Discovery and Characterization of a Novel Nematicidal Three-ORF <i>cry-like</i> 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 <i>cry-like</i> gene cluster containing three open reading frames (ORFs) was identified from <i>Bacillus</i> strain HSY144. The encoded proteins, ORF1, ORF2, and ORF3, showed sequence similarity to Cry65Aa1, Cry14Aa1, and Cry21Ha1, respectively. Heterologous expression in <i>Escherichia coli</i> and <i>Bacillus thuringiensis</i> confirmed the production of stable and alkali-soluble proteins. Bioassays demonstrated that the recombinant proteins exhibited strong nematicidal activity against <i>Caenorhabditis elegans</i> and <i>Meloidogyne incognita</i>, whereas toxicity toward the lepidopteran pest <i>Helicoverpa armigera</i> was negligible. Transcriptomic profiling of <i>C. elegans</i> 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 <i>cry-like</i> cluster with potent nematicidal properties, expanding the known diversity of Cry proteins and offering a promising biocontrol resource for nematode management in sustainable agriculture.