The Daisho Peptides Mediate Drosophila Defense Against a Subset of Filamentous Fungi

Abstract

Fungal infections, widespread throughout the world, affect a broad range of life forms, including agriculturally relevant plants, humans, and insects. In defending against fungal infections, the fruit fly Drosophila melanogaster employs the Toll pathway to induce a large number of immune peptides. Some have been investigated, such as the antimicrobial peptides (AMPs) and Bomanins (Boms); many, however, remain uncharacterized. Here, we examine the role in innate immunity of two related peptides, Daisho1 and Daisho2 (formerly IM4 and IM14, respectively), found in hemolymph following Toll pathway activation. By generating a CRISPR/Cas9 knockout of both genes, Δdaisho, we find that the Daisho peptides are required for defense against a subset of filamentous fungi, including Fusarium oxysporum, but not other Toll-inducible pathogens, such as Enterococcus faecalis and Candida glabrata. Analysis of null alleles and transgenes revealed that the two daisho genes are each required for defense, although their functions partially overlap. Generating and assaying a genomic epitope-tagged Daisho2 construct, we detected interaction in vitro of Daisho2 peptide in hemolymph with the hyphae of F. oxysporum. Together, these results identify the Daisho peptides as a new class of innate immune effectors with humoral activity against a select set of filamentous fungi.

Keywords: Drosophila; antifungal; humoral; innate immunity; toll.

Figure 1

Deletion of Drosophila daisho1 and daisho2 gene pair. (A) Alignment of mature Daisho1 and Daisho2 peptide sequences. Identical residues are highlighted. (B–E) Mass spectrometry analysis of Toll-induced hemolymph in linear (B,C) and reflectron (D,E) mode, illustrating loss of Daisho1 (Dso1, formerly IM4) and Daisho2 (Dso2, formerly IM14) signal in Δdaisho deletion mutant. The Dso1 signal overlaps with the BomS5 signal, which is readily apparent in the Δdaisho mutant analyzed in reflectron mode. Mtk, Metchnikowin; Drs, Drosomycin.

Figure 2

Survival of Δdaisho against E. faecalis (A), C. glabrata (B), E. cloacae (C), and F. oxysporum (D) infection. Shown is the combination of three independent experiments for each pathogen with 20-25 flies per genotype per experiment. Survival curves were compared using the Gehan-Breslow-Wilcoxon test. Significance is shown relative to w¹¹¹⁸ (***p < 0.0001; n.s., not significant; p > 0.01).

Figure 3

Survival of Δdaisho against F. verticillioides, (A), F. graminearum (B), A. parasiticus (C), A. flavus (D), A. fumigatus (E), B. cinerea (F), and N. crassa (G). The combination of three independent experiments for each pathogen with 20-25 flies per genotype per experiment is shown. Survival curves were compared using the Gehan-Breslow-Wilcoxon test. Significance is shown relative to w¹¹¹⁸ (***p > 0.0001; n.s., not significant; p > 0.01).

Figure 4

MALDI-TOF spectra for Δdso1 and Δdso2 hemolymph. (A,B) Mass spectrometry analysis of Toll-induced hemolymph in linear mode, highlighting loss of Dso1 (A) and Dso2 (B) in deletion mutants.

Figure 5

Survival of Δdso1 and Δdso2 against F. verticillioides. Shown is the combination of three independent experiments with 20–25 flies per genotype per experiment. Survival curves were compared using the Gehan-Breslow-Wilcoxon test. Significance is shown relative to w¹¹¹⁸ (***p < 0.0001).

Figure 6

Characterization of FLAG-Dso2 gene product. (A) Immunoblot stained with mouse α-FLAG M2 (1:500) and sheep α-mouse HRP (1:1,000). Two μl of Toll-induced hemolymph was loaded per lane. (B) MALDI-TOF analysis of FLAG-Dso2 Toll-induced hemolymph in linear mode.

Figure 7

Immunofluorescence staining of F. oxysporum hyphae. Images showing various staining patterns of hyphae incubated with FLAG-Dso2 (A–D) or w¹¹¹⁸ hemolymph (E–H) and then stained with mouse α-FLAG M2 (1:200) and donkey α-mouse Alexa 555 (1:400). DAPI marks fungal DNA. Scale bar is 10 μm. Images were generated as focused images from Z-stacks.