Roth Mitchell G, Chilvers Martin I
1Department of Plant, Soil and Microbial Sciences, Michigan State University, 1066 Bogue St., East Lansing, 48824 MI USA.
2Genetics Graduate Program, Michigan State University, 567 Wilson Rd., East Lansing, 48824 MI USA.
Fungal Biol Biotechnol. 2019 May 15;6:7. doi: 10.1186/s40694-019-0070-0. eCollection 2019.
Soybean production around the globe faces significant annual yield losses due to pests and diseases. One of the most significant causes of soybean yield loss annually in the U.S. is sudden death syndrome (SDS), caused by soil-borne fungi in the species complex. Two of these species, and , have been discovered in the U.S. The genetic mechanisms that these pathogens employ to induce root rot and SDS are largely unknown. Previous methods describing protoplast generation and transformation have been used to study gene function, but these methods lack important details and controls. In addition, no reports of protoplast generation and genetic transformation have been made for .
We developed a new protocol for developing fungal protoplasts in these species and test the protoplasts for the ability to take up foreign DNA. We show that wild-type strains of and are sensitive to the antibiotics hygromycin and nourseothricin, but strains transformed with resistance genes displayed resistance to these antibiotics. In addition, integration of fluorescent protein reporter genes demonstrates that the foreign DNA is expressed and results in a functional protein, providing fluorescence to both pathogens.
This protocol provides significant details for reproducibly producing protoplasts and transforming and . The protocol can be used to develop high quality protoplasts for further investigations into genetic mechanisms of growth and pathogenicity of and . Fluorescent strains developed in this study can be used to investigate temporal colonization and potential host preferences of these species.
全球大豆生产每年因病虫害面临显著的产量损失。在美国，每年大豆产量损失的最重要原因之一是猝死综合征（SDS），它由该物种复合体中的土传真菌引起。在美国已发现其中两个物种，即[物种名称1]和[物种名称2]。这些病原体用于诱导根腐病和猝死综合征的遗传机制在很大程度上尚不清楚。先前描述[真菌名称1]原生质体生成和转化的方法已用于研究基因功能，但这些方法缺乏重要细节和对照。此外，尚未有关于[真菌名称2]原生质体生成和遗传转化的报道。
我们开发了一种新方案，用于在这些[真菌名称]物种中培养真菌原生质体，并测试原生质体摄取外源DNA的能力。我们发现[真菌名称1]和[真菌名称2]的野生型菌株对潮霉素和诺尔丝菌素敏感，但用抗性基因转化的菌株对这些抗生素具有抗性。此外，荧光蛋白报告基因的整合表明外源DNA得到表达并产生功能性蛋白质，使两种病原体都发出荧光。
该方案为可重复地产生原生质体以及转化[真菌名称1]和[真菌名称2]提供了重要细节。该方案可用于培养高质量的原生质体，以进一步研究[真菌名称1]和[真菌名称2]生长及致病性的遗传机制。本研究中构建的荧光菌株可用于研究这些物种的时间定殖和潜在的宿主偏好。
