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. 2013 Apr 15;8(4):e62052.
doi: 10.1371/journal.pone.0062052. Print 2013.

dRYBP contributes to the negative regulation of the Drosophila Imd pathway

Ricardo Aparicio  1 , Claudine Neyen, Bruno Lemaitre, Ana Busturia
Affiliations

dRYBP contributes to the negative regulation of the Drosophila Imd pathway

Ricardo Aparicio et al. PLoS One. .

Abstract

The Drosophila humoral innate immune response fights infection by producing antimicrobial peptides (AMPs) through the microbe-specific activation of the Toll or the Imd signaling pathway. Upon systemic infection, the production of AMPs is both positively and negatively regulated to reach a balanced immune response required for survival. Here, we report the function of the dRYBP (drosophila Ring and YY1 Binding Protein) protein, which contains a ubiquitin-binding domain, in the Imd pathway. We have found that dRYBP contributes to the negative regulation of AMP production: upon systemic infection with Gram-negative bacteria, Diptericin expression is up-regulated in the absence of dRYBP and down-regulated in the presence of high levels of dRYBP. Epistatic analyses using gain and loss of function alleles of imd, Relish, or skpA and dRYBP suggest that dRYBP functions upstream or together with SKPA, a member of the SCF-E3-ubiquitin ligase complex, to repress the Imd signaling cascade. We propose that the role of dRYBP in the regulation of the Imd signaling pathway is to function as a ubiquitin adaptor protein together with SKPA to promote SCF-dependent proteasomal degradation of Relish. Beyond the identification of dRYBP as a novel component of Imd pathway regulation, our results also suggest that the evolutionarily conserved RYBP protein may be involved in the human innate immune response.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Genetic modulation of dRYBP expression levels affects AMP production.
(A, B, C, D, E). (A) All tested dRYBP mutants show an excessive immune response to Gram-negative infection. Adult females of indicated genotypes were left unchallenged (left panel) or infected by pricking with Ecc15 (heat-killed at 95°C during 15 min) and collected at 8 h (middle panel) and 24 h (right panel) after infection for Diptericin (Dpt) mRNA quantification. The complete data set (unchallenged, 8 h, 24 h) was analysed by two-way ANOVA with Bonferroni post-tests, using CanS as control. (B) dRYBP mutants also show excessive Imd activation after infection with another Gram-negative pathogen, E. coli. Adult females of indicated genotypes were treated as in (A). (C) Infection time-course in wild-type, heterozygous and homozygous dRYBPΔ55 mutants. Flies were treated as in (A). (D) Loss of dRYBP affects at least two Imd-dependent antimicrobial peptides. mRNA levels of Attacin-B (AttB) were quantified after infection as described in (A). (E) Overexpression of dRYBP reduces the Imd response to Gram-negative infection. Adult females were infected as in (A), then were heat-shocked for 1 h at 37°C and collected at 8 h and 24 h after infection for quantification of Dpt mRNA levels. (F) dRYBP expression levels are not affected by infection. Wild-type adult females were infected as in (A) and dRYBP mRNA levels were monitored over time. For all graphs, data represent mean + SEM of at least 3 biological repeats, and asterisks denote the following p values: *, 0.01<p<0.05; **, 0.001<p<0.01; *** p<0.001; ns, not significant.
Figure 2
Figure 2. Loss of dRYBP does not affect expression of canonical Imd pathway components.
(A) mRNA levels of indicated Imd pathway components were quantified in wild-type and dRYBP mutant flies under unchallenged conditions (A) or (B), 8 h after infection by pricking with Ecc15. Data in (A) and (B) represent mean + SEM of two pooled biological repeats. For each gene, expression is normalized to expression in unchallenged (uc) CanS. No significant difference in gene expression between wild-type and dRYBP mutants, by two-way ANOVA with Bonferroni post-test. (C) Overexpression of dRYBP does not affect Relish expression. Adult females were infected as in (B), then were heat-shocked for 1 h at 37°C and collected at 8 h and 24 h after infection for quantification of mRNA levels of the IMD-dependent transcription factor Relish. Data represent mean + SEM of 3 biological repeats.
Figure 3
Figure 3. dRYBP localizes to the nuclei of adult fat body cells.
(A) dRYBP protein is expressed in the nuclei of fat body cells (arrows). (B) Flies overexpressing Venus-dRYBP under the fat body specific driver c564-Gal4 were infected by pricking with Ecc15. Fat bodies were dissected 3 h after infection, fixed and stained with anti-GFP antibody and DAPI. Images are representative of several UAS-Venus-dRYBP insertion lines. (C) Fluorescence profile along arrow in (B) shows dRYBP is exclusively nuclear and excluded from nucleoli. AU (Arbitrary Units). Scale bar denotes 20 µm.
Figure 4
Figure 4. Epistatic relationships between dRYBP and components of the Imd pathway.
(A) dRYBP acts downstream of IMD. Flies overexpressing dRYBP, imd, or both under the control of a heat-shock-inducible promoter were kept at 18°C (OFF) or exposed to 37°C for 1 h, then shifted to 29°C for 7 h (ON), at which point Dpt mRNA levels were assessed to measure Imd pathway activity. (B, C) Overexpression of multiple UAS constructs does not lead to Gal4 dilution. (B) shows qRT-PCR quantification of mRNA levels of dRYBP expression. dRYBP expression is increased under induced conditions (compare hs-Gal4 to hs-Gal4;UAS-dRYBP) and does not significantly change when two UAS constructs are concomitantly expressed (compare hs-Gal4;UAS-dRYBP to hs-Gal4,UAS-imd/UAS-dRYBP). (C) shows qRT-PCR quantification of mRNA levels of imd expression. imd expression is increased under induced conditions (compare hs-Gal4 to hs-Gal4,UAS-imd) and does not significantly change when two UAS constructs are concomitantly expressed (compare hs-Gal4,UAS-imd to hs-Gal4,UAS-imd/UAS-dRYBP). (D) dRYBP acts downstream of Relish. Flies overexpressing dRYBP, Rel (Relish-His-6), or both under the control of a heat-shock-inducible promoter were kept at 18°C, which induces low overexpression of all constructs. Dpt mRNA levels were assessed as a measure of Relish-dependent transcriptional activation. (E) qRT-PCR quantification of mRNA levels of dRYBP expression in the same crosses. dRYBP expression is increased when overexpressed with hs-Gal4 in flies kept at 18°C (compare hs-Gal4 to hs-Gal4;UAS-dRYBP) and does not significantly change when two UAS constructs are concomitantly expressed (compare hs-Gal4;UAS-dRYBP to hs-Gal4/UAS-Relish-His-6;UAS-dRYBP). (F) dRYBP acts upstream of SKPA. Flies overexpressing dRYBP, RNAi against skpA, or both under the control of the fat-body specific driver c564-Gal4 were either kept at 18°C (OFF) or shifted to 29°C for 24 h (ON), at which point Dpt mRNA levels were assessed to measure Imd pathway activity. (G) qRT-PCR quantification of mRNA levels of dRYBP expression. dRYBP expression is increased under induced conditions (compare c564-Gal4 to c564-Gal4;UAS-dRYBP) and does not significantly change when two UAS constructs are concomitantly expressed (compare c564-Gal4;UAS-dRYBP to c564-Gal4;UAS-dRYBP/UAS-skpARNAi). Asterisks denote the following p values: *, 0.01<p<0.05; **, 0.001<p<0.01; *** p<0.001; ns, not significant. Data represent mean + SEM of 3 biological repeats.
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References

    1. Charroux B, Royet J (2010) Drosophila immune response: From systemic antimicrobial peptide production in fat body cells to local defense in the intestinal tract. Fly (Austin) 4: 40–47. - PubMed
    1. Lemaitre B, Hoffmann J (2007) The host defense of Drosophila melanogaster. Annu Rev Immunol 25: 697–743. - PubMed
    1. Ferrandon D, Imler JL, Hetru C, Hoffmann JA (2007) The Drosophila systemic immune response: sensing and signalling during bacterial and fungal infections. Nat Rev Immunol 7: 862–874. - PubMed
    1. Tzou P, Ohresser S, Ferrandon D, Capovilla M, Reichhart JM, et al. (2000) Tissue-specific inducible expression of antimicrobial peptide genes in Drosophila surface epithelia. Immunity 13: 737–748. - PubMed
    1. Uvell H, Engstrom Y (2007) A multilayered defense against infection: combinatorial control of insect immune genes. Trends Genet 23: 342–349. - PubMed

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