Wilson Salome, Schwessinger Benjamin
Research School of Biology, Australian National University, Acton, ACT, Australia.
Division of Plant Sciences, Research School of Biology, Australian National University, Acton, Australia.
Methods Mol Biol. 2025;2898:189-205. doi: 10.1007/978-1-0716-4378-5_11.
Fungal pathogens present a severe risk to food systems; however, complex crop-microbe interactions are challenging to study using tools developed for model species. In particular, efficient screening and rapid assessment of microbial effectors is hindered by a lack of cloned resistance (R) genes and difficulty in validating large numbers of predicted effector candidates. This chapter describes a protocol for preparing wheat protoplasts to enable positive identification of host defense induction without overexpression of a cloned R gene, increasing the available pool of host resistance genes for screening. The assay uses polyethylene glycol (PEG)-calcium-mediated transient transfection to introduce candidate effector gene constructs into wheat protoplasts, with a defense-activated reporter for inducing a positive readout with internal normalization, indicating host recognition. This protocol provides a valuable tool for the study of host-pathogen interactions in wheat, contributing to improved resources for the development of disease-resistant crops and genome-informed pathogen surveillance.
真菌病原体对粮食系统构成严重风险；然而，利用为模式物种开发的工具来研究复杂的作物-微生物相互作用具有挑战性。特别是，由于缺乏克隆的抗性（R）基因以及难以验证大量预测的效应子候选物，微生物效应子的高效筛选和快速评估受到阻碍。本章描述了一种制备小麦原生质体的方案，该方案能够在不克隆R基因过表达的情况下阳性鉴定宿主防御诱导，增加用于筛选的宿主抗性基因库。该检测方法使用聚乙二醇（PEG）-钙介导的瞬时转染将候选效应子基因构建体导入小麦原生质体，并使用防御激活报告基因进行内部标准化以诱导阳性读数，表明宿主识别。该方案为研究小麦中的宿主-病原体相互作用提供了一个有价值的工具，有助于为抗病作物的开发和基于基因组的病原体监测提供更好的资源。
