Development of a Pseudomonas syringae-Arabidopsis Suspension Cell Infection System for Investigating Host Metabolite-Dependent Regulation of Type III Secretion and Pattern-Triggered Immunity.
Development of a Pseudomonas syringae-Arabidopsis Suspension Cell Infection System for Investigating Host Metabolite-Dependent Regulation of Type III Secretion and Pattern-Triggered Immunity.
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开发丁香假单胞菌-拟南芥悬浮细胞感染系统，用于研究 III 型分泌和模式触发免疫的宿主代谢依赖性调节。
DOI：
10.1094/mpmi-10-18-0295-fi
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发表时间：
2019
期刊：
影响因子：
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通讯作者：
Jeffrey C. Anderson
中科院分区：
文献类型：
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作者：
Qing Yan;Conner J. Rogan;Jeffrey C. Anderson
通过对缺乏功能模式识别受体(PRR)和PRR激活下游信号元件的突变体的遗传研究，明确了模式触发免疫(PTI)在植物防御中的重要性。尽管对PRR介导的对病原体相关分子模式(PAMP)的信号反应有广泛的了解，但对这些反应中的哪些反应(如果有的话)直接限制细菌生长知之甚少。在这项工作中，我们建立了一种共培养细菌病原菌紫丁香假单胞菌的方法。番茄DC3000和拟南芥悬浮细胞。该系统密切反映了叶片中发生的感染过程，细菌依靠III型分泌系统(T3SS)实现最大生长，PAMP诱导的植物防御有效地限制了细菌的生长。为了证明该体系的实用性，我们研究了PAMP诱导生长抑制的分子基础，发现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基因表达降低的潜在机制，我们对悬浮细胞分泌物进行了代谢组学和生化分析，鉴定出14种代谢物在PAMP处理后显著增加或减少。柠檬酸是已知的DC3000中T3SS基因表达的诱导剂，是PAMP处理后植物细胞分泌物中减少的几种有机酸之一。外源柠檬酸增加了T3SS基因的表达，并在PAMP处理的细胞中部分恢复了DC3000的生长，表明PAMP诱导的防御系统中的一部分是减少了该代谢产物的胞外释放。我们预见，这种共培养系统明确的感染条件将对定量研究丁香假单胞菌的III型效应器传递有价值。此外，该系统提供了一种独特的“自上而下”的方法来揭示PTI抗丁香疫霉菌的分子基础。
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 Pseudomonas syringae pv. tomato DC3000 and Arabidopsis 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 P. syringae. Furthermore, this system provides a unique 'top-down' approach to unravel the molecular basis of PTI against P. syringae.