A VIGS-based leaf disc method reveals ethylene's crucial role in hydrangea leaf spot resistance

The bigleaf hydrangea (Hydrangea macrophylla) is an emerging fashionable flower. However, leaf spot disease caused by Corynespora cassiicola is a major fungal disease in this species, seriously limiting the high-quality development of the industry. Since potential C. cassiicola induced genes have been identified via RNA-seq data, we aimed to develop an efficient method to screen C. cassiicola resistant genes in hydrangea. Firstly, we established a C. cassiicola inoculation system for detached leaf discs (CISDLD) and applied it to assess leaf spot resistance in hydrangea cultivators. Then, HmPDS1 and HmPDS2 were selected as the reporter genes to test the silencing effect in both cuttings and detached leaf discs, through observing a significant reduction in gene expression and chlorophyll content. Furthermore, ethylene was identified as a positive regulator in hydrangea defense responding to C. cassiicola, as well as jasmonic acid. The results showed that ethylene synthesis genes (HmACS1 and HmACO3) and signaling transduction genes (HmEIN3 and HmERF001) were validated as the C. cassiicola resistant genes in hydrangea. The VIGS screen of C. cassiicola-induced ethylene related genes demonstrated the potential benefits of this method for the high-throughput identification of gene function. This study offers a rapid approach for characterizing C. cassiicola-related gene functions in hydrangea and provides a theoretical framework for high-throughput gene screening in other plant species. KEY MESSAGE: A rapid and effective method for gene function screening based on VIGS with detached leaf discs reveals the crucial role of ethylene in hydrangea leaf spot resistance.