Fig. 2: Plant-based platform for production of amidated AMPs.

a Schematic diagram of chimeric cassettes with different variants of bifunctional rat PAMs and the different domains: PHM, peptidylglycine α-hydroxylating monooxygenase domain; PAL, peptidyl-α-hydroxyglycine α-amidating lyase domain; A, region encoded by exon 16 separating the PHM and PHL domains; T, transmembrane domain; C, cytoplasmic domain. The HA epitope was added for immunodetection of PAMs. b In planta transient expression of PAM enzymes. Each plasmid was individually co-infiltrated in N. benthamiana leaves along with a plasmid encoding P19. Leaves were harvested 3 dpi, total proteins were extracted and analyzed by immunoblotting using a monoclonal anti-HA antibody. The predicted molecular masses were 120 kDa (PAM1), 105 kDa (PAM2) and 95 kDa (PAM3). The three black arrowheads indicate the bands corresponding to PAM1, PAM2, and PAM3 enzymes. Two independent blots were performed with similar results. c In planta transient co-expression of AMPs and PAM enzymes. Constructs encoding AMPs and PAMs were transiently co-expressed in N. benthamiana (1:1 ratio) and the extracts were immunoblotted using anti-HA antibody. Black arrowheads indicate the expected size of proteins and peptides, red arrowheads indicate to possible in planta modified AMPs, while the asterisk indicates the non-specific bands. Two independent blots were performed with similar results. d In planta amidation of AMPs in transgenic plants expressing PAM1. Transgenic N. benthamiana lines (T₄ generation) overexpressing a PAM1 variant were infiltrated with constructs encoding glycine-extended AMPs. AMPs were isolated with our established test method and subjected to separation in RP-HPLC using C8 with an acetonitrile gradient from 20 to 80% in 0.01 M HCl and monitored at the 215 nm wavelength. Purified peptides were eluted as a double peak, with the major peak belonging to amidated peptide with retention times of 10.2 min (AMP1), 9.7 min (AMP2), 10.2 min (AMP3) and the minor peak belonging to the non-amidated form with retention times 9.5 min (AMP1), 9.6 min (AMP2) and 9.7 min (AMP3). e Confirmation of AMP amidation via ESI-MS. Mass analysis of purified AMPs isolated from the PAM transgenic plants showing major peak belonging to amidated AMPs along the minor non-amidated peak. Source data are provided as a Source Data file.