Gene Expression and Interaction Analysis of FsWRKY4 and FsMAPK3 in Forsythia suspensa

Forsythia suspensa is a deciduous shrub that belongs to the family Myrtaceae, and its dried fruits are used as medicine. F. suspensa contains several secondary metabolites, which exert pharmacological effects. One of the main active components is forsythin, which exhibits free radical scavenging, antioxidant, anti-inflammatory, and anti-cancer effects. Mitogen-activated protein kinase (MAPKs) can increase the activity of WRKY family transcription factors in a phosphorylated manner, thereby increasing the content of secondary metabolites. However, the mechanism of interaction between MAPKs and WRKYs in F. suspensa remains unclear. In this study, we cloned the genes of FsWRKY4 and FsMAPK3, and performed a bioinformatics analysis. The expression patterns of FsWRKY4 and FsMAPK3 were analyzed in the different developmental stages of leaf and fruit from F. suspensa using real-time fluorescence quantitative PCR (qRT-PCR). Subcellular localization analysis of FsWRKY4 and FsMAPK3 proteins was performed using a laser scanning confocal microscope. The existence of interactions between FsWRKY4 and FsMPAK3 in vitro was verified by yeast two-hybridization. Results showed that the cDNA of FsWRKY4 (GenBank number: OR566682) and FsMAPK3 (GenBank number: OR566683) were 1587 and 522 bp, respectively. The expression of FsWRKY4 was higher in the leaves than in fruits, and the expression of FsMAPK3 was higher in fruits but lower in leaves. The subcellular localization results indicated that FsWRKY4 was localized in the nucleus and FsMAPK3 in the cytoplasm and nucleus. The prey vector pGADT7-FsWRKY4 and bait vector pGBKT7-FsMAPK3 were constructed and co-transferred into Y2H Glod yeast receptor cells. The results indicated that FsWRKY4 and FsMAPK3 proteins interact with each other in vitro. The preliminary study may provide a basis for more precise elucidation of the synthesis of secondary metabolites in F. suspensa.