Skip to main content

Advertisement

SpringerLink
Log in

Effect of mutations in the 2b protein of tomato aspermy virus on RNA silencing suppressor activity, virulence, and virus-induced gene silencing

  • Original Article
  • Published:
Archives of Virology Aims and scope Submit manuscript

Abstract

Tomato aspermy virus (TAV) and cucumber mosaic virus (CMV) belong to the genus Cucumovirus in the family Bromoviridae. The cucumovirus 2b protein is one of the first identified viral suppressors of RNA silencing (VSR). The cucumovirus 2b protein contains a conserved amino acid motif consisting of several highly conserved amino acid residues. Here, we demonstrate that the TAV 2b protein N-terminal region, Arg46, Ser40, and Ser42 as well as the CMV 2b protein are essential for VSR activity, virulence, and viral RNA accumulation. Furthermore, we developed the first TAV-induced gene silencing (VIGS) vector. In contrast to other cucumovirus vectors, such as CMV and peanut stunt virus, the TAV vector did not induce a silencing phenotype in Nicotiana benthamiana when 2b protein VSR activity was retained. These findings suggest that the cucumovirus 2b proteins share amino acid residues for VSR activity but may have different roles in VIGS induction.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. Palukaitis P, García-Arenal F (2003) Cucumoviruses. Adv Virus Res 62:241–323. https://doi.org/10.1016/S0065-3527(03)62005-1

    Article  CAS  PubMed  Google Scholar 

  2. Hayes RJ, Buck KW (1990) Complete replication of a eukaryotic virus RNA in vitro by a purified RNA-dependent RNA polymerase. Cell 63:363–368. https://doi.org/10.1016/0092-8674(90)90169-F

    Article  CAS  PubMed  Google Scholar 

  3. Ding S-W, Li W-X, Symons RH (1995) A novel naturally occurring hybrid gene encoded by a plant RNA virus facilitates long distance virus movement. EMBO J 14:5762–5772. https://doi.org/10.1002/j.1460-2075.1995.tb00265.x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Schwinghamer MW, Symons RH (1975) Fractionation of cucumber mosaic virus RNA and its translation in a wheat embryo cell-free system. Virology 63:252–262. https://doi.org/10.1016/0042-6822(75)90389-X

    Article  CAS  PubMed  Google Scholar 

  5. Shi B, Ding S, Symons RH (1997) Two novel subgenomic RNAs derived from RNA 3 of tomato aspermy cucumovirus. J Gen Virol 78:505–510. https://doi.org/10.1099/0022-1317-78-3-505

    Article  CAS  PubMed  Google Scholar 

  6. Baulcombe D (2004) RNA silencing in plants. Nature 431:356–363. https://doi.org/10.1038/nature02874

    Article  CAS  PubMed  Google Scholar 

  7. Csorba T, Kontra L, Burgyán J (2015) viral silencing suppressors: tools forged to fine-tune host-pathogen coexistence. Virology 479–480:85–103. https://doi.org/10.1016/j.virol.2015.02.028

    Article  CAS  PubMed  Google Scholar 

  8. Guo HS, Ding SW (2002) A viral protein inhibits the long range signaling activity of the gene silencing signal. EMBO J 21:398–407. https://doi.org/10.1093/emboj/21.3.398

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Goto K, Kobori T, Kosaka Y et al (2007) Characterization of silencing suppressor 2b of cucumber mosaic virus based on examination of its small RNA-binding abilities. Plant Cell Physiol 48:1050–1060. https://doi.org/10.1093/pcp/pcm074

    Article  CAS  PubMed  Google Scholar 

  10. Zhang X, Yuan Y-R, Pei Y et al (2006) Cucumber mosaic virus-encoded 2b suppressor inhibits Arabidopsis Argonaute1 cleavage activity to counter plant defense. Genes Dev 20:3255–3268. https://doi.org/10.1101/gad.1495506

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Duan C-G, Fang Y-Y, Zhou B-J et al (2012) Suppression of Arabidopsis ARGONAUTE1-mediated slicing, transgene-induced RNA silencing, and DNA methylation by distinct domains of the cucumber mosaic virus 2b Protein. Plant Cell 24:259–274. https://doi.org/10.1105/tpc.111.092718

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Cao X, Zhou P, Zhang X et al (2005) Identification of an RNA silencing suppressor from a plant double-stranded RNA virus. JVI 79:13018–13027. https://doi.org/10.1128/JVI.79.20.13018-13027.2005

    Article  CAS  Google Scholar 

  13. Chen H-Y, Yang J, Lin C, Yuan YA (2008) Structural basis for RNA-silencing suppression by tomato aspermy virus protein 2b. EMBO Rep 9:754–760. https://doi.org/10.1038/embor.2008.118

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Zhang X, Du P, Lu L et al (2008) Contrasting effects of HC-Pro and 2b viral suppressors from sugarcane mosaic virus and tomato aspermy cucumovirus on the accumulation of siRNAs. Virology 374:351–360. https://doi.org/10.1016/j.virol.2007.12.045

    Article  CAS  PubMed  Google Scholar 

  15. Ding S-W, Anderson BJ, Haase HR, Symons RH (1994) New overlapping gene encoded by the cucumber mosaic virus genome. Virology 198:593–601. https://doi.org/10.1006/viro.1994.1071

    Article  CAS  PubMed  Google Scholar 

  16. Kobori T, Ryang B-S, Natsuaki T, Kosaka Y (2005) A new technique to select mild strains of cucumber mosaic virus. Plant Dis 89:879–882. https://doi.org/10.1094/PD-89-0879

    Article  PubMed  Google Scholar 

  17. Mochizuki T, Yamazaki R, Wada T, Ohki ST (2014) Coat protein mutations in an attenuated cucumber mosaic virus encoding mutant 2b protein that lacks RNA silencing suppressor activity induces chlorosis with photosynthesis gene repression and chloroplast abnormalities in infected tobacco plants. Virology 456–457:292–299. https://doi.org/10.1016/j.virol.2014.04.010

    Article  CAS  PubMed  Google Scholar 

  18. Nemes K, Gellért Á, Almási A et al (2017) Phosphorylation regulates the subcellular localization of cucumber mosaic virus 2b protein. Sci Rep 7:13444. https://doi.org/10.1038/s41598-017-13870-7

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Nemes K, Gellért Á, Balázs E, Salánki K (2014) Alanine scanning of cucumber mosaic virus (CMV) 2B protein identifies different positions for cell-to-cell movement and gene silencing suppressor activity. PLoS ONE 9:e112095. https://doi.org/10.1371/journal.pone.0112095

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Burch-Smith TM, Anderson JC, Martin GB, Dinesh-Kumar SP (2004) Applications and advantages of virus-induced gene silencing for gene function studies in plants. Plant J 39:734–746. https://doi.org/10.1111/j.1365-313X.2004.02158.x

    Article  CAS  PubMed  Google Scholar 

  21. Hong JS, Rhee S-J, Kim E-J et al (2012) Application of a reassortant cucumber mosaic virus vector for gene silencing in tomato and chili pepper plants. Plant Pathol J 28:81–86. https://doi.org/10.5423/PPJ.NT.11.2011.0220

    Article  CAS  Google Scholar 

  22. Kim H, Onodera Y, Masuta C (2020) Application of cucumber mosaic virus to efficient induction and long-term maintenance of virus-induced gene silencing in spinach. Plant Biotechnol 37:83–88. https://doi.org/10.5511/plantbiotechnology.19.1227a

    Article  CAS  Google Scholar 

  23. Nagamatsu A, Masuta C, Senda M et al (2007) Functional analysis of soybean genes involved in flavonoid biosynthesis by virus-induced gene silencing. Plant Biotechnol J 5:778–790

    Article  CAS  Google Scholar 

  24. Otagaki S, Arai M, Takahashi A et al (2006) Rapid induction of transcriptional and post-transcriptional gene silencing using a novel cucumber mosaic virus vector. Plant Biotechnol 23:259–265. https://doi.org/10.5511/plantbiotechnology.23.259

    Article  CAS  Google Scholar 

  25. Tanase K, Matsushita Y, Mochizuki T (2019) Silencing of the Chalcone synthase gene by a virus vector derived from the cucumber mosaic virus in petunia. Hortic J 88:507–513. https://doi.org/10.2503/hortj.UTD-078

    Article  CAS  Google Scholar 

  26. Tasaki K, Terada H, Masuta C, Yamagishi M (2016) Virus-induced gene silencing (VIGS) in Lilium leichtlinii using the cucumber mosaic virus vector. Plant Biotechnol (Tokyo) 33:373–381. https://doi.org/10.5511/plantbiotechnology.16.1018a

    Article  CAS  Google Scholar 

  27. Tzean Y, Lee M-C, Jan H-H et al (2019) Cucumber mosaic virus-induced gene silencing in banana. Sci Rep 9:1–9. https://doi.org/10.1038/s41598-019-47962-3

    Article  CAS  Google Scholar 

  28. Yamagishi M, Masuta C, Suzuki M, Netsu O (2015) Peanut stunt virus-induced gene silencing in white lupin (Lupinus albus). Plant Biotechnology 32:181–191. https://doi.org/10.5511/plantbiotechnology.15.0521a

    Article  CAS  Google Scholar 

  29. Deleris A, Gallego-Bartolome J, Bao J et al (2006) Hierarchical action and inhibition of plant dicer-like proteins in antiviral defense. Science 313:68–71. https://doi.org/10.1126/science.1128214

    Article  CAS  PubMed  Google Scholar 

  30. Harries PA, Palanichelvam K, Bhat S, Nelson RS (2008) Tobacco mosaic virus 126-kDa protein increases the susceptibility of Nicotiana tabacum to other viruses and its dosage affects virus-induced gene silencing. Mol Plant Microbe Interact 21:1539–1548. https://doi.org/10.1094/MPMI-21-12-1539

    Article  CAS  PubMed  Google Scholar 

  31. Lim H-S, Vaira AM, Domier LL et al (2010) Efficiency of VIGS and gene expression in a novel bipartite potexvirus vector delivery system as a function of strength of TGB1 silencing suppression. Virology 402:149–163. https://doi.org/10.1016/j.virol.2010.03.022

    Article  CAS  PubMed  Google Scholar 

  32. Qiu W, Park J-W, Scholthof HB (2002) Tombusvirus P19-mediated suppression of virus-induced gene silencing is controlled by genetic and dosage features that influence pathogenicity. MPMI 15:269–280. https://doi.org/10.1094/MPMI.2002.15.3.269

    Article  CAS  PubMed  Google Scholar 

  33. Inoue S, Tamura M, Ugaki M, Suzuki M (2018) Complete genome sequences of three tomato aspermy virus isolates in Japan. Genome Announc. https://doi.org/10.1128/genomeA.00474-18

    Article  PubMed  PubMed Central  Google Scholar 

  34. Maddahian M, Massumi H, Heydarnejad J et al (2017) Characterization of Iranian tomato aspermy virus isolates with a variant 2b gene sequence. Trop Plant Pathol 42:475–484. https://doi.org/10.1007/s40858-017-0173-1

    Article  Google Scholar 

  35. Zhao XT, Liu XX (2015) Hong B (2015) Characterization of tomato aspermy virus isolated from chrysanthemum and elucidation of its complete nucleotide sequence. Acta Virol 59:204–206. https://doi.org/10.4149/av_2015_02_204

    Article  CAS  PubMed  Google Scholar 

  36. Inouye T, Asatani M, Mitsuhata K (1968) Studies on the viruses of plants in Compositae in Japan I. Tomato aspermy virus from chrysanthemum. Nogaku-kenkyu 52:55–64

    Google Scholar 

  37. Christie SR, Purcifull DE, Crawford WE, Ahmed NA (1987) Electron microscopy of negatively stained clarified viral concentrates obtained from small tissue samples with appendices on negative staining techniques. Bull Agric Exp Stn Univ Fla 872:1–45

    Google Scholar 

  38. Choi SK, Choi JK, Ryu KH, Park WM (2002) Generation of infectious cDNA clones of a Korean strain of tomato aspermy virus. Mol Cells 13:52–60

    CAS  PubMed  Google Scholar 

  39. Lindbo JA (2007) TRBO: a high-efficiency tobacco mosaic virus RNA-based overexpression vector. Plant Physiol 145:1232–1240. https://doi.org/10.1104/pp.107.106377

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Kawakami S, Watanabe Y (1997) Use of green fluorescent protein as a molecular tag of protein movement in vivo. Plant Biotechnol 14:127–130. https://doi.org/10.5511/plantbiotechnology.14.127

    Article  CAS  Google Scholar 

  41. Mochizuki T, Hirai K, Kanda A et al (2009) Induction of necrosis via mitochondrial targeting of Melon necrotic spot virus replication protein p29 by its second transmembrane domain. Virology 390:239–249. https://doi.org/10.1016/j.virol.2009.05.012

    Article  CAS  PubMed  Google Scholar 

  42. Jyothishwaran G, Kotresha D, Selvaraj T et al (2007) A modified freeze–thaw method for efficient transformation of Agrobacterium tumefaciens. Curr Sci 93:770–772

    CAS  Google Scholar 

  43. Von Arnim A, Frischmuth T, Stanley J (1993) Detection and possible functions of African cassava mosaic virus DNA B gene products. Virology 192:264–272. https://doi.org/10.1006/viro.1993.1029

    Article  Google Scholar 

  44. Symons RH, Shi BJ, Ding SW (1997) In vivo expression of an overlapping gene encoded by the cucumoviruses. J Gen Virol 78:237–241. https://doi.org/10.1099/0022-1317-78-1-237

    Article  PubMed  Google Scholar 

  45. González I, Martínez L, Rakitina DV et al (2010) Cucumber mosaic virus 2b protein subcellular targets and interactions: their significance to RNA silencing suppressor activity. Mol Plant Microbe Interact 23:294–303

    Article  Google Scholar 

  46. Rao ALN, Francki RIB (1981) Comparative studies on tomato aspermy and cucumber mosaic viruses. VI. Partial compatibility of genome segments from the two viruses. Virology 114:573–575. https://doi.org/10.1016/0042-6822(81)90238-5

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We are grateful to Dr. Kenji Kubota for his technical assistance on the VSR activity assay.

Author information

Authors and Affiliations

  1. Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1, Gakuen-cho, Naka-ku, Sakai, Osaka, 599-8531, Japan

    Hirotomo Murai, Kenta Atsumaru & Tomofumi Mochizuki

Authors
  1. Hirotomo Murai
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kenta Atsumaru
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Tomofumi Mochizuki
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Tomofumi Mochizuki.

Ethics declarations

Conflict of interest

All of the authors declare that they have no conflicts of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Additional information

Handling Editor: Ralf Georg Dietzgen.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (PDF 6615 KB)

Supplementary file2 (PDF 79 KB)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Murai, H., Atsumaru, K. & Mochizuki, T. Effect of mutations in the 2b protein of tomato aspermy virus on RNA silencing suppressor activity, virulence, and virus-induced gene silencing. Arch Virol 167, 471–481 (2022). https://doi.org/10.1007/s00705-021-05344-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00705-021-05344-z

Search

Navigation