Small RNA and Epigenetic Control of Plant Immunity

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This review highlights the roles of small RNAs in directing both transcriptional gene silencing and the post-transcriptional gene silencing, the importance of chromatin state in modulating immune gene accessibility, and the significance of DNA methylation dynamics in activating or repressing defense pathways.

Authors highlighted the plant primed state can be environmentally induced or inherited, offering an alternative to genetic modification  for engineering durable disease resistance in crops

The review is comprehensive, updated and well writte, The subject fits perfectly the scope of the journal. Therefore in my opinion it deserves to be published.

I have just two comments:

• Acronyms must be always accompained by an explanation when they are cited for the first time as the review is intended for a vast audience.
• Use italics for latin names of species throughout the text. 
• For other minor text editings see notes in the text (attached PDF file).

Comments for author File: Comments.pdf

Author Response

Reviewer 1

Comments and Suggestions for Authors

This review highlights the roles of small RNAs in directing both transcriptional gene silencing and the post-transcriptional gene silencing, the importance of chromatin state in modulating immune gene accessibility, and the significance of DNA methylation dynamics in activating or repressing defense pathways. Authors highlighted the plant primed state can be environmentally induced or inherited, offering an alternative to genetic modification for engineering durable disease resistance in crops. The review is comprehensive, updated and well written, The subject fits perfectly the scope of the journal. Therefore, in my opinion it deserves to be published.

Reply

We thank Reviewer 1 for the positive assessment and thoughtful comments on our manuscript. We are pleased to know that the topic aligns well with the journal’s scope and that the review was found to be comprehensive and well written. We have carefully addressed the points raised as follows:

I have just two comments:

• Acronyms must be always accompanied by an explanation when they are cited for the first time as the review is intended for a vast audience.

Reply: We have ensured that all acronyms and abbreviations are now defined at their first occurrence. Examples include “RNA-directed DNA methylation (RdDM),” “small interfering RNAs (siRNAs),” and “post-transcriptional gene silencing (PTGS).”

• Use italics for Latin names of species throughout the text. 

Reply: We have ensured that all acronyms and abbreviations are now clearly defined at their first occurrence throughout the manuscript. In addition to previously included terms, we have now introduced and defined acronyms such as “RNA-directed DNA methylation (RdDM),” “small interfering RNAs (siRNAs),” “post-transcriptional gene silencing (PTGS),” “clustered regularly interspaced short palindromic repeats/dead Cas9 (CRISPR/dCas9),” “long non-coding RNAs (lncRNAs),” “chromatin immunoprecipitation followed by sequencing (ChIP-seq),” and “assay for transposase-accessible chromatin using sequencing (ATAC-seq).” These changes were made to ensure clarity for a broad audience and improve the accessibility of the manuscript.

• For other minor text editing’s see notes in the text (attached PDF file).

Reply: We have reviewed the annotated PDF and incorporated all the suggested minor textual and formatting changes into the revised manuscript.

We sincerely thank Reviewer 1 for their encouraging remarks and valuable suggestions. We have carefully addressed all comments, including defining acronyms at first mention, italicizing Latin species names, and incorporating the minor textual edits noted in the annotated PDF. We believe these revisions have further improved the clarity and accessibility of our manuscript.

 

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

This is an excellent review of current knowledge of epigenetic mechanisms and it role in regulation of plant immunity which covers involvement of both DNA modifications (DNA methylation, histone modification, chromatin remodeling) and small RNA-mediated mechanisms (transcriptional and post-transcriptional gene silencing) in  plant immunity. This review is clearly structured, well-written, and cited 173 papers mostly published in the last decade. The figures are also appropriate and well presented. Therefore I am happy to recommend accepting this review in its current form. 

Minor comment:
Section 4 " Epigenetic memory and transgenerational immunity" should be left-aligned.

 

Author Response

comments and Suggestions for Authors

This is an excellent review of current knowledge of epigenetic mechanisms and it role in regulation of plant immunity which covers involvement of both DNA modifications (DNA methylation, histone modification, chromatin remodeling) and small RNA-mediated mechanisms (transcriptional and post-transcriptional gene silencing) in  plant immunity. This review is clearly structured, well-written, and cited 173 papers mostly published in the last decade. The figures are also appropriate and well presented. Therefore, I am happy to recommend accepting this review in its current form. 

We sincerely thank Reviewer 2 for their positive and encouraging feedback. We are pleased that the review was found to be comprehensive, well-structured, and up to date with relevant literature.

Minor comment:
Section 4 " Epigenetic memory and transgenerational immunity" should be left-aligned.

Response:
We appreciate the suggestion and have now corrected the alignment of Section 4 to ensure it is left-aligned in accordance with the formatting style of the rest of the manuscript.

Thank you again for your thoughtful review and recommendation to accept the manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript demonstrates a concise and well-organized review titled “Small RNA and Epigenetic Control of Plant Immunity” of the latest achievements about the role of small RNA and epigenetic regulation in plant immunity regulation. It also adds to the current discoveries in an area of small RNA pathways, DNA methylation, histone modifications, and chromatin remodelling in terms of plants-pathogen interactions and stress priming. However, some minor problems pertaining to clarity, formatting, consistency and citation are supposed to be done to enhance the overall quality and readability of the manuscript.

• Ensure in-text citations are properly formatted and match the reference list.
• Uniformity in author citations: some utilize "Zhang 2022" format, while others use numbered references.
• Some references may lack full details (e.g., [42], [45], [64]-[82]).
• Check that the full reference list is complete and formatted according to journal style.
• Table 1 may benefit from better alignment and consistent column spacing.
• Add a clear caption or footnote to explain acronyms and words, such as "tRF", phasiRNA", and "PR1".
• Include high-resolution figures (e.g., Figures 1 and 2) and cite them in the text as needed.
• Consider separating the legends from the main text to improve clarity.
• The review is well-organized, but certain sections may benefit from more seamless transitions. To better relate PTGS with chromatin crosstalk, consider including a bridging sentence to 3.4 at the end of Section 3.3.
• To emphasize the take-home message, include a summary of important obstacles and future directions in the conclusion.
• Line 287: "Pre-exposure of plants to biotic stress…" - explain the sort of stress or disease, for example.
• Italicize the genus and species names consistently on line 575, " syringae and Hyaloperonospora arabidopsidis".
• On line 662, explain the acronyms "dCas9-TET or dCas9-HAT fusions" or refer to prior sections where they are introduced.

Comments for author File: Comments.odt

Comments on the Quality of English Language

The document has a lot of grammatical mistakes and a few typos. A comprehensive language check is suggested.

• In line 108, "fi-ne-tunes" should be spelled "fine-tunes."
• Consider rephrasing "Marks H3K4me3 and H3K9ac…" to "Histone marks such as H3K4me3 and H3K9ac."
• Line 266 is as follows: "participates during plant immunity in chromatin remodeling" Consider rephrasing as "participates in chromatin remodeling during plant immunity."
• Some formatting errors found such as unnecessary remove dots (e.g., line 67 "This immune memory is mediated by stable changes…" and line 112 "infection..").
• Inconsistent spacing after citations, such as "[64]Enhances…" vs. "[64] Enhances…".
• Improve labeling and referencing of figures and tables in the text (e.g., "Figure 1," "Table 1").

Author Response

Reviewer 3

 

The manuscript demonstrates a concise and well-organized review titled “Small RNA and Epigenetic Control of Plant Immunity” of the latest achievements about the role of small RNA and epigenetic regulation in plant immunity regulation. It also adds to the current discoveries in an area of small RNA pathways, DNA methylation, histone modifications, and chromatin remodelling in terms of plants-pathogen interactions and stress priming. However, some minor problems pertaining to clarity, formatting, consistency and citation are supposed to be done to enhance the overall quality and readability of the manuscript.

Response:
We thank Reviewer 3 for the encouraging feedback and appreciation of the manuscript’s structure and content. We have thoroughly revised the manuscript to address all minor issues related to clarity, formatting, citation consistency, and readability. Specific improvements include grammar corrections, uniform citation style, clarification of technical terms, alignment of tables, and enhancement of figure quality and placement. We believe these revisions have strengthened the manuscript further, and we appreciate the reviewer’s suggestions that guided these refinements.

 

• The document has a lot of grammatical mistakes and a few typos. A comprehensive language check is suggested.

 

Response: A thorough language and grammar check has been performed across the manuscript to correct typos, improve sentence flow, and ensure clarity.

• In line 108, "fi-ne-tunes" should be spelled "fine-tunes."

Response: Corrected “fi-ne-tunes” to “fine-tunes” as suggested.

• Consider rephrasing "Marks H3K4me3 and H3K9ac…" to "Histone marks such as H3K4me3 and H3K9ac."
  Response: Revised to “Histone marks such as H3K4me3 and H3K9ac…” for improved clarity.

 

• Line 266 is as follows: "participates during plant immunity in chromatin remodeling" Consider rephrasing as "participates in chromatin remodeling during plant immunity."

 

Response: Rephrased to “participates in chromatin remodeling during plant immunity” as suggested.

• Some formatting errors found such as unnecessary remove dots (e.g., line 67 "This immune memory is mediated by stable changes…" and line 112 "infection..").

Response: Removed unnecessary dots and corrected punctuation throughout the manuscript.

• Inconsistent spacing after citations, such as "[64]Enhances…" vs. "[64] Enhances…".

Response: Standardized citation formatting across the manuscript to ensure consistent spacing and alignment with journal guidelines.

• Improve labeling and referencing of figures and tables in the text (e.g., "Figure 1," "Table 1").

Response: All figures and tables are now consistently cited in the text (e.g., “Figure 1,” “Table 1”) and labeled appropriately as per journal style.

• Ensure in-text citations are properly formatted and match the reference list.

Response: All in-text citations have been cross-checked and updated to match the full reference list.

 

• Uniformity in author citations: some utilize "Zhang 2022" format, while others use numbered references.

Response: All citations have been converted to the journal’s numbered referencing format. Author-year formats (e.g., “Zhang 2022”) were replaced with proper numerical citations.

• Some references may lack full details (e.g., [42], [45], [64]-[82]).

Response: All references have been reviewed and updated with complete bibliographic information according to the MDPI citation style, including DOI links where available.

• Check that the full reference list is complete and formatted according to journal style.

Response: The entire reference list has been revised for consistency, completeness, and alignment with MDPI guidelines.

• Table 1 may benefit from better alignment and consistent column spacing.

Response: Table 1 has been adjusted to improve alignment and maintain consistent spacing, while still conforming to the journal's table template.

• Add a clear caption or footnote to explain acronyms and words, such as "tRF", phasiRNA", and "PR1".

Response: Clarifying footnotes have been added below Table 1 to define tRF (tRNA-derived fragment), phasiRNA (phased small interfering RNA), and PR1 (Pathogenesis-Related Protein 1).

• Include high-resolution figures (e.g., Figures 1 and 2) and cite them in the text as needed.

Response: High-resolution versions of Figures 1 and 2 have been provided and embedded in the revised manuscript.

• Consider separating the legends from the main text to improve clarity.

Response: Figure legends have been placed directly below each respective figure and separated from the body text.

• The review is well-organized, but certain sections may benefit from more seamless transitions. To better relate PTGS with chromatin crosstalk, consider including a bridging sentence to 3.4 at the end of Section 3.3.

Response: A bridging sentence has been added at the end of Section 3.3 to improve the transition to Section 3.4 and to better establish the connection between PTGS and chromatin dynamics.

 

• To emphasize the take-home message, include a summary of important obstacles and future directions in the conclusion.

Response: The conclusion section has been expanded to include key challenges and opportunities, such as the need for field validation, advances in epigenome editing, and transgenerational studies for durable crop immunity.

• Line 287: "Pre-exposure of plants to biotic stress…" - explain the sort of stress or disease, for example.

Response:
We thank the reviewer for this helpful suggestion. We have revised the sentence to provide a specific example of biotic stress. The updated text now reads:
“For instance, pre-exposure of Arabidopsis thaliana to the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 has been shown to induce amplified resistance in the progeny, mediated through heritable epigenetic modifications such as histone acetylation and DNA hypomethylation at defense-related loci [51].

• Italicize the genus and species names consistently on line 575, "P. syringae and Hyaloperonospora arabidopsidis".

Response: All genus and species names have been consistently italicized throughout the text.

• On line 662, explain the acronyms "dCas9-TET or dCas9-HAT fusions" or refer to prior sections where they are introduced.

Response: A clarification has been added: “TET (ten-eleven translocation methylcytosine dioxygenase) and HAT (histone acetyltransferase) are fused to dCas9 for locus-specific DNA demethylation or histone acetylation, respectively.” A reference to prior sections where dCas9-based editing was introduced has also been added.

 

Recommendation:

Minor Revision
The manuscript is scientifically sound and well-documented. With minor revisions for language, consistency, formatting, and citation clarity, it will be suitable for publication.

 

Response:
We sincerely thank the reviewer for the positive evaluation and constructive feedback. We have carefully addressed all the minor revision points, including corrections to grammar and typographical errors, standardization of citation formats, clarification of scientific terms and acronyms, alignment of table formatting, enhancement of figure quality and referencing, and improvements in transition and flow between sections. We believe these changes have significantly improved the clarity and overall presentation of the manuscript. A detailed point-by-point response to each comment is provided above. We hope the revised version now meets the expectations for publication.