TRV-VIGS establishment and functional characterization identify cinnamoyl-CoA reductase 4 as a crucial enzyme for lignan biosynthesis in Phryma leptostachya.

Furofuran lignans are the primary insecticidal and medicinal bioactive compounds in Phryma leptostachya, yet their species-specific biosynthetic pathways remain incompletely deciphered. We established a Tobacco rattle virus-based virus-induced gene silencing system, identifying vacuum-assisted infiltration of Agrobacterium tumefaciens strain GV3101 at the four-leaf stage as the optimal inoculation condition. Silencing the PlCYP81Q38 gene via this system markedly decreased root accumulation of sesamin and 6-demethoxyleptostachyol acetate. Combined transcriptomic and metabolomic analyses revealed that PlCYP81Q38-silenced plants exhibited upregulated phenylpropanoid pathway genes, including phenylalanine ammonia lyase, cinnamoyl-CoA reductase (CCR), and dirigent protein 1, alongside elevated levels of lignan precursors (coniferyl alcohol, (+)-pinoresinol). Within this pathway, CCR enzymes catalyze hydroxycinnamoyl-CoA substrates across multiple branches. We characterized the PlCCR family and confirmed that PlCCR4 acts as a dedicated catalytic enzyme for lignan biosynthesis. It exhibited the highest in vitro catalytic efficiency for feruloyl-CoA (Kcat/Km = 244.90 min-1/nM) and was essential for lignan biosynthesis in vivo. Its homolog PlCCR5 exhibited significantly lower efficiency; homology modeling and molecular docking indicated that an Asp64-to-Glu64 substitution in the NADPH-binding region may alter the cofactor microenvironment, plausibly reducing its activity. This study identifies PlCCR4 as an important upstream enzyme, providing a valuable genetic target for engineering sustainable production of bioactive lignan insecticides.