Journal: PLoS ONE

Article Title: Rice Mitogen Activated Protein Kinase Kinase and Mitogen Activated Protein Kinase Interaction Network Revealed by In-Silico Docking and Yeast Two-Hybrid Approaches

doi: 10.1371/journal.pone.0065011

Figure Lengend Snippet: OsMKK3, OsMKK4, OsMKK6 and OsMKK10-2 were fused with GAL4 DNA-binding domain and were used as a bait against each of the fifteen rice MAPKs (OsMPK3, OsMPK4, OsMPK6, OsMPK7, OsMPK14, OsMPK16-1, OsMPK16-2, OsMPK17-1, OsMPK17-2, OsMPK20-1, OsMPK20-2, OsMPK20-3, OsMPK20-4, OsMPK20-5 and OsMPK21-2) fused with GAL4 activation domain as preys. OsMKK1 fused with GAL4 activation domain as prey and fifteen MAPKs fused to DNA-binding domain baits were used to study protein interactions. Plasmids for bait and prey were co-transformed in AH109 yeast strain in different combinations as mentioned and selected on nutrient medium lacking Leu and Trp (upper panels). Interaction of bait and prey combinations were checked by assessing growth of co-transformed colonies streaked on selective medium lacking Ade, His, Leu and Trp (lower panels). p53 and SV40 large T-antigen are two proteins that are known to interact in yeast were used as a positive control.

Article Snippet: For initial screening MAPKKs were fused with GAL4 DNA binding domain (vector pGBKT7, BD biosciences) while MAPKs with the GAL4 activation domain (vector pGADT7, BD biosciences).

Techniques: Binding Assay, Activation Assay, Transformation Assay, Positive Control

Journal: bioRxiv

Article Title: Evolution of taste processing shifts dietary preference

doi: 10.1101/2024.10.11.617601

Figure Lengend Snippet: A) Semi-natural 4-choice assay testing preference towards grape, banana, apple or noni fruit over 24 h. Spider plots: Percentage of flies (group of 20 males and 50 females) per quadrant 2 h (faded line) and 24 h (solid line) after assay start. Here and in other panels, sample sizes are indicated in the figure. D.sechellia = Drosophila Species Stock Center [DSSC] 14021-0248.07; D. simulans = DSSC 14021-0251.04; D. melanogaster = CantonS . On top: Phylogenetic relationship of the D. melanogaster , D. simulans , and D. sechellia trio. MYA = million years ago. Right: Distribution of near-anosmic D. sechellia ( Ir8a -/- /Orco 1-/- ) flies at the same time points as shown for wild-type. B) Feeding preference for two strains of each species ( D.sechellia = DSSC 14021-0248.07 and 14021-0248.28, D. simulans = DSSC 14021-0251.04 and DSSC 14021-0251.196, D. melanogaster = CantonS and Oregon R ) for noni or grape juice in a 72-well (left), petri dish (middle) or flyPAD (right) two-choice assay. In the first two assays, preference index = (number of flies feeding on A -number of flies feeding on B)/number of total flies feeding. In the flyPAD, preference index = (number of sips on A – number of sips on B)/number of total sips. Similar phenotypes are observed for mutant strains lacking eye pigmentation (see Sup. Fig. 1E ). For these and all other box plots, the center line represents the median, the box bounds the first and third quartiles, and the whiskers extend to the largest and smallest values within 1.5 x the interquartile range; individual data points are overlaid. For the 72-well and petri dish assays, a group of 80 or 20 males or females represents one datapoint, respectively. In the flyPAD, one individual female represents one datapoint. Kruskal-Wallis H test (72-well: H (5) =73.394, P=2.012e -14 ; petri dish: H (5) =64.468, P=1.445e -12 ; flyPAD: H (5) =238.45, P=2.2e -16 ). Pairwise comparisons were conducted using Dunn’s test with Bonferroni correction. For these and all other comparisons the results are shown in the figure, where groups sharing the same letter are not significantly different (P < 0.025). Exact p-values are listed in Sup. Table 5 . C) Left: Feeding preference of Orco-mutant animals after antennal removal (rendering them anosmic) for noni vs. grape juice in the petri dish assay. Kruskal-Wallis H test (H (2) =19.227, P=6.681e -05 ; Dunn’s test results are shown in the figure). Right: feeding preference of olfactory mutant flies ( D. melangoaster Ir8a -/- /Orco -/- /Ir25a -/- /Gr63a -/- , D. simulans Orco 1-/- , D. sechellia Ir8a -/- /Orco 1-/- ) for noni vs. grape juice in the flyPAD. Kruskal-Wallis H test (H (2) =16.537, P=0.0002564; Dunn’s test results are shown in the figure). D) Principal component analysis of flyPAD data ( B,C, Sup. Fig. 2 ) with clear species separation. D.sechellia = DSSC 14021-0248.07, 14021-0248.28, 14021-0248.30, Ir8a -/- /Orco 1-/- ; D. simulans = DSSC 14021-0251.04, DSSC 14021-0251.196, DSSC 14021-0251.195, Orco 1-/- ; D. melanogaster = CantonS, OregonR, w 1118 , Ir8a -/- /Orco -/- /Ir25a -/- /Gr63a -/- ). E) Preference index for feeding on noni juice vs. 200 mM sucrose in the flyPAD. Kruskal-Wallis H test (H (5) =93.908 P=2.2e -16 ; Dunn’s test results are shown in the figure). F) Preference index for feeding on noni juice vs. 200 mM sucrose in the petri dish assay. Kruskal-Wallis H test (H (2) =28.526 P=6.393e -07 ; Dunn’s test results are shown in the figure). G) Left: schematic of the fly labellum depicting taste pegs and taste bristles. Below: Molecular markers for different neuron types in the short (S), intermediate (I) and long (L) sensilla. L- and S-sensilla house four, I-sensilla two neurons each. Right: Preference index for noni vs. grape juice feeding in control ( UAS-Kir2.1 ) and experimental flies after silencing of selected taste cell populations. Kruskal-Wallis H test was performed among experimental and control flies for each taste cell population (Gr64f: H (3) =62.818, P=1.469e -13 ; Gr66a: H (3) =38.788, P=1.925e -08 ; Ir25a: H (3) =39.51, P=1.353e -08 ; ppk28: H (3) =15.84, P=0.001223; Ir94e: H (3) =24.31, P=2.152e -05 ; ppk23: H (3) =25.643, P=1.133e -05 ). Significant Dunn’s test results are shown in the figure (***P < 0.001) after Benjamini–Hochberg correction as pairwise comparison to Gal4-control.

Article Snippet: To generate Gal4 reporter alleles, a 2A-Gal4 expression vector (GeneScript) was synthesized and either D. sechellia , D. simulans or D. melanogaster specific homology arms generated via gene synthesis (GeneScript) were introduced via seamless cloning flanking the expression cassette.

Techniques: Mutagenesis, Petri Dish Assay, Control, Comparison

Journal: bioRxiv

Article Title: Evolution of taste processing shifts dietary preference

doi: 10.1101/2024.10.11.617601

Figure Lengend Snippet: A) Left: schematic of the fly brain depicting the subesophageal zone (SEZ) receiving taste input from the periphery. AL = antennal lobe. Middle: Immunofluorescence with anti-GFP (recognizing GCaMP6s) and nc82 (labelling neuropils) antibodies in the brain of D. sechellia Ir25a Gal4 transgenic flies expressing GCaMP6s. Scale bar = 25 µm. Next to the brain staining, the corresponding labelling in the labellum is shown (anti-GFP staining, scale bar = 25 µm). The inlet highlights taste pegs, scale bar = 5 µm. Right: Axonal innervation pattern in the SEZ. Axonal projections of taste bristle neurons target the PMS4 region; of taste peg neurons the AMS1 area. Scale bar = 10 µm. B) Immunofluorescence with anti-GFP and nc82 antibodies in the brain (left) or labellum (right, GFP only) of D. sechellia Gr64f Gal4 transgenic flies expressing GCaMP6s. Scale bars = 25 µm. C) Number of Gr64f + neurons in the labellum and legs of female D. melanogaster , D. simulans and D. sechellia based on transgenic labelling. In each panel, on the left a nuclear staining with an UAS-unc84:GFP reporter in the D. sechellia labellum and legs (scale bars = 25 µm). t5 – t2 = tarsal segments 5 – 2 (male cell numbers: Sup. Fig. 2C, Sup. Table 1 ). Here and in other panels, sample size is indicated in the figure. For these and all other cell number bar plots, the bar represents the mean, the error bar the standard error; individual data points are overlaid. Kruskal-Wallis H test (labellum: H (2) =14.05, P=0.0008894; foreleg: H (2) =17.631, P=0.0001484; midleg: H (2) =6.0282, P=0.04909; hindleg: H (2) =1.3862, P=0.5). Dunn’s test results (after Bonferroni correction) are shown in the figure. D) Left: Schematic of the calcium imaging set-up to characterize taste sensory neuron responses in the SEZ. Middle: Example image of raw fluorescence (top) and tastant evoked (0.5% hexanoic acid) calcium responses (bottom) in D. sechellia peg neurons (labeled by Ir25a Gal4 driven GCaMP6s expression in the AMS1 area, scale bar = 10 µm). The colour scale depicts relative fluorescent changes (ΔF/F). Right: Quantification of tastant evoked calcium responses in Ir25a + taste peg neuron projections in the SEZ of D. melanogaster (top) and D. sechellia (bottom), reported as normalised GCaMP6s fluorescence changes. For these and all other physiology box plots, the centre line represents the median, the box bounds represent the first and third quartiles, and whiskers depict at maximum 1.5 x the interquartile range; individual data points are overlaid. Wilcoxon rank-sum test. No significant differences to D. melanogaster responses are detected. E) Temporal fluorescence changes in taste peg neuron projections during labellum stimulation. The solid line connects mean values of consecutive timepoints, the shaded area around the line defines the standard error of the mean. The horizontal black bar indicates the interval of tastant application. F) Left: Example image of raw fluorescence (top) and tastant evoked (noni juice) calcium responses (bottom) in D. sechellia sweet taste bristle neuron projections (labelled by Gr64f Gal4 driven GCaMP6s expression, scale bar = 10 µm). The colour scale depicts relative fluorescent changes (ΔF/F). Right: Quantification of tastant evoked calcium responses in Gr64f + taste bristle neuron projections in the SEZ of D. melanogaster and D. sechellia , reported as normalised GCaMP6s fluorescence changes. Wilcoxon rank-sum test. No significant differences to D. melanogaster responses are detected. G) Temporal fluorescence changes in sweet sensing taste bristle neuron projections during labellum stimulation. The solid line connects mean values of consecutive timepoints, the shaded area around the line defines the standard error of the mean. The horizontal black bar indicates the interval of tastant application. H) Top: Dose dependent feeding preference for individual sugars across species in the petri dish assay, 18-20 females per datapoint; sample size for each condition is indicated below. Bottom: percentace of flies feeding in the petri dish assay. Kruskal-Wallis H test preference index (5 mM sucrose H (2) =0.56183, P=0.7551; 50 mM sucrose H (2) =1.1057, P=0.5753; 200 mM sucrose H (2) =4.3406, P=0.1141; 500 mM sucrose H (2) =1.6403, P=0.4404; 5 mM fructose H (2) =2.2974, P=0.317; 50 mM fructose H (2) =4.0143, P=0.1344; 200 mM fructose H (2) =7.3313, P=0.2559; 500 mM fructose H (2) =1.3846, P=0.5004; 5 mM glucose H (2) =4.0495, P=0.132; 50 mM glucose H (2) =0.54317, P=0.7622; 200 mM glucose H (2) =4.783, P=0.09149; 500 mM glucose H (2) =0.5432, P=0.76). Kruskal-Wallis H test percentage of feeding (5 mM sucrose H (2) =8.6161, P=0.01346; 50 mM sucrose H (2) =15.888, P=0.0003549; 200 mM sucrose H (2) =0.73588, P=0.6922; 500 mM sucrose H (2) =11, P=0.004087; 5 mM fructose H (2) =6.4043, P=0.04068; 50 mM fructose H (2) =12.093, P=0.002366; 200 mM fructose H (2) =1.91, P=0.3848; 500 mM fructose H (2) =10.954, P=0.004182; 5 mM glucose H (2) =4.4752, P=0.1067; 50 mM glucose H (2) =18.076, P=0.0001188; 200 mM glucose H (2) =8.5812, P=0.0137; 500 mM glucose H (2) =11.642, P=0.002964).

Article Snippet: To generate Gal4 reporter alleles, a 2A-Gal4 expression vector (GeneScript) was synthesized and either D. sechellia , D. simulans or D. melanogaster specific homology arms generated via gene synthesis (GeneScript) were introduced via seamless cloning flanking the expression cassette.

Techniques: Immunofluorescence, Transgenic Assay, Expressing, Staining, Imaging, Fluorescence, Labeling, Petri Dish Assay

Journal: bioRxiv

Article Title: Evolution of taste processing shifts dietary preference

doi: 10.1101/2024.10.11.617601

Figure Lengend Snippet: A) Anti-GFP immunofluorescence in the SEZ and labellum of DsecGr66a Gal4 >UAS-GCaMP6s transgenic flies. AL = antennal lobe. Scale bar = 25 µm. B) Number of Gr66a + neurons in the labellum and legs of female D. melanogaster , D. simulans and D. sechellia based on transgenic labelling (males in Sup. Fig. 4 A, scale bar = 25 µm). Here and in other panels, sample size is indicated in the figure. For these and all other cell counts bar plots, the bar represents the mean, the error bar is the standard error; individual data points are overlaid. Kruskal-Wallis H test (labellum: H (2) =13.614, P=0.001106; foreleg: H (2) =4.8756, P=0.08735; midleg: H (2) =3.0446, P=0.2182; hindleg: H (2) =3.4719, P=0.1762). Dunn’s test results (after Bonferroni correction) are shown in the figure. C) Left: Example image of raw fluorescence (top) and tastant evoked (noni juice) calcium responses (bottom) in D. sechellia bitter taste bristle neuron projections (labelled by DsecGr66a Gal4 > GCaMP6s , scale bar = 10 µm). The colour scale depicts relative fluorescent changes (ΔF/F). Right: Quantification of tastant evoked calcium responses in Gr66a+ bristles neuron projections of the fly labellum in the SEZ of D. melanogaster and D. sechellia , reported as normalised GCaMP6s fluorescence changes. Wilcoxon rank-sum test (***P < 0.001). Significant differences to D. melanogaster responses to the same stimulus are indicated in the figure ( Sup. Fig. 4C for D. simulans data). D) Temporal fluorescence changes in bitter sensing taste bristle neuron projections during labellum stimulation. The solid line connects mean values of consecutive timepoints, the shaded area around the line defines the standard error of the mean. The horizontal black bar indicates the interval of tastant application. Shown is a subset of the whole Gr66a dataset as not all recordings could be temporally aligned.

Article Snippet: To generate Gal4 reporter alleles, a 2A-Gal4 expression vector (GeneScript) was synthesized and either D. sechellia , D. simulans or D. melanogaster specific homology arms generated via gene synthesis (GeneScript) were introduced via seamless cloning flanking the expression cassette.

Techniques: Immunofluorescence, Transgenic Assay, Fluorescence

Journal: bioRxiv

Article Title: Evolution of taste processing shifts dietary preference

doi: 10.1101/2024.10.11.617601

Figure Lengend Snippet: A) Top: Schematics of the Gr39a locus with four splice isoforms (grey boxes) sharing the last three common exons (red boxes). Below: Anti-GFP (and nc82) immunofluorescence in the SEZ, labellum and foreleg of D. melanogaster Gr39a.a-Gal4 >UAS-GCaMP6s transgenic flies. Scale bars = 25 µm. Right: Expression of Gr39a (in red) in individual neurons of the fly proboscis and maxillary palp (data from the FlyCellAtlas 85 ). B) Of the four different transcripts of the Gr39a locus ( Gr39a.a , Gr39a.b , Gr39a.c , Gr39a.d ) a 8bp deletion in the most distal isoform (here called Gr39a.b following the Flybase nomenclature; in some studies Gr39a.a ), shown in a nucleotide alignment of all three species, leads to a frameshift and pre-mature stop codon after 33 residues in D. sechellia . Numbers indicate nucleotide position in the open reading frame. Right: The Gr39a.a protein carries a D. sechellia -specific three amino acid deletion (residues 202-204). Shown here a protein alignment of all three species. Numbers indicate protein residue number. In blue: divergent nucleotides and residues. C) Predicted protein structure of D. melanogaster Gr39a.a (monomer). The ligand binding domain faces the extracellular space next to the ion-conducting pore domain (PD). The intracellular anchor domain is important for tetramer assembly and the three amino acid deletion in D. sechellia is near this domain. TMD = transmembrane domain. The domain annotation is based on Ref. – . D) Quantification of caffeine evoked calcium responses in Gr39a.a + bristle neuron projections of the fly labellum in the SEZ of D. melanogaster in wild-type (top) and Gr39a -/- mutant animals re-expressing either the D. melanogaster ( DmelGr39a.a ), D. sechellia ( DsecGr39a.a ) or D. melanogaster orthologue carrying the D. sechellia specific 3 amino acid deletion ( DmelGr39a.a Δ3 ). Here and in other panels, sample size is indicated in the figure. Kruskal-Wallis H test (H (4) =26.343, P=2.698e -05 ). Dunn’s test results (after Bonferroni correction) are shown in the figure. E) Quantification of coumarin (top) and noni juice (bottom) evoked calcium responses in Gr39a.a + bristle neuron projections of the fly labellum in the SEZ of D. melanogaster of the indicated genotypes. Kruskal-Wallis H test (coumarin: H (4) =20.899, P=0.0003316 ; noni juice: H (4) =6.5233, P=0.1633). Dunn’s test results (after Bonferroni correction) are shown in the figure. F) Quantification of caffeine (top), coumarin (middle), and noni juice (bottom) evoked calcium responses in Gr66a + bristle neuron projections of the fly labellum in the SEZ of D. sechellia in wild-type and animals expressing D. melanogaster Gr39a.a ( DmelGr39a.a ). Wilcoxon rank-sum test (caffeine: W = 0.29167, p = 0.5892; coumarin: W = 3, p = 0.08326; noni juice: W = 8.9423, p = 0.01); (***P < 0.001; **P < 0.01). Dunn’s test results (after Bonferroni correction) are shown in the figure. G) Preference index for caffeine (10 mM) in 5 mM sucrose vs. 2 mM sucrose feeding in the petri dish assay for D. melanogaster of the indicated genotypes. Kruskal-Wallis H test (H (4) =31.118, P=2.896e -06 ). Dunn’s test results (after Bonferroni correction) are shown in the figure. H) Preference index for caffeine in 5 mM sucrose vs. 2 mM sucrose feeding in the petri dish assay for D. sechellia of the indicated genotypes. Kruskal-Wallis H test (H (2) =0.26381, P=0.8764). Dunn’s test results (after Bonferroni correction) are shown in the figure. I) Top: Preference index for feeding on increasing concentration of caffeine (left) or coumarin (right) in 50 mM sucrose vs. 5 mM sucrose in the petri dish assay for all three species. 18-20 females per datapoint; sample size for each condition is indicated below. Bottom: Percentage of flies feeding in the petri-dish assay. Kruskal-Wallis H test preference index (1 mM caffeine H (2) =1.2832, P=0.5264; 10 mM caffeine H (2) =5.1312, P=0.07687; 25 mM caffeine H (2) =9.291, P=0.009605; 50 mM caffeine H (2) =13.005, P=0.0015; 1 mM coumarin H (2) =6.719, P=0.03475; 5 mM coumarin H (2) =12.118, P=0.002336; 10 mM coumarin H (2) =9.8824, P=0.007146). Kruskal-Wallis H test percentage of feeding (1 mM caffeine H (2) =2.8511, P=0.2404; 10 mM caffeine H (2) =2.9132, P=0.233; 25 mM caffeine H (2) =2.8657, P=0.2386; 50 mM caffeine H (2) =7.4973, P=0.02355; 1 mM coumarin H (2) =4.4585, P=0.1076; 5 mM coumarin H (2) =6.5215, P=0.03836; 10 mM coumarin H (2) =6.0304, P=0.04904). (***P < 0.001; **P < 0.01; *P < 0.025).

Article Snippet: To generate Gal4 reporter alleles, a 2A-Gal4 expression vector (GeneScript) was synthesized and either D. sechellia , D. simulans or D. melanogaster specific homology arms generated via gene synthesis (GeneScript) were introduced via seamless cloning flanking the expression cassette.

Techniques: Immunofluorescence, Transgenic Assay, Expressing, Residue, Ligand Binding Assay, Mutagenesis, Petri Dish Assay, Concentration Assay