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. 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.

玉蜀黍黑粉菌（Ustilago maydis）是玉米黑粉病的致病菌，为二态真菌，可在腐生型单倍体出芽菌体与专性致病的丝状双核体两种形态间交替生长。玉米在感知玉蜀黍黑粉菌定殖后，会产生高度特化的肿瘤状结构，且该真菌仅能在这些植物瘿中完成产孢，形成黑色素化的有性孢子——冬孢子（teliospores）。此前本研究团队已鉴定得到一种隶属于APSES家族的转录因子调控蛋白，将其命名为Ust1；实验发现，当该蛋白在该真菌的酵母型菌体中缺失时，菌体将出现丝状生长，并在培养体系中产生高度色素化的类孢子结构。本研究中，我们对∆ust1缺失突变体的转录组进行了分析。为筛选潜在参与产孢程序的基因，我们以体外培养的单倍体玉蜀黍黑粉菌野生型菌株（1/2）与∆ust1突变株（14/25）为材料，开展了微阵列（microarray）分析。野生型与∆ust1突变菌株均于30℃下在马铃薯葡萄糖肉汤（potato dextrose broth, PDB）中培养48小时。为便于与玉蜀黍黑粉菌领域研究者已生成的其他微阵列数据进行有效比对，随后将菌体转接至液体微阵列培养基（成分为6.25% 霍利迪盐溶液（Holiday salt solution）、30mM L-谷氨酰胺、1%葡萄糖，pH 7.0，过滤除菌）中，于30℃继续培养。我们分别收集了在微阵列培养基中培养24小时（对应突变体的丝状生长阶段）与48小时（对应突变体的类孢子结构形成阶段）的野生型1/2与∆ust1突变菌株，使用Sigma Spectrum植物总RNA提取试剂盒（货号：STRN50）提取并纯化总RNA。将RNA样品寄送至NimbleGen公司，由其完成反转录合成cDNA并进行Cy3荧光标记。随后开展cDNA样品与微阵列芯片的杂交、芯片扫描，并通过合适的对照对原始数据进行标准化处理。