Extended Data Fig. 1: BIK1–GFP is functional in plants and undergoes endocytosis.
From: Ligand-induced monoubiquitination of BIK1 regulates plant immunity
a, BIK1–GFP is functional, as confirmed by BIK1 phosphorylation in 35S::BIK1-GFP-expressing Col-0 cotyledons after treatment with 1 μM flg22. 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 leaves upon flg22 treatment. Leaf discs from wild-type, bik1 and BIK1–GFP complementation plants (lines 1 and 2) were treated with 100 nM flg22 for ROS measurement using a luminometer over 50 min. Data are shown as mean ± s.e.m. (wild-type, 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 plasma membrane 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 depths (3, 6, 9, 12, 15, 18 and 21 μm) along with the maximum-intensity projection (MIP) of all 67 images through the epidermis. BIK1–GFP localizes to both plasma membrane and endosomal puncta (white arrows) within all sections. k–p, Method for quantification of BIK1–GFP puncta within MIPs of SDCM images. k, MIPs 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 (for example, stomata) were removed from the image using the line draw and crop functions. The total surface area (μm2) of the image was measured using the analyze measure function. m, Puncta within the cropped MIP were recognized using a customized model generated and applied with the Trainable Weka Segmentation plug-in for Fiji. The same model was applied to all images to generate binary images showing the physical locations of all BIK1–GFP puncta (black). n–o, Puncta within the size range 0.1–2.5 μm2 were highlighted in green (n) and counted (o) using the analyze particles function in Fiji. BIK1–GFP endocytosis was quantified as the number of puncta per 1,000 μm2. p, An overlay of the BIK1–GFP puncta (yellow highlight) over the cropped MIP confirmed correct identification of puncta. The experiments in a–c were repeated three times with similar results.
