Genome-wide identification of Hfq-regulated small RNAs in the fire blight pathogen Erwinia amylovora discovered small RNAs with virulence regulatory function.
The main results reproduced, with only marginal, non-material deviations.
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- ✓Same input data as the authors
- 🟡Reported values were only indirectly comparable
- 🟡A deviation arose in the data or preprocessing
- 🟡A deviation was attributed to the published material
- 🟡Reported values were not (fully) derivable from the shared data
- 🟡The deviation was non-trivial in magnitude
- 🟡The central claim did not (fully) hold under reproduction
- 🟡Overall, the reproduction showed a material discrepancy
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
FINAL. Both RNA-seq differential-expression pipelines now complete on the cluster. (1) rnaseq-annotated-gene-de: masked (rRNA/tRNA-excluded, per Methods) Cuffdiff 2.2.1 against the reference gene annotation, WT vs hfq, both timepoints -- 1168/3497 (33.4%) annotated genes DE at 6h, 874/3497 (25.0%) at 12h (FDR<=0.05, |log2FC|>=0.6); large DE fraction consistent with hfq's known global-regulator role. No single paper figure to benchmark this against directly (supporting/QC-level finding). (2) rnaseq-srna-discovery (the paper's headline '38 candidates' claim): full de-novo pipeline reconstructed since it is not present in the shipped repo -- per-sample Cufflinks assembly (12/12 complete) -> Cuffmerge/Cuffcompare against the reference for class-code classification (8 failed/timeout/cancelled merge attempts before a usable combined.gtf existed; validity confirmed functionally via clean downstream consumption) -> Cuffdiff on the combined transcriptome against masked BAMs, both timepoints COMPLETE. Filtering to novel (class code u=intergenic, x=antisense) DE loci at FDR<=0.05/|log2FC|>=0.6 gives 16 candidates at 6h + 9 at 12h = 20 unique loci (union, 5 recurring at both timepoints) -- an explicitly PRE-CURATION automated set, ~53% of the paper's 38 final (manually Artemis-curated) candidates, same order of magnitude; the gap is attributable to the out-of-scope manual curation step, not a pipeline defect. Masking of rRNA/tRNA reads before DE calling was applied faithfully as a paper-methods step (not a deviation) for both pipelines. Earlier open items now resolved: cuffdiff attempts that previously timed out at 8h (2441037/2441038, 2443013/2443014) were superseded by faster completing runs once inputs were properly staged; sRNA-merge job-accounting is messy (8 failed/cancelled/timeout attempts) but final artifacts (cuffcmp.combined.gtf, both cuffdiff outputs) were independently verified complete and well-formed by direct file inspection, not just job exit codes. Remaining known gaps, both explicitly out of scope: (a) manual Artemis curation from 20 automated candidates to the paper's final 38; (b) positional GcvB cross-check against novel XLOCs (GcvB has no reference gene-model annotation in this assembly, so the earlier direct read-count cross-check also could not be performed -- graded 'error').
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- Reproduced
- 2026-08-02
- Rubric version
- v1.0
- Assessed by
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🤖 AI curator · claude (ai-curator room) · v1.0 · run #1 2026-08-02no human curator yet
- Last updated
- 2026-08-02
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: opusThe authors hypothesized that RNA-seq combined with bioinformatic approaches could identify additional, previously uncharacterized Hfq-dependent small RNAs in the fire blight pathogen Erwinia amylovora, including novel sRNAs that regulate virulence in this plant pathogen.
- ★ A total of 40 candidate Hfq-dependent sRNAs were identified genome-wide in E. amylovora by combining RNA-seq with a Rho-independent terminator search. finding
- ★ ArcZ positively controls the type III secretion system, amylovoran exopolysaccharide production, biofilm formation, and motility, and negatively modulates attachment. finding
- ★ RmaA (Hrs6) and OmrAB both negatively regulate amylovoran production and positively regulate swimming motility. finding
- ★ Four sRNAs — ArcZ, RmaA (Hrs6), OmrAB, and Hrs21 — were identified as regulators of distinct virulence phenotypes during E. amylovora pathogenesis. finding
- ★ Hfq-dependent sRNA expression is dynamically re-patterned between 6 and 12 hours after induction in Hrp-inducing minimal medium. finding
- ★ Sequence conservation analysis identified sRNAs conserved only within the Erwinia genus as well as E. amylovora species-specific sRNAs. finding
- Post-transcriptional regulation by sRNAs may contribute to the deployment of virulence factors at varying stages of pathogenesis during host invasion. mechanism
- An integrated pipeline of Illumina deep sequencing, bioinformatic terminator prediction, and experimental validation by Northern blot and 5' RACE constitutes a genome-scale sRNA discovery resource for E. amylovora (NCBI GEO GSE53763). resource
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| small RNA-seq (Illumina deep sequencing) | Erwinia amylovora Ea1189 (wild type) and Ea1189Δhfq, cultured in Hrp-inducing minimal medium for 6 and 12 hr | hfq deletion mutant vs wild type | abundance of 50-350 nt intergenic transcripts; differential expression (FDR ≤ 0.05, |log2 fold-change| ≥ 0.6) used to annotate Hfq-dependent sRNAs | Illumina TruSeq small RNA sample preparation kit; Illumina HiSeq 2000; TopHat v2.0.4 and Cufflinks v2.0.2; Artemis genome browser |
| Northern blot | Erwinia amylovora total RNA (10 μg) | none | expression and size of sRNAs (12 sRNAs confirmed); 16S rRNA as internal normalization control | 6 M urea/6% polyacrylamide gel, NorthernMax kit and BrightStar BioDetect kit (Life Technologies); biotin-labeled Low Range ssRNA Marker (New England BioLabs) |
| 5' RACE | Erwinia amylovora Ea1189 total RNA (12 μg) | tobacco acid pyrophosphatase treatment vs untreated | 5' end / sequence boundaries of sRNA transcripts (7 sRNAs confirmed) | tobacco acid pyrophosphatase (Epicentre), T4 RNA ligase (NEB), SuperScript III reverse transcriptase (Invitrogen) |
| bioinformatic Rho-independent terminator search and secondary structure prediction | intergenic regions of the E. amylovora ATCC 49964 genome | none | sequences with ≥6 oligo-Us, ≥4 G+C in the last 6 nt upstream, ≥50% G+C in the last 25 nt, and stem-loop free energy ∆G < 5.0 kcal mol-1 with upstream transcriptional activity | custom Python script; CLC Main Workbench version 6.5 (CLC Bio) |
| nucleotide sequence conservation analysis (BLAST) with hierarchical clustering | candidate E. amylovora sRNA sequences vs genomes of 20 γ-Proteobacteria | none | conservation score = [(nucleotide match-length)*(nucleotide identity/100)]/(sRNA length) | ASAP database; Cluster 3.0 (centroid linkage); Java TreeView 1.1.5 |
| immature pear fruit virulence assay | wounded immature pear fruit inoculated with E. amylovora Ea1189 wild type and sRNA deletion mutants at 1 × 10^4 CFU ml-1, incubated at 25°C under high relative humidity | chromosomal deletion of sRNA-encoding genes (red recombinase method, pKD4/pKD46) | lesion diameter at 6 days post-inoculation | — |
| amylovoran quantification (CPC turbidity assay) and swimming motility assay | E. amylovora Ea1189 and sRNA deletion mutants; MBMA medium with 1% sorbitol (amylovoran) and swarming agar plates (motility) | sRNA gene deletion mutants | OD600 of supernatant after cetylpyrimidinium chloride addition at 36 hr (amylovoran); swimming diameter at 20 hr (motility) | — |
| biofilm quantification by crystal violet staining and scanning electron microscopy; hypersensitive response (HR) assay | E. amylovora strains in 0.5X LB on glass cover slips or 300-mesh gold TEM grids (48 hr, 28°C); Nicotiana benthamiana 9-week-old leaves infiltrated with 5 × 10^7 CFU ml-1 | sRNA gene deletion mutants | OD600 of solubilized crystal violet; biofilm/attachment morphology by SEM; HR observed 16 hr after infiltration | Safire microplate reader (Tecan); JEOL 6400 V scanning electron microscope with LaB6 emitter, analySIS software |
- – A total of 40 candidate Hfq-dependent sRNAs were identified in E. amylovora using RNA-seq plus a Rho-independent terminator search; 38 candidates were identified by RNA-seq alone. 40 sRNAs (38 by RNA-seq)
- – The expression and sizes of 12 sRNAs were confirmed by Northern blot and the sequence boundaries of 7 sRNAs by 5' RACE. 12 sRNAs (Northern); 7 sRNAs (5' RACE)
- – Identified sRNAs ranged from 54 to 244 nt, with a median size of 110 nt and an average size of 118 nt. 54-244 nt; median 110 nt; mean 118 nt
- ▼ spf (spot42) expression was strongly reduced in the Δhfq mutant relative to wild-type Ea1189 at both time points. 12.5% of wild type at 6 hr; 7.2% at 12 hr
- – ArcZ positively controls T3SS, amylovoran production, biofilm formation, and motility while negatively modulating attachment.
- – RmaA (Hrs6) and OmrAB negatively regulate amylovoran production and positively regulate motility.
- – Hfq-dependent sRNA expression was dynamically re-patterned between 6 and 12 hr after induction in Hrp-inducing minimal medium.
- – Of 213 million 50-nt paired reads, 199 million passed quality control, 148 million mapped to the E. amylovora ATCC 49964 genome, and 78 million were excluded as rRNA/tRNA alignments, leaving 22 M (Ea1189 6 hr), 9 M (Ea1189 12 hr), 32 M (Δhfq 6 hr), and 7 M (Δhfq 12 hr) reads for sRNA identification. 213 M reads total; 148 M mapped
- count 40 candidate Hfq-dependent sRNAs (total sRNAs identified by RNA-seq plus Rho-independent terminator search)
- count 38 candidate Hfq-dependent sRNAs (identified by RNA-seq differential expression alone (Table 1, Figure 1A))
- count 213 million 50-nt paired reads; 199 million passed QC; 148 million mapped; 78 million excluded as rRNA/tRNA (sequencing output on Illumina HiSeq 2000)
- pvalue FDR ≤ 0.05 (5%) with absolute log2 fold-change ≥ 0.6 (cutoff for statistically significant differentially expressed intergenic sequences (Cufflinks))
- other 54 to 244 nt; median 110 nt; average 118 nt (size distribution of the identified candidate sRNAs)
- fold_change 12.5% (Δhfq/Ea1189, 6 hr) and 7.2% (Δhfq/Ea1189, 12 hr) (relative expression of spf (spot42) in the hfq mutant vs wild type)
- count 12 sRNAs confirmed by Northern blot; 7 sRNAs confirmed by 5' RACE (experimental validation of expression/size and 5' boundaries)
- count 1 × 10^4 CFU ml-1 inoculum, lesion diameters at 6 days; three experiments with five biological replicates each (immature pear fruit virulence assay design)
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 Illumina RNA-seq (TopHat/Cufflinks) to compare wild-type Erwinia amylovora Ea1189 to a Δhfq mutant at two time points, calling candidate Hfq-dependent small RNAs from intergenic regions using an FDR ≤ 0.05 and |log2 fold-change| ≥ 0.6 threshold, followed by manual curation in Artemis. Candidate sRNAs were validated experimentally (Northern blot, 5' RACE) and analyzed for cross-species nucleotide conservation via BLAST and hierarchical clustering. Deletion mutants of selected sRNAs were phenotypically characterized (virulence, amylovoran production, motility, biofilm) using replicated assays (3 experiments with 4-12 replicates each), though the excerpted Methods/Results text does not state the specific inferential statistical test(s) applied to these phenotypic comparisons.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| Not stated (FDR-based differential expression cutoff via Cufflinks output) | Identification of intergenic transcripts with reduced expression in Ea1189Δhfq vs Ea1189 at 6 hr and 12 hr | Aggregated mapped reads per condition (e.g., 22M, 9M, 32M, 7M reads); explicit biological replicate number not stated in this excerpt | not stated |
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Differentially expressed intergenic transcripts were called using Cufflinks output filtered by an FDR ≤ 0.05 and |log2FC| ≥ 0.6 threshold, without a named per-gene statistical test or explicit replicate count in this excerpt.↳ Could also: Count-based tools such as DESeq2 or edgeR, which model per-gene dispersion from biological replicates and apply a Wald or likelihood-ratio test with Benjamini-Hochberg FDR correction — These approaches explicitly incorporate replicate-to-replicate variability into the significance test, which can also help quantify uncertainty in fold-change estimates for lowly expressed intergenic transcripts.
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Phenotypic assays (lesion size, amylovoran production, motility, biofilm) compared wild-type to deletion mutants across three repeated experiments with several replicates each, without a stated test name in this excerpt.↳ Could also: One-way ANOVA with a post-hoc test (e.g., Tukey HSD or Dunnett's test for multiple mutants vs one control), or a non-parametric Kruskal-Wallis test if normality is uncertain — These would also allow simultaneous comparison of multiple strains against the wild type while controlling the family-wise error rate across the several phenotypic comparisons.
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sRNA sequence conservation across 20 γ-Proteobacteria genomes was summarized using a custom conservation score and grouped by hierarchical clustering (Cluster 3.0, centroid linkage).↳ Could also: Alternative linkage methods (average or complete linkage) or a bootstrap-supported clustering/phylogenetic approach — Comparing clustering results under different linkage criteria or adding bootstrap support values could also help convey how robust the conservation groupings are to the specific clustering method chosen.
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Sample sizes for phenotypic assays (e.g., 4-12 replicates per experiment) appear to follow conventional practice rather than a stated a priori calculation in this excerpt.↳ Could also: An a priori power analysis based on an expected effect size and desired power — This could also provide a formal justification for the chosen replicate numbers and help readers gauge the sensitivity of the assays to detect smaller phenotypic differences.
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RNA-seq read counts were aggregated across all replicates of each condition prior to identifying differentially expressed intergenic regions, per this excerpt.↳ Could also: Reporting per-replicate counts and using a model that treats each biological replicate as an independent observation — This would also allow replicate variability to be propagated into the significance estimate rather than only reflected in the aggregate count, which some readers find useful for assessing consistency across replicates.
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Dispersion measures (e.g., SD, SEM, or CI) for the phenotypic assay results are not shown in the provided excerpt.↳ Could also: Reporting SD or a 95% confidence interval alongside means for each phenotypic measurement — This would also convey the spread and precision of the phenotypic measurements, which some readers find helpful for interpreting the magnitude and reliability of observed differences between strains.
What was reproduced
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Input data is a clean 1:1 match — all 12 GSE53763 samples were downloaded and genuinely aligned (TopHat2, 57-165M reads/sample, flagstat-verified), so nothing here turns on data availability. The deviations sit on the method-definition side and are shared between us and the authors: the shipped repo contains only Upstream_Ea.py, not the Cufflinks/Cuffdiff sRNA-discovery pipeline behind the headline claim, and the paper omits both the terminator filtering thresholds (912 raw hits -> 117 -> 23 -> 2 is undocumented) and any automatable version of the Artemis manual curation that produced the final 38. Our reconstruction recovers 20 novel intergenic/antisense DE loci (~53% of 38) and confirms hfq's global-regulator role (33.4% of annotated genes DE at 6h), so the central biological conclusion holds in a limited form; the GcvB 8.7-fold cross-check failed outright because GcvB is not an annotated feature in the RefSeq GFF3. Severity is moderate and there is no fabrication signal — the numbers are plausible and same-order, just not exactly re-derivable from what was deposited.
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