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PONE-D-22-05293A novel eukaryotic RdRP-dependent small RNA pathway represses antiviral immunity by controlling an ERK pathway component in the black-legged tickPLOS ONE

Dear Dr. Okamura,

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“Research in K.O.’s group was supported by the National Research Foundation, Prime Minister’s Office, Singapore under its NRF Fellowship Programme (NRF2011NRF-NRFF001-042), Temasek Life Sciences Laboratory core funding and the JSPS Fund for the Promotion of Joint International Research (Returning Researcher Development Research, 17K20145). Work in the T.T.’s group was supported by Takeda Science Foundation. The content is solely the responsibility of the authors and does not necessarily represent the official views of these agencies.”

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“. We thank ATCC for ISE6 cells and BGI for sequencing. Research in K.O.’s group was supported by the National Research Foundation, Prime Minister’s Office, Singapore under its NRF Fellowship Programme (NRF2011NRF-NRFF001-042), Temasek Life Sciences Laboratory core funding and the JSPS Fund for the Promotion of Joint International Research (Returning Researcher Development Research, 17K20145). Work in the T.T.’s group was supported by Takeda Science Foundation.”

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Reviewers' comments:

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Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #1: Yes

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: No

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors of this paper experimentally sought to test that RdRPs are directly involved in the production of sRNAs in the model black-legged tick ISE6 cell lines, and that sRNAs control viral transcripts through RNAi and regulation of the Dsor1 gene of the ERK pathway, which contains the 3'UTR of the RdRP-dependent target of repeat-derived sRNAs, and that knockdown of RdRP leads to down-regulation of viral transcripts in a Dsor1-dependent pathway. The authors therefore describe that black-legged tick RdRPs play a role in the biogenesis of specific sRNAs, and in gene regulation and control of viral transcript levels. Complementing the experimental studies on the involvement of RdRPs in RNA silencing in animals other than nematodes (in ticks), experimental evidence for a function in sRNA biogenesis is provided. Several of the following points may require further clarification and interpretation before publication.

1. The authors named the ISE6 cell lines RdRP as “IscRdRP1,3 and 4”, but thereafter, these RdRP names were shown as RdRP1, 3 and 4 throughout the manuscript. Please correct the names and make them consistently.

2. The name format is not consistent: in 1A are “Isc-Ago16” and “Isc-Ago30”; in 1C are “Ago16” and “Ago30”; in 1D are “Ago-16” and “Ago-30”; in the manuscript content are “IscAgo-16” and “IscAgo-30”. Same issue happens to other Ago, RdRp and Aub names. Please keep using one format throughout the manuscript and Figures.

3. In the section of sRNAs produced from repeats, for the description “Large overlaps were seen with Ago-16-RdRP3 and Ago-30-RdRP1 combinations, suggesting that the AGOs and RdRPs might work together to produce repeat associated sRNAs. Interestingly, very few loci overlapped between the three groups, similar to the observation with the sRNAs from coding genes (Figure3)”, however, no Ago-30 knockdown related data is shown in figure 3.

4. As Figure 4 showed that many genes were dependent on the RdRP1-Ago-30, but why RdRP1-Ago-30 combination is not reflected and mentioned in Figure 3A.

5. Quotation mark errors in Figure 6A caption, “Within its 3’UTR, there is a high peak of sRNAs(‘sRNA peak’’)”, please correct the (‘sRNA peak’’) to (“sRNA peak”).

6. Missing supportive evidence to prove that the downregulation of viral gene expression had no difference between Dsor1 single knockdown and Dsor1&RdRP1 knockdown together. The author should introduce a vector containing functional RdRP1 into Dsor1&RdRP1-knockdown cell, and then compare this recovered gene expression with Dsor1 single knockdown to clarify if the result still consistent.

7. The qPCR primer used to amplify Ago-96 cDNA, that noted in Figure S3A, should be listed in Table S5.

Reviewer #2: In this study, Feng et al. identified new factors (RdRP1, RdRP3) and homologs of known factors (Ago3 and Aub) involved in sRNA regulation in Ixodes scapularis ticks and further characterized their role in parallel with other argonaute-like genes previously identified (Ago-16, -30, -78, -96), highlighting synergy and specificity between each factor. The authors further characterize the role of RdRP1 in the regulation of MAPK Dsor1 involved in the antiviral immune response. Overall, they provide a nice, comprehensive and broad analysis of small RNA pathways in Ixodes ticks, through an extensive amount of work, providing valuable data and insights into tick sRNA biology to the scientific community.

While the paper is overall scientifically sound, some minor revisions are required and the author should significantly edit the manuscript to facilitate readability (especially in the abstract and introduction), clarify some elements and reflect the quality of the work performed. See comments for improvement below.

1. The manuscript should have line numbering to properly refer to the text.

2. Authors frequently refer to Figure ‘X’ and Supplementary Data without specifying which supplementary data they are referring to. Please clarify.

3. Authors included description of the data in figure legends. Example Fig 2A ‘Note the dramatic decrease of the “RNAP III” group’. Figure legends should generally only ‘provide a description of the figure that will allow readers to understand it without referring to the text’.

4. Authors provided supplementary information in the form of a 19 pages ‘supplementary PDF’ without describing much of its content. Authors should selectively format it with clear legends and reference clearly in the text.

5. In the abstract, the authors should also avoid generalizing the role of RdRPs as they have shown that RdRP1 and 3 to display different behavior. “RdRP-dependent sRNAs […] are mainly derived from RNA polymerase III-transcribed genes and repetitive elements” is misleading. While virtually all RNAPIII-derived sRNAs appear to be RdRP1-dependent, the regulation of repeat/transposable elements by RdRPs is only punctual and without overlap. Similarly in the second half of the abstract, only RdRP1 was shown to regulate Dsor1 and have antiviral activity, not RdRP3. Please correct throughout the manuscript. For example, in the discussion, page 14 “RdRPs might physically interact with RNAP III during transcription”.

6. Author summary, first sentence. “RdRPs […], but THEIR general importance […].”

7. Introduction, first sentence. Transposable elements cannot be considered foreign nucleic acids. They were integrated in the genome and are now part of the host. Please correct.

8. Introduction, second sentence. Cells do not use small RNAs to distinguish host/foreign nucleic acids. Please correct.

9. The relatively long description of CRISPR in the introduction is questionable as the paper focused solely on eucaryotes and never uses CRISPR technology. It should be shortened or removed.

10. Introduction, page 4, please clarify “worm-like secondary sRNAs”.

11. The authors should discuss the RdRP Ego-1 and other RBPs found by Kurscheid et al, 2009 (https://doi.org/10.1186/1471-2199-10-26).

12. Authors chose to express recombinant tick proteins in mammalian cells, which they acknowledge to have limitations. The authors mention page 20 ‘tick plasmids’, could they clarify? Do they have tick expression plasmids? Alternatively, authors could directly transfect mRNA into tick cells to circumvent the lack of expression vector.

13. In figure 2, sRNAs are shown as a fraction of a whole. Could the authors provide an absolute quantification, ideally normalized to a stable sRNA, for example the SRP RNA shown to be stable across all knockdowns Fig S5B?

14. Fig. 2E-F panels are unrelated to rest of Fig2 and its title. Those data could be associated with the rest of the data related to H. longicornis in Fig S6?

15. Fig. S4D, Ago3-2 knockdown induces a reduction of piRNA of about 50% compared to Ago3-1 knockdown. Also, the authors mentioned that both Ago3-1 and Ago3-2 have ‘very similar sequences’ without specifying percentage of identity. The authors should discuss this difference and if possible, investigate further? Are they both regulating the same piRNA?

16. Fig. S4D, the quantification of antisense piRNA in equivalent amount to sense piRNA is surprising, as piRNA are expected to be derived from the processing of single-stranded RNA. Could it be an artefact? This should be discussed. The author could check for 1U-/10A bias.

17. Page 11, “To test if repeat-associated sRNAs silence expression of repeats”, please rephrase.

18. Figure legends should be homogenous throughout the paper. For example, Fig 2B ‘Read count (RPM)’, 2F ‘Number of reads (RPM)’ and S4 “Normalized read count (RPM)”.

19. Fig. 3A and 4A are not clear. What are the ‘set size’ referring to? The intersection size could be expressed as a percent of effected genes? Also, in Fig. 3B and 4B, does the color code refers to genes regulated by both or either protein? For example, Fig3B shows 5 genes with red histograms, but Fig 3A denotes only 3 genes regulated by RdRP3/Ago-16.

20. Fig. 5B refers to ‘ISCI005428’ while it was stated that the gene is referred to as Dsor1 through the paper. Also, Dsor1 is sometimes referred to as IscDsor1 (discussion, page 15). Please use consistent nomenclature.

21. Description of both qPCR and method referred to as ‘sensor assay’ are missing from the material and methods.

22. The authors should clarify how the sensor assay is normalized. Is Dsor1 3’UTR destabilizing Fluc expression? RdRP1 knockdown only recovers ~20% of Fluc levels compared to the empty control? What is the efficiency of RdRP1 knockdown?

23. Knockdown experiments in Fig. 6-7 should be supported with controls of efficient knockdown similarly to Fig S2 A.

24. Distribution plots in Fig 2C/E, 6A and 7A + supplementary figures are missing a Y-axis legend. Please correct. It would also be best to use consistent scale and different colors for sense and antisense RNA.

25. Author should provide distribution plots of siRNA mapping to each virus mentioned in Fig 7. The authors do not show sufficient evidence to state that there are no virus-derived piRNA. In Fig 7B, they should either use a log scale or split y-axis as the scale only allow to observe the strong antiviral siRNA response and which may hide some processing of viral RNA by the piRNA machinery. This should also be done for each virus as the host response may change for each virus, as shown in Fig. 7C.

26. In the references, ref 71 does not appears to be linked to pEGFP cloning where it is mentioned in page 17. Also, please check conformity of reference formatting with PLOS policy. NCBI links are provided instead of DOI in several cases (ref. 19, 71, …).

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Reviewer #1: No

Reviewer #2: No

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A novel eukaryotic RdRP-dependent small RNA pathway represses antiviral immunity by controlling an ERK pathway component in the black-legged tick

PONE-D-22-05293R1

Dear Dr. Okamura,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Sébastien Pfeffer, PhD

Academic Editor

PLOS ONE

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