Alhawatema Mohammad S, Gebril Sayed, Cook Daniel, Creamer Rebecca
Molecular Biology Graduate Program, New Mexico State University, Las Cruces, NM, 88003, USA.
Tafila Technical University, Tafilah, Jordan.
World J Microbiol Biotechnol. 2017 Sep 20;33(10):179. doi: 10.1007/s11274-017-2346-y.
The fungus Slafractonia leguminicola, the causal agent of blackpatch disease of legumes produces two mycotoxins slaframine and swainsonine, causing slobbers' symptoms and locoism of grazing animals, respectively. The genetics of this important fungus is poorly understood. This work aimed to develop a genetic transformation system and evaluate the efficacy of RNA interference (RNAi) in S. leguminicola. In this study, S. leguminicola was transformed using a PEG-mediated method with a fungal construct that carries a hygromycin resistance cassette. To assess the use of RNAi, a silencing construct pSilentPKS1-AS was constructed which includes inverted repeat transgenes of the polyketide synthase gene (pks1) that is involved in melanin biosynthesis. Transformation of S. leguminicola with the IRT pks1 vector decreased pks1 transcripts levels 82-92% in knockdown mutants when compared with the wild type and was accompanied with a reduction in melanin and swainsonine production. These results demonstrate that RNAi can be a useful tool for studying gene function in S. leguminicola.
豆科植物黑块病的病原菌豆科丝核菌（Slafractonia leguminicola）会产生两种霉菌毒素——唾液酸和苦马豆素，分别导致放牧动物出现流涎症状和疯草中毒。人们对这种重要真菌的遗传学了解甚少。这项工作旨在开发一种遗传转化系统，并评估RNA干扰（RNAi）在豆科丝核菌中的效果。在本研究中，使用携带潮霉素抗性盒的真菌构建体，通过聚乙二醇（PEG）介导的方法对豆科丝核菌进行转化。为了评估RNAi的应用，构建了一个沉默构建体pSilentPKS1-AS，其中包含参与黑色素生物合成的聚酮合酶基因（pks1）的反向重复转基因。与野生型相比，用IRT pks1载体转化豆科丝核菌后，敲除突变体中的pks1转录本水平降低了82%-92%，同时黑色素和苦马豆素的产量也有所下降。这些结果表明，RNAi可以成为研究豆科丝核菌基因功能的有用工具。
