Functional Characterization And Mechanism Studies Of BmFAF, A Negative Immune Regulator In Silkworm, Bombyx Mori

Silkworm innate immunity mainly includes humoral immunity, cellular immunity and melanization. The hallmark of humoral immunity againsts the invading microbes is production of antimicrobial peptides, which is induced through the activation of the critical transcription factors Dorsal/Dif or Relish in Toll or Imd signaling pathway, respectively. The immune response is under both positive and negative regulation to maintain the immune homeostasis. Studies of Drosophila melanogaster, the model organism of Dipteran insects, discovered various negative immune regulators which reduce the activity of nuclear transcription factors or prevent them from nuclear translocation through mutation screening, knockout or transgenic technologies. There are only a few related reports in other insects. Using silkworm, Bombyx mori as a Lepidopteran model organism to study negative immune regulation is necessary for better understanding the mechanism of insect immunity.Our study focuses on functional characterization and mechiansm studies on BmFAF, which displays homology with Caspar, a negative regulator of fruitfly and is implicated in immunhomeostasis reported by a previous study. We evaluated its properties by spatio-temporal expression profiles and immune induction expression profiles, and further demonstrated it through RNA interference and overexpression at cell and individual level. At the same time, we explored the molecular mechanism and identified the key functional domains through biochemical assays including immunoprecipitation, Western blotting and luciferase assay. The main results are as follows:1. BmFAF is the immune regulatory molecule of Bombyx moriBmFAF (BGIBMGA008874) had a lower expression level in immune tissues such as midgut and fat body, and it was much more highly expressed in molting larvaes than in newly exuviated ones. At 0.5 h after microbial infection, BmFAF was significantly down regulated, whereas the expression level of antibacterial peptide CecropinAl was up regulated, implying that BmFAF was a negative regulatory molecule, In BmE cells, when BmFAF was overexpressed, the antibacterial peptides such as Moricin, Attacin and CecropinAl were significantly down regulated. If BmFAF was silenced by RNAi, the antibacterial peptides were remarkbly up regulated. RNAi at the individual level also resulted in up-regulation of the antibacterial peptides and higher survival rate. Overexpression and RNAi experiments further verifyed that BmFAF suppressed the production of antibacterial peptides.2. BmFAF promotes Relishact degradation by ubiquitination to suppress the IMD pathway.We used Western blotting and luciferase assay to verify that Dredd-dependent cleavage of Relish-FL into the active Relishact increases the expression level of the downstream antimicrobial peptides, while BmFAF would suppress the production of antimicrobial peptides. Further research showed that, BmFAF inhibited the activity of NF-κB by promotting Relishact degradation through ubiquitination pathway. GST pull-down and immunoprecipitation experiments demonstrated that BmFAF was capable of binding with ubiquitinated proteins, which led to the inhibition of the activity of Relishact.3. UBA domain is the key domain supress IMD pathway.We constructed five truncated mutatants of BmFAF and found that BmFAF negatively regulated the activity of Relishact via its UBA domain by western blotting and luciferase assay. And we performed immunoprecipitation to confirm that UBA domain has the potential to be ubiquinated.