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Seed-produced anti-globulin VHH-Fc antibodies retrieve globulin precursors in the insoluble fraction and modulate the Arabidopsis thaliana seed subcellular morphology

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Abstract

Key message

Nanobody-heavy chain (VHH-Fc) antibody formats have the potential to immunomodulate even highly accumulating proteins and provide a valuable tool to experimentally modulate the subcellular distribution of seed storage proteins.

Abstract

Recombinant antibodies often obtain high accumulation levels in plants, and thus, besides being the actual end-product, antibodies targeting endogenous host proteins can be used to interfere with the localization and functioning of their corresponding antigens. Here, we compared the effect of a seed-expressed nanobody-heavy chain (VHH-Fc) antibody against the highly abundant Arabidopsis thaliana globulin seed storage protein cruciferin with that of a VHH-Fc antibody without endogenous target. Both antibodies reached high accumulation levels of around 10% of total soluble protein, but strikingly, another significant part was present in the insoluble protein fraction and was recovered only after extraction under denaturing conditions. In seeds containing the anti-cruciferin antibodies but not the antibody without endogenous target, the amount of soluble, processed globulin subunits was severely reduced and a major part of the cruciferin molecules was found as precursor in the insoluble fraction. Moreover, in these seeds, aberrant vacuolar phenotypes were observed that were different from the effects caused by the depletion of globulins in knock-out seeds. Remarkably, the seeds with strongly reduced globulin amounts are fully viable and germinate with frequencies similar to wild type, illustrating how flexible seeds can retrieve amino acids from the stored proteins to start germination.

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References

  • Almquist KC, Niu Y, McLean MD, Mena FL, Yau KYF, Brown K, Brandle JE, Hall JC (2004) Immunomodulation confers herbicide resistance in plants. Plant Biotechnol J 2:189–197

    CAS  PubMed  Google Scholar 

  • Arcalis E, Stadlmann J, Marcel S, Drakakaki G, Winter V, Rodriguez J, Fischer R, Altmann F, Stoger E (2010) The changing fate of a secretory glycoprotein in developing maize endosperm. Plant Physiol 153:693–702

    CAS  PubMed  PubMed Central  Google Scholar 

  • Arcalis E, Ibl V, Hilscher J, Rademacher T, Avesani L, Morandini F, Bortesi L, Pezzotti M, Vitale A, Pum D, De Meyer T, Depicker A, Stöger E (2019) Russell-like bodies in plant seeds share common features with prolamin bodies and occur upon recombinant protein production. Front Plant Sci 10:777

    PubMed  PubMed Central  Google Scholar 

  • Artsaenko O, Peisker M, zur Nieden U, Fiedler U, Weiler EW, Müntz K, Conrad U (1995) Expression of a single-chain Fv antibody against abscisic acid creates a wilty phenotype in transgenic tobacco. Plant J 8:745–750

    CAS  PubMed  Google Scholar 

  • Baud S, Boutin J-P, Miquel M, Lepiniec L, Rochat C (2002) An integrated overview of seed development in Arabidopsis thaliana ecotype WS. Plant Physiol Biochem 40:151–160

    CAS  Google Scholar 

  • Baudisch B, Pfort I, Sorge E, Conrad U (2018) Nanobody-directed specific degradation of proteins by the 26S-proteasome in plants. Front Plant Sci 9:130

    PubMed  PubMed Central  Google Scholar 

  • Boonrod KJ, Galetzka D, Nagy PD, Conrad U, Krczal G (2004) Single-chain antibodies against a plant viral RNA-dependent RNA polymerase confer virus resistance. Nat Biotechnol 22:856–862

    CAS  PubMed  Google Scholar 

  • Brar HK, Bhattacharyya MK (2012) Expression of a single-chain variable-fragment antibody against a Fusarium virguliforme toxin peptide enhances tolerance to sudden death syndrome in transgenic soybean plants. Mol Plant-Microbe Interact 25:817–824

    CAS  PubMed  Google Scholar 

  • Cervera M, Esteban O, Gil M, Gorris MT, Martínez MC, Peña L, Cambra M (2010) Transgenic expression in citrus of single-chain antibody fragments specific to Citrus tristeza virus confers virus resistance. Transgenic Res 19:1001–1015

    CAS  PubMed  Google Scholar 

  • Cheng W, Li H-P, Zhang J-B, Du H-J, Wei Q-Y, Huang T, Yang P, Kong X-W, Liao Y-C (2015) Tissue-specific and pathogen-inducible expression of a fusion protein containing a Fusarium-specific antibody and a fungal chitinase protects wheat against Fusarium pathogens and mycotoxins. Plant Biotechnol J 13:664–674

    CAS  PubMed  Google Scholar 

  • Conrad U, Manteuffel R (2001) Immunomodulation of phytohormones and functional proteins in plant cells. Trends Plant Sci 6:399–402

    CAS  PubMed  Google Scholar 

  • De Buck S, Virdi V, De Meyer T, De Wilde K, Piron R, Nolf J, Van Lerberge E, De Paepe A, Depicker A (2012) Production of camel-like antibodies in plants. Methods Mol Biol 911:305–324

    PubMed  Google Scholar 

  • De Buck S, Nolf J, De Meyer T, Virdi V, De Wilde K, Van Lerberge E, Van Droogenbroeck B, Depicker A (2013) Fusion of an Fc chain to a VHH boosts the accumulation levels in Arabidopsis seeds. Plant Biotechnol J 11:1006–1016

    PubMed  Google Scholar 

  • De Jaeger G, De Wilde C, Eeckhout D, Fiers E, Depicker A (2000) The plantibody approach: expression of antibody genes in plants to modulate plant metabolism or to obtain pathogen resistance. Plant Mol Biol 43:419–428

    PubMed  Google Scholar 

  • De Meyer T, Depicker A (2014) Trafficking of endoplasmic reticulum-retained recombinant proteins is unpredictable in Arabidopsis thaliana. Front Plant Sci 5:473

    PubMed  PubMed Central  Google Scholar 

  • De Meyer T, Eeckhout D, De Rycke R, De Buck S, Muyldermans S, Depicker A (2014) Generation of VHH antibodies against the Arabidopsis thaliana seed storage proteins. Plant Mol Biol 84:83–93

    PubMed  Google Scholar 

  • De Meyer T, Laukens B, Nolf J, Van Lerberge E, De Rycke R, De Beuckelaer A, De Buck S, Callewaert N, Depicker A (2015) Comparison of VHH-Fc antibody production in Arabidopsis thaliana, Nicotiana benthamiana and Pichia pastoris. Plant Biotechnol J 13:938–947

    PubMed  Google Scholar 

  • De Wilde K, De Buck S, Vanneste K, Depicker A (2013) Recombinant antibody production in Arabidopsis seeds triggers an unfolded protein response. Plant Physiol 161:1021–1033

    PubMed  Google Scholar 

  • Eto J, Suzuki Y, Ohkawa H, Yamaguchi I (2003) Anti-herbicide single-chain antibody expression confers herbicide tolerance in transgenic plants. FEBS Lett 550:179–184

    CAS  PubMed  Google Scholar 

  • Fecker LF, Koenig R, Obermeier C (1997) Nicotiana benthamiana plants expressing beet necrotic yellow vein virus (BNYVV) coat protein-specific scFv are partially protected against the establishment of the virus in the early stages of infection and its pathogenic effects in the late stages of infection. Arch Virol 142:1857–1863

    CAS  PubMed  Google Scholar 

  • Feeney M, Kittelmann M, Menassa R, Hawes C, Frigerio L (2018) Protein storage vacuoles originate from remodeled preexisting vacuoles in Arabidopsis thaliana. Plant Physiol 177:241–254

    CAS  PubMed  PubMed Central  Google Scholar 

  • Gahrtz M, Conrad U (2009) Immunomodulation of plant function by in vitro selected single-chain Fv intrabodies. Methods Mol Biol 483:289–312

    CAS  PubMed  Google Scholar 

  • Ghannam A, Kumari S, Muyldermans S, Abbady AQ (2015) Camelid nanobodies with high affinity for broad bean mottle virus: a possible promising tool to immunomodulate plant resistance against viruses. Plant Mol Biol 87:355–369

    CAS  PubMed  Google Scholar 

  • Horsman J, McLean MD, Olea-Popelka FC, Hall JC (2007) Picloram resistance in transgenic tobacco expressing an anti-picloram scFv antibody is due to reduced translocation. J Agric Food Chem 55:106–112

    CAS  PubMed  Google Scholar 

  • Ibl V, Peters J, Stöger E, Arcalis E (2018) Imaging the ER and endomembrane system in cereal endosperm. Methods Mol Biol 1691:251–262

    CAS  PubMed  Google Scholar 

  • Jobling SA, Jarman C, Teh M-M, Holmberg N, Blake C, Verhoeyen ME (2003) Immunomodulation of enzyme function in plants by single-domain antibody fragments. Nat Biotechnol 21:77–80

    CAS  PubMed  Google Scholar 

  • Kolarich D, Altmann F (2000) N-Glycan analysis by matrix-assisted laser desorption/ionization mass spectrometry of electrophoretically separated nonmammalian proteins: application to peanut allergen Ara h 1 and olive pollen allergen Ole e 1. Anal Biochem 285:64–75

    CAS  PubMed  Google Scholar 

  • Kolarich D, Turecek PL, Weber A, Mitterer A, Graninger M, Matthiessen P, Nicolaes GAF, Altmann F, Schwarz HP (2006) Biochemical, molecular characterization, and glycoproteomic analyses of α1-proteinase inhibitor products used for replacement therapy. Transfusion 46:1959–1977

    CAS  PubMed  Google Scholar 

  • Leblanc N, David K, Grosclaude J, Pradier J-M, Barbier-Brygoo H, Labiau S, Perrot-Rechenmann C (1999) A novel immunological approach establishes that the auxin-binding protein, Nt-abp1, is an element involved in auxin signaling at the plasma membrane. J Biol Chem 274:28314–28320

    CAS  PubMed  Google Scholar 

  • Loos A, Van Droogenbroeck B, Hillmer S, Grass J, Pabst M, Castilho A, Kunert R, Liang M, Arcalis E, Robinson DG, Depicker A, Steinkellner H (2011) Expression of antibody fragments with a controlled N-glycosylation pattern and induction of endoplasmic reticulum-derived vesicles in seeds of Arabidopsis. Plant Physiol 155:2036–2048

    CAS  PubMed  PubMed Central  Google Scholar 

  • Malembic-Maher S, Le Gall F, Danet J-L, Dorlhac de Borne F, Bové J-M, Garnier-Semancik M (2005) Transformation of tobacco plants for single-chain antibody expression via apoplastic and symplasmic routes, and analysis of their susceptibility to stolbur phytoplasma infection. Plant Sci 168:349–358

    CAS  Google Scholar 

  • Morandini F, Avesani L, Bortesi L, Van Droogenbroeck B, De Wilde K, Arcalis E, Bazzoni F, Santi L, Brozzetti A, Falorni A, Stoger E, Depicker A, Pezzotti M (2011) Non-food/feed seeds as biofactories for the high-yield production of recombinant pharmaceuticals. Plant Biotechnol J 9:911–921

    CAS  PubMed  Google Scholar 

  • Muyldermans S (2013) Nanobodies: natural single-domain antibodies. Annu Rev Biochem 82:775–797

    CAS  PubMed  Google Scholar 

  • Nickel H, Kawchuk L, Twyman RM, Zimmermann S, Junghans H, Winter S, Fischer R, Prüfer D (2008) Plantibody-mediated inhibition of the Potato leafroll virus P1 protein reduces virus accumulation. Virus Res 136:140–145

    CAS  PubMed  Google Scholar 

  • Otegui MS, Herder R, Schulze J, Jung R, Staehelin LA (2006) The proteolytic processing of seed storage proteins in Arabidopsis embryo cells starts in the multivesicular bodies. Plant Cell 18:2567–2581

    CAS  PubMed  PubMed Central  Google Scholar 

  • Peschen D, Li H-P, Fischer R, Kreuzaler F, Liao Y-C (2004) Fusion proteins comprising a Fusarium-specific antibody linked to antifungal peptides protect plants against a fungal pathogen. Nat Biotechnol 22:732–738

    CAS  PubMed  Google Scholar 

  • Phillips J, Artsaenko O, Fiedler U, Horstmann C, Mock H-P, Müntz K, Conrad U (1997) Seed-specific immunomodulation of abscisic acid activity induces a developmental switch. EMBO J 16:4489–4496

    CAS  PubMed  PubMed Central  Google Scholar 

  • Sainsbury F, Thuenemann EC, Lomonossoff GP (2009) pEAQ: versatile expression vectors for easy and quick transient expression of heterologous proteins in plants. Plant Biotechnol J 7:682–693

    CAS  PubMed  Google Scholar 

  • Santos MO, Crosby WL, Winkel BSJ (2004) Modulation of flavonoid metabolism in Arabidopsis using a phage-derived antibody. Mol Breeding 13:333–343

    CAS  Google Scholar 

  • Sheedy C, Yau KYF, Hirama T, MacKenzie CR, Hall JC (2006) Selection, characterization, and CDR shuffling of naive llama single-domain antibodies selected against auxin and their cross-reactivity with auxinic herbicides from four chemical families. J Agric Food Chem 54:3668–3678

    CAS  PubMed  Google Scholar 

  • Shevchenko A, Tomas H, Havlis J, Olsen JV, Mann M (2006) In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc 1:2856–2860

    CAS  PubMed  Google Scholar 

  • Shimada T, Yamada K, Kataoka M, Nakaune S, Koumoto Y, Kuroyanagi M, Tabata S, Kato T, Shinozaki K, Seki M, Kobayashi M, Kondo M, Nishimura M, Hara-Nishimura I (2003) Vacuolar processing enzymes are essential for proper processing of seed storage proteins in Arabidopsis thaliana. J Biol Chem 278:32292–32299

    CAS  PubMed  Google Scholar 

  • Suzuki Y, Mizuno T, Urakami E, Yamaguchi I, Asami T (2008) Immunomodulation of bioactive gibberellin confers gibberellin-deficient phenotypes in plants. Plant Biotechnol J 6:355–367

    CAS  PubMed  Google Scholar 

  • Tavladoraki P, Benvenuto E, Trinca S, De Martinis D, Cattaneo A, Galeffi P (1993) Transgenic plants expressing a functional single-chain Fv antibody are specifically protected from virus attack. Nature 366:469–472

    CAS  PubMed  Google Scholar 

  • ten Hoopen P, Hunger A, Müller A, Hause B, Kramell R, Wasternack C, Rosahl S, Conrad U (2007) Immunomodulation of jasmonate to manipulate the wound response. J Exp Bot 58:2525–2535

    PubMed  Google Scholar 

  • Urakami E, Yamaguchi I, Asami T, Conrad U, Suzuki Y (2008) Immunomodulation of gibberellin biosynthesis using an anti-precursor gibberellin antibody confers gibberellin-deficient phenotypes. Planta 228:863–873

    CAS  PubMed  Google Scholar 

  • Van Droogenbroeck B, Cao J, Stadlmann J, Altmann F, Colanesi S, Hillmer S, Robinson DG, Van Lerberge E, Terryn N, Van Montagu M, Liang M, Depicker A, De Jaeger G (2007) Aberrant localization and underglycosylation of highly accumulating single-chain Fv-Fc antibodies in transgenic Arabidopsis seeds. Proc Natl Acad Sci USA 104:1430–1435

    PubMed  Google Scholar 

  • Villani ME, Roggero P, Bitti O, Benvenuto E, Franconi R (2005) Immunomodulation of cucumber mosaic virus infection by intrabodies selected in vitro from a stable single-framework phage display library. Plant Mol Biol 58:305–316

    CAS  PubMed  Google Scholar 

  • Wang G, Wang F, Wang G, Wang F, Zhang X, Zhong M, Zhang J, Lin D, Tang Y, Xu Z, Song R (2012) Opaque1 encodes a myosin XI motor protein that is required for endoplasmic reticulum motility and protein body formation in maize endosperm. Plant Cell 24:3447–3462

    CAS  PubMed  PubMed Central  Google Scholar 

  • Winichayakul S, Pernthaner A, Scott R, Vlaming R, Roberts N (2009) Head-to-tail fusions of camelid antibodies can be expressed in planta and bind in rumen fluid. Biotechnol Appl Biochem 53:111–122

    CAS  PubMed  Google Scholar 

  • Withana-Gamage TS, Hegedus DD, Qiu X, Yu P, May T, Lydiate D, Wanasundara JPD (2013) Characterization of Arabidopsis thaliana lines with altered seed storage protein profiles using synchrotron-powered FT-IR spectromicroscopy. J Agric Food Chem 61:901–912

    CAS  PubMed  Google Scholar 

  • Wu Y, Messing J (2010) RNA interference-mediated change in protein body morphology and seed opacity through loss of different zein proteins. Plant Physiol 153:337–347

    CAS  PubMed  PubMed Central  Google Scholar 

  • Wu Y, Holding DR, Messing J (2010) γ-Zeins are essential for endosperm modification in quality protein maize. Proc Natl Acad Sci USA 107:12810–12815

    CAS  PubMed  Google Scholar 

  • Xiao XW, Chu PWG, Frenkel MJ, Tabe LM, Shukla DD, Hanna PJ, Higgins TJV, Müller WJ, Ward CW (2000) Antibody-mediated improved resistance to ClYVV and PVY infections in transgenic tobacco plants expressing a single-chain variable region antibody. Mol Breeding 6:421–431

    CAS  Google Scholar 

  • Yajima W, Verma SS, Shah S, Rahman MH, Liang Y, Kav NNV (2010) Expression of anti-sclerotinia scFv in transgenic Brassica napus enhances tolerance against stem rot. New Biotechnol 27:816–821

    CAS  Google Scholar 

  • Zábrady M, Hrdinová V, Müller B, Conrad U, Hejátko J, Janda L (2014) Targeted in vivo inhibition of specific protein-protein interactions using recombinant antibodies. PLoS ONE 9:e109875

    PubMed  PubMed Central  Google Scholar 

  • Zimmermann S, Schillberg S, Liao Y-C, Fisher R (1998) Intracellular expression of TMV-specific single-chain Fv fragments leads to improved virus resistance in Nicotiana tabacum. Mol Breeding 4:369–379

    CAS  Google Scholar 

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Acknowledgements

The authors thank Annick Bleys for help in preparing the manuscript, Thomas Maggen for technical assistance, Prof. Ikuko Hara-Nishimura for the rabbit anti-globulin antibodies, Janitha Wanasundara and Dwayne Hegedus for the globulin knock-out lines and Prof. George Lomonosoff for the pEAQ-HT-DEST1 destination vector. TDM was indebted to the Agency for Innovation by Science and Technology (IWT) for a predoctoral fellowship. We also thank the European Cooperation in Science and Technology (COST) action FA0804 for conference reimbursements. The work was supported by a VIB fund for translational research and the Austrian Science Fund FWF (I2823-B25).

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  1. Thomas De Meyer and Elsa Arcalis have contributed equally to this work.

Authors and Affiliations

  1. VIB Center for Plant Systems Biology, 9052, Ghent, Belgium

    Thomas De Meyer, Katrien Maleux, Jonah Nolf & Ann Depicker

  2. Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium

    Thomas De Meyer, Katrien Maleux, Jonah Nolf & Ann Depicker

  3. Department of Applied Genetics and Cell Biology, BOKU University of Natural Resources and Life Sciences, Vienna, Austria

    Elsa Arcalis, Stanislav Melnik & Eva Stöger

  4. Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Austria

    Friedrich Altmann

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  1. Thomas De Meyer
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Contributions

IW, IY and TT designed the research; SA, TT and IW per- formed the computational analyses; IW, SS, HE, IY, YF, MA, MB and AD performed the experimental procedures; IW, IY and TT wrote the paper. TDM, EA, AD and ES designed the study. TDM, EA, AD and ES wrote the manuscript. TDM, EA, SM, KM, JN and FA performed the experiments. TDM and EA carried out analyses. AD and ES coordinated the project.

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Correspondence to Ann Depicker or Eva Stöger.

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De Meyer, T., Arcalis, E., Melnik, S. et al. Seed-produced anti-globulin VHH-Fc antibodies retrieve globulin precursors in the insoluble fraction and modulate the Arabidopsis thaliana seed subcellular morphology. Plant Mol Biol 103, 597–608 (2020). https://doi.org/10.1007/s11103-020-01007-w

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