Sclerotium rolfsii (teleomorph Athelia rolfsii) is one of the plant pathogenic basidiomycetes, which causes severe stem-rot disease in hundreds of plants and produces important metabolites, such as scleroglucan and TF-specific lectin. However, further molecular biological research on this filamentous fungus is severely plateaued out due to the lack of genetic methods. In this study, the A. tumefaciens strain LBA4404 harboring a binary vector containing the basta resistance gene fused with three reporters (DsRed, tdTomato, and GUSPlus) respectively, driven by the SrGPD promoter, was used for genetic transformation of S. rolfsii. The results showed that the three reporter genes were all effectively expressed in S. rolfsii. This study also showed that the intron of the SrGPD promoter is not necessary for transgene expression in this fungus. Besides, we showed that these reporters' signals could be observed easily but in a short time window. The efficient Agrobacterium-mediated transformation system and the three reporter gene plasmids for S. rolfsii developed in this study are of significance in overcoming current limitations of no available transformation and genetic manipulation techniques in S. rolfsii, facilitating further genetic manipulations and gene function exploration.

中文翻译：

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报告基因在根癌农杆菌介导的工业丝状真菌 Sclerotium rolfsii 中的高效表达

Sclerotium rolfsii (teleomorph Athelia rolfsii) 是植物病原担子菌之一，在数百种植物中引起严重的茎腐病，并产生重要的代谢物，如硬葡聚糖和 TF 特异性凝集素。然而，由于缺乏遗传方法，对这种丝状真菌的进一步分子生物学研究严重停滞。在这项研究中，根癌农杆菌菌株 LBA4404 含有一个二元载体，其中包含与三个报告基因（DsRed、tdTomato 和 GUSPlus）融合的 basta 抗性基因，由 SrGPD 启动子驱动，用于 S. rolfsii 的遗传转化。结果表明，三个报告基因均在 S. rolfsii 中有效表达。该研究还表明，SrGPD 启动子的内含子对于该真菌中的转基因表达不是必需的。此外，我们表明，这些记者的信号可以很容易地观察到，但在很短的时间窗口内。本研究开发的农杆菌介导的高效转化系统和三个报告基因质粒对于克服目前在 S. rolfsii 中没有可用转化和遗传操作技术的局限性具有重要意义，促进进一步的遗传操作和基因功能探索。