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.

Ustilago maydis，玉米黑粉病的病原体，是一种具有二态性的真菌，其在腐生性单倍体芽生形态与强制性致病性丝状二核相之间交替。玉米对U. maydis的侵染产生高度改造的肿瘤状结构作为响应，而真菌的孢子产生正是在这些植物瘤中进行的，从而形成了黑色的性孢子，即厚垣孢子。先前，我们鉴定出一种来自APSES转录因子家族的调控蛋白，我们将其命名为Ust1，其在酵母细胞中的缺失导致丝状生长以及在培养中产生高度色素化的类似孢子结构。在本研究中，我们对一个Δust1缺失突变体的转录组进行了分析。总体设计：为识别可能参与孢子形成程序的基因，我们进行了微阵列分析，比较了单倍体U. maydis野生型菌株（1/2）与ust1（14/25）体外培养。野生型和Δust1菌株在30℃的土豆葡萄糖肉汤（PDB）中培养48小时。为了有效地与其他U. maydis研究者生成的阵列数据进行比较，细胞随后被转移到液体阵列介质（6.25% Holiday盐溶液、30mM L-谷氨酰胺、1%葡萄糖、pH7.0、过滤灭菌）中，在30℃下培养。从培养24小时（突变体的丝状阶段）和48小时（突变体类似孢子结构形成阶段）的阵列介质中生长的WT 1/2和Δust1突变体菌株的总RNA样本被提取和纯化（Sigma Spectrum Plant Total RNA Kit，Cat.No.STRN50）。RNA被发送至NimbleGen，在那里它被逆转录为cDNA并Cy3标记。cDNA样本进行了杂交，微阵列芯片被扫描，并使用适当的对照对原始数据进行归一化。