Figure 7. The N-terminus of MoVAC8p contains a SH4 domain.

(A) SH4:GFP fusion proteins are anchored at the vacuole, septa and the plasma membrane in both conidia and appressoria. The first 21 amino acid residues of MoVAC8p were fused into the N-terminus of GFP to produce SH4:GFP and subcellular localisation of the GFP fusion analysed by epifluorescence microscopy. Guy11 conidia expressing SH4:GFP fusion proteins were stained with FM4–64 and allowed to germinate and undergo appressorium development. SH4:GFP fusions colocalise with the FM4–64-stained vacuole, septa and plasma membrane. (B) SH4:GFP fusion proteins associate with the vacuole, septa and the plasma membrane in penetration pegs and invasive hyphae. Penetration of onion epidermis was examined at indicated time points and Calcofluor White (CFW) used to stain the cell wall. SH4:GFP fusion proteins colocalise with the vacuole (as seen in the bright field image) and CFW-stained cell wall. (C) SH4:GFP fusion proteins were targeted to the vacuole, septa and the plasma membrane in vegetative hyphae. Vegetative hyphae expressing SH4:GFP fusion proteins were grown in CM for 24 h, and 200 µl of the cultures incubated with 7.5 µM FM4–64 at 26°C for 1 h, followed by CFW staining. In vegetative hyphae, SH4:GFP fusion proteins also colocalise with FM4–64- stained vacuoles and CFW-stained septa and cell walls. Scale bar = 10 µm.