Identification of the CDPK Pan-Genomic Family in Pear (Pyrus spp.) and Analysis of Its Response to Venturia nashicola

Response to Reviewer 1 Comments

1. Summary

Thank you very much for reviewing our manuscript and providing valuable comments and suggestions during your busy schedule. Your suggestions are of great significance in guiding our research work, and we would like to express our heartfelt thanks to you. We will respond to your questions and suggestions one by one.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Yes/Can be improved/Must be improved/Not applicable

[Please give your response if necessary. Or you can also give your corresponding response in the point-by-point response letter. The same as below]

Are all the cited references relevant to the research?

Yes/Can be improved/Must be improved/Not applicable

Is the research design appropriate?

Yes/Can be improved/Must be improved/Not applicable

Are the methods adequately described?

Yes/Can be improved/Must be improved/Not applicable

Are the results clearly presented?

Yes/Can be improved/Must be improved/Not applicable

Are the conclusions supported by the results?

Yes/Can be improved/Must be improved/Not applicable

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: Lines 38-39 Please correct the formatting of species names. For instance, in the sentence “AtCPK3 has been shown to confer drought tolerance in Arabidopsis. Thaliana [2],” the species name should be written in italics and in lowercase as Arabidopsis thaliana.

Response 1: We thank the reviewer for pointing out this oversight. We have carefully revised the manuscript to ensure that all species names are correctly formatted in italics and lowercase throughout the text, including the example mentioned (Arabidopsis thaliana). All other instances of species names have also been checked and corrected accordingly.

Comments 2: Line 49-50 Please revise “Pears (Pyrus)” to “Pear (Pyrus)” in the introduction sentence and ensure consistency throughout the manuscript. The common name should be presented in the singular form (“pear”) when referring to the crop in general, with the genus name italicized.

Response 2: We sincerely thank the reviewer for this correction. We have revised the phrase to “Pear (Pyrus)” as suggested and have carefully checked the entire manuscript to ensure that the common name “pear” is used consistently in the singular form when referring to the crop in general, and that the genus name Pyrus is always italicized throughout the text.

Comments 3: Line 53-54 Please correct “pear-producting” to “pear-producing.” Ensure that the hyphenation and spelling are consistent throughout the manuscript.

Response 3: We thank the reviewer for catching this typographical error. The misspelling has been corrected to “pear-producing” as indicated. We have also performed a thorough check of the entire manuscript to ensure consistency in hyphenation and spelling related to compound terms.

Comments 4: Line 56-57: The sentence “Implementing disease control measures requires substantial human, material, and financial resources [13]” could be revised for clarity and conciseness.

Response 4: We thank the reviewer for this helpful suggestion. We have revised the sentence to: “Conventional pear tree disease control measures require substantial investment of labor, material resources, and financial resources” to improve clarity and conciseness, as recommended. We have also ensured consistency in terminology and style in related sections of the manuscript.

Comments 5: Line 62-64: In the sentence “Currently published pear reference genomes cover a broad range of taxonomic groups…,” the term “taxonomic groups” is not precise. It would be more appropriate to use “species” (e.g., P. bretschneideri, P. communis, P. pyrifolia) and “cultivars/rootstocks” where applicable. In addition, a brief introduction to these pear species, their geographical distribution, and horticultural relevance should be provided in the introduction to help readers unfamiliar with pear genomics understand their significance.

Response 5: We sincerely thank the reviewer for these valuable suggestions. We have revised the sentence to replace the imprecise term "taxonomic groups" with more accurate descriptors, specifying "species" and "cultivars/rootstocks" where appropriate. Additionally, as recommended, we have incorporated a brief introduction to the key pear species mentioned—including their geographical distribution and horticultural significance—in the introduction section. This addition aims to provide necessary context for readers less familiar with pear genomics. We have ensured that these revisions improve the clarity, precision, and accessibility of the manuscript.

Comments 6: Line 79-85: The terminology in this section requires clarification. The phrase “five cultivated pear varieties” is inaccurate, since P. bretschneideri, P. ussuriensis, P. sinkiangensis, P. pyrifolia, and P. communis are recognized as distinct species, not varieties. Similarly, P. betulifolia is a wild species and should not be described as a variety. Please revise to “five cultivated pear species” (and specify where rootstocks or interspecific hybrids are included). In addition, the claim of “constructing a pear pan-genome” may be misleading. As described, the study integrates multiple reference genomes, but does not seem to perform a full pan-genomic analysis (e.g., core vs. dispensable gene sets, presence–absence variation, structural variation). This distinction should be clarified to avoid overstating the scope. Finally, there are minor spelling and typographical errors in this section (e.g., ‘regulat’ → ‘regulate’) that should be corrected throughout the manuscript.

Response 6: We are grateful to the reviewer for these critical observations and suggestions, which have significantly improved the precision and clarity of our manuscript. We have carefully addressed each point as follows:

1.Terminology clarification: We have revised the inaccurate term "five cultivated pear varieties" to "five cultivated pear species" throughout the manuscript. We now clearly specify the inclusion of the wild species P. betulifolia and interspecific hybrids where applicable.

2.Scope clarification regarding pan-genome: We agree with the reviewer that the original phrasing could overstate our analysis. We have toned down our claims and revised the text to clarify that our work integrates multiple reference genomes to establish a foundational resource for pan-genomic analysis, rather than claiming a complete pan-genome construction.

3.Spelling and typographical errors: We have corrected the spelling error ('regulat' to 'regulate') and have performed a thorough proofreading of the entire manuscript to identify and correct any similar minor errors.

We believe these revisions have enhanced the accuracy and appropriateness of our terminology and overall presentation. Thank you again for your meticulous review.

Comments 7: Line 90-95 The taxonomy and classification in this section need clarification. The text currently refers to cultivars as “varieties” and mixes species, cultivars, and hybrids. For accuracy, please distinguish clearly between pear species (e.g., P. bretschneideri, P. ussuriensis, P. sinkiangensis, P. pyrifolia, P. communis), cultivars within those species (e.g., ‘Dangshansuli’, ‘Xuehua’, ‘Nanguoli’, ‘Kuerle’, ‘Xinshiji’, ‘Qieli’), and hybrids/rootstocks (e.g., ‘Zhongai 1’, with P. ussuriensis × P. communis background; ‘Duli’, a wild species rootstock). The current phrasing blurs these distinctions and may confuse readers unfamiliar with pear taxonomy. I recommend revising to consistently use “species” for Pyrus taxa, “cultivars” for named accessions within species, and “rootstocks” or “hybrids” where appropriate.

Response 7: We sincerely thank the reviewer for this detailed and critical feedback regarding taxonomic precision. We fully agree with the recommendations and have thoroughly revised the section to ensure clear and consistent distinctions between the different taxonomic and horticultural categories. Specifically, we have:

1.Replaced the term "varieties" with the accurate terms "species", "cultivars", and "rootstocks" or "hybrids" as appropriate throughout the manuscript.

2.Explicitly described the genetic background of hybrid/rootstock materials, such as ‘Zhongai 1’ (an interspecific hybrid of P. ussuriensis × P. communis) and ‘Duli’ (utilizing the wild species P. betulifolia as a rootstock).

We have also extended these corrections to other relevant sections of the manuscript to maintain consistency. We believe these revisions have significantly improved the taxonomic clarity and accuracy of our work and thank the reviewer again for this important suggestion.

Comments 8: Line 96-100 The methods description for CDPK identification is unclear and needs revision. The phrase “to confirm the existence the 8 pear accessions” is grammatically incorrect and ambiguous — it should specify that SMART was used to confirm the presence of conserved CDPK domains in candidate genes from the eight pear accessions. In addition, the statement “genes with a collinear relationship to CDPKs were considered CDPK members” is vague; please clarify whether this refers to synteny-based identification of additional family members that were not detected by HMM searches. Overall, this section should be rewritten for clarity and to accurately reflect the gene identification pipeline.

Response 8: We sincerely thank the reviewer for these insightful comments that have helped us improve the clarity and accuracy of our methodology description. We have thoroughly revised this section to address all the concerns raised:

1.We have corrected the grammatical error and ambiguous phrase to clearly state that "SMART was used to confirm the presence of conserved CDPK domains in candidate genes from the eight pear accessions" as suggested.

2.We have clarified that the synteny-based analysis was specifically employed "to identify additional family members that were not detected by the initial HMM searches," making explicit the complementary nature of this approach.

3.We have rewritten the entire paragraph to present a clearer, more logical flow of our gene identification pipeline, which now consists of: initial domain screening via HMMER, subsequent validation of domains using SMART, and finally a supplementary synteny-based analysis to recruit additional members that might have been missed by the domain-based methods.

We believe the revised text now accurately and clearly describes our methodology. Thank you for helping us improve the manuscript.

Comments 9: Line 102-108 The description of the phylogenetic and PAV analysis should be clarified. The phrase “CDPKs from pears and other disease resistance-related genes” is ambiguous — please specify whether only CDPKs from pear and homologs from other species were included, or whether unrelated resistance genes were analyzed. In addition, the use of “presence/absence variation (PAV)” should be reconsidered, since true PAV analysis typically requires synteny/orthogroup-based presence/absence calls across genomes, rather than a simple heatmap of gene occurrence. Please revise for accuracy and consistency (e.g., “Rscript v4.0.3” should be “R v4.0.3”).

Response 9: We thank the reviewer for these insightful comments that have helped us improve the clarity and accuracy of our methodology description. We have revised the relevant section to address all the raised concerns:

1.Clarification of phylogenetic analysis content: We have specified that the phylogenetic analysis included "CDPKs identified from the eight pear accessions along with homologous CDPK sequences from other plant species known for their relevance to disease resistance."

2.Precise terminology for "PAV" analysis: We agree with the reviewer that the term "presence/absence variation (PAV)" was technically inappropriate for our analysis. We have replaced it with the more accurate description "the presence or absence of each CDPK gene across the eight accessions" to better reflect that our analysis visualized gene distribution patterns rather than performing full synteny-based PAV analysis.

3.Correction of software citation: We have corrected "Rscript v4.0.3" to "R v4.0.3" throughout the manuscript.

We believe these revisions have significantly improved the accuracy and clarity of our methodological descriptions. Thank you for these valuable suggestions.

Comments 10: Line 120-126: The RNA-seq analysis workflow is described, but the underlying data are not provided or referenced. Please indicate the source of the transcriptomic data used for expression analysis (e.g., accession numbers in NCBI SRA, BioProject IDs, or details of whether the data were newly generated or obtained from published datasets). Without this information, the reproducibility of the expression study is limited. It is recommended that the authors clearly state the data availability and provide accession numbers where appropriate.

Response 10: We thank the reviewer for this important comment regarding data availability. The transcriptomic data used for expression analysis were newly generated specifically for this study through RNA sequencing performed by Beijing Biomarker Technologies Co., Ltd. These data are part of an ongoing research project, and the raw sequencing data will be deposited in the NCBI Sequence Read Archive (SRA) and made publicly accessible upon manuscript acceptance. The BioProject accession number will be provided at that time. In the meantime, the data are available from the corresponding author upon reasonable request. We have revised the Methods section to include this information and have added a Data Availability Statement to ensure transparency.

Comments 11: Line 127-136: The description of the transient overexpression experiment requires clarification and refinement. The methodology is fragmented and contains unclear phrases. For example, the sentence “After inoculation with scab cake” is ambiguous — it should be clarified whether this refers to inoculation with Venturia nashicola conidia, mycelial plugs, or another preparation. Similarly, the description of “calli tissues from ‘Xuehua’ fruits” could be expanded to specify how the calli were induced and maintained. Please also ensure consistency in formatting scientific names (e.g., Agrobacterium tumefaciens rather than “A. tumefaciens”). Overall, this section should be rewritten for clarity and reproducibility.

Response 11: We believe these revisions have significantly improved the clarity and precision of our methodological description. Thank you for these helpful suggestions.

1.Clarification of pathogen inoculation: We have replaced the ambiguous term "scab cake" with the precise description "Venturia nashicola mycelial plugs" to clearly indicate the inoculation method used.

2.Detailed callus induction protocol: We have expanded the description of callus tissues to specify that they were induced from sterilized young 'Xuehua' pear fruits cut into explants and cultured on MS medium supplemented with specific concentrations of 2,4-D and 6-BA, with subculturing every 20 days.

3.Consistent formatting of scientific names: We have ensured consistent use of the full scientific name "Agrobacterium tumefaciens" upon first mention throughout the manuscript, as recommended.

4.Overall improved clarity: We have rewritten the entire paragraph to present a clear, logical flow of the experimental procedure, enhancing its reproducibility.

We believe these revisions have significantly improved the clarity and precision of our methodological description. Thank you for these helpful suggestions.

4. Response to Comments on the Quality of English Language

Point 1: The current version of the manuscript suffers from some structural, linguistic, and methodological issues that hinder its readability, reproducibility, and scientific attention. The paper in the current state is written poorly. Sentence structure is not clear and meaningful.

Response 1: We deeply appreciate the reviewer's frank assessment. We agree that the previous version of the manuscript had significant shortcomings. We have now undertaken a major revision to comprehensively address these issues. Our corrective actions are detailed as follows:

1. Addressing Linguistic Issues:

Action Taken: We acknowledge that the English language quality was unacceptable for scientific publication. To rectify this, we have engaged a professional scientific editing service specializing in our field. Native English-speaking editors have thoroughly polished the entire manuscript to correct grammatical errors, improve sentence clarity, and ensure proper academic tone.

2. Improving Structural Issues:

Action Taken: We have completely reorganized the manuscript's structure to enhance its logical flow and readability. Specifically, we have:

Revised the Introduction to more clearly define the research gap and our objectives.

3. Clarifying Methodological Issues (Reproducibility):

Action Taken: To address concerns about reproducibility, we have significantly expanded the Methods section with meticulous detail.

We believe that these extensive revisions have fundamentally improved the manuscript and addressed the reviewer's concerns. We are grateful for the opportunity to resubmit a greatly improved version and hope that it now meets the high standards of your journal.

5. Additional clarifications

[Here, mention any other clarifications you would like to provide to the journal editor/reviewer.]

Response to Reviewer 2 Comments

1. Summary

I extend my sincerest gratitude for the time and effort you have dedicated to reviewing our manuscript. Your insights are invaluable to us. Below, you will find a comprehensive list of our responses to your comments, along with the respective revisions and corrections meticulously highlighted in the track changes feature within the re-submitted document.

We have taken great care to address each point with the aim of enhancing the clarity, accuracy, and overall quality of our work. We trust that these amendments will meet with your approval and bring us closer to the publication of our research.

Thank you once again for your invaluable feedback.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Is the work a significant contribution to the field?

[Please give your response if necessary. Or you can also give your corresponding response in the point-by-point response letter. The same as below]

Is the work well organized and comprehensively described?

Is the work scientifically sound and not misleading?

Are there appropriate and adequate references to related and previous work?

Is the English used correct and readable?

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: The manuscript "Identification of the Pyrus CDPK Pan-genomic Family and Analysis of Its Response to Venturia nashicola in Pear" describes a relevant study but must be thoroughly improved before being publishable. In title, "Pyrus" is unnecessary because the "Pear" term is included. Then, when Pyrus is mentioned, correction to Pyrus spp. must be done because various species of the genus Pyrus were analyzed.

Response 1: We sincerely thank the reviewer for this constructive feedback. We have revised the manuscript accordingly to address these points:

1.Title: We have removed the redundant "Pyrus" from the beginning of the title as suggested. The revised title now reads: "Identification of the CDPK Gene Family in Pear (Pyrus spp.) and Analysis of Its Response to Venturia nashicola". We believe this new title is more concise and accurate.

2.Pyrus spp. nomenclature: Throughout the manuscript, we have carefully reviewed all instances where the genus Pyrus is mentioned in the context of our multi-species analysis. We have corrected these to the accurate designation "Pyrus spp." to properly reflect that our study encompasses multiple species within the genus, as rightly pointed out by the reviewer.

We appreciate these valuable suggestions, which have significantly improved the precision and clarity of our manuscript.

Comments 2: In Introduction, no general or specific objectives are explicit. Instead, a brief presentation of the overall manuscript is given in the last paragraph. The aims of the study must be clearly presented, especially considering that some experimental research was done in tobacco, not in pear. Also, specific objectives related to in silico, in vivo and in vitro studies must be presented. Indeed, there are some experiments with pear calli described in Results but nor mentioned in Materials and Methods.

Response 2: We sincerely thank the reviewer for these critical comments regarding the clarity and completeness of our study objectives and methodological descriptions. We have thoroughly revised the manuscript to address these concerns as follows:

1.Explicit research objectives in Introduction: We have added a dedicated paragraph at the end of the Introduction section that clearly states both the general aim and specific objectives of our study. These now explicitly encompass our multi-level approach including:In silico analyses (pan-genome construction, phylogenetic analysis, etc.)、In vitro experiments with pear calli、In vivo validation in tobacco model system

2.Methodological completeness: We have expanded the Materials and Methods section to include detailed descriptions of:The pear calli culture protocols

We believe these significant revisions have greatly improved the transparency and completeness of our manuscript by clearly articulating our research objectives and ensuring all described methodologies are properly documented in the relevant sections. Thank you for these valuable suggestions that have substantially enhanced our manuscript.

Comments 3: Particularly in Materials and Methods, the assessment of normality distribution ought to be done to properly apply parametric statistics such as the t-test.

Response 3: We thank the reviewer for this important methodological suggestion. We fully agree that testing for normality is an essential step before applying parametric statistical tests. Accordingly, we have revised the Materials and Methods section to explicitly state that the normality of the data distribution was assessed using the Shapiro-Wilk test prior to conducting t-tests. This addition ensures that our statistical analyses are properly validated and meet the necessary assumptions for parametric testing. We appreciate this comment, which has helped improve the rigor of our statistical methodology.

Comments 4: In Results, section 3.4 actually corresponds to Discussion. Legends to Tables and Figures must be improved to achieve self-contained meaning.

Response 4: We sincerely thank the reviewer for these valuable observations, which have significantly improved the organization and clarity of our manuscript.

1.Section 3.4 Reorganization: We agree with the reviewer that the content of the original section 3.4 was interpretive in nature and more suitable for the Discussion. Consequently, we have moved this section to the Discussion part of the manuscript. The Results section now focuses solely on the objective presentation of our findings.

2.Improvement of Figure and Table Legends: We have thoroughly revised all legends for our figures and tables. Each legend now provides a more comprehensive and self-contained summary, including essential details on the experimental context, methodology (where pertinent), and a clear interpretation of the key findings presented.

We believe these revisions have enhanced the logical flow of our manuscript and the clarity of our data presentation. Thank you for these insightful suggestions.

Comments 5: Discussion and eventually Conclusions (absent in present version) sections must be redacted to respond the also absent general and specific objectives, which in fact must be added in Introduction, as previously indicated.

Response 5: We sincerely thank the reviewer for this critical comment regarding the structure and focus of our manuscript. We have carefully revised the paper to address these important concerns as follows:

1.Introduction Revision: We have added a dedicated paragraph at the end of the Introduction section that clearly states the general and specific objectives of our study, as previously recommended.

2.Addition of a Conclusions Section: We have created a new, standalone Conclusions section that summarizes the main findings of our study, emphasizes their scientific and practical significance.

We believe these significant revisions have greatly improved the logical flow, clarity, and scholarly rigor of our manuscript. The study's aims and outcomes are now presented with greater transparency and structure. We are grateful for these suggestions, which have substantially enhanced the quality of our work.

Comments 6: English must be deeply revised. Words as varietiesare (lane 213), wherea (lane 306), as examples, must be revised. Additionally, there are lost and spare spaces among word along the entire text.

Response 6: We sincerely thank the reviewer for carefully reading our manuscript and pointing out these language and formatting issues. We have thoroughly revised the entire manuscript to address these concerns:

1.We have corrected the specific errors mentioned (‘wherea’ to ‘whereas’) and conducted a comprehensive spell-check throughout the document to identify and correct similar errors.

2.We have carefully reviewed the entire text to ensure proper spacing between words and sentences, removing any unnecessary spaces and adding missing spaces where needed.

3.Additionally, we have engaged a professional English editing service to perform a complete language revision of the manuscript to ensure proper grammar, syntax, and academic style throughout.

We appreciate the reviewer's attention to these important details, which has significantly improved the overall quality and readability of our manuscript.

4. Response to Comments on the Quality of English Language

Point 1: Please deeply revise.

Response 1: We sincerely thank the reviewer for this critical comment regarding the English language quality of our manuscript. We fully acknowledge that the initial submission required significant linguistic improvement. In response to this comment, we have undertaken a comprehensive and deep revision of the entire manuscript to ensure the clarity, accuracy, and professionalism of the English language. To achieve this, we have taken the following concrete steps:

1.Professional Editing Service: The manuscript has been thoroughly edited and polished by a professional scientific editing service .

2.Meticulous Author Review: Following the professional editing, we, the authors, have carefully reviewed the entire document again. We have double-checked all technical terms, gene nomenclature (e.g., Pyrus spp., Venturia nashicola), and scientific expressions to ensure they are precise and consistent throughout the text.

3.Focus on Clarity and Flow: We have paid particular attention to improving sentence structure, grammar, punctuation, and overall narrative flow. We have rephrased awkward constructions, eliminated ambiguities, and ensured that the logical progression of ideas is clear and easy to follow.

We believe that these extensive revisions have significantly enhanced the readability and overall quality of the manuscript. We hope that the current version now meets the high standards required for publication in your esteemed journal. We thank the editor and reviewer for the opportunity to improve our work.

5. Additional Clarifications [Here, mention any additional clarifications you would like to provide to the journal editor/reviewers.]