Song, S.-j. | Search bioRxiv

bioRxiv2025

Polyphenol oxidase silencing avoids protein crosslinking and enzymatic browning in Nicotiana benthamiana leaf extracts

Oxidation in extracts from agroinfiltrated leaves leads to browning and protein precipitation. Here we show that silencing of polyphenol oxidase (PPO) in Nicotiana benthamiana suppresses enzymatic browning and avoids crosslinking of endogenous proteins such as RuBisCo. PPO silencing does not impact plant growth or development and may also enhance transient gene expression. These results show that depletion of PPO activity could be highly beneficial for plant science applications and molecular pharming.

plant biology↗

bioRxiv2025

Immunity gene silencing increases transient protein expression in Nicotiana benthamiana

Agroinfiltration of Nicotiana benthamiana is routinely used for transient gene expression in plant sciences and molecular pharming. Here, we depleted transcripts of 21 different immunity-related genes through virus-induced gene silencing (VIGS) to identify genes that hamper transient expression in juvenile plants. These experiments uncovered that silencing of ethylene insensitive- 2 (EIN2), receptor-like kinase CERK1, transcription factor regulator nonexpressor of pathogenesis-related genes 1 (NPR1) and isochorismate synthase (ICS), increases transient GFP expression by 2-, 4-, 4- and 11-fold, respectively. Accordingly, the npr1a/npr1b double mutant of N. benthamiana does indeed facilitate increased protein accumulation when transiently expressed. These results indicate that glycan perception through CERK1, and ET and SA signaling pathways via EIN2 and NPR1/ICS, respectively, contribute to suppressed transient gene expression in frequently used juvenile N. benthamiana plants.

plant biology↗