6mA demethylase Mi-NMAD-1 modulates nematode development and parasitism through the transcription factor Mi-nhr-118

Abstract

N6-methyladenine (6mA) DNA methylation has emerged as an important epigenetic mark in eukaryotes, but its biological role in plant-parasitic nematodes remains largely unexplored. Here, we demonstrate that 6mA methylation is essential for the embryonic development and parasitic ability of Meloidogyne incognita. Silencing of the 6mA demethylase genes minmad-1 significantly reduced egg hatching, revealing that 6mA-mediated regulation is required for nematode development. Transcriptome analyses showed that minmad-1 knockdown downregulated genes involved in embryogenesis, RNA biosynthesis, and cell differentiation, suggesting a critical role of 6mA in developmental gene regulation. Among the transcription factors affected by minmad-1 silencing, Mi-nhr-118, a nuclear hormone receptor orthologous to C. elegans nhr-118, was identified as a key regulator of embryonic development. Functional assays confirmed that RNAi or host-induced gene silencing (HIGS) of Mi-nhr-118 led to developmental arrest, reduced gall and egg mass formation, and impaired infectivity across multiple Meloidogyne species. ChIP-seq analysis revealed that Mi-nhr-118 directly binds to promoters of genes associated with cellular regulation and developmental signaling. Several downstream targets, including transcription factors (Mi_23621.1, Mi_39647.1) and genes implicated in metabolic and signaling processes (Mi_03067.1, Mi_08398.1), were shown to be essential for both embryogenesis and parasitism. Functional enrichment analyses of Mi-nhr-118 target genes indicated suppression of immune-related signaling, carbohydrate recognition, and glycoprotein biosynthetic pathways following gene silencing. Collectively, these findings establish Mi-nhr-118 as a central regulator linking 6mA-mediated epigenetic control to development and parasitism in M. incognita.