JoVE Journal > Environment
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
Automatic Translation
Please note that all translations are automatically generated. Click here for the English version.
Environment

马铃薯病毒基于马铃薯的X型微RNA沉默（VbMS）。

Published: May 11, 2020
doi:
10.3791/61067
, ,
1Texas A&M AgriLife Research Center at Dallas,Texas A&M University System, 2Department of Plant Pathology, Microbiology,Texas A&M University

Summary

我们提出了基于马铃薯病毒X（PVX）的microRNA沉默（VbMS）系统的详细方案，以功能性地表征马铃薯中的内源性microRNA（miRNA）。将目标miRNA的靶向拟态（TM）分子整合到PVX载体中，并在马铃薯中瞬时表达，以沉默靶miRNA或miRNA家族。

Abstract

基于病毒的microRNA沉默（VbMS）是一种快速有效的工具，用于植物中microRNA（miRNA）的功能表征。VbMS系统已被开发并应用于各种植物物种，包括 Nicotiana benthamiana，番茄，拟南芥，棉花和单子叶植物，如小麦和玉米。在这里，我们描述了一个详细的方案，使用基于PVX的VbMS载体来沉默马铃薯中的内源性miRNA。为了降低特定miRNA的表达，设计了目标miRNA的靶向模拟（TM）分子，将其整合到植物病毒载体中，并通过 农杆菌 浸润在马铃薯中表达，以直接与感兴趣的内源性miRNA结合并阻断其功能。

Introduction

植物微RNA （miRNA） 的特征是 20–24 个核苷酸长、核编码的调节性 RNA1 ，在植物生物过程的几乎每个方面都起着重要作用，包括生长和发育2、3、光合作用和代谢4、5、6、7、激素合成和信号传导8、9、生物和非生物反应10，11，12，13，以及营养和能量调节14，15。植物miRNA的调节作用是良好编程的，通常在转录后水平上通过切割或翻译抑制靶标mRNA来实现。

在马铃薯中miRNA的鉴定、转录谱分析和靶标预测方面取得了巨大进展16、17、18、19、20、21。然而，由于缺乏高效和高通量的遗传方法，包括马铃薯在内的植物中miRNA的功能表征落后于其他生物体。通过标准功能丧失分析对单个miRNA进行功能分析具有挑战性，因为大多数miRNA属于具有相当遗传冗余的家族22。此外，单个miRNA可以控制多个靶基因23 ，几个不同的miRNA可以协同调节相同的分子途径24，25。这些特性使得难以表征特定miRNA或miRNA家族的功能。

miRNA的大部分功能分析在很大程度上依赖于具有明显局限性的功能增益方法。人工miRNA（amiRNA）方法利用内源性原代转录本（pri-miRNA）在高水平上产生miRNA，从而抑制靶基因表达26，27，28，29。然而，使用强本构35S启动子的活化标记和miRNA过表达通常会导致miRNA的表达升高，这些表达不代表体内条件，因此可能无法反映miRNA30的内源性功能。已经开发了一种替代方法，涉及表达在结合和/或切割位点中含有不易脱位突变的靶基因的miRNA抗性形式31，32，33。但是，由于转基因伪影，这种方法也可能导致对源自miRNA耐药靶基因的表型的误解。因此，应谨慎从这些功能获得研究中得出结论34。上述方法的另一个主要局限性是它们需要转换，这是劳动密集型和耗时的。此外，转基因依赖性方法几乎不适用于顽固的植物物种。因此，必须开发一种快速有效的功能丧失方法来解开miRNA的功能。

为了绕过转化过程的先决条件，通过将靶向模拟（TM）策略与病毒衍生载体相结合，建立了基于病毒的microRNA沉默（VbMS）。在VbMS系统中，人工设计的TM分子从病毒骨架瞬时表达，为解剖植物内源性miRNA的功能提供了一种功能强大，高通量和省时的工具35，36。VbMS最初是在边 楸 和番茄中与烟草拨浪鼓病毒（TRV）35，36，37 一起开发的，并且已经扩展到拟南芥，棉花，小麦和玉米，使用各种其他病毒表达系统，包括马铃薯病毒X（PVX）38，棉花屑病毒（ClCrV）39，黄瓜花叶病毒（CMV）40，41，42，中国小麦花叶病毒（CWMV）43和大麦条纹花叶病毒（BSMV）44，45。

马铃薯（Solanum tuberosum）是世界上第四大粮食作物，也是种植最广泛的非藜麦作物，主要是因为它具有高营养价值、高能量产量和相对较低的投入要求46。马铃薯的几个特点使其成为一种有吸引力的双子叶植物模型植物。它是一种营养繁殖的多倍体作物，具有高越交率，杂合性和遗传多样性。然而，迄今为止，还没有报告使用VbMS表征马铃薯中miRNA的功能。在这里，我们提出了一种基于结扎独立克隆（LIC）的马铃薯PVX的VbMS方法来评估miRNA在马铃薯植物中的功能38。我们选择了miR165/166家族来说明VbMS测定，因为miR165/166家族及其靶标mRNA和III类同源域/Leu拉链（HD-ZIP III）转录因子已被广泛表征22，47，48。 HD-ZIP III 基因是分生组织发育和器官极性的关键调节因子，miR165/166功能的抑制导致 HD-ZIP III 基因表达增加，导致顶端优势降低和叶极性异常模式等多向性发育缺陷22，35，38，41.与miRNA165/166沉默相关的易于评分的发育表型能够准确评估基于PVX的VbMS测定的有效性。

在这项研究中，我们证明了基于PVX的VbMS系统可以有效地阻断马铃薯中miRNA的功能。由于基于PVX的病毒诱导基因沉默（VIGS）系统已经在许多马铃薯品种中建立49，50，51，52，因此这种基于PVX的VbMS方法可能适用于广泛的二倍体和四倍体马铃薯品种。

Protocol

1. 种植马铃薯植物。 用Murashige和Skoog（MS）培养基加上Gamborg维生素（MS基盐混合物，Gamborg维生素，30g / L蔗糖，3.5 g / L琼脂，pH = 5.7）在培养管（25 x 150 mm）中繁殖马铃薯植物。将试管置于生长室中，温度为20-22°C，16小时光照/ 8小时黑暗光周期，光强度为120μmol/ m2∙s1。注意：新芽和根通常在植物的1-2周内发育。每月用新鲜的MS / Gamborg维生素培养基繁殖植物。 四周…

Representative Results

图2 显示了PVX-STTM165/166马铃薯植株（Katahdin），其叶组织从叶片的轴侧沿叶脉异位生长。还观察到更严重的表型，如喇叭形叶的形成。相比之下，在PVX控制工厂中没有观察到表型异常。这些结果表明，VbMS系统可有效抑制四倍体马铃薯植株的内源性miRNA功能，PVX-VbMS系统是确定特定miRNA或miRNA家族功能的强大遗传工具。 图3 显示了PVX-STTM…

Discussion

我们提出了一种基于PVX的miRNA沉默系统，通过将STTM设计整合到PVX载体中来表征马铃薯中内源性miRNA的功能。VbMS系统被证明可以有效地沉默马铃薯中的miRNA165/166，马铃薯是跨植物物种的高度保守的miRNA家族。

TM方法已被开发用于干扰基于人工miRNA靶标模拟的miRNA的表达，该拟态旨在在miRNA互补序列内的预期切割位点处创建不匹配环，从而导致靶向miRNA的封存并阻止其活性<sup class="xr…

Disclosures

The authors have nothing to disclose.

Acknowledgements

我们感谢清华大学的廖玉乐博士提供PVX-LIC载体。这项工作得到了德克萨斯州A&M AgriLife Research的启动基金和美国农业部国家食品和农业研究所的Hatch Project TEX0-1-9675的支持。

Materials

100 µM dATP and 100 µM dTTP Omega Bio-tek, Inc., Norcross, Norcross, GA 30071 , USA TQAC136
3 M Sodium acetate, pH 4.0. Teknova, Hollister, CA 95023, USA #S0297
Acetosyringone TCI America, Portland, OR 97203, USA D2666-25G
Agrobacterium tumefaciens strains: GV3101, GV2260 or EHA105.
Chloroform VWR Corporate, Radnor, PA 19087-8660, USA VWRV0757-950ML
Dimethyl sulfoxide, DMSO TCI America, Portland, OR 97203, USA D0798-25G
DTT VWR Corporate, Radnor, PA 19087-8660, USA VWRV0281-25G
E. coli DB3.1 for maintenance of PVX-LIC and pTRV2e containing the ccdB gene
E. coli DH5α for the destination constructs generated by LIC cloning
Fertilizer: Peters Peat Lite Special 15-0-15 Dark Weather Feed ICL Specialty Fertilizers, Summerville, SC 29483, USA G99260
High fidelity PCR reagents: KAPA HiFi DNA Polymerase with dNTPs Roche Sequencing and Life Science, Kapa Biosystems,
Wilmington, MA, USA		 7958960001
Isoamyl alcohol VWR Corporate, Radnor, PA 19087-8660, USA VWRV0944-1L
Koptec Pure Ethanol – 200 Proof Decon Labs, King of Prussia, PA 19406 , USA V1005M
MES TCI America, Portland, OR 97203, USA M0606-250G
MgCl2 ThermoFisher, Waltham, MA 02451, USA MFCD00149781
M-MuLV Reverse Transcriptase New England BioLabs, Ipswich, MA 01938-2723 USA M0253L
Nano-drop spectrometer: NanoDrop OneC Microvolume UV-Vis Spectrophotometer with Wi-Fi ThermoFisher, Waltham, MA 02451, USA ND-ONEC-W
PCR machine: Bio-Rad MyCycler PCR System Bio-Rad, Hercules, California 94547, USA 170-9703
PCR machine: Eppendorf Mastercycler pro Eppendorf, Hauppauge, NY 11788, USA 950030010
pH meter Sper Scientific, Scottsdale, AZ 85260, USA Benchtop pH / mV Meter – 860031
Phenol:chloroform:isoamyl alcohol (25:24:1), pH 6.7/8.0. VWR Corporate, Radnor, PA 19087-8660, USA VWRV0883-400ML
Phytagel: Gellan Gum Alfa Aesar, Tewksbury, MA 01876, USA J63423-A1
PVX VIGS vector: PVX-LIC Zhao et al., 2016
Real-time PCR machine: QuantStudio 6 Flex Real-Time PCR System ThermoFisher, Waltham, MA 02451, USA 4485697
Real-time PCR reagent: KAPA SYBR® FAST qPCR Master Mix (2x) Kit Roche Sequencing and Life Science, Kapa Biosystems,
Wilmington, MA 01887, USA		 7959389001
Restriction enzyme: SmaI New England BioLabs, Ipswich, MA 01938-2723 USA R0141S
Reverse transcription reagents: qScript cDNA SuperMix Quanta BioSciences, Gaithersburg, MD 20877 , USA 95107-100
RNA extraction Kit: E.Z.N.A. Plant RNA Kit Omega Bio-tek, Inc., Norcross, Norcross, GA 30071 , USA SKU: D3485-01
RNase Inhibitor Murine New England BioLabs, Ipswich, MA 01938-2723 USA M0314L
RNAzol RT Sigma-Aldrich, St. Louis, MO 63103, USA R4533
Soil: Metro-Mix 360 Sun Gro Horticulture, Agawam, MA 01001-2907, USA Metro-Mix 360
T4 DNA polymerase and buffer New England BioLabs, Ipswich, MA 01938-2723 USA M0203S
DOWNLOAD MATERIALS LIST

References

  1. Axtell, M. J., Meyers, B. C. Revisiting Criteria for Plant MicroRNA Annotation in the Era of Big Data. The Plant Cell. 30 (2), 272-284 (2018).
  2. Chen, X. Small RNAs and Their Roles in Plant Development. Annual Review of Cell and Developmental Biology. 25 (1), 21-44 (2009).
  3. Rubio-Somoza, I., Weigel, D. MicroRNA networks and developmental plasticity in plants. Trends in Plant Science. 16 (5), 258-264 (2011).
  4. Zhang, J. -. P., et al. MiR408 Regulates Grain Yield and Photosynthesis via a Phytocyanin Protein. Plant Physiology. 175 (3), 1175-1185 (2017).
  5. Gupta, O. P., Karkute, S. G., Banerjee, S., Meena, N. L., Dahuja, A. Contemporary Understanding of miRNA-Based Regulation of Secondary Metabolites Biosynthesis in Plants. Frontiers in Plant Science. 8 (374), (2017).
  6. May, P., et al. The effects of carbon dioxide and temperature on microRNA expression in Arabidopsis development. Nature Communications. 4 (1), 2145 (2013).
  7. Krützfeldt, J., Stoffel, M. MicroRNAs: A new class of regulatory genes affecting metabolism. Cell Metabolism. 4 (1), 9-12 (2006).
  8. Damodharan, S., Corem, S., Gupta, S. K., Arazi, T. Tuning of SlARF10A dosage by sly-miR160a is critical for auxin-mediated compound leaf and flower development. The Plant Journal. 96 (4), 855-868 (2018).
  9. Nizampatnam, N. R., Schreier, S. J., Damodaran, S., Adhikari, S., Subramanian, S. microRNA160 dictates stage-specific auxin and cytokinin sensitivities and directs soybean nodule development. The Plant Journal. 84 (1), 140-153 (2015).
  10. Chinnusamy, V., Zhu, J., Zhu, J. -. K. Cold stress regulation of gene expression in plants. Trends in Plant Science. 12 (10), 444-451 (2007).
  11. Covarrubias, A. A., Reyes, J. L. Post-transcriptional gene regulation of salinity and drought responses by plant microRNAs. Plant, Cell, Environment. 33 (4), 481-489 (2010).
  12. Wang, S., et al. Suppression of nbe-miR166h-p5 attenuates leaf yellowing symptoms of potato virus X on Nicotiana benthamiana and reduces virus accumulation. Molecular Plant Pathology. 19 (11), 2384-2396 (2018).
  13. Canto-Pastor, A., et al. Enhanced resistance to bacterial and oomycete pathogens by short tandem target mimic RNAs in tomato. Proceedings of the National Academy of Sciences. 116 (7), 2755-2760 (2019).
  14. Chiou, T. -. J., Lin, S. -. I. Signaling Network in Sensing Phosphate Availability in Plants. Annual Review of Plant Biology. 62 (1), 185-206 (2011).
  15. Sunkar, R., Chinnusamy, V., Zhu, J., Zhu, J. -. K. Small RNAs as big players in plant abiotic stress responses and nutrient deprivation. Trends in Plant Science. 12 (7), 301-309 (2007).
  16. Kwenda, S., Birch, P. R. J., Moleleki, L. N. Genome-wide identification of potato long intergenic noncoding RNAs responsive to Pectobacterium carotovorum subspecies brasiliense infection. BMC Genomics. 17 (1), 614 (2016).
  17. Lakhotia, N., et al. Identification and characterization of miRNAome in root, stem, leaf and tuber developmental stages of potato (Solanum tuberosum L.) by high-throughput sequencing. BMC Plant Biology. 14 (1), 6 (2014).
  18. Koc, I., Filiz, E., Tombuloglu, H. Assessment of miRNA expression profile and differential expression pattern of target genes in cold-tolerant and cold-sensitive tomato cultivars. Biotechnology, Biotechnological Equipment. 29 (5), 851-860 (2015).
  19. Zhang, N., et al. Identification of Novel and Conserved MicroRNAs Related to Drought Stress in Potato by Deep Sequencing. PLoS One. 9 (4), 95489 (2014).
  20. Xie, F., Frazier, T. P., Zhang, B. Identification, characterization and expression analysis of MicroRNAs and their targets in the potato (Solanum tuberosum). Gene. 473 (1), 8-22 (2011).
  21. Zhang, R., Marshall, D., Bryan, G. J., Hornyik, C. Identification and Characterization of miRNA Transcriptome in Potato by High-Throughput Sequencing. PLoS One. 8 (2), 57233 (2013).
  22. Yan, J., et al. Effective Small RNA Destruction by the Expression of a Short Tandem Target Mimic in Arabidopsis. The Plant Cell. 24 (2), 415-427 (2012).
  23. Roodbarkelari, F., Groot, E. P. Regulatory function of homeodomain-leucine zipper (HD-ZIP) family proteins during embryogenesis. New Phytologist. 213 (1), 95-104 (2017).
  24. Reichel, M., Millar, A. A. Specificity of plant microRNA target MIMICs: Cross-targeting of miR159 and miR319. Journal of Plant Physiology. 180, 45-48 (2015).
  25. Taylor, R. S., Tarver, J. E., Hiscock, S. J., Donoghue, P. C. J. Evolutionary history of plant microRNAs. Trends in Plant Science. 19 (3), 175-182 (2014).
  26. Schwab, R., Ossowski, S., Riester, M., Warthmann, N., Weigel, D. Highly Specific Gene Silencing by Artificial MicroRNAs in Arabidopsis. The Plant Cell. 18 (5), 1121-1133 (2006).
  27. Martin, A., et al. Graft-transmissible induction of potato tuberization by the microRNA miR172. Development. 136 (17), 2873-2881 (2009).
  28. Yang, L., et al. Overexpression of potato miR482e enhanced plant sensitivity to Verticillium dahliae infection. Journal of Integrative Plant Biology. 57 (12), 1078-1088 (2015).
  29. Tang, Y., et al. Virus-based microRNA expression for gene functional analysis in plants. Plant Physiology. 153 (2), 632-641 (2010).
  30. Voinnet, O. Origin, Biogenesis, and Activity of Plant MicroRNAs. Cell. 136 (4), 669-687 (2009).
  31. Teotia, S., Tang, G. To Bloom or Not to Bloom: Role of MicroRNAs in Plant Flowering. Molecular Plant. 8 (3), 359-377 (2015).
  32. Wu, G., Poethig, R. S. Temporal regulation of shoot development in Arabidopsis thaliana by miR156 and its target SPL3. Development. 133 (18), 3539-3547 (2006).
  33. Zhao, L., Kim, Y., Dinh, T. T., Chen, X. miR172 regulates stem cell fate and defines the inner boundary of APETALA3 and PISTILLATA expression domain in Arabidopsis floral meristems. The Plant Journal. 51 (5), 840-849 (2007).
  34. Li, J., Millar, A. A. Expression of a microRNA-Resistant Target Transgene Misrepresents the Functional Significance of the Endogenous microRNA: Target Gene Relationship. Molecular Plant. 6 (2), 577-580 (2013).
  35. Sha, A., et al. Virus-based microRNA silencing in plants. Plant Physiology. 164 (1), 36-47 (2014).
  36. Zhao, J., Liu, Y. Virus-based MicroRNA Silencing. Bio-protocol. 6 (2), 1714 (2016).
  37. Yan, F., et al. A virus-based miRNA suppression (VbMS) system for miRNA loss-of-function analysis in plants. Biotechnology Journal. 9 (5), 702-708 (2014).
  38. Zhao, J., et al. An efficient Potato virus X-based microRNA silencing in Nicotiana benthamiana. Scientific Reports. 6, 20573 (2016).
  39. Gu, Z., Huang, C., Li, F., Zhou, X. A versatile system for functional analysis of genes and microRNAs in cotton. Plant Biotechnology Journal. 12 (5), 638-649 (2014).
  40. Du, Z., et al. Using a viral vector to reveal the role of microRNA159 in disease symptom induction by a severe strain of cucumber mosaic virus. Plant Physiology. 164 (3), 1378-1388 (2014).
  41. Liao, Q., Tu, Y., Carr, J. P., Du, Z. An improved cucumber mosaic virus-based vector for efficient decoying of plant microRNAs. Scientific Reports. 5, 13178 (2015).
  42. Liu, X., et al. Analyses of MiRNA Functions in Maize Using a Newly Developed ZMBJ-CMV-2bN81-STTM Vector. Frontiers in Plant Science. 10, 1277 (2019).
  43. Yang, J., et al. Chinese Wheat Mosaic Virus-Induced Gene Silencing in Monocots and Dicots at Low Temperature. Frontiers in Plant Science. 9, 1627 (2018).
  44. Jiao, J., Wang, Y., Selvaraj, J. N., Xing, F., Liu, Y. Barley Stripe Mosaic Virus (BSMV) Induced MicroRNA Silencing in Common Wheat (Triticum aestivum L.). PLoS One. 10 (5), 0126621 (2015).
  45. Jian, C., et al. Virus-Based MicroRNA Silencing and Overexpressing in Common Wheat (Triticum aestivum L.). Frontiers in Plant Science. 8, 500 (2017).
  46. Barrell, P. J., Meiyalaghan, S., Jacobs, J. M. E., Conner, A. J. Applications of biotechnology and genomics in potato improvement. Plant Biotechnology Journal. 11 (8), 907-920 (2013).
  47. Peng, T., et al. A Resource for Inactivation of MicroRNAs Using Short Tandem Target Mimic Technology in Model and Crop Plants. Molecular Plant. 11 (11), 1400-1417 (2018).
  48. Teotia, S., Zhang, D., Tang, G., Kaufmann, M., Klinger, C., Savelsbergh, A. . Functional Genomics: Methods and Protocols. , 337-349 (2017).
  49. Dommes, A. B., Herbert, D. B., Kivivirta, K. I., Gross, T., Becker, A. Virus-induced gene silencing: empowering genetics in non-model organisms. Journal of Experimental Botany. 70 (3), 757-770 (2018).
  50. Lacomme, C., Chapman, S. Use of Potato Virus X (PVX)-Based Vectors for Gene Expression and Virus-Induced Gene Silencing (VIGS). Current Protocols in Microbiology. 8 (1), 11-16 (2008).
  51. Lim, H. -. S., et al. Efficiency of VIGS and gene expression in a novel bipartite potexvirus vector delivery system as a function of strength of TGB1 silencing suppression. Virology. 402 (1), 149-163 (2010).
  52. Gleba, Y., Klimyuk, V., Marillonnet, S. Viral vectors for the expression of proteins in plants. Current Opinion in Biotechnology. 18 (2), 134-141 (2007).
  53. Tang, G., et al. Construction of short tandem target mimic (STTM) to block the functions of plant and animal microRNAs. Methods. 58 (2), 118-125 (2012).
  54. Kozomara, A., Griffiths-Jones, S. miRBase: integrating microRNA annotation and deep-sequencing data. Nucleic Acids Research. 39, 152-157 (2010).
  55. Kozomara, A., Griffiths-Jones, S. miRBase: annotating high confidence microRNAs using deep sequencing data. Nucleic Acids Research. 42 (1), 68-73 (2013).
  56. Griffiths-Jones, S. The microRNA Registry. Nucleic Acids Research. 32, 109-111 (2004).
  57. Griffiths-Jones, S., Grocock, R. J., van Dongen, S., Bateman, A., Enright, A. J. miRBase: microRNA sequences, targets and gene nomenclature. Nucleic Acids Research. 34, 140-144 (2006).
  58. Griffiths-Jones, S., Saini, H. K., van Dongen, S., Enright, A. J. miRBase: tools for microRNA genomics. Nucleic Acids Research. 36, 154-158 (2007).
  59. Kozomara, A., Birgaoanu, M., Griffiths-Jones, S. miRBase: from microRNA sequences to function. Nucleic Acids Research. 47 (1), 155-162 (2018).
  60. Yin, K., Tang, Y., Zhao, J. Genome-wide characterization of miRNAs involved in N Gene-mediated Immunity in response to tobacco mosaic virus in Nicotiana benthamiana. Evolutionary Bioinformatics. , 1-11 (2015).
  61. Dunker, F., et al. Oomycete small RNAs invade the plant RNA-induced silencing complex for virulence. bioRxiv. , 689190 (2019).
  62. Green, M. R., Sambrook, J. . Molecular Cloning. A Laboratory Mannual 4th. , (2014).
  63. Sambrook, J., Russell, D. . Molecular Cloning: A Laboratory Manual. 3rd Edition. , (2001).
  64. Anderson, S., et al. Sequence and organization of the human mitochondrial genome. Nature. 290 (5806), 457-465 (1981).
  65. Sanger, F., Nicklen, S., Coulson, A. R. DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences. 74 (12), 5463-5467 (1977).
  66. Qian, L., et al. Hsp90 Interacts With Tm-22 and Is Essential for Tm-22-Mediated Resistance to Tobacco mosaic virus. Frontiers in Plant Science. 9 (411), (2018).
  67. Voinnet, O., Baulcombe, D. C. Systemic signalling in gene silencing. Nature. 389 (6651), 553 (1997).
  68. Li, C., et al. A cis Element within Flowering Locus T mRNA Determines Its Mobility and Facilitates Trafficking of Heterologous Viral RNA. Journal of Virology. 83 (8), 3540-3548 (2009).
  69. Chen, C., et al. Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Research. 33 (20), 179 (2005).
  70. Varkonyi-Gasic, E., Hellens, R. P., Kodama, H., Komamine, A. . RNAi and Plant Gene Function Analysis: Methods and Protocols. , 145-157 (2011).
  71. Varkonyi-Gasic, E., Wu, R., Wood, M., Walton, E. F., Hellens, R. P. Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs. Plant Methods. 3 (1), 12 (2007).
  72. Varkonyi-Gasic, E., Kovalchuk, I. . Plant Epigenetics: Methods and Protocols. , 163-175 (2017).
  73. Czimmerer, Z., et al. A Versatile Method to Design Stem-Loop Primer-Based Quantitative PCR Assays for Detecting Small Regulatory RNA Molecules. PLoS One. 8 (1), 55168 (2013).
  74. Dai, X., Zhuang, Z., Zhao, P. X. psRNATarget: a plant small RNA target analysis server (2017 release). Nucleic Acids Research. 46 (1), 49-54 (2018).
  75. Untergasser, A., et al. Primer3-new capabilities and interfaces. Nucleic Acids Research. 40 (15), 115 (2012).
  76. Livak, K. J., Schmittgen, T. D. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2−ΔΔCT Method. Methods. 25 (4), 402-408 (2001).
  77. Schmittgen, T. D., Livak, K. J. Analyzing real-time PCR data by the comparative CT method. Nature Protocols. 3 (6), 1101-1108 (2008).
  78. Todesco, M., Rubio-Somoza, I., Paz-Ares, J., Weigel, D. A Collection of Target Mimics for Comprehensive Analysis of MicroRNA Function in Arabidopsis thaliana. PLoS Genetics. 6 (7), 1001031 (2010).
  79. Franco-Zorrilla, J. M., et al. Target mimicry provides a new mechanism for regulation of microRNA activity. Nature Genetics. 39 (8), 1033-1037 (2007).
  80. Jiang, N., et al. Tomato lncRNA23468 functions as a competing endogenous RNA to modulate NBS-LRR genes by decoying miR482b in the tomato-Phytophthora infestans interaction. Horticulture Research. 6 (1), 28 (2019).
  81. Ivashuta, S., et al. Regulation of gene expression in plants through miRNA inactivation. PLoS One. 6 (6), 21330 (2011).
  82. Reichel, M., Li, Y., Li, J., Millar, A. A. Inhibiting plant microRNA activity: molecular SPONGEs, target MIMICs and STTMs all display variable efficacies against target microRNAs. Plant Biotechnology Journal. 13 (7), 915-926 (2015).
  83. Wong, G., Alonso-Peral, M., Li, B., Li, J., Millar, A. A. MicroRNA MIMIC binding sites: Minor flanking nucleotide alterations can strongly impact MIMIC silencing efficacy in Arabidopsis. Plant Direct. 2 (10), 00088 (2018).
  84. Paschoal, A. R., Lozada-Chávez, I., Domingues, D. S., Stadler, P. F. ceRNAs in plants: computational approaches and associated challenges for target mimic research. Briefings in Bioinformatics. 19 (6), 1273-1289 (2018).
  85. Faivre-Rampant, O., et al. Potato Virus X-Induced Gene Silencing in Leaves and Tubers of Potato. Plant Physiology. 134 (4), 1308-1316 (2004).
  86. Zhao, J., et al. Virus-Induced Gene Silencing in Diploid and Tetraploid Potata Species. Methods in Molecular Biology. , (2019).
  87. Leisner, C. P., et al. Genome sequence of M6, a diploid inbred clone of the high-glycoalkaloid-producing tuber-bearing potato species Solanum chacoense, reveals residual heterozygosity. The Plant Journal. 94 (3), 562-570 (2018).
  88. Aversano, R., et al. The Solanum commersonii Genome Sequence Provides Insights into Adaptation to Stress Conditions and Genome Evolution of Wild Potato Relatives. The Plant Cell. 27 (4), 954-968 (2015).
  89. The Potato Genome Sequencing, C. et al. Genome sequence and analysis of the tuber crop potato. Nature. 475, 189 (2011).
  90. Navarro, C., et al. Control of flowering and storage organ formation in potato by FLOWERING LOCUS T. Nature. 478 (7367), 119-122 (2011).
  91. Lehretz, G. G., Sonnewald, S., Hornyik, C., Corral, J. M., Sonnewald, U. Post-transcriptional Regulation of FLOWERING LOCUS T Modulates Heat-Dependent Source-Sink Development in Potato. Current Biology. 29 (10), 1614-1624 (2019).
  92. Natarajan, B., et al. MiRNA160 is associated with local defense and systemic acquired resistance against Phytophthora infestans infection in potato. Journal of Experimental Botany. 69 (8), 2023-2036 (2018).
  93. Li, F., et al. MicroRNA regulation of plant innate immune receptors. Proceedings of the National Academy of Sciences. 109 (5), 1790-1795 (2012).
  94. Weiberg, A., et al. Fungal Small RNAs Suppress Plant Immunity by Hijacking Host RNA Interference Pathways. Science. 342 (6154), 118-123 (2013).
  95. Huang, C. -. Y., Wang, H., Hu, P., Hamby, R., Jin, H. Small RNAs – Big Players in Plant-Microbe Interactions. Cell Host, Microbe. 26 (2), 173-182 (2019).
  96. Shahid, S., et al. MicroRNAs from the parasitic plant Cuscuta campestris target host messenger RNAs. Nature. 553 (7686), 82-85 (2018).
  97. Weiberg, A., Jin, H. Small RNAs-the secret agents in the plant-pathogen interactions. Current Opinion in Plant Biology. 26, 87-94 (2015).

Tags

Potato Virus XMicroRNA SilencingVbMSVirus-based MicroRNA SilencingMiRNAFunctional CharacterizationShort Tandem Target MimicPVXLigation Independent Cloning

Play Video
PDF
DOI
DOWNLOAD MATERIALS LIST
Cite This Article
Zhao, J., Rios, C. G., Song, J. Potato Virus X-Based microRNA Silencing (VbMS) In Potato.. J. Vis. Exp. (159), e61067, doi:10.3791/61067 (2020).
Download Citation
Reprints and Permissions

View Video

✖

To prove you're not a robot, please enter the text in the image below

CAPTCHA Image
Play CAPTCHA Audio
Refresh Image