Journal of Integrative Agriculture ›› 2026, Vol. 25 ›› Issue (10): 4236-4247.DOI: 10.1016/j.jia.2025.08.010

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基于AlphaFold3的单氨基酸替换，构建兼具除草剂抗性和遗传标记双重功能的筛选标签，并应用于十字花科植物病原菌希金斯炭疽菌研究

  

  • 收稿日期:2025-04-30 修回日期:2025-08-05 接受日期:2025-07-12 出版日期:2026-10-20 发布日期:2026-09-21

AlphaFold3-guided single-amino acid substitution creates a dual-functional selection marker for herbicide resistance and genetic tagging in the cruciferous plant pathogen Colletotrichum higginsianum

Yiming Zhu1, 2*, Weilun Chen1*, Wei Luo1, Zhihan Li1, Yingying Yang3, Jianye Chen3, Erxun Zhou1#   

  1. 1 Guangdong Provincial Key Laboratory of Microbial Signals and Disease Control, College of Plant Protection, South China Agricultural University, Guangzhou 510642, China

    2 Guangdong Provincial Key Laboratory of Utilization and Conservation of Food and Medicinal Resources in Northern Region/School of Biology and Agriculture, Shaoguan University, Shaoguan 512005, China

    3 Guangdong Provincial Key Laboratory of Postharvest Science of Fruits and Vegetables/Engineering Research Center of Southern Horticultural Products Preservation, Ministry of Education, College of Horticulture, South China Agricultural University, Guangzhou 510642, China

  • Received:2025-04-30 Revised:2025-08-05 Accepted:2025-07-12 Online:2026-10-20 Published:2026-09-21
  • About author:Yiming Zhu, E-mail: zhu_yiming1992@scau.edu.cn; Weilun Chen, E-mail: chenweilun0828@163.com; #Correspondence Erxun Zhou, Tel: +86-20- 85286089, E-mail: exzhou@scau.edu.cn * These authors contributed equally to this study.
  • Supported by:

    This work was supported by the Natural Science Foundation of Guangdong Province, China (2021A1515011166), and the Guangdong Basic and Applied Basic Research Foundation, China (2024A1515110091).

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摘要：

十字花科炭疽病菌（Colletotrichum higginsianum）是严重危害蔬菜作物的重要病原真菌，其遗传学研究长期受限于选择标记匮乏、外源基因干扰以及标记回收效率低等问题。针对该病原菌遗传操作工具缺乏的瓶颈，本研究利用AlphaFold3蛋白结构预测技术，解析了乙酰乳酸合成酶（ChALS）与除草剂氯嘧磺隆（CE）的结合机制。分子对接结果显示，ChALS的V191、A200和F201残基形成疏水性结合口袋，其中A200位于CE芳香环的关键作用位点。基于此，设计了A200D单点突变，并通过同源重组获得突变菌株。结果表明，突变体ChALSA200D在含CE培养基（100 ng/mL）中的生长速率与野生型无显著差异，且抗性强于对照突变体ChALSA195D。分子对接模拟分析显示，天冬氨酸侧链通过空间位阻破坏CE的结合，但酶整体结构保持稳定。表型分析证实，突变体的产孢量、附着胞形成及对寄主植物菜心（Brassica parachinensis）的致病力均未受影响。

基于ChALSA200D突变，创新性开发了以其为内源选择标记的模块化整合系统（pSFZY022系列载体），实现了细胞质（GFP）、细胞核（H1-GFP）、线粒体（Ech1-GFP）及过氧化物酶体（SKL-GFP）的精准定位。活体成像成功追踪到初级侵染阶段（40 hpi）病原菌在寄主表皮细胞内的营养生长动态，证明该遗传操作不会影响侵染生物学过程。

本研究首次将AlphaFold3结构预测与真菌遗传编辑结合，创建了基于内源ALS基因的双功能选择标记系统，克服了传统外源标记的局限性。研究揭示A200D突变通过疏水作用破坏而非抑制催化活性来实现CE抗性，为希金斯炭疽菌抗除草剂机制提供了新见解。该双功能选择标记系统支持多细胞器精准标记，且不影响病原菌致病性，为十字花科作物-希金斯炭疽菌互作研究提供了通用平台。此外，单一氨基酸替换（A200D）即可赋予强抗性，提示单一靶点抑制剂在病害防控中存在进化风险。

Abstract: Colletotrichum higginsianum, a plant pathogenic fungus threatening cruciferous crops, necessitates advanced genetic tools to study its pathogenesis and host interactions. However, selection markers available for fungal genome editing are often limited by scarcity and variable efficacy across species. Acetolactate synthase (ALS), a key enzyme in branched- chain amino acid (BCAA) biosynthesis, is the target of the herbicide chlorimuron ethyl (CE). Here, we utilized AlphaFold3- guided structural prediction to engineer ALS in C. higginsianum into a dual-functional endogenous selection marker. AlphaFold3-predicted ChALS structures revealed conserved catalytic regions and identified hydrophobic residues (V191, A200, F201) potentially critical for CE binding. Notably, introducing a single A200D substitution conferred the resistance of C. higginsianum to CE without impairing fungal growth, conidiation, or pathogenicity. Molecular docking simulations confirmed that the introduced aspartate side chain causes steric clashes that destabilize CE binding. Leveraging this mutation, we developed a homology-directed integration system enabling precise genetic tagging at the endogenous ChALS locus using the A200D allele for selection. Colletotrichum higginsianum strains expressing fluorescent proteins targeted to the nucleus, mitochondria, peroxisomes, or cytoplasm exhibited stable expression and unaltered infection dynamics in Chinese flowering cabbage (Brassica parachinensis). This work presents a dual-functional marker that addresses limitations in fungal genome editing tools. Moreover, the facility with which the A200D mutation confers resistance serves as a cautionary note on the potential evolutionary instability of single-target enzyme inhibitors in pathogen management.

Key words: AlphaFold3 , Colletotrichum higginsianum  ,  acetolactate synthase ,  plant–pathogen interaction