Characterization of the RNA-interference pathway as a tool for reverse genetic analysis in the nascent phototrophic endosymbiosis, Paramecium bursaria

Endosymbiosis was fundamental for the evolution of eukaryotic complexity. Endosymbiotic interactions can be dissected through forward- and reverse-genetic experiments, such as RNA-interference (RNAi). However, distinguishing small (s)RNA pathways in a eukaryote–eukaryote endosymbiotic interaction is challenging. Here, we investigate the repertoire of RNAi pathway protein-encoding genes in the model nascent endosymbiotic system, Paramecium bursaria–Chlorella spp. Using comparative genomics and transcriptomics supported by phylogenetics, we identify essential proteome components of the small interfering (si)RNA, scan (scn)RNA and internal eliminated sequence (ies)RNA pathways. Our analyses reveal that copies of these components have been retained throughout successive whole genome duplication (WGD) events in the Paramecium clade. We validate feeding-induced siRNA-based RNAi in P. bursaria via knock-down of the splicing factor, u2af1, which we show to be crucial to host growth. Finally, using simultaneous knock-down ‘paradox’ controls to rescue the effect of u2af1 knock-down, we demonstrate that feeding-induced RNAi in P. bursaria is dependent upon a core pathway of host-encoded Dcr1, Piwi and Pds1 components. Our experiments confirm the presence of a functional, host-derived RNAi pathway in P. bursaria that generates 23-nt siRNA, validating the use of the P. bursaria–Chlorella spp. system to investigate the genetic basis of a nascent endosymbiosis.


Recommendation? Accept with minor revision (please list in comments)
Comments to the Author(s) Jenkins et al. report about the genetic repertoire of RNAi components in Paramecium bursaria. This single celled organism is especially interesting to understand the evolution of symbiosis due to the ability to host intracellular chlorella algae. This work here describes on the one hand the evaluation of the ability to use the technique of RNAi by feeding by both, screening the genome for putative candidate genes and the direct application of dsRNA producing bacteria, and on the other hand an evaluation of sRNAs by NGS with a deeper analysis of recursive RNAi by double feeding with dsRNA of RNAi components themselves. By this approach the authors conclude that the siRNAs appear to derive from the host and not from the symbiont. The general work is extremely interesting and represents the basis for a deeper analysis of the symbiosis, e.g. for a further analysis of RNA transfer between symbiont and host. In addition it's more than worth for the scientific community that RNAi by feeding works in P.bursaria and especially the knowledge of the presence of individual RNAi components in different Paramecium species is an extremely important analysis and I appreciate the time consuming work invested. In fact, I cannot find many negative criticisms in this paper. All analysis appear more than solid, and the RNAi experiments including the subsequent analysis of phenotype and small RNAs involves the necessary controls and represents solid data. In addition the paper is well written and easy to follow the logic. I have only minor comments: • Line 138 dependent upon splicing for macronuclear generation and transcription: its not clear what macronuclear generation means in context of splicing • Line 154: E.coli vector uptake or better "dsRNA uptake" or "E.coli uptake". • Figure 1: "Stacked genes (single or unduplicated orthologues) represent putative local gene 312 duplication" this is not that clear to me: Is there one or two genes in P.bursaria with homology e.g. to Ptiwi08 and 14 of P.tetraurelia: I would assume only one, as 08 and 14 result from the last WGD in the aurelia complex and there will unlikely be two in bursaria: maybe the visualization is a bit confusing.. • Figure 3: sRNA seq: Does P.bursaria undergo autogamy and could the increased number of 25nt reads due to scnRNAs? The authors here normalized to total reads number. Maybe they could also try to normalize to total bursaria mapping read number to account for varying read numbers mapping to (i) chlorella and (ii) food bacteria. In the worst, but most interesting situation, Dicer silencing could affect a potential RNA interchange of host and symbiont and thus symbiont population alters, thus altering the mapping statistics. I would indeed also be interesting in a mapping statistic: what's the general percentage of reads mapping to the symbiont, the host, the food, the dsRNA, rRNA etc.

Review form: Reviewer 2
Is the manuscript scientifically sound in its present form? Yes Are the interpretations and conclusions justified by the results? Yes 1) In Figure 2C, It is not clear if there are any statistical differences in the P. bursaria cell number between the cultures fed with HT115 E. coli expressing u2af1 dsRNA (blue) and containing an empty vector control (grey). Although the figure legend indicates the difference by using three asterisks, these are not presented in the figure. In addition, I don't know if the use of blue and grey color is the best strategy in the figure. When the readers print out a copy of this paper with a black/white printer, it might be hard to tell the difference between these two colors.
2) In Figure 3C legend, the authors indicate "one-way analysis of variance (ANOVA)". Please make sure if this description is sufficient. My understanding is that they would need to use a different test (e.g., T-test) to tell the difference.

Decision letter (RSOS-210140.R0)
We hope you are keeping well at this difficult and unusual time. We continue to value your support of the journal in these challenging circumstances. If Royal Society Open Science can assist you at all, please don't hesitate to let us know at the email address below.

Dear Dr Jenkins
On behalf of the Editors, we are pleased to inform you that your Manuscript RSOS-210140 "Characterisation of the RNA-interference pathway as a Tool for Genetics in the Nascent Phototrophic Endosymbiosis, Paramecium bursaria" has been accepted for publication in Royal Society Open Science subject to minor revision in accordance with the referees' reports. Please find the referees' comments along with any feedback from the Editors below my signature.
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Both were highly supportive and found the study to be of interest and performed to a high standard.
There are minor suggestions relating to the statistical approach used requested.

Best Steven
Reviewer comments to Author: Reviewer: 1 Comments to the Author(s) Jenkins et al. report about the genetic repertoire of RNAi components in Paramecium bursaria. This single celled organism is especially interesting to understand the evolution of symbiosis due to the ability to host intracellular chlorella algae. This work here describes on the one hand the evaluation of the ability to use the technique of RNAi by feeding by both, screening the genome for putative candidate genes and the direct application of dsRNA producing bacteria, and on the other hand an evaluation of sRNAs by NGS with a deeper analysis of recursive RNAi by double feeding with dsRNA of RNAi components themselves. By this approach the authors conclude that the siRNAs appear to derive from the host and not from the symbiont. The general work is extremely interesting and represents the basis for a deeper analysis of the symbiosis, e.g. for a further analysis of RNA transfer between symbiont and host. In addition it's more than worth for the scientific community that RNAi by feeding works in P.bursaria and especially the knowledge of the presence of individual RNAi components in different Paramecium species is an extremely important analysis and I appreciate the time consuming work invested. In fact, I cannot find many negative criticisms in this paper. All analysis appear more than solid, and the RNAi experiments including the subsequent analysis of phenotype and small RNAs involves the necessary controls and represents solid data. In addition the paper is well written and easy to follow the logic. I have only minor comments: • Line 138 dependent upon splicing for macronuclear generation and transcription: its not clear what macronuclear generation means in context of splicing • Line 154: E.coli vector uptake or better "dsRNA uptake" or "E.coli uptake". • Figure 1: "Stacked genes (single or unduplicated orthologues) represent putative local gene 312 duplication" this is not that clear to me: Is there one or two genes in P.bursaria with homology e.g. to Ptiwi08 and 14 of P.tetraurelia: I would assume only one, as 08 and 14 result from the last WGD in the aurelia complex and there will unlikely be two in bursaria: maybe the visualization is a bit confusing.. • Figure 3: sRNA seq: Does P.bursaria undergo autogamy and could the increased number of 25nt reads due to scnRNAs? The authors here normalized to total reads number. Maybe they could also try to normalize to total bursaria mapping read number to account for varying read numbers mapping to (i) chlorella and (ii) food bacteria. In the worst, but most interesting situation, Dicer silencing could affect a potential RNA interchange of host and symbiont and thus symbiont population alters, thus altering the mapping statistics. I would indeed also be interesting in a mapping statistic: what's the general percentage of reads mapping to the symbiont, the host, the food, the dsRNA, rRNA etc.
Reviewer: 2 Comments to the Author(s) This manuscript represents a very interesting study. The authors investigated the repertoire of RNAi pathway protein-encoding genes in the model nascent endosymbiotic system, Paramecium bursaria-Chlorella spp. They identified essential proteome components of the small interfering RNA, scan RNA, and internal eliminated sequence RNA pathways by using comparative genomic and transcriptomic approaches that were supported by phylogentic analyses, Their results support the presence of a functional, host derived, exogenously-induced siRNA-based RNAi pathway in the P. bursaria-Chlorella spp. endosymbiotic system, dependent on Dcr1, Piwi and Pds1 protein function. The study seems to be well conducted and the manuscript is well written. The following are my minor comments or suggestions. Figure 2C, It is not clear if there are any statistical differences in the P. bursaria cell number between the cultures fed with HT115 E. coli expressing u2af1 dsRNA (blue) and containing an empty vector control (grey). Although the figure legend indicates the difference by using three asterisks, these are not presented in the figure. In addition, I don't know if the use of blue and grey color is the best strategy in the figure. When the readers print out a copy of this paper with a black/white printer, it might be hard to tell the difference between these two colors.

1) In
2) In Figure 3C legend, the authors indicate "one-way analysis of variance (ANOVA)". Please make sure if this description is sufficient. My understanding is that they would need to use a different test (e.g., T-test) to tell the difference.

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See Appendix A.
Decision letter (RSOS-210140.R1) We hope you are keeping well at this difficult and unusual time. We continue to value your support of the journal in these challenging circumstances. If Royal Society Open Science can assist you at all, please don't hesitate to let us know at the email address below.

Dear Dr Jenkins,
It is a pleasure to accept your manuscript entitled "Characterisation of the RNA-interference pathway as a Tool for Reverse Genetic analysis in the Nascent Phototrophic Endosymbiosis, Paramecium bursaria" in its current form for publication in Royal Society Open Science.
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Dear Editors and Reviewers
Thank you for the careful consideration of our manuscript, we are delighted with the positive appraisal and constructive responses provided. Please find attached a point-by-point response to all comments. Our responses are written in black font. The review provided is written in blue font.
Thank you for your submission, your manuscript has now been reviewed by two independent referees.
Both were highly supportive and found the study to be of interest and performed to a high standard.
There are minor suggestions relating to the statistical approach used requested.
Thank you for the positive appraisal of our work. We have addressed all reviewer comments below.
Reviewer comments to Author: Reviewer: 1 Comments to the Author(s) Jenkins et al. report about the genetic repertoire of RNAi components in Paramecium bursaria. This single celled organism is especially interesting to understand the evolution of symbiosis due to the ability to host intracellular chlorella algae. This work here describes on the one hand the evaluation of the ability to use the technique of RNAi by feeding by both, screening the genome for putative candidate genes and the direct application of dsRNA producing bacteria, and on the other hand an evaluation of sRNAs by NGS with a deeper analysis of recursive RNAi by double feeding with dsRNA of RNAi components themselves. By this approach the authors conclude that the siRNAs appear to derive from the host and not from the symbiont.
The general work is extremely interesting and represents the basis for a deeper analysis of the symbiosis, e.g. for a further analysis of RNA transfer between symbiont and host. In addition it's more than worth for the scientific community that RNAi by feeding works in P.bursaria and especially the knowledge of the presence of individual RNAi components in different Paramecium species is an extremely important analysis and I appreciate the time consuming work invested. In fact, I cannot find many negative criticisms in this paper. All analysis appear more than solid, and the RNAi experiments including the subsequent analysis of phenotype and small RNAs involves the necessary controls and represents solid data. In addition the paper is well written and easy to follow the logic.
We thank the Reviewer for their positive comments. These RNAi component surveys were only possible courtesy of the extensive sequence resources already available for these Paramecium species. It was our pleasure to add to this growing knowledge base.