Figure 2: A synthetic anchorage protein blocks fungal EE motility.
From: Long-distance endosome trafficking drives fungal effector production during plant infection
(a) Organization of synthetic linker EAP; N-terminal part of an immobile kinesin-1rigour protein (aa 1-739) fused to an EE-binding Phox domain (PX) of the putative t-SNARE Yup1 (ref. 23). (b) Contrast-inverted kymographs showing motility of GFP-Rab5a-labelled EEs. Cells were pre-grown in glucose-containing (no EAP) or arabinose-containing medium (+EAP). The linker blocks EE motility. Scale bars, 3 s (vertical) and 3 μm (horizontal). See Supplementary Movie 3. (c) Kymograph showing motility of kinesin-3-GFP (green, arrowheads), unbound to stationary EEs (red) in a hyphal cell expressing EAP for 5 h. Scale bars, 2 s (vertical) and 2 μm (horizontal). See Supplementary Movie 4. (d) Motile EEs in yeast-like (yeast) and hyphal cells (hypha) in absence (no EAP) or presence of EAP expression (+EAP). Mean±s.e. and sample size from two experiments is shown. ***Significance at P<0.001, unpaired Student’s t-test. (e) EE-independent movement of GFP-labelled kinesin-3 motors in the presence (+EAP) or absence of EAP (no EAP) in yeast-like cells and hyphae. Mean±s.e. and sample size n from two to three experiments is shown. **Significance at P=0.0066, ***significance at P=0.0004, unpaired Student’s t-test. (f) Flux of dynein in the presence (+EAP) or absence of EAP (no EAP). Mean±s.e. and sample size n from two experiments is shown. No significant difference at P=0.4030, unpaired Student’s t-test. (g) Velocity of retrograde dynein in the presence (+EAP) or absence of EAP (no EAP). Mean±s.e. and sample size n from two experiments is shown. No significant difference at P=0.823, unpaired Student’s t-test. (h) Contrast-inverted kymographs showing retrograde motility of dynein in the absence of EAP (no EAP) and after 15 h of expression of EAP (+EAP). Scale bars, 1 s (vertical) and 1 μm (horizontal). (i) Apical Mcs1-GFP3 signal intensity at the growth region of yeast-like and hyphal cells in the presence (+EAP) or absence of EAP (no EAP). Mean±s.e. and sample size n from one experiment is shown. The result was confirmed by a non-quantitative experiment. No significant difference at an error probability of P=0.372 and 0.336, unpaired Student’s t-test. (j) Apical Mcs1-GFP3 at the growth region of hyphal cells in the presence (+EAP) or absence of EAP (no EAP). Scale bar, 2 μm.
