RNAPII Degradation Factor Def1 Is Required for Development, Stress Response, and Full Virulence of Magnaporthe oryzae
and Weixiang Wang 1,*
Round 1
Reviewer 1 Report
Comments:
To the Editor Prof.
The manuscript “RNAPII Degradation Factor Def1 is Required for Development, Stress Response, and Full Virulence of Magnaporthe oryzae, investigate the role of Def1 during development and infection of the rice blast fungus Magnaporthe oryzae. The study is interesting and of great practical importance. The experimental studies are mostly carried out professionally. The article satisfies the criteria of the Journal of Fungi. As detailed data and interpretation, I recommend it for an international audience in this journal, however some points have to be précised and a minor revision is requested.
1. Materials and methods, Line 103, make M. oryzae italic.
2. Materials and methods, Line 103, "All strains used in this study' one strain or more than one were used.
With best regards
Author Response
The manuscript “RNAPII Degradation Factor Def1 is Required for Development, Stress Response, and Full Virulence of Magnaporthe oryzae, investigate the role of Def1 during development and infection of the rice blast fungus Magnaporthe oryzae. The study is interesting and of great practical importance. The experimental studies are mostly carried out professionally. The article satisfies the criteria of the Journal of Fungi. As detailed data and interpretation, I recommend it for an international audience in this journal, however some points have to be precised and a minor revision is requested.
Response: Thank a lot for your kind comments.
- Materials and methods, Line 103, make M. oryzae italic.
Response: Thanks, we have revised as the suggestion (Line 110).
- Materials and methods, Line 103, "All strains used in this study' one strain or more than one were used.
Response: Thank for the reminder. We used more than one strains, and the description was added in Line 109-112: “All strains used in this study including the wild type, deletion mutants, complementation strains, as well as point-mutation strains, .....”
Reviewer 2 Report
In the present study, authors have characterized the Def1 gene in Magnaporthe oryzae for its role in various activities such as mycelial growth, conidia formation and pathogenicity during infection process. They have also shown that the gene is involved in response to multiple stresses and plays important role as growth of the mutated M. oryzae is severely affected under stress conditions. It is a noteworthy attempt to characterize this important Def1 gene in M. oryzae for the first time.
I have few concerns that need to be clarified.
It is well known that the Def1 gene is involved in DNA repair, crossover recombination during meiosis, maintaining chromosome and genomic integrity, etc. Therefore, deleting this gene from the genome can severely affect these processes. As these are some of the fundamental processes of life, deleting this gene may lead to non survival of such fungi. Did the authors wonder how these functions are being carried out in mutant fungi? Have the authors studied any of these processes in this mutant fungi having deleted Def1? And if so whether these processes are normal or found any defects e.g. telomere length.
Major comment: There are several grammatical mistakes in language and need thorough correction before accepting the manuscript.
Abstract: Do not use abbreviations in abstract. Use full forms of RNAPII and DefI
Materials and methods
Line 103: write the scientific name in italics M. oryzae
Line 122: Oryza sativa make it italics
Line 123: Hordeum vulgare make it italics
Author Response
In the present study, authors have characterized the Def1 gene in Magnaporthe oryzae for its role in various activities such as mycelial growth, conidia formation and pathogenicity during infection process. They have also shown that the gene is involved in response to multiple stresses and plays important role as growth of the mutated M. oryzae is severely affected under stress conditions. It is a noteworthy attempt to characterize this important Def1 gene in M. oryzae for the first time.
I have few concerns that need to be clarified.
Response: Thank a lot for your kind comments.
It is well known that the Def1 gene is involved in DNA repair, crossover recombination during meiosis, maintaining chromosome and genomic integrity, etc. Therefore, deleting this gene from the genome can severely affect these processes. As these are some of the fundamental processes of life, deleting this gene may lead to non survival of such fungi. Did the authors wonder how these functions are being carried out in mutant fungi? Have the authors studied any of these processes in this mutant fungi having deleted Def1? And if so whether these processes are normal or found any defects e.g. telomere length.
Response: We thank the reviewer’s questions which are very interesting and important. Indeed, Def1 plays roles in key biological processes and a deletion could affect fundamental processes of life. But according to our results, the deletion mutants were still able to survive. However, due to the limited time and technical problems, at the present we are not able to evaluate the effects of Def1 deletion in fundamental processes such as telomere length. This is an interesting topic and is worth to be planned a new work in the future.
Major comment: There are several grammatical mistakes in language and need thorough correction before accepting the manuscript.
Abstract: Do not use abbreviations in abstract. Use full forms of RNAPII and Def1
Materials and methods
Line 103: write the scientific name in italics M. oryzae
Line 122: Oryza sativa make it italics
Line 123: Hordeum vulgare make it italics
Response: Thanks for the kind suggestion, we have tried our best to revise the grammatical mistakes in language with a thorough correction, hoping that it should be much better now.
Reviewer 3 Report
The manuscript identified Def1-like gene in M. oryzae based on its similarity to Def1 sequence found in S. cerevisiae. Def1 sequence was deleted from M. oryzae using gene displacement strategy. The knockout mutants were compared to the wild type strain for the mycelia growth, conidia formation, pathogenicity and stress response. Additionally, a O-GlcNAcylation site mutant was generated to determine the O-GlcNAc modification site in Def1 that results in a reduced protein stability of Def1. Overall, the results support the conclusion, but the materials and methods section needs to be more detailed. Too many typos or grammatical errors to list them all. Supplementary data needs to be improved to include a lot of information that was omit in the main text.
Figure 1b, please add the Def1 from M. oryzae in the phylogenetic tree. Italicize the fungal names in Fig 1b.
Line 106, how are the conidia harvested?
For Figure 3D, how many conidia were counted for different morphology? Change “conidinphore” in 3A description to “canidiophore. How was it quantified?
Line 183, change to “through a gene displacement strategy using a split-marker approach”. Same in Line 108, because the gene was replaced by a hygromycin B gene. How was monokaryotic deletion confirmed. Please list the primers in a supplementary table if PCR were done.
Line 113, please clarify which vector containing the neomycin resistant gene was used to complement the gene.
In section 2.2, please describe how protoplast was made and how transformation was done.
In Line 110, please provide primer sequences in a supplementary table.
Line 116, spell out what CM stands for.
Section 2.6, how are percent of cells stained estimated or counted?
Minor:
Many grammatical errors, please have an English editor look over throughout. Here are just a few examples from the first part of paragraph 1.
Line 36: Change “occur severely” to “experience severe”
Line 41: change “preferential” to “preferentially”
Line 44: change “evolutionary” to “evolutionarily”
Line 46: change “dissociate” to “dissociates”
Author Response
The manuscript identified Def1-like gene in M. oryzae based on its similarity to Def1 sequence found in S. cerevisiae. Def1 sequence was deleted from M. oryzae using gene displacement strategy. The knockout mutants were compared to the wild type strain for the mycelia growth, conidia formation, pathogenicity and stress response. Additionally, a O-GlcNAcylation site mutant was generated to determine the O-GlcNAc modification site in Def1 that results in a reduced protein stability of Def1. Overall, the results support the conclusion, but the materials and methods section needs to be more detailed. Too many typos or grammatical errors to list them all. Supplementary data needs to be improved to include a lot of information that was omit in the main text.
Response: Thank a lot for your kind comments.
Figure 1b, please add the Def1 from M. oryzae in the phylogenetic tree. Italicize the fungal names in Fig 1b.
Response: Thanks for the reminder, we have revised the phylogenetic tree as suggested.
Line 106, how are the conidia harvested?
Response: The conidia harvest method was described in Line 114-115: “Conidia from 7-day-old colonies cultured on OTA plates were washed down with 0.025% Tween 20 and adjusted to a proper concentration for inoculation. ”.
For Figure 3D, how many conidia were counted for different morphology? Change “conidinphore” in 3A description to “canidiophore. How was it quantified?
Response: Thanks for the kind reminder, relevant figure legend was added in Line 245: “For each strain, at least 100 conidia were counted.” And the typo of “conidiophore” has been revised.
Line 183, change to “through a gene displacement strategy using a split-marker approach”. Same in Line 108, because the gene was replaced by a hygromycin B gene. How was monokaryotic deletion confirmed. Please list the primers in a supplementary table if PCR were done.
Response: Thanks for the kind reminder, as suggested, we have added descriptions for deletion mutants confirmation. Because M. oryzae is haploid, so we only need to confirm the monokaryotic deletion by PCR, as described in Line 125-127: “The deletion transformants were selected by 250 μg/ml hygromycin B (Roche) and confirmed by PCR using the Def1 gene-up/gene-down, LCK/HCK-up, RCK/HCK-down primer pairs (Figure S1B; Table S1).”. We also added the supplementary table (Table S1) to list the primers used in this study, which can help the readers to understand.
Line 113, please clarify which vector containing the neomycin resistant gene was used to complement the gene.
Response: Thanks for the kind reminder, as suggested, vector containing the neomycin resistant gene was mentioned in Line 128-130: “For complementation, we inserted the 1.5 kb promoter region and gene-coding region of Def1 into pKN to construct the complementation vector, pKN-Def1, which was transformed into the Def1 deletion mutant.”.
In section 2.2, please describe how protoplast was made and how transformation was done.
Response: Thanks for the kind reminder, as suggested, how protoplast was made and how transformation was done were added in Line 118-125: “For protoplast preparation, the wild-type strain was incubated in liquid CM medium, around 1 g of mycelium was harvested for digestion by Lysing enzyme (Sigma, USA) for 2 h at 150 rpm. The digested protoplast was filtered with three-layer microscope lens papers and washed with 0.7M NaCl and resusmended with STC buffer (1.2 M Sorbitol, 10 mM Tris [pH7.5], 50 mM CaCl2) and adjusted to a concentration of 1x108/ml for transformation. For transformation, the split-PCR products were added into the protoplast (300 μl mixture) and add 2ml PTC (60% PEG 3350, 10 mM Tris [pH7.5], 50 mM CaCl2) dropwise. ”.
In Line 110, please provide primer sequences in a supplementary table.
Response: The primer sequences were listed in supplementary table (Table S1, see the above).
Line 116, spell out what CM stands for.
Response: Thanks, we have added full information of CM (line 128).
Section 2.6, how are percent of cells stained estimated or counted?
Response: Thanks for the comments, we observed the barley epidermis cells using a microscope after the treatment described in section 2.6. Then we counted the number of stained cells out of every 100 cells which had been infected by infection hypha of the fungi.
Minor:
Many grammatical errors, please have an English editor look over throughout. Here are just a few examples from the first part of paragraph 1.
Line 36: Change “occur severely” to “experience severe”
Line 41: change “preferential” to “preferentially”
Line 44: change “evolutionary” to “evolutionarily”
Line 46: change “dissociate” to “dissociates”
Response: Thanks for the kind suggestion, we have tried our best to revise the grammatical mistakes in language with a thorough correction, hoping that it should be much better now.
Reviewer 4 Report
The entitled “RNAPII Degradation Factor Def1 is Required for Development, Stress Response, and Full Virulence of Magnaporthe oryzae” conducted by Zhang et all. The experiment and finding appeared interesting and merit full for scientific community. Authors described the mutant Def1 strain unable to the trigger the virulence strongly compared to wild type, and claimed the “Def1 is required for full virulence of M. oryzae”. In this study, authors also required to consider about the expression study of some already characterized virulence factors for causing pathogenicity and its co-relationship with mutant Def1.
Some minor points presented below-
In introduction some recent publication required to describe for better understanding and recently work on hypothetical virulence factor in different host.
https://doi.org/10.1038/s41598-021-01980-2
and
https://doi.org/10.1016/j.pmpp.2023.101969 Characterization and validation of hypothetical virulence factors in recently sequenced genomes of Magnaporthe species
Author Response
The entitled “RNAPII Degradation Factor Def1 is Required for Development, Stress Response, and Full Virulence of Magnaporthe oryzae” conducted by Zhang et all. The experiment and finding appeared interesting and merit full for scientific community. Authors described the mutant Def1 strain unable to the trigger the virulence strongly compared to wild type, and claimed the “Def1 is required for full virulence of M. oryzae”. In this study, authors also required to consider about the expression study of some already characterized virulence factors for causing pathogenicity and its co-relationship with mutant Def1.
Some minor points presented below-
In introduction some recent publication required to describe for better understanding and recently work on hypothetical virulence factor in different host.
https://doi.org/10.1038/s41598-021-01980-2
and
https://doi.org/10.1016/j.pmpp.2023.101969 Characterization and validation of hypothetical virulence factors in recently sequenced genomes of Magnaporthe species
Response: Thanks for the kind suggestion, we have cited the suggested references in the introduction section. Ref. 42, 43.
Round 2
Reviewer 4 Report
Def1 also involved in development, stress and key role of this pathogen blast disease onset. The conideal development was reported reduced compared to wild type, which is indicative of underlying event of reduced virulence and disease occurrence. Therefore it is strongly advised to conduct the study of any development related and characterized virulence gene study also to assess the dependency of such events on Def1.
Author Response
Thanks a lot for the suggestion. But as the time is limited, we are not able to finish this experiment.
