Figure 5.

The Deletion of pmt4 Affects Appressoria Production.

(A) Filament formation of the solopathogenic SG200 and SG200 Δpmt4 strains that were spotted on PD-charcoal (PD-Ch) plates and incubated at 25°C for 24 h. The fuzzy spots indicate filament formation.

(B) Appressoria production was reduced in pmt4 mutants. A mixture of equal numbers of cells of SG200 and SG200 Δpmt4 strains tagged with CFP and YFP under the OMA promoter were used to inoculate 7-d-old maize seedlings. After 15 h, we scored appressoria formation by scoring for cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP) fluorescence. Three independent samples were analyzed. The error bars represent se. The deletion of pmt4 produced a decrease in appressoria formation.

(C) Visualization of appressorium morphology by deconvolution microscopy. Schematic XYZ drawing representing appressorium plant penetration. In the XY plane, we can see the appressorium structure on the leaf surface. Below, we showed an image without deconvolution. On the right, a series of images taken at different Z planes were reconstructed using maxim projection during deconvolution (see Methods for more details) to visualize fungal progression through plant tissue.

(D) Clamp-like cells are absent 24 h after infection in the pmt4 mutant. A mixture of equal numbers of cells for SG200 CFP and SG200 YFP Δpmt4, and SG200 YFP and SG200 CFP Δpmt4 strains were inoculated on 7-d-old maize seedlings. Twenty-four hours after infection, cells were observed by deconvolution microscopy in three channels: CFW, YFP, and CFP to identify each one of the strains. Deconvolved images of inoculated plants show that the deletion of pmt4 affects its capacity to form clamp-like cells, a structure associated with plant tissue penetration. Red asterisks indicate the site of penetration, and clamp cells in the wild type are marked with arrowheads.