A cellular and molecular atlas reveals the basis of chytrid development
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Evaluation Summary:
This work provides transcriptional profiling and structural insight into chytrid development and will very likely stimulate further work on this model.
(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.
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Abstract
The chytrids (phylum Chytridiomycota) are a major fungal lineage of ecological and evolutionary importance. Despite their importance, many fundamental aspects of chytrid developmental and cell biology remain poorly understood. To address these knowledge gaps, we combined quantitative volume electron microscopy and comparative transcriptome profiling to create an ‘atlas’ of the cellular and molecular basis of the chytrid life cycle, using the model chytrid Rhizoclosmatium globosum . From our developmental atlas, we describe the transition from the transcriptionally inactive free-swimming zoospore to the more biologically complex germling, and show that lipid processing is multifaceted and dynamic throughout the life cycle. We demonstrate that the chytrid apophysis is a compartmentalised site of high intracellular trafficking, linking the feeding/attaching rhizoids to the reproductive zoosporangium, and constituting division of labour in the chytrid cell plan. We provide evidence that during zoosporogenesis, zoospores display amoeboid morphologies and exhibit endocytotic cargo transport from the interstitial maternal cytoplasm. Taken together, our results reveal insights into chytrid developmental biology and provide a basis for future investigations into non-dikaryan fungal cell biology.
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Author Response:
Reviewer #1 (Public Review):
Major points
- Zoospores in several chytrids have been shown to be transcriptionally and translationally inactive, this means that the distribution of transcripts are maternally allocated. Although the authors do cite two papers on the topic in the discussion, this is a fundamental concept that might not be in the mind of non-specialist readers and the authors need to introduce and discuss from the beginning (see PMID: 4412066, 1259436, 3571161), as it provides key context for their finding that germlings have a wider range of transcriptional activity as this is consistent with Rg also being transcriptionally silent in the zoospore state. Finally, the language used to describe transcripts found in zoospores (the manuscript refers to them "expressing" particular genes) is confusing given …
Author Response:
Reviewer #1 (Public Review):
Major points
- Zoospores in several chytrids have been shown to be transcriptionally and translationally inactive, this means that the distribution of transcripts are maternally allocated. Although the authors do cite two papers on the topic in the discussion, this is a fundamental concept that might not be in the mind of non-specialist readers and the authors need to introduce and discuss from the beginning (see PMID: 4412066, 1259436, 3571161), as it provides key context for their finding that germlings have a wider range of transcriptional activity as this is consistent with Rg also being transcriptionally silent in the zoospore state. Finally, the language used to describe transcripts found in zoospores (the manuscript refers to them "expressing" particular genes) is confusing given this context.
As requested, we have added to the introduction on the biology of zoospores related to transcription/translation inactivity and maternally deposited mRNA (L 77-79) and have included the suggested references in the discussion section (L 444-445). We have checked and where appropriate revised the manuscript in terms of language used to describe transcripts in zoospores (e.g. 157, 161, 217, 229).
- The authors correlated structural changes observed with general KEGG pathway profiles obtained from transcriptomics. Unfortunately it is hard to pin down exactly what the authors are trying to say about this data because their observations are not placed with precision in the context of what is already known about chytrid development, and KEGG pathways are too broad to be very informative. Drawing inferences about chytrid biology from broad KEGG categories and link them to structural observation is not possible with more detailed molecular analysis. This comes up multiple times: (i) Correlation between an increase in endomembrane structures in a compartment and enrichment of KEGG categories of protein processing and ER etc is not enough to link these endomembrane systems with ER. This requires more direct evidence. (ii) High dynamic activity and endomembrane density in the apophysis is not evidence enough by itself to support the claim of the "apophysis acting as a cellular junction that regulates intracellular traffic." (iii) Although different lipid composition between zoospore and germling, and differences in KEGG categories of peroxisome activity on the other suggest important lipid metabolic changes, these correlations are is not hard enough evidence for the authors to call this process as a "biological characteristic" of the transition from zoospore to germling.
We have revised the manuscript to limit the proposed correlation between transcriptome data and the structural changes. We have also highlighted aspects of our work that requires future study. To complement the KEGG output, we also provide GOs as supplementary information so as not to add to the already data-rich manuscript (Figure 3 - Figure supplement 2-5). Please note that were relevant, we have highlighted specific transcripts and not only relied on KEGG categories.
- The claim that zoospores inside the sporangium undergo phagocytosis is not sufficiently supported by the data presented. To date there is only one case in which it a fungus undergoes a process akin to phagocytosis (i.e. Rozella), and finding a phagocytic fungus would be a very exciting result. Unfortunately, the authors provide no direct evidence to support this specific claim as (i) there are many ways one could imagine to explain the shapes seen in the EM data (perhaps the zoospores are squeezed around the the objects), and classic work on Allomyces and Blastocladiella zoosporogenesis indicates that cleavage vesicles can be orderly or very irregular before they align in continuous plates (sometimes concomitant with formation of ribosome aggregates), and that these cleavage planes are nearly complete, but not complete yet. (ii) The genes discussed are not specific to phagocytosis, but are used for a wide variety of other functions. Moreover, the authors appear to equate endocytosis and phagocytosis, and although there is some overlap in the proteins used for these processes, they are not equivalent.
We have revised the manuscript to limit claim about zoospores and emphasised that future work is needed on this topic. We have included Rozella as highlighted by the reviewer
- Although the author's findings about the complex endomembrane system in Rg apophysis is interesting, the details of the images provided do not support their interpretation of it being a "distinct subcellular structure". Such claims require detailed imaging of the "pseudo-septum", similar to what has been shown for "plasmodesmata" in Entophlyctis and Blastocladiella.
We agree that the complex endomembrane system in the apophysis is interesting and is one of the novel aspects of our study. In the revised manuscript, we have limited this claim and proposed future work.
Reviewer #2 (Public Review):
There are a lot of figures to present the data collected in this manuscript. It is a tour de force integrating methods though is a bit overwhelming on first or second read of the manuscript. But I think the primary and supplemental figures do provide necessary information to convey so I am not sure how I would suggest any other compaction of the presented material
As discussed above with Reviewer 1, in the revised version of the manuscript we have updated the figure. Figure 2 has been separated to two figures (now Figures 2 and 3).
There was more variation in the lipid fraction estimates for the germling and sometimes zoospore replicates. Does this suggest non-synchronization of the cells or just that there is a lot of variation of size and stage within the timepoint?
Based on our microscope assessments of the life stages (Figure 1 - Figure supplement 1), we are confident that stages 1 (zoospore), 2 (germling) and 3 (immature thallus) were synchronized.
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Evaluation Summary:
This work provides transcriptional profiling and structural insight into chytrid development and will very likely stimulate further work on this model.
(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.
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Reviewer #1 (Public Review):
Here Laundon et. al report a cellular "atlas" of the model chytrid Rhizoclosmatium globosum. The data presented include beautiful and informative 3D reconstructions of all four key life stages of this species, as well as transcriptional profiling of matched samples and analysis of lipid compositions. The data were collected from multiple biological replicates and represent a clearly important resource for the community. With this work, the goal of the authors is to link structural descriptions of chytrid morphology with molecular understanding: this is something that the field needs. The authors describe results in three areas that are very interesting to the field. Unfortunately, the evidence provided for these findings does not always support their conclusions. Additionally, discussion of the literature is …
Reviewer #1 (Public Review):
Here Laundon et. al report a cellular "atlas" of the model chytrid Rhizoclosmatium globosum. The data presented include beautiful and informative 3D reconstructions of all four key life stages of this species, as well as transcriptional profiling of matched samples and analysis of lipid compositions. The data were collected from multiple biological replicates and represent a clearly important resource for the community. With this work, the goal of the authors is to link structural descriptions of chytrid morphology with molecular understanding: this is something that the field needs. The authors describe results in three areas that are very interesting to the field. Unfortunately, the evidence provided for these findings does not always support their conclusions. Additionally, discussion of the literature is insufficient as previous work provides crucial context for interpreting the data presented.
Major points
1. Zoospores in several chytrids have been shown to be transcriptionally and translationally inactive, this means that the distribution of transcripts are maternally allocated. Although the authors do cite two papers on the topic in the discussion, this is a fundamental concept that might not be in the mind of non-specialist readers and the authors need to introduce and discuss from the beginning (see PMID: 4412066, 1259436, 3571161), as it provides key context for their finding that germlings have a wider range of transcriptional activity as this is consistent with Rg also being transcriptionally silent in the zoospore state. Finally, the language used to describe transcripts found in zoospores (the manuscript refers to them "expressing" particular genes) is confusing given this context.
2. The authors correlated structural changes observed with general KEGG pathway profiles obtained from transcriptomics. Unfortunately it is hard to pin down exactly what the authors are trying to say about this data because their observations are not placed with precision in the context of what is already known about chytrid development, and KEGG pathways are too broad to be very informative. Drawing inferences about chytrid biology from broad KEGG categories and link them to structural observation is not possible with more detailed molecular analysis. This comes up multiple times: (i) Correlation between an increase in endomembrane structures in a compartment and enrichment of KEGG categories of protein processing and ER etc is not enough to link these endomembrane systems with ER. This requires more direct evidence. (ii) High dynamic activity and endomembrane density in the apophysis is not evidence enough by itself to support the claim of the "apophysis acting as a cellular junction that regulates intracellular traffic." (iii) Although different lipid composition between zoospore and germling, and differences in KEGG categories of peroxisome activity on the other suggest important lipid metabolic changes, these correlations are is not hard enough evidence for the authors to call this process as a "biological characteristic" of the transition from zoospore to germling.
3. The claim that zoospores inside the sporangium undergo phagocytosis is not sufficiently supported by the data presented. To date there is only one case in which it a fungus undergoes a process akin to phagocytosis (i.e. Rozella), and finding a phagocytic fungus would be a very exciting result. Unfortunately, the authors provide no direct evidence to support this specific claim as (i) there are many ways one could imagine to explain the shapes seen in the EM data (perhaps the zoospores are squeezed around the the objects), and classic work on Allomyces and Blastocladiella zoosporogenesis indicates that cleavage vesicles can be orderly or very irregular before they align in continuous plates (sometimes concomitant with formation of ribosome aggregates), and that these cleavage planes are nearly complete, but not complete yet. (ii) The genes discussed are not specific to phagocytosis, but are used for a wide variety of other functions. Moreover, the authors appear to equate endocytosis and phagocytosis, and although there is some overlap in the proteins used for these processes, they are not equivalent.
4. Although the author's findings about the complex endomembrane system in Rg apophysis is interesting, the details of the images provided do not support their interpretation of it being a "distinct subcellular structure". Such claims require detailed imaging of the "pseudo-septum", similar to what has been shown for "plasmodesmata" in Entophlyctis and Blastocladiella.
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Reviewer #2 (Public Review):
The authors are developing and investigating a molecular atlas of the cell for the chytrid Rhizoclosmatium globosum. They are linking transcriptome and lipidome information data to cellular biology that can be observed through SBF-SEM. The detailed investigation allowed a development of a cell atlas and interpretation of cell wall and organelle dynamics.
The authors successfully explored several hypotheses about development in chytrids including that zoospores are provisioned with maternal mRNA. They interpret the composition of spores to include both essential machinery and instructions for cellular replication but also have more host- or substrate-interaction products primed for the cell to condition development on external signals.
They also explore whether cell wall genes are expressed in a fashion that …
Reviewer #2 (Public Review):
The authors are developing and investigating a molecular atlas of the cell for the chytrid Rhizoclosmatium globosum. They are linking transcriptome and lipidome information data to cellular biology that can be observed through SBF-SEM. The detailed investigation allowed a development of a cell atlas and interpretation of cell wall and organelle dynamics.
The authors successfully explored several hypotheses about development in chytrids including that zoospores are provisioned with maternal mRNA. They interpret the composition of spores to include both essential machinery and instructions for cellular replication but also have more host- or substrate-interaction products primed for the cell to condition development on external signals.
They also explore whether cell wall genes are expressed in a fashion that links to the cell wall-less spore stage, and seem to indicate there are at least one chitin synthase with high levels indicating preparation for dynamic growth and wall formation.
The RNASeq analysis/GO enrichment pointed to secondary metabolism enrichment in some of the comparisons, but there is little discussion of what types of genes these might be in the manuscript. Are these NRPS and siderophore or other product producing genes that contribute to that enrichment category?
The work will have an important impact in the field of cell biology of chytrids but also broadly to Fungi and perhaps also comparative biology of Opisthokonts. The detailed reconstruction and examination of the cellular structures in this lineage should be informative to how transitions occurred in the development of septa in other flagellated lineages (eg Blastocladiomycota) and in Dikarya. The development from encysting zoospore to thallus is clearly complex and this study gives a high resolution and a dynamic examination of the processes.
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