Sexual phases among pathogenic fungi are major determinants of morphological transitions linked to virulence. The sugarcane smut fungi, Sporisorium scitamineum, are an obligate bitrophic pathogen, whose molecular basis of sexual mating is poorly understood. Here we report the creation of a library of mating-defective mutants of S. scitamineum using Agrobacterium mediated random insertional mutagenesis in the quest to discover genes that drive the sexual process. A total of 10560 transfer DNA (T-DNA) insertional mutants, 27 of which were mating-defective, were generated. Southern blot analysis revealed 89% of mating-defective mutants to have single copy T-DNA insertion. Hi-tail polymerase chain reaction (PCR) gene isolation technique for three different mating-defective mutant isolates resulted in recovery of three unique amplicon fragments flanking T-DNA and sequencing the fragments followed by relevant bioinformatic approaches predicted in three different genes among three different mutants as follows; mutant 12, a gene coding for an uncharacterised protein N-terminal fragment located on chromosome 16; mutant 19, a gene coding for a protein related to FMS1- polyamine oxidase, converts spermine to spermidine located on chromosome 2 and mutant 24, a gene coding for a probable structural maintenance of chromosomes (SMC) segregation protein located on chromosome 2. We propose that mutant 19 and 24 are better candidates for gene functional analyses based on qRT-PCR data. Hypotheses are thus generated that either polyamine oxidase gene family or SMC chromosome segregation protein genes both residing on chromosome 2 of S. scitamineum have a role in the mating process.

Keywords: Sporisorium scitamineum, mating-defective mutants, insertional mutagenesis, hi-tail PCR, Agrobacterium.