Discovery and Characterization of a Novel Nematicidal Three-ORF cry-like Gene Cluster with Biocontrol Potential

The growing global impact of plant-parasitic nematodes on agricultural productivity highlights the urgent need for sustainable control strategies. In this study, a novel cry-like gene cluster containing three open reading frames (ORFs) was identified from Bacillus strain HSY144. The encoded proteins, ORF1, ORF2, and ORF3, showed sequence similarity to Cry65Aa1, Cry14Aa1, and Cry21Ha1, respectively. Heterologous expression in Escherichia coli and Bacillus thuringiensis confirmed the production of stable and alkali-soluble proteins. Bioassays demonstrated that the recombinant proteins exhibited strong nematicidal activity against Caenorhabditis elegans and Meloidogyne incognita, whereas toxicity toward the lepidopteran pest Helicoverpa armigera was negligible. Transcriptomic profiling of C. elegans exposed to the recombinant proteins revealed time-dependent regulation of genes involved in innate immunity, signaling transduction, and metabolic processes. Collectively, these findings identify a previously uncharacterized three-ORF cry-like cluster with potent nematicidal properties, expanding the known diversity of Cry proteins and offering a promising biocontrol resource for nematode management in sustainable agriculture.