Fig. 4

BIC development. a Tip to side BIC differentiation and first nuclear division event at 26 hpi. Shown are maximum intensity projections of confocal optical sections (18.9–28.5 μm in total depth). Images are of M. oryzae CKF1651, expressing cytoplasmic EYFP (green), nuclear mRFP (red), and BIC-localized mRFP (red; cytoplasmic effector Pwl2 fused to mRFP). Arrows = BICs. Arrowheads = nuclei. Asterisk = appressorium. Left: Tip BIC stage. Middle: Side BIC stage. Right: After nuclear division, the side BIC remained at the same position. Bars = 5 μm. b Representative measurement from the penetration point to the side BIC in M. oryzae CKF1616 at 29 hpi. Images show BIC-localized mCherry (red; cytoplasmic effector Pwl2 fused to mCherry:NLS), fungal hypha outlined by EGFP (green; apoplastic effector Bas4 fused to EGFP), and mCherry and EGFP overlapping in the side BIC (yellow). Top: A maximum intensity projection of confocal optical sections (23 μm in total depth) and 2-dimensional length (gray line; length = 31.4 μm) measured with the open Bezier tool in the Zen Black software. Bottom: A 3-dimensional reconstruction of the same infection site shows 3D length (gray line; distance = 33.2 μm) measured with the Imaris dendrite creation algorithm (still image of the last frame of Additional file 2: Movie S1). Bars = 5 μm. c Comparative graphical summary of measurements taken with 3D Imaris and with 2D Zen Black software of the distance between the penetration point to the side BIC. Individual infection sites (black dots) have both a 3D measurement (x-axis) and a corresponding 2D measurement (y-axis). The vertical distance between a data point and the 1-to-1 line (blue) on the graph corresponds to the difference in length between the 2D and 3D measurements associated with that data point