Regulators for Ustilago maydis mig gene expression during pathogenic development

玉米黑穗病病菌（Ustilago maydis）需要寄主玉米来发育和完成有性生命周期。我们已经鉴定出麦氏黑穗病菌基因mig1和mig2具有植物特异性表达谱。mig2基因被组织在一个由5个高度同源的基因组成的簇中，命名为mig2-1至mig2-5。在mig2-1和mig2-5基因的启动子中，已经绘制了不同的负顺式和正顺式作用区域。特别是，在mig2-5启动子中发现了一个高度保守的40bp区域，这是生物营养生长阶段基因激活所绝对需要的。在本提案中，我们希望描绘出各自的共识序列，并通过基于凝胶阻滞分析和酵母单杂交筛选的生化方法从U. maydis中分离出与该区域结合的转录调节因子。将分析编码结合活性的基因，并产生在已识别的mig2激活基因中缺失的U. maydis突变体，以研究它们对mig2基因调控和致病性的影响。此外，鉴定出的mig2激活基因将在诱导启动子的控制下表达，以评估过表达是否足以在无菌生长过程中触发mig2基因的表达。如果成功，条件激活菌株将利用微阵列分析筛选额外的靶基因。

The corn pathogen Ustilago maydis requires its host plant maize for development and completion of its sexual life cycle. We have identified the Ustilago maydis genes mig1 and mig2 that show a plant specific expression profile. The mig2 genes are organized in a cluster comprising five highly homologous genes, designated mig2-1 to mig2-5. In the promoters of the mig2-1 and mig2-5 genes distinct negatively- and positively cis-acting regions have been mapped. In particular, a highly conserved 40bp region has been identified in the mig2-5 promoter that is absolutely required for gene activation during the biotrophic growth phase. In this proposal we wish to delineate the respective consensus sequences and to isolate the transcriptional regulator(s) from U. maydis that bind(s) to this region by a biochemical approach based on gel retardation analysis and a yeast one-hybrid screen. The gene(s) encoding the binding activity will be analysed and U. maydis mutants deleted in identified mig2 activator genes will be generated to investigate their impact on mig gene regulation and pathogenicity. Furthermore, identified mig2 activator genes will be expressed under control of an inducible promoter to assess whether overexpression is sufficient to trigger mig gene expression during axenic growth. If successful, conditional activator strains will be exploited to screen for additional target genes by microarray analysis.