NLR4DS−1 enhances wheat resistance to stripe rust by upregulating multiple defense responses

Key message NLR 4DS−1 confers stripe rust resistance by enhancing multilayer physical and chemical defenses. This finding illuminates strategies for engineering durable resistance in wheat. Abstract Wheat stripe rust ( Puccinia striiformis f. sp. tritici , Pst ) is a major threat to global wheat production. The Yr28 locus for Pst resistance is derived from Aegilops tauschii subsp. strangulata . NLR 4DS−1 has been identified as the stripe rust resistance gene at the Yr28 locus, which encodes a coiled-coil nucleotide-binding leucine-rich repeat (NLR) protein, however, its molecular mode of action remains largely unknown. Here, we introduced the full-length genomic NLR 4DS−1 into the Pst susceptible wheat ‘Fielder’ by Agrobacterium -mediated transformation. Transgenic plants were resistant to Pst , with significantly reduced fungal biomass and elevated expression of pathogenesis-related genes. Integrated transcriptomic and metabolomic analysis revealed that NLR 4DS−1 orchestrates a multifaceted defense program involving a burst of reactive oxygen species, chlorophyll reduction, lignin accumulation, and the induction of flavonoid and terpenoid phytoalexins. In vitro, either the flavonoid quercetin or the terpenoid borneol can suppress spore germination and hyphal growth of Pst . Notably, NLR 4DS−1 expression did not compromise seedling growth or agronomic performance at the adult stage. Together, these findings identify NLR 4DS−1 as the stripe rust resistance gene for Yr28 locus, demonstrate its role in activating defensive pathways, and highlight its potential for engineering durable Pst resistance through gene stacking.