Rice gs3 allele and low-nitrogen conditions enrich rhizosphere microbiota that mitigate methane emissions and promote beneficial crop traits.

Methane emissions from rice paddies represent a critical environmental concern in agriculture. Although genetic strategies for mitigating emissions have gained attention, the specific microbial and molecular mechanisms remain underexplored. Here, we investigated how the gs3 loss-of-function allele in the near-isogenic rice line Milyang360 modulates rhizosphere and endosphere microbial communities under distinct nitrogen regimes. Field experiments revealed that Milyang360 consistently reduced methane emissions compared with its parental line Saeilmi particularly under low-nitrogen conditions. Integrated plant transcriptomic and rhizosphere metagenomic analyses, including the reconstruction of Metagenome-Assembled Genomes, demonstrated that the gs3 allele upregulated genes related to root hair elongation and promoting microbial nitrogen fixation. This physiological change limited substrate availability for methanogens and facilitated the colonization by beneficial microorganisms. Consequently, we observed a functional shift in the microbiome, characterized by the enrichment of methanotrophs and nitrogen-fixing bacteria. This microbial restructuring was most prominent under low-nitrogen conditions, indicating a strong genotype by environment interaction. Our findings highlight the gs3 allele's dual role in reducing methane emissions and improving nitrogen use efficiency by recruiting a beneficial microbiome. Our study provides a clear mechanistic link between a plant gene and rhizosphere ecology, offering a promising genetic target for developing sustainable, low emission rice cultivars.