First and fifth authors: State Key Laboratory of Crop Stress Biology for Arid Areas, College of Plant Protection, Northwest A&F University, Yangling 712100, China; first, second, third, and fifth authors: Department of Plant Pathology, College of Plant Protection, China Agricultural University, Beijing 100193, China; and first and fourth authors: Center for Genome Research and Biocomputing and Department of Botany and Plant Pathology, Oregon State University, Corvallis 97331.
Phytopathology. 2018 Dec;108(12):1412-1419. doi: 10.1094/PHYTO-01-18-0010-R. Epub 2018 Oct 31.
Oxathiapiprolin is a novel fungicide that was recently registered in a number of countries to control plant-pathogenic oomycetes such as Phytophthora capsici. In our previous study, point mutations G770V and G839W in oxysterol binding protein-related protein 1 (ORP1) were detected in oxathiapiprolin-resistant P. capsici isolates (PcORP1). Here, we used the CRISPR/Cas9 system to verify the effects of these two point mutations on P. capsici phenotypes. Transformants containing heterozygous G770V and G839W mutations in PcORP1 showed high levels of oxathiapiprolin resistance. The G770V transformants showed otherwise similar phenotypes compared with the wild-type isolate BYA5, including sporangia and zoospore production, cyst germination, and pathogenicity. However, two independent transformants with heterozygous G839W mutations in PcORP1 could not produce sporangia. Three transformants with an unexpected point mutation in PcORP1 (ΔN837) showed high oxathiapiprolin resistance, and either similar or significantly reduced fitness compared with BYA5. The same deletion (ΔN837) was confirmed to confer oxathiapiprolin resistance in P. sojae by using CRISPR/Cas9. These homozygous P. sojae mutants also showed either similar or strongly reduced fitness compared with the wild-type parent isolate P6497. These results improve our understanding of oxathiapiprolin resistance in Phytophthora spp., and will be useful for the development of novel oxysterol-binding protein homolog inhibitor fungicides.
唑菌酯是一种新型杀菌剂，最近在多个国家注册用于防治疫霉属等植物病原卵菌，如辣椒疫霉。在我们之前的研究中，在唑菌酯抗性辣椒疫霉分离株（PcORP1）中检测到甾醇结合蛋白相关蛋白 1（ORP1）中的点突变 G770V 和 G839W。在这里，我们使用 CRISPR/Cas9 系统验证了这两个点突变对辣椒疫霉表型的影响。含有 PcORP1 杂合 G770V 和 G839W 突变的转化体对唑菌酯表现出高水平的抗性。G770V 转化体与野生型分离株 BYA5 相比表现出相似的表型，包括游动孢子囊和游动孢子的产生、胞囊萌发和致病性。然而，PcORP1 中两个独立的杂合 G839W 突变的转化体不能产生游动孢子囊。PcORP1 中含有意外点突变（ΔN837）的三个转化体对唑菌酯表现出高抗性，与 BYA5 相比，其适应性要么相似，要么显著降低。通过使用 CRISPR/Cas9 证实相同的缺失（ΔN837）赋予了大豆疫霉对唑菌酯的抗性。这些纯合的大豆疫霉菌突变体与野生型亲本分离株 P6497 相比，适应性要么相似，要么显著降低。这些结果提高了我们对疫霉菌属中唑菌酯抗性的理解，并且将有助于新型甾醇结合蛋白同源物抑制剂杀菌剂的开发。
