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

Abstract 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 Nk Chit2b-1, demonstrating its capacity to confer broad-spectrum resistance against chitin-containing phytopathogenic fungi and insect pests. Biochemical assays revealed that Nk Chit2b-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 Nk Chit2b-1 conferred enhanced resistance to these pathogens in both model species (e.g., Arabidopsis ) and crop species (e.g., tobacco, tomato, and rice). Intriguingly, Nk Chit2b-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, Nk Chit2b-1 accelerated beet armyworm ( Spodoptera exigua ) egg hatching while delaying larval development. In addition, foliar application of Nk Chit2b-1 on Arabidopsis leaves conferred antifeedant activity against beet armyworm larvae in dual-choice assays. These results collectively indicate the exceptional potential of Nk Chit2b-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.