DOI: 10.5061/dryad.f4qrfj77k
title: Data from: Functional characterization of Paillotin: An immune peptide regulated by the Imd pathway with pathogen-specific roles in Drosophila immunity

description: Insects, such as Drosophila melanogaster, rely on innate immune defenses
 to combat microbial threats. Antimicrobial peptides (AMPs) play an
 important role in limiting pathogen entry and colonization. Despite
 intensive research into the regulation and biochemical properties of AMPs,
 their exact significance in vivo has remained uncertain due to the
 challenges of mutating small genes. Fortunately, recent technologies have
 enabled the mutation of individual AMP genes, overcome previous obstacles,
 and opened new avenues for research. In this study, we characterized one
 novel host-defense peptide, Paillotin (IM18, CG33706), using
 loss-of-function mutants. Paillotin is an ancient host defense peptide of
 Diptera, regulated by the Imd pathway. Loss of Paillotin does not impact
 the activity of either the Imd or Toll pathways. Importantly, we found
 that Paillotin mutants are viable but exhibit increased susceptibility to
 specific infections, particularly Providencia burhodogranariea. Paillotin
 was further found to contribute synergistically to defense against P.
 burhodogranariea when combined with other AMPs. However, we did not detect
 direct microbicidal activity of Paillotin in vitro in our hands. Taken
 together, our findings identify Paillotin as a novel host defense peptide
 acting downstream of Imd signaling, advancing our understanding of the
 Drosophila antimicrobial response.
description: # Functional characterization of Paillotin: An immune peptide regulated by
 the Imd pathway with pathogen-specific roles in *Drosophila* immunity
 Dataset DOI: [10.5061/dryad.f4qrfj77k](10.5061/dryad.f4qrfj77k) ##
 Description of the data and file structure This folder contains all the
 raw data from the experiments. Each ZIP archive includes the raw data
 corresponding to each figure, provided in either Prism or Excel format.
 The code used for the survival analysis is also included. **# Figure_1
 Data.zip** This folder contains annotated sequence files and alignment
 data supporting **Figure 1A** and **Figure 1B**, which illustrate the
 **genomic context**, **promoter structure**, and **sequence conservation**
 of the Paillotin (IM18) peptide across dipteran species. \## File
 Descriptions 1\. `Fig1a_Paillotin_promoter_region.gb` \- Genbank format
 file containing annotation of the **Pai-RA/CG10332-RA** and **Pai-RB**
 promoter regions. \- Highlights immune-related transcription factor
 binding motifs: \- **GGRDNNHHBS** (Relish-like) \- **GGGHNNNDVH**
 (Dif/Dorsal-like) \- Software: Can be opened with Geneious, SnapGene, or
 any GenBank-compatible viewer. 2\.
 `Fig1a_Paillotin_promoter_region.geneious` \- Native Geneious project file
 containing the full annotation track, motif prediction, and aligned
 promoter sequences. \- This file requires Geneious software (commercial).
 For open alternatives, use the .gb file above. 3\. `Fig1a_subfile_IM18
 region promoter.svg` \- Visualization output (e.g. from Geneious) showing
 binding site arrangement within the promoter. \- Software: View/edit with
 Inkscape (free), Adobe Illustrator, or web browser. 4\.
 `Fig1b_Paillotin_alignment_May1-2025_v1.02` \- FASTA or CLUSTAL format
 file with aligned Paillotin peptide sequences from &gt;20 dipteran
 species. -Open with Jalview, Geneious, or any alignment viewer supporting
 FASTA/NEXUS formats. **# Figure_2 Data.zip** This directory contains the
 qPCR data related to Figure 2 of the manuscript, focusing on the
 expression of **Paillotin** and **CG10332** genes in *Drosophila*. The
 data were obtained using quantitative PCR (qPCR), and include both the raw
 Ct values and the normalized expression values. \## File Description \-
 Contains the raw and processed qPCR data for **Paillotin** and **CG10332
 **expression upon *E. coli* and *M. luteus* bacterial infection. 1\.
 `CG10332-E.coli.csv` 2\. `CG10332-M.luteus.csv` \- Sheets include: \- Raw
 Ct: technical replicates for each condition \- Normalized: ΔCt and ΔΔCt
 values with fold-change \- Variables: \- Gene: target gene name \- Sample:
 genotype or treatment \- Ct: cycle threshold value \- Replicate:
 biological replicate ID -Formula: Relative expression was calculated :
 Expression ratio=(E ref)^Ct ref/(E target)^Ct target where Rpl32 served as
 the reference gene. For all runs the amplification efficiency (E) was
 assumed to be 2.00 unless stated otherwise. 3\. `Paillotin-E.coli.csv` 4\.
 `Paillotin-M.luteus.csv` \- Similar structure to the CG10332 file: raw Ct
 values, internal control (rpl32), and calculated expression values. 5\.
 `Figure 2-qpcr results.prism` \- GraphPad Prism project containing bar
 graphs and statistical analyses of qPCR results for Figure 2. Includes
 grouped bar charts with error bars and statistical test results. \- Note:
 Requires GraphPad Prism software (licensed); for users without Prism, data
 is fully accessible in .csv files. **# Figure_3 Data.zip** This folder
 contains the raw data, processed spectra, and amino acid sequence
 alignments related to Figure 3 of the manuscript, which characterizes the
 Paillotin peptide using MALDI-TOF mass spectrometry. \## Folder
 Description 1\. `TIC MALDI-TOF.csv` This file contains Total-Ion-Current
 (TIC) MALDI-TOF mass-spectrometry data from *Drosophila melanogaster*
 haemolymph collected 24 h after infection with *Erwinia carotovora* strain
 Ecc15. All spectra were interpolated onto a common 0.02667 Da grid
 covering 4400-4800 Da and then normalised by each sample’s TIC. -File
 layout (Column letters assume Excel begins at A) \- A  WT‑Reflectron m/z –
 raw, non‑uniform mass‑to‑charge axis for the WT spectrum recorded in
 reflectron mode. \- B  WT‑intensity – raw ion counts corresponding to
 column A. \- C  WT‑Reflectron m/z‑Interpolated – the common m/z grid in
 0.02667 Da steps used for all spectra. \- D  WT‑intensity‑Interpolated –
 WT intensities resampled onto the grid in column C. \-
 E  370‑intensity‑Interpolated – intensities for mutant 370 on the same
 grid. \- F  SFHI‑intensity‑Interpolated – intensities for mutant SFHI on
 the same grid. \- G  41‑intensity‑Interpolated – intensities for line 41
 on the same grid. \- H  WT‑Total‑Ion‑Current (TIC) – single‑cell constant
 (row 1 only) storing the WT spectrum’s total ion count; blank below. \-
 I  WT normalized intensity – WT intensities divided by the WT TIC
 (column H) and add a scaling factor 100000. \- J  370 TIC – constant TIC
 for mutant 370 (row 1 only). \- K  370 normalized intensity – 370
 intensities divided by column J and add a scaling factor 100000. \-
 L  SFHI TIC – constant TIC for mutant SFHI (row 1 only). \- M  SFHI
 normalized intensity – SFHI intensities divided by column L and add a
 scaling factor 100000. \- N  41 TIC – constant TIC for line 41 (row 1
 only). \- O  41 normalized intensity – line 41 intensities divided by
 column N and add a scaling factor 100000. \- Missing-data convention NA:
 not applicable– the field has no logical value for that record 2\. `Figure
 3-MALDI TOF.pzf` GraphPad Prism project file containing the normalized
 spectra and visualization shown in Figure 3(b), comparing the signal
 intensity of the Paillotin peptide (m/z ≈ 4626) in wild-type (*w^1118*)
 and three different mutant lines (Δ41, Δ370, ΔSFHI). 3\. `Pai amino acid
 sequence/` A folder containing amino acid sequences of Paillotin from
 wild-type (*Dmel R6*) and mutants (Δ41, Δ370, ΔSFHI), used to generate
 Figure 3(a). \- Software: Can be opened with Geneious or SnapGene.
 \###Data Processing Protocol Data were processed as summarized below: 1\.
 **Raw Data Conversion:** MALDI-TOF output files were initially exported as
 `.txt` format. 2\. **Pre-processing:** \- Files were imported into Origin
 for reformatting (space parsing, filtering m/z: 4400–4800). \- A new m/z
 axis was generated using a fixed step size (0.02667), with 15,000 points.
 3\. **Interpolation & Alignment:** \- Datasets with different m/z axes
 were interpolated using Origin (Analysis &gt; Mathematics &gt;
 Interpolate/Extrapolate). \- Intensities were aligned to the unified m/z
 axis. 4\. **Normalization:** \- Data were normalized across samples using
 TIC-based normalization. \- Final datasets were exported to Excel for
 Prism import. 5\. **Visualization:** \- Final spectra were plotted in
 GraphPad Prism. \- Figure 3(b) shows absence of the Paillotin peak in Δ41,
 Δ370, and ΔSFHI mutants relative to wild-type. **# Figure_4 Data.zip**
 This folder contains the qPCR data and visualization related to Figure 4
 of the manuscript, analyzing the transcriptional response of *DptA* and
 *Drosomycin* (Drs) in different *Drosophila* genotypes upon bacterial
 infections. \## Folder Description 1\. `Dpt A-Ecc15.csv` \- Contains the
 raw and processed qPCR data for **Diptericin A** expression upon *Ecc15*
 bacterial infection. \- Timepoints: **6 hours post-infection (hpi)** and
 **12 hpi**, alongside unchallenged controls (UC). \- Includes Ct values
 for DptA and reference gene (RpL32), and normalized ΔCt and ΔΔCt values
 with fold-change. \- Variables: \- Sample: genotype or treatment \- Ct:
 cycle threshold value \- Replicate: biological replicate ID 2\.
 `Drs-M.luteus.csv` \- Contains the raw and processed qPCR data for
 **Drosomycin (Drs)** expression following *Micrococcus luteus* bacterial
 infection. \- Timepoints: **8 hpi** and **24 hpi**, plus UC. \- Includes
 Ct values for Drs and reference gene (RpL32), and normalized ΔCt and ΔΔCt
 values with fold-change. \- Variables: the same as `Dpt A-Ecc15.csv`. 3\.
 `Figure 4.pzf` \- GraphPad Prism project file used to generate the bar
 charts shown in **Figure 4 (a–b)**. \- Subfigure (a): DptA expression (Imd
 pathway, Gram-negative bacteria). \- Subfigure (b): Drs expression (Toll
 pathway, Gram-positive bacteria). **# Figure_5 Data.zip** This folder
 contains data used to generate Figure 5, including survival records and
 CFU counts from *Drosophila* infection experiments. \## File Descriptions
 1.`P.burhodogranariea survival.prism` 2.`B.bassiana survival.pzfx`
 3.`E.faecalis survival.prism` These Prism files contain daily survival
 records of different fly genotypes after infection with *P.
 burhodogranariea*, *B. bassiana*, and *E. faecalis*. \- **1 = dead**, **0
 = alive** 4.`CFU of P.bruhog.csv` This Excel file contains colony-forming
 unit (CFU) counts after infection with *P. burhodogranariea*. \-
 Variables: genotypes, replicates, dilution factors, fly counts, and
 calculated CFU values. \- The calculation formula: =[Count] *
 [DilutionFactor] * [[Total originally]] / ( [[uL plated]] * [[Total
 flies]] ) 5.`CFU of P.bruhog.prism` Graphs and statistics generated from
 the .xls CFU data. **# Figure_6 Data.zip** This folder contains raw data,
 statistical models, and visualization files for the analysis of survival
 phenotypes in flies after infection with *Providencia burhodogranariea*.
 \## File Descriptions 1\. `Pai+GroupB_cnv_P.burhodog.csv` \- Excel file
 with original data used for survival analysis. \- Variables: \- `Time2`:
 time to death or censoring (in days) \- `Censored`: status (0 = censored,
 1 = dead) \- `Genotype`, `Sex`, `Experiment`: categorical variables \-
 `IM18(Paillotin)` and `GroupB`: expression levels or binary genotype
 indicators \- Missing-data convention \- n/a: not applicable– the field
 has no logical value for that record 2\. `GroupB_Pai_CoxPH-12-12-2024.R`
 \- R script that performs: \- **Kaplan–Meier survival analysis** with
 stratification by genotype \- **Cox proportional hazards models** (both
 fixed-effect and mixed-effect via `coxme`) \- Tests for
 **interaction/synergy** between IM18 and GroupB \- Export of data
 formatted for GraphPad Prism 3\. `Pai+GroupB_P.burhodog_survival.prism` \-
 GraphPad Prism file containing survival records for different genotypes.
 \- Survival data is binary-coded: \- **1 = dead** \- **0 = alive** **#
 Figure_7 Data.zip** This file contains the survival data used in **Figure
 7**, evaluating whether re-expression of **Paillotin (IM18)** can rescue
 susceptibility in IM18-deficient or immune-deficient mutant backgrounds
 upon *Providencia burhodogranariea* infection. \## File Description
 1.`Rescue survival.prism` \- GraphPad Prism file with **Kaplan–Meier
 survival data** for multiple genotypes. \- Experimental groups include: \-
 *Pai Δ41*, *Pai Δ370* mutants \- Rescue lines: Pai overexpression in
 **Paillotin mutant** and **Relish mutant** backgrounds \- Controls:
 *w^1118*, *Rel^E20* alone \- Flies were pricked with *P. burhodogranariea*
 (OD600 = 12), and survival was monitored daily. \- Data format: \- **1 =
 dead** \- **0 = alive** **# Figure_S1 Data.zip** Folder: **Pai nucleic
 acid sequence** contains FASTA-format DNA sequences of Paillotin (IM18)
 homologs from different strains. These sequences were used for comparative
 analyses and sequence alignments. \- **Pai-41.fasta** Nucleotide sequence
 of Paillotin from strain 41. \- **Pai-370.fasta** Nucleotide sequence of
 Paillotin from strain 370. \- **Pai-Dmel_R6.fasta** Reference IM18
 (Paillotin) gene sequence from *Drosophila melanogaster*, genome release
 R6. \- **Pai-SHFI.fasta** Nucleotide sequence of Paillotin from strain
 SHFI. \- Software: Can be opened with Geneious or SnapGene. **# Figure_S2
 Data.zip** Figure_S2 Data contains Prism and Excel files used for the
 validation of Paillotin (IM18) gene expression via qPCR. These data
 support Supplementary Figure S2 of the manuscript. \## File Description
 1.`Paillotin-Ecc15.csv` Excel spreadsheet contains: \- Raw Ct: technical
 replicates for each condition \- Normalized: ΔCt and ΔΔCt values with
 fold-change \- Variables: \- Gene: target gene name \- Sample: genotype or
 treatment \- Ct: cycle threshold value \- Replicate: biological replicate
 ID 2.`Figure S2.prism` GraphPad Prism file containing bar plots and
 statistical comparisons of qPCR results between wild-type and Paillotin
 mutant flies under different conditions. **# Figure_S3 Data.zip**
 Figure_S3 Data contains one Prism file showing survival curve of wt and
 Paillotin mutants after clean injury. **# Figure_S4 Data.zip** Figure_S4
 Data contains eight Prism files with survival curve of wt and Paillotin
 mutants after different pathogen infections corresponding to figure 4 in
 the main text. **# Figure_S5 Data.zip** Figure_S5 Data contains a Prism
 file including time-course OD600 measurements and growth curve analyses of
 bacteria cultured with or without antimicrobial peptides, including
 Paillotin (IM18) and Cecropin A. **# Figure_S6 Data.zip** Figure_S6 Data
 contains all supporting data and scripts used to generate Figure S6A–C.
 The experiments test whether the loss of Paillotin (IM18) and
 GroupB-related immune factors result in increased susceptibility to
 various pathogens, and whether they act synergistically. \## File
 Descriptions 1\. `FigureS6A.prism` GraphPad Prism file showing survival of
 different fly genotypes following infection with *P. rustigianii* (OD =
 2). 2\. `FigureS6B.prism` GraphPad Prism file containing survival data of
 different fly genotypes after infection with *P. vermicola* (OD = 0.1).
 3\. `FigureS6C.prism` GraphPad Prism file containing survival data of
 different fly genotypes after infection with *E. coli* (OD = 200). 4\.
 `GroupB_Pai_CoxPH-12-12-2024.R` R script used to: -Import survival data
 from Excel -Convert data to GraphPad-compatible format -Fit Cox
 proportional hazards and mixed-effect models -Test for interaction/synergy
 between IM18 and GroupB -Generate publication-quality Kaplan–Meier plots
 as PNG files 5\. `Pai+GroupB_cnv_P.rustigianii OD=2.csv` Raw survival data
 from *P. rustigianii* infection. \- Variables: \- `Time2`: time to death
 or censoring (in days) \- `Censored`: status (0 = censored, 1 = dead) \-
 `Genotype`, `Sex`, `Experiment`: categorical variables \-
 `IM18(Paillotin)` and `GroupB`: expression levels or binary genotype
 indicators \- Missing-data convention \- n/a: not applicable– the field
 has no logical value for that record 6\. `Pai+GroupB_cnv_P.vermicola
 OD=0.1.csv` Raw survival data from P. vermicola infection. \- Variables:
 \- `Time2`: time to death or censoring (in days) \- `Censored`: status (0
 = censored, 1 = dead) \- `Genotype`, `Sex`, `Experiment`: categorical
 variables \- `IM18(Paillotin)` and `GroupB`: expression levels or binary
 genotype indicators \- Missing-data convention \- n/a: not applicable– the
 field has no logical value for that record **# Figure_S7 Data.zip**
 Figure_S7 Data contains one Prism file showing survival curve of
 Paillotin-overexpressed flies after bacteria infection. **# Figure_S8
 Data.zip** Figure_S8 Data contains three Prism files showing survival
 curves of Paillotin mutants after different types of toxin infection
 corresponding Figure_S8 in the main text. **# Figure_S9 Data.zip**
 Figure_S9 Data contains the melanization rate and microbiota counting
 between wt and Paillotin mutants. \## File Descriptions 1\. `Figure
 S9A-Melanization.csv` Excel spreadsheet containing raw and summarized
 melanization scoring data for *Drosophila* infected with *P.
 burhodogranariea*. 2\. `Figure S9A-Melanization.pzfx` GraphPad Prism file
 used to generate bar graphs from melanization scores. 3\. `Figure
 S9B-Microbiota.csv` Excel file showing quantification of culturable gut
 microbiota of flies with different genotypes. Variables include: CFU
 counts, fly genotype, dilution factor, fly numbers, and average CFU per
 fly. Formula is included in the sheet. 4\. `Figure S9B-Microbiota.prism`
 GraphPad Prism project containing microbiota CFU plots. **# Table_S1
 Data.zip** This file summarizes natural genetic variation at the Paillotin
 (IM18) locus across multiple wild *Drosophila melanogaster* lines from the
 *Drosophila* Population Genomics Project Phase 3 (DPGP3). \## File
 Descriptions 1.`Table S1 DPGP3_summary.csv` Contains the full
 population-level dataset, pairing detailed location metadata with
 fine-scale INDEL counts across the Paillotin gene for 30 DPGP3
 populations. 2.`Table S1 DPGP3_summary-1.csv` A streamlined population
 summary that retains overall mutation statistics and adds sub-continent
 aggregates for rapid regional comparisons. 3.`Table S1
 DPGP3_summary-2.csv` Collapses the same data to six continent-level rows,
 reporting total mutations, sample sizes and derived frequencies for broad
 global analyses.