蛋白质中 Plasmodesmal 定位序列的识别在底中
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
Representative Results
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…
Divulgaciones
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 |
Referencias
- Lee, J. Y. Plasmodesmata: a signaling hub at the cellular boundary. Curr. Opin. Plant Biol. 27, 133-140 (2015).
- Kumar, D., Kumar, R., Hyun, T. K., Kim, J. Cell-to-cell movement of viruses via plasmodesmata. J. Plant Res. 128, 37-47 (2015).
- Kitagawa, M., Paultre, D., Rademaker, H. Intercellular communication via plasmodesmata. New Phytol. 205, 970-972 (2015).
- Jackson, D. Plasmodesmata spread their influence. F1000Prime Rep. 7, 25 (2015).
- Brunkard, J. O., Runkel, A. M., Zambryski, P. C. The cytosol must flow: intercellular transport through plasmodesmata. Curr. Opin. Cell Biol. 35, 13-20 (2015).
- Yadav, S. R., Yan, D., Sevilem, I., Helariutta, Y. Plasmodesmata-mediated intercellular signaling during plant growth and development. Front. Plant Sci. 5, 44 (2014).
- Sager, R., Lee, J. Y. Plasmodesmata in integrated cell signaling: insights from development and environmental signals and stresses. J. Exp. Bot. 65, 6337-6358 (2014).
- Jans, D. A., Xiao, C. Y., Lam, M. H. Nuclear targeting signal recognition: a key control point in nuclear transport?. BioEssays. 22, 532-544 (2000).
- Miyamoto, Y., et al. Different modes of nuclear localization signal (NLS) recognition by three distinct classes of NLS receptors. J. Biol. Chem. 272, 26375-26381 (1997).
- Nair, R., Carter, P., Rost, B. NLSdb: database of nuclear localization signals. Nucleic Acids Res. 31, 397-399 (2003).
- Lee, J. Y., Yoo, B. C., Lucas, W. J. Parallels between nuclear-pore and plasmodesmal trafficking of information molecules. Planta. 210, 177-187 (2000).
- Kim, J. Y., Rim, Y., Wang, J., Jackson, D. A novel cell-to-cell trafficking assay indicates that the KNOX homeodomain is necessary and sufficient for intercellular protein and mRNA trafficking. Genes Dev. 19, 788-793 (2005).
- Lucas, W. J., et al. Selective trafficking of KNOTTED1 homeodomain protein and its mRNA through plasmodesmata. Science. 270, 1980-1983 (1995).
- Chen, H., Jackson, D., Kim, J. Y. Identification of evolutionarily conserved amino acid residues in homeodomain of KNOX proteins for intercellular trafficking. Plant Signal. Behav. 9, e28355 (2014).
- Chen, H., Ahmad, M., Rim, Y., Lucas, W. J., Kim, J. Y. Evolutionary and molecular analysis of Dof transcription factors identified a conserved motif for intercellular protein trafficking. New Phytol. 198, 1250-1260 (2013).
- Thomas, C. L., Bayer, E. M., Ritzenthaler, C., Fernandez-Calvino, L., Maule, A. J. Specific targeting of a plasmodesmal protein affecting cell-to-cell communication. PLOS Biol. 6, e7 (2008).
- Zavaliev, R., Dong, X., Epel, B. L. Glycosylphosphatidylinositol (GPI) modification serves as a primary plasmodesmal targeting signal. Plant Physiol. 172, 1061-1073 (2016).
- Sasaki, N., Park, J. W., Maule, A. J., Nelson, R. S. The cysteine-histidine-rich region of the movement protein of Cucumber mosaic virus contributes to plasmodesmal targeting, zinc binding and pathogenesis. Virology. 349, 396-408 (2006).
- Kaido, M., Funatsu, N., Tsuno, Y., Mise, K., Okuno, T. Viral cell-to-cell movement requires formation of cortical punctate structures containing Red clover necrotic mosaic virus movement protein. Virology. 413, 205-215 (2011).
- Creager, A. N. H., Scholthof, K. B. G., Citovsky, V., Scholthof, H. B. Tobacco mosaic virus: pioneering research for a century. Plant Cell. 11, 301-308 (1999).
- Yuan, C., Lazarowitz, S. G., Citovsky, V. Identification of a functional plasmodesmal localization signal in a plant viral cell-to-cell movement protein. mBio. 7, e02052-e02015 (2016).
- Ueki, S., Citovsky, V. To gate, or not to gate: regulatory mechanisms for intercellular protein transport and virus movement in plants. Mol. Plant. 4, 782-793 (2011).
- Simpson, C., Thomas, C. L., Findlay, K., Bayer, E., Maule, A. J. An Arabidopsis GPI-anchor plasmodesmal neck protein with callose binding activity and potential to regulate cell-to-cell trafficking. Plant Cell. 21, 581-594 (2009).
- Maule, A. J. Plasmodesmata: structure, function and biogenesis. Curr. Opin. Plant Biol. 11, 680-686 (2008).
- Roberts, I. M., et al. Dynamic changes in the frequency and architecture of plasmodesmata during the sink-source transition in tobacco leaves. Protoplasma. 218, 31-44 (2001).
- Tzfira, T., et al. pSAT vectors: a modular series of plasmids for fluorescent protein tagging and expression of multiple genes in plants. Plant Mol. Biol. 57, 503-516 (2005).
- Chakrabarty, R., et al. pSITE vectors for stable integration or transient expression of autofluorescent protein fusions in plants: probing Nicotiana benthamiana-virus interactions. Mol. Plant-Microbe Interact. 20, 740-750 (2007).
- Chung, S. M., Frankman, E. L., Tzfira, T. A versatile vector system for multiple gene expression in plants. Trends Plant Sci. 10, 357-361 (2005).
- Lee, L. Y., Gelvin, S. B. T-DNA binary vectors and systems. Plant Physiol. 146, 325-332 (2008).
- Goderis, I. J., et al. A set of modular plant transformation vectors allowing flexible insertion of up to six expression units. Plant Mol. Biol. 50, 17-27 (2002).
- Walhout, A. J., et al. GATEWAY recombinational cloning: application to the cloning of large numbers of open reading frames or ORFeomes. Methods Enzymol. 328, 575-592 (2000).
- Tzfira, T., et al. Transgenic Populus: a step-by-step protocol for its Agrobacterium-mediated transformation. Plant Mol. Biol. Rep. 15 (3), 219-235 (1997).
- Woodman, M. E., Savage, C. R., Arnold, W. K., Stevenson, B. Direct PCR of intact bacteria (colony PCR). Curr. Protoc. Microbiol. 42 (3D), 1-7 (2016).
- Kapila, J., De Rycke, R., Van Montagu, M., Angenon, G. An Agrobacterium-mediated transient gene expression system for intact leaves. Plant Sci. , 101-108 (1997).
- Wroblewski, T., Tomczak, A., Michelmore, R. Optimization of Agrobacterium-mediated transient assays of gene expression in lettuce, tomato and Arabidopsis. Plant Biotechnol. J. 3, 259-273 (2005).
- Boyko, V., Ferralli, J., Ashby, J., Schellenbaum, P., Heinlein, M. Function of microtubules in intercellular transport of plant virus RNA. Nat. Cell Biol. 2, 826-832 (2000).
- Heinlein, M., Epel, B. L., Padgett, H. S., Beachy, R. N. Interaction of tobamovirus movement proteins with the plant cytoskeleton. Science. 270, 1983-1985 (1995).
- Oparka, K. J., Prior, D. A. M., Santa-Cruz, S., Padgett, H. S., Beachy, R. N. Gating of epidermal plasmodesmata is restricted to the leading edge of expanding infection sites of tobacco mosaic virus (TMV). Plant J. 12, 781-789 (1997).
- Crawford, K. M., Zambryski, P. C. Non-targeted and targeted protein movement through plasmodesmata in leaves in different developmental and physiological states. Plant Physiol. 125, 1802-1812 (2001).
- Kotlizky, G., et al. A dysfunctional movement protein of Tobacco mosaic virus interferes with targeting of wild-type movement protein to microtubules. Mol. Plant-Microbe Interact. 14, 895-904 (2001).
- Ueki, S., Lacroix, B., Krichevsky, A., Lazarowitz, S. G., Citovsky, V. Functional transient genetic transformation of Arabidopsis leaves by biolistic bombardment. Nat. Protoc. 4, 71-77 (2009).
- Giesman-Cookmeyer, D., Lommel, S. A. Alanine scanning mutagenesis of a plant virus movement protein identifies three functional domains. Plant Cell. 5, 973-982 (1993).
- Ausubel, F. M., et al. . Current Protocols in Molecular Biology. , (1987).
- Waigmann, E., Ueki, S., Trutnyeva, K., Citovsky, V. The ins and outs of non-destructive cell-to-cell and systemic movement of plant viruses. Crit. Rev. Plant Sci. 23, 195-250 (2004).
- Lee, M. W., Yang, Y. Transient expression assay by agroinfiltration of leaves. Methods Mol. Biol. 323, 225-229 (2006).
- Burch-Smith, T. M., Schiff, M., Liu, Y., Dinesh-Kumar, S. P. Efficient virus-induced gene silencing in Arabidopsis. Plant Physiol. 142, 21-27 (2006).
- Tian, G. W., et al. High-throughput fluorescent tagging of full-length Arabidopsis gene products in planta. Plant Physiol. 135, 25-38 (2004).
- Tian, G. W., Chen, M. H., Zaltsman, A., Citovsky, V. A pollen-specific pectin methylesterase involved in pollen tube growth. Dev. Biol. 294, 83-91 (2006).
- Hunter, C. C., et al. Multiple nuclear localization signals mediate nuclear localization of the GATA transcription factor AreA. Eukaryot. Cell. 13, 527-538 (2014).
