Fig. 2.

Co-visualization of fluorescent chitin and glucan synthase in hyphae of Z. tritici. (A) Co-visualization of fluorescent Chs5 (green) and the SPK, labelled with the dye FM4-64 (red) in a hypha of Z. tritici. Note that the fluorescent enzymes is not located in the SPK, which contradicts findings in N. crassa (Fajardo-Somera et al., 2015, Verdin et al., 2009). The edge of the cell is indicated in blue. Sale bar: 2 µm. (B) Co-visualization of eGFP₃-ZtChs5 (green) and mCherry₃-ZtGcs1 (red) at the tip of a Z. tritici hypha. Both cell wall synthases largely co-localize in the apical plasma membrane, resulting in a yellow signal. No apical accumulation of mCherry₃-ZtGcs1 is visible, suggesting that the putative β(1,3)-glucan synthase is not concentrating in the SPK. Again, this localization differs from reports in N. crassa (Sanchez-Leon et al., 2015a, Sanchez-Leon et al., 2015b). The edge of the cell is indicated in blue. Scale bar: 2 µm. (C) Kymographs showing directed co-motility of eGFP₃-ZtChs5 and mCherry₃-ZtGcs1 in Z. tritici. The direction relative to the hyphal tip is indicated by an arrow. Note that both signals move at the same speed and pause at the same time, suggesting that they are located to the same vesicle. Horizontal bar: 2 µm, vertical bar: 2 s. (D) Bar chart showing the spatial relationship of eGFP₃-ZtChs5 and mCherry₃-ZtGcs1 during directed motility in living cells. Most signals co-localize during motility in the cell. Bars represent mean ± SEM, sample size n is indicated. Statistical testing used a Student’s t-test; Triple asterisk indicate statistical difference to co-localization of both enzymes (Chs5 + Gcs1) at P < 0.0001. (E) Immuno-gold–labelling experiments, showing localization of eGFP₃-ZtChs5 (large nano-gold particles) and mCherry₃-ZtGcs1 (small nano-gold particles) on vesicles membranes. Both cell wall synthases localize to the same vesicles in various numbers. Note that signals are spatially separated, suggesting that the enzymes do not form super-complexes. Bar represents 50 nm. (F) Graph showing the diameter of cell wall synthase-carrying vesicles, estimated from immuno-gold preparations. Data given as a Whiskers' plot, with 25/75 percentiles indicated as blue lines, median as a red line, and minimum and maximum values as Whiskers ends; the sample sizes n is 28 from 3 preparations. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)