Divergent evolution of insecticidal protein families across bacteria and ferns

Insecticidal Cry proteins produced by the bacterial insect pathogen Bacillus thuringiensis possess conserved three domains (3D). Two other families of insecticidal proteins--Prb proteins from bacterial symbionts of entomopathogenic nematodes and a marine bacterial pathogen, and IPD113 proteins from ferns--are two-domain (2D) proteins structurally resembling domains I and II of Cry proteins. Despite the structural resemblance, the amino acid sequence identity among these families is only [~]10%, supporting the prevailing view that they evolved independently through convergent evolution. However, the possibility of remote homology and ancient horizontal gene transfer (HGT) has remained largely unexplored. Here, we present evidence supporting divergent evolution of these protein families. We found that proteins containing Cry-like two domains are distributed far more broadly across bacteria, eukaryotes, and archaea than previously recognized. Phylogenetic analyses separated the structurally similar proteins into five clades including Cry, IPD113, Prb, and 2D/3D-mixed clades. Domain-level trees revealed intermixing within domain II, with nematocidal Cry proteins frequently clustering with IPD113 and 2D/3D-mixed clades, suggesting domain swapping events across these clades. Conserved amino acid motifs and domain core structures across clades further support a shared evolutionary origin. Phylogenetic and structural analyses suggest a bacterial origin of the fern IPD113 gene and its horizontal transfer via fungal symbionts, as well as multiple HGT events into diverse terrestrial and marine eukaryotes and archaea. This work redefines the evolutionary landscape of the insecticidal protein families, revealing their unexpectedly wide taxonomic distribution, possibly shaped through bacterial ecological networks and close biological interactions such as symbiosis.