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