PPATHOGENS-D-26-00272

A TCER-1-siRNA Regulatory Axis Suppresses Antibacterial Innate Immunity in C. elegans

PLOS Pathogens

Dear Dr. Ghazi,

Thank you for submitting your manuscript to PLOS Pathogens. Your manuscript has been evaluated by members of the editorial board and three expert reviewers; all feel that the study is important and carefully implemented, but there are issues that you need to address before it can be published in PLOS Pathogens. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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We look forward to receiving your revised manuscript.

Kind regards,

Emily R. Troemel

Academic Editor

PLOS Pathogens

D. Scott Samuels

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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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: In this manuscript entitled “A TCER-1-siRNA Regulatory Axis Suppresses Antibacterial Innate Immunity in C. elegans,” the authors investigated how endogenous siRNA pathways modulate antibacterial immunity against Pseudomonas aeruginosa PA14. The authors identified endogenous small RNAs, WAGO Argonaute-associated 22G-RNAs, that repress antibacterial immunity. The authors found that multiple components required for WAGO 22G-RNA biogenesis suppress host resistance through TCER-1, which is transcription elongation and splicing factor. They also showed that TCER-1 targets a restricted set of immune-relevant effectors, such as scrm-4, rather than inducing widespread gene silencing. The authors highlighted a TCER-1-siRNA regulatory axis in which TCER-1 promotes WAGO 22G-RNA biogenesis, thereby repressing antibacterial immunity.

Reviewer #2: In this manuscript, Naim and colleagues provide new insights into the role of small interfering RNAs (siRNAs) in C. elegans antibacterial defense. They demonstrate that TCER-1, the C. elegans homolog of human transcription elongation and splicing factor TCERG1, suppresses host defense against Pseudomonas aeruginosa PA14 via the WAGO 22G-RNA pathway. The role of TCER-1 as a repressor of host defense against bacterial pathogens had been established in previous work by the same group. High-throughput small RNA and mRNA sequencing data from two tcer-1 mutants revealed that expression of WAGO 22G-RNA pathway genes was not down-regulated in tcer-1 mutants. Thus, TCER-1 probably does not directly affect the WAGO 22G-RNA pathway, but rather indirectly influences stability of 22G-RNAs or the processing of WAGO targets. The exact mechanism of the interaction remains unclear. However, the authors also find that the WAGO target scrm-4, which encodes a phospholipid translocase, contributes to pathogen defenses in a TCER-1-dependent manner.

These results are significant and novel. While the role of miRNAs in antibacterial host defense in Caenorhabditis elegans is well established, endogenous siRNAs have been primarily studied in the context of antiviral defense. Although emerging evidence suggests that endogenous siRNA pathways may influence antibacterial immunity, their precise contribution to antibacterial host defense remains unclear. This study provides direct evidence of endogenous siRNA participation in antibacterial pathways, via interaction with TCER-1 and the WAGO target gene scrm-4. It significantly broadens our understanding of the role of endo-siRNAs in host-pathogen interactions and is thus of high importance and broad interest to the community in this field.

The manuscript is clearly written, and the experiments have been performed rigorously and are presented in a convincing and comprehensive manner. I only have two comments, including a suggestion for an additional experiment. Other than that, publication of the presented results as they stand is in my opinion justified.

Reviewer #3: In this manuscript the authors link the 22GRNA pathway in C. elegans to pathogen resistance, via the factor TCER-1 which acts upstream of 22G pathways. They provide convincing evidence for the alteration of 22G-RNAs against a small number of genes when TCER-1 is inactive and, nicely, are able to trace the effect on pathogen resistance to a particular target, scrm-4. The genetics experiments, statistical and computational analyses are excellent and the paper is written clearly and logically, without overstating the results.

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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: 1. In Figure 1E, the authors conclude that TCER-1::GFP expression is unchanged by ppw-1 or rrf-1 mutations, but the evidence is presented mainly as representative images. I recommend that the authors include a quantitative analysis of GFP expression from the TCER-1::GFP translation reporter. This would strengthen the conclusion that ppw-1 or rrf-1 mutations do not alter TCER-1 expression.

2. Fourteen targets show strong anti-correlation between 22G-RNAs and mRNA levels, and three, including scrm-4, exhibit changes greater than four-fold. It remains unclear why scrm-4 was prioritized over the others. Please write the justification for that. The epistasis data indicate that scrm-4 is required for tcer-1 RNAi-mediated immunoresistance. Can they add similar experimental results using WAGO mutants? Overexpression study will also strengthen the role of scrm-4 as a downstream target. If the authors do not have such genetric tools for testing these, it will be nice to add discussion points regarding this issue.

Reviewer #2: The study focuses on WAGO Argonaute-associated siRNAs and is based on the intriguing observation that the ppw-1 mutant of the WAGO 22G-RNA pathway, which is used in C. elegans for germline- vs. soma-specific gene-knockdown by RNAi, exhibited clearly decreased susceptibility to infection with P. aeruginosa PA14. PPW-1 is a member of the WAGO clade in C. elegans. Of the 13 AGOs in the WAGO clade, SAGO-2 and PPW-1 were previously shown to be involved in defense against PA14 infection (both sago-2 and ppw-1 mutants are resistant to PA14 infection; Seroussi et al., 2023 https://doi.org/10.7554/eLife.83853). Therefore, it would be very interesting to know whether the authors tested if other WAGOs, in particular SAGO-2, interact with TCER-1 or if the interaction is specific for PPW-1?

Reviewer #3: There are two issues which I think the authors should consider.

1) I'm puzzled about the tissue specificity of the response. It's not clear whether scrm-4 is expressed in intestine or germline and how this relates to the tissue specificity of the 22Gs that regulate it (which is explored earlier in the manuscript). It would be nice if the authors could do tissue specific RNAi of scrm-4 to see where it is required for pathogen resistance and relate this to the localisation of the particular wagos that regulate it.

2) It would be good to consider a little more the mode of regulation of scrm-4. Is this a post-transcriptional or a transcriptional (or both) silencing response? It seems to me, from a cursory glance at the genome browser, that scrm-4 may be in a repressive chromatin domain (H3K27me3 marked) which would imply that the regulation by wago may be transcriptional. Whilst it is probably beyond the scope of the manuscript to perform ChIP-seq experiments before and after infection and in the tcer-1 mutant, it would be nice if they could test genetically a requirement for nuclear RNAi, such as nrde-2 or nrde-3 (if scrm4 is somatic in this response, hrde-1 if it is germline). Moreover, it would be good for the authors to do some genome-browser snapshots to clearly show what is known about the baseline chromatin environment using, for example, the Ahringer lab datasets on chromatin domains.

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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: 1. I recommend that the authors include the paper (Kim and Han, PMID: 39414082) in the “Introduction” section to support the statement regarding gene silencing by miRNAs. In addition, I recommend that the authors add the article (Lee et al., PMID: 38614390) to provide accessible background information of RNA-sequencing, enabling readers to better follow the interpretation of the study.

2. Please correct the significant errors and inconsistencies in the figure text.

- In Abstract, line 2, please correct “Small interfering RNA (siRNA)” to “Small interfering RNAs (siRNAs)”.

- On page 6, line 3, please correct “mut-16, mut-14 and smut-1” to “mut-16, mut-14, and smut-1”.

- On page 6, line 10, please correct “for required” to “required for”.

- On page 6, line 16, please correct “(Fig. 3C-D and Supplementary Table S4)” to “(Fig. 3C-D, Supplementary Table S4)”.

3. I recommend that the authors provide full terms at their first appearance in the manuscript to enhance clarity.

- On page 4, line 16, please provide full terms for “P. aeruginosa”.

- On page 7, line 3, please provide full terms for “E. coli”.

4. Please correct inconsistent space throughout the manuscript. Some examples are below:

- On page 4, line 11, please correct the space between “resistance” and “[36]”.

- On page 9, line 1, please correct the space between “ (Fig. 4D-E, Supplementary Tables S6-S7)” and “[58, 59]”.

- On page 10, line 6, please correct the space between “as well” and “[23, 51, 65]”.

- On page 11, line 22, please correct the space between “37” and “°C”.

Reviewer #2: Survival data from additional trials are easy to find and clearly presented in supplementary tables, which is great, but raw data are missing (or I did not find them).

Reviewer #3: (No Response)

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Reviewer #2: No

Reviewer #3: No

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Dear Dr. Ghazi,

We are pleased to inform you that your manuscript 'A TCER-1-siRNA Regulatory Axis Suppresses Antibacterial Innate Immunity in C. elegans' has been provisionally accepted for publication in PLOS Pathogens.

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Best regards,

Emily R. Troemel

Academic Editor

PLOS Pathogens

D. Scott Samuels

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: The authors addressed my concerns successfully.

Reviewer #2: The authors have satisfactorily addressed my comments, as well as the majority of the concerns raised by the other reviewers, resulting in a further improvement of the manuscript. I have no additional comments.

Reviewer #3: As in first review

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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: (No Response)

Reviewer #2: (No Response)

Reviewer #3: No remaining major issues- the authors have done a really nice job of addressing the comments that I made previously

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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: No minor issues

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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: Yes:  Peter Sarkies