epidermis. BIK1-GFP localizes to both PM and endosomal puncta (white arrows) within all sections. k-p. Quantification method for BIK1-GFP puncta within maximum-intensity projections of SDCM images. k. Maximum-intensity projections (MIP) were generated using Fiji distribution of ImageJ 1.51 (https://fiji.sc/) for each Z-series captured by SDCM imaging of BIK1-GFP cotyledons. l. Regions of MIP with non-pavement cells (e.g. stomata) are removed from the image using the Line Draw and Crop functions. The total surface area (μm²) of the image is measured using the Analyze Measure function. m. Puncta within the cropped MIP are recognized using a customized model generated and applied with the Trainable Weka Segmentation plug-in for Fiji. The same model is applied to all images to generate binary images showing the physical location of each BIK1-GFP puncta (black). n-o. Puncta within the size range of 0.1–2.5 μm² are highlighted in green (n) and counted (o) using the Analyze Particles function in Fiji. BIK1-GFP endocytosis is quantified as the number of puncta per 1000 μm². p. An overlay of the BIK1-GFP puncta (yellow highlight) over the cropped MIP confirms correct identification of puncta. The experiments in a-c were repeated three times with similar results.