DNA Methylation Shapes Seed‐Borne Microbiome and Proteome Responses During Early Maize‐Beneficial Bacteria Interactions

Microorganism and plant interactions are crucial for development and environmental adaptation. Plant growth promoting bacteria enhance agricultural productivity in a sustainable manner, while epigenetic modifications such as DNA methylation regulate gene expression and adaptive responses. The objective of this study is to determine how DNA hypomethylation influences early interactions between maize (Zea mays) and the endophytic diazotrophic bacterium Herbaspirillum seropedicae, particularly regarding plant growth, metabolism, and the root microbiome. Treatment with the hypomethylating agent 5-azacytidine (5-azaC) altered maize root morphology without affecting bacterial growth. Inoculation with H. seropedicae promoted plant growth and bacterial colonisation in root mucilage, with higher accumulation in 5-azaC treated roots. Global methylation analysis showed that bacterial inoculation modulates cytosine methylation in a manner similar to 5-azaC, suggesting a role in epigenetic regulation. Gene expression analysis of DNA methylation machinery confirmed that hypomethylation drives plant-microbe interactions. Root microbiome profiling revealed that 5-azaC disrupted microbial composition, which was partially restored by bacterial inoculation. Proteomic analysis identified 1,818 proteins and highlighted significant changes in metabolic pathways, especially carbon metabolism and the citric acid cycle. These findings demonstrate that DNA hypomethylation combined with bacterial interaction profoundly affects cellular and metabolic processes and provide insights for sustainable agricultural practices through epigenetic and microbial modulation.