Hog1-induced transcription of RTC3 and HSP12 is robust and occurs in cells lacking Msn2, Msn4, Hot1 and Sko1

Chen Bai; Masha Tesker; Dganit Melamed-Kadosh; David Engelberg; Arie Admon

doi:10.1371/journal.pone.0237540

Peer Review History

Original SubmissionMay 3, 2020
21 May 2020 Decision Letter - Juan Mata, Editor PONE-D-20-12974 Hog1-induced transcription of RTC3 and HSP12 is robust and occurs in cells lacking Msn2, Msn4, Hot1 and Sko1 PLOS ONE Dear Dr. Engelberg, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’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. The reviewers have a number of comments that should be addressed. This should be doable by modifying the text and by providing information that you probably already have (i.e., experiments should not be required as long as the qPCR experiments are biological replicates). Please submit your revised manuscript by Jul 05 2020 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 plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. 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, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols We look forward to receiving your revised manuscript. Kind regards, Juan Mata, Ph.D. Academic Editor PLOS ONE Journal requirements: When submitting your revision, we need you to address these additional requirements: 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at http://www.plosone.org/attachments/PLOSOne_formatting_sample_main_body.pdf and http://www.plosone.org/attachments/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ 3. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Partly Reviewer #2: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No Reviewer #2: No ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: In this manuscript, Bai et al describe a detailed analysis in which the individual contributions of different transcriptional activators was determined in the induction of four Hog1-regulated genes in budding yeast. Hot1, Sko1, and Msn2/4 are known to respond to osmotic stress, through activated Hog1, in order to induce expression of osmoresponse genes. Here, the specific contribution of each of these factors, at four specific target genes, is determined by transcription factor gene knock-out and promoter deletion analyses. The study is largely technically sound, well-controlled, and convincing. However, the following points should be addressed. 1. Starting on line 244, the authors refer to an analysis of a STRE-LacZ reporter that is not active in BY4741ras2Δ cells. Can they show this data? This is important for supporting the claim that Msn2/4 is not de-repressed in BY4741 cells. 2. Lines 305 – 309 refers to two Hot1-binding sites underlined in Fig. 1E between -707 to -389, but nothing is underlined in this region. Consequently, it’s not clear how this promoter region is used primarily by Hot1, as stated. Can the authors address this? 3. The authors indicate that the beta-gal assays were done in triplicate, but it’s not explicitly indicated that the qRT-PCR were also performed as independent replicates. Can the authors confirm this? Also, no statistical analysis (e.g. Students’ T-Test) is used. Although many of the differences are obvious as judged by eye, to be rigorous, p-values can be included, particularly where differences are not dramatic. 4. The authors refer to Table 1 containing primer sequences, but I did not find this table in the PDF package. 5. There’s a verb missing in the sentence starting on line 189: “In a previous study we already the RTC3 promoter …” 6. On line 250 “Figs 1D and D” should be “1C and D”. 7. On lines 308 and 312 “Fig 1D” should be “Fig 1E.” 8. On line 319, “backupping factors” is a bit awkward. “Backup factors” might be more appropriate. Reviewer #2: All the comments to the manuscripto have been uploaded in the attached file. A major revision, including manuscript structure, contents, graphics and statistical analysis, is required before publication in Plos One. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. Attachments Attachment Submitted filename: Peer review for PLOS ONE.pdf https://doi.org/10.1371/journal.pone.0237540.r001
Revision 1
30 Jun 2020 Author Response A point-by-point list of response to reviewers’ comments Reviewer 1: 1. The reviewer asks to show the data regarding STRE-LacZ activity – we thus added this data as an upper panel in Figure 1F. 2. The reviewer comments about sequences, which were underlined in Fig. 1E, and were not referred to properly in the text – we agree with the reviewer that the text in these lines and its relatedness to the figure were confusing. We now removed all lines from the sequence in Figure 1E and simplified the explanation in lines 316-325 (in the new version). 3. The reviewer asks whether qRT-PCR analysis was performed in three repeats just as β-galactosidase assays were – in the new version we more explicitly state this matter (lines 180-181) and explain the related statistical analysis (lines 181-183). 4. The reviewer noted that the primers Table is missing. This must have been a technical problem. In any case, as only 8 primers appeared in the Table we now simply added them as a list in Materials and Methods (lines 174-177). 5. The verb was added: cloned. Now in line 199. 6. The letter D was changed to E. We thank the reviewer for his/her so rigorous reading. 7. 1D was changed to 1E. Thanks again. 8. The word backupping was changed to backup factors (now line 334). Reviewer 2: 1. The reviewer suggests to add more information on the physiological functions of the genes studied – although the study deal with transcription of these genes, we accepted the recommendation and added: 1) information on the enzymatic activity of each of the gene products (in lines 195-205). Some information on the physiological functions of each gene products is provided now in lines 306-308, 404-406, 454-456, 496-498. 2. Figure legend and titles of legends were shortened. 3. Figures resolutions were improved. 4. Legends of graphs were shortened. 5. Many more references were added, including the one recommended by the reviewers. Added references are: 31, 33, 34, 38, 40, 41. 6. The order of authors is determined by the website – corrected now. 7. The letter s was added. 8. The word background is now spelled correctly. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0237540.r002
23 Jul 2020 Decision Letter - Juan Mata, Editor PONE-D-20-12974R1 Hog1-induced transcription of RTC3 and HSP12 is robust and occurs in cells lacking Msn2, Msn4, Hot1 and Sko1 PLOS ONE Dear Dr. Engelberg, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the minor points raised by reviewer 2. Please submit your revised manuscript by Sep 06 2020 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 plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. 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, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols We look forward to receiving your revised manuscript. Kind regards, Juan Mata, Ph.D. Academic Editor PLOS ONE [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed Reviewer #2: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: In this revised manuscript, the authors have now addressed all my comments to my satisfaction. I believe it is now suitable for publication in PLoS ONE. Reviewer #2: Authors addressed most of the points raised in the first round of revision. I still have some concerns on figures’ resolution, at least the ones I can see in the file PONE-D-20-12974_R1. Figs.1,2,3,4, including graphs, sequences and immunoblot analysis, continue to appear with poor resolution, especially when enlarged, and, in my personal opinion, could be further improved. Also the text of figures’legend requires a further improvement. Here follow some suggestions for a typical legend: -This title could be enough, other info are provided in the text: RTC3 promoter is regulated primarily by the Ras/cAMP/Msn2/4 pathway; -Cell growth conditions should be indicated; -RNA extraction should be mentioned since it precedes the quantification of mRNA levels by qRT-PCR, as in the case of Fig.1A, for example. ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0237540.r003
Revision 2
24 Jul 2020 Author Response Dr. Juan Mata Academic Editor, PLOS ONE Re: PONE-D-20-12974 Dear Dr. Mata, Many thanks for your e-mail of July 23rd that included the reviewers’ comments to our revised manuscript. We have prepared a revised version in which we addressed all comments and hope that this version will merit publication in PLoS One. A point-by-point list of answers to reviewers’ comments is appended below. Sincerely, David Engelberg A point-by-point list of response to reviewers’ comments Reviewer 2: 1. The reviewer raises the issue of figures resolution. I fully agree with the reviewer’s comment that was also raised in the first reviewing round. We must note that we upload high quality figures that passed all tests of the PLoS One submission system. The reduction in quality occurs when the PDF file in created. I noticed this problem even before the reviewer’s comment and approached PLoS One assistance system. Here is the correspondence that clearly suggest that the issue is in the hands of PLoS One editorial staff: Dear Dr. Engelberg, Thank you for your email. I'm sorry to hear that you're upset about the quality of your figures in the submission PDF. However, please be assured that the submission PDF is for reviewing purposes only and does not reflect the appearance of the final publication, should your manuscript be accepted. Instead, the figures will be typeset at the resolution of your original submitted figures. Please also note that editors and reviewers will have access to the original source files you’ve provided via the blue download link in the top right corner of the PDF rendering of your figure. For more information about our figure requirements, please see (https://journals.plos.org/plosone/s/figures). Thanks for considering PLOS ONE for your work. Please feel free to contact us if you have other questions. Best, Eoin O'Connor Editorial Office PLOS \| plos.org Empowering researchers to transform science 1160 Battery Street, Suite 225, San Francisco, CA 94111 Case Number: 06690049 ref:_00DU0Ifis._5004P1Bd6nK:ref --------------- Original Message --------------- From: David Engelberg [engelber@mail.huji.ac.il] Sent: 29/06/2020 15:42 To: plosone@plos.org Subject: Re: PLOS ONE Decision: Revision required [PONE-D-20-12974] - [EMID:501e99ac72418e0f] Hello, I have just uploaded the revised manuscript at the website. Two matters bother me: A. The list of authors as appears in the submission manager (with me as the first author) is not the correct one - the correct one is as appears in the manuscript title page with Bai Chen as the first author [one of the reviewers commented on it]. I hope that if the paper is accepted the proper order of authors will appear on it. B. Although I ran all figures through the Apex.PACE system, as requested, and although the quality is quite high as appears in the files, figures look less good on the final PDF file. I hope that, if the paper is accepted, this will not be the quality of the final publication. Thanks for dealing with our manuscript. Sincerely, David Engelberg 2. The reviewer suggest to further shorten the titles of figure legends and to add description of growth conditions and RNA extraction. We accepted the comments. All figure legend titles have been shortened: lines 337, 438, 490 and 535. Growth conditions are now added (lines 141-151) and the kit used for RNA extraction is mentioned now in line 147. Attachments Attachment Submitted filename: Response to Reviewers2.docx https://doi.org/10.1371/journal.pone.0237540.r004
29 Jul 2020 Decision Letter - Juan Mata, Editor Hog1-induced transcription of RTC3 and HSP12 is robust and occurs in cells lacking Msn2, Msn4, Hot1 and Sko1 PONE-D-20-12974R2 Dear Dr. Engelberg, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Juan Mata, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0237540.r005
Formally Accepted
3 Aug 2020 Acceptance Letter - Juan Mata, Editor PONE-D-20-12974R2 Hog1-induced transcription of RTC3 and HSP12 is robust and occurs in cells lacking Msn2, Msn4, Hot1 and Sko1 Dear Dr. Engelberg: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Juan Mata Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0237540.r006

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .