检测植物宿主中叶蝉载体的病毒和唾液蛋白
Summary
该协议演示了如何使用植物宿主来检测叶蝉的唾液蛋白和叶蝉载体释放的植物病毒蛋白。
Abstract
昆虫媒介水平传播许多具有农业重要性的植物病毒。超过一半的植物病毒是由具有刺穿吸口器的半翅目昆虫传播的。在病毒传播过程中，昆虫唾液桥接病毒载体宿主，因为唾液载体病毒和昆虫蛋白触发或抑制植物从昆虫到植物宿主的免疫反应。唾液蛋白的鉴定和功能分析正在成为虫媒病毒-宿主相互作用研究领域的新热点。该协议提供了一个使用植物宿主检测叶蝉唾液中蛋白质的系统。感染水稻矮病毒（RDV）的叶蝉载体Nephotettix cincticeps就是一个例子。在以辛克二头肌猪笼草为食的水稻植株中，可以同时检测到由辛克二头肌奈瑟菌唾液载体的RDV的卵黄原蛋白和主要外衣壳蛋白P8。该方法适用于检测昆虫摄食后暂时滞留在植物宿主中的唾液蛋白。相信这种检测系统将有利于半翅目-病毒-植物或半翅目-植物相互作用的研究。
Introduction
虫媒病毒的媒介-宿主传播方式是一个基本问题，处于生物科学的前沿。许多具有农业重要性的植物病毒通过昆虫媒介横向传播1。超过一半的植物病毒是由半翅目昆虫传播的，包括蚜虫、粉虱、叶蝉、飞虱和蓟马。这些昆虫具有独特的特征，使它们能够有效地传播植物病毒1。它们拥有刺吸口器，以韧皮部和木质部的汁液为食，分泌唾液1，2，3，4。随着技术的发展和改进，唾液成分的鉴定和功能分析正在成为深入研究的新焦点。唾液中已知的唾液蛋白包括许多酶，例如果胶酯酶、纤维素酶、过氧化物酶、碱性磷酸酶、多酚氧化酶和蔗糖酶等5，6，7，8，9，10，11，12，13.唾液中的蛋白质还包括触发宿主防御反应的诱发子，从而改变昆虫的性能，以及抑制宿主防御的效应子，其增强昆虫适应性和诱导宿主病理反应的成分14，15，16，17。因此，唾液蛋白是昆虫与宿主之间交流的重要材料。在病毒传播过程中，刺吸病毒昆虫唾液腺分泌的唾液也含有病毒蛋白。病毒成分利用唾液的流动将它们从昆虫释放到植物宿主。因此，昆虫唾液弥合了病毒-载体-宿主的三营养相互作用。研究含病毒昆虫分泌的唾液蛋白的生物学功能有助于了解病毒-载体-宿主的关系。
对于动物病毒，据报道，蚊子的唾液介导西尼罗河病毒（WNV）和登革热病毒（DENV）的传播和致病性。唾液蛋白AaSG34通过激活自噬促进登革热-2病毒复制和传播，而唾液蛋白AaVA-1通过激活自噬来促进登革热和寨卡病毒（ZIKV）传播18，19。蚊子的唾液蛋白D7可以通过与DENV病毒粒子和重组DENV包膜蛋白20的直接相互作用，在体外和体内抑制DENV感染。在植物病毒中，白栗病毒番茄黄叶卷曲病毒（TYLCV）诱导粉虱唾液蛋白Bsp9，抑制植物宿主WRKY33介导的免疫力，增加粉虱的偏好和性能，最终增加病毒的传播21。由于对昆虫唾液蛋白在植物宿主中的作用的研究落后于动物宿主，因此迫切需要一个稳定可靠的系统来检测植物宿主中的唾液蛋白。
被称为水稻矮病毒（RDV）的植物病毒由叶蝉Nephotettix cincticeps（半翅目：Cicadellidae）高效且持续繁殖22，23传播。RDV首先被报道通过昆虫媒介传播，并在亚洲引起严重的水稻病害24，25。病毒粒子为二十面体和双层球形，外层含有P8外衣壳蛋白22。RDV在蝴蝶奈瑟蝇中的循环传播周期为14天26，27，28，29，30。当RDV到达唾液腺时，病毒粒子通过胞吐样机制释放到唾液腺中储存唾液的腔中23。卵黄原蛋白（Vg）是雌性昆虫卵母细胞发育所必需的卵黄蛋白前体31，32，33。大多数昆虫物种至少有一个6-7 kb的Vg转录本，其编码约220 kDa的前体蛋白。Vg的蛋白质前体在进入卵巢之前通常可以裂解成大的（140至190 kDa）和小的（<50 kDa）片段18，19。先前的蛋白质组学分析显示，叶蝉Recilia dorsalis分泌的唾液中存在来自Vg的肽，尽管它们的功能尚不清楚（未发表的数据）。新报道，飞虱口服分泌的Vg起到效应作用，破坏植物的防御能力34。目前尚不清楚猪笼草的Vg是否也可以随唾液流释放到植物宿主，然后在植物中发挥作用干扰植物防御。为了解决猪笼草是否利用唾液蛋白（如Vg）来抑制或激活植物防御的问题，第一步是识别在摄食过程中释放到植物中的蛋白质。了解鉴定植物中存在的唾液蛋白的方法对于解释唾液蛋白的功能以及半翅目与植物之间的相互作用至关重要。
在这里介绍的协议中，以N . cincticeps 为例，提供一种方法来检查通过昆虫喂养引入的植物宿主中唾液蛋白的存在。该方案主要详细说明了唾液蛋白的收集和检测，有助于对大多数半翅目动物的进一步研究。
Protocol
非病毒性成虫叶蝉在中国福建农林大学媒介传播病毒研究中心繁殖。 1. 非病毒昆虫饲养 将成虫饲养在40厘米x 35厘米x 20厘米（长x宽x高）的立方体笼中。保持笼子的一侧盖上防虫网，以便通风。将带有叶蝉的笼子保存在包含内置湿度控制器的培养箱中，在26°C下，相对湿度为60-75%，光周期为16小时光照和8小时黑暗。 使用吸气器轻轻地将所有成虫从…
Representative Results
图1说明了该协议中的所有步骤：昆虫饲养，病毒获取，通过水稻喂养收集唾液蛋白以及蛋白质印迹。蛋白质印迹结果显示，在用Vg抗体孵育的膜上的喂养水稻和昆虫唾液腺的样品中观察到约220 kDa的特异性和预期条带。相比之下，在非饲料大米样品中未观察到条带。图2中的结果表明，Vg作为唾液蛋白释放到植物宿主中。在与RDV P8抗体?…
Discussion
刺吸昆虫唾液腺直接分泌的唾液起着举足轻重的作用，因为它将宿主组织和载体的跨王国生物因子预先消化和解毒到宿主1，3，4中。跨王国的生物因子，包括诱发子、效应子和小RNA，对于昆虫-宿主通讯至关重要14，15，16。因此，揭示唾液成分的更多种类和?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作得到了中国国家自然科学基金（31772124和31972239）和福建农林大学（KSYLX014）的资助。
Materials
| Reagents | |||
| Tris base | Roche | D609K69032 | For 5×Tris-glycine buffer and 10×TBS buffer preparation |
| glycine | Sigma-Aldrich | WXBD0677V | For 5×Tris-glycine buffer preparation |
| SDS | Sigma-Aldrich | SLCB4394 | For 5×Tris-glycine buffer preparation |
| NaCl | Sinopharm Chemical Reagent Co., Ltd | 10019318 | For 10×TBS buffer preparation |
| KCl | Sinopharm Chemical Reagent Co., Ltd | 10016318 | For 10×TBS buffer preparation |
| ß-mercaptoethanol | Xiya Reagent | B14492 | For 4× protein sample buffer preparation |
| bromophenol blue | Sigma-Aldrich | SHBL3668 | For 4× protein sample buffer preparation |
| glycerol | Sinopharm Chemical Reagent Co., Ltd | 10010618 | For 4× protein sample buffer preparation |
| methanol | Sinopharm Chemical Reagent Co., Ltd | 10014118 | For transfer buffer preparation |
| Tween 20 | Coolaber SCIENCE&TeCHNoLoGY | CT30111220 | For TBST preparation |
| non-fat dry milk | Becton.Dickinso and company | 252038 | For membrane blocking, antibodies dilution |
| goat anti-rabbit IgG | Sangon Biotech | D110058-0001 | Recognization of the primary andtibody |
| ECL Western kit | ThermoFisher Scientific | 32209 | Chemiluminescent substrate |
| nitrocellulose membrane | Pall Corporation | 25312915 | For proteins transfer |
| Buffers and Solutions | |||
| Buffer | Composition | Comments/Description | |
| 5×Tris-glycine buffer | 15.1 g Tris base 94 g glycine 5 g SDS in 1 L sterile water |
Stock solution | |
| 1×Tris-glycine buffer | 200 mL of 5×Tris-glycine buffer 800 mL sterile water |
Work solution, for SDS-PAGE | |
| 10×Tris-buffered saline (TBS) buffer | 80 g NaCl 30 g Tris base 2 g KCl in 1 L sterile water |
Stock solution | |
| TBS with Tween 20 (TBST) solution | 100 mL 10×TBS solution 3 mL Tween 20 900 mL sterile water |
Work solution | |
| 4× protein sample buffer | 8 g SDS 4 mL ß-mercaptoethanol 0.02 g bromophenol blue 40 mL glycerol in 40 mL 0.1 M Tris-HCl (pH 6.8) |
For protein extraction | |
| Transfer buffer | 800 mL Tris-glycine buffer 200 mL methanol |
For protein transfer | |
| Instruments | |||
| Bromophenol blue | Sigma-Aldrich | SHBL3668 | For 4x protein sample buffer preparation |
| Constant temperature incubator | Ningbo Saifu Experimental Instrument Co., Ltd. | PRX-1200B | For rearing leafhoppers |
| Electrophoresis | Tanon Science & Technology Co.,Ltd. | Tanon EP300 | For SDS-PAGE |
| Electrophoretic transfer core module | BIO-RAD | 1703935 | For SDS-PAGE |
| glycerol | Sinopharm Chemical Reagent Co., Ltd | 10010618 | For 4x protein sample buffer preparation |
| glycine | Sigma-Aldrich | WXBD0677V | For 5x Tris-glycine buffer preparation |
| goat anti-rabbit IgG | Sangon Biotech | D110058-0001 | Recognization of the primary andtibody |
| High-pass tissue grinding instrument | Shanghai Jingxin Industrial Development Co., Ltd. | JXFSIPRP-24 | For grinding plant tissues |
| KCl | Sinopharm Chemical Reagent Co., Ltd | 10016318 | For 10x TBS buffer preparation |
| methanol | Sinopharm Chemical Reagent Co., Ltd | 10014118 | For transfer buffer preparation |
| Mini wet heat transfer trough | BIO-RAD | 1703930 | For SDS-PAGE |
| NaCl | Sinopharm Chemical Reagent Co., Ltd | 10019318 | For 10x TBS buffer preparation |
| nitrocellulose membrane | Pall Corporation | 25312915 | For proteins transfer |
| non-fat dry milk | Becton.Dickinso and company | 252038 | For membrane blocking, antibodies dilution |
| Pierce ECL Western kit | ThermoFisher Scientific | 32209 | Chemiluminescent substrate |
| Protein color instrument | GE Healthcare bio-sciences AB | Amersham lmager 600 | For detecting proteins |
| SDS | Sigma-Aldrich | SLCB4394 | For 5x Tris-glycine buffer preparation |
| Tris base | Roche | D609K69032 | For 5x Tris-glycine buffer and 10×TBS buffer preparation |
| Tween 20 | Coolaber SCIENCE&TeCHNoLoGY | CT30111220 | For TBST preparation |
| Vertical plate electrophoresis tank | BIO-RAD | 1658001 | For SDS-PAGE |
| Water bath | Shanghai Jinghong Experimental equipment Co., Ltd. | XMTD-8222 | For boil the protein samples |
| β-mercaptoethanol | Xiya Reagent | B14492 | For 4x protein sample buffer preparation |
Riferimenti
- Hogenhout, S. A., Ammar el, D., Whitfield, A. E., Redinbaugh, M. G. Insect vector interactions with persistently transmitted viruses. Annual Review of Phytopathology. 46, 327-359 (2008).
- Cranston, P. S., Gullan, P. J., Resh, V. H., Carde, R. T. Phylogeny of insects. Encyclopedia of Insects. , (2003).
- Ammar el, D., Tsai, C. W., Whitfield, A. E., Redinbaugh, M. G., Hogenhout, S. A. Cellular and molecular aspects of rhabdovirus interactions with insect and plant hosts. Annual Review of Entomology. 54, 447-468 (2009).
- Wei, T., Li, Y. Rice reoviruses in insect vectors. Annual Review of Phytopathology. 54, 99-120 (2016).
- Hattori, M., Konishi, H., Tamura, Y., Konno, K., Sogawa, K. Laccase-type phenoloxidase in salivary glands and watery saliva of the green rice leafhopper, Nephotettix cincticeps. Journal of Insect Physiology. 51 (12), 1359-1365 (2005).
- Ma, R., Reese, J. C., William, I. V., Bramel-Cox, P. Detection of pectinesterase and polygalacturonase from salivary secretions of living greenbugs, schizaphis graminum (Homoptera: aphididae). Journal of Insect Physiology. 36 (7), 507-512 (1990).
- Miles, P. W. Dynamic aspects of the chemical relation between the rose aphid and rose buds. Entomologia Experimentalis et Applicata. 37 (2), 129-135 (2011).
- Urbanska, A., Tjallingii, W. F., Dixon, A., Leszczynski, B. Phenol oxidising enzymes in the grain aphid’s saliva. Entomologia Experimentalis et Applicata. 86 (2), 197-203 (1998).
- Miles, P. W., Peng, Z. Studies on the salivary physiology of plant bugs: detoxification of phytochemicals by the salivary peroxidase of aphids. Journal of Insect Physiology. 35 (11), 865-872 (1989).
- Will, T., van Bel, A. Physical and chemical interactions between aphids and plants. Journal of Experimental Botany. 57 (4), 729-737 (2006).
- Ma, R. Z., Reese, J. C., Black, W. C., Bramel-Cox, I. Chlorophyll loss in a greenbug-susceptible sorghum due to pectinases and pectin fragments. Journal of the Kansas Entomological Society. 71 (1), 51-60 (1998).
- Madhusudhan, V. V., Miles, P. W. Mobility of salivary components as a possible reason for differences in the responses of alfalfa to the spotted alfalfa aphid and pea aphid. Entomologia Experimentalis et Applicata. 86 (1), 25-39 (1998).
- Funk, C. J. Alkaline phosphatase activity in whitefly salivary glands and saliva. Archives of Insect Biochemistry & Physiology. 46 (4), 165-174 (2010).
- Hogenhout, S. A., Bos, J. I. Effector proteins that modulate plant-insect interactions. Current Opinion in Plant Biology. 14 (4), 422-428 (2011).
- Tomkins, M., Kliot, A., Maree, A. F., Hogenhout, S. A. A multi-layered mechanistic modelling approach to understand how effector genes extend beyond phytoplasma to modulate plant hosts, insect vectors and the environment. Current Opinion in Plant Biology. 44, 39-48 (2018).
- Huang, H. J., Lu, J. B., Li, Q., Bao, Y. Y., Zhang, C. X. Combined transcriptomic/proteomic analysis of salivary gland and secreted saliva in three planthopper species. Journal of Proteomics. , (2018).
- Hogenhout, S. A., Bos, J. I. Effector proteins that modulate plant–insect interactions. Current Opinion in Plant Biology. 14 (4), 422-428 (2011).
- Sun, P., et al. A mosquito salivary protein promotes flavivirus transmission by activation of autophagy. Nature Communications. 11 (1), 260 (2020).
- Sri-In, C., et al. A salivary protein of Aedes aegypti promotes dengue-2 virus replication and transmission. Insect Biochemistry and Molecular Biology. 111, 103181 (2019).
- Conway, M. J., et al. Aedes aegypti D7 saliva protein inhibits dengue virus infection. Plos Neglected Tropical Diseases. 10 (9), 0004941 (2016).
- Wang, N., et al. A whitefly effector Bsp9 targets host immunity regulator WRKY33 to promote performance. Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences. 374 (1767), 20180313 (2019).
- Omura, T., Yan, J. Role of outer capsid proteins in transmission of phytoreovirus by insect vectors. Advances in Virus Research. 54, 15-43 (1999).
- Chen, Q., Liu, Y., Long, Z., Yang, H., Wei, T. Viral release threshold in the salivary gland of leafhopper vector mediates the intermittent transmission of rice dwarf virus. Frontiers in Microbiology. 12, 639445 (2021).
- Fukushi, T. Further studies on the dwarf disease of rice plant. Journal of the Faculty of Agriculture, Hokkaido Imperial University. 45 (3), 83-154 (1940).
- Miyazaki, N., et al. The functional organization of the internal components of rice dwarf virus. Journal of Biochemistry. 147, 843-850 (2010).
- Wei, T., Shimizu, T., Hagiwara, K., Kikuchi, A., Omura, T. Pns12 protein of rice dwarf virus is essential for formation of viroplasms and nucleation of viral-assembly complexes. Journal of General Virology. 87, 429-438 (2006).
- Chen, Q., Zhang, L., Chen, H., Xie, L., Wei, T. Nonstructural protein Pns4 of rice dwarf virus is essential for viral infection in its insect vector. Virology Journal. 12, 211 (2015).
- Chen, Q., et al. Nonstructural protein Pns12 of rice dwarf virus is a principal regulator for viral replication and infection in its insect vector. Virus Research. 210, 54-61 (2015).
- Chen, Q., Zhang, L., Zhang, Y., Mao, Q., Wei, T. Tubules of plant reoviruses exploit tropomodulin to regulate actin-based tubule motility in insect vector. Scientific Reports. 7, 38563 (2017).
- Wei, T., et al. The spread of Rice dwarf virus among cells of its insect vector exploits virus-induced tubular structures. Journal of Virology. 80 (17), 8593-8602 (2006).
- Mao, Q., et al. Insect bacterial symbiont-mediated vitellogenin uptake into oocytes to support egg development. mBio. 11 (6), 01142 (2020).
- Tufail, M., Takeda, M. Molecular characteristics of insect vitellogenins. Journal of Insect Physiology. 54 (12), 1447-1458 (2008).
- Sappington, T. W., Raikhel, A. S. Molecular characteristics of insect vitellogenins and vitellogenin receptors. Insect Biochemistry and Molecular Biology. 28 (5-6), 277-300 (1998).
- Ji, R., et al. Vitellogenin from planthopper oral secretion acts as a novel effector to impair plant defenses. New Phytologist. , (2021).
Citazione di questo articolo
Wang, Y., Wang, X., Li, Z., Chen, Q. Detecting Virus and Salivary Proteins of a Leafhopper Vector in the Plant Host. J. Vis. Exp. (175), e63020, doi:10.3791/63020 (2021).