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  • Beibei Liu 1,†,
  • Jianing Wu 2,† and
  • Yue Li 1,2,*
  • et al.

Reviewer 1: Anonymous Reviewer 2: Anonymous Reviewer 3: Anonymous

Round 1

Reviewer 1 Report

The manuscript is well-structured, but some minor suggestions are included.

Graphical abstract was not detected.

The abstract should be limited to a maximum of 200 words.

It is suggested that the keywords be exactly different from those in the title of the work.

Line 40. I suggest that the text should be modified and written as follows, “…f. sp. tritici (Pst), …”

Lines 92-92. Were pots used? What volume of substrate did you use?

Line 102. Did you use liquid nitrogen to prevent the degradation of total RNA? Or how was the integrity of the RNA protected?

Line 123. The 2–ΔΔCt method requires the inclusion of a calibrator sample (usually uninoculated samples) for relative quantification. Was this considered in your analyses?

Line 22. According to the 2–ΔΔCt method, what was the calibrator sample?

Line 268. Usually, the relative expression value in the calibrator sample is equal to 1; therefore, values ​​less than 1 indicate downregulation. Mention this in the figure caption. In your relative expression data, did you have values ​​less than 1? If so, consider reviewing https://doi.org/10.1038/nprot.2008.73

Lines 294-296. Mention the number of biological or technical replicates.

Lines 347-351. Mention the number of biological or technical replicates.

Lines 493-496. To mention author contributions, you must follow the journal's instructions.

Lines 513-522. Delete these paragraphs of text.

No comment

Author Response

Graphical abstract was not detected.

Response: Thank you for your note regarding the graphical abstract. We apologize for any inconvenience caused. The graphical abstract was indeed included in our original submission. It is possible that a technical issue during file processing or preview prevented it from being displayed properly. To resolve this promptly, we have now re-uploaded the complete manuscript file in PDF format, ensuring all components, including the graphical abstract, are correctly embedded and accessible. Thank you for your patience and for bringing this to our attention.

 

The abstract should be limited to a maximum of 200 words.

Response: Thank you for your valuable comment. We sincerely appreciate your feedback and have carefully revised the abstract according to your suggestion. The word count has now been successfully reduced to under 200 words to meet the journal's requirement. We have ensured that all core elements—the study's background, objectives, key methods, major findings, and conclusions—remain clear and concise within the new limit. We thank you once again for your guidance, which has helped improve the clarity and conciseness of our manuscript.

 

It is suggested that the keywords be exactly different from those in the title of the work. Response: Thank you for your valuable suggestion. We have carefully reviewed the keywords and revised them to ensure that they are entirely distinct from the words used in the title of our work, as per your recommendation.

 

Line 40. I suggest that the text should be modified and written as follows, “…f. sp. tritici (Pst), …”

Response: Thank you for this attentive correction regarding the precise formatting of the pathogen's scientific name. We have revised line 40 as you suggested, ensuring the specific epithet is in lowercase to read "…f. sp. Tritici (Pst), …" This adjustment aligns with the standard nomenclatural format used in the field and in relevant literature. We appreciate your diligence in enhancing the manuscript's technical accuracy.

 

Lines 92-92. Were pots used? What volume of substrate did you use?

Response: Thank you for pointing out the need to clarify this important methodological detail. In response to your comment, we have revised Section 2.1 of the manuscript to specify that the plants were indeed grown in pots. The pots used measured 7 cm × 7 cm × 7 cm, and each was filled with substrate to approximately two-thirds of its volume. We appreciate your careful review, which has helped us make the experimental description more complete and precise.

 

Line 102. Did you use liquid nitrogen to prevent the degradation of total RNA? Or how was the integrity of the RNA protected?

Response: Thank you for your question regarding RNA preservation. Yes, to prevent RNA degradation and ensure its integrity, we specifically followed the protocol detailed in Section 2.2 of our manuscript. As stated there, the collected leaf samples were immediately snap-frozen in liquid nitrogen. This standard procedure effectively halts all enzymatic activity and protects the RNA from degradation until the extraction process. We appreciate your attention to this important methodological detail.。

 

Line 123. The 2–ΔΔCt method requires the inclusion of a calibrator sample (usually uninoculated samples) for relative quantification. Was this considered in your analyses? Response: Thank you for raising this important technical point regarding the 2–ΔΔCt method. Yes, the requirement for a calibrator sample was indeed carefully considered and incorporated into our experimental design and analysis. As specified in Section 2.3 of our manuscript, the urediniospore stage served as the calibrator for the relative gene expression quantification. We appreciate your meticulous attention to the methodological details, which ensures the robustness of the data analysis.

 

Line 22. According to the 2–ΔΔCt method, what was the calibrator sample? Thank you Response: for your question. You are absolutely right that the calibrator is a required component of the 2–ΔΔCt method. We followed this standard precisely; however, as you correctly noted, the abstract is intended to concisely summarize the main objectives and findings, so specific methodological details like the choice of calibrator are typically not included there. This information is provided in the dedicated Methods section. Specifically, as detailed in Section 2.3 of the manuscript, the urediniospore stage was explicitly used as the calibrator sample for all relative expression calculations. We hope this clarification addresses your question and thank you for your thorough review.

 

Line 268. Usually, the relative expression value in the calibrator sample is equal to 1; therefore, values ​​less than 1 indicate downregulation. Mention this in the figure caption. In your relative expression data, did you have values ​​less than 1? If so, consider reviewing https://doi.org/10.1038/nprot.2008.73

Response: Thank you for this precise methodological suggestion. We have followed your advice and updated the caption for the relevant figure to explicitly state that the relative expression in the calibrator sample is set to 1, and therefore values less than 1 indicate downregulation. Regarding your second question, yes, our relative expression data did include values less than 1. These values are consistent with the expected silencing effect of our treatment and support the conclusion of reduced PsAP1 expression. We are grateful for you sharing the link to the seminal protocol paper (doi:10.1038/nprot.2008.73) and will carefully review it to ensure our analysis and interpretation fully align with best practices.

 

Lines 294-296. Mention the number of biological or technical replicates.

Lines 347-351. Mention the number of biological or technical replicates.

Response: Thank you for this important suggestion to enhance the methodological transparency of our study. We have revised the manuscript as recommended and have now explicitly stated the number of biological and/or technical replicates used in the statistical analyses mentioned in Lines 294-296 and Lines 347-351. This addition provides essential context for assessing the robustness of our findings, and we appreciate your guidance in strengthening this aspect of the paper.

 

Lines 493-496. To mention author contributions, you must follow the journal's instructions.

Response: Thank you for reminding us to adhere to the journal's specific formatting requirements. We have carefully reviewed the journal's instructions for authors and have revised the author contributions section (Lines 493-496) accordingly to ensure it fully complies with the stipulated format.

 

Lines 513-522. Delete these paragraphs of text.

Response: Thank you for your suggestion. As advised, we have deleted the paragraphs of text in Lines 513-522 from the manuscript.

 

Reviewer 2 Report

Comprehensive work.

Review of the manuscript The AP-1 Sigma Subunit Gene PsAP1 Acts as a Key Pathogenicity Factor by Regulating Metabolic Reprogramming in Puccinia striiformis f. sp. tritici

 

In my opinion the manuscript is well written and brings important and interesting results. I haven't found there any serious problems. However, some minor technical errors exist there.

  1. 17: tritici (the same on l. 40 and 572).
  2. 20: PsAP1.
  3. 24: barley stripe mosaic virus (lowercased).
  4. 130: add N. benthamiana.
  5. 138: Pst (not in italics).
  6. 151, 152, 157: media SYM, CM, and CMC should be characterized.
  7. 180: it is not needed to repeat Pst.

Fig. 1: letters A, B, C should be better situated, especially C is a bit hidden inside of the figure.

Fig. 2A: there is a third peak at 48 hpi, is it clear what event it corresponds to (compare l. 248 and 249)? Could not this fluctuation correspond to night and day regime?

Fig. 5: legends for B and C are reversed.

  1. 399-423 and 463: I don't understand why eg. Thiamine metabolism or Glutathione metabolism are sometimes uppercased and sometimes lowercased.

References: not uniform:

  • Some article titles lowercased, others uppercased.
  • Some journals names abbreviated, others not (eg. l. 532), some uppercased, others not.
  • Not all Latin names of organisms are in italics.
  • Meaning of letters J or D behind article titles is not clear for me (are they needed?) Eg. on l. 536 it is missing.

(I haven't found link to the supplementary table 1 in the text, but it may exist.)

Author Response

 

17: tritici (the same on l. 40 and 572).

20: PsAP1.

24: barley stripe mosaic virus (lowercased).

130: add N. benthamiana.

138: Pst (not in italics).

151, 152, 157: media SYM, CM, and CMC should be characterized.

180: it is not needed to repeat Pst.

Fig. 1: letters A, B, C should be better situated, especially C is a bit hidden inside of the figure.

Response: Thank you for your meticulous review and for pointing out these important formatting and terminological details. We have carefully addressed each of the specific points you raised in Lines 17, 20, 24, 130, 138, 151, 152, 157, 180, and in Figure 1. All terms, such as the species name, gene symbols, virus names, and the designation of the pathogen isolate, have been corrected to adhere to standard scientific conventions. The descriptions for the specified culture media have been characterized as suggested. We have also repositioned the labels in Figure 1 to ensure they are clearly visible. We sincerely appreciate the time and expertise you have dedicated to improving the precision and presentation of our manuscript.

 

Fig. 2A: there is a third peak at 48 hpi, is it clear what event it corresponds to (compare l. 248 and 249)? Could not this fluctuation correspond to night and day regime?

Response: Thank you for this critical question regarding the interpretation of the results. The third peak at 48 hpi you pointed out indeed requires a clear biological explanation, and your thought to link it to the diurnal rhythm is highly insightful. We have re-examined the data and, based on the literature, believe a more robust explanation may involve the following aspects, according to which we will revise the manuscript:

  1. Association with Infection Stages: The peak may mark a transition in infection stages. 48 hpi is typically a key period when Pstcompletes substomatal vesicle formation and begins to establish primary haustorial mother cells for biotrophic nourishment. The expression peak could be closely tied to this active morphogenesis and establishment of the nutrient interface, which is more precise than the general "hyphal rapid growth."
  2. Coupling with Plant Defense Rhythms (Your suggested diurnal direction): Your insight is highly valuable. Plant defense responses (e.g., allocation of photosynthetic products, defense hormone signaling) are regulated by the circadian clock. The 48 hpi peak may reflect the synchronization of pathogen gene expression with the diurnal cycle of the host's specific physiological/immune status to maximize resource acquisition or suppress host defenses. This is a reasonable explanation worthy of in-depth discussion.
  3. Dynamics of Host-Pathogen Interaction: This peak could also correspond to a specific peak in host defense response (e.g., ROS burst or expression of specific defense genes), to which the pathogen correspondingly modulates the expression of this gene to cope with or suppress it.
  4. Consideration of Technical Replicates: We will re-check the biological replicate data at this time point to confirm its consistency and rule out technical variation.

We will revise the explanation in the Results section (around lines 248-249), shifting the focus from "hyphal growth" to: "This expression peak coincides with the active phase of the pathogen establishing biotrophic absorption structures and may be regulated by the host's diurnal physiological rhythms." Simultaneously, we will cite relevant literature in the Discussion to support the view that infection stage transitions or plant circadian rhythms can influence pathogen gene expression.

We sincerely appreciate this profound question, which has prompted us to provide a more rigorous and biologically in-depth interpretation of the data. If you find one of the above explanatory directions more reasonable, we will focus on refining it accordingly.

 

Fig. 5: legends for B and C are reversed.

Response: Thank you for catching this error in the figure legend. We have corrected Fig. 5 as you indicated, and the labels for parts B and C are now in their proper order.

 

399-423 and 463: I don't understand why eg. Thiamine metabolism or Glutathione metabolism are sometimes uppercased and sometimes lowercased.

Response: Thank you for highlighting this inconsistency in our manuscript. You are absolutely right to point it out, as maintaining uniform formatting is essential for clarity. We have carefully reviewed the text and have standardized the naming of all mentioned metabolic pathways, including "thiamine metabolism" and "glutathione metabolism", to be consistently written in lowercase throughout the manuscript (including in lines 399-423 and 463), unless they appear at the beginning of a sentence. We appreciate your attention to detail, which has helped us improve the consistency and professionalism of our work.

References: not uniform:

Some article titles lowercased, others uppercased.

Some journals names abbreviated, others not (eg. l. 532), some uppercased, others not.

Not all Latin names of organisms are in italics.

Meaning of letters J or D behind article titles is not clear for me (are they needed?) Eg. on l. 536 it is missing.

Response: Thank you for your thorough review and for pointing out the inconsistencies in the reference formatting. We sincerely apologize for these oversights and have carefully revised the entire reference list according to your comments. We have standardized the capitalization of article titles, ensured consistent abbreviation and formatting of journal names, italicized all Latin names of organisms, and clarified or removed any ambiguous letter designations following article titles as required. We appreciate your diligence in helping us ensure the references meet the journal's high standards.

 

I haven't found link to the supplementary table 1 in the text, but it may exist.

Response: Thank you for checking the details regarding the supplementary materials. You are correct that there is no active link to Supplementary Table 1 in the current main text, as this reference was removed during our revisions. The manuscript is complete in its current form without it. However, if you believe that Supplementary Table 1 contains data important to the review process, we would be happy to prepare and submit it for you. Thank you for your attention to ensuring the completeness of the submission.

Reviewer 3 Report

The article “The AP-1 Sigma Subunit Gene PsAP1 Acts as a Key Pathogenicity Factor by Regulating Metabolic Reprogramming in Puccinia striiformis f. sp. tritici” is devoted to the interesting and acute theme of the interaction of the pathogen with the host plant. Authors provide and analyze a lot of information on this theme and use modern methods to prove their hypothesis.
I recommend minor revision of the manuscript.
 
1) In my opinion in the title it should be defined, that authors investigate wheat/Pst system, may be “The AP-1 Sigma Subunit Gene PsAP1 Acts as a Key Pathogenicity Factor by Regulating Metabolic Reprogramming in Puccinia striiformis f. sp. Tritici when infecting wheat plants”
2) Authors investigate two different organisms and in all cases it must be specified what the parameter refers to wheat or Pst. It is particularly important in figure legends.
3) What strain of Pst was used? Who supplied it?
4) Figures 1 and 7 have unreadable text, it must be corrected or placed in Supplementary in full size.
5) What statistical methods were used? I have doubts that the analysis of data presented in fig 3b is correct.
6) In legend of fig 4 it must be defined that letters indicate significant differences between parameters on the same timepoint.
7) The paragraph “3.5 ROS Detection Post-Silencing” should be concretized carefully in all cases. It is far from clear that ROS were measured in wheat plants infected with Pst? Or a virus?
8) Covid-19 appearance in Fig 7 D must be explained.

Puccinia striiformis f. sp. Tritici replace with Puccinia striiformis f. sp. tritici
 Please, insert references on your previous articles (Lines 71-76)
 Please, add manufacturer and country in all cases (kits, software etc)

Author Response

1) In my opinion in the title it should be defined, that authors investigate wheat/Pst system, may be “The AP-1 Sigma Subunit Gene PsAP1 Acts as a Key Pathogenicity Factor by Regulating Metabolic Reprogramming in Puccinia striiformis f. sp. Tritici when infecting wheat plants”

Response: Thank you for your thoughtful suggestion regarding the manuscript title. We sincerely appreciate your effort to help make our title more precise and informative. We have carefully considered your proposed title, which explicitly mentions "when infecting wheat plants," and agree that it helpfully specifies the host-pathogen system.

Our rationale for retaining the original title is based on the standard practice in phytopathology and molecular plant-microbe interaction research, where the host is often implicitly defined by the pathogen's formal scientific name. Since Puccinia striiformis f. sp. tritici is an obligate pathogen that infects only wheat, the host is unambiguously indicated within the title to specialists in the field. The original title, therefore, concisely communicates the core finding—the role of PsAP1 in regulating metabolism within the pathogen—while remaining consistent with the style of related literature. We believe its current form is clear and appropriate for the target journal audience.

We are, of course, fully open to the final decision of you and the editorial team. If you consider the addition of the host plant to be essential for clarity to a broader readership, we will gladly adopt your suggested modification. Thank you again for this valuable discussion and for your guidance throughout the review process.

 

2) Authors investigate two different organisms and in all cases it must be specified what the parameter refers to wheat or Pst. It is particularly important in figure legends.

Response: Thank you for raising this critical point regarding the clarity of our data presentation. We fully agree that explicitly distinguishing whether a measured parameter refers to the wheat host or the Pst pathogen is essential for accurate interpretation. In response to your comment, we have systematically reviewed and revised the manuscript, with particular attention to all figure legends and relevant results sections. Each parameter (e.g., gene expression, hyphal length, ROS accumulation) now clearly specifies the organism of origin (wheat or Pst). We believe these revisions prevent any potential ambiguity and thank you for your guidance in enhancing the precision of our work.

3) What strain of Pst was used? Who supplied it?

Response: Thank you for your question regarding the origin of the pathogen strain. As stated in our manuscript, the Puccinia striiformis f. sp. tritici strain used in this study was CYR32. It was provided by the laboratory of the Institute of Plant Protection, Xinjiang Academy of Agricultural Sciences / Key Laboratory of Integrated Pest Management on Crops in Northwestern Oasis, Ministry of Agriculture and Rural Affairs, Urumqi 830091, China. This source has been clearly cited in the relevant section of the paper.

4) Figures 1 and 7 have unreadable text, it must be corrected or placed in Supplementary in full size.

Response: Thank you for your feedback regarding the readability of Figures 1 and 7. We have addressed this issue by replacing both figures with new, high-resolution versions to ensure all text and details are clearly legible. We appreciate your attention to this matter, which has helped improve the overall clarity of our manuscript.

5) What statistical methods were used? I have doubts that the analysis of data presented in fig 3b is correct.

Response: Thank you for raising this important methodological point regarding the statistical analysis in Fig. 3B. We appreciate the opportunity to clarify. The colony diameter data presented in Fig. 3B were obtained through direct measurement. For the statistical comparison between the relevant experimental groups, we performed an unpaired two-tailed Student's t-test using GraphPad Prism software. We have now revised the figure caption to detail this methodology explicitly. We are committed to ensuring the rigor of our data analysis. If you have specific concerns about the applicability of the t-test to our dataset or recommend an alternative statistical approach, we would be grateful for your guidance and are prepared to re-analyze the data accordingly.

 

6) In legend of fig 4 it must be defined that letters indicate significant differences between parameters on the same timepoint.

Response: Thank you for this precise and necessary correction to improve the clarity of our statistical presentation. We have revised the legend for Figure 4B as you recommended. It now explicitly states that the different lowercase letters shown on the bars indicate statistically significant differences between the compared parameters (i.e., treatment vs. control groups) at the same time point. The specific statistical test used is also noted. We appreciate your attention to detail, which has helped make our data interpretation more transparent and accurate.

 

7) The paragraph “3.5 ROS Detection Post-Silencing” should be concretized carefully in all cases. It is far from clear that ROS were measured in wheat plants infected with Pst? Or a virus?

Response: Thank you for your critical question regarding the ROS detection experiment, which helps us clarify a potentially ambiguous point. You are absolutely correct to seek precision. In the section “3.5 ROS Detection Post-Silencing”, the reactive oxygen species (ROS) were indeed detected and measured in wheat leaves infected with Puccinia striiformis f. sp. tritici (Pst). This assay was designed specifically to assess the plant's defense response (ROS burst) triggered by the fungal pathogen following gene silencing. To enhance methodological transparency, we have added a description of the specific DAB staining procedure in this section. However, we are fully aware of the need for conciseness. If you find these added details redundant or believe they disrupt the narrative flow, we will gladly remove them and keep the description focused on the core objective and result. We sincerely appreciate your guidance in helping us strike the right balance between detail and clarity.

 

8) Covid-19 appearance in Fig 7 D must be explained.

Response: Thank you for your insightful observation regarding the appearance of the "COVID-19" pathway term in our KEGG enrichment analysis (Fig. 7D). We appreciate the opportunity to clarify this point, which we agree could be surprising without proper context.

The appearance of this term is an indirect result of the functional conservation of genes across different biological systems. The KEGG pathway database annotates genes based on their known molecular functions. The set of differentially expressed genes in our study was enriched for functions related to core host immune and stress response mechanisms, such as receptor interaction, inflammatory signaling, and programmed cell death. In the KEGG classification, a significant subset of genes involved in these evolutionarily conserved processes is also cataloged under the specific model of the "COVID-19" pathway, which details host-virus interactions. Therefore, the enrichment signal reflects a shared underlying biology (e.g., host defense and apoptosis) rather than a direct link to the SARS-CoV-2 virus itself.

We did not emphasize this specific pathway in the manuscript because our study focuses on the plant-fungal interaction, and we considered its appearance as a bioinformatic artifact highlighting conserved cellular response modules. However, we acknowledge that its presence in the figure without explanation could be confusing. We are prepared to add a brief explanatory note in the results or figure legend (e.g., "Note: The 'COVID-19' pathway enrichment indicates genes involved in conserved host immune and stress responses, as classified by the KEGG database") if you consider it necessary for clarity. We are grateful for your attention to detail, which helps us present our data as clearly as possible.

 

Detailed comments

Puccinia striiformis f. sp. Tritici replace with Puccinia striiformis f. sp. tritici

 Please, insert references on your previous articles (Lines 71-76)

 Please, add manufacturer and country in all cases (kits, software etc)

Response: Thank you for these important corrections regarding nomenclature consistency, citation completeness, and methodological detail. We have carefully addressed all three points in the revised manuscript: 1) All instances of the pathogen's name have been standardized to the correct lowercase format "Puccinia striiformis f. sp. tritici". 2) The relevant references to our previous work have been inserted in the section covering Lines 71-76. 3) The manufacturer and country of origin have been added for all specified commercial kits, reagents, and software mentioned in the Methods section. We appreciate your thorough review, which has significantly enhanced the precision and reproducibility of our work.