Table_8_Pro-197-Ser Mutation in ALS and High-Level GST Activities: Multiple Resistance to ALS and ACCase Inhibitors in Beckmannia syzigachne.docx

American sloughgrass (Beckmannia syzigachne Steud.) is one of the most troublesome weeds infesting wheat and canola fields in China. Some biotypes cannot be controlled, either by acetolactate synthase (ALS) or acetyl coenzyme A carboxylase (ACCase) inhibitors, which are the main herbicides for controlling this weed. However, very few studies have investigated multiple resistance mechanism in B. syzigachne. In this study, a B. syzigachne biotype with a high resistance to ALS inhibitors we have reported was also showed relatively lower resistance to ACCase inhibitors, with a resistance index around 7. RNA-seq analysis was used to investigate the factors responsible for multiple resistance, and 60,108 unigenes were assembled by de novo transcriptome assembly and then annotated across eight databases. A Pro-197-Ser mutation was identified in the ALS gene by SNPs analysis and validated by PCR, while no mutation was identified in the ACCase gene. Nineteen candidate metabolic genes were screened and their overexpression was confirmed by qPCR. The expression of GST-T3 and GST-U6 in resistant plants ranged from 7.5- to 109.4-folds than that in susceptible ones at different times after two kinds of herbicide treatment. In addition, GST activities in resistant plants were 3.0–5.0 times higher than that in susceptible plants. Other novel resistance factors also showed high correlation with multiple resistance which included four genes encoding disease resistance proteins, a transcription factor (MYC3), and one gene conferring blight resistance. In this research, a B. syzigachne biotype was confirmed to have evolved multiple resistance to ACCase and ALS inhibitors. The Pro-197-Ser mutation in ALS gene and high-level GST activities were confirmed responsible for the multiple resistance. Characterized disease-resistance proteins, transcription factor, and blight-resistance proteins may play an essential role in these multiple herbicide resistance.

美国泥草（Beckmannia syzigachne Steud.）是我国小麦和大豆田中最为棘手的杂草之一。部分生物型对乙酰乳酸合成酶（ALS）或乙酰辅酶A羧化酶（ACCase）抑制剂均无抑制作用，而这两者是目前控制该杂草的主要除草剂。然而，关于B. syzigachne的多重抗性机制的研究却极为有限。本研究中，我们报道了一种对ALS抑制剂具有高度抗性的B. syzigachne生物型，同时也表现出对ACCase抑制剂相对较低的抗性，其抗性指数约为7。通过RNA-seq分析，我们探究了导致多重抗性的因素，并通过从头转录组组装，对8个数据库进行了注释，共组装出60,108个unigenes。通过SNPs分析，我们在ALS基因中鉴定出Pro-197-Ser突变，并通过PCR进行了验证，而ACCase基因中未发现突变。通过筛选，我们确定了19个候选代谢基因，并通过qPCR证实了其过表达。在两种除草剂处理后不同时间点，耐草植物中GST-T3和GST-U6的表达量是感草植物的7.5至109.4倍。此外，耐草植物中的GST活性比感草植物高3.0至5.0倍。其他新型抗性因素也与多重抗性高度相关，包括编码抗病蛋白的四个基因、一个转录因子（MYC3）以及一个赋予疫病抗性的基因。本研究证实，B. syzigachne生物型对ACCase和ALS抑制剂已进化出多重抗性。ALS基因中的Pro-197-Ser突变和高水平的GST活性被证实为多重抗性的原因。具有特征性的抗病蛋白、转录因子以及疫病抗性蛋白可能在这些多重除草剂抗性中发挥关键作用。