Functional chitinases enhancing Pinus massoniana resistance to Bursaphelenchus xylophilus

BACKGROUND Pine wilt disease (PWD), caused by the pinewood nematode Bursaphelenchus xylophilus , poses a severe threat to coniferous forests, particularly impacting Pinus massoniana . Chitinases, essential defense‐related enzymes, degrade chitin in pathogen cell walls to inhibit infection. However, their roles in conifer resistance against nematodes remain poorly understood. This study investigated the functions of two class IV chitinases, PmChia4‐1 and PmChia4‐4 , in P. massoniana and their potential to bolster defenses against B. xylophilus . RESULTS We cloned the full‐length coding sequences of PmChia4‐1 and PmChia4‐4 from P. massoniana and confirmed their significant upregulation in response to B. xylophilus infection. Subcellular localization analyses showed their secretion into the apoplast. In vitro bioassays demonstrated that recombinant PmChia4‐1 and PmChia4‐4 significantly reduced nematode egg hatching rates (33.6% and 28.0%, respectively, compared to 62.4% in controls), and increased juvenile mortality (20.23% and 21.40%, respectively, versus ≈2% in controls). Transgenic Arabidopsis thaliana plants overexpressing these genes exhibited significantly enhanced basal immune responses against B. xylophilus , characterized by increased reactive oxygen species accumulation and enhanced activities of defense enzymes, including chitinase, peroxidase and catalase. CONCLUSION Our results reveal critical roles for PmChia4‐1 and PmChia4‐4 in the defense responses of P. massoniana against B. xylophilus . These findings significantly advance the current understanding of chitinase‐mediated mechanisms in conifers, highlighting valuable molecular targets for breeding PWD‐resistant pine cultivars, with substantial implications for forestry practices and sustainable management strategies.