, 251:13 | Cite as

Transgenic expression of Hyp-1 gene from Hypericum perforatum L. alters expression of defense-related genes and modulates recalcitrance to Agrobacterium tumefaciens

  • Weina Hou
  • Rupesh Kumar Singh
  • Pan Zhao
  • Viviana Martins
  • Emmanuel Aguilar
  • Tomás Canto
  • Francisco TenlladoEmail author
  • Alberto Carlos Pires DiasEmail author
Original Article


Main conclusion

Phenolic oxidative coupling protein (Hyp-1) isolated from Hypericum perforatum L. was characterized as a defense gene involved in H. perforatum recalcitrance to Agrobacterium tumefaciens-mediated transformation


Hypericum perforatum L. is a reservoir of high-value secondary metabolites of increasing interest to researchers and to the pharmaceutical industry. However, improving their production via genetic manipulation is a challenging task, as H. perforatum is recalcitrant to Agrobacterium tumefaciens-mediated transformation. Here, phenolic oxidative coupling protein (Hyp-1), a pathogenesis-related (PR) class 10 family gene, was selected from a subtractive cDNA library from A. tumefaciens-treated H. perforatum suspension cells. The role of Hyp-1 in defense against A. tumefaciens was analyzed in transgenic Nicotiana tabacum and Lactuca sativa overexpressing Hyp-1, and in Catharanthus roseus silenced for its homologous Hyp-1 gene, CrIPR. Results showed that Agrobacterium-mediated expression efficiency greatly decreased in Hyp-1 transgenic plants. However, silencing of CrIPR induced CrPR-5 expression and decreased expression efficiency of Agrobacterium. The expression of core genes involved in several defense pathways was also analyzed in Hyp-1 transgenic tobacco plants. Overexpression of Hyp-1 led to an ample down-regulation of key genes involved in auxin signaling, microRNA-based gene silencing, detoxification of reactive oxygen species, phenylpropanoid pathway and PRs. Moreover, Hyp-1 was detected in the nucleus, plasma membrane and the cytoplasm of epidermal cells by confocal microscopy. Overall, our findings suggest Hyp-1 modulates recalcitrance to A. tumefaciens-mediated transformation in H. perforatum.


Agrobacterium tumefaciens-mediated expression Hypericum perforatum secondary metabolites Hyp-1 Plant defense responses PR-10 



Catharanthus roseus intracellular pathogenesis-related protein


Phenolic oxidative coupling protein




Virus-induced gene silencing



The author acknowledges the financial support provided by the FCT-Portuguese Foundation for Science and Technology, grant UID/BIA/04050/2013 (POCI-01-0145FEDER-007569), PhD grant (SFRH/BD/52561/2014) under the Doctoral Programme “Agricultural Production Chains—from fork to farm” (PD/00122/2012), and the Ministry of Economy and Competitiveness of Spain, grant BIO2016-75619-R (AEI/FEDER, UE) to T.C. and F.T. We gratefully acknowledge the advice of Prof. Vincent L. Chiang and Dr. Rui Shi (North Carolina State University) for microRNA detection. We gratefully thank Prof. Mariana Sottomayor and Dr Joana Guedes (University of Porto) for kindly providing pTRV2: CrChlh and advice.

Compliance with ethical standards

Conflict of interest

All authors declare that they have no conflict of interest.

Supplementary material

425_2019_3310_MOESM1_ESM.pdf (391 kb)
Supplementary material 1 (PDF 391 kb)
425_2019_3310_MOESM2_ESM.pdf (95 kb)
Supplementary material 2 (PDF 95 kb)
425_2019_3310_MOESM3_ESM.pdf (91 kb)
Supplementary material 3 (PDF 91 kb)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Weina Hou
    • 1
  • Rupesh Kumar Singh
    • 2
  • Pan Zhao
    • 3
  • Viviana Martins
    • 4
  • Emmanuel Aguilar
    • 5
  • Tomás Canto
    • 5
  • Francisco Tenllado
    • 5
    Email author
  • Alberto Carlos Pires Dias
    • 1
    • 4
    • 6
    Email author
  1. 1.Centre for the Research and Technology of Agro-Environmental and Biological Sciences (CITAB), Department of BiologyUniversity of MinhoBragaPortugal
  2. 2.Centro de Química de Vila Real (CQ-VR), UTADVila RealPortugal
  3. 3.National Key Laboratory of Plant GenomicsInstitute of Microbiology, Chinese Academy of SciencesBeijingChina
  4. 4.Centre of Molecular and Environmental Biology (CBMA), Department of BiologyUniversity of MinhoBragaPortugal
  5. 5.Departamento de Biotecnología Microbiana y de PlantasCentro de Investigaciones Biológicas, CSICMadridSpain
  6. 6.Center of Biological Engineering (CEB)University of MinhoBragaPortugal

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