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Immunology and Infection

蛋白质中 Plasmodesmal 定位序列的识别在底中

Published: August 15, 2017
doi:
10.3791/55301
, ,
1Department of Biochemistry and Cell Biology,State University of New York, 2Department of Plant Pathology and Plant-Microbe Biology,Cornell University

Summary

植物细胞间连接, 胞 (Pd), 在植物生理学和植物-病毒相互作用中扮演中心角色。对 pd 传输的关键是分拣信号, 直接蛋白质的 pd。然而, 我们对这些序列的了解还处于起步阶段。我们描述了一种在 pd 靶蛋白中识别 pd 定位信号的策略。

Abstract

胞 (Pd) 是细胞连接, 它的作用是通过在植物细胞之间传输小分子和大分子的通路。而 Pd 运输的小分子, 如离子和水, 被推定为被动发生, 细胞运输的生物大分子, 这种蛋白质, 最有可能发生通过一个积极的机制, 涉及特定的靶向信号的运输分子。识别的胞 (Pd) 定位信号 (PLSs) 的稀缺性严重限制了对植物细胞大分子运输和通讯所涉及的蛋白质分选途径的理解。从大量的植物内源性和病毒蛋白已知的交通通过 Pd, 迄今只有三 PLSs 报告, 所有这些来自内源植物蛋白。因此, 重要的是要建立一个可靠的, 系统的实验策略, 以确定一个功能 PLS 序列, 这是必要的和足够的 Pd 靶向, 直接在活植物细胞。在这里, 我们描述一个这样的战略使用作为一个范例的细胞运动蛋白 (MP) 的烟草马赛克病毒(TMV)。这些实验, 确定和特点的第一植物病毒 pls, 可以适应发现 pls 序列在多数 Pd 靶向蛋白质。

Introduction

胞 (Pd) 作为植物发育和形态发生的关键调控器的细胞间转运的导管, 从转录因子到 mRNA 和小 RNA 分子不等。此外, 大多数植物病毒利用 Pd 的大分子转运能力在感染过程中进行细胞间传播;通过 pd, 植物病毒进化出专门的蛋白质, 称为运动蛋白 (MPs), 专门针对 pd1,2,3,45,7. Pd 转运的分子通路最有可能是与特定的序列紧密相连的, 靶向转移的蛋白质进入这些通路。因此, 识别这些 pd 定位信号 (PLSs) 可能是对相应的 pd 转运通路的诊断。这是通过类比的 Pd 传输8, 例如, 不同的核进口通路, 可以具体为不同的核定位信号 (不规则) 序列9,10。从概念上讲, NLSs 和 PLSs 都表示 non-cleavable 的亚细胞靶序列是必要和足够的目标。但是, 与 NLSs11不同的是, 有关 PLSs 的序列信息受到严重限制。具体来说, 只有四蛋白序列涉及 Pd 靶向已经报告, 所有这些都来源于内源植物蛋白。第一个是由 KN112的同源域表示的, 这是一个转录因子, 从内部细胞层到植物叶的表皮,13 , 以及它的诺克斯同系物14。第二个也是从转录因子, 自由度, 其中包含一个假定 PLS 描述为细胞间贩运 (IT) 主题15。第三个序列是从 PDLP1 胞的1型膜蛋白, 它由跨膜域16表示。最后, 第四 Pd 靶序列最近报告了 glycosylphosphatidylinositol (GPI) 锚定蛋白, 它由 glycosylphosphatidylinositol (GPI) 修饰信号17表示。

有趣的是, 直到最近, 没有 PLSs 的病毒的 MPs 报告。先前的研究表明, 假设 pls 序列在植物病毒 MPs18,19, 但没有真正 pls,即, 一个极小的氨基酸序列必要和充足为 Pd 靶向不相关的货物分子 (e. g, CFP) 已在病毒 MP 中被识别。然而, 其中的一种蛋白质, 即烟草花叶病毒的 MP (TMV), 是 Pd 局部化和传输被证明的第一个20。

为了解决这一差距, 我们制定了一个实验策略, 以确定 TMV MP PLS。该策略基于三概念。(i) 我们定义 PLS 作为一个极小的氨基酸序列是必要和充足的蛋白质靶向 Pd21。(ii) 由于 TMV MP 首先瞄准 pd, 然后通过这些通道22translocates, 我们的目的是拆下这两种活动并确定真正的 PLS, 它只对 pd 目标起作用, 而不适用于随后的传输。(iii) 我们分析了在结构上或功能上对其 Pd 靶向活性有重要意义的氨基酸残留物。利用这种方法, 我们在 TMV MP 的氨基末端划定了一个 50-氨基氨基酸残留序列, 作为善意的 PLS。这是通过产生一系列的 TMV MP 片段, 饱和整个蛋白质的长度, 标记他们的羧基总站与 CFP 和瞬时表达他们在植物组织。每个测试片段的 pd 定位是通过 coexpressing 他们与 pd 标记蛋白, PDCB1 (pd 胼胝结合蛋白 1)23。最小的片段, 仍然本地化到 pd, 但没有遍历 pd, 被认为是代表 PLS。最后, PLS 是丙氨酸扫描, 以确定其结构和/或功能所需的关键氨基酸残留物。

在这里, 我们通过描述 TMV MP 的识别来说明这种方法, 它可能被用来发现 PLSs 在任何其他 Pd 靶蛋白, 无论是由植物病原体或植物本身编码;这是因为我们的方法没有利用病毒 MPs 的任何独特的特点, 关于他们的能力目标 Pd。

Protocol

1. 植物材料 植物种类的选择 使用的植物物种的利益蛋白,即, 其中一个编码这种蛋白质的内生蛋白或代表自然宿主的病原体的病毒蛋白。此外, 选定的植物种类必须服从选择的瞬态遗传转化方法。注意: 这些研究通常使用烟草烟, 它代表了 TMV 的良好宿主, 并通过农杆菌介导的遗传转化技术 (即, agroinfiltration (参见步骤 3) 有效地转换。N. 烟植物也很容易生?…

Representative Results

代表数据, 忠实地说明预期的结果从被描述的协议和辨认 TMV MP PLS, 适应从元et al.21.图 1A首先总结了表达全长 TMV mp (1-268) 的主要构造, TMV mp PLS (包括蛋白质的前50氨基酸残滓, 1-50) 和它的丙氨酸扫描 V4A 衍生物熔化了 CFP (生成作为在步骤2.2、5.2 和6中描述), 而图 1B对这些标记蛋白质的亚细胞定位进行了总结和量化。图 1C</…

Discussion

该协议有四核心成分: 确定一个序列的概念是必要的和足够的目标, 以 Pd, 系统划分的利益蛋白的片段, 逐步减少长度, 融合测试片段的 autofluorescent 蛋白, 既作为标签和高分子货物, 和功能测试的 Pd 靶向活植物组织后, 瞬态表达的检测融合蛋白。请注意, 农杆菌介导的瞬态表达式在相对较短的时间内生成数据,即, 与在类似研究中经常使用的转基因植物生产所需的月份相比, 是数天,1…

Disclosures

The authors have nothing to disclose.

Acknowledgements

由于篇幅不足, 我们主要引用了评论文章, 我们向我们的同事道歉, 他们的原创作品没有被引用。共实验室的工作得到 nih、NSF、美国农业部/NIFA、巴德和 BSF 共的资助, S.G.L. 实验室由国立卫生研究院和植物病理学和植物微生物生物学部门资助, S.G.L。

Materials

Confocal laser scanning microscope (CLSM) Zeiss LSM5 Any CLSM with similar capabilities is appropriate
Zen software for confocal microscope imaging Zeiss 2009 version The software should be compatible with the CLSM used
Quickchange II site-directed mutagenesis kit  Agilent 200523
Acetosyringone Sigma-Aldrich D134406
MES Sigma-Aldrich 69892
Syringes without needles BD 309659
MgCl2 FisherScientific M33-500
Spectinomycin  Sigma-Aldrich S4014
Rifampicin Sigma-Aldrich R3501
Ampicillin  Sigma-Aldrich A0166
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Tags

Keywords: PlasmodesmataLocalization SignalIntercellular TransportProtein TargetingNicotiana BenthamianaFluorescent TaggingAgrobacterium TumefaciensTransient Expression
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Yuan, C., Lazarowitz, S. G., Citovsky, V. Identification of Plasmodesmal Localization Sequences in Proteins In Planta. J. Vis. Exp. (126), e55301, doi:10.3791/55301 (2017).
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