Members of the Fusarium graminearum species (Fg) complex, which are homothallic ascomycetous species, carry two opposite mating-type (MAT) loci in a single nucleus for controlling sexual development. We investigated the roles of three (MAT1-1-1, MAT1-1-2, and MAT1-1-3) and two (MAT1-2-1 and MAT1-2-3) transcripts located at both loci in representative Fg complex species (F. graminearum and Fusarium asiaticum). In self-fertile F. graminearum strains, the transcript levels of MAT1-1-1, MAT1-2-1, and MAT1-2-3 peaked 2 days after sexual induction (dai) and then remained high until 12 dai, whereas MAT1-1-2 and MAT1-1-3 transcripts reached peak levels between 4 and 8 dai. In contrast, all of the MAT transcripts in self-sterile F. asiaticum strains accumulated at much lower levels than those in F. graminearum during the entire time. Targeted gene deletions confirmed that MAT1-1-1, MAT1-1-2, MAT1-1-3, and MAT1-2-1 were essential for self-fertility in F. graminearum, but MAT1-2-3 was not. All MAT-deleted strains (except ΔMAT1-2-3) produced recombinant perithecia when outcrossed to a self-fertile strain. These results indicate that developmental up-regulation of the individual MAT genes in both a proper fashion and quantity is critical for sexual development, and that alterations in the gene expression could be attributed to the variation in self-sterility among the Fg complex.

Protein-protein interactions play important roles in controlling many cellular events. To date, several techniques have been developed for detection of protein-protein interactions in living cells, among which split luciferase complementation has been applied in animal and plant cells. Here, we examined whether the split luciferase assay could be used in filamentous ascomycetes, such as Gibberella zeae and Cochliobolus heterostrophus. The coding sequences of two strongly interacting proteins (the F-box protein, FBP1, and its partner SKP1) in G. zeae, under the control of the cryparin promoter from Cryphonectria parasitica, were translationally fused to the C- and N-terminal fragments of firefly luciferase (luc), respectively. Each fusion product inserted into a fungal transforming vector carrying the gene for resistance to either geneticin or hygromycin B, was transformed into both fungi. We detected complementation of split luciferase proteins driven by interaction of the two fungal proteins with a high luminescence intensity-to-background ratio only in the fungal transformants expressing both N-luc and C-luc fusion constructs. Using this system, we also confirmed a novel protein interaction between transcription factors, GzMCM1 and FST12 in G. zeae, which could hardly be proven by the yeast two-hybrid method. This is the first study demonstrating that monitoring of split luciferase complementation is a sensitive and efficient method of studying in vivo protein-protein interactions in filamentous ascomycetes.