Nepenthes chitinase NkChit2b- 1 confers broad-spectrum resistance to chitin-containing pathogens and insects in plants

Chitinases play critical roles in plant-pathogen/insect interactions by degrading chitin, a key structural component of fungal cell walls and insect exoskeletons. However, current research lacks comprehensive studies on the broad-spectrum disease resistance of chitinases, and novel chitinases with higher enzymatic activity remain underexplored. Here, we report the prokaryotic expression and functional characterization of Nepenthes khasiana-derived chitinase NkChit2b-1, demonstrating its capacity to confer broad-spectrum resistance against chitin-containing phytopathogenic fungi and insect pests. Biochemical assays revealed that NkChit2b-1 exhibits high enzymatic activity within the optimal temperature range (28-42°C) for terrestrial plant growth and the pH range (5.0-6.0) encompassing pathogen-induced apoplastic alkalization in plants. This enzymatic profile correlates with its effective inhibition of mycelial growth in major phytopathogens including Sclerotinia sclerotiorum, Botrytis cinerea, and Magnaporthe oryzae. Exogenous application of NkChit2b-1 conferred enhanced resistance to these pathogens in both model species (e.g., Arabidopsis) and crop species (e.g., tobacco, tomato, and rice). Intriguingly, NkChit2b-1 pretreatment suppressed feeding activity of brown planthopper (BPH, Nilaparvata lugens) nymphs on rice phloem sap and induced mortality in adult BPH upon ingestion. Furthermore, NkChit2b-1 accelerated beet armyworm (Spodoptera exigua) egg hatching while delaying larval development. In addition, foliar application of NkChit2b-1 on Arabidopsis leaves conferred antifeedant activity against beet armyworm larvae in dual-choice assays. These results collectively indicate the exceptional potential of NkChit2b-1 as an eco-friendly "green pesticide". The exploration of novel chitinases and combinatorial chitinase strategies may overcome the limitations of single-enzyme formulations, thereby advancing chitinase applications in sustainable agriculture and plant protection.