Ghassemi Sara, Lichius Alexander, Bidard Fréderique, Lemoine Sophie, Rossignol Marie-Noëlle, Herold Silvia, Seidl-Seiboth Verena, Seiboth Bernhard, Espeso Eduardo A, Margeot Antoine, Kubicek Christian P
Research Division Biotechnology and Microbiology, Institute of Chemical Engineering, TU Wien, Vienna, 1060, Austria.
Mol Microbiol. 2015 Apr;96(2):405-18. doi: 10.1111/mmi.12944. Epub 2015 Mar 4.
The ascomycete Trichoderma reesei is an industrial producer of cellulolytic and hemicellulolytic enzymes, and serves as a prime model for their genetic regulation. Most of its (hemi-)cellulolytic enzymes are obligatorily dependent on the transcriptional activator XYR1. Here, we investigated the nucleo-cytoplasmic shuttling mechanism that transports XYR1 across the nuclear pore complex. We identified 14 karyopherins in T. reesei, of which eight were predicted to be involved in nuclear import, and produced single gene-deletion mutants of all. We found KAP8, an ortholog of Aspergillus nidulans KapI, and Saccharomyces cerevisiae Kap121/Pse1, to be essential for nuclear recruitment of GFP-XYR1 and cellulase gene expression. Transformation with the native gene rescued this effect. Transcriptomic analyses of Δkap8 revealed that under cellulase-inducing conditions 42 CAZymes, including all cellulases and hemicellulases known to be under XYR1 control, were significantly down-regulated. Δkap8 strains were capable of forming fertile fruiting bodies but exhibited strongly reduced conidiation both in light and darkness, and showed enhanced sensitivity towards abiotic stress, including high osmotic pressure, low pH and high temperature. Together, these data underscore the significance of nuclear import of XYR1 in cellulase and hemicellulase gene regulation in T. reesei, and identify KAP8 as the major karyopherin required for this process.
子囊菌里氏木霉是纤维素分解酶和半纤维素分解酶的工业生产菌,也是这些酶基因调控的主要模型。其大多数(半)纤维素分解酶都必须依赖转录激活因子XYR1。在此,我们研究了XYR1穿过核孔复合体的核质穿梭机制。我们在里氏木霉中鉴定出14种核转运蛋白,其中8种被预测参与核输入,并构建了所有这些蛋白的单基因缺失突变体。我们发现,构巢曲霉KapI和酿酒酵母Kap121/Pse1的直系同源物KAP8,对GFP-XYR1的核募集和纤维素酶基因表达至关重要。用天然基因转化可挽救这种效应。对Δkap8进行转录组分析发现,在纤维素酶诱导条件下,42种碳水化合物活性酶(CAZymes),包括所有已知受XYR1调控的纤维素酶和半纤维素酶,均显著下调。Δkap8菌株能够形成可育子实体,但在光照和黑暗条件下产孢均显著减少,并且对非生物胁迫,包括高渗透压、低pH值和高温,表现出增强的敏感性。总之,这些数据强调了XYR1核输入在里氏木霉纤维素酶和半纤维素酶基因调控中的重要性,并确定KAP8是该过程所需的主要核转运蛋白。