Deletion and complementation of the mating type (MAT) locus of the wheat head blight pathogen Gibberella zeae

Abstract

Gibberella zeae, a self-fertile, haploid filamentous ascomycete, causes serious epidemics of wheat (Triticum aestivum) head blight worldwide and contaminates grain with trichothecene mycotoxins. Anecdotal evidence dating back to the late 19th century indicates that G. zeae ascospores (sexual spores) are a more important inoculum source than are macroconidia (asexual spores), although the fungus can produce both during wheat head blight epidemics. To develop fungal strains to test this hypothesis, the entire mating type (MAT1) locus was deleted from a self-fertile (MAT1-1/MAT1-2), virulent, trichothecene-producing wild-type strain of G. zeae. The resulting MAT deletion (mat1-1/mat1-2) strains were unable to produce perithecia or ascospores and appeared to be unable to mate with the fertile strain from which they were derived. Complementation of a MAT deletion strain by transformation with a copy of the entire MAT locus resulted in recovery of production of perithecia and ascospores. MAT deletion strains and MAT-complemented strains retained the ability to produce macroconidia that could cause head blight, as assessed by direct injection into wheat heads in greenhouse tests. Availability of MAT-null and MAT-complemented strains provides a means to determine the importance of ascospores in the biology of G. zeae and perhaps to identify novel approaches to control wheat head blight.

FIG. 1.

(A) Wild-type G. zeae MAT locus; (B) MAT locus deletion vector, pGzMAT-H, shown in the linear form that would result from digestion with SalI; (C) MAT locus in which the 9.6-kb region with the four MAT genes has been deleted and replaced by a 5.1-kb fragment carrying the hygB and pCR XL TOPO portions of pGzMAT-H; (D) MAT locus complementation vector pGzMAT-Gen. Numbered arrows indicate relative positions and orientations of PCR primers used in this study. The black bars in panels A and C and the white bars in panels A and D indicate the regions of DNA used to prepare the hybridization probe for Southern analysis of transformants carrying pGzMAT-Gen.

FIG. 2.

(A) Southern blot analysis of G. zeae mat1-1/mat1-2 strains conducted by using BamHI/SalI-digested DNA and a mixture of probes prepared from fragments 2.8-up and 1.2-down as described in Materials and Methods. Lane 1, wild-type strain GZ3639; lanes 2 to 5, mat1-1/mat1-2 strains ΔMAT#9, ΔMAT#14, ΔMAT#40, and ΔMAT#43, respectively. The gel image has been modified from the original blot by deleting one lane between lanes 3 and 4. (B) Southern blot analysis of G. zeae mat1-1/mat1-2 strains transformed with the complementation vector pGzMAT-Gen, conducted by using BamHI/SalI-digested DNA and a mixture of probes described in Materials and Methods and illustrated in Fig. 1. Lane 1, wild-type strain GZ3639; lane 2, mat1-1/mat1-2 strain ΔMAT#78; lanes 3 to 5, MAT-complemented transformants 1, 2, and 9, respectively.

FIG. 3.

Self-fertility of G. zeae wild-type GZ3639 (A, D, and G), MAT deletion strain ΔMAT#78 (B and E), and a representative MAT add-back strain (C, F, and H). The top row shows the morphology and distribution patterns of perithecia on carrot agar plates. The middle row shows the contents of squashed perithecia and perithecium-like structures, stained with cotton blue. The bottom row shows a higher magnification view of asci and individual ascospores produced by wild-type and add-back strains.