A callus-based parenchymal sentinel (CaPS) system dissects the primordial defense mechanisms of Larix kaempferi against pine wilt nematode.

Pine wilt disease, instigated by the Bursaphelenchus xylophilus (also called pine wood nematode, PWN), poses a significant threat to coniferous forests across the globe, leading to widespread tree mortality and ecological disruption. While Japanese larch (Larix kaempferi) is a natural host of PWN, the molecular basis of its responses remains poorly understood. Here, we developed a callus-based parenchymal sentinel (CaPS) system mimicking xylem parenchyma-nematode interactions to bypass multi-tissue interference in traditional sapling studies. After five days of PWN inoculation, nematode proliferated 2.85-fold, while the callus exhibited water-soaked lesions and reduced cell viability, indicating a rapid defense activation. (1) Transcriptome analysis revealed 8,515 differentially expressed genes related to chitinase signaling, calcium-regulated immunity, and antimicrobial compound synthesis. (2) Metabolomic analysis identified 389 defense-related metabolites (e.g., alkaloids). (3) Integration of omics data uncovered 71 coordinated pathways categorized into eight functional groups, including reactive oxygen species burst and mitogen-activated protein kinase, and they formed a multi-layered defense network. Importantly, this CaPS system enabled five-day phenotyping cycles of transgenic callus, significantly accelerating evaluation compared to traditional sapling methods. Our work reveals early-stage conifer immunity against PWN and establishes an accelerated evaluation program for future screening of transgenic callus and breeding resistant larch varieties.