Virus Genes

, Volume 56, Issue 1, pp 67–77 | Cite as

Estimation of the functions of viral RNA silencing suppressors by apple latent spherical virus vector

  • Chunjiang Li
  • Makoto Ito
  • Ichiro Kasajima
  • Nobuyuki YoshikawaEmail author
Original Paper


Apple latent spherical virus (ALSV) is a latent virus with wide host range of plant species. In the present study, we prepared ALSV vectors expressing RNA silencing suppressors (RSSs) from eight plant viruses: P19 of carnation Italian ring spot virus (tombusvirus), 2b of peanut stunt virus (cucumovirus), NSs of tomato spotted wilt virus (tospovirus), HC-Pro of bean yellow mosaic virus (potyvirus), γb of barley stripe mosaic virus (hordeivirus), P15 of peanut clump virus (pecluvirus), P1 of rice yellow mottle virus (sobemovirus), or P21 of beet yellows virus (closterovirus). These vectors were inoculated to Nicotiana benthamiana to investigate the effects of RSSs on the virulence and accumulation of ALSV. Among the vectors, ALSV expressing NSs (ALSV-NSs) developed severe mosaic symptoms in newly developed leaves followed by plant death. Infection of ALSV-γb induced characteristic concentric ringspot symptoms on leaves, and plants infected with ALSV-HC-Pro showed mosaic and dwarf symptoms. Infection of the other five ALSV vectors did not show symptoms. ELISA and immunoblot assay indicated that virus titer increased in leaves infected with ALSV-NSs, γb, HC-Pro, or P19. RT-qPCR indicated that the amount of ALSV in plants infected with ALSV-NSs was increased by approximately 45 times compared with that of wtALSV without expression of any RSS. When ALSV-P19, NSs, or HC-Pro was inoculated to Cucumis sativus plants, none of these ALSV vectors induced symptoms, but accumulation of ALSV in plants infected with ALSV-NSs was increased, suggesting that functions of RSSs on virulence and accumulation of ALSV depend on host species.


Apple latent spherical virus RNA silencing suppressor Viral symptoms Viral accumulation 



This work was supported by the Programme for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry from the Ministry of Agriculture, Forestry, and Fisheries, Japan

Author contributions

CL and NY designed the study; CL and MI performed the experiments; IK analyzed the data; CL, IK, and NY drafted the manuscript; and all authors approved the final version.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest with respect to the data, authorship, or publication of this article.

Research involving in human and animal participants

This article does not contain any experiments with human participants or animals performed by any of the authors and is in compliance with ethical standards for research.


  1. 1.
    Bhushan L, Abraham A, Choudhury NR, Rana VS, Mukherjee SK, Savithri HS (2015) Demonstration of helicase activity in the nonstructural protein, NSs, of the negative-sense RNA virus, groundnut bud necrosis virus. Arch Virol 160:959–967PubMedGoogle Scholar
  2. 2.
    Bonneau C, Brugidou C, Chen L, Beachy RN, Fauquet C (1998) Expression of the rice yellow mottle virus P1 protein in vitro and in vivo and its involvement in virus spread. Virology 244:79–86PubMedGoogle Scholar
  3. 3.
    Bragg JN, Jackson AO (2004) The C-terminal region of the Barley stripe mosaic virus γb protein participates in homologous interactions and is required for suppression of RNA silencing. Mol Plant Pathol 5:465–481PubMedGoogle Scholar
  4. 4.
    Burgyán J, Hornyik C, Szittya G, Silhavy D, Bisztray G (2000) The ORF1 products of tombusviruses play a crucial role in lethal necrosis of virus-infected plants. J Virol 74:10873–11081PubMedPubMedCentralGoogle Scholar
  5. 5.
    Chapman EJ, Prokhnevsky AI, Gopinath K, Dolja VV, Carrington JC (2004) Viral RNA silencing suppressors inhibit the microRNA pathway at an intermediate step. Genes Dev 18:1179–1186PubMedPubMedCentralGoogle Scholar
  6. 6.
    Chiba M, Reed JC, Prokhnevsky AI, Chapman EJ, Mawassi M, Koonin EV, Carrington JC, Dolja VV (2006) Diverse suppressors of RNA silencing enhance agroinfection by a viral replicon. Virology 346:7–14PubMedGoogle Scholar
  7. 7.
    Csorba T, Kontra L, Burgyán J (2015) Viral silencing suppressors: tools forged to fine-tune host-pathogen coexistence. Virology 479–480:85–103PubMedGoogle Scholar
  8. 8.
    de Ronde D, Pasquier A, Ying S, Butterbach P, Lohuis D, Kormelink R (2014) Analysis of Tomato spotted wilt virus NSs protein indicates the importance of the N-terminal domain for avirulence and RNA silencing suppression. Mol Plant Pathol 15:185–195PubMedGoogle Scholar
  9. 9.
    Dhillon T, Chiera JM, Lindbo JA, Finer JJ (2009) Quantitative evaluation of six different viral suppressors of silencing using image analysis of transient GFP expression. Plant Cell Rep 28:639–647PubMedGoogle Scholar
  10. 10.
    Díaz-Pendón JA, Ding SW (2008) Direct and indirect roles of viral suppressors of RNA silencing in pathogenesis. Annu Rev Phytopathol 46:303–326PubMedGoogle Scholar
  11. 11.
    Donald RG, Jackson AO (1994) The barley stripe mosaic virus gamma b gene encodes a multifunctional cysteine-rich protein that affects pathogenesis. Plant Cell 6:1593–1606PubMedPubMedCentralGoogle Scholar
  12. 12.
    Fagoaga C, Pensabene-Bellavia G, Moreno P, Navarro L, Flores R, Peña L (2011) Ectopic expression of the p23 silencing suppressor of Citrus tristeza virus differentially modifies viral accumulation and tropism in two transgenic woody hosts. Mol Plant Pathol 12:898–910PubMedPubMedCentralGoogle Scholar
  13. 13.
    Hedil M, Sterken MG, de Ronde D, Lohuis D, Kormelink R (2015) Analysis of Tospovirus NSs proteins in suppression of systemic silencing. PLoS ONE 10:e0134517PubMedPubMedCentralGoogle Scholar
  14. 14.
    Huang CH, Hsiao WR, Huang CW, Chen KC, Lin SS, Chen TC, Raja JA, Wu HW, Yeh SD (2015) Two novel motifs of watermelon silver mottle virus NSs protein are responsible for RNA silencing suppression and pathogenicity. PLoS ONE 10:e0126161PubMedPubMedCentralGoogle Scholar
  15. 15.
    Igarashi A, Yamagata K, Sugai T, Takahashi Y, Sugawara E, Tamura A, Yaegashi H, Yamagishi N, Takahashi T, Isogai M, Takahashi H, Yoshikawa N (2009) Apple latent spherical virus vectors for reliable and effective virus-induced gene silencing among a broad range of plants including tobacco, tomato, Arabidopsis thaliana, cucurbits, and legumes. Virology 386:407–416PubMedGoogle Scholar
  16. 16.
    Kasajima I, Ito M, Yamagishi N, Yoshikawa N (2017) Apple latent spherical virus (ALSV) vector as a tool for reverse genetic studies and non-transgenic breeding of a variety of crops. In: Rajewsky N, Jurga S, Barciszewski J (eds) Plant epigenetics. Springer, Cham, pp 513–536Google Scholar
  17. 17.
    Kasschau KD, Cronin S, Carrington JC (1997) Genome amplification and long-distance movement functions associated with the central domain of tobacco etch potyvirus helper component-proteinase. Virology 228:251–262PubMedGoogle Scholar
  18. 18.
    Kasschau KD, Xie Z, Allen E, Llave C, Chapman EJ, Krizan KA, Carrington JC (2003) P1/HC-Pro, a viral suppressor of RNA silencing, interferes with Arabidopsis development and miRNA unction. Dev Cell 4:205–217PubMedGoogle Scholar
  19. 19.
    Kawai T, Gonoi A, Nitta M, Kaido M, Yamagishi N, Yoshikawa N, Tao T (2014) Virus-induced genes silencing in Apricot (Prunus armeniaca L.) and Japanese apricot (P. mume Siebold & Zucc.) with Apple latent spherical virus vector system. J Jpn Soc Hortic Sci 83:23–31Google Scholar
  20. 20.
    Kon T, Yoshikawa N (2014) Induction and maintenance of DNA methylation in plant promoter sequences by apple latent spherical virus-induced transcriptional gene silencing. Front Microbiol 5:595PubMedPubMedCentralGoogle Scholar
  21. 21.
    Lewsey M, Surette M, Robertson FC, Ziebell H, Choi SH, Ryu KH, Canto T, Palukaitis P, Payne T, Walsh JA, Carr JP (2009) The role of the Cucumber mosaic virus 2b protein in viral movement and symptom induction. Mol Plant Microbe Interact 22:642–654PubMedGoogle Scholar
  22. 22.
    Li C, Yoshikawa N, Takahashi T, Ito T, Yoshida K, Koganezawa H (2000) Nucleotide sequence and genome organization of apple latent spherical virus: a new virus classified into the family Comoviridae. J Gen Virol 81:541–547PubMedGoogle Scholar
  23. 23.
    Li C, Sasaki N, Isogai M, Yoshikawa N (2004) Stable expression of foreign proteins in herbaceous and apple plants using Apple latent spherical virus RNA2 vectors. Arch Virol 149:1541–1558PubMedGoogle Scholar
  24. 24.
    Li C, Yamagishi N, Kaido M, Yoshikawa N (2014) Presentation of epitope sequences from foreign viruses on the surface of apple latent spherical virus particles. Virus Res 190:118–126PubMedGoogle Scholar
  25. 25.
    Li C, Yoshikawa N (2015) Virus-induced gene silencing of N gene in tobacco by Apple latent spherical virus vectors. Methods Mol Biol 1236:229–240PubMedGoogle Scholar
  26. 26.
    Lokesh B, Rashmi PR, Amruta BS, Srisathiyanarayanan D, Murthy MR, Savithri HS (2010) NSs encoded by groundnut bud necrosis virus is a bifunctional enzyme. PLoS ONE 5:e9757PubMedPubMedCentralGoogle Scholar
  27. 27.
    Mascia T, Gallitelli D (2016) Synergies and antagonisms in virus interactions. Plant Sci 252:176–192PubMedGoogle Scholar
  28. 28.
    Mlotshwa S, Verver J, Sithole-Niang I, Prins M, van Kammen AB, Wellink J (2002) Transgenic plants expressing HC-Pro show enhanced virus sensitivity while silencing of the transgene results in resistance. Virus Genes 25:45–57PubMedGoogle Scholar
  29. 29.
    Nakamura K, Yamagishi N, Isogai M, Komori S, Ito T, Yoshikawa N (2011) Seed and pollen transmission of Apple latent spherical virus in apple. J Gen Plant Pathol 77:48–53Google Scholar
  30. 30.
    Netsu O, Hiratsuka K, Kuwata S, Hibi T, Ugaki M, Suzuki M (2008) Peanut stunt virus 2b cistron plays a role in viral local and systemic accumulation and virulence in Nicotiana benthamiana. Arch Virol 153:1731–1735PubMedGoogle Scholar
  31. 31.
    Okuda M, Taba S, Hanada K (2003) The S RNA segment determines symptom differences on Tetragonia expansa between two Watermelon silver mottle virus isolates. Physiol Mol Plant Pathol 62:327–332Google Scholar
  32. 32.
    Petty IT, French R, Jones RW, Jackson AO (1990) Identification of barley stripe mosaic virus genes involved in viral RNA replication and systemic movement. EMBO J 9:3453–3457PubMedPubMedCentralGoogle Scholar
  33. 33.
    Pruss G, Ge X, Shi XM, Carrington JC, Vance VB (1997) Plant viral synergism: the potyviral genome encodes a broad-range pathogenicity enhancer that transactivates replication of heterologous viruses. Plant Cell 9:859–868PubMedPubMedCentralGoogle Scholar
  34. 34.
    Pruss GJ, Lawrence CB, Bass T, Li QQ, Bowman LH, Vance V (2004) The potyviral suppressor of RNA silencing confers enhanced resistance to multiple pathogens. Virology 320:107–120PubMedGoogle Scholar
  35. 35.
    Pumplin N, Voinnet O (2013) RNA silencing suppression by plant pathogens: defence, counter-defence and counter-counter-defence. Nat Rev Microbiol 11:745–760PubMedGoogle Scholar
  36. 36.
    Rochow WF, Ross AF (1955) Virus multiplication in plants doubly infected by potato viruses X and Y. Virology 1:10–27PubMedGoogle Scholar
  37. 37.
    Savenkov EI, Valkonen JP (2001) Potyviral helper-component proteinase expressed in transgenic plants enhances titers of Potato leaf roll virus but does not alleviate its phloem limitation. Virology 283:285–293PubMedGoogle Scholar
  38. 38.
    Savenkov EI, Valkonen JP (2002) Silencing of a viral RNA silencing suppressor in transgenic plants. J Gen Virol 83:2325–2335PubMedGoogle Scholar
  39. 39.
    Scholthof HB, Scholthof KB, Jackson AO (1995) Identification of tomato bushy stunt virus host-specific symptom determinants by expression of individual genes from a potato virus X vector. Plant Cell 7:1157–1172PubMedPubMedCentralGoogle Scholar
  40. 40.
    Senanayake DM, Mandal B (2014) Expression of symptoms, viral coat protein and silencing suppressor gene during mixed infection of a N-Wi strain of potato virus Y and an asymptomatic strain of potato virus X. Virus Dis 25:314–321Google Scholar
  41. 41.
    Siddiqui SA, Sarmiento C, Truve E, Lehto H, Lehto K (2008) Phenotypes and functional effects caused by various viral RNA silencing suppressors in transgenic Nicotiana benthamiana and N. tabacum. Mol Plant Microbe Interact 21:178–187PubMedGoogle Scholar
  42. 42.
    Siddiqui SA, Sarmiento C, Kiisma M, Koivumäki S, Lemmetty A, Truve E, Lehto K (2008) Effects of viral silencing suppressors on tobacco ringspot virus infection in two Nicotiana species. J Gen Virol 89:1502–1508PubMedGoogle Scholar
  43. 43.
    Soitamo AJ, Jada B, Lehto K (2011) HC-Pro silencing suppressor significantly alters the gene expression profile in tobacco leaves and flowers. BMC Plant Biol 11:68PubMedPubMedCentralGoogle Scholar
  44. 44.
    Takahashi T, Sugawara T, Yamatsuta T, Isogai M, Natsuaki T, Yoshikawa N (2007) Analysis of the spatial distribution of identical and two distinct virus populations differently labeled with cyan and yellow fluorescent proteins in coinfected plants. Phytopathology 97:1200–1206PubMedGoogle Scholar
  45. 45.
    Takahashi T, Yoshikawa N (2008) Analysis of cell-to-cell and long-distance movement of apple latent spherical virus in infected plants using green, cyan, and yellow fluorescent proteins. Methods Mol Biol 451:545–554PubMedGoogle Scholar
  46. 46.
    Takeda A, Sugiyama K, Nagano H, Mori M, Kaido M, Mise K, Tsuda S, Okuno T (2002) Identification of a novel RNA silencing suppressor, NSs protein of Tomato spotted wilt virus. FEBS Lett 532:75–79PubMedGoogle Scholar
  47. 47.
    Taki A, Yamagishi N, Yoshikawa N (2013) Development of apple latent spherical virus-based vaccines against three tospoviruses. Virus Res 176:251–258PubMedGoogle Scholar
  48. 48.
    Tsuda S, Kubota K, Kanda A, Ohki T, Meshi T (2007) Pathogenicity of Pepper mild mottle virus is controlled by the RNA silencing suppression activity of its replication protein but not the viral accumulation. Phytopathology 97:412–420PubMedGoogle Scholar
  49. 49.
    Valli A, Gallo A, Calvo M, Pérez JJ, García JA (2014) A novel role of the potyviral helper component proteinase contributes to enhance the yield of viral particles. J Virol 88:9808–9818PubMedPubMedCentralGoogle Scholar
  50. 50.
    Vanitharani R, Chellappan P, Pita JS, Fauquet CM (2004) Differential roles of AC2 and AC4 of cassava geminiviruses in mediating synergism and suppression of posttranscriptional gene silencing. J Virol 78:9487–9498PubMedPubMedCentralGoogle Scholar
  51. 51.
    Voinnet O, Pinto YM, Baulcombe DC (1999) Suppression of gene silencing: a general strategy used by diverse DNA and RNA viruses of plants. Proc Natl Acad Sci USA 96:14147–14152PubMedGoogle Scholar
  52. 52.
    Wang MB, Masuta C, Smith NA, Shimura H (2012) RNA silencing and plant viral diseases. Mol Plant Microbe Interact 25:1275–1285PubMedGoogle Scholar
  53. 53.
    Wu HW, Lin SS, Chen KC, Yeh SD, Chua NH (2010) Discriminating mutations of HC-Pro of zucchini yellow mosaic virus with differential effects on small RNA pathways involved in viral pathogenicity and symptom development. Mol Plant Microbe Interact 23:17–28PubMedGoogle Scholar
  54. 54.
    Yaegashi H, Takahashi T, Isogai M, Kobori T, Ohki S, Yoshikawa N (2007) Apple chlorotic leaf spot virus 50 kDa movement protein acts as a suppressor of systemic silencing without interfering with local silencing in Nicotiana benthamiana. J Gen Virol 88:316–324PubMedGoogle Scholar
  55. 55.
    Yaegashi H, Yamatsuta T, Takahashi T, Li C, Isogai M, Kobori T, Ohki S, Yoshikaw N (2007) Characterization of virus-induced gene silencing in tobacco plants infected with apple latent spherical virus. Arch Virol 152:1839–1849PubMedGoogle Scholar
  56. 56.
    Yamagishi N, Yoshikawa N (2009) Virus-induced gene silencing in soybean seeds and the emergence stage of soybean plants with Apple latent spherical virus vectors. Plant Mol Biol 71:15–24PubMedGoogle Scholar
  57. 57.
    Yamagishi N, Sasaki S, Yamagata K, Komori S, Nagase M, Wada M, Yamamoto T, Yoshikawa N (2011) Promotion of flowering and reduction of a generation time in apple seedlings by ectopical expression of the Arabidopsis thaliana FT gene using the Apple latent spherical virus vector. Plant Mol Biol 75:193–204PubMedGoogle Scholar
  58. 58.
    Yamagishi N, Kishigami R, Yoshikawa N (2014) Reduced generation time of apple seedlings to within a year by means of a plant virus vector: a new plant-breeding technique with no transmission of genetic modification to the next generation. Plant Biotechnol J 12:60–68PubMedGoogle Scholar
  59. 59.
    Yelina NE, Savenkov EI, Solovyev AG, Morozov SY, Valkonen JP (2002) Long-distance movement, virulence, and RNA silencing suppression controlled by a single protein in hordei- and potyviruses: complementary functions between virus families. J Virol 76:12981–21291PubMedPubMedCentralGoogle Scholar
  60. 60.
    Yoon JY, Han KS, Park HY, Choi SK (2012) Comparative analysis of RNA silencing suppression activities between viral suppressors and an endogenous plant RNA-dependent RNA polymerase. Virus Genes 44:495–504PubMedGoogle Scholar
  61. 61.
    Yoshikawa N, Sasaki E, Kato M, Takahashi T (1992) The nucleotide sequence of apple stem grooving capillovirus genome. Virology 191:98–105PubMedGoogle Scholar
  62. 62.
    Zhai Y, Bag S, Mitter N, Turina M, Pappu HR (2014) Mutational analysis of two highly conserved motifs in the silencing suppressor encoded by tomato spotted wilt virus (genus Tospovirus, family Bunyaviridae). Arch Virol 159:1499–1504PubMedGoogle Scholar

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Authors and Affiliations

  1. 1.Faculty of AgricultureIwate UniversityMoriokaJapan
  2. 2.Agri-Innovation CenterIwate UniversityMoriokaJapan

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