Figure 8.

Forced Expression of GLS1 in Biotrophic Hyphae of C. graminicola Induces Defense Responses in Maize and Causes Reduced Fungal Virulence.

(A) Disease symptoms on nonwounded maize leaves after inoculation with the wild-type (WT) strain, two independent strains harboring a single ectopically integrated extra copy of GLS1 controlled by the trpC or toxB promoter of A. nidulans or P. tritici-repentis, respectively (ectopic, P_trpC:GLS; P_toxB:GLS), and strains with the GLS1 promoter exchanged by the trpC or toxB promoter (promoter exchange, P_trpC:GLS; P_toxB:GLS). Mock-inoculated leaves were treated with 0.01% (v/v) Tween 20.

(B) Appressorial penetration rates of the wild-type strain, two independent strains harboring a single ectopically integrated extra copy of GLS1 controlled by the trpC or toxB promoter, respectively (ectopic, P_trpC:GLS; P_toxB:GLS), and strains with the GLS1 promoter exchanged by the trpC or toxB promoter (promoter exchange, P_trpC:GLS; P_toxB:GLS).

(C) Quantification of fungal development on nonwounded (gray, WT; light blue, P_trpC:GLS; light green, P_toxB:GLS) and wounded maize leaves (black, WT; dark blue, P_trpC:GLS; dark green, P_toxB:GLS) by qPCR. Three independent measurements were performed for each strain in (B) and (C); bars are ±sd.

(D) Disease symptoms on wounded maize leaves after inoculation with the wild-type strain or representative promoter exchange or ectopic P_trpC:GLS or P_toxB:GLS strains. Mock inoculated leaves were treated with 0.01% (v/v) Tween 20.

(E) Both wild-type and P_trpC:GLS1 strains differentiated melanized appressoria (black arrowheads) and invaded intact maize leaves, but only the P_trpC:GLS1 strains caused whole-cell (white arrows) or cell wall fluorescence (white arrowheads) in maize under UV light, indicative of defense responses. Bars = 10 µm.

(F) Quantification of fluorescing maize cells decorated with single appressoria of the wild type or P_trpC:GLS1 strains. Three times 100 cells were measured. Bars represent ±sd.

(G) Maize cells infected by hyphae of P_trpC:GLS1 strains formed vesicles (arrowhead) that turned dark brown (insert) and densely decorated the invading hyphae (arrow). The wild-type strain formed hyphae (white arrow) rarely associated with vesicles. Bars = 10 µm.