A single synonymous nucleotide change impacts the male-killing phenotype of prophage WO gene wmk
Curation statements for this article:-
Curated by eLife
Evaluation Summary:
This study aims to find the genetic mechanisms underlying sex-ratio distortion through male-killing in Drosophila melanogaster flies infected with the endosymbiont Wolbachia. The endosymbiont carries the prophage WO, which is in the center of interested in this study. The key result of this study is that a synonymous mutation in a prophage gene can explain the differences between sex-ratio distorting and not distorting symbionts.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (eLife)
Abstract
Wolbachia are the most widespread bacterial endosymbionts in animals. Within arthropods, these maternally transmitted bacteria can selfishly hijack host reproductive processes to increase the relative fitness of their transmitting females. One such form of reproductive parasitism called male killing, or the selective killing of infected males, is recapitulated to degrees by transgenic expression of the prophage WO-mediated killing ( wmk ) gene. Here, we characterize the genotype-phenotype landscape of wmk- induced male killing in D. melanogaster using transgenic expression. While phylogenetically distant wmk homologs induce no sex-ratio bias, closely-related homologs exhibit complex phenotypes spanning no death, male death, or death of all hosts. We demonstrate that alternative start codons, synonymous codons, and notably a single synonymous nucleotide in wmk can ablate killing. These findings reveal previously unrecognized features of transgenic wmk -induced killing and establish new hypotheses for the impacts of post-transcriptional processes in male killing variation. We conclude that synonymous sequence changes are not necessarily silent in nested endosymbiotic interactions with life-or-death consequences.
Article activity feed
-
Author Response:
Reviewer #1:
This study aims to find the genetic mechanisms underlying sex-ratio distortion through male-killing in Drosophila melanogaster flies infected with the endosymbiont Wolbachia. The endosymbiont carries the prophage WO, which is in the center of interested in this study. The key result of this study is that a synonymous mutation in a prophage gene can explain the differences between sex-ratio distorting and not distorting symbionts. The study uses transgene technology to modify phage genes and to investigate which changes in the gene is involved in the phenotype. The finding, that a synonymous SNP plays a key role is not entirely novel in biology, but there are only few examples known of this type of genotype - phenotype associations. The study does not include experiments to show that the main finding is …
Author Response:
Reviewer #1:
This study aims to find the genetic mechanisms underlying sex-ratio distortion through male-killing in Drosophila melanogaster flies infected with the endosymbiont Wolbachia. The endosymbiont carries the prophage WO, which is in the center of interested in this study. The key result of this study is that a synonymous mutation in a prophage gene can explain the differences between sex-ratio distorting and not distorting symbionts. The study uses transgene technology to modify phage genes and to investigate which changes in the gene is involved in the phenotype. The finding, that a synonymous SNP plays a key role is not entirely novel in biology, but there are only few examples known of this type of genotype - phenotype associations. The study does not include experiments to show that the main finding is not limited to one particular background of the fly line used. An experiment including multiple genotypes would be needed to show this.
We agree that recapitulating the results in other backgrounds is intriguing and important for establishing a broader role of these findings. We thank the Reviewers and Editor for allowing us to pursue this line of investigation separately from this work, and we now discuss what experiments can be completed to answer these and other questions. We also edited the manuscript to tone down any conclusions that would imply generalizability of the findings at this point. For example:
"For example, we cannot conclude that the particular codon tested here is responsible for phenotype alterations in other host genetic backgrounds or species. It is possible that this codon plays a functional role only in a singular host genetic context. Here, we changed wmk sequences while holding the host genetic background fixed, but the reverse is required to conclude whether or not the particular codon plays a general role in other genotypes or natural contexts. Second, due to possible coevolution, various codons may or may not yield similar functional effects across different host backgrounds, and additional synonymous sites may contribute to the male-killing phenotype. Thus, the results here illuminate a previously unrecognized need for future research on the functional impacts of synonymous substitutions in endosymbionts. Future work may focus on determining if there is one specific synonymous codon that affects the male-killing function in all cases, if a more general feature exists where alteration of any or a subset of N-terminal or other wmk codons affects function, or if the effect of synonymous changes is specific to this background.”
Text summarizing the 06/21/2021 query to the Editor and Reviewers for further clarification: We believe there are several reasons why the results can stand on their own, while appropriately acknowledging caveats. First, we note the lack of genetic background testing on previous transgene experiments driving the major discoveries of Wolbachia genes involved in reproductive parasitism. This requirement would therefore hold the current work to a novel bar not previously applied by the field. In addition, the genetic background here is the same as used in previous work on these phenotypes, making it the most pertinent to test and inform previous and ongoing studies by many research groups. Second, the results shown here would still stand no matter the results of genetic background testing and would demonstrate that it is possible for synonymous changes to have functional relevance in the transgenic wmk phenotype. The major findings are still novel in the field, relevant to ongoing studies of reproductive parasitism, and informative regarding one of the most common genetic backgrounds. Finally, we note that two different lines with unique synonymous codon changes (the final experiment) independently created the same result that a synonymous codon change ablates phenotype, providing additional robustness to our findings. Doing additional experiments would be logistically difficult. Barriers include the relocation of the first author of the work to another lab for a postdoctoral position, completion of the funding for the project, remaining institutional COVID-19 restrictions, and lack of replacement personnel in the lab to continue the work. Notably, there is also the non-trivial requirement to create and test new transgene lines that would be costly and take nearly a year to complete (the experiments in the manuscript already took several years and the new fly lines would cost thousands to make).
The study is mostly clear and easy to follow, but requires a lot of attention. The authors choose to build up the story as I guess it was carried out in the lab. Thus, the reader is guided through every step of the process. While I see that this is appealing from the way the study was carried out, it results in a very long manuscript with a lot of material that would be much better placed in a supplement.
We thank the reviewer for pointing this out. We shortened the manuscript by removing redundant information and transferring some parts of the results to the supplement. We also removed about three pages of text from the discussion (before adding in new sections as requested by reviewers).
The introduction seems unfocused. It meanders around, jumping from topic to topic and does not give the reader a sense of where things will go.
We added a few topics into the Introduction as recommended in other comments, and we edited various portions of the Introduction to connect the ideas together more clearly. We hope the changes are now satisfactory, and we are of course happy to consider further feedback.
Fig. 1 gives an overview about the different aspects addressed here, but it is not used to guide the reader through the different lines of thought addressed in the introduction. If Fig. 1 will stay (I actually think it is not needed) it should be introduced earlier and used as a road map for the paper. Alternatively, the introduction could stay more general and only in the last paragraph the different ways the system is studied will be summarized.
We edited the final paragraph of the Introduction to more comprehensively cover the content of the figure and full direction of the paper. For readers not familiar with the biological system or questions, we believe this figure will serve as a gateway to the genetic alterations conducted in the experiments.
Along these lines, it would be good to have a better reasoning for the combination of experiments conducted. It is left to the reader to understand why certain types of experiments have been done.
It was not clear to us at the outset of these experiments what results would ultimately emerge and what follow-up experiments would be necessary as our initial hypotheses were proven wrong with many of the surprises from the work. So, there was no a priori reasoning for why experiments were done until we had the results of the previous experiments. We agree that this makes the reading a bit confusing. As such, we clarified the logic flow in the results section as the narrative progresses from experiment to experiment, and we reorganized some of the introduction to improve transition statements and offer a roadmap to readers earlier on.
On the other hand, the introduction misses a section on the biology of the phage and its interaction with the host(s). It is hard to understand the biology of the system without getting an understanding of the insect - Wolbachia - phage interactions. For non-specialist, understanding the role of the three players is essential for the system.
Thank you for the suggestion. We now add a section introducing phage WO and its relevance to the phenotypes tested here.
“The wmk gene and two cytoplasmic incompatibility factor (cif) genes that underlie cytoplasmic incompatibility (a parasitism phenotype whereby offspring die in crosses between infected males and uninfected females) occur in the eukaryotic association module (EAM) of prophage WO, which refers to the phage WO genome that is inserted into the bacterial chromosome. The EAM is common in WO phages across several Wolbachia strains and is rich in genes that are homologous to eukaryotic genes or annotated with eukaryotic functions. As such, the expression of reproductive parasitism genes from the EAM and tripartite interactions between phage WO, Wolbachia, and eukaryotic hosts are central to Wolbachia’s ability to interact with and modify host reproduction.”
The result section could be easily shortened by focusing on the essential experiments. Experiments that do not contribute to the final result can go into the supplement.
We removed redundant sentences and made some figures supplemental.
Also the discussion is much too long. I suggest to reduce it to half and focus on the important points and the take-home messages. Currently the discussion follows the way the results are presented in the result section. However, this is not needed. The important finding should be discussed first. Findings that are important in the development of the project, may not be important for the biology of the system overall. And they may not be important for the reader.
We reordered the discussion to cover the biggest findings first, and removed about a third of the original writing in the discussion.
Reviewer #2:
This study aims to unravel the genomic basis to wmk-induced male killing by transgenically expressing homologs of varying relatedness, with synonymous nucleotide changes, and predicted alternative start codons in D. melanogaster flies. The study builds on previous work showing that expression of wmk in fly embryos recapitulates several aspects of male killing. While more distantly related homologs did not induce male killing when expressed in D. melanogaster, more closely related wmk homologs induce either killing of both sexes or male killing only. However, the male-killing phenotype was not due to amino acid differences, but associated with RNA structural differences of the different wmk homologs. In addition, only one synonymous nucleotide change was sufficient to ablate the killing phenotype. These findings suggests that minor and even silent nucleotide differences impact on the expression of male killing in D. melanogaster. It is concluded that a new model incorporating the impacts of RNA structure and post-transcriptional processes in wmk-induced male killing needs to be developed.
The strength of the study lies in the systematic and carefully controlled approach to quantify the phenotypic effects of both sequence and structural changes to various wmk homologs for inducing the male-killing phenotype. Detailed dissection of the phenotypic impact of minor changes to the wmk homologs including sequence variation, silent nucleotide changes, and RNA structural differences was quantified. This approach reveals a complex genotype-phenotype relationship, but highlights the importance of including post-translational processes. The data is novel in that previous work have largely ignored structural changes and assumed that synonymous differences in codons has no effect on protein function, whereas the current study based on updated codon optimization algorithms reveal that this assumption is incorrect. The finding highlights the importance of considering also structural genetic variation for phenotypic expression differences. This suggestion is further corroborated by the lack of difference in wmk homologue expression levels, indicating that the functional differences are due to post-translational effects.
We thank the reviewer for the thoughtful comments.
There are limitations to the findings of this complex genotype-phenotype relationship. The current study only examined the phenotypic impact by expressing the different homologs in one D. melanogaster genetic background. Given the variability of the phenotypic pattern revealed based on minor changes to the wmk homologs, it will be critical to repeat some of the main findings in other D. melanogaster genotypes to determine the importance of the variation in the wmk homologs more generally. It is entirely plausible that the observed changes in the effect and strength of killing is due to an interaction between host and wmk genotype. This has implications for unravelling the underlying genetic basis to the male-killing phenotype more widely. It is as yet to be demonstrated whether wmk is involved in male killing in natural population, and to what extent there are shared patterns and mechanisms of male killing induced by other bacterial endosymbionts such as Spiroplasma.
We addressed this point in more detail above in the first response to the comments from Reviewer 1.
-
Evaluation Summary:
This study aims to find the genetic mechanisms underlying sex-ratio distortion through male-killing in Drosophila melanogaster flies infected with the endosymbiont Wolbachia. The endosymbiont carries the prophage WO, which is in the center of interested in this study. The key result of this study is that a synonymous mutation in a prophage gene can explain the differences between sex-ratio distorting and not distorting symbionts.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
-
Reviewer #1 (Public Review):
This study aims to find the genetic mechanisms underlying sex-ratio distortion through male-killing in Drosophila melanogaster flies infected with the endosymbiont Wolbachia. The endosymbiont carries the prophage WO, which is in the center of interested in this study. The key result of this study is that a synonymous mutation in a prophage gene can explain the differences between sex-ratio distorting and not distorting symbionts. The study uses transgene technology to modify phage genes and to investigate which changes in the gene is involved in the phenotype. The finding, that a synonymous SNP plays a key role is not entirely novel in biology, but there are only few examples known of this type of genotype - phenotype associations. The study does not include experiments to show that the main finding is not …
Reviewer #1 (Public Review):
This study aims to find the genetic mechanisms underlying sex-ratio distortion through male-killing in Drosophila melanogaster flies infected with the endosymbiont Wolbachia. The endosymbiont carries the prophage WO, which is in the center of interested in this study. The key result of this study is that a synonymous mutation in a prophage gene can explain the differences between sex-ratio distorting and not distorting symbionts. The study uses transgene technology to modify phage genes and to investigate which changes in the gene is involved in the phenotype. The finding, that a synonymous SNP plays a key role is not entirely novel in biology, but there are only few examples known of this type of genotype - phenotype associations. The study does not include experiments to show that the main finding is not limited to one particular background of the fly line used. An experiment including multiple genotypes would be needed to show this.
The study is mostly clear and easy to follow, but requires a lot of attention. The authors choose to build up the story as I guess it was carried out in the lab. Thus, the reader is guided through every step of the process. While I see that this is appealing from the way the study was carried out, it results in a very long manuscript with a lot of material that would be much better placed in a supplement.
The introduction seems unfocused. It meanders around, jumping from topic to topic and does not give the reader a sense of where things will go. Fig. 1 gives an overview about the different aspects addressed here, but it is not used to guide the reader through the different lines of thought addressed in the introduction. If Fig. 1 will stay (I actually think it is not needed) it should be introduced earlier and used as a road map for the paper. Alternatively, the introduction could stay more general and only in the last paragraph the different ways the system is studied will be summarized. Along these lines, it would be good to have a better reasoning for the combination of experiments conducted. It is left to the reader to understand why certain types of experiments have been done. On the other hand, the introduction misses a section on the biology of the phage and its interaction with the host(s). It is hard to understand the biology of the system without getting an understanding of the insect - Wolbachia - phage interactions. For non-specialist, understanding the role of the three players is essential for the system.
The result section could be easily shortened by focusing on the essential experiments. Experiments that do not contribute to the final result can go into the supplement.
Also the discussion is much too long. I suggest to reduce it to half and focus on the important points and the take-home messages. Currently the discussion follows the way the results are presented in the result section. However, this is not needed. The important finding should be discussed first. Findings that are important in the development of the project, may not be important for the biology of the system overall. And they may not be important for the reader.
-
Reviewer #2 (Public Review):
This study aims to unravel the genomic basis to wmk-induced male killing by transgenically expressing homologs of varying relatedness, with synonymous nucleotide changes, and predicted alternative start codons in D. melanogaster flies. The study builds on previous work showing that expression of wmk in fly embryos recapitulates several aspects of male killing. While more distantly related homologs did not induce male killing when expressed in D. melanogaster, more closely related wmk homologs induce either killing of both sexes or male killing only. However, the male-killing phenotype was not due to amino acid differences, but associated with RNA structural differences of the different wmk homologs. In addition, only one synonymous nucleotide change was sufficient to ablate the killing phenotype. These …
Reviewer #2 (Public Review):
This study aims to unravel the genomic basis to wmk-induced male killing by transgenically expressing homologs of varying relatedness, with synonymous nucleotide changes, and predicted alternative start codons in D. melanogaster flies. The study builds on previous work showing that expression of wmk in fly embryos recapitulates several aspects of male killing. While more distantly related homologs did not induce male killing when expressed in D. melanogaster, more closely related wmk homologs induce either killing of both sexes or male killing only. However, the male-killing phenotype was not due to amino acid differences, but associated with RNA structural differences of the different wmk homologs. In addition, only one synonymous nucleotide change was sufficient to ablate the killing phenotype. These findings suggests that minor and even silent nucleotide differences impact on the expression of male killing in D. melanogaster. It is concluded that a new model incorporating the impacts of RNA structure and post-transcriptional processes in wmk-induced male killing needs to be developed.
The strength of the study lies in the systematic and carefully controlled approach to quantify the phenotypic effects of both sequence and structural changes to various wmk homologs for inducing the male-killing phenotype. Detailed dissection of the phenotypic impact of minor changes to the wmk homologs including sequence variation, silent nucleotide changes, and RNA structural differences was quantified. This approach reveals a complex genotype-phenotype relationship, but highlights the importance of including post-translational processes. The data is novel in that previous work have largely ignored structural changes and assumed that synonymous differences in codons has no effect on protein function, whereas the current study based on updated codon optimization algorithms reveal that this assumption is incorrect. The finding highlights the importance of considering also structural genetic variation for phenotypic expression differences. This suggestion is further corroborated by the lack of difference in wmk homologue expression levels, indicating that the functional differences are due to post-translational effects.
There are limitations to the findings of this complex genotype-phenotype relationship. The current study only examined the phenotypic impact by expressing the different homologs in one D. melanogaster genetic background. Given the variability of the phenotypic pattern revealed based on minor changes to the wmk homologs, it will be critical to repeat some of the main findings in other D. melanogaster genotypes to determine the importance of the variation in the wmk homologs more generally. It is entirely plausible that the observed changes in the effect and strength of killing is due to an interaction between host and wmk genotype. This has implications for unravelling the underlying genetic basis to the male-killing phenotype more widely. It is as yet to be demonstrated whether wmk is involved in male killing in natural population, and to what extent there are shared patterns and mechanisms of male killing induced by other bacterial endosymbionts such as Spiroplasma.
-
-
-
-