Abstract
IKKγ/NEMO is the regulatory subunit of the IκB kinase (IKK) complex, which regulates the NF-κB signaling pathway. Within the IKK complex, IKKγ/NEMO is the non-catalytic subunit, whereas IKKα and IKKβ are the structurally related catalytic subunits. In this study, TmIKKγ was screened from the Tenebrio molitor RNA-Seq database and functionally characterized using RNAi screening for its role in regulating T. molitor antimicrobial peptide (AMP) genes after microbial challenges. The TmIKKγ transcript is 1521 bp that putatively encodes a polypeptide of 506 amino acid residues. TmIKKγ contains a NF-κB essential modulator (NEMO) and a leucine zipper domain of coiled coil region 2 (LZCC2). A phylogenetic analysis confirmed its homology to the red flour beetle, Tribolium castaneum IKKγ (TcIKKγ). The expression of TmIKKγ mRNA showed that it might function in diverse tissues of the insect, with a higher expression in the hemocytes and the fat body of the late-instar larvae. TmIKKγ mRNA expression was induced by Escherichia coli, Staphylococcus aureus, and Candida albicans challenges in the whole larvae and in tissues such as the hemocytes, gut and fat body. The knockdown of TmIKKγ mRNA significantly reduced the survival of the larvae after microbial challenges. Furthermore, we investigated the tissue-specific induction patterns of fourteen T. molitor AMP genes in TmIKKγ mRNA-silenced individuals after microbial challenges. In general, the mRNA expression of TmTenecin1, -2, and -4; TmDefensin1 and -2; TmColeoptericin1 and 2; and TmAttacin1a, 1b, and 2 were found to be downregulated in the hemocytes, gut, and fat body tissues in the TmIKKγ-silenced individuals after microbial challenges. Under similar conditions, TmRelish (NF-κB transcription factor) mRNA was also found to be downregulated. Thus, TmIKKγ is an important factor in the antimicrobial innate immune response of T. molitor.
Highlights
Unlike vertebrates, insects rely on their innate defense mechanisms for pathogen surveillance and immunity
The cDNA sequence of T. molitor IKKγ (TmIKKγ) was determined by a local tblastn search against the T. molitor genome and the expressed sequence tag database using the T. castanuem IKKγ (TcIKKγ) protein sequence (EEZ99267.2) as a query
The results suggest the requirement of TmIKKγ mRNA against bacterial and fungal challenges in T
Summary
Insects rely on their innate defense mechanisms for pathogen surveillance and immunity. AMPs are induced through the activation of two key signaling pathways, namely the toll and immune deficiency (IMD) pathways. In humans and most other vertebrates, the mode of action of the toll and IMD pathways are clearly identified. Ten (TLR1–10) and twelve (TLR1–9 and 11–13) functional toll-like receptors (TLRs) are known, respectively. TLRs provide protection against a wide variety of pathogens by regulating signaling through adaptor proteins, including the myeloid differentiation factor 88 (MyD88), TIR-domain-containing adapter-inducing interferon-β (TRIF), TRIF-related adaptor molecule (TRAM), and toll-interleukin 1 receptor domain containing adaptor protein (TIRAP). The toll and IMD pathways do not appear to share any intermediate components and regulate differential expression of AMP-encoding genes via distinct NF-κB-like transcription factors [14,15]
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