Functional characterization of squalene epoxidase genes in Rosa chinensis upregulated by infection with Podosphaera pannosa

• Squalene epoxidase gene RcSE1 is very important in the indirect plant-mediated interactions between phytopathogen and insect on Rosa chinensis . • RcSE1 is upregulated 29.1-fold after the infection of roses by Podosphaera pannosa . • RcSE1 can directly activate the metabolic pathway critical for the resistance of roses against Spodoptera exigua by elevating the insect-resistant triterpenoids, including maslinic acid. • Key substrate-binding residues (VAL67, TYR78) identified by molecular docking are critical for catalytic function of RcSE1. • The results elucidate the molecular biological mechanism of the interactions. Rose ( Rosa chinensis ), as the most popular cut flower in the world, is frequently attacked by rose powdery mildew ( Podosphaera pannosa ) and beet armyworm ( Spodoptera exigua ). Indirect plant-mediated interactions between these two pests consistently occur in the ternary system of these three organisms, but little is known about the functions of rose genes of triterpenoid-related key enzymes for the interactions. In this study, we investigated the functional characteristics of the squalene epoxidase (SE) gene, which was upregulated in roses after infection with the mildew. The two SE genes ( LOC112173431 and LOC112174075 ) were cloned from healthy (control) and infected rose leaves, and real-time PCR results showed that these genes were upregulated 29.1-fold and 46.8-fold, respectively, when compared to the controls. Bioinformatic analysis revealed that the conserved domains are critical for squalene binding and catalytic activity, while molecular docking identified key residues involved in substrate interaction (e.g., VAL67, TYR78). Heterologous expression of RcSE1 ( LOC112173431 ) in Escherichia coli confirmed its enzymatic activity with a specific activity of 15.43 IU/L. In Saccharomyces cerevisiae , overexpression of RcSE1 led to a 45% reduction in squalene accumulation, and alongside an increase in the sterol contents. The transgenic Arabidopsis thaliana plants with overexpression of RcSE1 showed significant increases in the contents of the S.exigua -resistant triterpenoids, including maslinic acid by 46.87 times, betulonic acid by 8.12 times, ganoderic acid B by 1.07 times, and ursolic acid by 2.86 times in the T2 generation, suggesting that RcSE1 can directly activate the metabolic pathway critical for insect resistance without the mildew infection or hormone signals. The results indicate that RcSE1 plays a critical role in the interactions.