Author Response
The following is the authors’ response to the original reviews.
Response to Reviewer 1 Comments (Public Review):
Point 1: While the authors provided a large amount of data regarding the genes involved in the TOR pathway, it is highly descriptive and mostly confirmative data, as numerous papers have already shown that the TOR pathway plays essential roles in a myriad of biological processes in multiple fungi.
Response 1: Thank you for your comment. The target of rapamycin (TOR) signal pathway plays critical roles in various eukaryotic organisms. However, its specific role in controlling the development and virulence of opportunistic pathogenic fungi like A. flavus has remained unclear. Additionally, the underlying mechanism of the TOR pathway remains elusive in the A. flavus. As such, our study provides a useful contribution, as it is the first to comprehensively investigate the majority of genes in the conserved TOR signaling pathway in A. flavus.
Point 2: The authors seemed to perform a series of parallel studies in several genes involved in the TOR pathway in other fungi. However, their data are not properly interconnected to understand the TOR signaling pathway in this fungal pathogen. The authors frequently drew premature conclusions from basic phenotypic observations. For instance, based on their finding that sch9 mutant showed high calcium stress sensitivity, they concluded that Sch9 is the element of the calcineurin-CrzA pathway. Furthermore, based on their finding that the sch9 mutant show weak rapamycin sensitivity and increased Hog1 phosphorylation, they concluded that Sch9 is involved in TOR and HOG pathways. To make such conclusions, the authors should provide more detailed mechanistic data.
Response 2: Yes, we agree with the reviewer's comment. We have carefully reviewed the manuscript and made necessary revisions to eliminate arbitrary conclusions. For example, we have removed the statement that "Sch9 is the element of the calcineurin-CrzA pathway". Furthermore, we have rephrased our conclusions to better reflect our findings. "these results reflected that Sch9 regulates osmotic stress response via the HOG pathway in A. flavus"(Lines 279-280, page 13). We appreciate the reviewer's input, which has contributed to the clarity and accuracy of our work.
Point 3: In the section "Tor kinase plays important roles in A. flavus", some parts of their data are confusing. The authors said they identified a single Tor kinase ortholog, which is orthologous to S. cerevisiae Tor2. And then, they said failed to obtain a null mutant, but constructed a single copy deletion strain delta Tor1+/Tor2-. What does this mean? Does this mean A. flavus diploid strain? So is this heterozygous TOR/tor mutant? Otherwise, does the haploid A. flavus strain they used contain multiple copies of the TOR gene within its genome? What is the real name of A. flavus Tor kinase (Tor1 or Tor2?). "tor1+/tor2-" is the wrong genetic nomenclature. What is the identity of detalTor1+/Tor2-? Please provide detailed information on how all these mutants were generated. A similar issue was found in the analysis of TapA, which is speculated to be essential (what is the deltaTapA1+/TapA2-?). I couldn't find any detailed information even in Materials and Methods. The authors should provide southern blot data to validate all their mutants.
Response 3: Thank you for your comments. We acknowledge the confusion in our presentation and will ensure that accurate genetic nomenclature is used consistently throughout the paper.
In response to your queries, we have included a section in the Materials and Methods, titled "Detection of tor and tapA genes copy number in strains" (Lines 615-621, page 29), to provide details on how we determined the copy numbers of the tor and tapA genes in the strains. Our findings revealed that both the tor and tapA genes are present in double copies in our strains, which guided our decision to construct single-copy deletion strains using homologous recombination. We have verified these copy numbers using absolute quantification PCR (Table S1).
The use of the abbreviation '+/-' for the single copy knockout strains, such as tor+/- and tapA+/-, is consistent with common fungal literature practice. We apologize for any confusion caused by this nomenclature.
Although we did not employ southern blot data for validation, we conducted PCR and gene sequencing to confirm the mutants. We appreciate your comments to improve the clarity and accuracy of our manuscript.
Point 4: How were the FRB domain deletion mutants constructed? If the FKBP12-rapamycin binding (FRB) domain is specifically deleted in the Tor kinase allele, should it be insensitive and resistant to rapamycin? However, the authors showed that the FRB domain deleted TOR allele was indeed non-functional.
Response 4: We appreciate the reviewer's attention to the construction of the Fkbp12-rapamycin binding (FRB) domain deletion mutants and the discrepancy between the expected and observed results.
For the knockout of the FRB domain, we used the homologous recombination method, but because tor genes are double-copy genes, there are also double copies in the FRB domain. Despite our efforts, we encountered challenges in precisely determining the location of the other copy of the tor gene.
We speculate the common expectation that the deletion of the FRB domain should result in insensitivity and resistance to rapamycin, as it disrupts the binding site for Fkbp-rapamycin. However, we observed that the FRB domain-deleted mutant was more sensitive to rapamycin. This intriguing result suggests that there are additional factors or complexities involved in TOR signaling pathway regulation in A. flavus. We hypothesize that this result is related to the double copy of the tor gene. The reviewer's keen observation and comment have contributed to our efforts to better understand and explain this intriguing result.
Point 5: In Figure 4C, the authors should monitor Hog1 phosphorylation patterns under stressed conditions, such as NaCl treatment, and provide quantitative measurements. Similar issues were found in the western blot analysis of Slt2 (Fig. 8D).
Response 5: We agree with the reviewer that we should monitor Hog1 phosphorylation patterns under stressed conditions. In response to this valuable suggestion, we conducted additional experiments to examine Hog1 phosphorylation patterns under NaCl treatment for 30 minutes. The quantitative measurements of Hog1 phosphorylation levels under stress have been added to Figure 4E in the revised manuscript. Similarly, we have addressed the issue raised regarding Slt2 in Figure 8D.
Point 6: For all the deletion mutants generated in this study, the authors should generate complemented strains to validate their data.
Response 6: We appreciate the reviewer's suggestion to generate complemented strains for all the deletion mutants in our study to validate our data. However, due to the extensive number of genes involved in this research, it is hard to create complemented strains for each individual deletion mutant. As suggested by the reviewer, we have constructed complemented strains for several key deletion mutants, such as ΔsitA-C and Δppg1-C.
Response to Reviewer 1 Comments (Recommendations For The Authors):
Point 1: Overall, this manuscript was very poorly organized and not presented logically. It requires extensive English language editing.
Response 1: We appreciate the reviewer's feedback regarding the organization and language quality of our manuscript. To address these concerns, we have restructured the manuscript to improve its logical flow and coherence. We thank the reviewer for their constructive criticism, which has been instrumental in the manuscript's refinement.
Point 2: The authors did not present their figures in the order of description. For example, the authors suddenly described Figure 9A data in lines 128-130 in the middle of describing Figure 1. Furthermore, Figures 1D and 1F were described earlier than Figures 1B and 1C. In addition, Figure S2 was shown earlier than Figure S1. Please check this throughout the manuscript.
Response 2: We thank the reviewer for their insightful observation. We acknowledge the importance of a logical and coherent figure sequence for reader comprehension. After careful review, we have rearranged the text and images throughout the entire document to enhance the reading experience. The revised manuscript now presents figures in a consistent and logical order, following the sequence of descriptions. We believe this improvement will enhance the overall readability and comprehension of our research.
Point 3: The authors should follow the standard genetic nomenclature rules.
Response 3: Thank you for your suggestion. We have revised our manuscript to ensure that we are following the standard genetic nomenclature rules throughout. This includes the correct naming of genes, proteins, and mutations, as well as the use of appropriate italicization and formatting. We follow the rules: gene symbols are typically composed of three lowercase italicized letters, while protein symbols are not italicized, with an initial capital letter followed by lowercase letters.
Point 4: These are just a few examples. Besides the ones that I mentioned, I found numerous grammatically wrong or awkward sentences throughout the manuscript. So this manuscript requires extensive English proofreading.
Response 4: We apologize for the problem of our manuscript. We have asked an English native speaker to enhance the overall language quality and readability of the text. We believe that these improvements will significantly enhance the manuscript's overall quality and make it more accessible to a broader audience.
Response to Reviewer 2 Comments (Public Review):
Point 1: However, findings have not been deeply explored and conclusions mostly are based on parallel phenotypic observations. In addition, there are some concerns that exist surrounding the conclusions.
Response 1: We are grateful for the suggestion. We conduct additional experiments and analyses to delve more deeply into our findings and ensure a more robust basis for our conclusions.
Response to Reviewer 2 Comments (Recommendations For The Authors):
Point 1: Verification for mutants: a single copy deletion strain ΔTor1+/Tor2(containing one copy of the Tor gene), however, in the table of strain list, it seems like null mutants. There are no further verifications for relative genes' expression and no complementary strains.
A. Flavus ΔTor: Δku70; ΔniaD; ΔTor::pyrG
A. Flavus ΔTapA Δku70; ΔniaD; ΔTapA::pyrG
As described in pp208, "While we failed to obtained a null mutant, we constructed a single copy deletion strain ΔTor1+/Tor2- (containing one copy of the Tor gene) constructed by homologous recombination)"? But the authors think there was only one Tor kinase ortholog (AFLA_044350). It is hard to understand for this mutant What is the evidence to verify phenotypes of the ΔTor1+/Tor2- strain resulted from deletion of Tor2, no detail for how to make ΔTor1+/Tor2- strain.
Response 1: Thank you for your important comments and suggestion. We apologize for the confusion caused by genetic nomenclature. We make the necessary corrections in the table of strain lists to accurately reflect the genotypes of the strains (Table S3).
Multicopy variation of genes has not been explored in detail in fungi, especially in A. flavus, but is a commonly known phenomenon in mammalian genomes[1-2]. In yeast, the presence of two tor genes, tor1 and tor2, whereas in higher eukaryotes such as plants, animals, and filamentous fungi, there is only one tor gene[3-4]. The homology comparison results show that the genome of A. flavus contains only one tor gene. However, the tor gene in A. flavus exhibited varying copy numbers, as was confirmed by absolute quantification PCR at the genome level (Table S1).
In this study, we constructed a single copy deletion strain, tor+/-, through homologous recombination. This strain contains one copy of the tor gene. We provide a more detailed and explicit description of the methods used to detect of the genes copy number in strains (Lines 615-621, page 29). We thank the reviewer for pointing out these important issues.
Point 2: For a point mutant strain TORS1904L, they found that the sensitivity to rapamycin is consistent with the WT strain, it could not tell anything. It should be moved to Suppl.
Response 2: Thanks for your important comments. We acknowledge that these results may not provide significant insights. In response to this suggestion, we delete the data related to the TORS1904L point mutant strain and its sensitivity to rapamycin to ensure that the main manuscript focuses on the most pertinent and informative findings. Corresponding modifications have been made in the revised manuscript.
Point 3: For subtitle "Sch9 is correlate with the HOG and TOR pathways "What is the meaning for "correlate" similarly?
Response 3: Thank you for this comment. We apologize for the unclear wording. To enhance clarity, we revise the subtitle to more explicitly convey this conclusion, for example, "The Sch9 kinase is involved in aflatoxin biosynthesis and the HOG pathway". (Lines 242, page 12).
Point 4:for the ΔTapA 1+/TapA 2- strain (containing one copy of the TapA gene). It should have the complementary strain to verify the specific role of TapA. In FigS1B, ΔTOR and ΔTapA it could not tell TOR gene has been edited. Did you test mRNA of TOR gene?
Response 4: Thanks for your important comments. Due to the large number of genes involved, we did not perform a complementation experiment. However, we used PCR and sequencing to verify the editing of our gene. Additionally, we conducted copy number and mRNA analyses to verify its function. The transcriptional level of the tor gene in the tor+/- mutant was downregulated compared to the level in the wild-type strain (Fig. S6).
Response to Reviewer 3 Comments (Public Review):
Point 1: As for many results, I miss the re-complementation of the created mutants throughout the manuscript. This is standard praxis.
Response 1: Thanks for your suggestions. We acknowledge that re-complementation is a standard practice for validating the effects of gene deletions. However, due to the large number of genes involved in our study, we have performed supplementary experiments on a selection of them, such as ΔsitA-C and Δppg1-C. We are grateful to the reviewer for your understanding of this practical consideration.
Point 2: Fig. 1: cultures were grown for 48 h before measuring the transcript level. The authors show that brlA, abaA, and some sexual regulators are less expressed. In my opinion, this does not allow the conclusion that there is a direct control through rapamycin. Since the colonies grow very slowly in the presence of rapamycin, the authors should add rapamycin and follow gene expression after 15, 30, 60, 90 min. The figure legend needs to be more detailed. Which type of cultures were used, liquid, solid medium? Etc.
Response 2: We deeply appreciate the reviewer’s suggestion. Since we found that there were no significant differences in gene expression changes following shorter treatment times, we extended the treatment duration. We conduct additional experiments to examine the gene expression levels at longer time intervals (3, 6, and 9 h) after the addition of rapamycin (Figure 1H-1J). These time points allow us to capture the dynamic changes in gene expression in response to rapamycin more effectively. Additionally, we enhance the figure legend to provide a more comprehensive description that specifies the type of cultures used in the experiments.
Point 3: Why in chapter one Fig. 9 is already cited? Those data should then be included in Fig. 1 for the general phenotype.
Response 3: Thank you for the suggestion. We have reordered the figures in the updated version of the manuscript to ensure that the data for consistent and clarity.
Point 4: The authors wrote that radial growth and conidiation were gradually reduced with increasing rapamycin concentrations. This is not true. There is no gradient! However, it should be tested if there is a gradient if lower concentrations are used. The current data imply that there is a threshold concentration, so either there is 100 % growth or a reduction to 25 %. This looks strange.
Response 4: Thank you for underlining this deficiency. We agree that a threshold concentration versus a gradient is an important distinction that needs to be clarified. Our results show that the addition of excessive quantities of rapamycin does not increase the inhibition of A. flavus growth. As the concentration of the FK506 drug increases, there is a gradual decrease in the growth and cell production of A. flavus. This phenomenon could potentially be attributed to varying mechanisms of action exhibited by the drugs. Therefore, we have revised these confused sentences. ( Lines 120-121, Page 5)
Point 1: There are many wrong spellings:
Fig. 1. Before washed, before washing; RelaTEtive gene expERSion should read relative gene expression. Sclerotial should be sclerotia. See also Fig. 5 F, H, Fig. 6 E. 6D colon diameter should be colony diameter.
Fig. 4E. The expressED level... should read Expression level..... (also without article) Also in A, F, H.
Fig. 6C. TLC detection of WT.... The authors mean AF detection in extracts of WT.....
AF was extracted and analyzed by TLC.....
Labelling of axes in one figure should be uniform.
Response 1: Thank you for your reminder. We apologize for the oversights, and we carefully address and correct all the mentioned spelling issues to ensure the accuracy and clarity of the manuscript.
Point 2: If the authors refer to the genes, I think they should be in small letters and italics, if it is the protein, the first letter should be capitalised tap1 (italics) and Tap1.
Response 2: We appreciate this suggestion. We have carefully checked the entire manuscript and revised follow the standard genetic nomenclature rules. We follow the naming conventions for microbial genes and proteins, where gene symbols are typically composed of three lowercase italicized letters, and protein symbols are not italicized, with an initial capital letter followed by lowercase letters.
Point 3: Very often articles are used where I would not use them.
Response 3: Thanks for your careful checks. We are sorry for our carelessness. Based on your comments, we have made the corrections to make the articles harmonized within the whole manuscript. We value the reviewer's feedback, which will contribute to the overall quality of our writing.
References:
[1] Handsaker R, Van Doren, V, Berman, J. et al. Large multiallelic copy number variations in humans. Nat Genet 47, 296–303 (2015).
[2] Wang Y, Wang S, Nie X. et al. Molecular and structural basis of nucleoside diphosphate kinase-mediated regulation of spore and sclerotia development in the fungus Aspergillus flavus. J Biol Chem. 2019 Aug 16;294(33):12415-12431.
[3] Kim DH, Sarbassov DD, Ali SM, et al. mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery. Cell. 2002; 110(2): 163-75.
[4] Fu L, Liu Y, Qin G, et al. The TOR-EIN2 axis mediates nuclear signalling to modulate plant growth. Nature. 2021; 591(7849): 288-292.
