Alignment results from each RNA-Seq samples.

Nonhost resistance (NHR) serves as a fundamental defense response in plants against non-adapted pathogens, yet its underlying molecular mechanisms remain poorly understood. This study investigates the rice-Pst (Puccinia striiformis f. sp. tritici) interaction using integrated transcriptomic and metabolomic analyses to unravel the temporal dynamics of gene expression and metabolite changes associated with NHR. Our findings reveal a temporally coordinated activation of defense responses, with early induction of receptor-like kinases (RLKs) and hypersensitive response proteins, followed by later activation of jasmonic acid and systemic acquired resistance pathways, along with the accumulation of amino acids and other phenolic compounds. Notably, metabolic pathways related to cell wall reinforcement were significantly upregulated during Pst infection, highlighted by enhanced lignin biosynthesis (phenylpropanoid pathway), nucleotide sugar metabolism, and tryptophan pathways. Rice mutants deficient in genes involved in lignin biosynthesis (OsPAL3, Os4CL3, Os4CL5, and OsCCoAOMT) displayed reduced lignin deposition at infection sites and compromised resistance to Pst, underscoring a critical role of lignin-based physical barriers in NHR. This study provides novel insights into the molecular framework of rice NHR, emphasizing the pivotal role of structural defenses in plant immunity.