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Author:
Date:
2015-11-20
[Abstract] Based on gene expression data after biotic stress, the GTPase RabA4c has been suggested to regulate pathogen-induced callose biosynthesis in the model organism Arabidopsis thaliana. We studied the function of RabA4c in its native and dominant negative (dn) isoform. In planta, RabA4c overexpression prevented penetration of the virulent powdery mildew Golovinomyces cichoracearum into epidermal leaf cells. This penetration resistance was caused by enhanced callose deposition at sites of attempted fungal penetration at early time points of infection. By contrast, RabA4c (dn) overexpression did not increase callose deposition or penetration resistance.
In this protocol, we describe the expression, purification and ...
[摘要] 基于生物胁迫后的基因表达数据，已经建议GTP酶RabA4c 在模拟生物拟南芥中调节病原体诱导的胼lose质生物合成。我们研究了其天然和显性负性（dn）同种型中的RabA4c 的功能。 ，RabA4c 过表达阻止了毒性白粉病菌向鸡冠状叶细胞的侵入。这种穿透阻力是由于在感染早期试图真菌穿透的部位处的胼cal质沉积增加引起的。相反，RabA4c（dn）过表达不增加胼cal质沉积或穿透抗性。在本方案中，我们描述了异源表达的GTPase RabA4c的表达，纯化和活性测定从 thaliana ，基于出版物Ellinger 等人（2014）。我们将< em> RabA4c</em>与荧光团mCitrine融合，并在酵母菌株巴斯德毕赤酵母GS115中表达该蛋白质。为了纯化RabA4c，我们使用特异性结合GFP衍生物如mCitrine的GFP-Trap_A 试剂盒（Chromo Tek）。通过使用来自Innova Biosciences的 GTPase测定试剂盒进行酶活性测定。一般来说，我们遵循制造商的指示。
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