A highly efficient grapevine mesophyll protoplast system for transient gene expression and the study of disease resistance proteins
A highly efficient grapevine mesophyll protoplast system for transient gene expression and the study of disease resistance proteins
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DOI：
10.1007/s11240-015-0928-7
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发表时间：
2016-04-01
影响因子：
3
通讯作者：
Wen, Ying-Qiang
中科院分区：
文献类型：
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作者：
Zhao, Feng-Li;Li, Ya-Juan;Wen, Ying-Qiang
植物原生质体是一个多功能的瞬时基因表达系统，已被广泛应用于多种植物物种的基因功能表征和多种信号通路的研究。然而，由于大规模分离葡萄原生质体的挑战，这种系统尚未开发用于葡萄（Vitis vinifera L.）。在这里，我们报告了一种从葡萄幼苗叶片中获得高产和良好活力的分离原生质体的简化方法。此外，本文还对葡萄叶肉原生质体的分离和转染条件进行了改进，证明该体系适合于蛋白质表达、蛋白质亚细胞定位和蛋白质-蛋白质相互作用的研究。此外，我们在葡萄原生质体中以荧光融合蛋白的形式异源和瞬时表达了拟南芥抗病蛋白RPW8.2，该蛋白先前被报道具有对不同植物科几种生物营养病原体的广谱抗性。我们观察到RPW8.2融合蛋白的表达在外源水杨酸和葡萄霜霉病（Plasmopara viticola）侵染后被诱导。这些结果说明了这个高效的叶肉原生质体系统在葡萄瞬时基因表达和抗病蛋白活性研究中的潜力。
Plant protoplasts constitute a versatile system for transient gene expression and have been widely used with several plant species for the functional characterization of genes and studies of diverse signaling pathways. However, such a system has not been developed for grapevine (Vitis vinifera L.) due to the challenges of large-scale isolation of viable grapevine protoplasts. Here, we report a simplified method for obtaining high yields and excellent viability of isolated protoplasts from young grapevine leaves. In addition, both the conditions for isolation and transfection of grapevine mesophyll protoplasts were modified, and the system was shown to be suitable for protein expression and studies of protein subcellular localization and protein-protein interactions. In addition, we heterologously and transiently expressed the Arabidopsis thaliana disease resistance protein RPW8.2, which has previously been reported to confer broad-spectrum resistance to several biotrophic pathogens in different plant families, as a fluorescent fusion protein in grapevine protoplasts. We observed that expression of the RPW8.2 fusion protein was induced in response to application of exogenous salicylic acid and following infection by the grapevine downy mildew pathogen, Plasmopara viticola. These results illustrate the potential of this highly efficient mesophyll protoplast system for transient gene expression and investigation of the activity of disease resistance proteins in grapevine.