The chromatin remodeling factor Arp9 modulates multidrug-resistance and plays a key role in aflatoxins biosynthesis under mammalian-physiological-temperature in Aspergillus flavus

PLOS Pathogens

Dear Dr. Zhuang,

Thank you for submitting your manuscript to PLOS Pathogens. After careful consideration, we feel that it has merit but does not fully meet PLOS Pathogens's publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript within 60 days Dec 19 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plospathogens@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/ppathogens/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Haoping Liu

Academic Editor

PLOS Pathogens

Alex Andrianopoulos

Section Editor

Editor-in-Chief

PLOS Pathogens

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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Reviewers' Comments:

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: The authors have addressed all my queries satisfactorily. The revised manuscript is much improved. Hence, I think the manuscript can be now accepted for publication.

Reviewer #2: I have reviewed this manuscript previously and have comments for this part. In general, novelty of this study is good.

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Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: (No Response)

Reviewer #2: In general, authors have responded all comments point by point. Authors really tried to make efforts to improve manuscript quality such as they did swi1, sfh1, and snf2 deletion mutants and found they all exhibited increased resistance to VOR compared to the WT which is consistent with that of dArp9, suggesting Arp9 and its putative complex partners had similar functions to them but we still could not conclude they are partners since there are no Co-IP biochemistry data or other direct evidence data.

However, function of Arp9 as a chromatin remodeling factor still is not very solid only based on transcriptome analysis, qRT-PCR, ChART-qPCR. Why the deletion of Arp9 exacerbated their resistance to VOR and what is molecular mechanism? No working model to be understood for the function of Arp9. In addition, there are still have some confused conclusions such as

as they demonstrated that swi1, sfh1, and snf2 deletion mutants exhibited increased resistance to VOR compared to the WT (Fig. 8C). However, these mutants displayed the opposite phenotype to AMB compared to the ΔArp9? how they have opposite functions for their targeted genes transcription?

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: (No Response)

Reviewer #2: minor comments:

in the title: Arp9 modulates multidrug-resistance. However, AMB has an opposite effect with that of VOR for Arp9 mutants

Figure 3G. what is indication for red cycle of H&E staining for observation

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Figure resubmission:

Reproducibility:

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PPATHOGENS-D-25-02077R1

The chromatin remodeling factor Arp9 modulates drug-resistance and plays a key role in aflatoxins biosynthesis under mammalian-physiological-temperature in Aspergillus flavus

PLOS Pathogens

Dear Dr. Zhuang,

Thank you for submitting your manuscript to PLOS Pathogens. After careful consideration, we feel that it has merit but does not fully meet PLOS Pathogens's publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

1. While you performed the suggested experiments to address questions regarding the arp9 phenotype in the context of the SWI/SNF and RSC complexes, you did not clearly distinguish between these two complexes in the writing. Line 108 should provide more information on Arp9, such as: "Arp9 (actin-related protein 9) is a member of both the SWI/SNF and RSC chromatin remodeling complexes in fungi (refs)." In the results section that describes Fig. 8S and Fig. 8C, please clarify: "Sfh1 (Snf5 homolog 1) is a key component of the yeast RSC complex, but not part of the SWI/SNF complex (ref). Swp82 is a component of the SWI/SNF complex (ref)."

2. Please expand the abbreviation ChART-qPCR (Chromatin Accessibility by Real-Time PCR) upon first use in the text and define relative CAI (chromatin accessibility index) in the Fig. 5E legend. Line 370 should read "qRT-PCR and ChART-qPCR." Typically, ChART-qPCR uses specific primer pairs tiling across a gene of interest. It is not clear how you determined the location of just one primer pair for each promoter examined in Fig. 5E. Additionally, Line 672 in the methods section states "ChART-rtPCR was performed to analyze gene expression levels," which is misleading, as ChART-qPCR measures chromatin accessibility, not gene expression.

3. Lines 441-448: The new Fig. S9 presents data that are not MNase-seq or ATAC-seq and therefore cannot provide direct information regarding nucleosome sliding. This content would be better suited for the discussion section.

4. The abstract should be updated to reflect the revisions. The writing throughout the entire manuscript should be reviewed to improve clarity and expression. For example, Lines 122-123 use the phrase "individual level," which is unclear in this context and should be clarified.

Please submit your revised manuscript by Mar 08 2026 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plospathogens@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/ppathogens/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

* A letter that responds to each point raised by the editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. This file does not need to include responses to any formatting updates and technical items listed in the 'Journal Requirements' section below.

* A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

* An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, competing interests statement, or data availability statement, please make these updates within the submission form at the time of resubmission. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

We look forward to receiving your revised manuscript.

Kind regards,

Haoping Liu

Academic Editor

PLOS Pathogens

Michal Olszewski

Section Editor

PLOS Pathogens

Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

Journal Requirements:

If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

1) In the online submission form, you indicated that your data will be submitted to a repository upon acceptance. We strongly recommend all authors deposit their data before acceptance, as the process can be lengthy and hold up publication timelines. Please note that, though access restrictions are acceptable now, your entire minimal dataset will need to be made freely accessible if your manuscript is accepted for publication. This policy applies to all data except where public deposition would breach compliance with the protocol approved by your research ethics board. If you are unable to adhere to our open data policy, please kindly revise your statement to explain your reasoning and we will seek the editor's input on an exemption.

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Reviewers' Comments:

Figure resubmission:

After uploading your figures to PLOS’s NAAS tool - https://ngplosjournals.pagemajik.ai/artanalysis, NAAS will process the files provided and display the results in the "Uploaded Files" section of the page as the processing is complete. If the uploaded figures meet our requirements (or NAAS is able to fix the files to meet our requirements), the figure will be marked as "fixed" above. If NAAS is unable to fix the files, a red "failed" label will appear above. When NAAS has confirmed that the figure files meet our requirements, please download the file via the download option, and include these NAAS processed figure files when submitting your revised manuscript.

Reproducibility:

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PPATHOGENS-D-25-02077R2

The chromatin remodeling factor Arp9 modulates drug-resistance and plays a key role in aflatoxins biosynthesis under mammalian-physiological-temperature in Aspergillus flavus

PLOS Pathogens

Dear Dr. Zhuang,

Thank you for submitting your manuscript to PLOS Pathogens. After careful consideration, we feel that it has merit but does not fully meet PLOS Pathogens' publication criteria as it currently stands. Therefore, we invite you to submit a revised manuscript that addresses the points raised during the review process. Please ensure that the requested corrections are finalized to avoid additional rounds of review.

Please submit your revised manuscript by Apr 12 2026 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plospathogens@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/ppathogens/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

* A letter that responds to each point raised by the editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. This file does not need to include responses to any formatting updates and technical items listed in the 'Journal Requirements' section below.

* A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

* An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, competing interests statement, or data availability statement, please make these updates within the submission form at the time of resubmission. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

We look forward to receiving your revised manuscript.

Kind regards,

Haoping Liu

Academic Editor

PLOS Pathogens

Michal Olszewski

Section Editor

PLOS Pathogens

Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

Additional Editor Comments:

The revision inadequately addresses the Abstract. The authors do not seem to agree that the methods they used to assess promoter accessibility are outdated and non-conclusive, yet they still included them in the abstract.

1. Replace “Transcriptome analysis, qRT-PCR, ChART-qPCR (Chromatin Accessibility by Real-Time PCR) and HPLC assay determined” with “We show”.

2. Remove “Micrococcal nuclease (MNase) digestion assays revealed that Arp9 deficiency impairs the rate of nucleosome release from chromatin, suggesting that Arp9 may play a critical role in maintaining chromatin accessibility.”

Here is a revised abstract with additional changes in red:

Aspergillus flavus is the second most prevalent species of Aspergillus causing invasive aspergillosis, but its treatment has been hindered by the continuous emergence of drug-resistant fungal strains, while the underlying mechanisms remain largely unexplored. In this study, we investigated the role of the chromatin remodeling factor Arp9 in A. flavus drug-resistance. We show that Arp9 up-regulates the chromatin accessibility of the Erg3 and Erg6 promoters, thereby increasing their transcription levels and enhancing ergosterol synthesis. Therefore, the absence of Arp9 enhances A. flavus sensitivity to amphotericin B (AMB). Additionally, by down-regulating the chromatin accessibility of the Erg11A gene promoter, Arp9 deletion decreases its transcription levels and subsequently reduces A. flavus resistance to voriconazole (VOR). Co-immunoprecipitation analysis revealed that Arp9 exists in both SWI/SNF and RSC complex. Drug susceptibility test results indicated that the drug sensitivity response induced by Arp9 may be unique to Arp9, as neither SWP82 of the SWI/SNF nor Sth1 of the RSC is not required. The role of Arp9 in drug-resistance was also confirmed using the Galleria mellonella model. Furthermore, we found that VOR induces aflatoxin B1 (AFB1) biosynthesis in an Arp9-dependent manner at 35°C and 37°C, and the effect is dramatically magnified in the VOR-resistant A. flavus strain. This study demonstrates that Arp9 plays a critical role in regulating fungal drug-resistance in vitro and in vivo and reveals that Arp9 is an important factor in enhancing AFB1 biosynthesis under Mammalian physiological temperatures. This study provides potential new insights for the control of infections caused by filamentous pathogenic fungi.

3. The revision does not address the methodological issues used to conclude promoter accessibility.

Line 690, the ChART-qPCR method referenced publication # 76 (published 1997), but this paper only provides a method for nuclease digestion and DNA extraction. This publication did not use qPCR or name their method ChART-qPCR. A publication that established the ChART-qPCR method is needed if the author followed the published method. The author still have not address or provide experimental evidence for how they selected the primer set used in Figure 5E.

Line 448 -450, change “Furthermore, to investigate… “ to “Furthermore, we performed an MNase digestion assay. “

Journal Requirements:

If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

1) In the online submission form, you indicated that your data will be submitted to a repository upon acceptance. We strongly recommend all authors deposit their data before acceptance, as the process can be lengthy and hold up publication timelines. Please note that, though access restrictions are acceptable now, your entire minimal dataset will need to be made freely accessible if your manuscript is accepted for publication. This policy applies to all data except where public deposition would breach compliance with the protocol approved by your research ethics board. If you are unable to adhere to our open data policy, please kindly revise your statement to explain your reasoning and we will seek the editor's input on an exemption.

Reviewers' Comments:

Figure resubmission:

After uploading your figures to PLOS’s NAAS tool - https://ngplosjournals.pagemajik.ai/artanalysis, NAAS will process the files provided and display the results in the "Uploaded Files" section of the page as the processing is complete. If the uploaded figures meet our requirements (or NAAS is able to fix the files to meet our requirements), the figure will be marked as "fixed" above. If NAAS is unable to fix the files, a red "failed" label will appear above. When NAAS has confirmed that the figure files meet our requirements, please download the file via the download option, and include these NAAS processed figure files when submitting your revised manuscript.

Reproducibility:

To enhance the reproducibility of your results, we recommend that authors of applicable studies deposit laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

Dear Dr. Zhuang,

We are pleased to inform you that your manuscript 'The chromatin remodeling factor Arp9 modulates drug-resistance and plays a key role in aflatoxins biosynthesis under mammalian-physiological-temperature in Aspergillus flavus' has been provisionally accepted for publication in PLOS Pathogens.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Haoping Liu

Academic Editor

PLOS Pathogens

Michal Olszewski

Section Editor

PLOS Pathogens

Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************************************************

Reviewer Comments (if any, and for reference):