Domains rearranged methyltransferases (DRMs)‐mediated DNA methylation plays key roles in modulating gene expression and maintaining transposable element silencing in soybean

The domains rearranged methyltransferases (DRMs) play a critical role in the RNA‐directed DNA methylation (RdDM) pathway in plants. However, the effects of inactivating the RdDM pathway on gene expression, transposable element (TE) activity, and phenotype in soybean remain unexplored. Here, we employed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein 9 gene editing to generate a quintuple mutant line in soybean ( Gmdrm2a −/− 2b −/− 2c −/− 3a −/− 3b −/− , designated Gmdrm ). Gmdrm exhibited severe developmental abnormalities, including dwarfism and delayed growth, albeit remaining viable and fertile; however, the fully homozygous mutant could be maintained for a limited number of generations (T0–T3). Whole genome bisulfite sequencing revealed a significant reduction in DNA methylation across all cytosine sequence contexts, with an average loss of 10%. The loss of m C was biased toward euchromatic regions, which is in contrast to the chromomethylase mutant. Transcriptome profiling identified 1,685 up‐regulated genes, including photosynthesis‐related genes, accompanied with altered chloroplast ultrastructure. Additionally, a cluster of resistance ( R ) genes on chromosome 16 was significantly up‐regulated, coinciding with their reduced non‐CG methylation. We also observed 3,164 differentially expressed TEs (DETs), of which, 2,655 were up‐regulated and hypomethylated along their entire length. A substantial reduction in the abundance of 24‐nt small interfering RNAs (siRNAs) in the Gmdrm mutant was detected by small RNA sequencing. Of note, the DRM‐targeted TEs typically display higher levels of 24‐nt siRNA abundance, shorter lengths, and are more AT‐rich compared to chromomethylase‐targeted TEs, highlighting 24‐nt siRNAs as key determinants of DRM‐dependent TE regulation. Together, this study documents a critical role of DRM‐mediated DNA methylation in regulating gene expression, TE silencing, and normal development in soybean.