Figure 6. Expression of general control nonderepressible 2 (GCN2) in the gut is necessary for L. plantarum (Lp) to rescue the delay due to amino acid (AA) imbalance.

(A–I) Developmental timing experiments. The graphs show five replicates per condition (mean and standard deviation). Each replicate consists in one tube containing 40 larvae. (A–G) Developmental timing of larvae raised in germ-free (GF) condition (grey circles) or Lp-associated conditions (green circles), in a control background (Mex>mCherry^RNAi, filled circles) or in a knock-down background in enterocytes (empty circles). The graphs represent the total fraction of emerged pupae over time as a percentage of the final number of pupae. When indicated, we used a Cox proportional hazards model to compare the effect of Lp in the control background and in the GCN2 knock-down background. n.s.: non-significant. **: p-value <0.01. ***: p-value <0.001. (A–F) GCN2 knock-down in the enterocytes on imbalanced diet (–60% Val) (A), balanced diet (B), or imbalanced diet: –60% Ile (C), –60% Lys (D), –60% Thr (E), –60% Trp (F). (G) ATF4 knock-down in the enterocytes (triangles) and 4E-BP knock-down in enterocytes (crossed circles) on imbalanced diet (–60% Val). (H–I) Developmental timing of GF larvae (grey), larvae associated with Lp:Tn_r/tRNA (black), and larvae associated with Lp:Tn_control (green) on imbalanced diet (–60% Val) in a control background (Mex>mCherry^RNAi) (H) or in a GCN2 knock-down in the enterocytes (Mex>GCN2^RNAi) (I). We used a Cox proportional hazards model to compare the effect of Lp:Tn_control association with Lp:Tn_r/tRNA association and GF condition. n.s.: non-significant, **: p-value <0.01, ***: p-value <0.001.

Figure 6—figure supplement 1. Expression of GCN2 in the gut, but not in the fat body nor expression of TOR is necessary for L. plantarum to promote growth on an AA-imbalanced diet.

(A–B) Developmental timing of germ-free (GF) larvae (grey) or L. plantarum (Lp)-associated larvae (green) from a control background (Mex>mCherry^RNAi, filled circles) or GCN2 knock-down larvae (Mex>GCN2^RNAi, empty circles). The graph represents the total fraction of emerged pupae over time as a percentage of the final number of pupae. The graphs show five replicates per condition (mean and standard deviation). Each replicate consists in one tube containing 40 larvae. Larvae were reared on imbalanced diet (FLY AA –60% Val). We used the lines #BL 35355 (A) and #BL 67215 (B). (C–E) Expression of GCN2 in the anterior midgut of control larvae (Mex>mCherry^RNAi) or GCN2 knock-down larvae (Mex>GCN2^RNAi-1 (C) -2 or -3 (D)). Expression is normalised with the expression of rp49 using the formula 2^{Cq(rp49)-Cq(GCN2)}. We performed a Mann-Whitney test to compare the conditions, *p-value <0.05. (E) Colonisation of the gut by Lp in control larvae (Mex>mCherry^RNAi, filled circles) or GCN2 knock-down larvae (Mex>GCN2^RNAi, empty circles) fed an imbalanced diet (FLY AA –60% Val). The graph shows the quantity of colony-forming units (CFUs) of Lp per larva. We performed a Mann-Whitney test to compare the two conditions. **: p-value <0.01. (F) Developmental timing of GF larvae (grey) or Lp-associated larvae (green) from a control background (C564>mCherry^RNAi, filled circles) or GCN2 knock-down in the fat body (C564>GCN2^RNAi, empty circles). The graph represents the total fraction of emerged pupae over time as a percentage of the final number of pupae. The graphs show five replicates per condition (mean and standard deviation). Each replicate consists in one tube containing 40 larvae. Larvae were reared on imbalanced diet (FLY AA –60% Val). (G, H) Developmental timing of GF larvae (grey) or Lp-associated larvae (green) from a control background (mCherry^RNAi, filled circles) or TOR knock-down (TOR^RNAi, empty circles) in the fat body (G) or in the enterocytes (H). The graph represents the total fraction of emerged pupae over time as a percentage of the final number of pupae. Larvae were reared on imbalanced diet (FLY AA –60% Val). GF C564>TOR^RNAi larvae did not reach pupariation. (I) Developmental timing of GF larvae (grey) or Lp-associated larvae (green) from a control background (yw, filled circles) or two lines of 4E-BP deletion (del-1: empty circles, del-2: triangles) on imbalanced diet (FLY AA –60% Val). The graphs show five replicates per condition (mean and standard deviation). Each replicate consists in one tube containing 40 larvae. GF 4E-BP^del-2 larvae did not reach pupariation.

Figure 6—figure supplement 2. Unlike L. plantarum, the symbiont Acetobacter pomorum does not activate GCN2 in the host's enterocytes.

(A) Developmental timing of larvae raised in germ-free (GF) condition (grey circles), Acetobacter pomorum (Ap)-associated conditions (purple circles), or L. plantarum (Lp)-associated conditions (green circles) on an imbalanced diet (FLY AA –60% Val).The graph represents the total fraction of emerged pupae over time as a percentage of the final number of pupae. The graph shows five replicates per condition (mean and standard deviation). Each replicate consists in one tube containing 40 larvae. (B) Developmental timing of larvae raised in GF condition (grey circles) or Ap-associated conditions (purple circles), in a control background (Mex>mCherry^RNAi, filled circles) or in a GCN2 knock-down background (Mex>GCN2^RNAi, empty circles) on an imbalanced diet (FLY AA –60% Val). The graph represents the total fraction of emerged pupae over time as a percentage of the final number of pupae. The graph shows five replicates per condition (mean and standard deviation). Each replicate consists in one tube containing 40 larvae. (C,E) Representative pictures of the anterior midgut of 4E-BP^introndsRed larvae. Cyan: DAPI. Magenta: 4E-BP^introndsRed reporter. Scale bar: 200 µm. Left panel: GF larva. Middle panel: Ap-associated larva. Right panel: Lp-associated larva. Larvae were reared on imbalanced diet (C) or balanced diet (E). (D,F) Quantification of the signal: proportion of enterocytes positive for 4E-BP^introndsRed reporter’s activity in the anterior midgut. Each dot represents one larva. The bar shows the mean. We performed a Kruskal-Wallis test followed by post hoc Dunn’s tests to compare GF and Ap to Lp. ***: p-value <0.001. Larvae were reared on imbalanced diet (FLY AA –60% Val) (D) or balanced diet (FLY AA) (F).