The filamentous fungus Trichoderma reesei is a major source of cellulytic enzymes in biofuel production. Despite its economic relevance, our understanding of its secretory pathways is fragmentary. A major challenge is to visualise the dynamic behaviour of secretory vesicles in living cells. To this end, we establish a location juxtaposing the succinate dehydrogenase locus as a “soft-landing” site for controlled expression of 4 green-fluorescent and 5 red-fluorescent protein-encoding genes (GFPs, RFPs,). Quantitative and comparative analysis of their fluorescent signals in living cells demonstrates that codon-optimised monomeric superfolder GFP (TrmsGFP) and codon-optimised mCherry (TrmCherry) combine highest signal intensity with significantly improved signal-to-noise ratios. Finally, we show that integration of plasmid near the sdi1 locus does not affect secretion of cellulase activity in RUT-C30. The molecular and live cell imaging tools generated in this study will help our understanding the secretory pathway in the industrial fungus T. reesei.

丝状真菌里氏木霉是生物燃料生产中纤维素分解酶的主要来源。尽管它与经济有关，但我们对其分泌途径的理解是零碎的。一个主要的挑战是可视化活细胞中分泌囊泡的动态行为。为此，我们建立了一个将琥珀酸脱氢酶位点并置的位置，作为“软着陆”位点，以控制表达4个绿色荧光和5个红色荧光蛋白编码基因（GFP，RFP）。对活细胞中其荧光信号的定量和比较分析表明，密码子优化的单体超级文件夹GFP（TrmsGFP）和密码子优化的mCherry（TrmCherry）结合了最高的信号强度和显着改善的信噪比。最后，我们证明了sdi1附近质粒的整合基因座不影响RUT-C30中纤维素酶活性的分泌。这项研究中产生的分子和活细胞成像工具将有助于我们了解工业真菌里氏木霉的分泌途径。