PPATHOGENS-D-25-00024

Stylet cuticular gene-directed mutagenesis impairs the pea aphid vector capacity to transmit a plant virus

PLOS Pathogens

Dear Dr. Uzest,

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PLOS Pathogens

Savithramma Dinesh-Kumar

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PLOS Pathogens

Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

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Michael Malim

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PLOS Pathogens

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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: The manuscript describes an extensive analysis of Cas9 mutants of the stylet proteins which participate in CaMV virus transmission. The results are internally consistent and appropriate.

The novelty of this work is repeatedly noted – though the generation of the mutants was previously published.

The authors have done a good job of making quantitative presentation of qualitative observations.

Reviewer #2: Aphids are serious agricultural pests, notorious for transmitting hundreds of plant viruses, including Cauliflower mosaic virus (CaMV). CaMV transmission by aphids relies on a helper mechanism involving the viral non-structural protein P2 and its receptors on the surface of the acrostyle, a specialized structure at the tip of the insect’s stylet. Stylin proteins are prime candidates for these receptors. In their study, Fu et al. developed two mutant aphid lines with edits to the Stylin-01 gene: one is a complete knockout, and the other produces a modified version of the protein with changes to its exposed C-terminal domain. Using these mutants, the researchers showed that fully functional Stylin-01 is essential for efficient CaMV transmission by aphids. This research significantly enhances our understanding of the critical role this cuticular component plays in plant-virus-vector interactions. This manuscript is well-written and only a few minor modifications and discussion are required.

Reviewer #3: The long-standing question of the biochemical basis of how plant viruses are transmitted by insects to the host plant is key to the development of new, environmentally approaches to controlling their transmission. These viruses can replicate within the insect host while others have a direct transmission route from infected stylets or other mouthparts.

Previous studies had used RNAi approaches to down-regulate target genes however these are, by nature, transient, variable and somewhat inconclusive. Here the authors utilized two previously generated CRISPR/Cas9 mutants in a stylet gene to determine the role of this gene in virus transmission. One was essentially a completely dysfunctional mutation whole the second had retained most of the amino-terminal sequence but has lost the carboxy-terminal domain.

The experiments were well conducted and clearly show the role that the stylin-1 gene plays in transmission of the virus. They implicate the ability of the acrostyle to successfully bind the P2 protein of the virus and also show up-regulation effects on another stylin in these mutants.

I recommend the manuscript be accepted. It is short and concise, but will be of significant interest in the field.

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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 raw data for the figures should be made available via a data repository, or provided as a supplemental spreadsheet / document. This is particularly true given the nature of statistical packages used for the analysis.

Reviewer #2: 1. In lines 119-121, the authors mention that the Sty01-Cter line lacks the 11 C-terminal amino acids that form the surface-exposed domain of the acrostyle, which is thought to interact with viruses. Did the researchers test whether this domain is essential for binding to CaMV? To properly verify this interaction, co-immunoprecipitation (Co-IP) or immunofluorescence co-localization experiments should have been conducted.

2. Fig 3 demonstrates a reduction in the number of positively labeled stylets for both mutant lines compared to WT aphids. However, it appears that a significant majority of the mutant aphids (over 80%) still retain the ability to bind CaMV P2 proteins and thus can transmit the virus. This raises the question of whether Stylin-01 is indeed the most critical protein regulating viral transmission. While the gene knockout has a statistically significant effect, the impact on virus transmission does not seem substantial enough to conclusively establish Stylin-01 as the primary regulator. This observation suggests that other factors or proteins might also play crucial roles in CaMV transmission, which deserves discussion.

Reviewer #3: (No Response)

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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 writing style is at times cumbersome with excessive use of comas and prepositions. The PDF is provided with several dozen suggested edits.

The presentation of statistical includes non-parametric tests associated with non-normal distribution. Additional detail, including the assessment of normality would seem appropriate, even though the work understandably has limited numbers (making normality tests problematic). This statistical analysis seems to rely on the authors use of packages, more so than an understanding of the underlying statistical tests.

The discussion is quite redundant with the results, including reiterated references to the figures rather than extending the analysis to a broader perspective. This section could be considerably shortened.

The effort made in high quality presentation of graphics and tables (including supplemental) is noted. The overall quality and quantity of research represents a excellent addition to the literature.

Reviewer #2: 1. The antibodies used for western blotting and immunofluorescent localization, particularly those against Sty01-Cter and Sty01-KO in Fig 1, 3, and 5, should be clearly indicated either in the figures themselves or thoroughly described in the figure legends. This will help readers better understand and interpret the data presented in the figures.

2. Fig 3B presents the number of stylets observed for each group from a single experiment batch. To establish the reproducibility of these results, it is a standard practice to conduct at least 3 independent experimental batches. Without multiple batches, the results may lack statistical significance and robustness. It is crucial to know whether the authors conducted different experimental batches to validate their findings.

Reviewer #3: (No Response)

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Reviewer #1: Yes:  Wayne R. Curtis

Reviewer #2: No

Reviewer #3: No

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Dear Dr Uzest,

We are pleased to inform you that your manuscript 'Stylet cuticular gene-directed mutagenesis impairs the pea aphid vector capacity to transmit a plant virus' has been provisionally accepted for publication in PLOS Pathogens.

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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,

Christophe Ritzenthaler

Academic Editor

PLOS Pathogens

Savithramma Dinesh-Kumar

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):