Kangara Ngonidzashe, Kurowski Tomasz J, Radhakrishnan Guru V, Ghosh Sreya, Cook Nicola M, Yu Guotai, Arora Sanu, Steffenson Brian J, Figueroa Melania, Mohareb Fady, Saunders Diane G O, Wulff Brande B H
Crop Genetics Department, John Innes Centre, Norwich, United Kingdom.
The Bioinformatics Group, Cranfield Soil and Agrifood Institute, Cranfield University, Bedford, United Kingdom.
Front Plant Sci. 2020 Sep 16;11:570180. doi: 10.3389/fpls.2020.570180. eCollection 2020.
Wheat stem rust caused by the fungus f. sp. (), is regaining prominence due to the recent emergence of virulent isolates and epidemics in Africa, Europe and Central Asia. The development and deployment of wheat cultivars with multiple stem rust resistance () genes stacked together will provide durable resistance. However, certain disease resistance genes can suppress each other or fail in particular genetic backgrounds. Therefore, the function of each gene must be confirmed after incorporation into an -gene stack. This is difficult when using pathogen disease assays due to epistasis from recognition of multiple avirulence (Avr) effectors. Heterologous delivery of single effectors can circumvent this limitation, but this strategy is currently limited by the paucity of cloned . To accelerate gene cloning, we outline a procedure to develop a mutant population of spores and select for gain-of-virulence mutants. We used ethyl methanesulphonate (EMS) to mutagenize urediniospores and create a library of > 10,000 independent mutant isolates that were combined into 16 bulks of ~658 pustules each. We sequenced random mutants and determined the average mutation density to be 1 single nucleotide variant (SNV) per 258 kb. From this, we calculated that a minimum of three independently derived gain-of-virulence mutants is required to identify a given gene. We inoculated the mutant library onto plants containing , , or and obtained 9, 4, and 14 mutants with virulence toward , , or , respectively. However, only mutants identified on and maintained their virulence when reinolculated onto the lines from which they were identified. We further characterized 8 mutants with virulence toward . These also maintained their virulence profile on the stem rust international differential set containing 20 genes, indicating that they were most likely not accidental contaminants. In conclusion, our method allows selecting for virulent mutants toward targeted resistance () genes. The development of a mutant library from as little as 320 mg spores creates a resource that enables screening against several genes without the need for multiple rounds of spore multiplication and mutagenesis.
由真菌禾柄锈菌小麦专化型（Puccinia graminis f. sp. tritici）引起的小麦秆锈病，由于近期在非洲、欧洲和中亚出现了毒性分离株和病害流行，正重新受到关注。将多个抗秆锈病（Sr）基因堆叠在一起的小麦品种的开发和推广将提供持久抗性。然而，某些抗病基因可能会相互抑制，或者在特定的遗传背景下失效。因此，每个Sr基因在整合到多基因堆叠中后，其功能必须得到确认。当使用病原菌病害检测方法时，由于对多种无毒（Avr）效应子的识别存在上位性，这一过程变得困难。单个Avr效应子的异源递送可以规避这一限制，但目前这一策略受到已克隆的Avr基因数量稀少的限制。为了加速Sr基因克隆，我们概述了一个开发P. graminis孢子突变群体并选择毒性增强突变体的程序。我们使用甲磺酸乙酯（EMS）诱变夏孢子，创建了一个由超过10,000个独立突变分离株组成的文库，这些分离株被合并成16个库，每个库约有658个夏孢子堆。我们对随机突变体进行测序，确定平均突变密度为每258 kb有1个单核苷酸变异（SNV）。据此，我们计算出至少需要三个独立获得的毒性增强突变体才能鉴定出一个给定的Sr基因。我们将突变体文库接种到含有Sr2、Sr33或Sr50的植株上，分别获得了对Sr2、Sr33或Sr50有毒性的9个、4个和14个突变体。然而，只有在Sr2和Sr50上鉴定出的突变体在重新接种到鉴定它们的品系上时仍保持其毒性。我们进一步对8个对Sr50有毒性的突变体进行了表征。这些突变体在含有20个Sr基因的秆锈病国际鉴别品种上也保持了它们的毒性特征，这表明它们很可能不是偶然的污染物。总之，我们的方法允许选择针对目标抗性（Sr）基因的毒性突变体。从仅320 mg孢子中开发突变体文库创造了一种资源，使得无需多轮孢子繁殖和诱变就能够针对多个Sr基因进行筛选。
