Detecting N-myristoylation and S-acylation of host and pathogen proteins in plants using click chemistry
Detecting N-myristoylation and S-acylation of host and pathogen proteins in plants using click chemistry
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DOI：
10.1186/s13007-016-0138-2
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发表时间：
2016-08-03
期刊：
影响因子：
5.1
通讯作者：
Martin, Gregory B.
中科院分区：
文献类型：
--
作者：
Boyle, Patrick C.;Schwizer, Simon;Martin, Gregory B.
背景：植物质膜是植物与病原菌斗争的关键战场。植物利用传感蛋白检测质膜上病原体的存在，其中许多蛋白通过脂肪酸修饰的方式靶向这种亲脂区域。病原菌分泌效应蛋白进入植物细胞，抑制植物的防御机制。这些效应物能够通过劫持宿主的脂肪酰化装置进入并干扰植物质膜上的监视机制。尽管蛋白质脂肪酰化在植物免疫和病原体毒力机制中都有重要作用，但对这种修饰在植物与病原体相互作用中的作用知之甚少。我们对这种缺乏理解主要是由于缺乏监测植物细胞中蛋白质脂肪酸修饰的方法。结果：我们描述了一种快速检测候选蛋白质的两种主要形式的脂肪酰化，n -肉豆肉酰化和s -酰化的方法，使用炔脂肪酸类似物结合点击化学。我们用我们的方法证实并决定性地证明了典型的模式识别受体FLS2、特征明确的病原体效应物AvrPto和研究得最好的细胞内抗性蛋白之一Pto都经历了植物介导的脂肪酰化。除了提供一种容易测定候选蛋白的脂肪酰化，特别是肉豆肉酰化的方法外，该方法还适用于各种表达系统。我们利用拟南芥原生质体和稳定的转基因拟南芥植株证明了这一点，并利用农杆菌介导的benthamiana叶片中的瞬时表达作为高通量评估候选蛋白的手段。结论：蛋白质脂肪酰化是植物及其病原体采用的一种靶向策略。利用炔脂肪酸类似物和点击化学的代谢标记方法有可能提供在植物和病原体之间的战斗中在宿主质膜上使用的分子策略的机制细节。
Background: The plant plasma membrane is a key battleground in the war between plants and their pathogens. Plants detect the presence of pathogens at the plasma membrane using sensor proteins, many of which are targeted to this lipophilic locale by way of fatty acid modifications. Pathogens secrete effector proteins into the plant cell to suppress the plant's defense mechanisms. These effectors are able to access and interfere with the surveillance machinery at the plant plasma membrane by hijacking the host's fatty acylation apparatus. Despite the important involvement of protein fatty acylation in both plant immunity and pathogen virulence mechanisms, relatively little is known about the role of this modification during plant-pathogen interactions. This dearth in our understanding is due largely to the lack of methods to monitor protein fatty acid modifications in the plant cell.Results: We describe a rapid method to detect two major forms of fatty acylation, N-myristoylation and S-acylation, of candidate proteins using alkyne fatty acid analogs coupled with click chemistry. We applied our approach to confirm and decisively demonstrate that the archetypal pattern recognition receptor FLS2, the well-characterized pathogen effector AvrPto, and one of the best-studied intracellular resistance proteins, Pto, all undergo plant-mediated fatty acylation. In addition to providing a means to readily determine fatty acylation, particularly myristoylation, of candidate proteins, this method is amenable to a variety of expression systems. We demonstrate this using both Arabidopsis protoplasts and stable transgenic Arabidopsis plants and we leverage Agrobacterium-mediated transient expression in Nicotiana benthamiana leaves as a means for high-throughput evaluation of candidate proteins.Conclusions: Protein fatty acylation is a targeting tactic employed by both plants and their pathogens. The metabolic labeling approach leveraging alkyne fatty acid analogs and click chemistry described here has the potential to provide mechanistic details of the molecular tactics used at the host plasma membrane in the battle between plants and pathogens.