A FnWRKY17-FnFLA16 regulatory module controls leaf curling in Fragaria nilgerrensis

Moderate leaf curling is a desirable trait in crop breeding and has attracted increasing research interest. A curled leaf (cl) mutant was obtained from a previously developed ethyl methanesulfonate (EMS) mutant library of the strawberry Fragaria nilgerrensis; however, the regulatory frameworks and interaction networks responsible for this phenotype remain largely undefined.

Modifications in cell wall architecture and cellulose content, along with related biological features, were detected in the cl mutant. Transcriptomic RNA sequencing was performed to compare gene expression between the cl mutant and the wild type (WT). Functional verification was carried out through genetic manipulation. Yeast one- hybrid (Y1H), dual- luciferase (LUC), and β- glucuronidase (GUS) assays were used to test transcriptional regulation. Transient expression assays were also conducted to confirm the role of the identified regulator.

Transcriptomic analysis revealed substantial differential expression of fasciclin- like arabinogalactan (FLA) genes between the cl mutant and WT. Functional verification showed that FnFLA16 altered cellulose- associated biological properties, thereby promoting leaf curling. Subsequent correlation analyses and quantitative assays identified critical transcriptional regulators. FnWRKY17 was demonstrated to directly bind to the FnFLA16 promoter, as confirmed by Y1H, LUC, and GUS assays. Transient expression assays further confirmed that FnWRKY17 operates within the FnFLA16- cellulose regulatory framework controlling leaf curling.

These findings reveal a previously unreported FnFLA16- centered working model underlying leaf curling in F. nilgerrensis. This study provides new insights into the regulatory mechanisms of leaf curling and identifies potential targets forbreeding programs.