Intestinal GCN2 controls Drosophila systemic growth in response to Lactiplantibacillus plantarum symbiotic cues encoded by r/tRNA operons

Abstract

Symbiotic bacteria interact with their host through symbiotic cues. Here, we took advantage of the mutualism between Drosophila and Lactiplantibacillus plantarum (Lp) to investigate a novel mechanism of host-symbiont interaction. Using chemically defined diets, we found that association with Lp improves the growth of larvae-fed amino acid-imbalanced diets, even though Lp cannot produce the limiting amino acid. We show that in this context Lp supports its host's growth through a molecular dialogue that requires functional operons encoding ribosomal and transfer RNAs (r/tRNAs) in Lp and the general control nonderepressible 2 (GCN2) kinase in Drosophila's enterocytes. Our data indicate that Lp's r/tRNAs are packaged in extracellular vesicles and activate GCN2 in a subset of larval enterocytes, a mechanism necessary to remodel the intestinal transcriptome and ultimately to support anabolic growth. Based on our findings, we propose a novel beneficial molecular dialogue between host and microbes, which relies on a non-canonical role of GCN2 as a mediator of non-nutritional symbiotic cues encoded by r/tRNA operons.

Keywords: D. melanogaster; GCN2; Lactobacillus; developmental biology; infectious disease; microbiology; symbiosis.

Figure 1.. L. plantarum (Lp) rescues the developmental delay due to amino acid (AA) imbalance.

(A,B) Developmental timing of germ-free (GF) larvae (grey) and Lp-associated larvae (green) on FLY AA diets with a –70% (A) or –60% (B) decrease in each essential AA (EAA). Boxplots show maximum, minimum, and median D50 (median time of pupariation) of five replicates. Each replicate consists in one tube containing 40 larvae. L2: larvae stalled at the L2 stage. L3: larvae stalled at the L3 stage. For each diet in (B), we used a Cox proportional hazards model to test the effect of the diet, association with Lp, and the interaction between these two parameters. We show the p-values of the interactions between diet and association with Lp after correction by the FDR method. n.s.: non-significant, **: p-value <0.01, ***: p-value <0.001. (C) Developmental timing of larvae raised on balanced diet (FLY AA, filled circles) or imbalanced diet (FLY AA –60% Val, empty circles). 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. We used a Cox proportional hazards model to test the effect of the diet, the association with Lp, and the interaction between these two parameters. (D) Developmental timing of larvae raised on balanced diet (FLY AA, filled circles), on imbalanced diet (FLY AA Val –60%, empty circles), on imbalanced diet adjusted with His (FLY AA Val –60%+His, triangles) or on imbalanced diet adjusted with Leu (FLY AA Val –60%+Leu, squares). 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. (E) Valine concentration in the haemolymph of larvae. The graph shows the mean and standard deviation of three replicates. Each replicate consists in the haemolymph of 10 larvae pooled together. We used an ANOVA followed by post hoc Dunnett’s test to compare the mean of each condition to the mean of the condition GF on imbalanced diet. **: p-value <0.01.

Figure 1—figure supplement 1.. Characterization of the growth promoting effect of L. plantarum on AA-imbalanced diet.

(A–D) Developmental timing and survival rate: the graphs show five replicates (mean and standard deviation). Each replicate consists in one tube containing 40 larvae. (A) Developmental timing of larvae raised on balanced diet (FLY AA, filled circles) or on severely imbalanced diets (FLY AA –80% Val, empty circles, FLY AA –90% Val, empty triangles) in germ-free (GF) condition (grey) or L. plantarum (Lp)-associated conditions (green). The graph represents the total fraction of emerged pupae over time as a percentage of the final number of pupae. GF larvae reared on severely imbalanced diets (–80% or –90% Val) did not reach pupariation. (B) Developmental timing of larvae raised on balanced diet (FLY AA, filled circles) or on imbalanced diet due to excess Val (FLY AA +60% Val, empty circles) in GF condition (grey) or Lp-associated conditions (green). (C) Egg-to-pupa survival of GF larvae (in grey) and Lp-associated larvae (green) on balanced diet and imbalanced diets. Survival was calculated as the final number of pupae divided by the initial number of eggs (i.e. 40 per replicate). We performed a Kruskal-Wallis test followed by post hoc Dunn’s tests to compare each condition to the condition GF on balanced diet and found no statistically significant difference. (D) Developmental timing of larvae raised on imbalanced diet (FLY AA Val –60%) in GF conditions (grey), in live Lp-associated conditions (filled green circles) or after supplementation with the same quantity of heat-killed (HK) Lp (empty green circles). We used a Cox proportional hazards model to compare the effect of Lp and HK Lp to the GF condition. (E–F) Food intake of GF larvae (grey) or Lp-associated larvae (green) reared on balanced diet (E) or imbalanced diet (F). Larvae were transferred on coloured food, and food intake was calculated as the optical density (OD) of the food ingested by the larvae over time. The graph shows the mean absorbance and standard deviation of five replicates per condition. Each replicate consists in 5 larvae.

Figure 2.. The operons encoding ribosomal and transfer RNAs (r/tRNAs) in L. plantarum (Lp) are necessary for Lp to rescue the delay due to amino acid (AA) imbalance.

(A) Representation of the genetic screen. (B) Result of the screen: for each Lp mutant (X-axis), we calculated the median time of development of associated larvae on a severely imbalanced diet (FLY AA –80% Val) and normalised it into a z-score (Y-axis). We selected the 32 candidates that yielded a z-score >2.5. (C) Developmental timing of germ-free (GF) larvae (grey) and larvae associated with Lp:Tn_control (green) or the 32 candidate mutants from the genetic screen, on a severely imbalanced diet (FLY AA –80% Val). GF larvae and larvae associated with mutant F09.11 did not reach pupariation. Boxplots show maximum, minimum, and median of D50 (median time of pupariation) of five replicates per condition. Each replicate consists in one tube containing 40 larvae. We performed a Kruskal-Wallis test followed by post hoc Dunn’s tests to compare all mutants to Lp:Tn_control. In red: statistically significant difference with Lp:Tn_control (p-value <0.05). (D) Growth of the nine candidates on imbalanced diet (FLY AA –60% Val), in association with larvae. The graph shows the quantity of colony-forming units (CFUs) of Lp over time (mean and standard deviation of three replicates). (E) Colonisation of the larval gut by the six remaining candidates, on imbalanced diet (FLY AA –60% Val). The graph shows the quantity of CFUs of Lp per larva (mean and standard deviation of 5 replicates). We performed a Kruskal-Wallis test followed by post hoc Dunn’s tests to compare each candidate to Lp:Tn_control and found no statistically significant difference. (F) Developmental timing of larvae raised on imbalanced diet (FLY AA –60% Val), in GF condition or in association with each one of the six candidates or with Lp:Tn_control. 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. We used a Cox proportional hazards model to compare the effect of each candidate to the effect of Lp:Tn_control. The p-values were adjusted by the Tukey method. n.s.: non-significant. ***: p-value <0.001. (G) Representation of the six transposon insertions. Tn: transposon. rspC: 16S rRNA methyltransferase. gnd2: phosphogluconate dehydrogenase. Of note, C09.09 and F07.08 show two independent insertions in the same r/tRNA operon.

Figure 2—figure supplement 1.. L. plantarum mutants for r/tRNA operon show a decrease in the abundance of r/tRNAs, but no effect on growth or host colonization.

(A,B) Expression of 16S (A) and 23S (B) rRNA of Lp:Tn_control and Lp:Tn_r/tRNA. Expression is normalised with the expression of gyrB using the formula 2^{Cq(gyrB)-Cq(rRNA)}. We performed a Student’s t-test to compare the two conditions. *: p-value <0.05. (C) Developmental timing of larvae raised on imbalanced diet (FLY AA –60% Val), in germ-free (GF) condition (grey) or in association with Lp (dark green), Lp:Tn_control (light green), LpΔop_r/tRNA (dark blue), or Lp:Tn_r/tRNA (black). 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. We used a Cox proportional hazards model to compare the effect of each candidate to the effect of L. plantarum (Lp). The p-values were adjusted by the Tukey method. n.s.: non-significant. ***: p-value <0.001. (D) Developmental timing of larvae raised on FLY AA –70% Leu, in GF condition (grey) or in association with Lp WT (dark green) or LpΔop_r/tRNA (dark blue). 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. (E) Growth of Lp WT (dark green) and LpΔop_r/tRNA (dark blue) on imbalanced diet (FLY AA –60% Val), in association with larvae. The graph shows the quantity of colony-forming units (CFUs) of Lp over time. We performed a Mann-Whitney test at each time point and found no statistically significant difference. (F) Colonisation of the larva gut by Lp (dark green) and LpΔop_r/tRNA (dark blue) on imbalanced diet (FLY AA –60% Val). The graph shows the quantity of CFUs of Lp per larva. We performed a Mann-Whitney test and found no statistically significant difference. (G,H) Expression of 16S (G) and 23S (H) rRNA of Lp WT or LpΔop_r/tRNA. Expression is normalised with the expression of gyrB using the formula 2^{Cq(gyrB)-Cq(rRNA)}. We performed a Student’s t-test to compare the two conditions. *: p-value <0.05; **: p-value <0.01. (I) Expression of Thr-tRNAs of Lp WT or LpΔop_r/tRNA from small RNAseq. Expression is normalised with the total number of reads. tRNA15, tRNA57, and tRNA69 are outside the deleted operon. tRNA5 is inside the deleted operon.

Figure 3.. L. plantarum (Lp) produces extracellular vesicles containing ribosomal and transfer RNAs (r/tRNAs).

(A) Representative transmission electronic microscopy images of extracellular vesicles purified from Lp’s supernatant. The text in red shows the diameter in nm. The scale bar indicates 200 nm. (B–D) RT-qPCR quantification of 16S rRNA, 23S rRNA, and Thr-tRNA from total bacterial cells (B), purified extracellular vesicles (C), or purified extracellular vesicles treated with RNAse (C), normalised with sterile supernatant. RNA quantity is shown as 2^{Cq(sterile supernatant)-Cq(cells)} or 2^{Cq(sterile supernatant)-Cq(extracellular vesicles)}. The dotted line shows the level of RNA detected in sterile supernatant. The graph shows the mean of two replicates and standard deviation.

Figure 4.. Association with L. plantarum (Lp) activates general control nonderepressible 2 (GCN2) in the larval anterior midgut.

(A,C,E) Representative pictures of the anterior midgut of 4E-BP^introndsRed larvae. 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. Each dot represents an independent replicate. The bar shows the mean. (A) Larvae in germ-free (GF) conditions (left) or Lp-associated conditions (right) on imbalanced (FLY AA –60% Val, top) or balanced (FLY AA, bottom) diet. Quantification of the signal in (B): GF larvae (grey) or Lp-associated larvae (green) fed an imbalanced (top) or balanced (bottom) diet. We performed a Mann-Whitney test to compare the GF and Lp-associated conditions. **: p-value <0.01. ***: p-value <0.001. (C–E) Control larvae (Mex-Gal4x4E-BP^introndsRed reporter) (top panel) and GCN2 knock-down (Mex-Gal4x4E-BP^introndsRed reporter, UAS-GCN2^RNAi) (bottom panel) in GF conditions (left) and Lp-associated conditions (right) fed an imbalanced diet (FLY AA –60% Val) (C) or a balanced diet (FLY AA) (E). Quantification of the signal in (D,F): GF larvae (grey circles) or Lp-associated larvae (green circles) fed an imbalanced diet (D) or a balanced diet (F). Filled circles: control condition (Mex-Gal4x4E-BP^introndsRed reporter). Empty circles: GCN2 knock-down (Mex-Gal4x4E-BP^introndsRed reporter, UAS-GCN2^RNAi). Each dot represents one larva. We performed a Kruskal-Wallis test followed by post hoc Dunn’s tests to compare all conditions together. a: the conditions are not significantly different from each other (p-value >0.05). b: the condition is significantly different from other conditions (p-value <0.01).

Figure 4—figure supplement 1.. L. plantarum activates GCN2 in the larval anterior midgut.

(A) Representative picture of the full gut of a larva associated with Lp^GFP. Cyan: DAPI. Yellow: GFP. Scale bar: 500 µm. (B) Representative pictures of the full gut of a germ-free (GF) larva (left panel) and a L. plantarum (Lp)-associated larva (right panel) fed an imbalanced diet (FLY AA –60% Val, top panel) or a balanced diet (FLY AA, bottom panel). Cyan: DAPI. Magenta: 4E-BP^introndsRed reporter. The red squares show the region of the anterior midgut where Lp activates the 4E-BP^introndsRed reporter. Scale bar: 500 µm. (C) 4E-BP expression in the anterior midgut of larvae reared on imbalanced diet (FLY AA –60% Val) in GF conditions (grey) or Lp-associated conditions (green). Expression is normalised with the expression of rp49 using the formula 2^{Cq(rp49)-Cq(4E-BP)}. We performed a Mann-Whitney test to compare the two conditions. *: p-value <0.05.

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.

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).

Figure 7.. The anterior midgut gut of L. plantarum (Lp)-associated larvae display transcriptomic signatures of remodelling and metabolic switch.

(A) Experimental design of the RNAseq strategy. We looked for genes differentially expressed (DE, red, orange, and yellow lines) or not differentially expressed (not DE, grey line) between conditions. Arrowheads indicate the control condition in each comparison. (B, C) Venn diagram showing the number of genes from each filter. We further selected genes from filter 1 with a Log2 fold change (LogFC) of less than –0.5 for genes down-regulated (B) and more than 0.5 for genes up-regulated (C) for functional enrichment analyses. (D, E) Dot plot of Gene Ontology (GO) enrichment analysis of genes down-regulated (D) or up-regulated (E) by Lp association in a general control nonderepressible 2 (GCN2)-dependent, ATF4-independent, and ribosomal and transfer RNA (r/tRNA) operon-dependent manner. The size of the dots represents the number of DE genes in each category, x-axis represents the gene ratio enrichment in comparison to the complete gene set of D. melanogaster; colour indicates the significance in terms of BH-adjusted p-values. (F,G) Corresponding LogFC plot from filter 1 (control Lp vs control germ-free [GF]) of genes belonging to the GO terms categories in (D) and (E), respectively.

Figure 7—figure supplement 1.. 3D principal component analysis (PCA) plot showing the first three components, calculated with a variance stabilising transformation of normalised counts from DESeq2.

(A) PCA of the condition’s Mex>control ^RNAi germ-free (GF) (grey circles) and L. plantarum (Lp) (green filled circles), Mex>GCN2^RNAi Lp (empty green circles) and Mex>ATF4^RNAi Lp (green squares). (B) PCA of the conditions Mex>control^RNAi associated with Lp:Tn_control (grey diamonds) with Lp:Tn_r/tRNA (green diamonds).

Figure 8.. L. plantarum (Lp) inhibits the expression of the growth repressor fezzik in a general control nonderepressible 2 (GCN2)-dependent manner.

(A) Expression of fezzik in germ-free (GF) larvae (grey) or larvae associated with Lp WT (dark green), Lp:Tn_r/tRNA (black), or Lp:Tn_control (light green) in a control background (Mex>mCherry^RNAi) or in larvae knocked down for GCN2 (Mex>GCN2^RNAi) or ATF4 (Mex>ATF4^RNAi) in enterocytes. The data is from the RNAseq described in Figure 7. (B) Developmental timing of GF larvae (grey) or larvae associated with Lp WT larvae (green) or LpΔop_r/tRNA in a control background (Mex>mCherry^RNAi, filled circles) or fezzik knock-down larvae (Mex>fezzik^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 a Cox proportional hazards model to compare the effect of fezzik knock-down in Lp, LpΔop_r/tRNA, and GF larvae. For each microbial condition, we used a post hoc Tukey test to compare the fezzik knock-down to control. ***: p-value <0.001.

Figure 8—figure supplement 1.. fezzik RNAi efficiently represses the expression of fezzik in enterocytes.

Expression of fezzik in the anterior midgut of germ-free (GF) larvae on imbalanced diet (FLY AA –60% Val) in a control background (Mex>mCherryRNAi, filled circles) or in a fezzik knock-down in enterocytes (Mex>fizz^RNAi). Expression is normalised with the expression of rp49 using the formula 2^{Cq(rp49)-Cq(fezzik)}. We performed a Mann-Whitney test. *p-value <0.05.

Author response image 1.. Representative images of the anterior midgut of larvae stained for Lp’s 16S rRNA (yellow) and DAPI (cyan).

Scale bar: 50 µm.

Author response image 2.. Representative confocal picture of the anterior midgut of a Lp-associated larva.

We selected a plane showing putative inclusions of Lp’s rRNA inside the enterocytes. Scale bar: 50 µm. Right picture: the red line depicts the border between the lumen (on the top, characterized by DAPI-positive bacteria) and the gut epithelium (on the bottom). The red arrows show putative inclusion of Lp’s 16S rRNA in the enterocytes.

Author response image 3.