Involvement of RAV-like 9 in sex-specific responses of <i>Taxus mairei</i> to fungal infection

Dioecious plants often exhibit dimorphism in morphology, physiology, and environmental adaptation. As a dioecious gymnosperm, Taxus is well known for its ornamental and ecological value. However, the sexual dimorphism in the responses of Taxus mairei to fungal infection remains unclear. In the present study, we investigated the effect of sex on the responses of T. mairei to S01, a fungus belonging to the Aspergillus genus, using untargeted metabolomic analysis. Although there is no significant difference in the contents of eight analyzed flavonoid monomers between female and male T. mairei plants under normal condition, a significant difference emerges under fungal infection. We identified 15 members of the abscisic acid insensitive3/viviparous1 (RAV)-like gene subfamily in the T. mairei genome. Subsequently, a RAV-like transcription factor (TF) gene, RAV-like 9, which is responsive to S01 infection, was identified to be involved in flavonoid metabolism based on Pearson's correlation analysis. To identify the genome-wide binding sites of RAV-like 9, DNA affinity purification sequencing (DAP-seq) was performed, yielding 3993 overlapping peaks. Motif enrichment analysis identified several de novo motifs, providing new insights into RAV TF recognition sites. After searching the peak pool, two flavonoid biosynthesis-related target genes were detected: ANS (ctg19199_gene.2) and IRL1 (ctg9900_gene.5). Quantitative reverse transcription polymerase chain reaction analysis confirmed the differential expression of ANS and IRL1 between female and male T. mairei under S01 infection. Our data suggest that RAV-like 9 may play an important regulatory role in sex-specific responses of flavonoid biosynthesis to fungal infection by targeting the ANS and IRL1 genes.