State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, No. 8 BeiErTiao, ZhongGuanCun, Beijing 100080, China.
Fungal Biol. 2013 Jun;117(6):422-30. doi: 10.1016/j.funbio.2013.04.006. Epub 2013 May 1.
To gain insight into the regulatory mechanisms of oxidative stress responses in filamentous fungi, the genome-wide transcriptional response of Neurospora crassa to menadione was analysed by digital gene expression (DGE) profiling, which identified 779 upregulated genes and 576 downregulated genes. Knockout mutants affecting 130 highly-upregulated genes were tested for menadione sensitivity, which revealed that loss of the transcription factor siderophore regulation (SRE) (a transcriptional repressor for siderophore biosynthesis), catatase-3, cytochrome c peroxidase or superoxide dismutase 1 copper chaperone causes hypersensitivity to menadione. Deletion of sre dramatically increased transcription of the siderophore biosynthesis gene ono and the siderophore iron transporter gene sit during menadione stress, suggesting that SRE is required for repression of iron uptake under oxidative stress conditions. Contrary to its phenotype, the sre deletion mutant showed higher transcriptional levels of genes encoding reactive oxygen species (ROS) scavengers than wild type during menadione stress, which implies that the mutant suffers a higher level of oxidative stress than wild type. Uncontrolled iron uptake in the sre mutant might exacerbate cellular oxidative stress. This is the first report of a negative regulator of iron assimilation participating in the fungal oxidative stress response. In addition to SRE, eight other transcription factor genes were also menadione-responsive but their single gene knockout mutants showed wild-type menadione sensitivity. Two of them, named as mit-2 (menadione induced transcription factor-2) and mit-4 (menadione induced transcription factor-4), were selected for double mutant analysis. The double mutant was hypersensitive to menadione. Similarly, the double mutation of mit-2 and sre also had additive effects on menadione sensitivity, suggesting multiple transcription factors mediate oxidative stress resistance in an additive manner.
为了深入了解丝状真菌氧化应激反应的调控机制，我们采用数字基因表达（DGE）谱分析方法研究了 N. crassa 对亚甲蓝的全基因组转录反应，鉴定出 779 个上调基因和 576 个下调基因。对影响 130 个高上调基因的敲除突变体进行了亚甲蓝敏感性测试，结果表明转录因子铁载体调节（SRE）（铁载体生物合成的转录抑制剂）、CATase-3、细胞色素 c 过氧化物酶或超氧化物歧化酶 1 铜伴侣缺失会导致对亚甲蓝敏感。sre 缺失显著增加了亚甲蓝胁迫下铁载体生物合成基因 ono 和铁载体铁转运基因 sit 的转录，表明 SRE 是在氧化应激条件下抑制铁摄取所必需的。与表型相反，sre 缺失突变体在亚甲蓝胁迫下表现出比野生型更高的活性氧（ROS）清除剂基因转录水平，这意味着突变体比野生型遭受更高水平的氧化应激。sre 突变体中铁的不受控制摄取可能加剧细胞氧化应激。这是第一个参与真菌氧化应激反应的铁同化负调节因子的报道。除了 SRE 之外，还有其他八个转录因子基因也对亚甲蓝有反应，但它们的单个基因敲除突变体表现出野生型对亚甲蓝的敏感性。其中两个，命名为 mit-2（亚甲蓝诱导转录因子-2）和 mit-4（亚甲蓝诱导转录因子-4），被选为双突变体分析。该双突变体对亚甲蓝敏感。同样，mit-2 和 sre 的双突变也对亚甲蓝敏感性有累加效应，表明多个转录因子以累加的方式介导氧化应激抗性。
