PPATHOGENS-D-26-00129

Exposure to herbivore-induced plant volatiles primes JA-dependent gossypol defenses in cotton

PLOS Pathogens

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Melissa Bredow

Academic Editor

PLOS Pathogens

Shou-Wei Ding

Section Editor

PLOS Pathogens

Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

Additional Editor Comments :

Dear Dr. Yunhe Li,

The reviewers uniformly agree that the work presented fills an important knowledge gap in the field and have recommended publication, but also suggest some minor revisions to the manuscript. Therefore, I invite you to respond to the reviewers' comments and revise your manuscript accordingly.

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Reviewers' Comments:

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: This manuscript by Zhu et al. demonstrates that cotton bollworm-induced plant volatiles can prime the defense responses of neighboring plants. Specifically, exposure to these volatiles enhances the accumulation of herbivory-induced jasmonic acid and the biosynthesis of the defensive metabolite gossypol.

Reviewer #2: Zhu et al. demonstrated that cotton bollworm-induced volatiles can serve as defense-priming stimuli in neighboring cotton plants, both in laboratory and semi-field conditions. These volatiles were shown to prime JA signaling and promote gossypol accumulation in neighboring plants. Furthermore, the priming effect was abolished when JA signaling or gossypol biosynthesis was inhibited in the receiver plants. Given that volatile-mediated signaling is known to be context- and species-specific, studies conducted in crop systems such as cotton are of particular interest. I have only a few minor suggestions for this otherwise well-written manuscript.

Reviewer #3: Zhu et al. base their work on practical agricultural production needs and employ an experimental system combining laboratory and semi-field conditions. They clearly demonstrate that volatiles induced by cotton bollworm (CBW) infestation enhance the defensive resistance of recipient cotton plants against subsequent herbivore attack, effectively reducing feeding damage. Building on this, the study reveals that CBW-induced volatiles prime anti-herbivore defense in cotton by potentiating the JA signaling pathway and promoting gossypol biosynthesis upon actual herbivore challenge. These findings fill a critical knowledge gap in cotton HIPV communication research by showing how defense signals are transduced into chemical defenses. The regulatory mechanism elucidated here also provides important theoretical support and practical reference for reducing chemical pesticide use in sustainable agriculture and for developing novel biocontrol strategies. However, there are still some drawbacks that should be addressed.

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Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: The study on the gossypol-deficient mutant gl2gl2gl3gl3 clearly demonstrates that gossypol contributes to the volatile-induced priming effect. Given the authors' proposed model that "HIPV perception sensitizes the JA pathway, enabling rapid gossypol biosynthesis upon herbivore attack" (Lines 205-206)�， I am curious to know whether the enhanced JA induction they observed is also detectable in these gossypol-free lines.

Reviewer #2: (No Response)

Reviewer #3: (1) I have concerns regarding the strength of the evidence supporting the conclusion that "CBW-induced volatiles prime anti-herbivore resistance in cotton by potentiating the JA signaling pathway, which in turn enhances gossypol biosynthesis upon actual herbivore attack." While the study successfully demonstrates that volatile exposure activates the JA pathway and promotes gossypol accumulation, a critical gap remains: it does not fully establish that the JA pathway serves as the causal mediator linking volatile perception to increased gossypol production. I recommend either providing additional mechanistic evidence or rephrasing this conclusion to avoid ambiguity.

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Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: The sample size for the herbivore bioassay (n ≈ 10) is insufficient for a reliable larval growth assay, given the inherent natural variation in such experiments.

Reviewer #2: 1. In the Abstract (line 24), the statement “the molecular basis for this phenomenon remains poorly understood” could be made more specific by explicitly mentioning cotton.

2. The results for the first (line 105) and third (line 133) main experiments are described too briefly. Please add clarifying details to these sections, such as a short description of the experimental design and the names of the JA inhibitors used.

3. In Figure 1, the number of replicates differs between the Figure legend and the Methods section. For instance, the lab assays used “four uniformly sized CBW larvae,” yet the replicates were 13 or 8-9. A similar discrepancy appears in the semi-field assay data.

4. In Figure 2B, CBW-induced JA-Ile levels showed no significant difference between the control and HIPV-treated groups at 24 h treatment. However, Figure 3B demonstrates a significant increase in JA-Ile in the HIPV-treated group. Please discuss this discrepancy.

5. Although it is well established that gossypol biosynthesis is regulated by JA signaling, to further support the conclusion that primed JA signaling correlates with gossypol biosynthesis in neighboring plants, I recommend either: (1) quantifying gossypol accumulation in receiver plants treated with buffer or JA inhibitors, or (2) examining the expression of gossypol biosynthesis-related genes in HIPV-exposed plants versus control plants treated with CBW, as illustrated in Fig. 2.

Reviewer #3: I recommend improving the logical coherence of the Introduction section. For example, in Lines 65-80, the text abruptly transitions from introducing constitutive defense compounds (pigment glands, gossypol) that can be induced by pests to discussing HIPV-induced defense responses. It is suggested to clarify the relationship between HIPV-mediated jasmonic acid (JA) signaling pathway and downstream defense compounds (such as gossypol) to enhance the logical flow of the Introduction.

Given that gene expression, phytohormone, and gossypol levels were evaluated by two-way ANOVA, the figures should therefore present the results of this analysis. Please include the P values for treatment effect, time effect, and their interaction in each figure panel or legend.

Line 72：Please add the definite article "the" before "lepidopteran larva" .

Line 87: correct the typo "plans" to "plants".

Line 109 and line 158 ：Please confirm that the degrees of freedom (df) are integers, and unify the number of decimal places throughout the manuscript.

In Figure 1: Only the final body weight of cotton bollworm (CWB) was presented in Figures 1B and 1D. Even with uniform initial selection, the milligram-scale body weight of the larvae means that minor initial differences could have affected the results. It is recommended that authors record the initial larval weight and use weight gain as the evaluation index to improve the reliability of the results.

In Figure 2: The data for JA-Ile at 12 h show high variability. I recommend verifying these results. Furthermore, JA pathway-related gene expression was only measured at 3 h and 24 h, whereas significant differences in JA content were observed at 12 h and 24 h. To address this temporal gap, including gene expression data at or before the 12 h time point is suggested.

Line 237: Please clarify the exact temperature of warm water in the Methods.

Lines 295-296: The relative gene expression was calculated using the 2-△Ct method. Please use the 2-△△Ct method to ensure valid and reliable results.

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Reviewer #2: No

Reviewer #3: No

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Dear Dr. Li,

We are pleased to inform you that your manuscript 'Exposure to herbivore-induced plant volatiles primes JA-dependent gossypol defenses in cotton' has been provisionally accepted for publication in PLOS Pathogens.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Melissa Bredow

Academic Editor

PLOS Pathogens

Shou-Wei Ding

Section Editor

PLOS Pathogens

Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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Reviewer Comments (if any, and for reference):

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: Zhu et al. demonstrated that cotton bollworm-induced volatiles can serve as systemic signal trigger neighboring plants' priming effects. This priming effect is depending on JA signaling and gossypol accumulation in neighboring plants. consistently, the priming effect was abolished when JA signaling or gossypol biosynthesis was inhibited in the receiver plants.

Reviewer #2: The authors have satisfactorily addressed all my concerns through additional experiments and revisions. I therefore recommend accepting this manuscript for publication in PLOS Pathogens.

Reviewer #3: The authors have addressed my concerns and the manuscript is acceptable now.

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Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: Authors addressed all my comments, and I don't have further concern.

Reviewer #2: (No Response)

Reviewer #3: NA

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Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

Reviewer #3: NA

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PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review?   For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No