Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.
Mol Cell Proteomics. 2012 Oct;11(10):945-56. doi: 10.1074/mcp.M112.020214. Epub 2012 Jul 9.
Grapevine (Vitis vinifera) is an economically important fruit crop that is subject to many types of insect and pathogen attack. To better elucidate the plant response to Lobesia botrana pathogen infection, we initiated a global comparative proteomic study monitoring steady-state protein expression as well as changes in N-glycosylation, phosphorylation, and Lys-acetylation in control and infected mesocarp and exocarp from V. vinifera cv Italia. A multi-parallel, large-scale proteomic approach employing iTRAQ labeling prior to three peptide enrichment techniques followed by tandem mass spectrometry led to the identification of a total of 3059 proteins, 1135 phosphorylation sites, 323 N-linked glycosylation sites and 138 Lys-acetylation sites. Of these, we could identify changes in abundance of 899 proteins. The occupancy of 110 phosphorylation sites, 10 N-glycosylation sites and 20 Lys-acetylation sites differentially changed during L. botrana infection. Sequence consensus analysis for phosphorylation sites showed eight significant motifs, two of which containing up-regulated phosphopeptides (X-G-S-X and S-X-X-D) and two containing down-regulated phosphopeptides (R-X-X-S and S-D-X-E) in response to pathogen infection. Topographical distribution of phosphorylation sites within primary sequences reveal preferential phosphorylation at both the N- and C termini, and a clear preference for C-terminal phosphorylation in response to pathogen infection suggesting induction of region-specific kinase(s). Lys-acetylation analysis confirmed the consensus X-K-Y-X motif previously detected in mammals and revealed the importance of this modification in plant defense. The importance of N-linked protein glycosylation in plant response to biotic stimulus was evident by an up-regulated glycopeptide belonging to the disease resistance response protein 206. This study represents a substantial step toward the understanding of protein and PTMs-mediated plant-pathogen interaction shedding light on the mechanisms underlying the grape infection.
葡萄（Vitis vinifera）是一种经济上重要的水果作物，易受到多种昆虫和病原体的攻击。为了更好地阐明葡萄对 L. botrana 病原体感染的反应机制，我们启动了一项全球比较蛋白质组学研究，监测了 V. vinifera cv Italia 的中果皮和外果皮对照和感染组织的稳态蛋白表达以及 N-糖基化、磷酸化和 Lys-乙酰化的变化。采用 iTRAQ 标记，结合三种肽富集技术，随后进行串联质谱分析，采用多平行、大规模蛋白质组学方法，共鉴定到 3059 种蛋白质、1135 个磷酸化位点、323 个 N-连接糖基化位点和 138 个 Lys-乙酰化位点。其中，我们可以鉴定到 899 种蛋白丰度的变化。在 L. botrana 感染过程中，有 110 个磷酸化位点、10 个 N-糖基化位点和 20 个 Lys-乙酰化位点的占有率发生了变化。磷酸化位点序列共识分析显示，有 8 个显著基序，其中 2 个包含上调的磷酸肽（X-G-S-X 和 S-X-X-D），2 个包含下调的磷酸肽（R-X-X-S 和 S-D-X-E），对病原体感染有响应。磷酸化位点在一级序列中的拓扑分布显示，在 N 端和 C 端都有优先磷酸化，并且在响应病原体感染时，C 端磷酸化明显偏好，表明诱导了区域特异性激酶。Lys-乙酰化分析证实了先前在哺乳动物中检测到的 X-K-Y-X 基序的重要性，并揭示了该修饰在植物防御中的重要性。N-连接蛋白糖基化在植物对生物刺激的反应中的重要性通过属于抗病反应蛋白 206 的上调糖肽得到证实。这项研究是朝着理解蛋白和 PTMs 介导的植物-病原体相互作用迈出的重要一步，揭示了葡萄感染的机制。
