1.8.54
ISOLATION OF THE MAT-2 MATING TYPE GENE FROM SPECIES OF CERATOCYSTIS SENSU STRICTO

RC WITTHUHN1, TC HARRINGTON3, BD WINGFIELD2, DL McNEW3 and MJ WINGFIELD1,2

1Tree Pathology Co-operative Programme, Department of Microbiology and Biochemistry, University of the Orange Free State, P0 Box 339, Bloemfontein, 9300, South Africa; 2Tree Pathology Co-operative Programme, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa; 3Department of Plant Pathology, 351 Bessey Hall, Iowa State University, Ames, Iowa 50011, USA

Background and objectives
Single ascospore strains of filamentous ascomycetes are generally one of two opposite mating types. This mating type system is reflected within Ceratocystis sensu stricto by the strictly heterothallic species Ceratocystis eucalypti. In the case of C. coerulescens, C. virescens and most other Ceratocystis species, the MAT-2 strains are self-fertile and capable of selfing due to a uni-directional mating type switching. The MAT-1 strains are self-sterile and grow more slowly than the MAT-2 strains. Half of the progeny of a selfing are self-sterile and have the MAT-1 phenotype [2]. The current hypothesis is that self-sterility is due to the deletion of the MAT-2 mating type idiomorph and the resulting expression of the MAT-l idiomorph. The aim of this study was to amplify and sequence the MAT-2 gene of Ceratocystis species and to test whether the MAT-2 gene is deleted during mating type switching.

Materials and methods
A portion of the MAT-2 gene of isolates of C. eucalypti, C. coerulescens and C. fimbriata was amplified using degenerate primers designed from the conserved HMG DNA-binding motif flanking the mta mating type idiomorph in Neurospora crassa [1]. The expected 0.3-kb PCR products were cloned and sequenced. Specific primers, nested within the degenerate primers, were designed for the amplification of a portion of the MAT-2 idiomorph of each of the three species. The DNA flanking regions of the HMG box for C. eucalypti were obtained by using the TAIL-PCR technique [1].

Results and conclusions
The expected 0.3-kb fragment was only amplified in MAT-2 strains of the heterothallic C. eucalypti and in the self-fertile strains (MAT-2) of the homothallic C. coerulescens and C. fimbriata. Only MAT-2 progeny of C. eucalypti had the MAT-2 idiomorph based on amplification with the specific MAT-2 primers. Single-ascospore progeny were recovered from selfings of MAT-2 isolates of C. coerulescens, C. virescens and C. fimbriata. In each of these species, only the parents and the self-fertile (MAT-2) progeny had the MAT-2 idiomorph, and no PCR product was obtained from the self-sterile (MAT-1) progeny using the specific MAT-2 primers. Thus the newly designed specific primers only produced PCR fragments in the MAT-2 strains and not in the MAT-1 strains, showing that at least a portion of the MAT-2 idiomorph is deleted during mating type switching. Using TAIL-PCR, the known sequence of the MAT-2 idiomorph in C. eucalypti was increased to 1.3 kb.

References
1. Arie T, Christiansen K, Yoder OC, Turgeon BG, 1997. Fungal Genetics and Biology 21, 118-130.
2. Harrington TC, McNew DL, 1997. Current Genetics 32, 52-59.