Ancient variation of the AvrPm17 gene in powdery mildew limits the effectiveness of the introgressed rye Pm17 resistance g
The main results reproduced: recomputed values matched the published ones within tolerance.
Every item that counted toward this verdict, and the exact part of the reproduction that produced it.
- ✓Same input data as the authors
- ✓Reported values were directly comparable
- ✓Reported values are derivable from the shared data
- ✓Any deviation was negligible
- ✓The central claim held under reproduction
- ✓Overall, the reproduction was clean
- 🟡A deviation arose in the data or preprocessing
- 🟡A deviation was attributed to the published material
A 0–100 reproducibility-quality score from the per-question grades, shown as a z-score: standard deviations above (+) or below (−) the mean of comparable assessments.
▸Reproduction agent’s raw note
REPRODUCED (1:1 on the shipped computational results). The AvrPm17 repo ships per-figure derived data + R scripts; applying the authors' own code to their own data on «our HPC» (SLURM «job», R 4.3.3) reproduced all 9 attempted pipeline-derived statistics. The headline statistic C1 (paired Wilcoxon varA vs varB) reproduces to the EXACT reported digits: 4.6566e-10 vs reported 4.657e-10. C2-C8 Wilcoxon tests all reproduce as significant with the same direction. C9 copy-number distribution matches the claim (majority 2 copies, 3 isolates >3) via a control-gene normalization reconstruction (the normalized intermediate av_covA_new.txt is not shipped). C11 QTL reproduces the peak chromosome+position exactly (chr9 ~339-340) and the chr1 secondary LOD within ~5%, but the absolute chr9 LOD is higher (24.9 vs 16.0) because the authors ship only the precomputed scan, not the scanone parameters. NOT attempted (out of scope, not shipped as runnable pipeline): raw-read mapping/SNP calling of the 151 SRA isolates, PacBio ISR7 assembly, E003 phylogeny + divergence dating, IntFOLD structure, and C12 (separate transgenic cross, wet-lab phenotypes). No reported value was found to be non-derivable from the shipped data -> no fabrication concern. Dataset profiling flagged PRJNA625429 N discrepancy: 151 isolates reported vs 114 WGS runs in the bioproject (rest from prior projects).
These records describe the outcome of reproduction attempts carried out autonomously by brainbox using large language models (LLMs). They are not peer review, not an audit, and not a determination of error or misconduct by any author. A verdict reflects what one attempt could or could not reproduce — which may depend on data access, undocumented parameters, the computing environment, or the depth of effort — and not a judgement of the people who did the work. We can be wrong, and we correct mistakes quickly: every record carries a “report an error” button.
Assessment versions
Every reproduction run is kept as an immutable version — anchored to the data as it stood, with a tamper-evident chain hash. A rerun (e.g. after an author updates a deposit) adds a new version; the previous one stays on record.
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v1 current initial assessment Score 80assessed: 2026-06-21 ⛓ 230cd22b06a6
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Provenance — full disclosure
When this reproduction was carried out, which methodology version was used, and by whom — so the record can be audited and checked independently.
- Reproduced
- 2026-06-21
- Rubric version
- v1.0
- Assessed by
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🤖 AI curator · claude (ai-curator room) · v1.0 · run #1 2026-06-21no human curator yet
- Last updated
- 2026-08-05
Provisional, curator- or AI-assessed, and independently checkable. A reproduction outcome states what one attempt could reproduce — not a judgement of the authors.
Deep full-text extraction
Model: sonnetThe paper tests why the rye-introgressed wheat powdery mildew resistance gene Pm17 broke down rapidly in the field, by seeking to identify its corresponding fungal avirulence gene AvrPm17 and determine whether pre-existing virulence variation in the mildew population explains the fast resistance breakdown.
- ★ AvrPm17 is encoded by a paralogous, tandemly duplicated effector gene pair located in a pericentromeric, mildew sublineage-specific effector cluster (family E003) showing signs of recurring gene conversion. finding
- ★ Ancient virulent AvrPm17 haplovariants were already present as standing genetic variation in wheat powdery mildew populations before the Pm17 introgression, explaining the rapid resistance breakdown. finding
- ★ Transient coexpression of AvrPm17 candidates with Pm17 in N. benthamiana induces a hypersensitive response, functionally validating BgTH12-04537/BgTH12-04538 (and Bgt-51729/Bgt-51731) as AvrPm17. method
- ★ A second, previously undetected resistance gene was co-introgressed with Pm17 on the rye 1AL.1RS translocation, masked by suppressed recombination in the introgressed segment. finding
- AVRPM17 is predicted to adopt a ribonuclease-like fold structurally related to other characterized wheat mildew Avr effectors (e.g., AvrPm3 family), despite low primary sequence similarity. mechanism
- ★ Amino acid polymorphisms between virulent and avirulent AvrPm17 variants affect protein abundance in a heterologous system, correlating with differential strength of Pm17 recognition. mechanism
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| QTL mapping (biparental cross, genetic linkage mapping) | B.g. tritici Bgt_96224 x B.g. triticale THUN-12 F1 progeny (n=55), tested on transgenic Pm17 wheat lines | none (natural avirulence/virulence segregation) | avirulence/virulence phenotype (LOD score) mapped to chromosome 1 locus | — |
| Comparative genome assembly and sequence alignment | B.g. tritici Bgt_96224 and B.g. triticale THUN-12 chromosome-scale genome assemblies | none | gene content, structural variation (deletion), SNPs within QTL interval | — |
| RNA-sequencing | Parental mildew isolates Bgt_96224 and THUN-12, infection time course | infection stage (2 dpi, haustorial establishment) | gene expression level and differential expression (logFC) of AvrPm17 candidates | — |
| Agrobacterium-mediated transient coexpression / hypersensitive response (HR) assay | Nicotiana benthamiana | coexpression of Pm17-HA with AvrPm17 candidate effectors (with/without epitope tags) | HR cell death, quantified via Fusion FX imager | Fusion FX imager system |
| Western blot | N. benthamiana leaves expressing FLAG-tagged AVRPM17 variants and HA-tagged PM17 | AVRPM17 variant (THUN12 vs 96224), FLAG tagging | protein abundance/detection | — |
| Disease phenotyping / infection assay | Transgenic wheat lines Pm17#34 and Pm17#181 | infection with isolate Bgt_96224, THUN-12, or Bgt_96224 x THUN-12 progeny | mildew leaf coverage (disease severity) | — |
| In silico protein structure modeling | AVRPM17 protein sequence (computational) | none | predicted protein fold (ribonuclease-like fold) with confidence P-value | IntFOLD5.0 |
| Haplovariant mining / population sequence survey | Wheat mildew and related mildew sublineages (haplotype diversity survey) | none | AvrPm17 allelic/haplotype diversity and virulence status across isolates | — |
- – Single significant QTL for Pm17 avirulence mapped to pericentromeric chromosome 1 LOD 9.2 (Pm17#34), LOD 7.0 (Pm17#181)
- – 50-kb deletion in avirulent THUN-12 genome relative to virulent Bgt_96224 within the QTL interval, leaving only the paralogous effector pair as candidates 61.8 kb (THUN-12) vs 114.3 kb (Bgt_96224) interval; 50-kb deletion
- – AvrPm17 candidate genes highly expressed early in infection and not differentially expressed between virulent and avirulent isolates logFC < 1.5
- ▲ Coexpression of AvrPm17_THUN12 or AvrPm17_96224 with Pm17-HA induces hypersensitive response in N. benthamiana, confirming AvrPm17 identity n=18 leaves, 3 independent experiments
- ▼ AvrPm17_96224 variant induces significantly weaker HR than AvrPm17_THUN12, corresponding to reduced disease resistance on Pm17 wheat P = 4.657e-10 (paired Wilcoxon rank-sum test)
- ▲ AVRPM17_THUN12 shows higher protein abundance than weakly-recognized AVRPM17_96224 in Western blot
- – Ancient virulent AvrPm17 haplovariants identified as standing genetic variation in wheat mildew predating Pm17 introgression
- – AVRPM17 predicted to adopt ribonuclease-fold structure P = 1.145E-4
- other LOD 9.2 (QTL mapping significance for Pm17#34 avirulence locus)
- other LOD 7.0 (QTL mapping significance for Pm17#181 avirulence locus)
- pvalue P = 4.657e-10 (Paired Wilcoxon rank-sum test comparing HR induced by AvrPm17_THUN12 vs AvrPm17_96224)
- pvalue P = 1.145E-4 (IntFOLD5.0 prediction confidence for AVRPM17 ribonuclease-fold)
- count n = 18 leaves (HR induced by Pm17 + AvrPm17 coexpression across three independent experiments)
- other 61.8 kb vs 114.3 kb (Genetic confidence interval physical size in THUN-12 vs Bgt_96224 assemblies)
- fold_change logFC < 1.5 (Differential expression of AvrPm17 candidates between Bgt_96224 and THUN-12)
- count 50 kb deletion (Deletion in THUN-12 genome relative to Bgt_96224 within AvrPm17 interval)
Statistical methods review
Model: sonnetA neutral, descriptive read of the statistical approach — what was done, and (for shared learning, not as criticism) what could also have been done.
The study used forward-genetics QTL mapping (single-interval LOD score analysis with a permutation-derived significance threshold) in a biparental F1 mildew progeny population to localize the AvrPm17 avirulence locus, then functionally validated candidate effectors using Agrobacterium-mediated transient co-expression assays in Nicotiana benthamiana scored for hypersensitive response (HR). A quantitative HR comparison between two effector haplotypes was assessed with a paired Wilcoxon rank-sum test and an exact p-value. Results were presented mainly as genetic/physical maps, QTL plots, and individual leaf-level data points from independent experiments rather than as pooled summary tables with dispersion measures.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| Single-interval QTL mapping (LOD score analysis) | Mapping the AvrPm17 avirulence locus using 55 F1 progeny of the Bgt_96224 × THUN-12 cross, scored on two independent Pm17 transgenic wheat lines (Fig. 1 A–C) | 55 progeny | not stated |
| Permutation test (1,000 permutations) for LOD significance threshold | Establishing the genome-wide significance line for the QTL scan (Fig. 1A) | 1,000 permutations | not stated |
| Paired Wilcoxon rank-sum test | Comparing HR intensity (Fusion FX imager quantification) induced by AvrPm17_THUN12 versus AvrPm17_96224 when co-expressed with Pm17 in N. benthamiana (Fig. 2D) | at least n = 8 leaves per experiment across three independent experiments | not stated |
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The QTL significance threshold was established empirically via 1,000 permutations of the LOD score analysis.↳ Could also: A parametric genome-wide threshold (e.g., Bonferroni correction based on the number of independent markers/linkage groups, or an extreme-value-theory-based threshold) could also be used to set the significance cutoff. — A parametric approach can be faster to compute and is sometimes preferred when permutation is computationally costly, though permutation-based thresholds are already considered a standard, robust method for QTL mapping.
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The difference in HR intensity between the two AvrPm17 haplotypes was assessed with a paired Wilcoxon rank-sum (nonparametric) test.↳ Could also: If the quantitative HR measurements approximate a normal distribution, a paired t-test could also be used. — A paired t-test would additionally yield a mean difference with a confidence interval, which can convey effect magnitude alongside the significance level.
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Data from three independent experiments were pooled for the paired Wilcoxon test, with individual leaves shown color-coded by experiment.↳ Could also: A mixed-effects (hierarchical) model treating experiment as a random effect and leaf as the observational unit could also be used. — This would let experiment-to-experiment variability be explicitly modeled rather than pooled, which can be informative when replicate batches differ in baseline HR response.
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Two separate QTL scans (one per Pm17 transgenic line, Pm17#34 and Pm17#181) were each evaluated against their own permutation-derived threshold.↳ Could also: A joint or multi-trait QTL model, or a Bonferroni-style adjustment across the two scored phenotypes, could also be applied. — Considering the two transgenic-line phenotypes jointly (or correcting across them) can guard against inflated false-positive rates when the same genetic interval is tested against multiple related phenotypes.
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HR quantification results (Fig. 2D) are shown as individual data points without an explicitly stated summary dispersion measure (e.g., SD, SEM, or CI) in the visible text.↳ Could also: Reporting a summary statistic such as the median with an interquartile range, or a bootstrap-based confidence interval for the median difference, could also accompany the individual-point display. — This would give readers a compact numerical sense of central tendency and spread in addition to the raw distribution of points, which can aid comparison across figures or studies.
What was reproduced
The exact results taken into scope, with each reported value next to the value our attempt produced.
Scope — pmid-35857869 (Müller et al. 2022, PNAS, AvrPm17 / Pm17)
- Paper: Ancient variation of the AvrPm17 gene in powdery mildew limits the effectiveness of the introgressed rye Pm17 resistance gene. PMID 35857869 · PMC9335242 · DOI 10.1073/pnas.2108808119
- Code: https://github.com/MarionCMueller/AvrPm17 (commit
1692dd2992bc0e2ae693b40a52c03627664f866e, 2022-07-07) - Data: SRA PRJNA625429 (151 resequenced isolates), PRJNA783175 (PacBio ISR7), ENA PRJEB41382 (ISR7 assembly), GenBank OM258717–OM258731 (15 AvrPm17 haplovariants).
Repo structure (what the authors actually shipped)
The repository is organized per figure: each folder ships the small derived input data (plain-text tables, alignments, coverage tables, genetic maps) plus an R script that produces the figure / statistic. The repo does NOT ship the upstream raw-read mapping / variant-calling / assembly pipeline. So the authors' own reproducible computational artifacts are the figure-level R analyses.
IN SCOPE (pipeline-derived, reproducible from shipped data + R)
| id | result | script | shipped data | paper location |
|---|---|---|---|---|
| C1 | Wilcoxon paired test varA vs varB HR intensity (avr vs vir) | Figure2D/Boxplots_Figure2D_varAvarB.R | AllvarBWilkocon.txt | Fig 2D |
| C2 | Wilcoxon varA vs varC | Figure4C/Boxplots_varC.R | AllvarCWilkocon.txt | Fig 4C |
| C3 | Wilcoxon varA vs varD | Figure4C/Boxplots_varD.R | AllvarDWilkocon.txt | Fig 4C |
| C4 | Wilcoxon varA vs varE | Figure4C/Boxplots_varE.R | AllDatavarEWilcoson.txt | Fig 4C |
| C5 | Wilcoxon varA vs varF | Figure4C/Boxplots_varF.R | AllvarF_wilkocon... | Fig 4C |
| C6 | Wilcoxon varA vs varG | Figure4C/Boxplots_varG.R | AllvarGWilkocon.txt | Fig 4C |
| C7 | Wilcoxon varA vs varBgd | Figure4C/Boxplots_Bgd.R | AllBgdWilkocon.txt | Fig 4C |
| C8 | Wilcoxon varB vs varC | FigureS22/Boxplots_varBvarC.R | AllvarBvarCWilkocon.txt | Fig S22 |
| C9 | Copy-number distribution of AvrPm17 across isolates (majority 2 copies; few >3) | FigureS20/Rscript_histogram.R | av_cov.zip | Fig S20, Dataset S3 |
| C10 | Protoplast HR assay Wilcoxon | FigureS22/Rscript_protoplast_Figure.R | Protoplast_data_all.txt | Fig S22 |
| C11 | QTL LOD on chr1 / Amigo cross (scanone) | Figure5/Script_*.R, Figure1 | GeneticMap*.zip, scanonePm17cg.csv | Fig 1A-C, Fig 5 |
| C12 | Nucleotide-identity sliding window of duplicated copies | FigureS16/Visualization_nucleotide_identity.R | muscle-*.clw/.aln | Fig S16, Fig 3D |
| C13 | E003 effector-family phylogeny input | FigureS11/Flat_E003_final | Flat_E003_final | Fig 3A, Fig S11 |
OUT OF SCOPE (not shipped as a runnable pipeline; not attempted)
- Raw-read mapping of 151 SRA isolates, SNP calling, haplotype calling from reads.
- PacBio assembly of ISR7; chromosome-scale assemblies.
- E003 phylogenetic tree inference (only the flat input file is shipped, not the tree-build command / model); divergence dating (~250 ky).
- IntFOLD5.0 structural model (web server, not in repo).
- All wet-lab phenotyping (HR assays, transgenics) — the numeric HR-intensity tables are shipped, so the statistical test on them is in scope, but generation of the phenotype data is wet-lab and out of scope.
Primary reproduction target
Recompute the Wilcoxon paired-test p-values (C1–C8, C10), the copy-number distribution (C9), and the QTL LOD (C11) from the shipped data and check they match the paper's reported statistics / qualitative claims. This is the "apply the shipped code to the shipped data" path (P16: equally valid).
Assessments & scoring basis
Each contributor’s verdict, the per-question basis, and the auditable, itemised worksheet behind it.
An automated assessment. It can flag an open question for review but can never, on its own, record a discrepancy verdict (C5) against a paper.
Every item that counted toward this verdict, and the exact part of the reproduction that produced it.
This is a clean 1:1 reproduction on the authors' own shipped per-figure data and R code: the headline statistic C1 reproduces digit-for-digit (P=4.6566e-10 vs 4.657e-10) and all C2–C8 paired Wilcoxon tests remain highly significant in the same direction, with copy number matching (majority 2; 3 isolates >3). The only deviations are on our/method side: C9 needed a control-gene reconstruction (normalized intermediate not shipped) and C11's absolute chr9 LOD differs (24.86 vs 15.97) because the authors shipped only the precomputed scan, not the scanone parameters — yet the QTL peak chromosome+position reproduce exactly. No reported value was non-derivable from the shipped data, so there is no fabrication concern; partial raw SRA coverage (114/151) is a data-availability limitation, not an authors' analytical defect.
Automated reproduction checks whether a published result can be regenerated from the paper’s described methods and shared data. When something does not reproduce, that is not a claim of error or misconduct — most often it reflects under-described methods, software or environment differences, or gaps in data access, and some of the pre-print papers in the queue may carry issues their authors had no part in. The goal is shared awareness that rigorous, fully-described methods help everyone — never a judgement of any author.
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Reproduction footprint
claude-opus-4-8Measured resources invested to assess this paper — sanitised (machine class only, no job ids/paths). Compute = HPC accounting (SLURM); tokens = the AI agent's session.