The role of WRKY transcription factors, FaWRKY29 and FaWRKY64, for regulating Botrytis fruit rot resistance in strawberry (Fragaria x ananassa Duch.)

Background The cultivated strawberry (Fragaria x ananassa Duch.) is one of the most economically important horticultural crops worldwide. Botrytis fruit rot (BFR) caused by the necrotrophic fungal pathogen Botrytis cinerea is the most devasting disease of cultivated strawberries. Most commercially grown strawberry varieties are susceptible to BFR, and controlling BFR relies on repeated applications of various fungicides. Despite extensive efforts, breeding for BFR resistance has been unsuccessful, primarily due to lack of information regarding the mechanisms of disease resistance and genetic resources available in strawberry. Results Using a reverse genetics approach, we identified candidate genes associated with BFR resistance and screened Arabidopsis mutants using strawberry isolates of B. cinerea. Among the five Arabidopsis T-DNA knockout lines tested, the mutant line with AtWRKY53 showed the greatest reduction in disease symptoms of BFR against the pathogen. Two genes, FaWRKY29 and FaWRKY64, were identified as orthologs in the latest octoploid strawberry genome, 'Florida Brilliance'. We performed RNAi-mediated transient assay and found that the disease frequencies were significantly decreased in both FaWRKY29- and FaWRKY64-RNAi fruits of the strawberry cultivar, 'Florida Brilliance'. Furthermore, our transcriptomic data analysis revealed significant regulation of genes associated with ABA and JA signaling, plant cell wall composition, and ROS in FaWRKY29 or FaWRKY64 knockdown strawberry fruits in response to the pathogen. Conclusion Our study uncovered the foundational role of WRKY transcription factor genes, FaWRKY29 and FaWRKY64, in conferring resistance against B. cinerea. The discovery of susceptibility genes involved in BFR presents significant potential for developing resistance breeding strategies in cultivated strawberries, potentially leveraging CRISPR-based gene editing techniques.