Integrative sRNA, DNA Methylation, and Transcriptomics Reveals Dynamic Epigenetic Reprogramming of <i>Meloidogyne javanica</i>-Induced Galls in Arabidopsis

Root knot nematodes (RKNs) induce galls, containing multinucleated giant cells (GCs) to nourish them. The differentiation of precursor cells to galls/GCs involves extensive cellular reprogramming with multiple layers of regulation. Epigenetic regulation during the early stages of infection indicates that RNA-directed DNA methylation (RdDM) and microRNA-dependent gene silencing contribute to transcriptional and post-transcriptional reprogramming during gall organogenesis. Although later stages of galls/GC development are crucial for nematode life-cycle maintenance, epigenetic reprogramming events remain largely unexplored. An integrative analysis of sRNAs, DNA methylation, and transcriptomic dynamics in galls induced by <i>Meloidogyne javanica</i> revealed that enrichment of 24 nt sRNAs represents a gall hallmark across early and late developmental stages. Fewer gall-distinctive sRNAs were detected at mid-to-late stages than at early stages, alongside a pronounced spatial reorganization of rasiRNA accumulation. At early stages, gall-distinctive rasiRNAs preferentially accumulated in pericentromeric retrotransposon-rich regions, whereas, at mid-to-late stages, they predominantly localized to chromosome arms, matching DNA transposons, promoters, and gene bodies. A decline in the regulatory influence of miRNAs was observed as infection progressed, possibly reflecting a transition toward specialized regulatory states associated with gall maintenance. Moreover, three regulatory modules, miR2111-5p/HOLT, miR172/AP2, and miR156/SPL10, were tightly but oppositely regulated at 3 and 14 days post-infection. Furthermore, miR156/SPL10 showed crucial functions during gall formation and/or maintenance, possibly influenced by hormonal cues involving ARF8 among other ARFs. Our results highlight stage-specific patterns involving sRNA dynamics, DNA methylation, and transcriptomic changes underlying nematode feeding site development and maintenance.