Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331, U.S.A.
Mol Plant Microbe Interact. 2019 May;32(5):527-539. doi: 10.1094/MPMI-10-18-0295-FI. Epub 2019 Mar 29.
The importance of pattern-triggered immunity (PTI) in plant defense has been clearly established through genetic studies of mutants lacking functional pattern recognition receptors (PRRs) and signaling components downstream of PRR activation. Despite extensive knowledge of PRR-mediated signaling responses to pathogen-associated molecular patterns (PAMPs), little is known about which of these responses, if any, are directly responsible for limiting bacterial growth. In this work, we established a protocol for coculturing the bacterial pathogen pv. DC3000 and suspension cells. The system closely mirrors infection processes that occur in leaves, with bacteria relying on the type III secretion system (T3SS) for maximal growth and PAMP-induced plant defenses effectively limiting bacterial growth. To demonstrate the utility of this system, we investigated the molecular basis of PAMP-induced growth inhibition and discovered that T3SS-associated genes are inhibited when DC3000 is cocultured with PAMP-treated plant suspension cells. To determine the underlying mechanism of decreased T3SS gene expression, we performed metabolomics and biochemical analyses of suspension cell exudates and identified 14 metabolites that significantly increased or decreased following PAMP treatment. Citric acid, a known inducer of T3SS gene expression in DC3000, was among several organic acids decreased in exudates from PAMP-treated plant cells. Exogenous addition of citric acid increased T3SS gene expression and partially recovered growth of DC3000 in the presence of PAMP-treated cells, indicating that a portion of PAMP-induced defense in this system is decreased extracellular release of this metabolite. We envision that the well-defined infection conditions of this coculture system will be valuable for quantitative studies of type III effector delivery by . Furthermore, this system provides a unique 'top-down' approach to unravel the molecular basis of PTI against .
模式触发免疫（PTI）在植物防御中的重要性已经通过缺乏功能模式识别受体（PRR）和 PRR 激活下游信号成分的突变体的遗传研究得到了明确确立。尽管对 PRR 介导的信号反应到病原体相关分子模式（PAMP）有广泛的了解，但对于这些反应中哪些反应（如果有）直接负责限制细菌生长却知之甚少。在这项工作中，我们建立了共培养细菌病原体 pv. DC3000 和悬浮细胞的方案。该系统紧密模拟了在叶片中发生的感染过程，细菌依赖于 III 型分泌系统（T3SS）来实现最大生长，而 PAMP 诱导的植物防御有效地限制了细菌的生长。为了证明该系统的实用性，我们研究了 PAMP 诱导的生长抑制的分子基础，并发现当 DC3000 与 PAMP 处理的植物悬浮细胞共培养时，T3SS 相关基因受到抑制。为了确定 T3SS 基因表达下降的潜在机制，我们对悬浮细胞分泌物进行了代谢组学和生化分析，并鉴定出 14 种代谢物在 PAMP 处理后显著增加或减少。柠檬酸是 DC3000 中 T3SS 基因表达的已知诱导剂，是几种从 PAMP 处理的植物细胞分泌物中减少的有机酸之一。外源性添加柠檬酸可增加 T3SS 基因表达，并部分恢复了 PAMP 处理细胞存在时 DC3000 的生长，表明该系统中部分 PTI 是通过减少该代谢物的细胞外释放来实现的。我们设想，该共培养系统明确的感染条件将对定量研究 III 型效应物的传递非常有价值。此外，该系统为解开 PTI 针对的分子基础提供了一种独特的“自上而下”方法。
