Characterization of O-methyltransferases in the biosynthesis of phenylphenalenone phytoalexins based on the telomere-to-telomere gapless genome of Musella lasiocarpa.

Phenylphenalenones (PhPNs), phytoalexins in wild bananas (Musaceae), are known to act against various pathogens. However, the abundance of PhPNs in many Musaceae plants of economic importance is low. Knowledge of the biosynthesis of PhPNs and the application of biosynthetic approaches to improve their yield is vital for fighting banana diseases. However, the processes of PhPN biosynthesis, especially those involved in methylation modification, remain unclear. Musella lasiocarpa is a herbaceous plant belonging to Musaceae, and due to the abundant PhPNs, their biosynthesis in M. lasiocarpa has been the subject of much attention. In this study, we assembled a telomere-to-telomere gapless genome of M. lasiocarpa as the reference, and further integrated transcriptomic and metabolomic data to mine the candidate genes involved in PhPN biosynthesis. To elucidate the diversity of PhPNs in M. lasiocarpa, three screened O-methyltransferases (Ml01G0494, Ml04G2958, and Ml08G0855) by phylogenetic and expressional clues were subjected to in vitro enzymatic assays. The results show that the three were all novel O-methyltransferases involved in the biosynthesis of PhPN phytoalexins, among which Ml08G0855 was proved to function as a multifunctional enzyme targeting multiple hydroxyl groups in PhPN structure. Moreover, we tested the antifungal activity of PhPNs against Fusarium oxysporum and found that the methylated modification of PhPNs enhanced their antifungal activity. These findings provide valuable genetic resources in banana breeding and lay a foundation for improving disease resistance through molecular breeding.