Identifying Critical Regulators in the Viral Stress Response of Wheat (Triticum aestivum L.) Using Large-Scale Transcriptomics Data

Round 1

Reviewer 1 Report

The authors have studied the response of wheat to virus inoculation in different set of experiments including single an mixed inoculation of important viruses of wheat. The study is important as it is expected that in future, the plants will get infected simultaneously by several viruses and even by other pathogens.  So, it is necessary to understand the response of plants to such multiple inoculations to develop the plants resistant to them. The manuscript is well written. But I have few concerns as below.

The RNA-Seq was performed with only one sample infected with American isolate of WSMV. The other single and mixed infection data was used from NCBI database. To exclusively identify the genes in response to viruses, it would be important to compare the transcriptome data of virus inoculated samples with the tissue that are only attacked by insect vectors (without virus). This will help to remove out the genes expressed in response to insect attack.

Line 165: The lines are copied from template are there as such, kindly delete these lines.

Scientific names should be in italicized font throughout the manuscript.

Quality of figure 7(a) is not good. The name of the genes in the network are not readable.

The expression level of genes should be validated by qRT-PCR to support the claims in the results that the genes actually play important role in virus defense response.

The title of the manuscript says that the “Allene oxide synthase genes are the common key regulators in viral stress response of wheat (Triticum aestivum L.)”, however, there is nothing much about this gene in results. It is not clear how the authors landed upon this gene. Further, based on only the gene expression data in microarray experiments, it is very preliminary to conclude the results, until and unless, the gene is validated either by functional characterization using overexpression or gene knock out or by at least expression study by qRT-PCR.

Author Response

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Reviewer 2 Report

Comments for “Allene oxide synthase genes are the common key regulators in

viral stress response of wheat (Triticum aestivum L.)”

 

This study attempted to expoit the common key regulators during viral stress response of wheat (Triticum aestivum L.). To address this question, the authors conducted transcriptome profiling under mono- (WSMV, BSMV, or BMV), bi- (BMV&BSMV, BMV&WSMV, and BSMV&WSMV), and tripartite (WSMV, BSMV, and BMV) infections. The study showed that a defense-related gene like allene oxide synthase (AOS) gene served as a crucial hub in the tripartite infection.

 

 

Major comments:

1. In section 3.4 was not a validation process. Instead, here was using sub-sampling to replicate the result from the whole set. A regular validation process required an independent dataset, or a experimental validation, for showing the robustness of the results.

2. The authors should clarify what is the mono-, bi-partite and  tri-partite infections. It seems like they simply defined them as the additive of two or three virus infections as the bi-partite and  tri-partite infections.

3. Simply combined the DEGs from two different treatments did not have any biological or practical relevance. Each of the treatment should have their specific factors which could only be detected in this scenarios. The authors should focus more on the shared or specific DEGs.

 

 

Minor comments:

1. In section 2.1, cutoff for significant DEGs should be given. 

2. In section 2.1, how the microarray data was processed should be given for clarifying how the data from different studies were harmonized. 

3. Legend of Figure 1 was not appropriated. Here the condition for differently expressed should be clarified.

4.  The legends of fig1, fig2 and fig3 are the same. It is wired that the legends of fig1 to fig3 were the same one.

5. In line 222, the author should explain what is “ene ontology”

6. Potential typo in line 179

Author Response

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Reviewer 3 Report

The manuscript entitled "Allene oxide synthase genes are the common key regulators in viral stress response of wheat (Triticum aestivum L.)" is a nice attaemt to identify key gene in the viral response.

Authors have nicely used the publically available datasets and designed the experiment to validate their hypothesis and succeeded too.

There are few suggestions for improvement in the manuscript for better acceptability to the readers.

One graphical abstract summarising whole experiment can attract larger number of readers.

Minor improvement is needed in sentences. Re-phrasing in few paragraph needed.

Author Response

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Reviewer 4 Report

Shahriari et al through in-silico analysis investigated how the host transcriptome is affected in the presence of single, double, and triple infection. This study is useful for understanding what molecular factors play a critical role in viral infection when multiple viruses are infecting simultaneously which occurs frequently in nature. Given the importance of this study, some critical gaps are mentioned below which need to be added to increase the significance of the study:

Major critical comment:

1- lines 107 to 109: Please submit all the accession numbers properly. ERP128185 is mentioned twice. It is not clear how the authors obtained the data for all the combinational viral treatments, as the one mentioned here does not have all the treatments. I would kindly request the authors to provide a proper description of what treatments were obtained from which accession numbers. This would make the study reproducible and better understandable.

2- lines 69 to 75: These citations are quite old given that mixed infections in nature are frequently reported currently and are much more widespread than previously thought. I would request authors to update it with more current citations: doi: 10.1093/ve/veac095

 

3- Allene oxide synthase is a major enzyme in the oxylipin pathway which also has a role in vector attraction. Viruses modulate plant metabolic pathways to attract vectors to increase their host range needs to be discussed. Moreover, JA also has a role in regulating protease inhibitors which is one of the defense responses against potyvirus. It was not clear why the antagonistic pathway was discussed between JA and SA as none of the results show any presence of cross-talk. I would kindly request the authors to have a better discussion which increases the broad impact of this study. Some citations are mentioned here but would request authors to add more as seems fit: 10.1007/s10886-020-01157-7; 10.3390/v14061341.

 

English is easy to understand and clear. 

Author Response

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Round 2

Reviewer 1 Report

The authors have revised the manuscript in a significant way. However, I have opinion tha the title is still misleading and if possible it need to be changed. There is no validation by qRT-PCR or by functional characterization of any allene oxide synthase gene and therefore, this title is not justified.

Author Response

We are glad to see that the manuscript has been improved remarkably. The title was revised accordingly. Although experimental validation was hindered by time and financial constraints, and also, we didn’t have access to the multiple viral isolates to inoculate wheat plants and validate the hub genes using qPCR, therefore, we validated our results using a 10-fold cross-validation approach. and experimental validation was suggested in the “Discussion”. As explained in the first round of revision, this approach has been used in several studies similarly.

 

Reviewer 2 Report

I still can not be convinced by the way that combining differently expressed genes (DEGs) from different treatments (even from different studies) to exploit biological relevance underlying both treatments.  DEGs from each of the different studies/treatments had their specific pattern relative to that specific study. Combining treatments should have different results from the combination of multiple single treatments. 

Author Response

Dear Reviewer 

Thank you for your input. Given that this approach is a widely accepted method for identifying key genes, and numerous articles have employed it, the results we've achieved hold great potential for advancing our understanding of the wheat viral stress response.

Overall, it’s an in-silico analysis on various experiments with high-throughput data to evaluate wheat responses to the single and mixed viral infections that could bioinformatically evaluate the mixed infection of viruses from each different viral treatments to identify differentially and common expressed virus-responsive genes and unique and conserved pathways in plant-virus interaction as a potential candidate for further analysis. The similar analysis for single and mixed viral infections was also previously conducted in another study; Microarray analysis of Arabidopsis thaliana exposed to single and mixed infections with Cucumber mosaic virus and turnip viruses (DOI: 10.1007/s12298-021-00925-3).

 

 

 

Reviewer 4 Report

No further revisions required

Author Response

Dear Reviewer 

Thanks for your positive view towards the manuscript.  

 

Round 3

Reviewer 2 Report

I still can not be persuaded by the methodology that the authors used in the study. RNA seq is kind of technical that is sensitive to external environments. Similar experimental treatments even can't reproduce similar results. The authors employed data from different studies with different plant materials and treatments. Variations among the studies were generally explained by technical issues rather than biological. A convincing approach should be the authors obtained another data and showed the replicatabiligy of their genes in that data. 

Author Response

Dear Reviewer 

Thank you for your feedback. While it is accurate that each study conducted under distinct conditions, it's important to highlight that each study was evaluated alongside its respective control samples. The genes identified in each distinct analysis signify their role in the viral stress response. In essence, we initially scrutinized each experiment independently, and subsequently, we amalgamated the findings. This approach allows us to discern which genes are triggered under varying stress conditions and provides a comprehensive overview of this subject.

 

The similar analysis was also previously conducted in other studies and published in peer-reviewed journals;

Microarray analysis of Arabidopsis thaliana exposed to single and mixed infections with Cucumber mosaic virus and turnip viruses (DOI: 10.1007/s12298-021-00925-3).

Integrative system biology analysis of transcriptomic responses to drought stress in soybean (Glycine max L.) (doi.org/10.3390/genes13101732).

Transcriptome meta-analysis identifies candidate hub genes and pathways of pathogen stress responses in Arabidopsis thaliana (10.3390/biology11081155).

Meta-Analysis of common and differential transcriptomic responses to biotic and abiotic stresses in Arabidopsis thaliana (DOI: 10.3390/plants11040502).

A comprehensive meta-analysis to identify transcriptional signatures of abiotic stress responses in barley (Hordeum vulgare) (10.1007/s42976-020-00107-z).

Comparison of transcriptional response of C3 and C4 plants to drought stress using meta-analysis and systems biology approach (10.3389/fpls.2021.668736).

 Integrative meta-analysis of transcriptomic responses to abiotic stress in cotton (10.1016/j.pbiomolbio.2019.02.005).