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Specific region affects the difference in accumulation levels between apple food allergen Mal d 1 and birch pollen allergen Bet v 1 which are expressed in vegetative tissues of transgenic rice

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Abstract

Key message

Specific domain of the Mal d 1 was identified to be mainly involved in higher accumulation level in vegetative tissues of transgenic rice than the Bet v 1.

Abstract

Apple food allergen Mal d 1 and birch pollen allergen Bet v 1 belong to the same pathogen related protein 10 (PR10) family. When green fluorescent protein (GFP) fused to either of these allergens was expressed as a secretory protein in transgenic rice by ligating an N terminal signal peptide and a C terminal KDEL ER retention signal under the control of the maize ubiquitin constitutive promoter, the GFP:Mald1 highly accumulated in various tissues, whereas accumulation level of the GFP:Betv1 was remarkably reduced in vegetative tissues except for seed. Analysis by RT-PCR exhibited that there was little difference in their transcript levels, indicating the involvement of post-transcriptional regulation. To investigate the cause of such difference in accumulation levels, deletion analysis of the Mal d 1 and domain swapping between them were carried out in transgenic rice. The results showed that the region between positions 41–90 in the Mal d 1 is predominantly implicated in higher level accumulation in vegetative tissues as well as seed as compared with the Bet v 1. The GFP:Mald1 was localized in oligomeric form within ER lumen or ER-derived particles in vegetative tissues, whereas in seed mainly deposited into novel huge ER-derived protein bodies with the size of 5–10 µm in aleurone cells.

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Abbreviations

Cys:

Cysteine

BiP:

Binding protein

DTT:

Dithiothreitol

ER:

Endoplasmic reticulum

ELP:

Elastin-like polypeptide

GFP:

Green fluorescence protein

OAS:

Oral allergy syndrome

PB:

Protein body

2-MER:

2-Mercaptoethanol

SDS-PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

RT-PCR:

Reverse transcription-polymerase chain reactions

PR10:

Pathogen related protein 10

PDIL:

Protein disulfide isomerase like

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Acknowledgements

We thank Ms. K. Miyashita, Y. Ikemoto and H. Yajima for technical assistance and Dr. Kenjirou Ozawa for encourage of this research.

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Authors and Affiliations

  1. Plant Molecular Farming Unit, Division of Biotechnology, Institute of Agrobiological Sciences, National Agriculture and Food Research Organization, Owashi 1-2, Tsukuba, Ibaraki, 305-8634, Japan

    Fumio Takaiwa, Yuko Ogo & Yuhya Wakasa

  2. Institute of Crop Science, National Agriculture and Food Research Organization, Kannondai 2-1-2, Tsukuba, Ibaraki, 305-8602, Japan

    Yuko Ogo

Authors
  1. Fumio Takaiwa
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  2. Yuko Ogo
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FT carried out the experimental design, data analysis and wrote the manuscript. YO and YW participated in experiments and contributed the data analysis.

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Correspondence to Fumio Takaiwa.

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Takaiwa, F., Ogo, Y. & Wakasa, Y. Specific region affects the difference in accumulation levels between apple food allergen Mal d 1 and birch pollen allergen Bet v 1 which are expressed in vegetative tissues of transgenic rice. Plant Mol Biol 98, 439–454 (2018). https://doi.org/10.1007/s11103-018-0789-7

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