Department of Agricultural Biotechnology, Seoul National University, Seoul, Republic of Korea.
Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul, Republic of Korea.
Mol Plant Pathol. 2021 Nov;22(11):1427-1435. doi: 10.1111/mpp.13118. Epub 2021 Aug 13.
Fusarium graminearum is an important plant-pathogenic fungus that causes Fusarium head blight on wheat and barley, and ear rot on maize worldwide. This fungus has been widely used as a model organism to study various biological processes of plant-pathogenic fungi because of its amenability to genetic manipulation and well-established outcross system. Gene deletion and overexpression/constitutive expression of target genes are tools widely used to investigate the molecular mechanism underlying fungal development, virulence, and secondary metabolite production. However, for fine-tuning gene expression and studying essential genes, a conditional gene expression system is necessary that enables repression or induction of gene expression by modifying external conditions. Until now, only a few conditional expression systems have been developed in plant-pathogenic fungi. This study proposes a new and versatile conditional gene expression system in F. graminearum using the promoter of a copper-responsive gene, designated F. graminearum copper-responsive 1 (FCR1). Transcript levels of FCR1 were found to be greatly affected by copper availability conditions. Moreover, the promoter (P ), 1 kb upstream of the FCR1 open reading frame, was sufficient to confer copper-dependent gene expression. Replacement of a green fluorescent protein gene and FgENA5 promoter with a P promoter clearly showed that P could be used for fine-tuning gene expression in this fungus. We also demonstrated the applicability of this conditional gene expression system to an essential gene study by replacing the promoter of FgIRE1, an essential gene of F. graminearum. This enabled the generation of FgIRE1 suppression mutants, which allowed functional characterization of the gene. This study reported the first conditional gene expression system in F. graminearum using both repression and induction. This system would be a convenient way to precisely control gene expression and will be used to determine the biological functions of various genes, including essential ones.
镰刀菌禾谷种是一种重要的植物病原真菌，可引起小麦和大麦的镰孢穗枯病，以及玉米的穗腐病。由于其易于遗传操作和成熟的异交系统，该真菌已被广泛用作研究植物病原真菌各种生物学过程的模式生物。基因缺失和靶基因的过表达/组成型表达是广泛用于研究真菌发育、毒力和次生代谢产物产生的分子机制的工具。然而，对于精细调节基因表达和研究必需基因，需要一个条件性基因表达系统，该系统可以通过改变外部条件来抑制或诱导基因表达。到目前为止，只有少数几种条件性表达系统在植物病原真菌中得到了发展。本研究提出了一种新的、多功能的条件性基因表达系统，该系统使用铜反应基因 F. graminearum 铜反应 1（FCR1）的启动子在 F. graminearum 中。发现 FCR1 的转录水平受铜可用性条件的显著影响。此外，FCR1 开放阅读框上游 1kb 的启动子（P）足以赋予铜依赖性基因表达。用 P 启动子替换绿色荧光蛋白基因和 FgENA5 启动子清楚地表明，P 可用于该真菌的精细基因表达调控。我们还通过用必需基因 FgIRE1 的启动子替换 P 启动子，证明了该条件性基因表达系统在必需基因研究中的适用性。这使得 FgIRE1 抑制突变体的产生成为可能，从而能够对该基因的功能进行特征分析。本研究首次在 F. graminearum 中使用抑制和诱导报告了一种条件性基因表达系统。该系统将是一种精确控制基因表达的便捷方式，将用于确定各种基因的生物学功能，包括必需基因。
