RfGNBP5 Negatively Regulates Innate Immunity of Red Palm Weevil, Rhynchophorus ferrugineus, Against Distinctive Pathogens

Insects are continuously exposed to diverse microorganisms, including symbionts and pathogens, and rely on pattern-recognition receptors (PRRs) to initiate innate immune responses. Gram-negative bacteria-binding proteins (GNBPs) constitute an important class of the pivotal PRRs in insect immunity. However, whether GNBPs participate in immune responses against taxonomically distinct pathogens remains poorly understood. Red palm weevil (RPW), Rhynchophorus ferrugineus, is a notorious stem-boring insect pest that has caused huge economic loss worldwide. Our preliminary data indicated that the expression of RfGNBP5 could be induced dramatically upon the challenge of non-entomopathogenic bacteria Escherichia coli, Staphylococcus aureus and entomopathogenic fungus Beauveria bassiana. Here, it has been found that RfGNBP5 only possesses two conserved domains, a signal peptide and an intact glycoside hydrolase 16 (GH16) domain, but without the canonical carbohydrate-binding module (CBM39), implying that it might mediate insect immunity via its hydrolytic activity. RT-qPCR revealed that RfGNBP5 was expressed at the highest level in the hemolymph compared with other tissues and was significantly upregulated after exposure to pathogens, such as Escherichia coli, Staphylococcus aureus and Beauveria bassiana. Furthermore, the knockdown of RfGNBP5 potentiated the clearance efficiency of hemolymph against the invading pathogenic bacteria and fungi by increasing the expression level of two antimicrobial peptide genes, including RfAttacin in fat body and RfColeoptericin in gut, and augmenting the phenoloxidase (PO) activity in hemolymph. Taken together, these results suggested that RfGNBP5 acts as a negative regulator of RPW immunity, and it might be a potential candidate for further evaluation in RNAi-based pest management.