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)gwt and 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). (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). Statistical analyses with ANOVA for multiple comparisons, using Tukey-Kramer approach with *** p≤0.001, 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. 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=12).

Representative Su(H)gwt lymph glands after infestation are shown above, co-stained for nuclear Pzg (in blue). Scale bars 100 µm. Statistical analyses with ANOVA using Dunnet’s approach relative to Su(H)gwt control; only significant differences are indicated (*** p≤0.001).

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. 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≤0.01.

(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) or not with L. boulardi, were isolated by RFP-Trap precipitation and probed in Western blots. The α-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 serves as loading control, probed with α-mCherry antibodies, revealing the typical Su(H) protein pattern in all lanes; the lowest band presumably stems from degradation (open arrowhead). 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 supplement Table 1. (B) In vitro assay screening 245 human Ser/Thr kinases for their ability to phosphorylate the BTD domain of Su(H); see supplement Table 2. (C) In vivo screen of 44 different Drosophila kinase mutants for crystal cell occurrence in third instar larvae; see supplement Table 4. (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 supplement 1.

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, commercially available, active PKCα very efficiently phosphorylates the pseudo-substrate PS and the Su(H) Swt peptides, but less efficiently the SSA mutant peptide. Activity is given as percentage of the auto-active kinase without substrate. (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 triggers phosphorylation of PS and Swt but not of SSA. (E) Addition of PMA does not change Pkc53EDDD activity.

PMA induced activation of Pkc53E inhibits Su(H) transcriptional activity in vitro and crystal cell formation in vivo

(A) 2xMyc-Su(H)-VP16 (SuH)VP16), transfected into RBPjko HeLa cells, activates expression of a luciferase-reporter gene independent of Notch activity. Luciferase activity is given relative to the value of the reporter construct alone; the Su(H)-VP16 value is taken as 100%. Addition of PMA reduces Su(H)-VP16 dependent transcriptional activity to about 40%, which is reversed by the inhibitor Staurosporine (STAU). STAU itself increases Su(H)-VP16 activity nearly twofold. Six independent experiments were performed. Bars show the mean, error bars indicate standard deviation. Statistical analysis was performed with a two-way ANOVA for multiple comparisons, Dunnet’s test with *** p≤0.001, ** p≤0.01 relative to Su(H)-VP16 alone.

(B) Number of melanized larval crystal cells determined in the last two segments of larvae fed with normal fly food, or with fly food plus PMA (n=20). Note strong drop of crystal cell numbers in the Su(H)gwt control fed with PMA, in contrast to the Su(H)S269A mutant larvae, where numbers drop to only control level. Representative animals are shown above; scale bar 250 µm. Statistical analysis by two-way ANOVA for multiple comparisons, Tukey-Kramer approach (*** 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 knock down 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= 20-25). Representative 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.

(B) Melanized crystal cells enumerated from the last two segments of larvae with the given genotype (n=45-70). Representative examples of the evaluated last segments of Pkc53EΔ28, lz::Pkc53-RNAi and lz::Cas9 sgPkc53E are shown above. Scale bar 250 µm. ANOVA for multiple comparisons, Dunnet’s approach 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) and is immune compromised

(A) Larval crystal cell number and (B) crystal cell indices in lymph glands were determined in the given genotypes. Each dot represents one analysed larva (n=33-70) in (A) or lymph gland lobus (n=12) in (B). ANOVA for multiple comparisons, Tukey-Kramer’s approach relative to controls with *** p≤0.001; p>0.05 ns (not significant).

(C,D) Co-immuno-precipitation of Pkc53EHA with Su(H)gwt-mCh protein. RFP-Trap IP was performed on protein extracts from 400 heads (C) and 25 third instar larvae (D), respectively. UAS-Pkc53E-HA expression was induced with Gmr-Gal4 in the head or hml-Gal in the hemolymph. Endogenous mCherry-tagged Su(H) was trapped and detected with α-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 describes the Trap with only agarose beads as a control. M, pre-stained protein ladder; protein size is given in kDa.

(E,F) Quantification of lamellocytes labelled with the atilla-GFP reporter in the circulating hemolymph (E) or in the lymph glands (F), in un-infested conditions or upon wasp infestation as indicated. (E) 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=12 experiments). (F) Lamellocyte index, i.e. number of GFP-labelled cells relative to the size of the lymph gland (n=15). Statistical analysis by ANOVA multiple comparisons with Tukey-Kramer’s test; *** p≤0.001.