Weina Hou1, Rupesh Kumar Singh2, Pan Zhao3, Viviana Martins4, Emmanuel Aguilar5, Tomás Canto5, Francisco Tenllado6, Alberto Carlos Pires Dias7,8,9. 1. Centre for the Research and Technology of Agro-Environmental and Biological Sciences (CITAB), Department of Biology, University of Minho, 4710-057, Braga, Portugal. 2. Centro de Química de Vila Real (CQ-VR), UTAD, 5000-801, Vila Real, Portugal. 3. National Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China. 4. Centre of Molecular and Environmental Biology (CBMA), Department of Biology, University of Minho, 4710-057, Braga, Portugal. 5. Departamento de Biotecnología Microbiana y de Plantas, Centro de Investigaciones Biológicas, CSIC, 28040, Madrid, Spain. 6. Departamento de Biotecnología Microbiana y de Plantas, Centro de Investigaciones Biológicas, CSIC, 28040, Madrid, Spain. tenllado@cib.csic.es. 7. Centre for the Research and Technology of Agro-Environmental and Biological Sciences (CITAB), Department of Biology, University of Minho, 4710-057, Braga, Portugal. acpdias@bio.uminho.pt. 8. Centre of Molecular and Environmental Biology (CBMA), Department of Biology, University of Minho, 4710-057, Braga, Portugal. acpdias@bio.uminho.pt. 9. Center of Biological Engineering (CEB), University of Minho, 4710-057, Braga, Portugal. acpdias@bio.uminho.pt.
Abstract
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.
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.