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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\u2010associated protein 9 gene editing to generate a quintuple mutant line in soybean (<jats:italic>Gmdrm2a<\/jats:italic><jats:sup><jats:italic>\u2212\/\u2212<\/jats:italic><\/jats:sup><jats:italic>2b<\/jats:italic><jats:sup><jats:italic>\u2212\/\u2212<\/jats:italic><\/jats:sup><jats:italic>2c<\/jats:italic><jats:sup><jats:italic>\u2212\/\u2212<\/jats:italic><\/jats:sup><jats:italic>3a<\/jats:italic><jats:sup><jats:italic>\u2212\/\u2212<\/jats:italic><\/jats:sup><jats:italic>3b<\/jats:italic><jats:sup><jats:italic>\u2212\/\u2212<\/jats:italic><\/jats:sup>, designated <jats:italic>Gmdrm<\/jats:italic>). <jats:italic>Gmdrm<\/jats:italic> 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\u2013T3). 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 <jats:sup>m<\/jats:sup>C was biased toward euchromatic regions, which is in contrast to the chromomethylase mutant. Transcriptome profiling identified 1,685 up\u2010regulated genes, including photosynthesis\u2010related genes, accompanied with altered chloroplast ultrastructure. Additionally, a cluster of resistance (<jats:italic>R<\/jats:italic>) genes on chromosome 16 was significantly up\u2010regulated, coinciding with their reduced non\u2010CG methylation. We also observed 3,164 differentially expressed TEs (DETs), of which, 2,655 were up\u2010regulated and hypomethylated along their entire length. A substantial reduction in the abundance of 24\u2010nt small interfering RNAs (siRNAs) in the <jats:italic>Gmdrm<\/jats:italic> mutant was detected by small RNA sequencing. Of note, the DRM\u2010targeted TEs typically display higher levels of 24\u2010nt siRNA abundance, shorter lengths, and are more AT\u2010rich compared to chromomethylase\u2010targeted TEs, highlighting 24\u2010nt siRNAs as key determinants of DRM\u2010dependent TE regulation. Together, this study documents a critical role of DRM\u2010mediated DNA methylation in regulating gene expression, TE silencing, and normal development in soybean.<\/jats:p>","DOI":"10.1111\/jipb.13883","type":"journal-article","created":{"date-parts":[[2025,3,11]],"date-time":"2025-03-11T16:39:38Z","timestamp":1741711178000},"page":"1501-1514","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":15,"title":["Domains rearranged methyltransferases (DRMs)\u2010mediated DNA methylation plays key roles in modulating gene expression and maintaining transposable element silencing in soybean"],"prefix":"10.1111","volume":"67","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2409-2299","authenticated-orcid":false,"given":"Hongwei","family":"Xun","sequence":"first","affiliation":[{"name":"Key Laboratory of Molecular Epigenetics of MOE Northeast Normal University Changchun 130024 China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-1151-3034","authenticated-orcid":false,"given":"Lijie","family":"Lian","sequence":"additional","affiliation":[{"name":"National Key Laboratory of Crop Genetic Improvement, College of Plant Science and Technology, Hubei Hongshan Laboratory Huazhong Agricultural University Wuhan 430070 China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4472-7741","authenticated-orcid":false,"given":"Jing","family":"Yuan","sequence":"additional","affiliation":[{"name":"National Key Laboratory of Agricultural Microbiology Huazhong Agricultural University Wuhan 430070 China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-7776-280X","authenticated-orcid":false,"given":"Jianhui","family":"Hong","sequence":"additional","affiliation":[{"name":"Key Laboratory of Molecular Epigenetics of MOE Northeast Normal University Changchun 130024 China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-2632-7701","authenticated-orcid":false,"given":"Shanmeng","family":"Hao","sequence":"additional","affiliation":[{"name":"National Key Laboratory of Crop Genetic Improvement, College of Plant Science and Technology, Hubei Hongshan Laboratory Huazhong Agricultural University Wuhan 430070 China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-5236-5572","authenticated-orcid":false,"given":"Haonan","family":"Zhao","sequence":"additional","affiliation":[{"name":"National Key Laboratory of Crop Genetic Improvement, College of Plant Science and Technology, Hubei Hongshan Laboratory Huazhong Agricultural University Wuhan 430070 China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0004-6910-805X","authenticated-orcid":false,"given":"Shuhan","family":"Liu","sequence":"additional","affiliation":[{"name":"Key Laboratory of Molecular Epigenetics of MOE Northeast Normal University Changchun 130024 China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-5206-4454","authenticated-orcid":false,"given":"Wanjie","family":"Feng","sequence":"additional","affiliation":[{"name":"National Key Laboratory of Crop Genetic Improvement, College of Plant Science and Technology, Hubei Hongshan Laboratory Huazhong Agricultural University Wuhan 430070 China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6635-2055","authenticated-orcid":false,"given":"Huanran","family":"Yin","sequence":"additional","affiliation":[{"name":"National Key Laboratory of Crop Genetic Improvement, College of Plant Science and Technology, Hubei Hongshan Laboratory Huazhong Agricultural University Wuhan 430070 China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5481-1675","authenticated-orcid":false,"given":"Bao","family":"Liu","sequence":"additional","affiliation":[{"name":"Key Laboratory of Molecular Epigenetics of MOE Northeast Normal University Changchun 130024 China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4625-7659","authenticated-orcid":false,"given":"Xutong","family":"Wang","sequence":"additional","affiliation":[{"name":"National Key Laboratory of Crop Genetic Improvement, College of Plant Science and Technology, Hubei Hongshan Laboratory Huazhong Agricultural University Wuhan 430070 China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2025,3,7]]},"reference":[{"key":"e_1_2_9_2_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cub.2003.11.052"},{"key":"e_1_2_9_3_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0960-9822(02)00925-9"},{"key":"e_1_2_9_4_1","doi-asserted-by":"crossref","unstructured":"Cao X. andJacobsen S.E.(2002b). 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