Guangdong Provincial Key Laboratory of Plant Resources, State Key Laboratory of Biocontrol, MOE Key Laboratory of Gene Function and Regulation, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, China.
J Integr Plant Biol. 2020 Jun;62(6):761-776. doi: 10.1111/jipb.12859. Epub 2019 Oct 14.
Plant cells mount plenty of pattern-recognition receptors (PRRs) to detect the microbe-associated molecular patterns (MAMPs) from potential microbial pathogens. MAMPs are overrepresented by proteinaneous patterns, such as the flg22 peptide from bacterial flagellin. Identification of PRR receptor complex components by forward or reverse genetics can be time/labor-consuming, and be confounded by functional redundancies. Here, we present a strategy for identifying PRR complex components by engineering plants to inducibly secrete affinity-tagged proteinaneous MAMPs to the apoplast. The PRR protein complexes bound to self-secreted MAMPs are enriched through affinity purification and dissected by mass spectrometry. As a proof of principle, we could capture the flg22 receptor FLS2 and co-receptor BAK1 using Arabidopsis plants secreting FLAG-tagged flg22 under estradiol induction. Moreover, we identified receptor-like kinases LIK1 and PEPR1/PEPR2 as potential components in the FLS2 receptor complex, which were further validated by protein-protein interaction assays and the reverse genetics approach. Our study showcases a simple way to biochemically identify endogenous PRR complex components without overexpressing the PRR or using chemical cross-linkers, and suggests a possible crosstalk between different immune receptors in plants. A modest dose of estradiol can also be applied to inducing enhanced immunity in engineered plants to both bacterial and fungal pathogens.
植物细胞会装配大量的模式识别受体（PRR）来识别潜在微生物病原体的微生物相关分子模式（MAMP）。MAMP 以蛋白相关模式为主，例如细菌鞭毛蛋白的 flg22 肽。通过正向或反向遗传学鉴定 PRR 受体复合物的组成部分既耗时又费力，并且会受到功能冗余的影响。在这里，我们提出了一种通过工程改造植物来诱导分泌亲和标签蛋白相关 MAMP 到质外体的策略，从而鉴定 PRR 复合物组成部分。通过亲和纯化富集与自身分泌的 MAMP 结合的 PRR 蛋白复合物，然后通过质谱进行分析。作为原理验证，我们可以使用在雌二醇诱导下分泌 FLAG 标记的 flg22 的拟南芥植物来捕获 flg22 受体 FLS2 和共受体 BAK1。此外，我们还鉴定出类受体激酶 LIK1 和 PEPR1/PEPR2 可能是 FLS2 受体复合物中的组成部分，通过蛋白-蛋白相互作用测定和反向遗传学方法进一步验证。我们的研究展示了一种无需过表达 PRR 或使用化学交联剂即可在生化水平上鉴定内源性 PRR 复合物组成部分的简单方法，并提示植物中不同免疫受体之间可能存在串扰。适量的雌二醇也可以应用于诱导工程植物对细菌和真菌病原体的增强免疫。
