Mechanistic insights into the role of Ca 2+ -stimulated AMPK in the secretion of cellulases during carbon stress
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Abstract
The response of filamentous fungi towards recalcitrant carbohydrates is majorly governed by transcriptional activators of cellulase genes; however, little is known about the downstream events beyond transcription. We show here in Penicillium funiculosum that increasing the expression of a transcriptional activator CLR-2 in the catabolically derepressed strain, ΔMig1, didn’t exhibit a synergistic effect on cellulase production unless Ca 2+ was simultaneously increased. The RNA-seq screen for Ca 2+ -activated kinases identified SNF1-AMPK and SSP1-AMPKK as being specific to cellulose induction. Deletion of snf1 led to negligible secretion of cellulase upon induction. Quantitative whole-cell proteomics followed by chemical-genetic experiments with snf1- deleted strain showed that Ca 2+ -signaling channelizes carbon, nitrogen and energy sources towards cellulase production. Further, Ca 2+ -signaling phosphorylates SNF1-AMPK via SSP1, which in turn downregulates the phospho-HOG1 levels, leading to stimulus for cellulase secretion during carbon stress. The findings reported here are significant for understanding fungal pathology and developing second-generation biorefineries.
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Reply to the reviewers
Reviewer #1 (Evidence, reproducibility and clarity (Required)):
*Randhawa and co-authors have studied various aspects of the regulation of lignocellulose degradation by the filamentous ascomycete fungus Penicillium funiculosum. Over-expression of the well-known transcription factor clr2 (which regulates cellulase gene expression in Neurospora and other ascomycetes) in a delta-mig1 strain did not result in an increase in cellulase activity. However, when combined with an increased Ca2+ concentration the cellulase activity in the medium did increase. Using RNA-Seq, the authors have identified a candidate regulator: Snf1. Indeed, a knockout confirms that this …
Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Reply to the reviewers
Reviewer #1 (Evidence, reproducibility and clarity (Required)):
*Randhawa and co-authors have studied various aspects of the regulation of lignocellulose degradation by the filamentous ascomycete fungus Penicillium funiculosum. Over-expression of the well-known transcription factor clr2 (which regulates cellulase gene expression in Neurospora and other ascomycetes) in a delta-mig1 strain did not result in an increase in cellulase activity. However, when combined with an increased Ca2+ concentration the cellulase activity in the medium did increase. Using RNA-Seq, the authors have identified a candidate regulator: Snf1. Indeed, a knockout confirms that this gene is involved in the posttranscriptional regulation of cellulase production, specifically by regulating the secretion of the cellulases. *
Major comments:
In general, the topic and results are interesting. There are a few issues that need to be addressed, however. The manuscript would benefit from some careful proofreading. For example, articles ('the', 'a') are frequently missing. Very informal language is sometimes used ('zilch effect'). Put a space between '1000bp', etc. It is 'kDa', not 'kD', etc.
Response – Thank you very much for the encouraging remarks. We have thoroughly checked the manuscript and have added the articles at appropriate places. We have also improved the manuscript’s language and removed any informal language used.
I am a bit puzzled by the choice of calcium source: CaCO3, up to 10 g/L. Calcium carbonate does not efficiently dissolve in water unless the pH is low. Fungi generally acidify their culture medium during growth. As such, calcium carbonate likely has a pH buffering effect. Therefore, the described effects may also be attributed to a more neutral pH of the medium, and not necessarily to an increase in calcium ions.
Response – We completely agree with the reviewer and had the same thought that the pH buffering effect of CaCO3 could be the reason for increased cellulase production. We ruled out this by using 50 mg/l CaCl2 solely in rest of the experiments performed in Fig. 3 and afterwards. We have also mentioned the same in the manuscript (lines 175-178).
The authors have performed RNA-Seq, but as far as I can tell the data has not been made publicly available. At least, the raw reads should be deposited in the Short Read Archive of NCBI (or a similar repository), and preferably also the expression values in GEO of NCBI (or a similar repository).
Response – We will comply and deposit the raw reads in the short read archive of NCBI. We will also be providing the differential analysis of transcription factors expressed under glucose and Avicel in NCIM1228 and ∆Mig1 in the supplementary information.
P21. Very little information is provided in the M&M regarding the gene expression analysis. Provide references to all the tools, as well as the version numbers. Were any non-default parameters used?
Response – We have added the complete information on tools and procedures used for RNA-seq data analysis. For differential expression profiling, all FPKM values were normalized to the library size using the R package, Edge R. The expression value for the transcript was calculated using the reads aligned & normalised it on library size (Total sequencing reads generated) & transcript length giving us FPKM value (Fragments Per Kilobase of transcript per Million mapped reads), and TPM value (Transcript per million reads), which is regarded as normalized expression value for a particular transcript. We have taken the number of reads which got aligned to the conserved transcripts (Present in both the comparison group i.e Wild Type Glu & Cellulose samples (S1, S2, S7 & S8) Vs MIG1 glu & Cellulose sample (S3, S4, S5 & S6) and performed the differential gene expression between the two groups. The excel sheet having differential expression profiling of transcription factors is available as supplementary data.
The authors claim that SSP1 CaMKK phosphorylates SNF1 AMPK (last title of the Results section). I don't see any evidence for a direct interaction between these two proteins. I will believe that they are in the same pathway, but if the authors want to claim a direct interaction then additional experiments will be required. E.g. Y2H.
Response – Ssp1 is known to phosphorylate SNF1 during nutritional stress in S. pombe and they were found to interact directly by Co-IP studies. Based on the literature, we planned to over-express Ssp1 in P. funiculosum.
Minor comments:
- Please add line numbers to the manuscript, this facilitates the review process.*
Response - Line numbers have been added.
*P14 "in all yeasts and filamentous fungi". I doubt that all fungi have been tested. *
Response - The phrase has been modified.
P18. "in diverse yeasts and fungi". Yeasts are also fungi.
Response - The phrase has been modified.
P16. "solves dual purpose". I think this is meant: "serves a dual purpose"?
Response - The phrase has been modified.
*P17, first paragraph: this seems very speculative to me, so it should probably be labeled as such. *
Response - The phrase has been modified.
P21. What reference genome is used? Please cite the paper.
Response - We have our own reference genome in lab which is yet to be published.
Fig 1B. These are reported as volcano plots, but to me it looks like an empty graph (no data points), only a number of genes.
Response - The pictures have been changed.
Fig 1D. What do the colors on the right represent? The colors on the right represents k-means clustering of the genes of transcription factors.
Response - The same has been added to the figure legend also.
On various places in the manuscript the term "three times in triplicate" is used. What is meant here, three technical replicates of each of the three biological replicates?
Response - Yes we mean the same and the phrase has been modified.
P46. "We aimed to sought"
Response - The phrase has been modified.
Abstract: The sentence "Further, Ca2+-signaling" should be rewritten, because currently is seems to suggest that SSP1 downregulates the phospho-HOG1 levels.
Response – As suggested by the Western blot in the Fig.4b, Snf1 gets phosphorylated only when dual signal of calcium and cellulose are present. Since we observed upregulated Ssp1 expression in Avicel (Fig. 4a), and increased Ssp1 expression could increase the phosphorylated Snf1 in the cell (Fig. 7i), our data suggests that Ssp1 phosphorylates Snf1 in a Ca2+-dependent manner. Further Hog1 was found in hyperphosphorylated state in ∆Snf1 (Fig 6e), thus we believe Snf1 AMPK downregulates phospho-Hog1 levels.
Reviewer #1 (Significance (Required)):
*In general, the topic and results are interesting. There are a few issues that need to be addressed, however. The manuscript would benefit from some careful proofreading. *
Response – We highly appreciate the encouraging words of the reviewer. We have addressed all the issues raised by the reviewer. The major ones included the language and readability of the text, which has been improvised. We have replaced the volcano plot figures, and will be uploading the RNA-seq data to the SRA database of NCBI and excel sheet of differential expression analysis of transcription factor will be added as a supplementary file to the manuscript.
Reviewer #2 (Evidence, reproducibility and clarity (Required)):
- Randhawa et al. study the effect of loss of function of Snf1 kinase and calcium on the production of enzymes related to cellulose degradation in the fungus Penicillium funiculosum. *
- The manuscript is well structured and the researchers have done an enormous amount of work in constructing a number of mutant strains in this fungus. *
- Transcriptomics and proteomics support the conclusions reached with the strains generated.*
Response - Thank you very much for showing confidence in our research work, we are highly obliged by positive remarks on the manuscript.
The manuscript is long and suffers from an excess of results presented in figures. My main criticism focuses on the presentation of data on the cellular distribution of the ER and Golgi apparatus. The micrographs are inconclusive and it is not really clear what the authors are trying to show in these experiments. These results are not really necessary for the article and I suggest that they be removed from the article.
Response – We agree with the reviewers comments on data on the cellular distribution of the ER and Golgi apparatus. We have removed the micrograph data on the cellular distribution of the ER and Golgi apparatus (earlier Figure 3j and Figure 4r).
Reviewer #2 (Significance (Required)):
The authors have done an excellent job in producing a large number of strains carrying null alleles. In addition, they have used two broad analysis techniques that allow them to establish coherent hypotheses and corroborate them with the results.
Response – Thank you very much for the positive comments
The manuscript is difficult to understand in some sections because of the excessive amount of data and panels in the figures. The names designating each strain and given in full length in the graphs do not help either.
Response – Thank you very much for the valuable suggestion. We have reduced the number of graphs by including all enzymes assays in one concise graph in Figure 4. We have also shortened the names of strains and enzymes, in all the figures.
This work is of interest to all researchers interested in the integrity of signaling and regulatory pathways on extracellular enzymes of biotechnological interest.
*My interests focus on the cell biology of filamentous fungi, in particular on the molecular mechanisms and subcellular localization of elements involved in intracellular transport, signaling against environmental stresses and changes in transcriptional regulatory patterns. *
Response – Thank you once again for the encouraging remarks.
Reviewer #3 (Evidence, reproducibility and clarity (Required)):
The manuscript submitted by Randhawa et al focus on the mechanism of cellulases secretion, the very important and basal question in the filamentous fungi, particularly for cellulases biotechnology. As the author said the molecular basis of cellulases production previously study mainly focuses on regulation mechanism at transcription level, the study of molecular mechanism of cellulases translation and secretion are much rare. Therefore the submitted work is very impressive me on the progress of this area. What they presented shown the Ca2+ is critical for the regulation of cellulases secretion by SNF-1, SSP1 and HOG1. The regulation might be caused by affecting the protein trafficking in ER and Golgi, the manuscript found the development of ER and Golgi shown changes by staining by ER-tracker and Bodipy under different conditions and mutants. The manuscript constructed a model about regulatory mechanism of Ca2+ on cellulases translation and secretion level. The present study is close to make significant progress in the cellulases regulation area.
Response – We appreciate the positive comments of the reviewer.
Major comments: I am really impressive for the great work in the manuscript, however, I think the more work do need for give the conclusion of paper.
1.In terms of dynamics development of ER and Golgi of strains, the very critical data for the conclusion of the paper, the current data is only by chemical staining. It is not robust, it will be needed by other methods, for example, GFP-labeling the marker of ER or Golgi.
Response – The manuscript focuses on the signaling events governing cellulase production, and secretion. Since ER and Golgi are the sites of protein production and secretion, we hypothesized, if the Ca2+ signaling affects post-transcriptional events, it must have had some impact on the dynamics of these organelles; and microscopy experiments suggested us the same. In the next set of experiments, we proved our hypothesis with the proteomics and functional analysis of Snf1, Ssp1, and Hog1 MAPK. Hog1 MAPK pathway is known to regulate protein trafficking and secretion in yeast. We here showed that Ca2+- dependent regulation of Hog1 MAPK and its downregulation by Snf1 AMPK is crucial to cellulase secretion.
2.Also the author try to suggest the cellulases were detained in the ER, not went into Golgi, therefore the secretome protein decreased. It is very much possibly but the evidence is not robust either, to trafficking the GFP-labelled CBH1 might be a good experiment to make it clear.
Response – Thank you very much for raising the query. The manuscript majorly focuses on the role of calcium signaling on cellulase translation and secretion. Further, we have studied two signaling proteins, Snf1 AMPK and Hog1 MAPK which are downstream to calcium signaling, and we found their crosstalk vital to cellulase secretion. We have not talked about cellulases being detained in the ER or Golgi, rather we focused on the signaling events regulating cellulase production and transport.
Since we had already ruled out the role of calcium in cellulase transcriptional activation, and ER and Golgi being major site of protein production in the cell; we performed microscopy experiments to see if the calcium signaling modifies ER and Golgi morphology during carbon stress. We found under-developed Golgi in the absence of calcium in wild type. This experiment helped us to build a hypothesis that calcium signaling might have role in downstream events like protein translation, and secretion. The hypothesis was proved by functional analysis of signaling proteins, Western blot and proteomics experiments. Further, microscopy experiments further strengthened our observation that Snf1 AMPK is downstream target of calcium signaling and has no role in the cellulase translation, but cellulase secretion.
Considering that we are not focusing on the protein trafficking of cellulase, the confocal microscopy experiments are not decisive, rather build supporting evidence for our hypothesis, as suggested by the second reviewer. We have proved our hypothesis of Ca2+-dependent post-transcriptional regulation of cellulase by proteomics, and other biochemical experiments. Nevertheless, we plan to perform the confocal experiments again to achieve pictures with higher resolution.
1.On page 9, please indicate the fold changes of the kinases genes talked about, snf1 and so on.
Response – We have added the Fold change in the expression of Snf1 and Ssp1 (line number 221).
2.The quality of microscopic figure is not good, should have one with higher resolution, even consider to present the electron microscope picture to give the er and Golgi dynamics changes the manuscript talked about(optional).
Response: We agree with the reviewer’s suggestion to add high resolution confocal images of mycelia in Fig 3j and Fig. 4o. We are in the process of repeating the confocal microscopy experiment. We will update the manuscript with improved microscopic pictures.
*3. The quality of Western plot need to be improved, particularly figure 4f，figure 7i， it is hard to give the conclusion based on the picture presented *
Response – We have replaced the pictures of western blots (Fig 4f, and Fig 7i) with high resolution images.
Reviewer #3 (Significance (Required)):
The manuscript submitted by Randhawa et al focus on the mechanism of cellulases secretion, the very important and basal question in the filamentous fungi, particularly for cellulases biotechnology. As the author said the molecular basis of cellulases production previously study mainly focuses on regulation mechanism at transcription level, the study of molecular mechanism of cellulases translation and secretion are much rare. Therefore the submitted work is very impressive me on the progress of this area. What they presented shown the Ca2+ is critical for the regulation of cellulases secretion by SNF-1, SSP1 and HOG1. The regulation might caused by affecting the protein trafficking in ER and Golgi, the manuscript found the development of ER and Golgi shown changes by staining by ER-tracker and Bodipy under different conditions and mutants. The manuscript constructed a model about regulatory mechanism of Ca2+ on cellulases translation and secretion level. The present study is close to make significant progress in the cellulases regulation area.
Response - Thank you for the positive comments on the manuscript.
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Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #3
Evidence, reproducibility and clarity
The manuscript submitted by Randhawa et al focus on the mechanism of cellulases secretion, the very important and basal question in the filamentous fungi, particularly for cellulases biotechnology. As the author said the molecular basis of cellulases production previously study mainly focuses on regulation mechanism at transcription level, the study of molecular mechanism of cellulases translation and secretion are much rare. Therefore the submitted work is very impressive me on the progress of this area. What they presented shown the Ca2+ is critical for the regulation of cellulases secretion by SNF-1, SSP1 and HOG1. The …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #3
Evidence, reproducibility and clarity
The manuscript submitted by Randhawa et al focus on the mechanism of cellulases secretion, the very important and basal question in the filamentous fungi, particularly for cellulases biotechnology. As the author said the molecular basis of cellulases production previously study mainly focuses on regulation mechanism at transcription level, the study of molecular mechanism of cellulases translation and secretion are much rare. Therefore the submitted work is very impressive me on the progress of this area. What they presented shown the Ca2+ is critical for the regulation of cellulases secretion by SNF-1, SSP1 and HOG1. The regulation might caused by affecting the protein trafficking in ER and Golgi, the manuscript found the development of ER and Golgi shown changes by staining by ER-tracker and Bodipy under different conditions and mutants. The manuscript constructed a model about regulatory mechanism of Ca2+ on cellulases translation and secretion level. The present study is close to make significant progress in the cellulases regulation area.
Major comments
I am really impressive for the great work in the manuscript, however, I think the more work do need for give the conclusion of paper.
1.In terms of dynamics development of ER and Golgi of strains, the very critical data for the conclusion of the paper, the current data is only by chemical staining. It is not robust, it will be needed by other methods, for example, GFP-labeling the marker of ER or Golgi 2.Also the author try to suggest the cellulases were detained in the ER, not went into Golgi，therefore the secretome protein decreased. It is very much possibly but the evidence is not robust either, to trafficking the GFP-labelled CBH1 might be a good experiment to make it clear
Minors
1.On page 9, please indicate the fold changes of the kinases genes talked about, snf1 and so on. 2.The quality of microscopic figure is not good, should have one with higher resolution, even consider to present the electronmicroscope picture to give the er and Golgi dynamics changes the manuscript talked about(optional) . 3.The quality of western plot need to be improved, particularly figure 4f，figure 7i， it is hard to give the conclusion based on the picture presented
Significance
The manuscript submitted by Randhawa et al focus on the mechanism of cellulases secretion, the very important and basal question in the filamentous fungi, particularly for cellulases biotechnology. As the author said the molecular basis of cellulases production previously study mainly focuses on regulation mechanism at transcription level, the study of molecular mechanism of cellulases translation and secretion are much rare. Therefore the submitted work is very impressive me on the progress of this area. What they presented shown the Ca2+ is critical for the regulation of cellulases secretion by SNF-1, SSP1 and HOG1. The regulation might caused by affecting the protein trafficking in ER and Golgi, the manuscript found the development of ER and Golgi shown changes by staining by ER-tracker and Bodipy under different conditions and mutants. The manuscript constructed a model about regulatory mechanism of Ca2+ on cellulases translation and secretion level. The present study is close to make significant progress in the cellulases regulation area.
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Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #2
Evidence, reproducibility and clarity
Randhawa et al. study the effect of loss of function of snf1 kinase and calcium on the production of enzymes related to cellulose degradation in the fungus Penicillium funiculosum. The manuscript is well structured and the researchers have done an enormous amount of work in constructing a number of mutant strains in this fungus. Transcriptomics and proteomics support the conclusions reached with the strains generated. The manuscript is long and suffers from an excess of results presented in figures. My main criticism focuses on the presentation of data on the cellular distribution of the ER and Golgi apparatus. The micrographs are …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #2
Evidence, reproducibility and clarity
Randhawa et al. study the effect of loss of function of snf1 kinase and calcium on the production of enzymes related to cellulose degradation in the fungus Penicillium funiculosum. The manuscript is well structured and the researchers have done an enormous amount of work in constructing a number of mutant strains in this fungus. Transcriptomics and proteomics support the conclusions reached with the strains generated. The manuscript is long and suffers from an excess of results presented in figures. My main criticism focuses on the presentation of data on the cellular distribution of the ER and Golgi apparatus. The micrographs are inconclusive and it is not really clear what the authors are trying to show in these experiments. These results are not really necessary for the article and I suggest that they be removed from the article.
Significance
The authors have done an excellent job in producing a large number of strains carrying null alleles. In addition, they have used two broad analysis techniques that allow them to establish coherent hypotheses and corroborate them with the results. The manuscript is difficult to understand in some sections because of the excessive amount of data and panels in the figures. The names designating each strain and given in full length in the graphs do not help either. This work is of interest to all researchers interested in the integrity of signalling and regulatory pathways on extracellular enzymes of biotechnological interest.
My interests focus on the cell biology of filamentous fungi, in particular on the molecular mechanisms and subcellular localisation of elements involved in intracellular transport, signalling against environmental stresses and changes in transcriptional regulatory patterns.
-
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
Randhawa and co-authors have studied various aspects of the regulation of lignocellulose degradation by the filamentous ascomycete fungus Penicillium funiculosum. Over-expression of the well-known transcription factor clr2 (which regulates cellulase gene expression in Neurospora and other ascomycetes) in a delta-mig1 strain did not result in an increase in cellulase activity. However, when combined with an increased Ca2+ concentration the cellulase activity in the medium did increase. Using RNA-Seq, the authors have identified a candidate regulator: Snf1. Indeed, a knockout confirms that this gene is involved in the …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
Randhawa and co-authors have studied various aspects of the regulation of lignocellulose degradation by the filamentous ascomycete fungus Penicillium funiculosum. Over-expression of the well-known transcription factor clr2 (which regulates cellulase gene expression in Neurospora and other ascomycetes) in a delta-mig1 strain did not result in an increase in cellulase activity. However, when combined with an increased Ca2+ concentration the cellulase activity in the medium did increase. Using RNA-Seq, the authors have identified a candidate regulator: Snf1. Indeed, a knockout confirms that this gene is involved in the posttranscriptional regulation of cellulase production, specifically by regulating the secretion of the cellulases.
Major comments:
In general, the topic and results are interesting. There are a few issues that need to be addressed, however. The manuscript would benefit from some careful proofreading. For example, articles ('the', 'a') are frequently missing. Very informal language is sometimes used ('zilch effect'). Put a space between '1000bp', etc. It is 'kDa', not 'kD', etc.
I am a bit puzzled by the choice of calcium source: CaCO3, up to 10 g/L. Calcium carbonate does not efficiently dissolve in water unless the pH is low. Fungi generally acidify their culture medium during growth. As such, calcium carbonate likely has a pH buffering effect. Therefore, the described effects may also be attributed to a more neutral pH of the medium, and not necessarily to an increase in calcium ions. The authors have performed RNA-Seq, but as far as I can tell the data has not been made publicly available. At least, the raw reads should be deposited in the Short Read Archive of NCBI (or a similar repository), and preferably also the expression values in GEO of NCBI (or a similar repository). P21. Very little information is provided in the M&M regarding the gene expression analysis. Provide references to all the tools, as well as the version numbers. Were any non-default parameters used? The authors claim that SSP1 CaMKK phosphorylates SNF1 AMPK (last title of the Results section). I don't see any evidence for a direct interaction between these two proteins. I will believe that they are in the same pathway, but if the authors want to claim a direct interaction then additional experiments will be required. Eg Y2H.
Minor comments:
Please add line numbers to the manuscript, this facilitates the review process.
P14 "in all yeasts and filamentous fungi". I doubt that all fungi have been tested.
P18. "in diverse yeasts and fungi". Yeasts are also fungi.
P16. "solves dual purpose". I think this is meant: "serves a dual purpose"?
P17, first paragraph: this seems very speculative to me, so it should probably be labeled as such.
P21. What reference genome is used? Please cite the paper.
Fig 1B. These are reported as volcano plots, but to me it looks like an empty graph (no data points), only a number of genes.
Fig 1D. What do the colors on the right represent?
On various places in the manuscript the term "three times in triplicate" is used. What is meant here, three technical replicates of each of the three biological replicates?
P46. "We aimed to sought"
Abstract: The sentence "Further, Ca2+-signaling" should be rewritten, because currently is seems to suggest that SSP1 downregulates the phosphor-HOG1 levels.
Significance
In general, the topic and results are interesting. There are a few issues that need to be addressed, however. The manuscript would benefit from some careful proofreading.
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