Transcriptome Sequence Analysis of Defense Response of Resistant and Susceptible Bottle Gourd to Powdery Mildew

Round 1

Reviewer 1 Report

The authors should know that powdery mildew is a fungal disease, not a bacterial disease. Also, the scientific name of the fungal pathogen used should be stated.

The authors should describe analytical methods in more detail. Statistical analysis results should be adequately described, i.e., multiple comparisons but not pair-wise comparisons should be shown for ANOVA. All figures should be called out.

Because the English language and the styles must be corrected before submission, authors must ask some experts, including a native English speaker, for help preparing the manuscript.

Author Response

Response to Reviewer 1 Comments

Dear reviewer.

Thank you very much for your comments and professional advice. These opinions help to improve the academic rigor of our article. Based on your suggestion and request, we have made corrected modifications to the revised manuscript. Meanwhile, the manuscript had been reviewed and edited by a native English speaker. We hope that our work can be improved again. Furthermore, we would like to show the details as follows:

Point 1: The authors should know that powdery mildew is a fungal disease, not a bacterial disease. Also, the scientific name of the fungal pathogen used should be stated.

Response 1: We thank the reviewer for pointing this out. The bacteria were Podosphaera xanthii. We’ve added this to our paper.

Point 2: The authors should describe analytical methods in more detail. Statistical analysis results should be adequately described, i.e., multiple comparisons but not pair-wise comparisons should be shown for ANOVA. All figures should be called out.

Response 2: We appreciate the reviewer’s insightful suggestion and agree that it would be useful. We added all figures in Figure 5.

Point 3: Because the English language and the styles must be corrected before submission, authors must ask some experts, including a native English speaker, for help preparing the manuscript.

Response 3: Thank you for your advice. Meanwhile, the manuscript had been reviewed and edited by a native English speaker.

Thank you very much for your attention and time. Look forward to hearing from you.

Yours sincerely,

Kuanhong Wang

20 Apr., 2023

Author Response File: Author Response.docx

Reviewer 2 Report

Powdery mildew is a major disease on cucurbits and the resistance to this disease is largely unknown. The author deployed RNAseq technology to study the transcriptome analysis after inoculation with PM spores. This study will help understanding the interaction between bottle gourd and PM.

Major concern is reverse description of resistance of G3 and G6 in Material and Result parts. Inoculation method need detailed information.

Line 19 Non-disease-resistant varieties, how to define?

Line 20 “Bottle” capitalize B

Line 39 covered with

Line 47 “At present”->Currently

Line 54 Double check the linkage distance, if it is 9.6cM, the marker isn’t suitable for assisted breeding.

Line 60 Biotic stress

Line 63 Sensitive ->susceptible

Line 66 F-1 isolated? Or F-1 derived?

Line 70 Sensitive materials or materials with resistance or susceptibility

Line 72 fungal infection

Line 79 Advanced inbred lines. Any relationship between G3 and G6. Please indicate more information of these two lines if available.

Line 84 Sown

Line 85 how about the light condition? For how long before inoculation?

Line 89 which method used to inoculate on plants? Spray inoculation with a spray gun or? About how much inoculum on each plant? Plants were kept with misting for 7 days with 95% humidity?

Line 93 Clarify-> detect?

Line 94 G3 is resistant to PM, would you consider it is compatible interaction?

Line 131-134 G6 is more resistant to PM than G3, which is opposite to description in material part.

Line 189 which bacteria?

Author Response

Response to Reviewer 2 Comments

Dear reviewer.

Thank you very much for your comments and professional advice. These opinions help to improve the academic rigor of our article. Based on your suggestion and request, we have made corrected modifications to the revised manuscript. Meanwhile, the manuscript had been reviewed and edited by a native English speaker. We hope that our work can be improved again. Furthermore, we would like to show the details as follows:

Point 1: Major concern is reverse description of resistance of G3 and G6 in Material and Result parts. Inoculation method need detailed information.

Response 1: We apologize for our error. We've corrected the description of the resistance of G3 and G6 in Material 2.1. We have added the vaccination method, please refer to point 15 for detail.

Point 2: Line 19 Non-disease-resistant varieties, how to define?

Response 2: ‘Non-disease-resistant varieties’ refers to powdery mildew susceptible variety G3. We have changed ‘Non-disease-resistant varieties’ to ‘susceptible variety(G3)’.

Point 3-5: Line 20 “Bottle” capitalize B; Line 39 covered with; Line 47 “At present”->Currently

Response 3-5: We apologize for our error. We've corrected these.

Point 6: Line 54 Double check the linkage distance, if it is 9.6cM, the marker isn’t suitable for assisted breeding.

Response 6: We appreciate the reviewer’s insightful suggestion. We have double-checked the linkage distance in the original literature. We will pay attention to this indicator in future molecular marker screening.

Point 7-9,11,13: Line 60 Biotic stress; Line 63 Sensitive ->susceptible; Line 66 F-1 derived? Line 72 fungal infection; Line 84 Sown

Response 7-9,11,13: We thank the reviewer for pointing this out. We have revised it.

Point 10: Line 70 Sensitive materials or materials with resistance or susceptibility

Response 10: We have made the change. The new sentence reads as follows, ’we selected the susceptible materials of the gourd respectively’.

Point 12: Line 79 Advanced inbred lines. Any relationship between G3 and G6. Please indicate more information of these two lines if available.

Response 12: The original germplasm resources of G3 and G6 are from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences (Crop Germplasm Resources Platform Vegetable Germplasm Resources Sub platform). Subsequently, they conducted multiple self-investigations at the Zhuang Xing experimental station and obtained the materials used in this experiment. Apart from this, there was no other correlation between G3 and G6.

Point 14: Line 85 how about the light condition? For how long before inoculation?

Response 14: We have revised the text to address your concerns and hope that it is now clearer. We’ve added [an illumination of 16h/8h(day/night)] on page 2, line 85.

Point 15: Line 89 which method used to inoculate on plants? Spray inoculation with a spray gun or? About how much inoculum on each plant? Plants were kept with misting for 7 days with 95% humidity?

Response 15: We thank the reviewer for pointing this out. We have revised the text to address your concerns. We used the spraying method to inoculate plants. By using a spray gun to spray until it covers the entire leaf as a standard. Plants were kept with misting for 12h with 95% humidity to simulate the onset conditions. Please see page 2 of the revised manuscript, lines 90-94 for detail revise.

Point 16: Line 93 Clarify-> detect?

Response 16: This observation is correct. We have changed.

Point 17: Line 94 G3 is resistant to PM, would you consider it is compatible interaction?

Response 17: We apologize for our error that G3 is susceptible to PM. We’ve corrected this.

Point 18: Line 131-134 G6 is more resistant to PM than G3, which is opposite to description in material part.

Response 18: We apologize for our error that G3 is susceptible to PM and G6 is resistance to PM. We checked and changed the description of G3 and G6 resistance in the material part.

Point 19: Line 189 which bacteria?

Response 19: The bacteria were Podosphaera xanthii. We thank the reviewer for pointing this out.

Thank you very much for your attention and time. Look forward to hearing from you.

Yours sincerely,

Kuanhong Wang

20 Apr., 2023

Author Response File: Author Response.docx

Reviewer 3 Report

Comments for author File: Comments.pdf

Author Response

Response to Reviewer 3 Comments

Dear reviewer.

Thank you very much for your comments and professional advice. These opinions help to improve the academic rigor of our article. Based on your suggestion and request, we have made corrected modifications to the revised manuscript. Meanwhile, the manuscript had been reviewed and edited by a native English speaker. We hope that our work can be improved again. Furthermore, we would like to show the details as follows:

Point 1: Firstly authors should fix the typing error through the manuscript specially use of space. For example- “the clean reads were mapped to the bollte gourd reference genome(http://cucurbit-111 genomics.org/organism/13).FPKM” a space between genome( and ).FPKM

Response 1: We apologize for our error. We've corrected these.

Point 2: Table 2: Authors identified the DEGs between different periods and samples. In this case, authors should fix the two different direction one is the effective time point and another is the between lines; and descibe that in a specific way. My point of view is to use venn diagram to represent it in a better way.

Response 2: We appreciate the reviewer’s insightful suggestion. Therefore, we added three Venn diagrams to represent two different directions. We have also added a description of the Venn diagrams on page 5 line 169-171.

Figure 2. (A) Venn diagram illustrating DEGs of 0h, 12h, 24h, 48h and 72h of G3 line. (B) Venn diagram illustrating DEGs of 0h, 12h, 24h, 48h and 72h of G6 line. (C) Venn diagram illustrating DEGs of G3 and G6 lines at the same time of 0h, 12h, 24h, 48h and 72h. (D)Principal component analysis (PCA) plots of transcripts identified by RNA-seq of bottle gourd leaves of G6 and G3 at 0, 12, 24, 48, and 72h after PM treatment. Transcriptomes of bottle gourd leaves under PM treatment.

Point 3: Compare the DEGs of 0h, 12h, 24h, 48h and 72h of G3 line using a venn, again for G6 line using another venn. You will have some common DEGs of powdery mildew in 12h, 24h, 48h and 72h that might have better resistant mechanism pathways.

Response 3: We appreciate the reviewer’s insightful suggestion. We agree with the reviewer that further elaborating on this point using new data would be helpful. However, when we analyzed the venn diagrams, we found that the analysis results in the time dimension were consistent with the previous results. For this reason, we chose not to make this change, but we added the following sentence to line 169 in page 5: ‘Which is consistent with the PCA display results.’

Point 4: Then try to compare between G3 and G6 in each timepoint using venn.

Response 4: Thanks for your kind suggestion, which is valuable for improving the accuracy of the manuscript. We’ve added this figure and the corresponding analysis to our manuscript.

Point 5: What is the need of Figure 3?

Response 5: Thanks for your question. We think that Figure 3 can provide a more intuitive display of 44 genes expression. In addition, through cluster analysis, 44 genes can be more clearly classified.

Point 6: Table 3: It is very general to use the TOP10 Gos, but focusing on TOP10 might not always give you the better outsight. You should look on some other GOs that might give you some GO like “response to stress”.

Response 6: Thank you for your kind suggestion. We use more detailed GO function entries in this manuscript. Therefore, GO like ‘response to stress’ was included in our analysis such as ‘response to oxidative stress’.

 Point 7: Figure 3: Expression pattern between resistant and suceptable lines are quite similar especially for first 19 genes. You need to recheck your analysis for the identification of 44 genes. You have also selected most of the genes from the bottom of the Figure 3 for validation.

Response 7: We thank the reviewer for pointing this out. We rechecked our analysis for the 44 genes. Although the expression trends between resistant and susceptible lines are quite similar for the first 19 genes, the expression level of resistant and susceptible lines in these genes were significantly different, which can be proved in QPCR validation.

Point 8: The pathway analysis does not give any important resistant mechanism pathway.

Response 8: Thanks a lot for your comment. More descriptions have been included in the revised manuscript, line 208-214.

Point 9: Line 214-215: where did you get this pathways?

Response 9: Thanks a lot for your question. These pathways were picked from figure 4. We added the source of the pathways in line 225.

Point 10: Finally, you have concluded in four points. How did you come to colclude in these points. For exampleLine 321-323: (3) activates plant signaling pathways, including the activation of diacylglycerol second messengers, and plant hormones include SA, JA, ET, and BA. Your results does not suggested this conclusion. Similarly, other three points as well.

Response 10: Thank the reviewer for the constructive comments and suggestions. The four points in the summary correspond to the three paragraphs in the conclusion, among which point 3 was derived from the KEGG pathway analysis. As it is not the focus of the analysis in this article and has not been verified, the relevant analysis was not reflected in the article. We are sorry for mistakenly including it and have removed this point in the conclusion.

Thank you very much for your attention and time. Look forward to hearing from you.

Yours sincerely,

Kuan-hong Wang

20 Apr., 2023

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Although I suggested rejection for the original manuscript, the revised version has not been significantly improved as far as my comments on the original one are concerned.

Author Response

Thanks again for your suggestions. We feel sorry for our poor writing. In this modification, we used the editing service in MDPI. Due to the English editors' help, this manuscript was edited extensively. And we hope the revised manuscript could be acceptable to you.

Author Response File: Author Response.docx

Reviewer 3 Report

Please check the attached file.

Comments for author File: Comments.docx

Author Response

Dear reviewer.

We sincerely thank you for your valuable feedback that we have used to improve the quality of our manuscript. Please refer to the document for specific modification responses. We hope that the correction will meet with approval. Should you have any questions, please contact us without hesitate. 

Once again, thank you very much for your comments and suggestions.

Yours Sincerely,

Kuanhong Wang

Author Response File: Author Response.docx