Skip to main content

Advertisement

SpringerLink
Log in

Seed coat pigmentation in transgenic soybean expressing the silencing suppressor 2b gene of Cucumber mosaic virus

  • Original Paper
  • Published:
Plant Cell Reports Aims and scope Submit manuscript

Abstract

Key message

Soybean expressing the Cucumber mosaic virus 2b gene manifests seed coat pigmentation due to suppression of endogenous RNA silencing but no other morphological abnormality. This gene may help prevent transgene silencing.

Abstract

RNA silencing is an important mechanism for gene regulation and antiviral defense in plants. It is also responsible for transgene silencing, however, and thus hinders the establishment of transgenic plants. The 2b protein of Cucumber mosaic virus (CMV) functions as a suppressor of RNA silencing and therefore might prove beneficial for stabilization of transgene expression. We have now generated transgenic soybean that harbors the 2b gene of a CMV-soybean strain under the control of a constitutive promoter to investigate the effects of 2b expression. No growth abnormality was apparent in 2b transgenic plants, although the seed coat was pigmented in several of the transgenic lines. Genes for chalcone synthase (CHS), a key enzyme of the flavonoid pathway, are posttranscriptionally silenced by the inhibitor (I) locus in nonpigmented (yellow) soybean seeds. The levels of CHS mRNA and CHS small interfering RNA in strongly pigmented 2b transgenic seed coats were higher and lower, respectively, than those in the seed coat of a control transgenic line. The expression level of 2b also correlated with the extent of seed coat pigmentation. On the other hand, introduction of the 2b gene together with the DsRed2 gene into somatic embryos prevented the time-dependent decrease in transient DsRed2 expression. Our results indicate that the 2b gene alone is able to suppress RNA silencing of endogenous CHS genes regulated by the I locus, and that 2b is of potential utility for stabilization of transgene expression in soybean without detrimental effects other than seed coat pigmentation.

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
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Brigneti G, Voinnet O, Li W-X, Ji L-H, Ding S-W, Baulcombe DC (1998) Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana. EMBO J 17:6739–6746

    Article  PubMed  CAS  Google Scholar 

  • Chiera JM, Bouchard RA, Dorsey SL, Park E, Buenrostro-Nava MT, Ling PP, Finer JJ (2007) Isolation of two highly active soybean (Glycine max (L.) Merr.) promoters and their characterization using a new automated image collection and analysis system. Plant Cell Rep 26:1501–1509

    Article  PubMed  CAS  Google Scholar 

  • Chiera JM, Lindbo JA, Finer JJ (2008) Quantification and extension of transient GFP expression by the co-introduction of a suppressor of silencing. Transgenic Res 17:1143–1154

    Article  PubMed  CAS  Google Scholar 

  • Della Vedova CB, Lorbiecke R, Kirsch H, Schulte MB, Scheets K, Borchert LM, Scheffler BE, Wienand U, Cone KC, Birchler JA (2005) The dominant inhibitory chalcone synthase allele C2-Idf (Inhibitor diffuse) from Zea mays (L.) acts via an endogenous RNA silencing mechanism. Genetics 170:1989–2002

    Article  PubMed  CAS  Google Scholar 

  • 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 28:639–647

    Article  PubMed  CAS  Google Scholar 

  • Ding S-W, Anderson BJ, Haase HR, Symons RH (1994) New overlapping gene encoded by the cucumber mosaic virus genome. Virology 198:593–601

    Article  PubMed  CAS  Google Scholar 

  • 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

    PubMed  CAS  Google Scholar 

  • El-Shemy HA, Teraishi M, Khalafalla MM, Katsube-Tanaka T, Utsumi S, Ishimoto M (2004) Isolation of soybean plants with stable transgene expression by visual selection based on green fluorescent protein. Mol Breed 14:227–238

    Article  CAS  Google Scholar 

  • Finer JJ, Nagasawa A (1988) Development of an embryogenic suspension culture of soybean (Glycine max Merrill). Plant Cell Tissue Organ Cult 15:125–136

    Article  CAS  Google Scholar 

  • González I, Martínez L, Rakitina DV, Lewsey MG, Atencio FA, Llave C, Kalinina NO, Carr JP, Palukaitis P, Canto T (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  PubMed  CAS  Google Scholar 

  • Goto K, Kobori T, Kosaka Y, Natsuaki T, Masuta C (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

    Article  PubMed  CAS  Google Scholar 

  • Hamera S, Song X, Su L, Chen X, Fang R (2012) Cucumber mosaic virus suppressor 2b binds to AGO4-related small RNAs and impairs AGO4 activities. Plant J 69:104–115

    Article  PubMed  CAS  Google Scholar 

  • Hong JS, Masuta C, Nakano M, Abe J, Uyeda I (2003) Adaptation of Cucumber mosaic virus soybean strains (SSVs) to cultivated and wild soybeans. Theor Appl Genet 107:49–53

    PubMed  CAS  Google Scholar 

  • Ishimoto M, Rahman SM, Hanafy MS, Khalafalla MM, El-Shemy HA, Nakamoto Y, Kita Y, Takanashi K, Matsuda F, Murano Y, Funabashi T, Miyagawa H, Wakasa K (2010) Evaluation of amino acid content and nutritional quality of transgenic soybean seeds with high-level tryptophan accumulation. Mol Breed 25:313–326

    Article  CAS  Google Scholar 

  • Ji L-H, Ding S-W (2001) The suppressor of transgene RNA silencing encoded by Cucumber mosaic virus interferes with salicylic acid-mediated virus resistance. Mol Plant–Microbe Interact 14:715–724

    Article  PubMed  CAS  Google Scholar 

  • Kasai A, Kasai K, Yumoto S, Senda M (2007) Structural features of GmIRCHS, candidate of the I gene inhibiting seed coat pigmentation in soybean: implications for inducing endogenous RNA silencing of chalcone synthase genes. Plant Mol Biol 64:467–479

    Article  PubMed  CAS  Google Scholar 

  • Kasai A, Ohnishi S, Yamazaki H, Funatsuki H, Kurauchi T, Matsumoto T, Yumoto S, Senda M (2009) Molecular mechanism of seed coat discoloration induced by low temperature in yellow soybean. Plant Cell Physiol 50:1090–1098

    Article  PubMed  CAS  Google Scholar 

  • Khalafalla MM, Rahman SM, El-Shemy HA, Nakamoto Y, Wakasa K, Ishimoto M (2005) Optimization of particle bombardment conditions by monitoring of transient sGFP(S65T) expression in transformed soybean. Breed Sci 55:257–263

    Article  Google Scholar 

  • Kinney AJ, Jung R, Herman EM (2001) Cosuppression of the α subunits of β-conglycinin in transgenic soybean seeds induces the formation of endoplasmic reticulum-derived protein bodies. Plant Cell 13:1165–1178

    PubMed  CAS  Google Scholar 

  • Kurauchi T, Kasai A, Tougou M, Senda M (2011) Endogenous RNA interference of chalcone synthase genes in soybean: formation of double-stranded RNA of GmIRCHS transcripts and structure of the 5′ and 3′ ends of short interfering RNAs. J Plant Physiol 168:1264–1270

    Article  PubMed  CAS  Google Scholar 

  • Kusaba M, Miyahara K, Iida S, Fukuoka H, Takano T, Sassa H, Nishimura M, Nishio T (2003) Low glutelin content1: a dominant mutation that suppresses the glutelin multigene family via RNA silencing in rice. Plant Cell 15:1455–1467

    Article  PubMed  CAS  Google Scholar 

  • Lewsey M, Robertson FC, Canto T, Palukaitis P, Carr JP (2007) Selective targeting of miRNA-regulated plant development by a viral counter-silencing protein. Plant J 50:240–252

    Article  PubMed  CAS  Google Scholar 

  • 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 22:642–654

    Article  PubMed  CAS  Google Scholar 

  • Lewsey MG, Murphy AM, MacLean D, Dalchau N, Westwood JH, Macaulay K, Bennett MH, Moulin M, Hanke DE, Powell G, Smith AG, Carr JP (2010) Disruption of two defensive signaling pathways by a viral RNA silencing suppressor. Mol Plant–Microbe Interact 23:835–845

    Article  PubMed  CAS  Google Scholar 

  • Lim H-S, Ko T-S, Lambert KN, Kim H-G, Korban SS, Hartman GL, Domier LL (2005) Soybean mosaic virus helper component-protease enhances somatic embryo production and stabilizes transgene expression in soybean. Plant Physiol Biochem 43:1014–1021

    Article  PubMed  CAS  Google Scholar 

  • Llave C (2010) Virus-derived small interfering RNAs at the core of plant–virus interactions. Trends Plant Sci 15:701–707

    Article  PubMed  CAS  Google Scholar 

  • Moissiard G, Voinnet O (2004) Viral suppression of RNA silencing in plants. Mol Plant Pathol 5:71–82

    Article  PubMed  CAS  Google Scholar 

  • Nishizawa K, Kita Y, Kitayama M, Ishimoto M (2006) A red fluorescent protein, DsRed2, as a visual reporter for transient expression and stable transformation in soybean. Plant Cell Rep 25:1355–1361

    Article  PubMed  CAS  Google Scholar 

  • Ohnishi S, Funatsuki H, Kasai A, Kurauchi T, Yamaguchi N, Takeuchi T, Yamazaki H, Kurosaki H, Shirai S, Miyoshi T, Horita H, Senda M (2011) Variation of GmIRCHS (Glycine max inverted-repeat CHS pseudogene) is related to tolerance of low temperature-induced seed coat discoloration in yellow soybean. Theor Appl Genet 122:633–642

    Article  PubMed  Google Scholar 

  • Phillips JR, Dalmay T, Bartels D (2007) The role of small RNAs in abiotic stress. FEBS Lett 581:3592–3597

    Article  PubMed  CAS  Google Scholar 

  • Praveen S, Mangrauthia SK, Singh P, Mishra AK (2008) Behavior of RNAi suppressor protein 2b of Cucumber mosaic virus in planta in presence and absence of virus. Virus Genes 37:96–102

    Article  PubMed  CAS  Google Scholar 

  • Price ML, Van Scoyoc S, Butler LG (1978) A critical evaluation of the vanillin reaction as an assay for tannin in sorghum grain. J Agric Food Chem 26:1214–1218

    Article  CAS  Google Scholar 

  • Reddy MSS, Dinkins RD, Collins GB (2003) Gene silencing in transgenic soybean plants transformed via particle bombardment. Plant Cell Rep 21:676–683

    PubMed  CAS  Google Scholar 

  • Rodriguez TO, Rojas Rodas F, Oyoo ME, Senda M, Takahashi R (2013) Inverted repeat of chalcone synthase 3 pseudogene is associated with seed coat discoloration in soybean. Crop Sci 53:518–523

    Article  CAS  Google Scholar 

  • Roossinck MJ, Zhang L, Hellwald K-H (1999) Rearrangements in the 5′ nontranslated region and phylogenetic analyses of cucumber mosaic virus RNA 3 indicate radial evolution of three subgroups. J Virol 73:6752–6758

    PubMed  CAS  Google Scholar 

  • Ruiz-Ferrer V, Voinnet O (2009) Roles of plant small RNAs in biotic stress responses. Annu Rev Plant Biol 60:485–510

    Article  PubMed  CAS  Google Scholar 

  • Sahoo PK, Soltani S, Wong AKC (1988) A survey of thresholding techniques. Comput Vis Graph Image Process 41:233–260

    Article  Google Scholar 

  • Samoylov VM, Tucker DM, Thibaud-Nissen F, Parrott WA (1998) A liquid-medium-based protocol for rapid regeneration from embryogenic soybean cultures. Plant Cell Rep 18:49–54

    Article  CAS  Google Scholar 

  • Senda M, Kasai A, Yumoto S, Akada S, Ishikawa R, Harada T, Niizeki M (2002) Sequence divergence at chalcone synthase gene in pigmented seed coat soybean mutants of the Inhibitor locus. Genes Genet Syst 77:341–350

    Article  PubMed  CAS  Google Scholar 

  • Senda M, Masuta C, Ohnishi S, Goto K, Kasai A, Sano T, Hong J-S, MacFarlane S (2004) Patterning of virus-infected Glycine max seed coat is associated with suppression of endogenous silencing of chalcone synthase genes. Plant Cell 16:807–818

    Article  PubMed  CAS  Google Scholar 

  • Shi B-J, Palukaitis P, Symons RH (2002) Differential virulence by strains of Cucumber mosaic virus is mediated by the 2b gene. Mol Plant–Microbe Interact 15:947–955

    Article  PubMed  CAS  Google Scholar 

  • Shi B-J, Miller J, Symons RH, Palukaitis P (2003) The 2b protein of Cucumoviruses has a role in promoting the cell-to-cell movement of pseudorecombinant viruses. Mol Plant–Microbe Interact 16:261–267

    Article  PubMed  CAS  Google Scholar 

  • Soards AJ, Murphy AM, Palukaitis P, Carr JP (2002) Virulence and differential local and systemic spread of Cucumber mosaic virus in tobacco are affected by the CMV 2b protein. Mol Plant–Microbe Interact 15:647–653

    Article  PubMed  CAS  Google Scholar 

  • Takahashi K, Nagasawa J, Tanaka T, Iizuka N (1987) Reaction of soybean varieties to the strains of soybean mosaic virus and soybean stunt virus in Japan (in Japanese). Misc Publ Tohoku Natl Agric Exp Stn 7:1–35

    Google Scholar 

  • Thévenaz P, Ruttimann UE, Unser M (1998) A pyramid approach to subpixel registration based on intensity. IEEE Trans Image Process 7:27–41

    Article  PubMed  Google Scholar 

  • Todd JJ, Vodkin LO (1993) Pigmented soybean (Glycine max) seed coats accumulate proanthocyanidins during development. Plant Physiol 102:663–670

    PubMed  CAS  Google Scholar 

  • Tsubokura Y, Hajika M, Kanamori H, Xia Z, Watanabe S, Kaga A, Katayose Y, Ishimoto M, Harada K (2012) The β-conglycinin deficiency in wild soybean is associated with the tail-to-tail inverted repeat of the α-subunit genes. Plant Mol Biol 78:301–309

    Article  PubMed  CAS  Google Scholar 

  • Tuteja JH, Clough SJ, Chan W-C, Vodkin LO (2004) Tissue-specific gene silencing mediated by a naturally occurring chalcone synthase gene cluster in Glycine max. Plant Cell 16:819–835

    Article  PubMed  CAS  Google Scholar 

  • Vance V, Vaucheret H (2001) RNA silencing in plants—defense and counterdefense. Science 292:2277–2280

    Article  PubMed  CAS  Google Scholar 

  • Wang C-S, Vodkin LO (1994) Extraction of RNA from tissues containing high levels of procyanidins that bind RNA. Plant Mol Biol Rep 12:132–145

    Article  CAS  Google Scholar 

  • Yamada T, Takagi K, Ishimoto M (2012) Recent advances in soybean transformation and their application to molecular breeding and genomic analysis. Breed Sci 61:480–494

    Article  PubMed  CAS  Google Scholar 

  • Ye J, Qu J, Zhang J-F, Geng Y-F, Fang R-X (2009) A critical domain of the Cucumber mosaic virus 2b protein for RNA silencing suppressor activity. FEBS Lett 583:101–106

    Article  PubMed  CAS  Google Scholar 

  • Zhang X, Yuan Y-R, Pei Y, Lin S–S, Tuschl T, Patel DJ, Chua N-H (2006) Cucumber mosaic virus-encoded 2b suppressor inhibits Arabidopsis Argonaute1 cleavage activity to counter plant defense. Genes Dev 20:3255–3268

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by the Program for Promotion of Basic and Applied Research for Innovations in Bio-oriented Industry (BRAIN) and in part by a grant (Genomics for Agricultural Innovation, GMZ-1004) from the Ministry of Agriculture, Forestry, and Fisheries of Japan.

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Author notes

  1. Kyoko Takagi

    Present address: National Agriculture and Food Research Organization (NARO) Agricultural Research Center, 3-1-1 Kannondai, Tsukuba, Ibaraki, 305-8666, Japan

  2. Keito Nishizawa

    Present address: NARO Institute of Crop Science, 2-1-18 Kannondai, Tsukuba, Ibaraki, 305-8518, Japan

Authors and Affiliations

  1. National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602, Japan

    Kyoko Takagi & Masao Ishimoto

  2. National Agricultural Research Center for Hokkaido Region, 1 Hitsujigaoka, Toyohira, Sapporo, Hokkaido, 062-8555, Japan

    Kyoko Takagi, Keito Nishizawa, Aya Hirose & Masao Ishimoto

  3. Faculty of Agriculture and Life Science, Hirosaki University, Bunkyo-cho 3, Hirosaki, Aomori, 036-8581, Japan

    Tasuku Kurauchi & Mineo Senda

  4. Graduate School of Agriculture, Hokkaido University, Kita 9, Nishi 9, Kita-ku, Sapporo, 060-8589, Japan

    Chikara Masuta

Authors
  1. Kyoko Takagi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Keito Nishizawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Aya Hirose
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Tasuku Kurauchi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Mineo Senda
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Chikara Masuta
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Masao Ishimoto
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Masao Ishimoto.

Additional information

Communicated by H. Ebinuma.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PPT 153 kb)

Supplementary material 2 (DOC 34 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Takagi, K., Nishizawa, K., Hirose, A. et al. Seed coat pigmentation in transgenic soybean expressing the silencing suppressor 2b gene of Cucumber mosaic virus . Plant Cell Rep 32, 1903–1912 (2013). https://doi.org/10.1007/s00299-013-1502-6

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00299-013-1502-6

Keywords

Search

Navigation