PbeVAMP724 Alleviates Cell Death and Enhances Resistance to Valsa Canker in Pyrus betulifolia

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The main question addressed by this study is whether PbeVAMP724 functions as a positive regulator of pear (Pyrus betulifolia) resistance to Valsa canker, and if so, through which defense-related mechanisms (hormone signaling, ROS accumulation, and activation of defense responses) it contributes to alleviating pathogen-induced cell death and enhancing immunity.

The topic is relevant and timely for the fields of plant pathology, plant immunity, and molecular breeding, as Valsa canker is a destructive disease and resistant germplasm is of high practical importance. The manuscript is original in its focus on PbeVAMP724, a VAMP family member, and its role in regulating resistance to Valsa canker in pear.
The study addresses a specific gap in the field: although vesicle trafficking proteins (including VAMPs) are known to participate in plant defense, their functional roles and regulatory mechanisms in woody fruit crops, particularly pear–Valsa interactions, remain insufficiently characterized. Therefore, the work provides useful mechanistic insight and candidate targets for resistance improvement.

Compared with previous studies on general defense pathways or single hormone-related resistance mechanisms, this manuscript adds value by:
- Identifying PbeVAMP724 as a functional component contributing to Valsa canker resistance in pear.
- Linking a vesicle trafficking-related protein to hormone signaling integration and ROS burst regulation, which are central to immune responses.
- Providing evidence that this gene may be a promising molecular target for breeding or engineering disease resistance, which enhances the translational relevance of the work.
Overall, the study contributes a new candidate gene and a plausible mechanistic framework that can support future resistance breeding programs.

The methodology is generally clear and appropriate; however, the following improvements would strengthen reproducibility and rigor:
- Statistical analysis clarity: Please provide more details on the statistical analyses (as noted for line 201), including: exact datasets compared by t-test; whether assumptions were checked (normality/homoscedasticity); number of biological replicates and technical replicates for each assay.
- Consistency of formatting and nomenclature: Ensure gene/protein names and species names are formatted consistently (italicization, abbreviations, and first full mention).

Additionally, a Conclusion section is missing and should be included to summarize key findings and implications.
The manuscript would also benefit from:
- a clearer statement of scientific limitations (e.g., boundaries of inference from the current experimental system),
-and a short, explicit future perspectives paragraph (as previously suggested), indicating how this work could be extended toward breeding applications and deeper mechanistic dissection.

The references appear appropriate and relevant to the topic, covering plant defense signaling and pathogen interactions. No major concerns were identified; however, the authors should ensure that citations adequately support statements regarding: VAMP family roles in immunity, hormone signaling crosstalk, and ROS-related defense mechanisms.

Specific Suggestions and Comments

Abstract L15: Give in full at first mention: ABA, SA, JA.
L96: V. pyri
L145: cefatothin sodium
L201: Please provide more details on the statistical analyses. Specify exactly which datasets were analyzed using the t-test.
L213 and later in the text: PbeVAMP724 — this was previously italicized; please ensure consistent formatting.
L221 (Figure 1): PbeVAMP724 — give in full at first mention and format in italics.
L225: Nicotiana benthamiana (italicize)
L286: Pyrus bretschneideri (italicize)
L353: Pyrus betulifolia (italicize)
L410: P. betulifolia
L441–442: The rows need to be merged.
L496: Valsa sordida (italicize)
L554: Valsa mali (italicize)
L557: Piper colubrinum and Phytophthora capsici (italicize)

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

That's a very nice paper, very descriptive. I think you can point more the novelty throughout the paper. I have some minor considerations to imporve clarity and reproducibility:

1. Title: "PbeVAMP724 alleviates cell death and enhances resistance to Valsa canker in Pyrus betulifolia" feels nicer
2. Abstract: Add a clear novelty statement that distinguishes this work from prior gene-function studies in the same pear Valsa canker experimental system, and explicitly cite closely related functional studies in Pyrus betulifolia ‘Duli-G03’/‘Huangguan’ fruit assays to avoid an overbroad novelty claim. Also, adjust to reflect any signifficant changes in the manuscript
3. Throuout the manuscript: Correct taxonomy and terminology (capitalize genus, consistent species names, avoid ambiguous “from valsa species”), and ensure species references match the pathogen actually used (Valsa pyri).
4. L34: Add concrete, properly referenced evidence for the specific risks and resistance development claims in the Valsa canker context (not generic statements), or narrow the claim.
5. L61: Clarify what is known specifically for VAMP72/724 homologs versus broader SNARE generalities, and cite primary functional studies relevant to VAMP72 family roles in immunity and secretion.
6. L68: Specify the gap with precision (species, trait, pathogen system, assays), and cite the most directly related published pear/apple Valsa canker resistance-gene functional studies to situate novelty and prevent redundancy. 
7. L73: Identify the dataset unambiguously (where published, accession, sample design), and predefine what “differential expression” means (effect size, FDR threshold).
8. L83: Provide objective criteria for fruit selection and handling (harvest maturity indices, storage duration/temperature, surface sterilization, wounding protocol, number of fruits per treatment), to ensure supermarket sourcing as biologically reproducible.
9. L87: Specify identification method, strain deposition/availability, and inoculum preparation details.
10. L90: Add essential details for reproducibility: culture conditions, filtration/sterilization method (not only centrifugation), and clarify “bacteria were removed” wording.
11. L90:  Define percent as v/v of clarified filtrate in what base medium, and justify dose selection.
12. L98: Define the experimental unit for each assay, and distinguish biological versus technical replicates across qRT-PCR, ROS, and lesion assays.
13. L101: Specify exact genome/annotation versions used for Pyrus betulifolia, promoter extraction coordinates/length, and how orthologs were selected.
14. L108: Fix tupo and report alignment method/parameters, model, bootstrap replicates, and justify NJ versus ML; include accession IDs for all sequences used.
15. L119-125: Specify promoter/terminator, tag presence/absence, selectable markers, cloning strategy, and provide primer sequences and verification via Sanger if possible.
16. L120: Provide fragment selection rationale, where the fragment maps in the gene, and include a positive silencing control to validate efficiency in tissue.
17. L132: Provide buffer composition, injection volume per site, number and position of sites per fruit, incubation humidity, and randomization/blinding for lesion measurement.
18. L136: Define how lesion diameter/area was measured (tool/software, single or two-axis, thresholding), and specify whether measurements were averaged per fruit or per site.
19. L144-149 Correct antibiotic name and units, specify selection agent for plant cells and concentration, define independent transformation events, and describe how many clones were screened.
20. L152-156: Replace ambiguous terminology (“bacterial cake”), specify pathogen inoculum type, and whether the assay measures fungal growth, host cell death zone, or both.
21. L158-163: Provide dye concentration, loading buffer, washing steps, normalization approach (per cell number/protein/volume), and include dye-only and ROS quencher controls to validate specificity, and define whether live pathogen or metabolites were used in ROS assays.
22. L165-175: Report library type, read length, single/paired-end, depth, QC/trimming software and versions, mapping parameters, genome build, gene model version, quantification method, and normalization approach used for downstream statistics. Also, state how counts/abundance were derived and avoid FPKM for differential expression; report multiple-testing correction method. Replace the untested “significant” label with explicit p-values and multiple-testing correction across genome-wide correlations, and justify the threshold choice.
23. L180-183: Specify background gene universe, enrichment test, p-value adjustment, and cutoffs; report exact versions of eggNOG-mapper and clusterProfiler.
24. “L190-199: Provide amplicon sizes, primer efficiencies, whether melt curves and no-RT/no-template controls were used, and justify/validate the reference gene(s) stability under treatments.
25. L201: Replace with an analysis plan appropriate for multi-group, multi-timepoint designs (ANOVA/mixed models), include assumption checks, specify two-sided versus one-sided tests, define alpha, and apply multiple-testing correction across timepoints and multiple genes/figures.
26. L207: Reconcile this with the classification as a VAMP (typically membrane-anchored) and with the membrane localization claim; report the specific prediction tools and outputs, and correct the domain interpretation if a C-terminal TM helix exists.
27. L217-220: Quantify localization, and add appropriate compartment markers to distinguish plasma membrane from other endomembranes.
28. L230: Avoid implying functional responsiveness from motif prediction alone; ensure the Methods fully describe hormone treatment design.
29. L398: Replace “n≥3” with exact n for each panel/timepoint, define biological replicate units, and report exact p-values or corrected significance thresholds.
30. L258: Ensure the qRT-PCR timecourse and the RNA-seq timecourse are directly comparable, and report whether the transient 3 h increase is statistically significant and how multiple comparisons were handled.
31. L270-278: Clarify that these are transiently infiltrated fruits rather than stable “lines,” and define how many fruits/sites were analyzed, how independence was ensured, and whether experimenters were blinded. For the lesion, provide the distribution of lesion sizes, confirm equal variances/normality or use nonparametric tests, and apply multiplicity correction for multiple timepoints and comparisons.
32. L298-300: Report whether each line represents an independent transformation event, and propagate all downstream phenotyping (viability, ROS, defense gene expression) across multiple independent lines rather than only OE1.
33. L301-307: Provide the quantitative MTT method rather than only representative images, and define what constitutes “survival rate.” Clarify what is being measured and include a pathogen-only growth control to disentangle host resistance from altered medium effects.
34. L432: Avoid implying that higher ROS is necessarily beneficial; add evidence that ROS changes are not simply reflecting differential viability, and clarify whether ROS is upstream or downstream of the observed defense gene induction.
35. L456: Discuss the biological plausibility given the suspension cell system, include corrected enrichment statistics (FDR), and avoid elevating co-expression to regulation without perturbation/causality tests.
36. L463: Rephrase claims to match the evidence level (association, positive regulator in assay contexts), and explicitly separate findings based on Vp metabolites from findings based on live pathogen inoculation.
37. Figure 2: Replace “n≥3” with exact n, add exact statistical tests and multiplicity handling, and ensure y-axis units and normalization basis are stated.
38. Figure 5: Clarify what “lesion” means in this assay, add scale bars/units consistently, and state whether images are representative of independent experiments with defined n.
39. Figure 7: define the baseline for “value… set as 1” for each gene, and report multiplicity correction across the multi-gene panel.
40. Figure 8: Add corrected p-values/FDR in the plot or legend, state background universe, and avoid implying pathway-level regulation from co-expression edges alone; ensure correlation coefficients are accompanied by significance criteria.
41. Table A1 Add amplicon sizes and expected melting temperatures, verify gene naming consistency (Actin versus Tub-b2 as reference genes in different figures), and ensure all primers correspond to the exact gene models used (IDs and annotation version).

Comments on the Quality of English Language

English is fine, there's some typos to fix but I don't need a certified translator is required for that.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Respected authors:

The manuscript entitled “PbeVAMP724 mediates the alleviation of cell death and enhances Valsa canker resistance in Pyrus betulifolia” addresses a relevant topic in plant physiology and phytopathology, particularly concerning the molecular mechanisms that regulate resistance to Valsa canker in Pyrus species. The study is valuable, and overall, the results presented indicate that PbeVAMP724 functions as a positive regulator of Valsa canker resistance, acting as a mediator in the ABA, SA, JA, and ROS signaling pathways, and participating in processes associated with vascular development that contribute to the immune response. These findings represent a significant contribution with potential utility for breeding programs aimed at improving resistance.

However, the manuscript requires several improvements to strengthen its clarity, methodological rigor, and scientific robustness. Specific observations are annotated directly in the revised manuscript attached to this report.

Comments for author File: Comments.pdf

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

I acknowledge the effort made to strengthen the quality of the manuscript, which has incorporated the comments made during the revision of the original document. Please receive cordial greetings

Author Response

Dear Reviewer,       Thank you very much for your valuable suggestions and contributions to my manuscript.    

Author Response File: Author Response.pdf