A guide to Agrobacterium-mediated transformation of the chytrid fungus Spizellomyces punctatus
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Chytrid fungi play key ecological roles in aquatic ecosystems, and some species cause a devastating skin disease in frogs and salamanders. Additionally, chytrids occupy a unique phylogenetic position– sister to the well-studied Dikarya (the group including yeasts, sac fungi, and mushrooms) and related to animals– making chytrids useful for answering important evolutionary questions. Despite their importance, little is known about the basic cell biology of chytrids. A major barrier to understanding chytrid biology has been a lack of genetic tools with which to test molecular hypotheses. Medina and colleagues recently developed a protocol for Agrobacterium -mediated transformation of Spizellomyces punctatus . In this manuscript, we describe the general procedure including planning steps and expected results. We also provide in-depth, step-by-step protocols and video guides for performing the entirety of this transformation procedure on protocols.io (dx.doi.org/10.17504/protocols.io.x54v9dd1pg3e/v1).
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In this revised version of the manuscript "A guide to Agrobacterium-mediated transformation of the chytrid fungus Spizellomyces punctatus", the authors have addressed all the reviewers' comments and suggestions. Thus, this manuscript is now suitable for publication. I would like to thank the authors for their diligence in amending the manuscript. Congratulations!
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In summary, the study presents a detailed description of method for Agrobacterium-mediated transformation of the chytrid fungus Spizellomyces punctatus. After the peer-review process, several changes have been proposed and suggested to improve the quality of the manuscript prior to publication. Please, consider all the reviewers’ suggestions and comments thoroughly, especially those concerning indicating information about the transformation frequency, detection of transformant and stability of the modifications. Additionally, the reviewers have suggested some point to enrich the discussion section that may be of interest for prospective readers. Please provide a revised manuscript containing all changes and a point-by-point response to all the reviewers’ comments within 1 month.
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Comments to Author
The ms by Prostak et al. represents a detailed protocol and guide to Agrobacterium-mediated transformation of the chytrid fungus Spizellomyces punctatus as was published by the same group of authors before (in less practical detail) in Curr. Biol. (ref.1). This protocol will be helpful to scientists interested in applying this protocol, especially when not experienced in Agrobacterium-mediated transformation of other fungal species. The results ms starts with a short general introduction to the field, followed by the step-by-step protocol. In the protocol I miss indications as to how many transformants are generally obtained, how presence of the T-DNA is assessed in the hygromycin resistant colonies obtained (by PCR?) and whether transformation is stable as indicated by T-DNA presence in the genome. …
Comments to Author
The ms by Prostak et al. represents a detailed protocol and guide to Agrobacterium-mediated transformation of the chytrid fungus Spizellomyces punctatus as was published by the same group of authors before (in less practical detail) in Curr. Biol. (ref.1). This protocol will be helpful to scientists interested in applying this protocol, especially when not experienced in Agrobacterium-mediated transformation of other fungal species. The results ms starts with a short general introduction to the field, followed by the step-by-step protocol. In the protocol I miss indications as to how many transformants are generally obtained, how presence of the T-DNA is assessed in the hygromycin resistant colonies obtained (by PCR?) and whether transformation is stable as indicated by T-DNA presence in the genome. In general, the presentation is clear. I have also the following detailed comments: line 69: Although there are two papers indicating that sea urchin and human cells are transformed by Agro, these studies do not exclude that this occurred through uptake of DNA from lysed bacteria. Also they have never seen a follow-up and AMT is sofar NOT used for animals. Therefore, in this ms it would be more in balance to refer to plants and fungi, for which AMT is in use worldwide. line 89: is growth at 28 C really necessary. In fact Agro grows even faster at 30 C, but transformation occurs preferably at lower temp (20-25 C). line 120: would more gentle shaking not be more appropriate immediately after electroporation? line 136: 40M MES? line 146: mixed instead of co-cultured. Co-cultivation occurs after this step lines 135-152 co-cultivation. Traditionally, co-cultivation of Agro and fungi is performed on membrane filters which are placed on the medium in the petri dish. Why did the authors chose to perform co-cultivation directly on the medium in a depresion in the medium? This is much more laborious and maybe less efficient. Did they test filters and didn't the procedure work with filters? line 146/149 and throughout: here the abbreviation Agro is used instead of Agrobacterium in full. And also Sp instead of the full name of the fungus. This should be explained and be in accordance with journal style. I would prefer a similar abbreviation, if allowed. for example: A.t. for Agroabcterium tumefaciens and S.p. for the fungus. line 3 protocol4: DNA instead of DAN line 4 protocol4: acetosyringone is NOT a hormone, but a vir-inducer p11 line 10, 11: Carb and Tet abbreviations are not explained
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Very good
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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Comments to Author
This 'methods' paper presents a protocol for the transformation of the chytrid species Spizellomyces punctatus with T-DNA delivered from Agrobacterium tumefaciens. To date, S. punctatus is the only species in amongst a suite of early-diverging lineages of the fungi that can be stably transformed, a tool that can open up this and other species to direct testing of gene functions or other molecular biology discoveries. 1. Methodological rigour, reproducibility and availability of underlying data The paper is structured as a conventional article that provides a brief overview of the methods, which include generating a suitable Agrobacterium strain, culturing bacteria and fungi, the transformation media and process, and then selection of transgenic S. punctatus lines. The previous studies have reported …
Comments to Author
This 'methods' paper presents a protocol for the transformation of the chytrid species Spizellomyces punctatus with T-DNA delivered from Agrobacterium tumefaciens. To date, S. punctatus is the only species in amongst a suite of early-diverging lineages of the fungi that can be stably transformed, a tool that can open up this and other species to direct testing of gene functions or other molecular biology discoveries. 1. Methodological rigour, reproducibility and availability of underlying data The paper is structured as a conventional article that provides a brief overview of the methods, which include generating a suitable Agrobacterium strain, culturing bacteria and fungi, the transformation media and process, and then selection of transgenic S. punctatus lines. The previous studies have reported on the success of the transformation method (i.e. Medina et al. 2020). It would be interesting to know how often the authors have had success or failure using the method, and if it works on more than the single S. punctatus strain reported to date. 2. Presentation of results The steps for transformation are illustrated with four figures, and a fifth shows the outcome of transformation as colonies on a petri dish and a micrograph of a transformed line expressing a fluorescent protein. These will help readers trying the method for the first time. 3. How the style and organization of the paper communicates and represents key findings One query on presentation is that in addition to the paper, there is also a 40 minute video that has been recorded and a more detailed set of methods that have been deposited to protocols.io. These are likely the primary resources investigators would first use, rather than the paper, so it would be worth considering how best to integrate those resources into the manuscript, e.g. would this be through upload to the journal website or links off the paper? 4. Literature analysis or discussion As a methods paper on the one chytrid that can be transformed, there is little need for extensive text. However, the Discussion section is brief. That paragraph can be summarized as suggesting the next steps are to use the method in S. punctatus and then develop the same transformation method in other chytrids. There is scope to consider the practicalities of the next steps, especially given it is now well over three years since the original transformation method was deposited in bioRxiv. As someone contemplating trying this method, a consideration would be why has it not been reported again since the first report? Questions could be: Will ploidy or nucleus numbers of chytrid lineages or cell types, and the implications this would have on gene manipulation? That is, how easy would it be to generate lines homozygous for gene disruptions, or would something like RNAi have to be used to impair gene expression (and if so, do these species have the RNAi machinery)? Or is another alternative a gene editing approach (but that might then just need electroporation)? Will the same plasmids and selection media work for other species, or will they need changing, e.g. different promoters to drive the selectable marker? It seems that Swafford et al. 2020 Sci Rep might offer some answers. 5. Any other relevant comments A few minor typographical or other points for consideration are as follows. Lines 15 and 36: recommend editing the phrase 'yeasts, sac fungi, and their multicellular relatives' as this is an odd definition of the Dikarya. Line 47: 'motile cilia' reflects my ignorance wherein I think of cilia on things like Paramecium and flagella on chytrids. Are these the same structures, and are there non-motile versions as well? Perhaps it would be good to expand the text here for those more familiar with the Dikarya. Line 137: add a space between 'acetyosringone (Sigma'. Lines 156-157: 'conical'. Does this mean a conical flask or a 50 ml Falcon tube? Throughout the text: check correct use of the micron symbol instead of 'u', e.g. on lines 154, 160, 168 and on figure 2 and 5B. Line 207: delete 'of growth'. Line 212: add italics to 'Sp'. Line 264: missing species name. Line 271: delete 'table of contents'. Figure 1: spelling of 'selection'. Line 358: spelling of 'co-culturing'.
Please rate the manuscript for methodological rigour
Very good
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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