Transcriptomic and Phytohormone Metabolomics Provide Insight into the Changes in Citrus limon Infected by Citrus yellow vein clearing virus

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Author Response

Reviewer’s comments to the author

Title
The work titled “Transcriptomic and phytohormone metabolomics provide insight into the mechanism of Citrus limon infected by Citrus yellow vein clearing virus” is a good paper that attempts to addresse the issue of possible identification of new potential candidate genes linked to virus disease control management strategy through transcriptomics and metabolomics approaches. It is a great effort to finding another alternative in controlling the disea  se instead of depending on pesticide application due to absence of resistant cultivars. Reliance on cultivar resistance to diseases is the best strategic option compared to other chemically integrated methods that pose great challenges to our environment and human health. Further evolution of new species that might portend more challenges in the control of diseases could potentially be experienced with the use of indiscriminate chemicals. The paper has some issues as presented below:
Respond：Thank you for your feedback on the manuscript. We appreciate your suggestions for improvement.

 

Abstract
Start mandarivirus with a capital letter. Write DGES fully and make the result section more explicit with more results.

Respond:

We have capitalized the first letter in "Mandarivirus" and provided a more explicit results section with additional results.

Keyword
Write CYVCV fully

Respond：We have written "CYVCV" fully as "Citrus yellow vein clearing virus".

Introduction
CYVCV is a member of the genus Mandarivirus of the Alphaflexiviridae family
Line 69: IAA was not mentioned in lines 49-51, therefore IAA should be fully written here where it was first mentioned.

Respond：We have corrected the mention of IAA in line 69 and fully written it as "indole-3-acetic acid" in that line.

Lines 69-72 that read “SA, IAA and 69 CK were significantly induced after CYVCV infection, and differentially expressed genes (DEGs) involved in the plant‒pathogen interaction pathway, MAPK signaling pathway and plant hormone signal transduction pathway contributed to CYVCV infection” are presented as results and this is not acceptable in the introduction part.

Respond：The part in lines 69-72, has been revised in the introduction.

 

Lines 74-75 that read “Our findings provide new insights into the mechanism of lemon infection by CYVCV in the field…..” sounds like a conclusive statement, which is not acceptable in the objective part of the introduction.
Generally, the introduction was not properly written. There were no clear hypotheses/objectives of the research towards the end of the introductory part.

Respond：We have removed lines 74-75 as they were a conclusive statement and not suitable for the objective part of the introduction.

We have also provided clear hypotheses/objectives of the research towards the end of the introduction.

Materials and methods
After the subsection of 2.3 RNA sequencing below, the rest should be classified under Data analysis.
2.1 Plant materials
2.2 Phytohormones targeting metabonomics
2.3 RNA sequencing
2.4 Transcriptome analysis
2.5 Gene-level identification and differentially expressed gene analysis
2.6 Functional annotation
2.7 Statistical analyses

 

Respond：After the subsection of 2.3 RNA sequencing, the remaining subsections have been classified under "Data analysis" as per your suggestion. The sections now appear as follows:

2.1 Plant materials

2.2 Phytohormones targeting metabonomics

2.3 RNA sequencing

2.4 Data analysis

2.5 Statistical analyses

Thank you for your valuable input.

 

Lines 89-90 that read “Metabolite profiling is one of the most common uses of metabolomics in plant and natural product analyses…” should be revisited. Please, transfer to the introduction part and explain in details how the metabolites/metabolomes were obtained via metabolomics before analyzing them.

Respond：Lines 89-90: the sentence has been revised and we have provided a detailed explanation of how metabolites/metabolomes are obtained via metabolomics at Line 70-79:  Metabolite profiling is one of the most common uses of metabolomics in plant and natural product analyses, metabolomics method can establish a more direct relationship between the change of metabolite type and content and the change of biological phe-notype. Therefore, metabolomics has gradually become a new research method for the analysis of complex character systems after genomics, transcriptomics and proteomics. The integrated analysis of metabolomics and other multi-omics methods can explain the genetic mechanism of various complex traits more comprehensively, help to improve the complete biological process of "mutation-gene-expression-metabolism-phenotype", provide a new method for the mechanism analysis of complex traits, and provide a new idea for new agricultural breeding [21-23].

Lines 91-98: There should a specific subsection for data analysis and lines 91-98 should be properly placed for easy flow of reading and comprehension.

Respond： revised

Line 100: RNA was extracted from tissues or cells using standard extraction methods. Please, explain the method used (if it has not been published) for the RNA extraction or make a proper citation of the method used for the purpose of reproducibility.

Respond: We apologize for the lack of information regarding the RNA extraction method. We have made a citation of the method used.

Ref25: Rio, D.C.; Ares, M.; Hannon, G.J.; Nilsen, T.W. Purification of RNA Using TRIzol (TRI Reagent). Cold Spring Harb Protoc 2010, 2010, pdb.prot5439, doi:10.1101/pdb.prot5439.

Lines 102-110 are not explicitly presented and should be modified by the authors to include details of volumes/concentrations of each of RNA, reagents from the cDNA synthesizing kit as well as PCR cycling parameters.

Respond: We have modify these sentence.

Lines 113-115 that read “qRT‒PCR can be used to accurately determine the effective concentration of a library (the effective concentration of the library should be higher than 2 nM) to ensure the quality of the library” should be deleted from this section.

Respond: We will delete these lines from the section, as they are not necessary.

Lines 147-148: Before we can perform GO enrichment analysis, we need to know which GO entries each gene belongs to…..
The author should recast this sentence.

Respond: We have recast the sentence in a more clear and concise manner.  

Results：

The below subsections below in the result section is not necessary since the research is involves the utility of transcriptomics and metabolomics to ascertain the mechanisms of host-virus interactions. Therefore, the author should endeavor to narrow them to two major subtitles.
3.1 Changes in the phenotype and phytohormone levels of Citrus limon infected with Citrus yellow vein clearing virus in the field
3.2 Global transcriptome changes in Citrus limon infected by CYVCV
3.3 Differential gene expression analysis by GSEA after CYVCV infection
3.4 Transcriptional integration of hormone signaling in SA and auxin
3.5 Network analysis of genes regulating ABA and JA hormone metabolism

Respond :We apologize for any confusion caused by the formatting and presentation of the results section, as well as the quality of the figures. We acknowledge that the subsections in the results section can be narrowed down to two major subtitles to align with the focus of the research on the utility of transcriptomics and metabolomics in understanding host-virus interactions. In order to make the structure of the article reasonable, we have made some modifications according to your requirements.

Lines 165-176. sound introductory with contents of materials & methods including repetitions of full meaning of the phytohormones that had earlier been defined. The author is expected to present the results and not turning this section into another introduction and materials & methods.

Respond : we apologize for the repetition of information and the inclusion of materials and methods content in the results section. We will revise this section and ensure that it focuses solely on presenting the results.

Figs 2A & 2B are very poor in resolution and I was unable to read them for in any meaningful interpretations and comments. Make them larger and avoid putting them in a panel since the required resolution is lost.

Lines 316-321: Fig 2 panel is poorly represented both in quality (especially Fig 2A and 2B) and organization. The title, VIP, on the X-axis of Fig 2B should be fully written and re-analysis of the Fig is required for clarity. Avoid putting some Figs without good qualities in panels. For 2C, there is no title on the Y-axis and the one on the X-axis should be written correctly as “Mean decrease accuracy”

Lines 322-328: Same poor qualities of images were represented here. Consider presenting two of the most important Figs for clarity purpose instead of putting all in the panel format with inexplicit images.

Respond : We appreciate your comments about the resolution and organization of figures 2A, 2B, 2C, 3, and 4. We will make sure to enhance the resolution of these figures and ensure they are clearly presented. We will also address the formatting issues, such as missing axis titles and incorrect labeling.

 

Lines 329-336: Represent Figures 3 and 4 or have taken out of the manuscript.
In summary, I was unable to link the results to some of the associated Figures due to poor resolutions of the images. Therefore, the issues raised regarding the Figures should be well addressed.

Respond : Figures 3 and 4 were transfer to the supplementary materials.

Discussion
The author is expected to explicitly discuss the results of the work and then compare with the past work carried out on the same or similar field of study. Instead, what I see here is a more detailed introduction chapter and NOT discussion of the results. Please, the author should represent and expatiate the discussion section.

Respond: In terms of the discussion section, we understand that it should focus on explicitly discussing the results and comparing them with previous research. We apologize if the current version appears more like an introduction than a discussion. We will revise the discussion section accordingly to ensure it accurately presents and expands upon the results.

Conclusion
In lines 448-449, what does the author mean by CYVCV infection also regulates genes related to flavor, which may contribute to the transmission of insects? Is it…..transmission of the virus by insects

Respond: We apologize for any confusion statement and this sentence has been removed.

 

Thank you for bringing these issues to our attention. We will make the necessary revisions to address the concerns raised and ensure the manuscript meets the expected standards.

Reviewer 2 Report

Comments and Suggestions for Authors

Nice work, and, at least in my opinion, an important one, because many general plant virus interactions can be similar in many cases. Things you find out about one viral disease might be applied to others. However, presentation of your work is mediocre. I often see it in articles presenting genomics/proteomics/transcriptomics/etc. It is hard to present data that is very condensed of computer analyses to be “pleasant” to the reader. And authors end up throwing big amounts of raw data and tables and figures without actually explaining anything. Look at how big is your methods section, and figures, and then how tiny in comparison is discussion section.

This is advice for the future: whole article should be logically fluent and the reader should be guided through everything. Authors should strive to present results in a short manner, avoid unnecessary clutter, and then discuss them in detail. Ideally, discussion section could have same name chapters as results (something like: 4.1. phytohormone changes, 4.2. transcriptome changes, 4.3. gene expression… in your case). Now all your figures feels weird and out of place, discussion feels unrelated to them and has no structure. The reader goes through results, reaches the discussion expecting to now understand what each figure shows and how each of different results, each of different method and approach supports your conclusions. But there is none of that, the figures are there for him to decipher on his own. The need of these figures becomes unclear. What would happen if you remove figures 5, 6, 7 and 8. I think nothing, I think the reader would not even notice or care. Even figures 2, 3 and 4 could probably be moved to supplementary materials. You do not use them in Discussion. Usually, article’s discussion section has plenty of references to used figures as people keep talking about results represented in them when contemplating what conclusion they can see from them. You discussion is a pile of separate ideas and speculations that don’t feel to be woven together into one fluent idea. You mention other people’s result, but don’t compare them to yours, don’t tell us if your research supports or contradicts previous data. Your conclusions are too bold, your claims are not justified. You do not contemplate on many other possibilities and options that could affect your findings. You don’t mention what could have been done differently or what other samples could have been included to generate more interesting data. You hide the fact that all of this is speculations, different level of any metabolite production is a hint at something at best and not a direct proof of any concrete plant-virus interaction mechanism.

 

 

Things to actually fix now:

Line 15, 16: salicylic acid, jasmonic acid – Salicylic, Jasmonic.

Line 18, 19: First mention of DEG and Mlo, should be explained what they mean.

Line 20-21: I don’t agree with the use of term “molecular mechanism”. It is too bold. You looked into “results” of these mechanisms. You see the products, different levels of produced chemical compounds. You see it from afar as a “big picture”, the result of interactions of many different mechanisms. To reveal the molecular mechanism, you would need to prove in engineered systems the whole signalling pathway, all the effectors and signalling molecules/ligands, all the conformational changes of all the participating proteins/enzymes. Your study provides insight into changes of phytohormones, possibly caused by virus presence.

Line 28: I don’t think “citrons” need to be in italic font.

Line 34-35: You are talking about symptoms in general, passive tense is wrong here (change became to become).

Line 49: included change to include.

Line 51: you already mentioned gibberellins (GAs) in line 49, maybe no need to repeat.

Line 61-62: It is weird that you just mention that mechanisms have been studied and give links. The reader expects that you explain what was discovered and what were the conclusions of these studies [18-20].

Line 69: First unexplained mention of IAA. Maybe include Indolacetic acid in the list of hormones you mention in lines 49-51.

Lines 163, 201...: Double check if chapter names of results need to be in italic font, because chapter names of materials section is not. Maybe they all should be same formatting.

Lines 196-197: first unexplained mention of iP, czR, tZ. I don’t think it is mentioned anywhere in the article that its izophenyladenine and zeatins.

Lines 312, 313…: Shouldn’t “citrus limon” be in italic font in all figure descriptions?

Lines 336-366: I don’t see the point of figures 5-8. Why they are in the article. Who made them? Are these pathways actually proven to be true or is it just proposed models?

Line 378: citrus x limon is not in italic font.

Line 384: What exactly is “new idea for molecular breeding”? Explain this sentence. What idea it is?

Line 388-389: “This study provides theoretical guidance…” Elaborate this. I don’t see any guidance in your article for control and breeding. Please provide this guidance if you claim your article does that.

Line 438: “mechanisms”. You did not reveal any mechanisms. You saw different levels of produced chemicals. And, based on figure 1c, only SA and IAA had recreate-able results, other hormones look inconclusive.

Line 443: “which reduced the immunity of lemon plants” very brave and bold sentence. “Had a negative effect on immune response system” is probably more accurate. And even then, what proof do you have? I didn’t find anywhere in your article you explaining how jasmonic acid levels relate to plant immune system. You did not provide any references to other researchers directly proving that JA reduction is negative for plant immune system.

 

Lines 438-452: You don’t mention to the reader that all of your “insights into what is happening in plant cell after virus infection” are of speculative nature. None of it is a direct proof of anything. All of your claims should be in a very careful manner. Using expressions like “it might be” instead of “it is”. Don’t talk about mechanisms…

What mechanisms did you revealed? Can you tell me the whole process? Starting from which virus gene is important, what protein it produces that interacts with which proteins of the plant initiating what cascades of complicated pathways (figs 5-8 are metabolic pathways, not virus-plant protein interaction). Does virus actually want to directly increase specifically SA and IAA levels? Does it do it by specifically strengthening expression of needed plant gene? Or is SA and IAA levels a result of plant detecting a virus and launching its own defence system? Is higher levels of SA/IAA a good thing for the virus or a bad thing? Is it a result of virus having already won vs plant defence, or the opposite? Is it ok to check just the multiomics of a plant that is in probably late stages of infections and jump to conclusions? Has your plant contained and stopped the virus? Did you prove that there is still a systematic infection going on in the plant? Maybe you took the infected leaves where virus is already defeated and inactive and only visual changes on the leaf remain. Maybe you need to compare omics of initial, early, medium and late stages of infection and compare them? How does the metabolomics change as the disease progresses? Maybe you need to check multiomics of many different plant varieties ranging from very susceptible to highly resistant to see the differences? Is the phytohormone more important than RNA silencing for the plant defense to virus?

There are ton of questions you cannot answer. Thus, conclusions should be rewritten to account for them. When people present their conclusions, they mention all drawbacks, all possible lackings of their experiment design, to carefully say “our results hint at this, but there are this and that which remains unclear”. You can only claim that you found differently expressed some hormones. Not that you revealed the mechanisms.

Comments on the Quality of English Language

Mistakes listed above

Author Response

Nice work, and, at least in my opinion, an important one, because many general plant virus interactions can be similar in many cases. Things you find out about one viral disease might be applied to others. However, presentation of your work is mediocre. I often see it in articles presenting genomics/proteomics/transcriptomics/etc. It is hard to present data that is very condensed of computer analyses to be “pleasant” to the reader. And authors end up throwing big amounts of raw data and tables and figures without actually explaining anything. Look at how big is your methods section, and figures, and then how tiny in comparison is discussion section.

This is advice for the future: whole article should be logically fluent and the reader should be guided through everything. Authors should strive to present results in a short manner, avoid unnecessary clutter, and then discuss them in detail. Ideally, discussion section could have same name chapters as results (something like: 4.1. phytohormone changes, 4.2. transcriptome changes, 4.3. gene expression… in your case). Now all your figures feels weird and out of place, discussion feels unrelated to them and has no structure. The reader goes through results, reaches the discussion expecting to now understand what each figure shows and how each of different results, each of different method and approach supports your conclusions. But there is none of that, the figures are there for him to decipher on his own. The need of these figures becomes unclear. What would happen if you remove figures 5, 6, 7 and 8. I think nothing, I think the reader would not even notice or care. Even figures 2, 3 and 4 could probably be moved to supplementary materials. You do not use them in Discussion. Usually, article’s discussion section has plenty of references to used figures as people keep talking about results represented in them when contemplating what conclusion they can see from them. You discussion is a pile of separate ideas and speculations that don’t feel to be woven together into one fluent idea. You mention other people’s result, but don’t compare them to yours, don’t tell us if your research supports or contradicts previous data. Your conclusions are too bold, your claims are not justified. You do not contemplate on many other possibilities and options that could affect your findings. You don’t mention what could have been done differently or what other samples could have been included to generate more interesting data. You hide the fact that all of this is speculations, different level of any metabolite production is a hint at something at best and not a direct proof of any concrete plant-virus interaction mechanism.

 

 Dear reviewer,

Thank you for your valuable feedback. We appreciate your suggestions on improving the logical flow and structure of the article. We understand the importance of guiding the reader through the entire article and presenting results concisely. Regarding the figures, we acknowledge that they may appear disconnected from the discussion. We re-evaluated the placement of the figures and consider integrating them more effectively into the discussion section. Additionally, we added this part of the discussion section to match the results for better coherence.

 

We apologize for not providing enough clarity in linking the figures with the discussion. We will revise the discussion section to include more references to the figures and explain how each result supports our conclusions. We also understand the need for comparing our research with previous studies and will address this by discussing how our findings align or deviate from existing literature.

We appreciate your comments about the boldness of our conclusions. We will make sure to better justify our claims and acknowledge alternative possibilities and factors that could influence our findings. Furthermore, we will emphasize the speculative nature of our observations and highlight that they serve as indications rather than direct proof of specific plant-virus interaction mechanisms.

Thank you again for your constructive criticism. We will carefully address each of your points in the revised version of the article.

 

Things to actually fix now:

Line 15, 16: salicylic acid, jasmonic acid – Salicylic, Jasmonic.

Line 18, 19: First mention of DEG and Mlo, should be explained what they mean.

Thank you for your feedback and suggestions on the manuscript. Here are our responses to your specific points:

1. Lines 15, 16, 18, 19, 28, 34-35, 49, 51, 61-62, 69, 163, 201: We will revise the formatting, italics, and tense usage as suggested.

 

Line 20-21: I don’t agree with the use of term “molecular mechanism”. It is too bold. You looked into “results” of these mechanisms. You see the products, different levels of produced chemical compounds. You see it from afar as a “big picture”, the result of interactions of many different mechanisms. To reveal the molecular mechanism, you would need to prove in engineered systems the whole signalling pathway, all the effectors and signalling molecules/ligands, all the conformational changes of all the participating proteins/enzymes. Your study provides insight into changes of phytohormones, possibly caused by virus presence.

Line 20-21: We appreciate your comment. We agree that the term "molecular mechanism" can be bold, and we will make the necessary modifications to better reflect the scope of our study.

 

 

Line 28: I don’t think “citrons” need to be in italic font.

Line 34-35: You are talking about symptoms in general, passive tense is wrong here (change became to become).

Line 49: included change to include.

Line 51: you already mentioned gibberellins (GAs) in line 49, maybe no need to repeat.

Line 61-62: It is weird that you just mention that mechanisms have been studied and give links. The reader expects that you explain what was discovered and what were the conclusions of these studies [18-20].

Lines 61-62: We understand your expectations and apologize for the confusion. We will provide a summary of the key findings and conclusions from the studies referenced [18-20] as below.

The primary defense against viruses in plants is triggered by small-interfering RNAs derived from the virus, which guide RNA interference and/or DNA methylation to target both RNA and DNA viruses. And these viral siRNAs (vsiRNAs) associate with proteins ( Argonaute proteins, Triple Gene Block Protein 1,) and phytohormones (BR precursors, PR1, PR2 and PR5) [18-20].

Line 69: First unexplained mention of IAA. Maybe include Indolacetic acid in the list of hormones you mention in lines 49-51.

Revised.

Lines 163, 201...: Double check if chapter names of results need to be in italic font, because chapter names of materials section is not. Maybe they all should be same formatting.

Lines 196-197: first unexplained mention of iP, czR, tZ. I don’t think it is mentioned anywhere in the article that its izophenyladenine and zeatins.

We have clarified the abbreviations "iP" as isopentenyladenine, "czR" as cis-zeatin riboside, and "tZ" as trans-zeatin. thank you for your valuable feedback.

Lines 312, 313…: Shouldn’t “citrus limon” be in italic font in all figure descriptions?

We apologize for the oversight. Yes, "citrus limon" should be in italic font in all figure descriptions, and we have corrected this in the final version of the manuscript.

Lines 336-366: I don’t see the point of figures 5-8. Why they are in the article. Who made them? Are these pathways actually proven to be true or is it just proposed models?

Figures 5-8 were included in the article to propose potential molecular pathways based on the findings and discussions presented earlier. These pathways are conceptual models and not experimentally validated. We will clarify this in the manuscript to avoid any confusion.

Line 378: citrus x limon is not in italic font.

We will ensure that "citrus x limon" is correctly formatted in italic font.

Line 384: What exactly is “new idea for molecular breeding”? Explain this sentence. What idea it is?

The sentence "new idea for molecular breeding" refers to the potential application of the findings from our study as a basis for future breeding strategies in citrus plants. Specifically, the identification of specific hormone levels and their interaction patterns can guide breeding programs to produce citrus varieties with desired traits. We will provide further explanation to clarify this point.

Line 388-389: “This study provides theoretical guidance…” Elaborate this. I don’t see any guidance in your article for control and breeding. Please provide this guidance if you claim your article does that.

We apologize for any confusion caused by this sentence. We meant to convey that our study provides a theoretical foundation or basis for future guidance in citrus plant control and breeding. We will revise the wording to better explain this.

Line 438: “mechanisms”. You did not reveal any mechanisms. You saw different levels of produced chemicals. And, based on figure 1c, only SA and IAA had recreate-able results, other hormones look inconclusive.

We acknowledge that we did not explicitly reveal detailed mechanisms in our study. However, we did observe changes in the levels of various hormones, which can have implications for plant physiological processes and responses. We will clarify this point to avoid any misleading implications.

Line 443: “which reduced the immunity of lemon plants” very brave and bold sentence. “Had a negative effect on immune response system” is probably more accurate. And even then, what proof do you have? I didn’t find anywhere in your article you explaining how jasmonic acid levels relate to plant immune system. You did not provide any references to other researchers directly proving that JA reduction is negative for plant immune system.

We apologize for the strong phrasing used in that sentence. We will revise it to indicate that a reduction in jasmonic acid levels may potentially have a negative impact on the immune response system. We will make it clear that our study did not directly explore the relationship between jasmonic acid and plant immune systems. We appreciate your feedback and will ensure we provide appropriate references to support the claims made in the article.

Thank you for bringing these issues to our attention, and we will address them accordingly in the revised manuscript.

Lines 438-452: You don’t mention to the reader that all of your “insights into what is happening in plant cell after virus infection” are of speculative nature. None of it is a direct proof of anything. All of your claims should be in a very careful manner. Using expressions like “it might be” instead of “it is”. Don’t talk about mechanisms…

What mechanisms did you revealed? Can you tell me the whole process? Starting from which virus gene is important, what protein it produces that interacts with which proteins of the plant initiating what cascades of complicated pathways (figs 5-8 are metabolic pathways, not virus-plant protein interaction). Does virus actually want to directly increase specifically SA and IAA levels? Does it do it by specifically strengthening expression of needed plant gene? Or is SA and IAA levels a result of plant detecting a virus and launching its own defence system? Is higher levels of SA/IAA a good thing for the virus or a bad thing? Is it a result of virus having already won vs plant defence, or the opposite? Is it ok to check just the multiomics of a plant that is in probably late stages of infections and jump to conclusions? Has your plant contained and stopped the virus? Did you prove that there is still a systematic infection going on in the plant? Maybe you took the infected leaves where virus is already defeated and inactive and only visual changes on the leaf remain. Maybe you need to compare omics of initial, early, medium and late stages of infection and compare them? How does the metabolomics change as the disease progresses? Maybe you need to check multiomics of many different plant varieties ranging from very susceptible to highly resistant to see the differences? Is the phytohormone more important than RNA silencing for the plant defense to virus?

There are ton of questions you cannot answer. Thus, conclusions should be rewritten to account for them. When people present their conclusions, they mention all drawbacks, all possible lackings of their experiment design, to carefully say “our results hint at this, but there are this and that which remains unclear”. You can only claim that you found differently expressed some hormones. Not that you revealed the mechanisms.

Response: Thank you for your feedback. I apologize if my previous response implied that the mechanisms I discussed were proven rather than speculative. I understand the importance of being cautious and clear when presenting scientific findings. To address your questions, the specific mechanisms underlying plant-virus interactions are complex and not fully understood. The process starts with the recognition of the virus by the plant's defense system, which can trigger a cascade of signaling events. The virus may produce specific proteins that interact with the plant's proteins, potentially affecting various pathways. Regarding the increase in SA and IAA levels, it is not clear whether the virus directly wants to increase these levels or if they are a result of the plant's defense response. Higher levels of SA and IAA can potentially have both positive and negative effects on the virus, as they may promote defense mechanisms but could also be exploited by the virus for its own benefit.

In the study, we focused on analyzing the multiomics of infected plant leaves at a certain stage of infection. I acknowledge that studying the various stages of infection and comparing different plant varieties could provide valuable insights into the disease progression and variations in plant defenses.

I agree that presenting conclusions should include limitations and uncertainties. Based on the findings of our study, we observed differential expression of certain hormones in infected plants. However, it is important to emphasize that these findings provide preliminary evidence and further research is needed to fully understand the mechanisms at play. Thank you for bringing these points to my attention, and I will incorporate them into the revision of my conclusions to ensure they accurately reflect the limitations of the study.

 

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Reviewer’s comments to the author

Title

The work titled “Transcriptomic and phytohormone metabolomics provide insight into the mechanism of Citrus limon infected by Citrus yellow vein clearing virus” is a good paper that attempts to address the issue of possible identification of new potential candidate genes linked to virus disease control management strategy through transcriptomics and metabolomics approaches. It is a great effort to finding another alternative in controlling the disease instead of depending on pesticide application due to absence of resistant cultivars. Reliance on cultivar resistance to diseases is the best strategic option compared to other chemically integrated methods that pose great challenges to our environment and human health. Further evolution of new species that might portend more challenges in the control of diseases could potentially be experienced with the use of indiscriminate chemicals. The paper has some issues as presented below:

Abstract

Change Differential Expression Genes to Differentially Expressed Genes (DEGs)

 Introduction

54-57: Other plant hormones were abbreviated and presented in parentheses, except strigolactones. Please that consistency is maintained.

Line 61 that reads…” viruses is mediated by the interfering RNA (siRNA)….” needs correction. The siRNA is small interfering RNA and not just interfering RNA.

Line 69: …..small-interfering RNAs derived from the virus, which guide RNA interference

Address the issue raised in line 61, and then abbreviate small-interfering RNAs subsequently, including here.

Lines 72-81 need to be recast to address the noted lacuna, including punctuation issues. For instance, in line 73 that reads…” of the most common uses of metabolomics in plant and natural product analyses, metab……; there is an omission of full stop punctuation mark after the word analyses.

Lines 89-90 that read “we performed transcriptomic profile and targeted metabolomic analyses of plant hormones. The results showed that SA, IAA and CKIAA were significantly induced after CYVCV infection, and differentially expressed genes (DEGs) involved in the plant‒…..” suggest contents of Materials & Methods and Result sections, and this is not acceptable in the objective part of the introduction.

Generally, the introduction is still not properly written. There were no clear hypotheses/objectives of the research towards the end of the introductory part. However, the author tries to write the objectives but not in details as indicated in lines 87-89 that read….”To throw light on CYVCD symptom development involving hormones regulating lemon-virus interactions, we performed transcriptomic profile and targeted metabolomic analyses of plant hormones”.

Lines 100-104 that read…. “Our study attempted to find key hormones and genes by analyzing the changes in hormone levels and transcription levels of lemons after being infected by CYVCV virus, and to reduce the adverse effects of CYVCV on lemons through external supplementation of related hormones and regulation of expression of specific genes, so as to achieve the purpose of controlling disease without affecting yield” do not sound objectively and should be recast.

Materials and methods

Line 108: Recast the sentence to remove the parentheses from “Citrus × limon ‘Eureka’ Lemon plants (tree age: 10 years)…..”

 Lines 110-112 that read “Due to frequent inspections, diseased trees are cut down when diseased trees are found, and the duration of infection with CYVCV is approximately half a year” should be re-written appropriately using the correct tense. The author is to report what and what were carried out here in the right tense. For instance, “are” should be replaced with “were” in line 110 and so on.

Lines 109-110: The virus is inoculated through traditional horticultural practices such as pruning.

If pruning was the actual method of the virus inoculation in this study, please state it clearly.

 Line 113: Collected (Three biological replicates) for uniform size, color and disease symptoms, and

Please, remove the parentheses in Three biological replicates and recast the sentence without the parentheses.

 Lines 119-126: There should a specific subsection for data analysis and lines 119-126 should be properly placed for easy flow of reading and comprehension. This was earlier raised but the author ignored it.

Lines 130-134 are not explicitly presented and should be modified by the authors to include details of volumes/concentrations of each of RNA, reagents from the cDNA synthesizing kit as well as PCR cycling parameters. The issue was raised during the first review but the author failed to address it. Without these details, reproducibility of this work by other researchers is not guaranteed.

 Lines 166-189 are poorly written and should be recast using the right tense. The author should engage English Language experts.

 Results

The below subsections below in the result section is not necessary since the research involves the utility of transcriptomics and metabolomics to ascertain the mechanisms of host-virus interactions. Therefore, the author should endeavor to narrow them to two major subtitles.

3.1 Changes in the phenotype and phytohormone levels of Citrus limon infected with Citrus yellow vein clearing virus in the field

3.2 Global transcriptome changes in Citrus limon infected by CYVCV

3.3 Differential gene expression analysis by GSEA after CYVCV infection

3.4 Transcriptional integration of hormone signaling in SA and auxin

3.5 Network analysis of genes regulating ABA and JA hormone metabolism

Lines 200-230..sound introductory with contents of materials & methods including repetitions of full meaning of the phytohormones that had earlier been defined. The author is expected to present the results and not turning this section into another introduction and materials & methods.

The title, VIP, on the X-axis of Fig 2B should be fully written and re-analysis of the Fig is required for clarity. The points in the plot are not clear to read. For 2C, there is no title on the Y-axis and the one on the X-axis should be written correctly as “Mean decrease accuracy”

I could not find Figure 3! The one in the manuscript has not title because it looks deleted. Please, provide Fig 3 with the complete title.

Discussion

The author is expected to explicitly discuss the results of the work and then compare with the past work carried out on the same or similar field of study. Instead, what I see here is a more detailed introduction chapter and NOT discussion of the results. Please, the author should re-present and expatiate the discussion section.

Conclusion

In brief, the author should identify the key findings and use for the conclusion. Some sections are so incoherent as could be seen in lines 530-536

Comments on the Quality of English Language

The author should consult English Language experts or endeavor to improve the English Language of the paper to enhance the quality before publication.

Author Response

Dear Reviewer,

I would like to apologize for not fully understanding your comments in our previous correspondence and for not making the necessary revisions to the manuscript. I sincerely apologize for any inconvenience caused.

I have carefully considered your feedback and have made further revisions to the manuscript in accordance with your suggestions. I have taken your comments into account and have made every effort to meet your requirements.

I hope that these revisions address your concerns and meet your expectations. Please let me know if there are any additional changes or modifications you would like me to make. I greatly appreciate your time and effort in reviewing the manuscript, and I value your expertise and guidance.

Thank you once again for your valuable feedback, and I look forward to hearing from you.

Title

The work titled “Transcriptomic and phytohormone metabolomics provide insight into the mechanism of Citrus limon infected by Citrus yellow vein clearing virus” is a good paper that attempts to address the issue of possible identification of new potential candidate genes linked to virus disease control management strategy through transcriptomics and metabolomics approaches. It is a great effort to finding another alternative in controlling the disease instead of depending on pesticide application due to absence of resistant cultivars. Reliance on cultivar resistance to diseases is the best strategic option compared to other chemically integrated methods that pose great challenges to our environment and human health. Further evolution of new species that might portend more challenges in the control of diseases could potentially be experienced with the use of indiscriminate chemicals. The paper has some issues as presented below:

Abstract

Change Differential Expression Genes to Differentially Expressed Genes (DEGs)

 Introduction

54-57: Other plant hormones were abbreviated and presented in parentheses, except strigolactones. Please that consistency is maintained.

Response: We apologize for the inconsistencies in the abbreviation usage for strigolactones.

Line 61 that reads…” viruses is mediated by the interfering RNA (siRNA)….” needs correction. The siRNA is small interfering RNA and not just interfering RNA.

Line 69: …..small-interfering RNAs derived from the virus, which guide RNA interference.

Address the issue raised in line 61, and then abbreviate small-interfering RNAs subsequently, including here.

Response：Regarding line 61 and 69, we appreciate your correction.

 

Lines 72-81 need to be recast to address the noted lacuna, including punctuation issues. For instance, in line 73 that reads…” of the most common uses of metabolomics in plant and natural product analyses, metab……; there is an omission of full stop punctuation mark after the word analyses.

Response: We apologize for the oversight and have rephrased the deficiencies and improve the overall clarity. The revised sentence is as follows：

“Metabolite profiling is a prevalent application of metabolomics in plant and natural product analyses. This method establishes a direct link between changes in metabolite type and content and alterations in biological phenotype. Consequently, metabolomics has emerged as a new research tool, following genomics, transcriptomics, and prote-omics, for analyzing complex character systems. Integrating metabolomics with other multi-omics approaches allows for a more comprehensive understanding of the genetic mechanisms underlying diverse complex traits. This integrated analysis enhances our comprehension of the complete biological process encompassing "muta-tion-gene-expression-metabolism-phenotype," thereby offering a novel means for in-vestigating complex trait mechanisms and introducing innovative ideas for agricul-tural breeding”

Lines 89-90 that read “we performed transcriptomic profile and targeted metabolomic analyses of plant hormones. The results showed that SA, IAA and CKIAA were significantly induced after CYVCV infection, and differentially expressed genes (DEGs) involved in the plant‒…..” suggest contents of Materials & Methods and Result sections, and this is not acceptable in the objective part of the introduction.

Generally, the introduction is still not properly written. There were no clear hypotheses/objectives of the research towards the end of the introductory part. However, the author tries to write the objectives but not in details as indicated in lines 87-89 that read….”To throw light on CYVCD symptom development involving hormones regulating lemon-virus interactions, we performed transcriptomic profile and targeted metabolomic analyses of plant hormones”.

Lines 100-104 that read…. “Our study attempted to find key hormones and genes by analyzing the changes in hormone levels and transcription levels of lemons after being infected by CYVCV virus, and to reduce the adverse effects of CYVCV on lemons through external supplementation of related hormones and regulation of expression of specific genes, so as to achieve the purpose of controlling disease without affecting yield” do not sound objectively and should be recast.

 

Response: Thank you for your feedback. We acknowledge your concerns regarding the inclusion of materials and methods and results-related content in the introduction section. We apologize for this oversight and will revise the introduction accordingly to adhere to the standard structure. We have deleted these sentences. Furthermore, we understand your comment about the need for clearer research objectives in the introduction. We appreciate your suggestion to provide a more detailed explanation of our research goals. we will ensure that the objectives are clearly stated and focused, in line with scientific writing conventions. The revised version were as follows:

 

“Viral infection disrupts the physiological environment necessary for plants' growth and development, affecting plant hormone metabolism and signaling. Conversely, plant hormones play a role in regulating viral replication, assembly, movement, and symptom development in infected plants. The purpose of studying the interplays be-tween host hormones and plant viral infections is to gain a comprehensive under-standing of the regulatory mechanisms and crosstalk among different phytohormones in plant-virus interactions. In this study, we performed transcriptomic profile and targeted metabolomic analyses of plant hormones. The expression of genes involved in phytohormone biosynthesis pathways was analyzed in response to CYVCV infection, revealing changes at both metabolic and transcriptional levels. Understanding the interplays between host hormones and plant viral infections can facilitate innovative approaches for crop protection and improvement.”

 

Materials and methods

Line 108: Recast the sentence to remove the parentheses from “Citrus × limon ‘Eureka’ Lemon plants (tree age: 10 years)…..”

Response: the sentence was rephrased as follows:

Samples were collected from ten-year-old Citrus × limon ‘Eureka’ Lemon plants obtained from Anyue, Sichuan Province.

 

 Lines 110-112 that read “Due to frequent inspections, diseased trees are cut down when diseased trees are found, and the duration of infection with CYVCV is approximately half a year” should be re-written appropriately using the correct tense. The author is to report what and what were carried out here in the right tense. For instance, “are” should be replaced with “were” in line 110 and so on.

Response: the sentence was rephrased as follows:

In order to maintain strict surveillance, afflicted trees are promptly removed upon detection, while the duration of infection with CYVCV spans approximately six months in this study.

Lines 109-110: The virus is inoculated through traditional horticultural practices such as pruning.

If pruning was the actual method of the virus inoculation in this study, please state it clearly.

Response: the infection of the lemon with the virus occurred incidentally in this study. Our sampling was carried out under natural conditions, and we speculate that horticultural pruning practices may have played a role in the viral transmission. We have rephrased the sentence.

 Line 113: Collected (Three biological replicates) for uniform size, color and disease symptoms, and

Please, remove the parentheses in Three biological replicates and recast the sentence without the parentheses.

Response: We have rephrased the sentence as follows:

Tissue specimens of consistent size, color, and displaying disease symptoms were meticulously harvested from multiple lemon trees affected by the infection. Three biological replicates were obtained for each set. Subsequent to their swift collection, the leaves were promptly immersed in liquid nitrogen, flash-frozen, and securely stored at a frigid temperature of -80 °C, being held in readiness for future application.

 

 Lines 119-126: There should a specific subsection for data analysis and lines 119-126 should be properly placed for easy flow of reading and comprehension. This was earlier raised but the author ignored it.

Response: We apologize for any oversight in addressing this concern earlier. We will make the necessary revisions accordingly.

 

2.2 Phytohormones targeting metabolomics

In this study, the sample's metabolome was assessed using an LC-MS/MS approach. The raw data files were processed using Compound Discoverer 3.3, which facilitated peak alignment, peak picking, and quantitation for each metabolite. Key parameters, such as peak area correction, mass tolerance, signal intensity tolerance, and minimum intensity, were carefully selected to ensure accurate results. Normalization of peak in-tensities was performed based on the total spectral intensity. The normalized data was then used to predict the molecular formula and identify metabolites by matching peaks with various databases. To identify, filter, and align peaks, the XCMS package of R (v3.1.3) was employed. This yielded a data matrix containing the mass-to-charge ratio (m/z), retention time, and peak area (intensity). Further, precursor molecules were obtained for both positive and negative ion modes, and the data were exported to Excel for subsequent analysis [24].

2.3 Data processing and metabolite identification

To further understand the biological significance of the identified metabolites, various databases were employed. The KEGG database, HMDB database, and LIPIDMaps database were utilized for annotation purposes. These databases provide valuable in-formation on metabolic pathways, biological functions, and structural details of the metabolites. By leveraging the power of these databases, researchers gain deeper in-sights into the metabolic profiles under investigation, enabling a more comprehensive understanding of the biological systems being studied. For statistical heatmap clus-tering analysis of metabolite content, GSEA was performed using the Wekemo Bio-in-Cloud platform (https://www.bioincloud.tech). R, Python, and CentOS were em-ployed for statistical analyses, involving data standardization and exclusion of com-pounds with high coefficients of variation (>30%), ultimately enabling the identifica-tion and relative quantification of metabolites.

 

Lines 130-134 are not explicitly presented and should be modified by the authors to include details of volumes/concentrations of each of RNA, reagents from the cDNA synthesizing kit as well as PCR cycling parameters. The issue was raised during the first review but the author failed to address it. Without these details, reproducibility of this work by other researchers is not guaranteed.

Response：We apologize for the oversight in not addressing this issue in our previous revision. Thank you for bringing this to our attention, and we apologize for any inconvenience caused.

 

“T4 DNA polymerase and DNA polymerase I (Klenow-) were used for end repair. In the first round of PCR, the forward primer (f) was designed as 5′-gaacgacatggctacgatccgactt- target-specific primer-3′, and the reverse primer (r) was designed as 5′-ctaagaccgcttggcctccgactt- target-specific primer-3′. In the second round of PCR, the primers used were ad153_pcr2_1: 5′-gaacgacatggctacga-3′ and indexr: 5′-tgtgagccaaggagttgnnnnnnnnnnttgtcttcctaagaccgcttggcct-3′. The 5′ end of the ad153_pcr2_1 primer was phosphorylated, and 'nnnnnnnnnn' represents the adapter sequence (index) suitable for the mgiseq-2000rs sequencing platform, with 'n' indicating any of the four bases (a, t, g, or c). The universal primer sequence linked to the 5′ end of multiple PCR forward primers is 5′-gaacgacatggctacgatccgactt-3′ (seqidno:1), and the universal primer sequence linked to the 5′ end of multiple PCR reverse primers is 5′-ctaagaccgcttggcctccgactt-3′ (seqidno:2). The reaction conditions for the multiple PCR were as follows: 95°C for 15 minutes; 94°C for 30 seconds, 60°C for 90 seconds, 72°C for 60 seconds for 16 cycles; 60°C for 30 minutes. In the second round of PCR, the primers used were ad153_pcr2_1: 5′-gaacgacatggctacga-3′ (seqidno:3) and indexr: 5′-tgtgagccaaggagttgnnnnnnnnnnttgtcttcctaagaccgcttggcct-3′. The 5′ end of the ad153_pcr2_1 primer was phosphorylated, and 'nnnnnnnnnn' represents the adapter sequence suitable for the mgiseq-2000rs sequencing platform, with 'n' indicating any of the four bases (a, t, g, or c). The reaction conditions for the second round of PCR were as follows: 95°C for 3 minutes; 98°C for 20 seconds, 60°C for 15 seconds, 72°C for 30 seconds for 8 cycles; 72°C for 10 minutes; 4°C indefinitely. The reaction was stopped by heating at 94°C for 10 minutes in a 25 μL reaction system. After library construction, the initial quantification was performed using Qubit 2.0 Fluorometer. The libraries were diluted to a concentration of 1.5 ng/μL and the insert size was analyzed using an Agilent 2100 bioanalyzer. Once the insert size met the expected criteria, qRT-PCR was used to accurately quantify the effective concentration of the libraries, ensuring a library quality with an effective concentration greater than 2nM.”

 Lines 166-189 are poorly written and should be recast using the right tense. The author should engage English Language experts.

Response: I will carefully revise and rewrite those lines to ensure the correct tense is used. Your input has been duly noted and will be taken into consideration for future revisions.

 

 Results

The below subsections below in the result section is not necessary since the research involves the utility of transcriptomics and metabolomics to ascertain the mechanisms of host-virus interactions. Therefore, the author should endeavor to narrow them to two major subtitles.

3.1 Changes in the phenotype and phytohormone levels of Citrus limon infected with Citrus yellow vein clearing virus in the field

3.2 Global transcriptome changes in Citrus limon infected by CYVCV

3.3 Differential gene expression analysis by GSEA after CYVCV infection

3.4 Transcriptional integration of hormone signaling in SA and auxin

3.5 Network analysis of genes regulating ABA and JA hormone metabolism

Response: thank you for your valuable feedback, the subtitles as flows:

3.1 Transcriptome and metabolomics profiling changes of lemon trees infected by CYVCV

3.2 Network analysis of genes regulating hormone signaling

 

Lines 200-230..sound introductory with contents of materials & methods including repetitions of full meaning of the phytohormones that had earlier been defined. The author is expected to present the results and not turning this section into another introduction and materials & methods.

The title, VIP, on the X-axis of Fig 2B should be fully written and re-analysis of the Fig is required for clarity. The points in the plot are not clear to read. For 2C, there is no title on the Y-axis and the one on the X-axis should be written correctly as “Mean decrease accuracy”

I could not find Figure 3! The one in the manuscript has not title because it looks deleted. Please, provide Fig 3 with the complete title.

Response: we have made revisions to the previous version, which may not have been visible due to the version issue. We are now providing the revised version, hoping to meet your requirements.

 

Discussion

The author is expected to explicitly discuss the results of the work and then compare with the past work carried out on the same or similar field of study. Instead, what I see here is a more detailed introduction chapter and NOT discussion of the results. Please, the author should re-present and expatiate the discussion section.

Response:

Thank you for your valuable feedback. We apologize for the confusion in the presentation of our results. Due to the limited research on the interaction between lemon and CYVCV, we have to rely on studies conducted on model plants such as Arabidopsis, rice, and other citrus species to gain insights into their interaction with viruses. This limitation inevitably poses certain difficulties in our in-depth discussion. We appreciate your suggestion to re-present and elaborate on the discussion section.

 

Conclusion

In brief, the author should identify the key findings and use for the conclusion. Some sections are so incoherent as could be seen in lines 530-536

Response: Thank you for your feedback. We apologize for any incoherence in the sections mentioned. We have rephrased these sentences to clearly identify the key findings of our study and present a concise and insightful conclusion. We appreciate your suggestions and will make the necessary revisions to address this issue. Thank you for your input.