Expression of the lectin-24A reporter VenusLP437.

(A) Expression of VenusLP437 24 hours post-infection (hpi) with different pathogens, left to right: Leptopilina boulardi parasitoid wasps, Escherichia coli bacteria, and Drosophila C virus. The relative fluorescence intensity represents the fluorescence intensity normalized with total protein quantity of each sample. Fluorescence was measured in homogenized larvae with a plate reader in a 96-well plate. Each point represents a pool of 15 larvae. (B) Expression pattern of the reporter VenusLP437 in the Drosophila larval fat body. Microscopy imaging of larval fat bodies dissected from uninfected and infected larvae, at 0, 12, 24, and 48 hpi. Fat bodies are shown with posterior pointing up unless indicated by the direction of white arrows. Green represents Venus expression, and blue represents Hoescht nuclear staining. Scale bars represent 500μm.

RNA sequencing of larval fat body sections.

(A) Dissection of male larval fat bodies along the representative red lines into anterior and posterior sections, where the developing gonads were removed, for RNA sequencing. (B) Differentially expressed genes, with absolute log2FC above 1 and False Discovery Rate (FDR) below 0.05, between anterior and posterior larval fat body sections under infected condition. (C) Venn diagram showing overlap of significantly differentially expressed genes with absolute log2FC above 1 after infection in anterior and posterior fat body sections.

Gene expression patterns of components from major immune pathways in anterior and posterior sections with and without infection.

Expression shown as Reads Per Kilobase of transcript per Million mapped reads (RPKM). Labels denote significant (genome-wide false discovery rate < 0.05) differential expression with absolute log2FC of at least 1 between treatment groups. Only significant differences are shown.

Transcription factor binding motifs (TFBMs) upstream of lectin-24A.

(A) Truncated lectin-24A reporter Venus expression compared to the full-length reporter constructs with the susceptible DGRP-892 (LP892; blue) and resistant DGRP-437 (LP437; red) alleles. (B) Yeast one-hybrid screen results showing proteins that bind to the sequence 444 bp upstream of the resistant DGRP-437 lectin-24A allele and/or the susceptible DGRP-892 lectin-24A allele. Dif ¦ dl represents a heterodimer. (C) Predicted TFBMs of transcription factors from the JAK/STAT, NF-κB, and GATA pathways upstream of lectin-24A. The STAT consensus sequence shown corresponds to the conserved STAT binding site “TT(n)AA”, with 5-6 variable n sites in the middle. (D) Expression of the reporter constructs with the 314 bp upstream sequence of lectin-24A where predicted NF-κB, GATA, and three of the STAT TFBMs are scrambled. The scrambled STAT sites are indicated with asterisks in (C). Each point represents a pool of 10 larvae. The relative fluorescence intensity represents the fluorescence intensity normalized with total protein quantity of each sample.

The role of the JAK/STAT pathway in the regulation of lectin-24A.

In all panels the VenusLP437 reporter expression was measured. (A) Larvae with knockdown of Stat92E using RNAi, compared to the knockdown of the white gene as a control. (B) Larvae carrying a mutant or wildtype (WT) form of the JAK/STAT negative regulator et. (C) Larvae expressing a dominant negative form of the JAK/STAT receptor Dome (DomeΔCYT) in the fat body and hemocytes under the control of the Cg-GAL4 driver, with the ubiquitous da-GAL4 driver, and with no driver. As the parental stock expressing UAS-DomeΔCYT also expresses the JAK/STAT negative regulator et under UAS control on a balanced chromosome II, a portion of the larvae also expresses UAS-et. Larvae expressing GAL4 drivers also expressed tub-GAL80ts. (D) Larvae expressing the constitutively active hop (hopTum) compared to larvae expressing the wild type (WT) hop. (E) Larvae expressing Act-Cas9 and guide RNA targeting hop compared to control larvae. Each point represents a pool of 10 larvae. The relative fluorescence intensity represents the fluorescence intensity normalized with total protein quantity of each sample. All infected larvae were collected at 24 hpi and uninfected age-matched larvae were collected for the controls. Tukey’s test for each panel, P < 0.05 between groups.

The role of the GATA transcription factor, Pannier, in the regulation of lectin-24A.

The VenusLP437 reporter expression was measured. (A) Larvae expressing the dominant positive form of Pnr (PnrD4) under UAS control driven by the fat body and hemocyte-specific Cg-GAL4 driver or no driver. (B) Larvae expressing Act-Cas9 and guide RNA targeting pnr compared to control larvae. Each point represents a pool of 10 larvae. The relative fluorescence intensity represents the fluorescence intensity normalized with the total protein quantity of each sample. Tukey’s test for each panel, P < 0.05 between groups.

The role of NF-κB transcription factors in the regulation of lectin-24A.

The VenusLP437reporter expression was measured. (A) Larvae expressing Act-Cas9 and guide RNA targeting dl compared to control larvae. The same WT control samples were used here as in Figure 6B. (B) Larvae expressing Act-Cas9 and guide RNA targeting Dif compared to control larvae. (C) Larvae expressing dl or Rel under UAS control driven by the fat body and hemocyte-specific Cg-GAL4 driver or no driver. F1 heterozygote larvae were collected. As the parental line expressing UAS-dl carried a balancer chromosome that could not be identified at the larval stage in F1 progeny, larvae collected from the UAS-dl crosses were a mix of Cg-GAL4/UAS-dl and Cg-GAL4/+. Each point represents a pool of 10 larvae. The relative fluorescence intensity represents the fluorescence intensity normalized with the total protein quantity of each sample. Tukey’s test for each panel, P < 0.05 between groups.

Schematic overview of Drosophila humoral defenses to microbial pathogens and parasitoid.

Antimicrobial responses in the fat body are regulated by the NF-κB pathways, Toll and Imd. The Imd pathway is mostly activated by Gram-negative bacteria through the PGRP-LC receptor, whereas the Toll pathway is largely activated by fungi and Gram-positive bacteria through the Toll receptor. Activation of the Imd pathway leads to the NF-κB transcription factor Relish translocating into the nucleus after its cleavage and activation, and the transcription factors Dif and Dorsal are activated through Toll pathway signaling. These NF-κB transcription factors along with the GATA transcription factor Serpent induce the expression of genes encoding antimicrobial effectors in the fat body via closely linked GATA and κB binding sites in the upstream promoter region. The Drosophila humoral anti-parasitoid response is regulated by a combination of inputs from JAK/STAT, GATA and NF-κB pathways. After recognition of parasitism, hemocytes produce the cytokines, Upd2 and Upd3, which bind to the receptor Dome to activate JAK/STAT pathway signaling in the fat body. Dome dimerization after ligand binding leads to trans-phosphorylation of Hop, which then activates the transcription factor STAT92E. STAT92E binds the upstream region of anti-parasitoid genes including lectin-24A. ET and SOCS36E are negative regulators of the JAK/STAT pathway that suppress this signaling. STAT, GATA, and NF-κB TFBMs are found to be enriched in the upstream sequences of parasitism-induced genes. The GATA transcription factor Pannier and the NF-κB transcription factor Dorsal also participate in the regulation of anti-parasitoid genes in the larval fat body by binding to their respective binding sites in the promoter region.