In all self-sterile F. asiaticum strains examined, the MAT1-1-1, MAT1-2-1, and MAT1-2-3 expression was also highly induced at the early stage, similar to those in F. graminearum described above, but the transcript levels during the entire sexual cycles were c. 10- to 20-fold lower than those in F. graminearum (Fig. 1, Table S2). The later sexual stage-specific patterns of MAT1-1-2 and MAT1-1-3 shown in F. graminearum were significantly altered in F. asiaticum. Neither MAT1-1-2 nor MAT1-1-3 was significantly induced at any time point during the sexual development compared with those during the vegetative growth (Fig. 1, Table S2). Integration of a transforming DNA construct
for gene deletion into the fungal genome via a double cross-over resulted IDO inhibitor in a F. graminearum Z3643 or Z3639 strain lacking individual MAT genes (designated ΔMAT1-1-1, ΔMAT1-1-2, ΔMAT1-1-3, ΔMAT1-2-1, and ΔMAT1-2-3; Fig. 2a). Targeted gene deletion was verified
by DNA blot hybridization (Fig. 2b). In carrot agar cultures of the wild-type Z3643 or Z3639 strains, protoperithecia began to form at 3 dai and developed into fully fertile perithecia after 6–7 dai, which carried asci containing eight ascospores. However, those formed in the ΔMAT1-1-1, ΔMAT1-1-2, and ΔMAT1-1-3 strains were smaller than normal perithecia from wild-type cultures, and carried neither asci nor ascospores even 4 weeks after perithecial induction (Fig. 3). Barren perithecia in the ΔMAT1-1-1 strains were smaller than those in the ΔMAT1-1-2 and ΔMAT1-1-3 strains, but the numbers of barren perithecia from selleck inhibitor all of these ΔMAT strains were similar to those of fertile wild-type strains (Fig. 3). In addition, the ΔMAT1-2-1 strain (T43ΔM2-2) produced no perithecia on carrot agar, as reported previously (Lee et al., 2003). Unlike these MAT deletion strains, the ΔMAT1-2-3 strains produced a similar number of normal fertile perithecia to Z3643, demonstrating that MAT1-2-3 are dispensable for perithecia formation in F. graminearum
(Fig. 3). The phenotypes of all of the MAT-deleted strains examined, other than Quisqualic acid fertility (e.g. mycelial growth, pigmentation, and conidiation), were not different from those of their wild-type progenitor (data not shown). To determine whether self-sterile ΔMAT strains retain the ability to outcross, we set up sexual crosses of a transgenic F. graminearum (FgGFP-1) carrying a GFP gene to each of the ΔMAT1 strains, wherein the ΔMAT strains were forced to act as the female parent. All outcrosses except that of the ΔMAT1-2-3 strain produced morphologically normal mature perithecia with asci containing eight ascospores; the numbers of perithecia formed in the outcrosses were reduced to c. 30% of the level of the self or wild-type strains based on examination of more than 100 perithecia. All perithecia from each outcross examined yielded eight tetrads, of which four fluoresced (Fig. 4), indicating the occurrence of normal meiosis for production of recombinant progeny.