PONE-D-22-20188The ‘Candidatus Phytoplasma mali’ effector protein SAP11CaPm interacts with MdTCP16, a class II CYC/TB1 transcription factor that is highly expressed during phytoplasma infection.PLOS ONE

Dear Dr. Janik,

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 current submission does not meet PLOS ONE publication criteria primarily in areas of a sufficient description of the experimental materials and methods used, some issues with analyses of the data, and that significant conclusions are not adequately supported by the data provided. All of the reviewers' comments are relevant and should be addressed in a revised manuscript. It is particularly important that you address Reviewer 2's concerns about the interpretation of MdTCP16 expression. 

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PLOS ONE

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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: Yes

Reviewer #2: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: I Don't Know

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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: No

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Reviewer #1: Yes

Reviewer #2: Yes

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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: The MS “The ‘Candidatus Phytoplasma mali’ effector protein SAP11CaPm interacts with MdTCP16, a class II CYC/TB1 transcription factor that is highly expressed during phytoplasma infection.” (PONE-D-22-20188) is a very nice little story which deals with an interesting mechanism of effector protein interacting with transcription factors.

The MS is written very well and is also well structured. Nevertheless, I have some critical points which should be considered for revision. The M+M part have to be improved as I could not found the protocol for protoplastation and also the growth conditions of the apple trees (field and greenhouse) are missing. Which apple cultivars are used and did the authors used the same cultivar for grafting (rootstock and scion)? Which Ca. P mali accession did the authors used in the grafted trees? (virulent or avirulent?) Can the interaction of SAP11 and MdTCP16 be different if the tree is infected with a virulent or avirulent accession? What about the phytoplasma titre of the trees, should be shown as suppl. table. Fig. 2: YFP signal seems also be in the membrane. That this is not the case should be checked by colocalization.

Reviewer #2: In this study, Mittelberger et al. investigate the interactions of the Candidatus phytoplasma mali effector protein SAP11 with two host proteins: the transcription factor MdTCP16 and NYC1, a chloroplast reductase. The paper also analyses the expression of three different Malus domestica transcription factors (MdTCP16, MdTCP24 and MdTCP25) during phytoplasma infection over the plant vegetative season. The main focus of the study is improving the knowledge about SAP11 role in Ca. P. mali infection identifying new interactors and describing the expression dynamic of SAP11 and its host targets between spring and autumn.

The manuscript is generally well-written and easy to follow. The two approaches adopted to study SAP11 targets congruently identify MdTCP16 as a new interactor of the effector protein. However, some issues with the methods and analysis of the gene expression studies need to be addressed.

Below are more specific comments by section:

Title, Abstract and Introduction:

- The title appropriately summarizes the content and results of the paper.

- The abstract is detailed, concise, and appropriately condenses the manuscript content.

- The introduction provides enough information to understand the background and the relevance of the study.

Methods:

- A description of the different plant growing conditions would help understand and interpret the results. Since BRC1 are influenced by photoperiod and temperature fluctuations, it is important to know if the plants in greenhouse were maintained in controlled conditions or not. I suggest introducing one or more sections to describe the plant materials and the plant growth conditions in the greenhouse (light, temperature). The sampling strategies, and the sampling size for naturally infected and greenhouse plants need also to be provided to better interpret the results and statistical analysis. See also comment to lines 221-224.

Results

- Line 193: NYC1 and TCP16 do not share common features, do not belong to the same protein family and are localized in different subcellular compartments. The co-expression of SAP11 and MdNYC1 further confirms the lack of interaction between these two proteins already suggested by the Y2H experiment. Therefore, I would not consider this result as the negative control but part of the experiment, since the BiFC experiment was conducted to validate the results obtained by the Y2H. Moreover,

- Line 221-224: the discrepancy mentioned in line 223 seems to refer to greenhouse vs naturally infected plants. Nevertheless, the authors suggested that the discrepancy “might be due to the season-dependent phytoplasma colonization”. This statement needs to be detailed and moved to the Discussion paragraph.

- Figure 3 Panel A and B. The plots show no difference in MdTCP16 expression between healthy and infected plants in spring and autumn. The fact that MdTCP16 expression increases during the season, such as the phytoplasma titer (panel D), is not enough to prove a direct correlation between the two phenomena. Moreover, the fact that MdTCP16 has the same trend of expression in healthy and infected plants shows that the increase of MdTCP16 transcription is correlated to environmental or plant physiological conditions rather than phytoplasma infection.

Panels C and D. It is unclear what samples are plotted here. The size of the data set differs between the two panels. It would be important to report and describe the data sets used to build the plots.

- Lines 251-252 See comment to Figure 3.

Discussion

- Line 286-290 The authors suggest that TCP16 might play a role in the early bud break during the spring. Nevertheless, in springtime TCP16 expression level is the lowest registered during the vegetative season. Moreover, in spring, the expression level of SAP11 was not detectable in the open field and was extremely low in the greenhouse.

A further concern is that the expression analysis of all the genes involved in the study was conducted on leaf samples, but the MdTCP16 is mainly expressed in buds, where it is supposed to play a crucial role. Similarly, the expression of SAP11 in bud cells was not investigated. Consequently, the hypothesis of the involvement of SAP11-TCP16 interaction in the induction of early bud break of phytoplasma infected trees is speculative and not supported by the data presented.

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Reviewer #1: Yes: Alexandra C. U. Furch

Reviewer #2: No

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PONE-D-22-20188R1

The ‘Candidatus Phytoplasma mali’ effector protein SAP11CaPm interacts with MdTCP16, a class II CYC/TB1 transcription factor that is highly expressed during phytoplasma infection.

PLOS ONE

Dear Dr. Janik,

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.

Please submit your revised manuscript by Dec 09 2022 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: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Keith R. Davis

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments:

Although you did a nice job of addressing many of the reviewers' concerns, Reviewer 2 still has some legitimate questions about the data analysis presented in Figure 3. I agree that combining data from treatments as currently presented is questionable and needs to be addressed. Once this is adequately done, the manuscript can be recommended for publication.

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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: (No Response)

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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: Partly

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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

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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

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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: Dear authors,

Thank you very much for the revised version. All my raised comments were adequately addressed.

Good luck for your future work.

Reviewer #2: The authors have clarified most of the issues I raised during my last review. I still have a few points about Figure 3 that need to be addressed:

Figure 3C: Since the samples plotted here come from different growing conditions (naturally vs graft infected and greenhouse vs foil tunnel) and, as shown in panels A and B, have different expression profiles, they shouldn't be analyzed as a single set of samples. The expression level of MdTCP16 in grafted plants is more than ten times lower than the transcription level in naturally infected trees. Similarly, the correlation analysis should be performed separately.

Figure 3D: Five grafted inoculated samples were plotted in figure 3B, but in figure 3D only three samples for spring and four for autumn were plotted. What is the rationale behind the exclusion of some of the samples?

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Reviewer #1: No

Reviewer #2: No

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The ‘Candidatus Phytoplasma mali’ effector protein SAP11CaPm interacts with MdTCP16, a class II CYC/TB1 transcription factor that is highly expressed during phytoplasma infection.

PONE-D-22-20188R2

Dear Dr. Janik,

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.

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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,

Keith R. Davis

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Thank you for your patience as we worked our way through the reviews. I hope you agree that the manuscript is improved and more useful for the research community. Best of luck in your future studies.

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 #2: All comments have been addressed

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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 #2: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

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 #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 #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 #2: The authors did a good job and addressed all my concerns.

Only one little comment below:

Lines 272-274: "Lines in graphs C, D, E and F show linear regression of the respective samples either from the greenhouse (dashed line) or from the foil tunnel (solid line)": In Figures 3C-E, the regression lines are all solid and dashed lines are missing

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Reviewer #2: No

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