Transcriptomic and Metabolomic Analyses Reveal Differing Phytohormone Regulation in Rhododendron Cultivars in Response to Azalea Lace Bug (Stephanitis pyrioides)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript is dedicated to the study of stress response mechanisms of two Rhododendron cultivars treated with azalea lace bug. The plant's response is examined through changes in phytohormonal patterns analysed using transcriptomic and metabolomic methods. The research topic  is interesting and deserves attention. However, despite noteworthy results obtained by the authors some additional information, analyses and speculation would improve the  manuscript value and deepen understanding of the Rhododendron stress mechanism induced by insect.

The additional information, except " 'YZM' (susceptible) and 'TL' (resistant) ", about two cultivars used in the study could explain their choice for analysis and improve understanding of the results. 

Line 97: Please, provide more details in Section "2.2 Transcriptome Sequencing and DEG Analysis" (the analysis was performed for individual plants or as a bulk sample, how many plants / leaves were taken for analysis, how RNA was isolated, cDNA synthesize etc)

Line 124: Section "3.1 Transcriptomic Sequencing Data Analysis and DEGs Screening" - it would be valuable to compare the control groups of two cultivars: to detect specific profiles. Could cultivar-specific gene expression patterns reflect differences in stress response (in terms of DEGs)?

Figures 2C, 4A-C, 5A-D: altering the colours would be appreciated: the same colour for group and heat map (green). Could it be possible to put definition of Clusters, at least the most significant? Or explain abbreviations (SAUR, BSK etc)?

Figure 2: Please, provide explanation of heat legends, padj.

Line 183: please, provide explanation or reference to term "traditional Chinese medicine (TCM)-related metabolites"

Figure 3C: are these data for both cultivars taken together? Could the colours be more sharpen, probably, darker?

Figures 4D,E,F and 5E,F,G,H - what was the purpose of these figures? No reference in the text.

The authors' opinion of revealed differences in defense response should be discussed in Discussion sections as well as possible regulation mechanisms of hormonal synthesis.

In Conclusion, please, define common and specific activity (in terms of DEGs and metabolites) of two cultivars that are the most significant. If there are any.

 

 

Comments on the Quality of English Language

I am not qualified to evaluate English

Author Response

Dear Reviewer,

We thank you for having reviewed our paper , and appreciate the reviewers’ valuable comments on parts of the manuscript.  

Here we submit our revised manuscript with the title ‘Transcriptomic and Metabolomic Analyses Reveal Divergent Phytohormone Regulation in Rhododendron Cultivars in Response to Azalea Lace Bug (Stephanitis pyrioides)’, which has been modified according to the reviewers’ suggestions. We have provided a detailed point-by-point response to the reviewers’ comments, such as the altering the figure colours, adding the heat legends, and some expressions have been changed appropriately in the revised manuscript. We believe we have been able to answer all comments in a satisfactory fashion and look forward to your decision on this version of the manuscript.

The following attachment is a point-by-point response to the reviewers’ comments.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The article highlights some relevant genes involved in the interaction with the ALV. However, I believe its impact is limited because these responses are extensively studied and similar to other research in this area. Considering these genes and proteins under phytopathological conditions would likely be of interest to the reader and enhance the study's significance. For instance, demonstrating that proteins involved in the response serve as crucial molecules for understanding infection conditions, managing them, and determining the appropriate control measures. On the other hand, Im concerned about some results showed in Figures 4 and 5. Results about phytohormones tend to produce confusion, especially in phytohormone graphs, which are the key results from the paper.

For example, in the graph of phytohormones such as salicylic acid, no significant differences are seen between the control and treated plants. However, transcriptomic analyses and supplemental results reveal other results with slight differences between their treatments. On the other hand, it would be appropriate to place letters to the results of relative expression for both graphs 4 and 5, concerning the statistical differences.

Author Response

Dear Reviewer,

We thank you for having reviewed our paper , and appreciate the reviewers’ valuable comments on parts of the manuscript.  

Here we submit our revised manuscript with the title ‘Transcriptomic and Metabolomic Analyses Reveal Divergent Phytohormone Regulation in Rhododendron Cultivars in Response to Azalea Lace Bug (Stephanitis pyrioides)’, which has been modified according to the reviewers’ suggestions. We have provided a detailed point-by-point response to the reviewers’ comments, such as the altering the figure colours, adding the heat legends, and some expressions have been changed appropriately in the revised manuscript. We believe we have been able to answer all comments in a satisfactory fashion and look forward to your decision on this version of the manuscript.

The following attachment is a point-by-point response to the reviewers’ comments.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors significantly improved the manuscript by considering suggested comments. Thank you. 

Lines 133-153: Section "3.1 Transcriptomic Sequencing Data Analysis and Screening of DEGs ". Certainly, the authors emphisize on the commonalities between the two 
different pest-resistant cultivars in their response to the stress. Though the differences in  plant hormone expression between these cultivars without  infestations of the azalea lace bug could play significant role in stress-specific response. A mention of the necessary consideration of the phytohormonal status of healthy cultivars would be appreciated.

 

Author Response

Dear Reviewer:

Thank you for your insightful comments and for acknowledging the significant improvements we have made in our manuscript. We appreciate your thorough review and have carefully considered your comment regarding the differences in plant hormone expression between two cultivars without infestation of the azalea lace bug.

While the baseline differences in genes and pathways between the cultivars without infestation are noted in our data, we have added the description of the pre-existing genes expression differences in both the results and supplementary figures in this revised manuscript. Under healthy condition, 19,366 differentially expressed genes (DEGs) were found between the two rhododendron cultivars, reflecting significant genotypic differences. Of these, 9,305 DEGs were up-regulated and 10,061 were down-regulated (Figure S2). KEGG enrichment analysis of TL_CK vs YZM_CK revealed no significant involvement of plant hormone signal transduction pathway. However, after ALB infestation (TL_SH vs YZM_SH), DEGs were primarily enriched in this pathway, suggesting a rapid induction of hormone-related defense mechanism in response to stress (Figure S3).

This addition aims to provide a more comprehensive understanding of plant defense mechanisms and highlights the critical role of considering the hormonal background of plants in pest resistance evaluation.

We are grateful for the opportunity to clarify these points and believe that these revisions will further strengthen our manuscript. We appreciate your expertise and the guidance you have provided.

 

Thank you once again for your valuable feedback.

Sincerely,

 

Jing Mao

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

According to the arrangements presented, it is appropriate for publication; however, I would have preferred to see more detailed information regarding some of the symptoms the plants showed at the time of collection plant material for the RNA sequencing studies.

Author Response

Dear Reviewer:

Thank you for your insightful comments and for acknowledging the improvements we have made in our manuscript. We appreciate your thorough review and have carefully considered your comment regarding the symptoms the plants showed at the time of collection plant material for the RNA sequencing studies.

We've added photographs of the sampled leaves to the supplementary figures, which visually and descriptively detail the symptoms like localized sucking damage trace and small sucking spots by the feeding of azalea lace bug observed at the time of collection. We hope these additions provide the detailed information you requested and enhance the manuscript.

We are grateful for the opportunity to clarify these points and believe that these revisions will further strengthen our manuscript. We appreciate your expertise and the guidance you have provided.

Thank you once again for your valuable feedback.

Sincerely,

Jing Mao

Author Response File: Author Response.pdf