Su(H)S269A mutants are compromised in their response to parasitoid wasp infestation

(A) Quantification of melanized crystal cells from the last two segments of Su(H)gwtand Su(H)S269A larvae with and without wasp infestation as indicated. In the control, wasp parasitism causes crystal cell numbers to drop to a level of about 50%, whereas in Su(H)S269A mutants the number settles at the un-infested Su(H)gwt level. Each dot represents one analysed larva (n=70-100 as indicated). (B) Crystal cell index in larval lymph glands is given as ratio of Hnt-positive crystal cells per 1° lobe relative to the size of the lobe. Each point represents one analysed lobus (n=15). (A,B) Statistical analyses Kruskal-Wallis test, followed by Dunn’s test with *** p<0.001, ** p<0.01, * p<0.05, ns (not significant p≥0.05).

(C-F) Quantification of larval lamellocytes in the circulating hemolymph (C,D) or in lymph glands (E,F) before and after wasp infestation in Su(H)gwt versus Su(H)S269A. Lamellocytes were marked with either PPO3-Gal4::UAS-GFP (C,E) or atilla-GFP (D,F) as indicated. (C,D) The fraction of GFP-labelled lamellocytes of the total number of DAPI-labelled blood cells isolated from hemolymph is given; each dot represents ten pooled larvae (n=8-10 as shown). Representative image of labelled control hemolymph is shown above (DAPI-labelled nuclei in light blue, GFP in green). Scale bars 50 µm. (E,F) Lamellocyte index is given as number of GFP-labelled lamellocytes per area in the 1° lobe of the lymph gland. Each dot represents the lamellocyte index of one lobus (n=15). Representative Su(H)gwt lymph glands after wasp infestation are shown, co-stained for nuclear Pzg (in blue). Scale bars 100 µm. Statistical analyses with unpaired Student’s t-test; only significant differences are indicated (*** p<0.001). (A-F) Representative images for each genotype and condition are shown in supplemental Figure 2.

(G) qRT-PCR analyses measuring expression of NRE-GFP (left panel) and atilla (right panel). Transcript levels were quantified from hemolymph isolated from infested larvae at 0-6 h or 24-30 h post-infestation as indicated, relative to the untreated Su(H)gwtcontrol. Tbp and cyp33 served as reference genes. Shown data were gained from four biological and two technical replicates each. Left panel: Immediately after wasp infection, NRE-GFP expression dropped significantly in the Su(H)gwt control, and even further to about 30% 24-30 h post-infection, whereas it remained at 60%-70% in the infested Su(H)S269Amutants. Right panel: atilla transcripts remained stable at first in the Su(H)gwt control, to rise dramatically 24-30 h post-infection, in contrast to Su(H)S269A. Mini-max depicts 95% confidence, mean corresponds to expression ratio. Exact p-values are given in the raw data table. Significance was tested using PFRR from REST (*p<0.05).

Resistance to wasp infestation and phosphorylation at S269

(A) Resistance of Su(H)gwt and Su(H)S269A to the infestation with parasitic wasp strains L. boulardi and L. heterotoma (both family Figitidae) and A. japonica (family Braconidae), as indicated. Numbers of eclosed flies versus wasps as well as of dead pupae are presented in relation to the total of infested pupae. Left two columns are from non-infested controls. At least three independent experiments were performed, n=number of infested pupae. Statistically significant difference determined by Student’s t-test is indicated with *p<0.05.

(B) Su(H) is phosphorylated at Serine 269 upon parasitoid wasp infestation. Protein extracts from Su(H)gwt-mCh (wt) and Su(H)S269A-mCh (SA) larvae, respectively, infested (wtinf, SAinf) with L. boulardi or uninfested, were isolated by RFP-Trap precipitation and probed in Western blots. The anti-pS269 antiserum specifically detects wild type Su(H) protein only in wasp infested larvae (arrowhead), but not the Su(H)S269A isoform. The blot on the right served as loading control, probed with anti-mCherry antibodies, revealing the typical Su(H) protein pattern in all lanes; the lowest band presumably stems from degradation (open asterisk). M, prestained protein ladder, protein size is given in kDa.

Pipeline of screening procedures for kinase candidates triggering phosphorylation at Su(H)S269

(A) In silico screening of database(s) predicting kinase recognition motif in Su(H)S269; see Table 1 supplement. (B) In vitro assay screening 245 human Ser/Thr kinases for their ability to phosphorylate the BTD domain of Su(H); see Table 2 supplement. (C) In vivo screen of 44 different Drosophila kinase mutants for crystal cell occurrence in third instar larvae; see Table 4 supplement. (D) NanoLC/ESI mass spectrometry with active human kinases monitoring their ability to phosphorylate the given Su(H) peptide. PKCα phosphorylates S269, whereas AKT1, CAMK2D and S6 kinase prefer T271. Spectra are shown in Figure 3_ supplement 2.

Kinase assays using activated PKCα and Drosophila Pkc53E variants

(A) Right, schema of ADP-GloTM assay to quantify kinase activity. The wild type (Swt) and mutant (SSA) Su(H) peptides 262-276 offered as specific kinase substrates are indicated above. Left, the commercially available, active human PKCα very efficiently phosphorylates the pseudo-substrate PS and the Su(H) Swt peptide, but less efficiently the SSA mutant peptide. Activity is given as percentage of the auto-active kinase without substrate. Each dot represents one experiment. (B) Bacterially expressed Pkc53E has no activity on any of the offered substrates PS, Swt or SSA. (C) PMA raised Pkc53E activity to nearly 125% for PS and Swt but not for SSA. (D) Activated Pkc53EDDD phosphorylates PS and Swt but not of SSA. (E) Addition of PMA does not change Pkc53EDDD activity. Statistical analyses were performed with Kruskal-Wallis test followed by Dunn’s test (A,C,E) or ANOVA followed by Tukey’s approach in (B,D) with ** p<0.01, ns (not significant p≥0.05).

PMA inhibits Su(H) transcriptional activity in vitro and crystal cell formation in vivo

(A) Expression of NRE-luciferase reporter gene in RBPjko HeLa cells, transfected with 2xMyc-Su(H)-VP16 [Su(H)VP16]. Luciferase activity is given relative to the reporter construct normalized to Su(H)-VP16 set to 100%. Addition of PMA causes reduction of Su(H)-VP16 dependent transcriptional activity to about 40%, which is reversed by the kinase inhibitor Staurosporine (STAU). STAU itself results in increased Su(H)-VP16 activity. Each dot represents one experiment (n=6). Statistical analysis was performed with ANOVA followed by Dunnet’s multiple comparison test with *** p<0.001, ** p<0.01 relative to Su(H)-VP16 alone (black asterisks) or to Su(H)-VP16 plus PMA (blue asterisks).

(B) Number of melanized larval crystal cells determined in the last two segments of larvae fed with fly food plus 10% DMSO (control), or with fly food supplemented with 1mM PMA, or 1 mM PMA plus 0.2 mM STAU (n=20 or 30 as indicated). Note strong drop of crystal cell numbers in the Su(H)gwt control fed with PMA, and a reversal by STAU addition even above control levels. In contrast, PMA has a small effect on crystal cell number in the Su(H)S269Amutant, which is reversed by STAU. Representative animals are shown above; scale bar 250 µm. Statistical analysis was performed by ANOVA followed by Tukey’s multiple comparison test (*** p<0.001), significant differences are colour coded.

Loss of Pkc53E causes a gain of crystal cell number

Depletion of Pkc53E activity in the Pkc53EΔ28 mutant or after knockdown by Pkc53E-RNAi or sgPkc53E with the help of the Gal4-UAS system using lz-Gal4 (A) or hml-Gal4 (B). Controls as indicated. (A) Crystal cell index in lymph glands; each dot represents the value of an analysed lobus (n> 20 as indicated). Examples of Pkc53EΔ28, lz::Pkc53-RNAi and lz::Cas9 sgPkc53E are shown above. Crystal cells are labelled with Hnt (green), the lobe is stained with α-Pzg (blue). Scale bar 50 µm. Representative images of lymph glands for each genotype are shown in supplemental Figure S2A. Statistical analysis by ANOVA followed by Tukey’s multiple comparison test relative to controls with *** p<0.001.

(B) Melanized crystal cells enumerated from the last two segments of larvae with the given genotype (n=45-70 as indicated). Examples of respective Pkc53EΔ28, lz::Pkc53-RNAi and for each genotype are shown in supplemental Figure S2B. Statistical analysis by Kruskal-Wallis test, followed by Dunn’s test relative to controls with *** p<0.001. Note that there were no significant differences between any of the controls shown in black.

Pkc53E interacts with Su(H) at a genetic and a physiological level

(A) Larval crystal cell numbers and (B) crystal cell indices in lymph glands were determined in the given genotypes. Each dot represents one analysed larva (n, as indicated) (A) or lymph gland lobus (n=12) (B Statistical analysis by Kruskal-Wallis test, followed by Dunn’s test relative to controls with *** p<0.001; p≥0.05 ns (not significant). Representative images of sessile crystal cells and of lymph glands for each genotype are shown in Figure supplement S1.

(C,D) Co-immuno-precipitation of Pkc53EHA with Su(H)gwt-mCh protein. RFP-Trap IP was performed with protein extracts from 400 heads (C) or 25 third instar larvae (D), respectively. UAS-Pkc53E-HA expression was induced with Gmr-Gal4 in the head or with hml-Gal4 in the hemolymph. Endogenous mCherry-tagged Su(H) was trapped and detected with anti-mCherry antibodies (black arrowheads). The lowest band from the hemolymph is presumably a degradation product (open arrowhead in (D)). HA-tagged Pkc53E was specifically co-precipitated as detected with anti-HA antibodies (arrow). 10% of the protein extract (PE) used for the IP-Trap was loaded for comparison. BC corresponds to the Trap with only agarose beads as a control. M, pre-stained protein ladder; protein size is given in kDa.

Pkc53E-eGFP is expressed in the cytoplasm of all hemocytes

Hemocytes derived from Pkc53E-eGFP expressing larvae, either infested or not infested with L. boulardi were stained with the antibodies and compounds indicated. GFP was detected directly without further antibody staining. (A) Pkc53E-eGFP is present in the cytoplasm of hemocytes independent of wasp infection. Note complete overlap with Hemese (red) marking all types of blood cells; Putzig (blue) labels nuclei. Asterisk denotes lamellocyte in hemolymph of infected larvae. Arrows point to nuclei expressing Pkc53E-eGFP. (B) Pkc53E-eGFP is expressed in the cytoplasm of a lamellocyte (asterisk), labelled with Myospheroid (blue) and Rhodamine-coupled Phalloidin (red). (C) Pkc53E-eGFP is enriched in the cytoplasm of crystal cells (arrow), labelled either with Hnt (red) or PPO1 (red), as indicated. Wheat germ agglutinin (WGA, blue) served to label nuclear lamina. Scale bar, 25 µm.

The Pkc53EΔ28 null mutant is immune compromised

(A) Resistance of Pkc53EΔ28 and the double mutant Su(H)S269A Pkc53EΔ28, compared to Su(H)gwt and Su(H)S269A for control, to the infestation with parasitic wasp strain A. japonica. Numbers of eclosed wasps versus flies as well as of dead pupae are presented in relation to the total of infested pupae (n, number of infested pupae). Statistical analysis by ANOVA followed by Tukey’s multiple comparison test relative to control Su(H)gwt; * p<0.05; *** p<0.001. (B,C) Quantification of lamellocytes labelled with the atilla-GFP reporter in the circulating hemolymph (B) or in the lymph glands (C), in un-infested conditions or upon wasp infestation as indicated. Representative images of hemolymph and of lymph glands for each genotype are shown in the supplement S2. (B) Fraction of GFP-positive lamellocytes relative to the total of DAPI-stained hemocytes in the pooled hemolymph from ten larvae. Each dot represents one larval pool (n, number of experiments as shown). (C) Lamellocyte index, i.e. number of GFP-labelled cells relative to the size of the lymph gland (n=15). Statistical analysis by ANOVA followed by Tukey’s multiple comparison test; *** p<0.001; significant differences are colour coded.