A callus-based parenchymal sentinel system dissects the primordial defense mechanisms of Larix kaempferi against pine wood nematode

Abstract 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 5 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. (i) Transcriptome analysis revealed 8515 differentially expressed genes related to chitinase signaling, calcium-regulated immunity and antimicrobial compound synthesis. (ii) Metabolomic analysis identified 389 defense-related metabolites (e.g., alkaloids). (iii) 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 5-day phenotyping cycles of transgenic callus, significantly accelerating evaluation compared with 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.