New Insights into the Regulatory Non-Coding RNAs Mediating Rice–Brown Planthopper Interactions

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Strengths:

1. Relevance of the Topic:
   The manuscript addresses a highly relevant topic in agriculture, particularly for rice production, which is a staple crop for global food security. The interaction between rice and the pest Nilaparvata lugens (BPH) is a significant issue, and the exploration of the role of non-coding RNAs (ncRNAs) in this interaction is both timely and innovative.

2. Multidisciplinary Approach:
   The work integrates knowledge from genomics, molecular biology, and entomology, offering a comprehensive view of the molecular mechanisms involved in rice resistance to BPH and the pest's adaptation to plant defenses. The detailed review of the roles of miRNAs, lncRNAs, and circRNAs in both species is a strong point, highlighting the complexity of plant-insect interactions.

3. Solid Experimental Foundation:
   The manuscript provides an extensive review of recent studies, including high-throughput sequencing data and genetic experiments, which validate the functions of ncRNAs in rice resistance and BPH adaptation. The inclusion of studies demonstrating cross-kingdom RNAi regulation is particularly interesting and suggests new approaches for pest control.

4. Potential Applications:
   The work not only advances scientific knowledge but also suggests practical applications, such as the development of BPH-resistant rice varieties and RNAi-based pest control strategies. This is especially relevant given the need to reduce chemical pesticide use and promote more sustainable agricultural practices.

5. Organization and Clarity:
   The manuscript is well-organized, with a clear introduction, a detailed review of the roles of ncRNAs in rice-BPH interactions, and a conclusion that effectively synthesizes the main findings and future perspectives. The language is technical but accessible to the specialized target audience.

Weaknesses and Suggestions for Improvement:

1. Depth in Mechanism Discussion:
   While the manuscript covers various molecular mechanisms, some points could be explored in greater depth. For example, the discussion on how ncRNAs regulate specific defense pathways in rice (such as the jasmonic acid pathway) could be more detailed. Additionally, the interaction between ncRNAs and resistance (R) genes in rice could be further explored.

Some other sugestions to expand the discussion:

A. Expanding the Discussion on ncRNA Regulation of Defense Pathways:

Example: Jasmonic Acid (JA) Pathway

• Current Content: The manuscript briefly mentions that ncRNAs, such as miR156, regulate JA biosynthesis and its role in BPH resistance. However, the discussion is somewhat superficial.

• Suggested Expansion:

  • Mechanistic Details: Provide a more detailed explanation of how miR156 and other ncRNAs (e.g., miR319, miR396) interact with key components of the JA pathway, such as LOX (lipoxygenase), AOS (allene oxide synthase), and OPR (12-oxophytodienoate reductase). For instance, how do these ncRNAs modulate the expression of these genes, and what are the downstream effects on JA levels and defense responses?

  • Cross-Talk with Other Hormones: Discuss how ncRNAs might mediate cross-talk between the JA pathway and other hormonal pathways, such as salicylic acid (SA) or ethylene, which are also critical for plant defense. For example, does miR156 indirectly affect SA signaling through its regulation of JA biosynthesis?

  • Experimental Evidence: Include examples from recent studies that have experimentally validated the role of specific ncRNAs in the JA pathway. For instance, studies showing that overexpression or silencing of miR156 alters JA levels and BPH resistance could be highlighted.

Example: Flavonoid Biosynthesis Pathway

• Current Content: The manuscript mentions that miR396 regulates flavonoid biosynthesis through the "miR396-OsGRF8-OsF3H-flavonoid" module but does not delve deeply into the mechanistic details.

• Suggested Expansion:

  • Regulatory Network: Provide a more detailed description of how miR396 and its target gene OsGRF8 influence the expression of OsF3H (flavanone 3-hydroxylase), a key enzyme in flavonoid biosynthesis. How do changes in flavonoid levels affect BPH feeding behavior or survival?

  • Downstream Effects: Discuss the role of flavonoids in plant defense, such as their antioxidant properties, their role in deterring herbivores, or their impact on BPH reproduction. Are there specific flavonoids that are more effective against BPH, and how are they regulated by ncRNAs?

  • Comparative Analysis: Compare the roles of different ncRNAs (e.g., miR396, miR159) in regulating flavonoid biosynthesis and their relative contributions to BPH resistance.

B. Exploring the Interaction Between ncRNAs and Resistance (R) Genes:

Example: ncRNA Regulation of R Genes

• Current Content: The manuscript mentions that ncRNAs target NBS-LRR (nucleotide-binding site leucine-rich repeat) genes, which are a major class of R genes, but does not provide much detail on how this regulation occurs.

• Suggested Expansion:

  • Specific Examples: Highlight specific ncRNAs that regulate well-known R genes in rice, such as BPH6, BPH9, or BPH14. For instance, does miR160 or miR167 directly target these R genes, and how does this affect their expression and function?

  • Mechanism of Action: Discuss the molecular mechanisms by which ncRNAs regulate R genes. For example, do lncRNAs act as competing endogenous RNAs (ceRNAs) to sequester miRNAs that target R genes, thereby enhancing R gene expression?

  • Functional Consequences: Explore the functional consequences of ncRNA-mediated regulation of R genes. For example, how does the upregulation or downregulation of specific R genes by ncRNAs affect rice's ability to recognize and respond to BPH infestations?

Example: ncRNAs and Effector-Triggered Immunity (ETI)

• Current Content: The manuscript does not explicitly discuss the role of ncRNAs in effector-triggered immunity (ETI), a key component of plant defense against pests.

• Suggested Expansion:

  • ETI Pathways: Explain how ncRNAs might modulate ETI pathways, which involve the recognition of BPH effectors by R proteins. For example, do lncRNAs or circRNAs regulate the expression of R proteins or downstream signaling components, such as MAPKs (mitogen-activated protein kinases) or WRKY transcription factors?

  • Case Studies: Include examples from recent studies where ncRNAs have been shown to influence ETI. For instance, are there specific miRNAs that target WRKY70, a transcription factor involved in both JA and SA signaling, and how does this affect rice's defense against BPH?

C. Cross-Kingdom RNAi and Its Mechanistic Implications:

Example: Rice-Derived miRNAs Targeting BPH Genes

• Current Content: The manuscript briefly mentions that rice-derived miRNAs, such as miR162a and miR5795, can target BPH genes, but the discussion is limited.

• Suggested Expansion:

  • Mechanism of Uptake: Provide more details on how rice-derived miRNAs are taken up by BPH during feeding. Are these miRNAs packaged into extracellular vesicles, and how do they survive the insect's digestive system?

  • Gene Silencing in BPH: Discuss the molecular mechanisms by which rice-derived miRNAs silence BPH genes. For example, how does miR162a target the NITOR (Target of Rapamycin) gene in BPH, and what are the downstream effects on BPH reproduction and survival?

  • Experimental Validation: Include examples of experimental studies that have validated the cross-kingdom RNAi mechanism, such as transgenic rice plants expressing modified miRNAs (e.g., miR162a-m1) and their effects on BPH populations.

Example: BPH-Derived miRNAs Targeting Rice Genes

• Current Content: The manuscript mentions that BPH-derived miRNAs, such as miR-7-5P, can suppress rice immunity, but the discussion is brief.

• Suggested Expansion:

  • Mechanism of Action: Provide a more detailed explanation of how BPH-derived miRNAs suppress rice immunity. For example, how does miR-7-5P target OsbZIP43, a bZIP transcription factor involved in defense signaling, and what are the downstream effects on rice's ability to resist BPH infestations?

  • Systemic Effects: Discuss whether BPH-derived miRNAs have systemic effects in rice, spreading beyond the feeding site to suppress immunity in other parts of the plant.

  • Host-Pathogen Coevolution: Explore the evolutionary implications of cross-kingdom RNAi. For example, how might the coevolution of rice and BPH have shaped the development of these miRNA-mediated interactions?

D. Integration of Multi-Omics Data:

Example: Transcriptomic and Proteomic Data

• Current Content: The manuscript mentions the importance of integrating multi-omics data but does not provide specific examples.

• Suggested Expansion:

  • Case Studies: Include examples of studies that have integrated transcriptomic and proteomic data to identify ncRNA-mRNA-protein networks involved in rice-BPH interactions. For instance, how do changes in miRNA expression correlate with changes in protein levels of their target genes?

  • Network Analysis: Discuss how network analysis tools, such as weighted gene co-expression network analysis (WGCNA), can be used to identify key ncRNA-mRNA interactions in rice defense pathways.

2. Integration of Multi-Omics Data:
The manuscript mentions the importance of integrating multi-omics data (transcriptomics, proteomics, and metabolomics) but does not deeply explore how this integration can be practically achieved. The authors should include a more detailed discussion on how combining these approaches could lead to a more holistic understanding of rice-BPH interactions.

3. Future Perspectives:
The conclusions and future perspectives section could be expanded to include a more detailed discussion on the practical implications of the findings. For example, how ncRNAs can be used in the development of transgenic rice varieties or in the field application of RNAi. Additionally, it would be interesting to discuss the technical and regulatory challenges associated with these applications.

4. Tables and Figures:
The manuscript lacks tables and figures that could visually summarize the main findings. Including a table summarizing the key ncRNAs involved in rice-BPH interactions, their targets, and functions would be very helpful for readers. Additionally, a schematic diagram showing the regulatory pathways mediated by ncRNAs in both species could facilitate understanding of the discussed mechanisms.

Conclusion:

The manuscript "New Insights Into How Non-coding RNAs Mediate Rice–Brown Planthopper Interactions" is a comprehensive and well-written review that addresses a highly relevant topic for the scientific community and agriculture. The review of the roles of ncRNAs in rice-BPH interactions is current and well-supported, with the potential to significantly contribute to the field.

However, the lack of original data and the need for a deeper discussion of some molecular mechanisms and practical applications are areas that could be improved. With the suggested revisions, particularly the inclusion of visual data (tables and figures) and an expanded discussion on practical implications and future research directions, the manuscript has the potential to be published in a high-impact scientific journal, such as Agronomy or Plant Biotechnology Journal.

Recommendation:
The manuscript can be accepted for publication after minor revisions, particularly regarding the inclusion of visual data (tables and figures) and the expansion of the discussion on practical implications and future research directions.

Author Response

Response to Reviewer #1

 

Comment 1：

Strengths:

1. Relevance of the Topic:

The manuscript addresses a highly relevant topic in agriculture, particularly for rice production, which is a staple crop for global food security. The interaction between rice and the pest Nilaparvata lugens (BPH) is a significant issue, and the exploration of the role of non-coding RNAs (ncRNAs) in this interaction is both timely and innovative.

2. Multidisciplinary Approach:

The work integrates knowledge from genomics, molecular biology, and entomology, offering a comprehensive view of the molecular mechanisms involved in rice resistance to BPH and the pest’s adaptation to plant defenses. The detailed review of the roles of miRNAs, lncRNAs, and circRNAs in both species is a strong point, highlighting the complexity of plant-insect interactions.

3. Solid Experimental Foundation:

The manuscript provides an extensive review of recent studies, including high-throughput sequencing data and genetic experiments, which validate the functions of ncRNAs in rice resistance and BPH adaptation. The inclusion of studies demonstrating cross-kingdom RNAi regulation is particularly interesting and suggests new approaches for pest control.

4. Potential Applications:

The work not only advances scientific knowledge but also suggests practical applications, such as the development of BPH-resistant rice varieties and RNAi-based pest control strategies. This is especially relevant given the need to reduce chemical pesticide use and promote more sustainable agricultural practices.

5. Organization and Clarity:

The manuscript is well-organized, with a clear introduction, a detailed review of the roles of ncRNAs in rice-BPH interactions, and a conclusion that effectively synthesizes the main findings and future perspectives. The language is technical but accessible to the specialized target audience.

Response: Thank you very much for such favorable and encouraging comments on the value of our work.

 

Weaknesses and Suggestions for Improvement:

1. Depth in Mechanism Discussion:

While the manuscript covers various molecular mechanisms, some points could be explored in greater depth. For example, the discussion on how ncRNAs regulate specific defense pathways in rice (such as the jasmonic acid pathway) could be more detailed. Additionally, the interaction between ncRNAs and resistance (R) genes in rice could be further explored.

Some other sugestions to expand the discussion:

 

Comment 2：

1. Expanding the Discussion on ncRNA Regulation of Defense Pathways:

Example: Jasmonic Acid (JA) Pathway

Current Content: The manuscript briefly mentions that ncRNAs, such as miR156, regulate JA biosynthesis and its role in BPH resistance. However, the discussion is somewhat superficial.

Suggested Expansion:

Mechanistic Details: Provide a more detailed explanation of how miR156 and other ncRNAs (e.g., miR319, miR396) interact with key components of the JA pathway, such as LOX (lipoxygenase), AOS (allene oxide synthase), and OPR (12-oxophytodienoate reductase). For instance, how do these ncRNAs modulate the expression of these genes, and what are the downstream effects on JA levels and defense responses?

Cross-Talk with Other Hormones: Discuss how ncRNAs might mediate cross-talk between the JA pathway and other hormonal pathways, such as salicylic acid (SA) or ethylene, which are also critical for plant defense. For example, does miR156 indirectly affect SA signaling through its regulation of JA biosynthesis?

Experimental Evidence: Include examples from recent studies that have experimentally validated the role of specific ncRNAs in the JA pathway. For instance, studies showing that overexpression or silencing of miR156 alters JA levels and BPH resistance could be highlighted.

Example: Flavonoid Biosynthesis Pathway

Current Content: The manuscript mentions that miR396 regulates flavonoid biosynthesis through the "miR396-OsGRF8-OsF3H-flavonoid" module but does not delve deeply into the mechanistic details.

Suggested Expansion:

Regulatory Network: Provide a more detailed description of how miR396 and its target gene OsGRF8 influence the expression of OsF3H (flavanone 3-hydroxylase), a key enzyme in flavonoid biosynthesis. How do changes in flavonoid levels affect BPH feeding behavior or survival?

Downstream Effects: Discuss the role of flavonoids in plant defense, such as their antioxidant properties, their role in deterring herbivores, or their impact on BPH reproduction. Are there specific flavonoids that are more effective against BPH, and how are they regulated by ncRNAs?

Comparative Analysis: Compare the roles of different ncRNAs (e.g., miR396, miR159) in regulating flavonoid biosynthesis and their relative contributions to BPH resistance.

Response: Thank you for your helpful suggestions. In response, we have expanded the discussion to include a more detailed exploration of how miR156 regulates JA biosynthesis and then regulates the rice resistance to BPH (see highlighted section in red, lines 126-130). Please refer to the highlighted sections in the revised manuscript for further details.

In addition, we have also expanded the discussion to include a more detailed exploration of how miR396 and its target gene OsGRF8 influence the expression of OsF3H (flavanone 3-hydroxylase) and how do changes in flavonoid levels affect BPH feeding behavior or survival (see highlighted section in red, lines 137-140). Please refer to the highlighted sections in the revised manuscript for further details.

 

Comment 3：

1. Exploring the Interaction Between ncRNAs and Resistance (R) Genes:

Example: ncRNA Regulation of R Genes

Current Content: The manuscript mentions that ncRNAs target NBS-LRR (nucleotide-binding site leucine-rich repeat) genes, which are a major class of R genes, but does not provide much detail on how this regulation occurs.

Suggested Expansion:

Specific Examples: Highlight specific ncRNAs that regulate well-known R genes in rice, such as BPH6, BPH9, or BPH14. For instance, does miR160 or miR167 directly target these R genes, and how does this affect their expression and function?

Mechanism of Action: Discuss the molecular mechanisms by which ncRNAs regulate R genes. For example, do lncRNAs act as competing endogenous RNAs (ceRNAs) to sequester miRNAs that target R genes, thereby enhancing R gene expression?

Functional Consequences: Explore the functional consequences of ncRNA-mediated regulation of R genes. For example, how does the upregulation or downregulation of specific R genes by ncRNAs affect rice's ability to recognize and respond to BPH infestations?

Example: ncRNAs and Effector-Triggered Immunity (ETI)

Current Content: The manuscript does not explicitly discuss the role of ncRNAs in effector-triggered immunity (ETI), a key component of plant defense against pests.

Suggested Expansion:

ETI Pathways: Explain how ncRNAs might modulate ETI pathways, which involve the recognition of BPH effectors by R proteins. For example, do lncRNAs or circRNAs regulate the expression of R proteins or downstream signaling components, such as MAPKs (mitogen-activated protein kinases) or WRKY transcription factors?

Case Studies: Include examples from recent studies where ncRNAs have been shown to influence ETI. For instance, are there specific miRNAs that target WRKY70, a transcription factor involved in both JA and SA signaling, and how does this affect rice's defense against BPH?

Response: Thank you for your helpful suggestion. In response, we have expanded the discussion to include a more detailed exploration of how miRNA regulates R genes expression to regulate the resistance of rice to brown planthopper (see highlighted section in red, lines 150-155).

In addition to the revisions made in response to your earlier recommendations in section A regarding the discussion on hormones and secondary metabolism, we have further expanded the discussion to include R genes, all of which fall under the ETI category (see highlighted sections in red, lines 126-130, 137-140, and 150-155). This section serves as a more detailed exploration of how miRNAs modulate ETI pathways. Please refer to the highlighted sections in the revised manuscript for further details.

 

Comment 4：

1. Cross-Kingdom RNAi and Its Mechanistic Implications:

Example: Rice-Derived miRNAs Targeting BPH Genes

Current Content: The manuscript briefly mentions that rice-derived miRNAs, such as miR162a and miR5795, can target BPH genes, but the discussion is limited.

Suggested Expansion:

Mechanism of Uptake: Provide more details on how rice-derived miRNAs are taken up by BPH during feeding. Are these miRNAs packaged into extracellular vesicles, and how do they survive the insect's digestive system?

Gene Silencing in BPH: Discuss the molecular mechanisms by which rice-derived miRNAs silence BPH genes. For example, how does miR162a target the NITOR (Target of Rapamycin) gene in BPH, and what are the downstream effects on BPH reproduction and survival?

Experimental Validation: Include examples of experimental studies that have validated the cross-kingdom RNAi mechanism, such as transgenic rice plants expressing modified miRNAs (e.g., miR162a-m1) and their effects on BPH populations.

Example: BPH-Derived miRNAs Targeting Rice Genes

Current Content: The manuscript mentions that BPH-derived miRNAs, such as miR-7-5P, can suppress rice immunity, but the discussion is brief.

Suggested Expansion:

Mechanism of Action: Provide a more detailed explanation of how BPH-derived miRNAs suppress rice immunity. For example, how does miR-7-5P target OsbZIP43, a bZIP transcription factor involved in defense signaling, and what are the downstream effects on rice's ability to resist BPH infestations?

Systemic Effects: Discuss whether BPH-derived miRNAs have systemic effects in rice, spreading beyond the feeding site to suppress immunity in other parts of the plant.

Host-Pathogen Coevolution: Explore the evolutionary implications of cross-kingdom RNAi. For example, how might the coevolution of rice and BPH have shaped the development of these miRNA-mediated interactions?

Response: Thank you for your valuable suggestions. In response, we have expanded the discussion on rice-derived miRNAs, such as miR162a (including miR162a-m1), and BPH-derived miRNAs, such as miR-7-5P, to provide more detailed insights. Specifically, we have provided examples of how ncRNAs could be applied in developing transgenic rice varieties or RNAi-based field applications, clarifying their practical relevance (see highlighted sections in red, lines 288-294 and 315-319). This expansion aims to provide a deeper understanding for readers, as well as to address the mechanisms of cross-kingdom RNAi and the coevolution of rice and BPH. Please refer to the highlighted sections for further details.

 

Comment 5：

1. Integration of Multi-Omics Data:

Example: Transcriptomic and Proteomic Data

Current Content: The manuscript mentions the importance of integrating multi-omics data but does not provide specific examples.

Suggested Expansion:

Case Studies: Include examples of studies that have integrated transcriptomic and proteomic data to identify ncRNA-mRNA-protein networks involved in rice-BPH interactions. For instance, how do changes in miRNA expression correlate with changes in protein levels of their target genes?

Network Analysis: Discuss how network analysis tools, such as weighted gene co-expression network analysis (WGCNA), can be used to identify key ncRNA-mRNA interactions in rice defense pathways.

Response: Thank you for your helpful suggestion. In response, we have added a new section to expand the discussion to include a more detailed exploration of how integrating multi-omics data (transcriptomics, proteomics, and metabolomics) can provide a more comprehensive understanding of rice-BPH interactions (see highlighted section in red, lines 324-340). Additionally, we have added a brief section on future research directions related to this integration (see highlighted section in red, lines 397-400). Please refer to the highlighted sections in the revised manuscript for further details.

 

Comment 6：

2. Integration of Multi-Omics Data:

The manuscript mentions the importance of integrating multi-omics data (transcriptomics, proteomics, and metabolomics) but does not deeply explore how this integration can be practically achieved. The authors should include a more detailed discussion on how combining these approaches could lead to a more holistic understanding of rice-BPH interactions.

Response: Thank you for your insightful comment. Similar to your previous suggestion, in response, we have further expanded the discussion on the practical integration of multi-omics data (transcriptomics, proteomics, and metabolomics) and how this approach can lead to a more holistic understanding of rice-BPH interactions. Specifically, we now explore how combining these data types can provide deeper insights into the molecular mechanisms underlying rice's defense responses to BPH by linking gene expression, protein function, and metabolic changes. Please refer to the updated sections in the manuscript (see highlighted section in red, lines 324-340, 397-400) for further details.

 

Comment 7：

3. Future Perspectives:

The conclusions and future perspectives section could be expanded to include a more detailed discussion on the practical implications of the findings. For example, how ncRNAs can be used in the development of transgenic rice varieties or in the field application of RNAi. Additionally, it would be interesting to discuss the technical and regulatory challenges associated with these applications.

Response: Thank you for your valuable suggestions. In response, we have added a section discussing the practical implications of our findings in more detail (see highlighted section in red, lines 341-356). Additionally, we have included a brief discussion on future research directions related to the practical implications, such as the potential use of new technologies like CRISPR/Cas to study and manipulate ncRNAs for improving resistance to rice pests (see highlighted section in red, lines 408-411). We also provided specific examples of how ncRNAs could be applied in the development of transgenic rice varieties or RNAi-based field applications, helping to clarify their practical relevance (highlighted sections in red, lines 288-294 and 315-319). Furthermore, we have addressed the regulatory challenges of technical and economic related to these applications (highlighted section in red, lines 357-385). Please refer to the highlighted sections in the revised manuscript for further details.

 

Comment 8：

4. Tables and Figures:

The manuscript lacks tables and figures that could visually summarize the main findings. Including a table summarizing the key ncRNAs involved in rice-BPH interactions, their targets, and functions would be very helpful for readers. Additionally, a schematic diagram showing the regulatory pathways mediated by ncRNAs in both species could facilitate understanding of the discussed mechanisms.

Response: Thank you for your helpful suggestion. In response, we have added more information of circRNAs mentioned in section 2.1.2 to Table 1. Additionally, we have included a new schematic diagram “Figure 1” that illustrates the regulatory pathways mediated by ncRNAs in both rice and BPH, which we believe will enhance the understanding of the discussed mechanisms. We also have moved Table 1 and the newly added Figure 1 to an earlier section of the article for better organization. Please refer to the revised manuscript for these changes (see highlighted section in red, lines 77-79, 413-433, Table 1, Figure 1).

 

Comment 9：

Conclusion:

The manuscript "New Insights Into How Non-coding RNAs Mediate Rice–Brown Planthopper Interactions" is a comprehensive and well-written review that addresses a highly relevant topic for the scientific community and agriculture. The review of the roles of ncRNAs in rice-BPH interactions is current and well-supported, with the potential to significantly contribute to the field.

Response: Thank you very much for such favorable and encouraging comments on the value of our work.

 

Comment 10：

However, the lack of original data and the need for a deeper discussion of some molecular mechanisms and practical applications are areas that could be improved. With the suggested revisions, particularly the inclusion of visual data (tables and figures) and an expanded discussion on practical implications and future research directions, the manuscript has the potential to be published in a high-impact scientific journal, such as Agronomy or Plant Biotechnology Journal.

Response: Thank you for your insightful comments. In response to your suggestions, we have addressed the areas that could be improved. Specifically, we have included additional visual data, such as newly modified table and newly added figure, to better illustrate the key findings. Additionally, we have expanded the discussion on molecular mechanisms, practical implications, and future research directions as you suggested before to provide a more comprehensive analysis. We believe these revisions significantly enhance the manuscript’s quality. Please refer to the revised manuscript for further details.

 

Comment 11：

Recommendation:

The manuscript can be accepted for publication after minor revisions, particularly regarding the inclusion of visual data (tables and figures) and the expansion of the discussion on practical implications and future research directions.

Response: Thank you very much for your positive and encouraging comments regarding the value of our work. We appreciate your recommendation to include visual data and expand the discussion on practical implications and future research directions. In response, we have added additional visual data, including a modified table and a newly added figure, to better illustrate the key findings. Additionally, we have expanded the discussion on practical implications and future research directions as you suggested before to further enhance the manuscript. Please refer to the revised manuscript for further details.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The authors of the present manuscript reviewed the studies on the important rice pest brown planthoppers mediated by non-coding RNAs in the past 20 years.

This is the first time to review such topic and the manuscript is comprehensively included almost every study on it. It is suggested to be considered for publication after minor English edition

The authors of the present manuscript reviewed the studies on the important rice pest brown planthoppers mediated by non-coding RNAs in the past 20 years.

This is the first time to review such topic and the manuscript is comprehensively included almost every study on it. It is suggested to be considered for publication after minor English edition.

Comments on the Quality of English Language

The manuscript is suggested to be minor English edited.

Author Response

Response to Reviewer #2

 

Comment 1：

The authors of the present manuscript reviewed the studies on the important rice pest brown planthoppers mediated by non-coding RNAs in the past 20 years.

 

This is the first time to review such topic and the manuscript is comprehensively included almost every study on it. It is suggested to be considered for publication after minor English edition

Response: Thank you very much for such favorable and encouraging comments on the value of our work. In response to your suggestion, we have requested a careful review of the manuscript by an experienced editor, whose first language is English and who specializes in editing papers. We believe this will help ensure the manuscript meets the required language standards for publication.

 

Comment 2：

The authors of the present manuscript reviewed the studies on the important rice pest brown planthoppers mediated by non-coding RNAs in the past 20 years.

 

This is the first time to review such topic and the manuscript is comprehensively included almost every study on it. It is suggested to be considered for publication after minor English edition.

Response: Thank you very much for such favorable and encouraging comments on the value of our work. In response to your suggestion, we have requested a careful review of the manuscript by an experienced editor, whose first language is English and who specializes in editing papers written by non-native English-speaking scientists. We believe this will help ensure the manuscript meets the required language standards for publication.

 

Comment 3：

Comments on the Quality of English Language: The manuscript is suggested to be minor English edited.

Response: Thank you for your suggestion regarding the quality of the English language. In response to your suggestion, we have requested a careful review of the manuscript by an experienced editor, whose first language is English and who specializes in editing papers. We believe this will help ensure the manuscript meets the required language standards for publication.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Lu et al. present a review summarizing recent discoveries on the role of non-coding RNAs in insect–plant interactions, specifically in the brown planthopper. Research on regulatory ncRNAs in insect–plant interactions is an emerging field with fundamental importance, making this review timely and relevant. However, the manuscript's organization and writing need significant improvement. Below are some specific suggestions for revision.

The title mentioned non-coding RNAs, however, this paper focuses more on the regulatory RNAs including microRNAs, lncRNAs, and circRNAs. The title should be modified to a more accurate one to describe the scope of the review. “New Insights Into How Non-coding RNAs Mediate Rice– Brown Planthopper Interactions” change to “New Insights into Regulatory Non-coding RNAs mediated Rice-Brown Planthopper Interactions”

The introduction section is too simplistic, and some descriptions of ncRNAs and their classification are inaccurate. Additional details on regulatory non-coding RNAs, including miRNAs, lncRNAs, and circRNAs, their functions, and a clearer classification are needed to improve the manuscript.

The first paragraph of the introduction should better frame the rice–brown planthopper interaction within the broader context of insect–plant interactions. This would help highlight its significance and potential mechanisms. Additionally, it would be beneficial to emphasize how regulatory ncRNA-mediated mechanisms emerge as a promising and important area of research in this field.

Page 2, lines 47-48, siRNAs are not regulatory ncRNAs, they play roles in silencing gene expression. While piRNAs (PIWI-interacting RNAs) are a class of small non-coding RNAs that play a crucial role in transposon silencing, genome stability, and germline development in animals, including insects and mites.

Section 2. Role of Non-coding RNAs in Rice–BPH Interactions

Change the first sentence “ncRNAs regulate the resistance of rice to BPHs and the adaptation of BPHs to host defenses.” To “In rice, ncRNAs play roles in regulating the resistance to BPHs. While in BPH, ncRNAs are involve in the adaptation of host plant defenses.” And move this sentence after “ncRNAs regulate key physiological processes involved in both plant defenses 61 and insect adaptation [24-26].”

Page 3, line 106 “Genetic evidence also indicates that miRNAs mediate BPH resistance.” Change to “Genetic evidence also indicates that miRNAs play roles in mediating BPH resistance.”

Page 4, Section 2.2 Roles of ncRNAs in BPH: add “adaption to rice”

Page 4, line 170, add “to rice” after “The roles of miRNAs in BPH adaptation”

Page 6, 2.3.1. Rice-Derived miRNAs Targeting BPH. These two paragraphs should merge to one paragraph.

Page 6, line 268. Change “BPH secretes salivary miRNAs into rice, which suppress plant immunity.” to “BPH secretes salivary miRNAs into rice, which serves as a salivary effector targeting multiple host genes and suppress plant immunity.”

Page 6, section 2.3.2. BPH-Derived miRNAs Targeting Rice. Some parts of these two paragraphs are overlapping; please reorganize them for better clarity and flow.

Page 6. Section 3. Conclusions and Perspectives

Change “ncRNAs regulate rice–BPH interactions.” to “In this review, we summarized the latest advances in ncRNA-mediated regulation of rice–BPH interactions.”

Conclusion (Section 3): This section should focus on summarizing the key points discussed earlier and highlighting future research directions, rather than repeating detailed information from previous sections. Please revise this section by removing redundant paragraphs.

Table 1. The lncRNAs and circRNAs mentioned in sections 2.1.2 and 2.2.2 are missing. Please add these ncRNAs in the table as well. Additionally, this table should be placed earlier in the article for better organization.

Comments on the Quality of English Language

The manuscript's organization and writing need significant improvement. I provided some specific suggestions for revision in my comments.

Author Response

Response to Reviewer #3

 

Comment 1：

Liang et al. present a review summarizing recent discoveries on the role of non-coding RNAs in insect–plant interactions, specifically in the brown planthopper. Research on regulatory ncRNAs in insect–plant interactions is an emerging field with fundamental importance, making this review timely and relevant. However, the manuscript’s organization and writing need significant improvement. Below are some specific suggestions for revision.

Response: In response to your suggestions, we have not only requested a thorough review by an experienced English editor, whose first language is English and who specializes in editing papers, but also made substantial revisions to improve the manuscript’s organization and overall writing. We have reorganized certain sections for better logical flow and clarity, and streamlined the presentation of our ideas to enhance readability. We believe these changes, along with the language improvements, will significantly strengthen the manuscript. Please refer to the revised manuscript for these changes highlighted in red.

 

Comment 2：

The title mentioned non-coding RNAs, however, this paper focuses more on the regulatory RNAs including microRNAs, lncRNAs, and circRNAs. The title should be modified to a more accurate one to describe the scope of the review. “New Insights Into How Non-coding RNAs Mediate Rice– Brown Planthopper Interactions” change to “New Insights into Regulatory Non-coding RNAs mediated Rice-Brown Planthopper Interactions”

Response: Thank you for your helpful suggestion. In response, we have modified the title to “New Insights into the Regulatory Non-coding RNAs mediating Rice–Brown Planthopper Interactions” (see highlighted section in red, lines 2-3).

 

Comment 3：

The introduction section is too simplistic, and some descriptions of ncRNAs and their classification are inaccurate. Additional details on regulatory non-coding RNAs, including miRNAs, lncRNAs, and circRNAs, their functions, and a clearer classification are needed to improve the manuscript.

Response: Thank you for your valuable suggestion. In accordance with your suggestion, we have expanded the introduction to include more detailed information on miRNAs, lncRNAs, and circRNAs, their functions, and a clearer classification (see highlighted section in red, lines 47 and 49-65). We believe these additions enhance the manuscript and provide a more comprehensive understanding of regulatory non-coding RNAs.

 

Comment 4：

The first paragraph of the introduction should better frame the rice–brown planthopper interaction within the broader context of insect–plant interactions. This would help highlight its significance and potential mechanisms. Additionally, it would be beneficial to emphasize how regulatory ncRNA-mediated mechanisms emerge as a promising and important area of research in this field.

Response: Thank you for your constructive suggestion. In response, we have revised the first paragraph of the introduction to better frame the rice–brown planthopper interaction within the broader context of insect–plant interactions to help highlight its significance and potential mechanisms (see highlighted section in red, lines 30-33). We have also emphasized how regulatory ncRNA-mediated mechanisms emerge as a promising and important area of research in this field (see highlighted section in red, lines 34-38). Additionally, we integrated the first and second paragraphs to improve cohesiveness and eliminate redundancy. We hope the revised introduction now more effectively outlines the complexity of BPH resistance and the potential of ncRNAs in advancing pest control strategies.

 

Comment 5：

Page 2, lines 47-48, siRNAs are not regulatory ncRNAs, they play roles in silencing gene expression. While piRNAs (PIWI-interacting RNAs) are a class of small non-coding RNAs that play a crucial role in transposon silencing, genome stability, and germline development in animals, including insects and mites.

Response: Thank you for your valuable feedback. We apologize for any confusion caused by our previous statement. We have clarified that siRNAs, which are classified as small interfering RNAs (siRNAs) in plants and endogenous siRNAs (endo-siRNAs) in insects, play important roles in gene silencing in the revised manuscript (see highlighted section in red, lines 54-60). We also appreciate your suggestion regarding piRNAs. We have included piRNAs in line 47, and further elaborated on their roles in transposon silencing, genome stability, and germline development in animals, including insects and mites the revised manuscript (see highlighted section in red, lines 51-54).

 

Section 2. Role of Non-coding RNAs in Rice–BPH Interactions

 

Comment 6：

Change the first sentence “ncRNAs regulate the resistance of rice to BPHs and the adaptation of BPHs to host defenses.” To “In rice, ncRNAs play roles in regulating the resistance to BPHs. While in BPH, ncRNAs are involve in the adaptation of host plant defenses.” And move this sentence after “ncRNAs regulate key physiological processes involved in both plant defenses and insect adaptation [24-26].”

Response: Thank you for your suggestion. Follow your suggestion, we have changed the first sentence to “In rice, ncRNAs play roles in regulating the resistance to BPHs. While in BPH, ncRNAs are involve in the adaptation of host plant defenses.” and move this sentence after “ncRNAs regulate key physiological processes involved in both plant defenses and insect adaptation [24-26].” Please refer to the revised manuscript for these changes (see highlighted section in red, lines 77-79).

 

Comment 7：

Page 3, line 106 “Genetic evidence also indicates that miRNAs mediate BPH resistance.” Change to “Genetic evidence also indicates that miRNAs play roles in mediating BPH resistance.”

Response: Thank you for your suggestion. Follow your suggestion, we have changed “Genetic evidence also indicates that miRNAs mediate BPH resistance.” to “Genetic evidence also indicates that miRNAs play roles in mediating BPH resistance.” Please refer to the revised manuscript for this change (see highlighted section in red, line 123).

 

Comment 8：

Page 4, Section 2.2 Roles of ncRNAs in BPH: add “adaption to rice”

Response: Thank you for your suggestion. Follow your suggestion, we have changed “Roles of ncRNAs in BPH” to “Roles of ncRNAs in the adaptation of BPHs to rice”. Please refer to the revised manuscript for these changes (see highlighted section in red, line 188).

 

Comment 9：

Page 4, line 170, add “to rice” after “The roles of miRNAs in BPH adaptation”

Response: Thank you for your suggestion. Follow your suggestion, we have added “to rice” after “The roles of miRNAs in BPH adaptation”. Please refer to the revised manuscript for these changes (see highlighted section in red, line 201).

 

Comment 10：

Page 6, 2.3.1. Rice-Derived miRNAs Targeting BPH. These two paragraphs should merge to one paragraph.

Response: Thank you for your suggestion. Follow your suggestion, we have merged these two paragraphs to one paragraph. Please refer to the revised manuscript for these changes (see highlighted section in red, lines 281-284).

 

Comment 11：

Page 6, line 268. Change “BPH secretes salivary miRNAs into rice, which suppress plant immunity.” to “BPH secretes salivary miRNAs into rice, which serves as a salivary effector targeting multiple host genes and suppress plant immunity.”

Response: Thank you for your suggestion. Follow your suggestion, we have changed “BPH secretes salivary miRNAs into rice, which suppress plant immunity.” to “BPH secretes salivary miRNAs into rice, which serve as salivary effectors that target multiple host genes and suppress plant immunity.” Please refer to the revised manuscript for these changes (see highlighted section in red, lines 305-306).

 

Comment 12：

Page 6, section 2.3.2. BPH-Derived miRNAs Targeting Rice. Some parts of these two paragraphs are overlapping; please reorganize them for better clarity and flow.

Response: Thank you for your suggestion. After reviewing the section, we agree with your observation that some parts of the two paragraphs overlap. Specifically, the content in the first paragraph, “miR-7-5P targets rice immune-related genes, such as OsbZIP43. Silencing of miR-7-5P reduces BPH feeding efficiency, indicating that it helps overcome host defenses [71],” is repeated in the second paragraph, which provides a more detailed description of miR-7-5P. To address this, we have removed the redundant sentence and merged the two paragraphs into one for better clarity and flow. (see highlighted section in red, lines 306-309).

 

Page 6. Section 3. Conclusions and Perspectives

 

Comment 13：

Change “ncRNAs regulate rice–BPH interactions.” to “In this review, we summarized the latest advances in ncRNA-mediated regulation of rice–BPH interactions.”

Response: Thank you for your suggestion. Follow your suggestion, we have changed “ncRNAs regulate rice–BPH interactions.” to “In this review, we summarized the latest advances in the ncRNA-mediated regulation of rice–BPH interactions.” Please refer to the revised manuscript for these changes (see highlighted section in red, lines 387-388).

 

Comment 14：

Conclusion (Section 3): This section should focus on summarizing the key points discussed earlier and highlighting future research directions, rather than repeating detailed information from previous sections. Please revise this section by removing redundant paragraphs.

Response: Thank you for your suggestion. Following your recommendation, we have revised the “3. Conclusions and Perspectives” section to focus on summarizing the key points discussed earlier and highlighting future research directions, while removing redundant paragraphs. We hope this revision addresses your concern and improves the clarity of the section. Please refer to the revised manuscript for these changes (see highlighted section in red, lines 386-411).

 

Comment 15：

Table 1. The lncRNAs and circRNAs mentioned in sections 2.1.2 and 2.2.2 are missing. Please add these ncRNAs in the table as well. Additionally, this table should be placed earlier in the article for better organization.

Response: Thank you for your suggestion. Following your suggestion, we have added the missing lncRNAs XLOC_042442 and XLOC_028297 mentioned in section 2.1.2 to Table 1. Regarding your comment about potential missing circRNAs in section 2.1.2 and missing lncRNAs and circRNAs in section 2.2.2, due to the limited and insufficient research currently available on this topic, no additional lncRNAs or circRNAs are missing from these sections.

Additionally, we have included a new schematic diagram “Figure 1” that illustrates the regulatory pathways mediated by ncRNAs in both rice and BPH, which we believe will enhance the understanding of the discussed mechanisms.

Following your suggestion, we have moved Table 1 and the newly added Figure 1 to an earlier section of the article for better organization. Please refer to the revised manuscript for these changes (see highlighted section in red, lines 77-79, 413-433, Table 1, Figure 1).

 

Comment 16：

The manuscript’s organization and writing need significant improvement. I provided some specific suggestions for revision in my comments.

Response: Thank you for your valuable feedback and specific suggestions on the manuscript’s organization and writing. In response to your suggestions, we have addressed the areas that could be improved: we have expanded the discussion on molecular mechanisms, practical implications, and future research directions as you suggested before to provide a more comprehensive analysis.

Specifically, we have included additional visual data, such as newly modified table and newly added figure, to better illustrate the key findings. We believe these revisions significantly enhance the manuscript’s quality.

Additionally, we have requested a thorough review by an experienced English editor, whose first language is English and who specializes in editing papers.

We believe these revisions will greatly improve the manuscript’s organization and writing, and ensure it meets the required standards for publication. Please refer to the highlighted sections in the revised manuscript for further details.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

The article deals in a relevant and detailed way with the interactions between rice and the insect Nilaparvata lugens mediated by non-coding RNA (ncRNA). It is an original work with a well-structured overview based on recent studies that emphasizes the importance of ncRNAs for pest control and rice resistance. The language used is clear and concise, the figures and tables are useful and well designed, and the references are up-to-date and relevant.

The language used is clear and concise, with a technical level suitable for a scientific audience. The figures and tables, particularly the table summarizing ncRNAs and their functions, are well designed and usefully complement the information. The references are up-to-date and relevant and cover a wide range of recent studies on the subject. No ethical issues are raised as no new experimental data are presented. The article appropriately mentions the funding received and has no conflicts of interest.

 

Below are some recommendations to improve the manuscript

• Include a section on the current limitations of the practical application of ncRNAs in pest control:
• It would be useful to provide a more balanced perspective that addresses the existing challenges in implementing these tools in practise.
• Expand the section on “cross-kingdom interactions”:
• I recommend enriching this section with specific examples of the application of these findings in commercial or experimental crops to help readers better understand the practical implications.
• Discuss the practical implications and potential technical or economic challenges:
• Deepening the practical implications of using ncRNAs in integrated pest management strategies and highlighting potential technical or economic barriers would enrich the discussion and add value for researchers and practitioners in the agricultural sector.
• Add a section on future research directions:
• It would be interesting to include a brief discussion on future research directions, such as the use of new technologies like CRISPR/Cas to study and manipulate ncRNAs to improve resistance to rice pests.
• Review and complete tables and figures: It is recommended to ensure that all tables and figures contain full explanations and are accessible to readers from different scientific disciplines. This would improve clarity and facilitate understanding of the content.

Comments on the Quality of English Language

The English could be improved to more clearly express the research.

Author Response

Response to Reviewer #4

 

Comment 1：

The article deals in a relevant and detailed way with the interactions between rice and the insect Nilaparvata lugens mediated by non-coding RNA (ncRNA). It is an original work with a well-structured overview based on recent studies that emphasizes the importance of ncRNAs for pest control and rice resistance. The language used is clear and concise, the figures and tables are useful and well designed, and the references are up-to-date and relevant.

 

The language used is clear and concise, with a technical level suitable for a scientific audience. The figures and tables, particularly the table summarizing ncRNAs and their functions, are well designed and usefully complement the information. The references are up-to-date and relevant and cover a wide range of recent studies on the subject. No ethical issues are raised as no new experimental data are presented. The article appropriately mentions the funding received and has no conflicts of interest.

Response: Thank you very much for such favorable and encouraging comments on the value of our work.

 

Below are some recommendations to improve the manuscript.

 

Comment 2：

Include a section on the current limitations of the practical application of ncRNAs in pest control: It would be useful to provide a more balanced perspective that addresses the existing challenges in implementing these tools in practice.

Response: Thank you for your valuable suggestions. In response, we have added a section discussing the current limitations of the practical application of ncRNAs in pest control (see highlighted section in red, lines 341-385). Please refer to the highlighted sections in the revised manuscript for further details.

 

Comment 3：

Expand the section on “cross-kingdom interactions”:

I recommend enriching this section with specific examples of the application of these findings in commercial or experimental crops to help readers better understand the practical implications.

Response: Thank you for your valuable suggestions. Follow your suggestion, we have expanded the section on “cross-kingdom interactions” by including specific examples, rice-derived miRNAs, such as miR162a (including miR162a-m1), and BPH-derived miRNAs, such as miR-7-5P, to provide more detailed insights. Specifically, we have provided examples of how ncRNAs could be applied in commercial or experimental crops to help readers better understand the practical implications (see highlighted sections in red, lines 288-294 and 315-319). Please refer to the highlighted sections for further details.

 

Comment 4：

Discuss the practical implications and potential technical or economic challenges:

Deepening the practical implications of using ncRNAs in integrated pest management strategies and highlighting potential technical or economic barriers would enrich the discussion and add value for researchers and practitioners in the agricultural sector.

Response: Thank you for your valuable suggestions. Follow your suggestion, we have added a section discussing the practical implications and potential technical or economic challenges (see highlighted section in red, lines 341-385). Please refer to the highlighted sections in the revised manuscript for further details.

 

Comment 5：

Add a section on future research directions:

It would be interesting to include a brief discussion on future research directions, such as the use of new technologies like CRISPR/Cas to study and manipulate ncRNAs to improve resistance to rice pests.

Response: Thank you for your valuable suggestions. Follow your suggestion, we have added a brief discussion on future research directions, including the potential use of new technologies such as CRISPR/Cas to study and manipulate ncRNAs for improving resistance to rice pests (see highlighted section in red, lines 408-411). Please refer to the highlighted sections in the revised manuscript for further details.

 

Comment 6：

Review and complete tables and figures: It is recommended to ensure that all tables and figures contain full explanations and are accessible to readers from different scientific disciplines. This would improve clarity and facilitate understanding of the content.

Response: Thank you for your valuable suggestions. Follow your suggestion, we have reviewed the table and figure and ensured that they now contain comprehensive explanations (see highlighted section in red, lines 413-433, Table 1, Figure 1). Additional captions and clarifications have been added where necessary to make the content more accessible and clearer to a wider audience, please refer to the revised manuscript for these changes.

 

Comment 7：

The English could be improved to more clearly express the research.

Response: Thank you for your suggestions. In response, we have requested a thorough review by an experienced English editor, whose first language is English and who specializes in editing papers. We believe this will greatly enhance the manuscript’s clarity and ensure it meets the required language standards for publication.

Author Response File: Author Response.pdf