Study on the Mechanism of Resistance of Pepper Cultivars Against Phytophthora Blight via Transcriptome Analysis

Response to Reviewer X Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

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

We have revised the manuscript according to the reviewers' suggestions. In addition, we have carefully polished other parts of the text to improve clarity and coherence.

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: It is poorly described; the growing conditions of the plants (humidity and fertilization) are not specified.

Response 1: Thank you for pointing this out. We agree with this comment. Therefore, we have refined the plant cultivation methods by providing additional details on the conditions, such as humidity and fertilization in line 105-110.

Comments 2: The methodology for qRT-PCR analysis to assess the reliability of RNA-Seq was not described.

Response 2: Agree. We have, accordingly, added a new section (Section 2.4) entitled "Primer design, qRT-PCR, and data analysis" to Chapter 2.

Comments 3: It is not described how the relative expression in plants was determined (Fig. 1D), nor is the result described in the corresponding section.

Response 3: Thank you for pointing this out. We agree with this comment. Therefore, we have added text to the Results section in line 189-194 to enhance the clarity of our findings.

Comments 4: Line 194 and 209: Figures are not appropriately referenced.

Response 4: Thank you for pointing this out. Agree. We have carefully revised the in-text citations for all figures to ensure correct numbering and improved contextual relevance.

Comments 5: The authors indicate that the resistance screenings were performed at 0, 2, and 5 dpi; however, in line 154, it is stated that disease symptoms were recorded at 7 dpi. Please explain.

Response 5: Thank you for pointing this out. While the disease incidence is typically assessed at 7 dpi according to Yu’s method (as mentioned in Section 2.1). However, based on our findings (Section 3.1., Figures 1B-1D), we defined 2 dpi and 5 dpi as the early and late infection stages for transcriptomic sequencing.

Comments 6: Table 1: I suggest changing Resistance by phenotype.

Response 6: Thank you for this thoughtful suggestion. We have used the term "Resistance" because the classifications (S, MR, R, and HR) are based on a widely accepted scoring system established by Yu’s study. To avoid any potential ambiguity, we have clearly described this established grading standard in the Methods section (Section 2.1.). Given that "Resistance" is the standard term used in this specific context within our field, we believe retaining it is appropriate for clarity and consistency with the cited literature. Therefore, we have kept the original header but ensured the criteria are explicitly stated in the text.

Comments 7: Please take a look at Figure Legend 1, where the left panel corresponds to the non-inoculated plants.

Response 7: Thank you for pointing this out. We sincerely apologize for the errors in the original version. The figure legend has been amended, and it has undergone a comprehensive revision to enhance its accuracy and readability.

Comments 8: The description of the results does not match the figures 3 and 4. Line 254. It is not clear where this data was obtained from (DEGs number); it does not match the Venn diagrams in the Figure.

Response 8: We sincerely thank the reviewer for this astute observation. The discrepancy arises because we employed two different fold-change thresholds for distinct analytical purposes, which was not clearly stated in the original manuscript. We apologize for the lack of clarity.

For Figures 3 and 4: The analysis aimed to provide a broad overview of functional enrichment. Therefore, we used a relatively inclusive threshold of |log2FC| ≥ 1 (corresponding to a 2-fold change) to identify a comprehensive set of DEGs for Gene Ontology (GO) and KEGG pathway analysis.

For the analysis in Line 254 (and surrounding text): The goal was to identify a high-confidence set of genes with dramatic expression changes that are most critical for the contrasting resistance responses between cultivars. For this focused discovery, we applied a more stringent threshold of |log2FC| ≥ 5 (corresponding to a 32-fold change) to isolate the most significantly upregulated and downregulated genes.

We have now revised the manuscript to explicitly state the specific fold-change thresholds used in each section. The Materials and Methods section has been updated to include both criteria in line 152 and the Results section (Section 3.5.) has been revised to enhance its scientific rigor. In addition, the descriptions of the Venn diagrams have been refined to provide a more precise interpretation of the overlaps and unique gene sets. We believe this clarification resolves the discrepancy and strengthens the narrative of our study.

4. Response to Comments on the Quality of English Language

Point 1: Review the entire manuscript, as there are some misspelled words or repeated phrases, for example:
Line 69: Transcriptomixs
Line 157: Table 1, Figure 1A
Line 180

Line 226

Line 397

Response 1:    We sincerely apologize for these errors and thank the reviewer for bringing them to our attention. The specific issues listed (e.g., the misspelling in line 69) have been corrected.

To ensure the highest quality, we have performed a word-by-word review of the manuscript to eliminate any remaining spelling, grammatical, or redundant phrases.

Response to Reviewer X Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

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

We have revised the manuscript according to the reviewers' suggestions. In addition, we have carefully polished other parts of the text to improve clarity and coherence.

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

Dear authors, your manuscript presents an interesting study, however, here are some comments and suggestions:

Comments 1: The presentation of the article looks sloppy. The figure captions are not aligned. Please be more careful with the presentation.

Response 1: We sincerely apologize for the oversight in the presentation of our manuscript and thank the reviewer for bringing this to our attention. We have thoroughly revised the entire document to ensure a professional and consistent format. Specifically, all figure captions have been carefully aligned and uniformly formatted. Additionally, we have checked the consistency of fonts, spacing, and layout throughout the manuscript to eliminate any appearance of carelessness. We thank the reviewer again for this suggestion, which has  undoubtedly improved the overall quality of our work.

Comments 2: Figure 1 is very confusing, with letters that are unclear whether they are subsections or treatments. The figure could be divided or each subfigure could be better identified. In addition, the figure caption is not formatted.

Response 2: We thank the reviewer for this valuable feedback regarding the clarity and presentation of Figure 1. We sincerely apologize for the lack of clarity in the original version.

We have carefully considered the suggestion to divide Figure 1. After thorough deliberation, we believe that keeping the figure as a single entity is scientifically preferable because it provides a comprehensive and sequential visual summary of the entire disease progression and the rationale for time-point selection. Dividing it might disrupt the narrative flow and make it harder for readers to grasp the complete picture.

However, we fully agree with the reviewer's core concern about the figure's clarity. Therefore, we have implemented the following comprehensive revisions to address this issue:

1.       Completely Rewritten Figure Legend: The figure legend has been thoroughly reformatted and expanded into a detailed, point-by-point description. It now explicitly and concisely explains what each panel (A, B, C, etc.) represents, ensuring the reader can easily interpret the figure without referring back to the main text.

2.       Revised Corresponding Text: As suggested, we have enhanced the description of the results in Section 2.1 to more clearly guide the reader through the figure and its key findings.

We are confident that these significant revisions have substantially improved the clarity, readability, and professional presentation of Figure 1, successfully resolving the confusion mentioned by the reviewer. We believe the figure in its current integrated form, supported by the detailed legend and text, now effectively communicates our findings.

Comments 3: As a recommendation for presenting comparative graphs between RNAseq and qRT-PCR results, present the RNAseq results as lines above the qRT-PCR bars, as they are presented as if they were different treatments.

Response 3: Thank you for pointing this out. We agree with this comment. The figure has been revised accordingly and is now presented as Figure 8 in the revised manuscript.

Comments 4: Improving the discussion and conclusions is very simple.

Create a diagram integrating the main metabolic pathways differentially affected and correlate it with the processes of resistance and infection.

Response 4: Thank you for pointing this out. We have incorporated this diagram into the Conclusion section to summarize the key findings of the study.

4. Response to Comments on the Quality of English Language

Point 1: Review the wording; there are several errors with some words and in the grammar.

Response 1: We sincerely apologize for these errors and thank the reviewer for bringing them to our attention. To ensure the highest quality, we have performed a word-by-word review of the manuscript to eliminate any remaining spelling, grammatical, or redundant phrases.

Response to Reviewer X Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

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

We have revised the manuscript according to the reviewers' suggestions. In addition, we have carefully polished other parts of the text to improve clarity and coherence.

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: The present paper titled “Study on the mechanism of pepper's resistance against pepper blight via transcriptome analysis” presents a transcriptomic analysis of the interaction between pepper and Phytophthora capsici. In the first part of the paper, the authors present an inoculation assay on 21 different pepper varieties to classify them into resistant or susceptible. This first part is the strong suit of the paper. From these 21 varieties the authors select 1 resistant and 1 susceptible to perform the transcriptomic analysis. The authors conclude that several pathways are differentially expressed focusing on Photosynthesis, Signal transduction, Secondary metabolism, Lipid metabolism, Cell wall and Stress response. However, the authors should address some comments/suggestions to improve the readability of the paper.

Response 1: We sincerely thank the reviewer for the positive assessment of our work, particularly regarding the phenotypic screening of the 21 pepper varieties, and for the constructive comments aimed at improving the readability and overall quality of our manuscript. We have carefully considered all the comments and suggestions provided and have revised the manuscript accordingly. Our point-by-point responses to the specific recommendations are detailed below.

Comments 2: The main concern is the writing of the text. The text is hard to read and difficult to follow. For example, un line 397 there is the sentence “In our RNA-seq analysis, uninoculated plants from both the resistant (19K23) and susceptible (QM) varieties.” What does this sentence mean? It seems incomplete and it follows with “To address this, our subsequent qRT-PCR validation included dedicated, time-matched uninoculated controls” which also is difficult to understand because the “problem” laid on the previous sentence is missing. Probably the authors mean that there is no uninoculated control at 2dpi and 5dpi, but it is not clear. The authors should thoroughly rewrite the text to increase the readability.

Response 2: We thank the reviewer for this critical comment and sincerely apologize for the lack of clarity in our original writing. The reviewer is absolutely correct; the sentence in line 397 was incomplete and the logical flow was broken. We have thoroughly revised the entire manuscript to rectify this issue. Specifically, we have completed the incomplete sentences and carefully rewritten the sections with logical jumps to ensure a clear and seamless narrative. The specific paragraph in question has been entirely rewritten for clarity and logical coherence. We believe these efforts have significantly improved the readability of the manuscript.

Comments 3: The lack of uninoculated controls mentioned above is another concern. Seedlings 2 and 5 days older will have differences in gene expression due to developmental process unrelated to the Phytophthora infection. The authors should indicate in the text how they will discern between DEGs related to development and the DEGs related to the infection.

Response 3: Thank you for pointing this out. We fully agree that distinguishing gene expression changes due to development from those due to pathogen infection is essential.

We thank the reviewer for raising this critical methodological point. We fully agree that distinguishing gene expression changes due to development from those due to pathogen infection is essential.

The reviewer's concern is precisely why we included dedicated, time-matched mock-inoculated control plants for each cultivar (19K23 and QM) at each time point in our qRT-PCR measurement. In our qRT-PCR analysis, the groups labeled "H2" and "H5" (for the resistant cultivar) and "S2" and "S5" (for the susceptible cultivar) were each compared against their own corresponding mock-inoculated controls at the same developmental stage (2- or 5-days-old). This means that the differential expression analysis intrinsically filters out gene expression changes attributable solely to normal growth over 2 or 5 days.

As the reviewer astutely noted, the original text failed to clearly state this rationale. We have now thoroughly revised the Methods section (2.2) to explicitly describe the use of these time-matched mock controls. Furthermore, we have added a clarification in the Discussion to emphasize that our experimental design allowed us to control for ontogenetic variation, thereby strengthening the conclusion that the identified DEGs are primarily linked to the immune response against Phytophthora capsici. The strong correlation between our RNA-seq and qRT-PCR results further validates this approach.

Comments 4: In figure 1D, what does it mean “Relative expression of Phytophthora”. It is unclear if this means the authors are quantifying the pathogen in planta. Please, incorporate a clarification in M&M and indicate the gen/marker being used.

Response 4: Thank you for pointing this out. We agree with this comment. Therefore, we have added text to M&M (Section 2.4.) and the Results section in line 189-194 to enhance the clarity of our findings.

Comments 5: Please, indicate the number of experiments performed for the inoculation assay and the number of plants per experiment.

Response 5: Thank you for pointing this out. We agree with this comment. Therefore, we have added text to M&M (Section 2.1.) in line 113-116.

Comments 6: In line 179 the authors indicated that “A total of 827,516,272 raw data were obtained.” What does raw data mean, reads? Bases? Please specify.

Response 6: We are grateful to the reviewer for this insightful comment. To avoid any ambiguity, we have revised the manuscript to consistently use the term "raw reads" instead of the more generic "raw data" when referring to the initial sequencing output. This change has been applied globally to ensure terminological precision.

Comments 7: In line 140 DEGs are defined as “adjusted p-value < 0.01 and FC ≥ 2” but in line 192 the criteria is changed to “p-value ≤ 0.05 and |log2 FC| ≥ 1”. Please, explain why two different criteria are used to define DEGs.

Response 7: Thank you for pointing this out. We apologize for this oversight in our writing. In fact, a consistent threshold of p-value ≤ 0.01 and |log₂FC| ≥ 1 was applied to all our analyses. The manuscript has been corrected to accurately reflect this criterion.

Comments 8: Text in the figures (figure 2, 3, 4 and 7) is hard to read. Please, increase fond size.

Response 8: We appreciate the reviewer's feedback on the readability of the figures. We have now significantly increased the font size in Figures 2, 3, 4, and 7. Furthermore, all figures have been re-exported as high-resolution images to ensure that the text and labels remain clear and sharp upon publication.

Comments 9: In line 241 the authors indicate that “The DEGs which were proven to be involved in disease resistance were then intensively analyzed in each genotype”. Please, explain what does it mean “proven”, did the authors perform additional experiments not mentioned in the text? Is a external citation needed? What do the authors mean with “intensively analyzed”? did the authors perform additional measurements or do they mean the heatmap?

Response 9: Thank you for raising these points. In response, we have taken the following actions to improve clarity:

1.       Regarding the statement on disease resistance-related genes, we have added specific citations (see line 442-445) in the Discussion section to support our claim.

2.       The relevant sections of the text have been revised to provide a more precise and comprehensive explanation of these analyses. The term "intensively analyzed" has been clarified to explicitly refer to the detailed examination of expression patterns presented in the heatmap.

Comments 10: In formation in figures is hard to read. In some figures the information seems not to even match with the description. In figure 7, line 351 the authors indicate that “In this figure, panels A-B: Photosynthesis”. However, panel B, very hard to read, seems to indicate “chitinase activity”. Please, explain how the chitinase activity is related to Photosynthesis.

Response 10: We sincerely thank the reviewer for this critical observation and for pointing out the significant error in the labeling of Figure 7. We sincerely apologize for this mistake and the confusion it has caused.

The reviewer is absolutely correct. Panel B in Figure 7 relates to chitinase activity, which is a well-known defense-related enzyme in plant-pathogen interactions, and it is not related to photosynthesis. The description in the original manuscript ("panels A-B: Photosynthesis") was incorrect due to an error during figure assembly and labeling.

We have taken the following corrective actions:

1.       Corrected Figure 7: We have revied the figure to ensure all text is clear and legible. Most importantly, we have corrected the figure legend and the in-text description to accurately reflect the content of each panel. The figure now correctly identifies Panel A as related to photosynthesis and Panel B as related to chitinase activity.

2.       Revised the Text: The text in the Results section (Section 3.5.1) has been thoroughly rewritten to correctly describe the findings presented in each panel of Figure 7.

3.       Full Manuscript Check: We have conducted a comprehensive review of all figures and their corresponding legends and in-text descriptions throughout the manuscript to ensure consistency and prevent similar errors.

We are grateful to the reviewer for their meticulous review, which has been essential in improving the accuracy and clarity of our manuscript.

Comments 11: In line 253 the authors compare the resistant and the susceptible cultivars to find shared and unique genes. However, the authors mention that the cutoff for the DEGs is changed: “The absolute values of log2FC were set as equal to or greater than 5”. Please explain, why the authors decided to change the log2FC cutoff.

Response 11: We sincerely thank the reviewer for this astute observation. The discrepancy arises because we employed two different fold-change thresholds for distinct analytical purposes, which was not clearly stated in the original manuscript. We apologize for the lack of clarity.

For Section 3.3. and Section 3.4.: The analysis aimed to provide a broad overview of functional enrichment. Therefore, we used a relatively inclusive threshold of |log2FC| ≥ 1 (corresponding to a 2-fold change) to identify a comprehensive set of DEGs for Gene Ontology (GO) and KEGG pathway analysis.

For the analysis in Line 254 (Section 3.5.): The goal was to identify a high-confidence set of genes with dramatic expression changes that are most critical for the contrasting resistance responses between cultivars. For this focused discovery, we applied a more stringent threshold of |log2FC| ≥ 5 (corresponding to a 32-fold change) to isolate the most significantly upregulated and downregulated genes.

We have now revised the manuscript to explicitly state the specific fold-change thresholds used in each section. The Materials and Methods section has been updated to include both criteria in line 152 and the Results section (Section 3.5.) has been revised to enhance its scientific rigor. In addition, the descriptions of the Venn diagrams have been refined to provide a more precise interpretation of the overlaps and unique gene sets. We believe this clarification resolves the discrepancy and strengthens the narrative of our study.

Comments 12: In line 260, the legend of the figure indicates that “Venn diagram of unique DGEs of comparison group of H2, S2, H5, H5”. Please, explain what do “H2, S2, H5, H5” stand for. These abbreviations are not used in the figure, why do the authors mention then? The authors introduce the definition in line 209 (H2: RK2 vs. RK0), they should indicate this information in the legend or the figure. Btw, probably “H5” is duplicated and the authors meant “S5” and by DGEs they mean DEGs. The text is plagued with these mistakes, please revise carefully the text.

Response 8: Thank you for pointing out these issues. We have now annotated all occurrences of the labels H2, S2, H5, and S5 in the respective figure legends. Additionally, we have thoroughly proofread the entire manuscript to correct numerous spelling, grammatical, and formatting errors.

4. Response to Comments on the Quality of English Language

Point 1:

The text is hard to read and difficult to follow. There are incomplete sentences (missing the verb or the subject) and the following sentences seem to rely on information that has not been previously presented. Please, see specific examples in the comments.

Response 1:  We sincerely apologize for these errors and thank the reviewer for bringing them to our attention. The specific issues listed have been corrected.

To ensure the highest quality, we have performed a word-by-word review of the manuscript to eliminate any remaining spelling, grammatical, or redundant phrases.

Response to Reviewer X Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

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

We have revised the manuscript according to the reviewers' suggestions. In addition, we have carefully polished other parts of the text to improve clarity and coherence.

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

Thank you for following the recommendations.

Comments 1: Line 111: spore concentration is not indicated; please include it.

Response 1: We thank the reviewer for this constructive comment. The spore concentration (1×10⁴ spores/mL) has been included in the revised Methods section.

Response to Reviewer X Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

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

We have revised the manuscript according to the reviewers' suggestions. In addition, we have carefully polished other parts of the text to improve clarity and coherence.

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

Dear authors, thank you for taking the comments into account. The work now looks more complete. However, there are still a few things I would recommend:

Comments 1: Figure 1. Add a name to the figure.

Response 1: We thank the reviewer for this valuable feedback regarding the clarity and presentation of Figure 1. As suggested, we have now provided a concise title for Figure 1 that captures the overall finding of the figure. The updated figure legend reads:"Figure 1. Disease assessment and pathogen dynamics in resistant and susceptible plant cultivars. A: Disease symptoms observed in 21 test materials; B-C: Disease progression in resistant and susceptible cultivars at 0, 2, and 5- days post-inoculation (dpi); D: Relative expression of Phytophthora in resistant and susceptible cultivars at 0, 2, and 5 dpi.”

Comments 2: In line 194, they mention: These findings are consistent with previous observations by Dunn et al. [26]. Move this to the discussion section.

Response 2: We thank the reviewer for this constructive comment. We have moved the sentence to the Discussion section, as it provides a better context for interpreting our results in relation to the existing literature.

Comments 3: Although Figure 1 is now presented in a more understandable way, the text is now difficult to follow. Please describe in order: 1a, 1b, etc.

Response 3: We sincerely thank the reviewer for this critical comment. We have thoroughly revised the corresponding text in the Results section to strictly follow the logical sequence of the subpanels (Figure 1a, 1b, 1c, and 1d). Furthermore, the figure legend has been refined to be more self-contained and to facilitate better understanding of each subpanel. The description now guides the reader through the figure in a clear manner.

Comments 4: Please give the figure captions an appropriate format.

Response 4: Thank you for pointing this out. We have carefully reviewed and reformatted all figure captions throughout the manuscript to ensure they conform to the journal's required style.

Comments 5: I suggest replacing Table 3 with a figure or diagram that integrates this information.

Response 5: We thank the reviewer for this constructive suggestion. We have replaced Table 3 with a new figure (now Figure 9), which provides a more intuitive visual summary of the expression patterns across key biological pathways.