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Rapid Screening of Myzus persicae (Green Peach Aphid) RNAi Targets Using Tobacco Rattle Virus

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RNAi Strategies for Pest Management

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2360))

Abstract

Plant-mediated RNA interference (RNAi) can be used to reduce the growth of insect pests, including Myzus persicae (green peach aphid), a prolific pest of numerous dicot crop species. In one approach, viruses that have been engineered to carry an aphid gene fragment are used to infect plants and thereby silence target gene expression in the aphids feeding on these plants, a process called virus-induced gene silencing, or VIGS. Tobacco Rattle Virus (TRV) in the model plant, Nicotiana benthamiana, was the first of many VIGS systems that have been developed for different plant species. In this chapter, we describe a method for silencing M. persicae gene expression using an established TRV-VIGS vector that infects and spreads in N. benthamiana. The two parts of the TRV genome, RNA1 and RNA2, have been cloned into Agrobacterium T-DNA vectors for initiation of plant infections. The RNA2 construct is modified with a Gateway-compatible cloning site to allow insertion of aphid genes. When feeding on TRV-infected N. benthamiana plants, aphids ingest dsRNAs that silence specific target genes. TRV-VIGS of aphid genes allows rapid identification of essential gene targets that can be used for the control of M. persicae by this and other RNAi methods.

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References

  1. Baum JA, Bogaert T, Clinton W et al (2007) Control of coleopteran insect pests through RNA interference. Nat Biotechnol 25(11):1322–1326. https://doi.org/10.1038/nbt1359

    Article  CAS  PubMed  Google Scholar 

  2. Mao YB, Cai WJ, Wang JW et al (2007) Silencing a cotton bollworm P450 monooxygenase gene by plant-mediated RNAi impairs larval tolerance of gossypol. Nat Biotechnol 25(11):1307–1313. https://doi.org/10.1038/nbt1352

    Article  CAS  PubMed  Google Scholar 

  3. Blackman RL, Eastop VF (2000) Aphids on the World’s crops. Wiley, Chichester

    Google Scholar 

  4. Pitino M, Coleman AD, Maffei ME et al (2011) Silencing of aphid genes by dsRNA feeding from plants. PLos One 6(10):e25709. https://doi.org/10.1371/journal.pone.0025709

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Pitino M, Hogenhout SA (2013) Aphid protein effectors promote aphid colonization in a plant species-specific manner. Mol Plant Microbe Interact 26(1):130–139. https://doi.org/10.1094/Mpmi-07-12-0172-Fi

    Article  CAS  PubMed  Google Scholar 

  6. Elzinga DA, De Vos M, Jander G (2014) Suppression of plant defenses by a Myzus persicae (green peach aphid) salivary effector protein. Mol Plant-Microbe Interact 27(7):747–756. https://doi.org/10.1094/MPMI-01-14-0018-R

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Naessens E, Dubreuil G, Giordanengo P et al (2015) A secreted MIF cytokine enables aphid feeding and represses plant immune responses. Curr Biol 25(14):1898–1903. https://doi.org/10.1016/j.cub.2015.05.047

    Article  CAS  PubMed  Google Scholar 

  8. Tzin V, Yang X, Jing X et al (2015) RNA interference against gut osmoregulatory genes in phloem-feeding insects. J Insect Physiol 79:105–112. https://doi.org/10.1016/j.jinsphys.2015.06.006

    Article  CAS  PubMed  Google Scholar 

  9. Rauf I, Asif M, Amin I et al (2019) Silencing cathepsin L expression reduces Myzus persicae protein content and the nutritional value as prey for Coccinella septempunctata. Insect Mol Biol 28(6):785–797. https://doi.org/10.1111/imb.12589

    Article  CAS  PubMed  Google Scholar 

  10. Guo HY, Song XG, Wang GL et al (2014) Plant-generated artificial small RNAs mediated aphid resistance. PLoS One 9(5):e97410. https://doi.org/10.1371/journal.pone.0097410

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Bhatia V, Bhattacharya R, Uniyal PL et al (2012) Host generated siRNAs attenuate expression of serine protease gene in Myzus persicae. PLoS One 7(10):e46343. https://doi.org/10.1371/journal.pone.0046343

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Mao JJ, Zeng FR (2014) Plant-mediated RNAi of a gap gene-enhanced tobacco tolerance against the Myzus persicae. Transgenic Res 23(1):145–152. https://doi.org/10.1007/s11248-013-9739-y

    Article  CAS  PubMed  Google Scholar 

  13. Faisal M, Abdel-Salam EM, Alatar AA et al (2019) Genetic transformation and siRNA-mediated gene silencing for aphid resistance in tomato. Agronomy 9(12):893. https://doi.org/10.3390/agronomy9120893

    Article  CAS  Google Scholar 

  14. Hayward A, Padmanabhan M, Dinesh-Kumar SP (2011) Virus-induced gene silencing in Nicotiana benthamiana and other plant species. Methods Mol Biol 678:55–63. https://doi.org/10.1007/978-1-60761-682-5_5

    Article  CAS  PubMed  Google Scholar 

  15. Senthil-Kumar M, Mysore KS (2014) Tobacco rattle virus-based virus-induced gene silencing in Nicotiana benthamiana. Nat Protoc 9(7):1549–1562. https://doi.org/10.1038/nprot.2014.092

    Article  CAS  PubMed  Google Scholar 

  16. MacFarlane SA (1999) Molecular biology of the tobraviruses. J Gen Virol 80:2799–2807. https://doi.org/10.1099/0022-1317-80-11-2799

    Article  CAS  PubMed  Google Scholar 

  17. Ratcliff F, Martin-Hernandez AM, Baulcombe DC (2001) Tobacco rattle virus as a vector for analysis of gene function by silencing. Plant J 25(2):237–245. https://doi.org/10.1046/j.0960-7412.2000.00942.x

    Article  CAS  PubMed  Google Scholar 

  18. Liu YL, Schiff M, Dinesh-Kumar SP (2002) Virus-induced gene silencing in tomato. Plant J 31(6):777–786. https://doi.org/10.1046/j.1365-313X.2002.01394.x

    Article  CAS  PubMed  Google Scholar 

  19. Senthil-Kumar M, Hema R, Anand A et al (2007) A systematic study to determine the extent of gene silencing in Nicotiana benthamiana and other Solanaceae species when heterologous gene sequences are used for virus-induced gene silencing. New Phytol 176(4):782–791. https://doi.org/10.1111/j.1469-8137.2007.02225.x

    Article  CAS  PubMed  Google Scholar 

  20. Mulot M, Boissinot S, Monsion B et al (2016) Comparative analysis of RNAi-based methods to down-regulate expression of two genes expressed at different levels in Myzus persicae. Viruses 8(11):316. https://doi.org/10.3390/v8110316

    Article  CAS  PubMed Central  Google Scholar 

  21. Ryu CM, Anand A, Kang L et al (2004) Agrodrench: a novel and effective agroinoculation method for virus-induced gene silencing in roots and diverse Solanaceous species. Plant J 40(2):322–331. https://doi.org/10.1111/j.1365-313X.2004.02211.x

    Article  CAS  PubMed  Google Scholar 

  22. Mathers TC, Chen Y, Kaithakottil G et al (2017) Rapid transcriptional plasticity of duplicated gene clusters enables a clonally reproducing aphid to colonise diverse plant species. Genome Biol 18(1):27. https://doi.org/10.1186/s13059-016-1145-3

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(T)(-Delta Delta C) method. Methods 25(4):402–408. https://doi.org/10.1006/meth.2001.1262

    Article  CAS  PubMed  Google Scholar 

  24. Ramsey JS, Elzinga DA, Sarkar P et al (2014) Adaptation to nicotine feeding in Myzus persicae. J Chem Ecol 40:869–877. https://doi.org/10.1007/s10886-014-0482-5

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Ramsey JS, Wilson AC, De Vos M et al (2007) Genomic resources for Myzus persicae: EST sequencing, SNP identification, and microarray design. BMC Genomics 8:423

    Article  PubMed  PubMed Central  Google Scholar 

  26. Feng H, Acosta-Gamboa L, Kruse LH, et al. (2020) An improved Nicotiana benthamiana strain for aphid and whitefly research. bioRxiv. https://doi.org/10.1101/2020.08.04.237180

  27. Schneider CA, Rasband WS, Eliceiri KW (2012) NIH image to ImageJ: 25 years of image analysis. Nat Methods 9(7):671–675. https://doi.org/10.1038/nmeth.2089

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

We thank Mariko Alexander and Michelle Heck for sharing of a TRV1 Agrobacterium culture, and Robyn Roberts and Greg Martin for providing TRV2-PDS control construct. Thanks to the previous and current members of the Jander lab for their help in developing the aphid TRV-VIGS protocol. This research was supported by US Department of Agriculture Biotechnology Risk Assessment Grant 2017-33522-27006 to G. J.

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Authors and Affiliations

  1. Boyce Thompson Institute, Ithaca, NY, USA

    Honglin Feng & Georg Jander

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  1. Honglin Feng
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  2. Georg Jander
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Correspondence to Georg Jander .

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Editors and Affiliations

  1. Oncativo, Argentina

    Luis María Vaschetto

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© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Feng, H., Jander, G. (2022). Rapid Screening of Myzus persicae (Green Peach Aphid) RNAi Targets Using Tobacco Rattle Virus. In: Vaschetto, L.M. (eds) RNAi Strategies for Pest Management. Methods in Molecular Biology, vol 2360. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1633-8_10

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  • DOI: https://doi.org/10.1007/978-1-0716-1633-8_10

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1632-1

  • Online ISBN: 978-1-0716-1633-8

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