Transcriptome analysis of a Ustilago maydis ust1 deletion mutant

Ustilago maydis, the causal agent of corn smut disease, is a dimorphic fungus alternating between a saprobic haploid budding form, and an obligate pathogenic filamentous dikaryon. Maize responds to U. maydis colonization by producing highly modified tumorous structures and it is only within these plant galls that the fungus sporulates giving rise to melanized sexual spores, the teliospores. Previously we identified a regulatory protein from the APSES family of transcription factors, which we named Ust1, whose absence in yeast cells led to filamentous growth and the production of highly pigmented spore-like structures in culture. In this study, we analyzed the transcriptome of a ∆ust1 deletion mutant. Overall design: To identify genes potentially involved in the sporulation program, we carried out microarray analysis comparing a haploid U. maydis WT strain (1/2) and ust1 (14/25) in vitro. Wild-type and ∆ust1 strains were grown in potato dextrose broth (PDB) for 48 h at 30oC. To allow effective comparison with other array data generated by U. maydis researchers, cells were then transferred to liquid array medium (6.25% Holiday salt solution, 30mM L-glutamine, 1% glucose, pH7.0, filter sterilized) at 30 C. Total RNA samples from WT 1/2 and ∆ust1 mutant strains grown in array medium for 24 h (mutant’s filamentous phase), and 48 h (mutant’s spore-like structure formation phase) were extracted and purified (Sigma Spectrum Plant Total RNA Kit, Cat.No.STRN50). RNA was sent to NimbleGen, where it was reverse transcribed to cDNA and Cy3 labeled. The cDNA samples were hybridized, microarray chips scanned, and raw data normalized with appropriate controls.