Advances in Plant Antiviral RNAi: From Host DCLs/RDRs to Diversified Viral Counteracting Strategies

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This review paper considers several aspects related to phenomenon of RNA silencing as a fundamental antiviral defense in plants. First, it describes modern data on the structure, evolution and activities of DICER-like endonucleases (DCLs), Argonaute proteins (AGOs) and RNA-dependent RNA polymerases (RDRs). Second, it summarizes the modern data on positive and negative modulators of silencing by antiviral RNAi and includes the information on newly identified mechanisms of viral RNA silencing suppressors and endogenous RNA silencing suppressors. 

Generally the review is well-written and provides very useful information for readers. However, to improve the manuscript and its impact for readers I strongly suggest to add special table (Table 3) summarizing activities of well-known and novel silencing suppressors in the form presented in Table 2.   

Author Response

We would like to sincerely thank the reviewers for their constructive and insightful comments. We have revised the manuscript accordingly, and our point-by-point responses are provided below. All revisions have been highlighted in the revised manuscript.

This review paper considers several aspects related to phenomenon of RNA silencing as a fundamental antiviral defense in plants. First, it describes modern data on the structure, evolution and activities of DICER-like endonucleases (DCLs), Argonaute proteins (AGOs) and RNA-dependent RNA polymerases (RDRs). Second, it summarizes the modern data on positive and negative modulators of silencing by antiviral RNAi and includes the information on newly identified mechanisms of viral RNA silencing suppressors and endogenous RNA silencing suppressors.

Generally the review is well-written and provides very useful information for readers. However, to improve the manuscript and its impact for readers I strongly suggest to add special table (Table 3) summarizing activities of well-known and novel silencing suppressors in the form presented in Table 2.

Our response: We thank the reviewer for the positive evaluation of our manuscript and for this constructive suggestion. We agree that a concise summary table would greatly enhance clarity and accessibility for readers, particularly given the rapidly expanding diversity of viral RNA silencing suppressors and their mechanisms of action. Accordingly, we have added a new table (Table 3) that systematically summarizes both classical and recently identified viral RNA silencing suppressors. This table is explicitly referenced in Section 3.1 to provide an overview of the diverse mechanisms by which viral suppressors interfere with antiviral RNA interference.

Reviewer 2 Report

Comments and Suggestions for Authors

Li et al. present a comprehensive and well-structured review that provides a timely synthesis of current knowledge on plant antiviral RNA interference mechanisms and viral counter-defenses.

The manuscript is clearly written, and logically organized.

My primary suggestion for improvement concerns the abstract, which states that the authors "outline translational opportunities for improving virus resistance in crops through breeding, biotechnological approaches, and RNA-based applications". However, this aspect is not really developed in the current version. Aligned with the abstract and to strengthen the manuscript relevance by bridging fundamental mechanisms with real-world applications, I recommend adding a brief section (such as "4. Translational opportunities") that highlights how advances in antiviral RNAi research can be leveraged for crop protection and biotechnology:

- Breeding, for instance moving here the content of lines 262-271, discussing the Ty-1 and Ty-3 resistance alleles, to illustrate how natural variation in RNAi components can be exploited in breeding programs.

- Biotechnologies, including VIGS and recent advances such as viral delivery of short RNAs (24–32 nt) for functional genomics and targeted RNAi in crops (Plant Biotechnology Journal, 2025, doi:10.1111/pbi.70254).

- RNA-based applications, such as emerging strategies like topical application of dsRNA for antiviral control (Nature Plants, 2017, doi:10.1038/nplants.2016.207).

In addition, please:

- L55: revise to read as "against DNA viruses such as those of the Geminiviridae", since another DNA virus (CaMV, family Caulimoviridae) is also discussed in the text.

- L123: Correct the typo "desmontartes"

- Ensure consistent formatting of abbreviations and nomenclature, for instance, "Triticum mosaic virus" should be abbreviated as TriMV (not TMV, L365). The calmodulin-like protein currently appearing as "RgsCaM", "rgsCaM", and "rgs-CaM".

Author Response

We would like to sincerely thank the reviewers for their constructive and insightful comments. We have revised the manuscript accordingly, and our point-by-point responses are provided below. All revisions have been highlighted in the revised manuscript.

Li et al. present a comprehensive and well-structured review that provides a timely synthesis of current knowledge on plant antiviral RNA interference mechanisms and viral counter-defenses.

The manuscript is clearly written, and logically organized.

My primary suggestion for improvement concerns the abstract, which states that the authors "outline translational opportunities for improving virus resistance in crops through breeding, biotechnological approaches, and RNA-based applications". However, this aspect is not really developed in the current version. Aligned with the abstract and to strengthen the manuscript relevance by bridging fundamental mechanisms with real-world applications, I recommend adding a brief section (such as "4. Translational opportunities") that highlights how advances in antiviral RNAi research can be leveraged for crop protection and biotechnology:

- Breeding, for instance moving here the content of lines 262-271, discussing the Ty-1 and Ty-3 resistance alleles, to illustrate how natural variation in RNAi components can be exploited in breeding programs.

- Biotechnologies, including VIGS and recent advances such as viral delivery of short RNAs (24–32 nt) for functional genomics and targeted RNAi in crops (Plant Biotechnology Journal, 2025, doi:10.1111/pbi.70254).

- RNA-based applications, such as emerging strategies like topical application of dsRNA for antiviral control (Nature Plants, 2017, doi:10.1038/nplants.2016.207).

Our response: We thank the reviewer for this constructive and insightful suggestion. In response, we have added a new dedicated section entitled “4. Translational opportunities for antiviral RNAi in crop protection” to explicitly bridge fundamental antiviral RNAi mechanisms with real-world applications.

In addition, please:

- L55: revise to read as "against DNA viruses such as those of the Geminiviridae", since another DNA virus (CaMV, family Caulimoviridae) is also discussed in the text.

Our response: We thank the reviewer for this clarification. The sentence has been revised as suggested to avoid ambiguity.

- L123: Correct the typo "desmontartes"

Our response: We apologize for this typographical error, which has now been corrected.

- Ensure consistent formatting of abbreviations and nomenclature, for instance, "Triticum mosaic virus" should be abbreviated as TriMV (not TMV, L365). The calmodulin-like protein currently appearing as "RgsCaM", "rgsCaM", and "rgs-CaM".

Our response: We appreciate the reviewer’s careful attention to nomenclature consistency. All abbreviations and gene/protein names have now been standardized throughout the manuscript. Specifically, Triticum mosaic virus is consistently abbreviated as TriMV. We note that different studies have adopted slightly different nomenclature for this protein (e.g., RgsCaM, rgsCaM, or rgs-CaM). To avoid confusion and to improve clarity and consistency throughout the manuscript, we have standardized the nomenclature to rgsCaM in all instances, regardless of the original formatting used in individual references.