A protoplast generation and transformation method for soybean sudden death syndrome causal agents Fusarium virguliforme and F. brasiliense
A protoplast generation and transformation method for soybean sudden death syndrome causal agents Fusarium virguliforme and F. brasiliense
复制标题
DOI：
10.1186/s40694-019-0070-0
复制
发表时间：
2019-05-15
影响因子：
--
通讯作者：
Chilvers, Martin I.
中科院分区：
文献类型：
--
作者：
Roth, Mitchell G.;Chilvers, Martin I.
背景地球仪的大豆生产由于病虫害而面临严重的年产量损失。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），由茄病镰刀菌物种复合体中的土传真菌引起。其中两个物种，F。virguliforme和F.这些病原体诱导根腐病和SDS的遗传机制在很大程度上是未知的。以前的方法描述F. virguliforme原生质体的产生和转化已被用于研究基因功能，但这些方法缺乏重要的细节和控制。此外，还没有F.巴西的。结果我们开发了一种新的方案，在这些镰刀菌属真菌原生质体的发展和测试的能力，采取外源DNA的原生质体。我们表明，野生型菌株的F。virguliforme和F. brasiliense对潮霉素和诺尔丝菌素敏感，但转化了抗性基因的菌株对这些抗生素表现出抗性。此外，荧光蛋白报告基因的整合表明外源DNA表达并产生功能蛋白，为两种病原体提供荧光。结论该方法为F. virguliforme和F.巴西的。该方法可用于获得高质量的原生质体，为进一步研究镰刀菌生长和致病的遗传机制奠定基础。virguliforme和F.巴西的。本研究中开发的荧光菌株可用于研究这些物种的时间定殖和潜在宿主偏好。
BackgroundSoybean 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 Fusarium solani species complex. Two of these species, F. virguliforme and F. brasiliense, 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 F. virguliforme 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 F. brasiliense. ResultsWe developed a new protocol for developing fungal protoplasts in these Fusarium species and test the protoplasts for the ability to take up foreign DNA. We show that wild-type strains of F. virguliforme and F. brasiliense 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. ConclusionsThis protocol provides significant details for reproducibly producing protoplasts and transforming F. virguliforme and F. brasiliense. The protocol can be used to develop high quality protoplasts for further investigations into genetic mechanisms of growth and pathogenicity of F. virguliforme and F. brasiliense. Fluorescent strains developed in this study can be used to investigate temporal colonization and potential host preferences of these species.