Transient expression of PRISEs and Trichoderma-mediated elicitation promote iridoid production in Nepeta sibirica L

The genus Nepeta is the sole representative in the plant kingdom that produces nepetalactones, a group of iridoids with a unique stereochemistry, which play key roles in plant defense and ecological interactions. This study investigates N. sibirica L., a species rich in cis,trans-nepetalactone and 1,5,9-epi-deoxyloganic acid, aiming to enhance production of these bioactive iridoids by two alternative strategies: transient expression of key iridoid biosynthesis-related genes and fungal elicitation. In vitro treatments with Trichoderma harzianum and T. viride promoted iridoid production in N. sibirica leaves. It appears that regulatory proteins COI1, MYC2, and YABBY5 provoke coordinated upregulation of the early iridoid pathway genes (NsGPPS, NsGES, NsG8H, Ns8HGO), and of NsMLPL, thus stimulating metabolic flux through the iridoid pathway and providing substrates for the downstream steps mediated by NsISY, NsNEPS1, and NsNEPS2. The N. sibirica PRISE orthologue (NsPRISE) is closely related phylogenetically to the Family 1 isoforms known as P5βRs. However, its ISY-like activity was confirmed through in vitro assays with recombinant proteins expressed heterologously in E. coli. Transient overexpression experiments, which comparatively analysed in planta function of homologous NsPRISE and previously characterized ISY and PRISE orthologues from other Nepeta species, suggested possible in vivo residual ISY-like activity of NsPRISE and its involvement in iridoid production. The current study recognized N. sibirica as a plant susceptible to agroinfiltration, with iridoid metabolism that can be induced by pathogen attack, making it an ideal candidate for developing scalable systems for bioactive compounds production.