Transcription Factor Forkhead Regulates Expression of Antimicrobial Peptides in the Tobacco Hornworm, Manduca sexta

Area of Science:

• Insect immunity
• Molecular biology
• Genetics

Background:

• Antimicrobial peptides (AMPs) are crucial for insect defense against microbial infections.
• NF-κB signaling pathways, involving factors like Dorsal, Dif, and Relish, primarily regulate AMP expression.
• Previous research indicated NF-κB and GATA-1 factors are essential for activating the moricin promoter in Manduca sexta.

Purpose of the Study:

• To investigate the role of forkhead (Fkh) transcription factors in regulating insect AMP gene expression.
• To identify and characterize Fkh-binding sites in the moricin and lysozyme promoters.
• To elucidate the interaction between Fkh factors and NF-κB signaling components in AMP activation.

Main Methods:

• Identification of Fkh-binding sites within the moricin and lysozyme promoter regions.
• Demonstration of Fkh factor-dependent activation of moricin and lysozyme promoters using mutant constructs.
• Analysis of Fkh mRNA expression in Drosophila S2 cells.
• Investigation of protein-protein interactions between Manduca sexta Fkh (MsFkh) and NF-κB Relish-Rel-homology domain (RHD) using co-expression studies.
• Dual luciferase assays to assess promoter activity.

Main Results:

• Three Fkh-binding sites were identified in the moricin promoter and several in the lysozyme promoter.
• Fkh-binding sites were essential for the activation of both moricin and lysozyme promoters by Fkh factors.
• Fkh mRNA was not detected in Drosophila S2 cells.
• MsFkh interacted with Relish-RHD but not Dorsal-RHD.
• Co-expression of MsFkh and Relish-RHD did not show an additive effect on moricin promoter activity, indicating independent regulation.

Conclusions:

• Forkhead (Fkh) factors play a significant role in activating insect AMP genes, such as moricin and lysozyme.
• Fkh factors regulate AMP gene expression independently of the NF-κB pathway, particularly under non-infectious conditions.
• This Fkh-mediated activation may be vital for protecting insects from microbial infections during critical developmental stages like molting and metamorphosis.