Figure 5. L. plantarum (Lp) mutants for ribosomal and transfer RNA (r/tRNA) operon fail to activate general control nonderepressible 2 (GCN2) in the anterior midgut.

(A,C) Representative pictures of the anterior midgut of 4E-BP^introndsRed larvae in germ-free (GF) conditions (left panels), in association with LpΔop_r/tRNA (middle panels) or in association with Lp WT (right panels). Cyan: DAPI. Magenta: 4E-BP^introndsRed reporter. Scale bar: 200 µm. (B,D) Quantification of the signal: proportion of enterocytes positive for 4E-BP^introndsRed reporter’s activity in the anterior midgut of GF larvae (grey), larvae associated with LpΔop_r/tRNA (blue) and larvae associated with Lp WT (green). Each dot represents one larva. The bar shows the mean. We performed Mann-Whitney test to compare association with Lp WT and association with LpΔop_r/tRNA. *: p-value <0.05. (A) Larvae fed an imbalanced diet (FLY AA –60% Val), signal quantified in (B). (C) Larvae fed a balanced diet (FLY AA), signal quantified in (D). (E) Representative images of 4E-BP^introndsRed GF larvae (top panel) and 4E-BP^introndsRed GF larvae fed 625 µg of bacterial tRNAs (bottom panel) on balanced diet (FLY AA). Cyan: DAPI. Magenta: 4E-BP^introndsRed reporter. (F) Quantification of the signal: proportion of enterocytes positive for 4E-BP^introndsRed reporter’s activity in the anterior midgut of GF larvae (grey), GF larvae fed with increasing concentrations of bacterial tRNAs (blue) on balanced diet (FLY AA). Each dot represents one larva. We performed a Kruskal-Wallis test followed by post hoc Dunn’s tests to compare each condition to GF. n.s.: non-significant. *: p-value <0.05.

Figure 5—figure supplement 1. The L. plantarum (Lp) insertion mutants for ribosomal and transfer RNA (r/tRNA) operon identified in the genetic screen fail to activate general control nonderepressible 2 (GCN2) in the anterior midgut.

(A,C,E) Representative pictures of the anterior midgut of 4E-BP^introndsRed larvae in germ-free (GF) conditions (left panels), in association with Lp:Tn_r/tRNA (middle panels) or in association with Lp:Tn_control (right panels). Cyan: DAPI. Magenta: 4E-BP^introndsRed reporter. Scale bar: 200 µm. (B,D,F) Quantification of the signal: proportion of enterocytes positive for 4E-BP^introndsRed reporter’s activity in the anterior midgut of GF larvae (grey), larvae associated with Lp:Tn_r/tRNA (black), and larvae associated with Lp:Tn_control (black). 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 the conditions together. n.s.: non-significant. *: p-value <0.05. **: p-value <0.01. (A) Larvae fed an imbalanced diet (FLY AA –60% Val), signal quantified in (B). (C) Larvae fed a balanced diet (FLY AA), signal quantified in (D). (E) Larvae fed an imbalanced diet (FLY AA –60% Val) upon short-term association with L. plantarum (Lp), signal quantified in (F). (G) Proportion of enterocytes positive for 4E-BP^introndsRed reporter’s activity in the anterior midgut of GF larvae (grey), GF larvae fed with increasing concentrations of bacterial tRNAs (blue), yeast tRNAs (orange), or Lp-associated larvae (green) on balanced diet (FLY AA). Each dot represents one larva. We performed a Kruskal-Wallis test followed by post hoc Dunn’s tests to compare each condition to GF. n.s.: non-significant. *: p-value <0.05. **: p-value <0.01. ***: p-value <0.001.