Bacterial flagellin protein is a potent microbe-associated molecular pattern. Immune responses are triggered by a 22-amino-acid epitope derived from flagellin, known as flg22, upon detection by the pattern recognition receptor FLAGELLIN-SENSING2 (FLS2) in multiple plant species. However, increasing evidence suggests that flg22 epitopes of several bacterial species are not universally immunogenic to plants. We investigated whether flg22 immunogenicity systematically differs between classes of the phylum Proteobacteria, using a dataset of 2,470 flg22 sequences. To predict which species encode highly immunogenic flg22 epitopes, we queried a custom motif (¹¹[ST]xx[DN][DN]xAGxxI²¹) in the flg22 sequences, followed by sequence conservation analysis and protein structural modeling. These data led us to hypothesize that most flg22 epitopes of the γ- and β-Proteobacteria are highly immunogenic, whereas most flg22 epitopes of the α-, δ-, and ε-Proteobacteria are weakly to moderately immunogenic. To test this hypothesis, we generated synthetic peptides representative of the flg22 epitopes of each proteobacterial class, and we monitored their ability to elicit an immune response in Arabidopsis thaliana. The flg22 peptides of γ- and β-Proteobacteria triggered strong oxidative bursts, whereas peptides from the ε-, δ-, and α-Proteobacteria triggered moderate, weak, or no response, respectively. These data suggest flg22 immunogenicity is not highly conserved across the phylum Proteobacteria. We postulate that sequence divergence of each taxonomic class was present prior to the evolution of FLS2, and that the ligand specificity of A. thaliana FLS2 was driven by the flg22 epitopes of the γ- and β-Proteobacteria, a monophyletic group containing many common phytopathogens.

Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

细菌鞭毛蛋白是一种有效的微生物相关分子模式。在多种植物物种中被模式识别受体 FLAGELLIN-SENSING2 (FLS2) 检测到后，免疫反应由源自鞭毛蛋白的 22 个氨基酸的表位触发，称为 flg22。然而，越来越多的证据表明，几种细菌物种的 flg22 表位对植物并非普遍具有免疫原性。我们使用 2,470 个 flg22 序列的数据集研究了 flg22 免疫原性是否在变形杆菌门的类别之间存在系统差异。为了预测哪些物种编码高免疫原性 flg22 表位，我们查询了一个自定义基序 ( ¹¹ [ST]xx[DN][DN]xAGxxI ²¹) 在 flg22 序列中，然后是序列保守性分析和蛋白质结构建模。这些数据使我们假设 γ- 和 β- Proteobacteria的大多数 flg22 表位具有高度免疫原性，而 α-、δ- 和 ε- Proteobacteria 的大多数 flg22 表位具有弱至中度免疫原性。为了验证这一假设，我们生成了代表每个变形菌纲的 flg22 表位的合成肽，并且我们监测了它们在拟南芥中引发免疫反应的能力。γ- 和 β- Proteobacteria的 flg22 肽引发强烈的氧化爆发，而来自 ε-、δ- 和 α- Proteobacteria 的肽分别触发中度、微弱或无响应。这些数据表明 flg22 免疫原性在变形菌门中不是高度保守的。我们假设每个分类学类别的序列差异在 FLS2 进化之前就存在，并且拟南芥FLS2的配体特异性是由 γ- 和 β- Proteobacteria的 flg22 表位驱动的，这是一个包含许多常见植物病原体的单系群.

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