Volume 4, Issue 5, Pages 827-837 (November 1999) Relish, a Central Factor in the Control of Humoral but Not Cellular Immunity in Drosophila  Marika Hedengren, BengtÅsling, Mitchell S Dushay, Istvan Ando, Sophia Ekengren, Margareta Wihlborg, Dan Hultmark  Molecular Cell  Volume 4, Issue 5, Pages 827-837 (November 1999) DOI: 10.1016/S1097-2765(00)80392-5

Figure 1 Genomic Map of the 85C Region and of Relish Deletion Mutants (A) The transcripts of Relish and neighboring genes. The coordinates are in kb, in the upper panel as in Kim-Ha et al. 1991 and Macdonald 1992 and in the expanded part using the EcoRI site in Relish as origin. Transcripts in the Relish region were mapped by comparing the sequenced part of cosmid 1698 (thick line) with cDNA sequences. The 3′ ends of the Mst85C cDNA clones were not sequenced, and the structure in this part is based on the assumption that no further introns occur. The positions of other transcripts are from Boulianne et al. 1991, Kim-Ha et al. 1991, Macdonald 1992, Price and Laughon 1993, Sonnenfeld et al. 1997. (B) A detailed map of the deletion mutants and of the Relish and Nmdmc transcripts. In (B), the coding parts of the transcripts are shown as boxes, and conserved domains in the Relish open reading frame are indicated: the Rel homology domain (RHD) and ankyrin repeats (Ank). Molecular Cell 1999 4, 827-837DOI: (10.1016/S1097-2765(00)80392-5)

Figure 2 Expression of the Relish and Nmdmc Genes in the Different Excision Lines (A) The expression of Relish was assayed by Northern blot. Each lane contains 15 μg of total RNA from a batch of female (F) or male (M) flies that are either untreated (−) or induced by a bacterial injection (+). Canton-S (CS) or mutant flies were used as indicated. In the upper panel, the filter is probed for Relish expression. In the lower panel, the same filter was reprobed with an actin probe as loading control. (B) Nmdmc transcripts A and B were assayed by RT-PCR, using RNA samples from induced female flies. A control, C, with inactivated reverse transcriptase and RNA from E23 was included to check that the amplified bands do not derive from genomic DNA in the RNA samples. Molecular Cell 1999 4, 827-837DOI: (10.1016/S1097-2765(00)80392-5)

Figure 3 Expression of Immune Genes in Different Relish Mutants The induction of different immune genes is assayed by Northern blot. (A) shows the effect of Relish mutants on the expression of antimicrobial genes. (B) shows the rescue of the immune response after Relish overexpression, and (C) shows Dif expression in the mutant flies. In (A), each lane contains 6 μg of total RNA from female (F) or male (M) flies, untreated (−) or induced by a bacterial injection for 6 hr (+). In (B) and (C), we loaded 15 μg RNA from equal numbers of males and females, and the time of induction was 3 hr. Canton-S (CS), the indicated Relish mutants, or RelishE20 with a Relish transgene (Rel) were used. Probes: Cecropin A1, CecA1; Diptericin, Dpt; Attacin A, AttA; Drosomycin, Drs; Metchnikowin, Mtk; Actin 5C, Act5C; Relish; Dif. Molecular Cell 1999 4, 827-837DOI: (10.1016/S1097-2765(00)80392-5)

Figure 4 Survival of Wild-Type and Relish Mutant Flies after Injections of Different Doses of Bacteria Batches of 150–200 adult flies, 2–5 days old, were injected with suspensions of live E. cloacae β12 at different concentrations and incubated at 22°C–24°C. The number of bacterial cells received per fly was approximately 2 × 104 (line with diamonds); 2 × 103 (line with triangles); 2 × 102 (line with asterisks); 20 (line with plus symbols); 2 (line with minus symbols); or 0.2 (line with boxes). Controls were injected with 0.1 × L broth + 1.5 μg/ml nalidixic acid (line with crosses). There was some initial lethality due to the injection procedure itself, and the number of living flies 1 hr after injection was therefore set to 100%. Molecular Cell 1999 4, 827-837DOI: (10.1016/S1097-2765(00)80392-5)

Figure 5 Survival of Wild-Type and Relish Mutant Flies after Injections of Different Fungi Batches of 50 flies, 3–5 days old, were injected with approximately 100–200 spores of Geotrichum candidum, Dipodascopsis uninucleata, Beauveria bassiana, or Metarhizium anisopliae and incubated at 25°C. Ringer's solution was injected as a control. The number of living flies at 41 hr was set to 100%. Molecular Cell 1999 4, 827-837DOI: (10.1016/S1097-2765(00)80392-5)

Figure 6 Phagocytosis of Bacteria by Hemocytes of Mutant and Wild-Type Larvae Third instar larvae were injected with FITC-labeled dead Staphylococcus saprophyticus. Larvae were bled, and live cells were observed in bright field (left) or in fluorescent light after quenching the fluorescence of extracellular bacteria with ethidium bromide (right). Similar results were observed with Escherichia coli and Enterobacter cloacae. Practically all plasmatocytes contain phagocytosed bacteria. Molecular Cell 1999 4, 827-837DOI: (10.1016/S1097-2765(00)80392-5)

Figure 7 Possible Roles of Relish in the Signal Pathways of the Humoral Immune Response Two hypothetical models are presented. In (A), Relish is required for signaling in each of three independent pathways, whereas in (B) only one pathway depends on Relish. The Enterobacter injections we have used may have preferentially stimulated the imd pathway, as indicated in (B). This would be in agreement with the observation that the choice of microorganism and route of delivery can influence the relative stimulation of the different pathways (Lemaitre et al. 1997). The models are simplified, and further elaboration is required to accommodate all known facts. For instance, since dominant active Toll mutations do not activate Cecropin gene expression (Lemaitre et al. 1996), it is possible that Cecropin requires two independent signals for induction. Dorsal may play a redundant role with Dif, at least in larvae (Manfruelli et al. 1999). The information is still incomplete for Metchnikowin, Drosocin, and Defensin, and therefore these genes were not included in the scheme. Molecular Cell 1999 4, 827-837DOI: (10.1016/S1097-2765(00)80392-5)