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. 2018 Jul 9;28(13):2136–2144.e7. doi: 10.1016/j.cub.2018.05.014

Figure 2.

Figure 2

FORMIN4 Transport and Delivery Require Actin, but Not Its Endogenous FH1-FH2 Domain

(A) Mosaics consisting of multiple confocal laser scanning microscope images combined to show wide areas of dark grown FORMIN4-GFP transformant hypocotyls. Left: the corresponding transmission image to the mock-treated sample. Right: FORMIN4-GFP signal after 4 hr elicitation. Cells expressing FORMIN4-GFP after elicitation showed an average number of 1.98 (±1.16) FORMIN4-GFP regions per GFP-positive cell (n = 64). The scale bars represent 100 μm.

(B) Aniline blue (magenta) staining shows that the majority of FORMIN4-GFP regions are associated with centralized callose regions (88.9% ± 8.6%). The scale bar represents 10 μm.

(C) Typical TIRF microscopy image of regional FORMIN4-GFP accumulation after 4 hr of elicitation. The scale bar represents 10 μm.

(D) FORMIN4-GFP expression is induced and delivery achieved four hours after immune system stimulation. Kymographs (taken along the lengths of indicated red boxes) show that FORMIN4-GFP-labeled vesicular bodies can be detected in the cytoplasmic stream. Latrunculin A and B treatment disrupts streaming, but plasma membrane-associated FORMIN4-GFP remains in a stable pattern. Red boxes are 25 μm in length. See also Figure S2.

(E) Fluorescence recovery after photobleaching (FRAP) experiments performed using total internal reflection fluorescence (TIRF) microscopy demonstrate that the site-specific delivery process is active four hours after stimulation. See also Figure S2. The scale bar represents 10 μm.

(F) The delivery process is interrupted by latrunculin B treatment as measured using pixel fluorescence recovery and recovery of object number. Asterisk indicates a significant difference of p < 0.05. Error bars indicate the SE. See also Figure S2.

(G) Removal of the FORMIN4 cytosolic domain (that includes the FH1-FH2 domain) does not prevent FORMIN4 localization to the infection site either in wild-type plants or in plants where all group 1e formins have been genetically disrupted. The scale bar represents 5 μm.