Characterization and Expression Analysis of the Sterol C-5 Desaturase Gene PcERG3 in the Mycobiont of the Lichen Peltigera canina Under Abiotic Stresses

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The article study the function of Sterol C-5 Desaturase Gene PcERG3 in the Mycobiont of Lichen Peltigera canina, and reveal the expression levles under abiotic stresses. The design of the study is not complex, and describe relatively well. Sterols is an important compound. It is meaningful to study its synthase in this article, from lichen especially with a high degree of unsaturated Sterols. I have some suggestions:
1. The description of the experimental method should be more detailed.
2. Can you provide photos of lichens in the article?
3. PcERG3 protein have a low homology withe Arabidopsis thaliana (NP_186908.1). Is it correct? Could you use the PcERG3 protein to blast Arabidopsis genome, to get the finest protein sequence.
4. Please explain why the experiment use CuSO4 for stress treatment.
5. The discussion can be improved. The discussion section is a bit like describing the research results.
6. iThenticate check showed a higher Percent match: 31%. Please fix this problem.

Author Response

Reviewer 1

The article study the function of Sterol C-5 Desaturase Gene PcERG3 in the Mycobiont of Lichen Peltigera canina, and reveal the expression levles under abiotic stresses. The design of the study is not complex, and describe relatively well. Sterols is an important compound. It is meaningful to study its synthase in this article, from lichen especially with a high degree of unsaturated Sterols. I have some suggestions:

1. The description of the experimental method should be more detailed.

We added more details in our Methods.

2. Can you provide photos of lichens in the article?

We added a photo of Peltigera canina (as Figure 1).

3. PcERG3 protein have a low homology withe Arabidopsis thaliana (NP_186908.1). Is it correct? Could you use the PcERG3 protein to blast Arabidopsis genome, to get the finest protein sequence.

Yes, there is low homology of PcERG3 with corresponding protein of Arabidopsis thaliana, and this seems to be logical considering that the majority of lichen metabolites including sterols originate from mycobiont. Indeed, quite often fungal genes have rather low homologies with their Angiosperm homologues. However, the amino acid sequence of the PcERG3 showed close relationships with ERG3 proteins from other lichens and free-living fungi.

4. Please explain why the experiment use CuSO4 for stress treatment.

We treated lichen with CuSO4 for several reasons. First, this treatment simulates heavy metal stress, enabling us to study the response of lichens to pollution. Second, copper is an inducer of oxidative stress and can promote the generation of reactive oxygen species, which can in turn damage proteins, lipids, and DNA. Lichens activate their antioxidant defenses, and this was confirmed in our study by the upregulation of a gene encoding superoxide dismutase 1 (Fig. 5). We also know from our earlier work that Cu can affect lichen sterol metabolism.

5. The discussion can be improved. The discussion section is a bit like describing the research results.

We improved our Discussion.

6. iThenticate check showed a higher Percent match: 31%. Please fix this problem.

Unfortunately, the reviewer did not indicate which part of the m/s contains matches. Most probably this is mostly in our methods protocols, which are standard and were described earlier. Other parts of the m/s are original.

 

Reviewer 2 Report

Comments and Suggestions for Authors

Review attached 

Comments for author File: Comments.pdf

Author Response

Reviewer 2

Overall: This manuscript reports on the examination of the expression of a gene in lichen fungi that is involved in stress response.  The authors chose to study a Sterol Desaturase gene that has been demonstrated in other non-lichen fungi (and other organisms) to be directly involved in the stress response.  This manuscript is the first report of a detailed examination of this particular gene (named PcERG3) in a lichen mycobiont.  The authors sequence the gene and demonstrate that it clades nicely with other characterized genes from non-lichen fungi, suggesting that it does indeed carry out the proposed function.  The authors then conduct an examination of the expression levels of PcERG3 as well as other genes PcHSP20 and PcHSP98 andPcSOD1 under conditions of abiotic stress.  

This is a very useful addition to the research on lichen fungi mycobionts as it does shed some useful insight into the role of abiotic stress in regulating gene expression in lichen fungi. 

However, the authors should add some information to the manuscript that would greatly strengthen the results and raise the impact of the paper in the community.  The authors should add some specific details about the timing of the experiments that they conducted for abiotic stress.  For example, the authors carry out experiments at both – 20 °C and at +40 °C but there is no information about how long the lichen thallus was held at those temperatures and how long after removal from those conditions were the thalli extracted for mRNA content.  Similar time domain information for the CuSO4 and dehydration and rehydration experiments should be included.  How long after CuSO4 exposure were the thalli extracted?  And how long after ‘rehydration’ were the thalli extracted.  A detield discussions on the timing of the experiment would be very helpful.  This should not require additional experimentation and is not required. This should be information that the authors already possess in their records and can and should be included in the manuscript. 

We are grateful to a reviewer for encouraging comments and will add more details about timing of the experiments. As indicated in the Methods, the time of the exposure of lichen thalli to -20oC and +40^oC was 3 h. Immediately after treatment thalli were fixed in liquid nitrogen and stored in -80^oC until RNA was extracted. Details of the timing of the exposure to CuSO₄ and of the dehydration and rehydration experiments have now been made more explicit.

The time course information is essential as there is little known about the timing of gene expression in lichen fungi and these experiments will provide some very important information about the timing of the stress response in particular. 

 

Specific Comments: 

Section 3.2 the authors refer to the genes PcHSP20 and PcHSP98 and do mention that they are heat shock proteins but do not give much context as to why they are useful benchmarks ot compare the expression of PcERG3 against.  Some description of their role would be helpful.

An important reaction to thermal stress is the expression of genes encoding heat shock proteins (HSPs). HSPs are able to unfold and refold proteins which become misfolded because of heat exposure (Albrecht et al. 2010). The heat-induced upregulation of HSPs has been shown in many organisms including prokaryotes and eukaryotes. There are several families of HSPs classified based on their molecular weight, which ranges from 8 to 200 kDa. HSPs are usually divided into the following families: HSP100, HSP90, HSP70, HSP60, HSP40, and low-molecular-weight HSPs. In the present study, to confirm that the lichens were indeed heat stressed, we analysed the expression of a gene encoding a small HSP (PcHSP20) and a high-molecular-weight HSP from the HSP100 family (PcHSP98). Small HSPs are ATP-independent chaperones that delay the onset of protein misfolding and the initiation of aggregation. Small HSPs are involved in the response of the free-living fungus Aspergillus nidulans to various stresses such as thermal/cold, osmotic, and oxidative stress (Wu et al., 2016). Furthermore, small HSPs are important in maintaining membrane functioning during stress (Nakamoto & Vígh, 2007), playing similar roles to those of the membrane sterol component. The higher molecular mass proteins from the HSP100 family, including HSP98, play important roles in reactivating aggregated proteins by resolubilizing non-functional protein aggregates, which facilitates the degradation of irreversibly damaged polypeptides (Kraft et a., 2022). We have explained our rationale for our study of specific HSPs in our amended text, and included an expanded discussion of their roles.

 

References:

Albrecht D, Guthke R, Brakhage AA, Kniemeyer O (2010) Integrative analysis of the heat shock response in Aspergillus fumigatus. BMC Genomics 11, 32.

Wu, J.; Wang, M.; Zhou, L.; Yu, D. Small heat shock proteins, phylogeny in filamentous fungi and expression analyses in Aspergillus nidulans. Gene 2016, 575, 675–679.

Do JH, Yamaguchi R, Miyano S (2009) Exploring temporal transcription regulation structure of Aspergillus fumigatus in heat shock by state space model. BMC Genomics 10, 306.

Steinhäuser SS, Andrésson ÓS, Pálsson A, Werth S (2016) Fungal and cyanobacterial gene expression in a lichen symbiosis: effect of temperature and location. Fungal Biology 120, 1194–1208.

Nakamoto H., Vígh, L. 2007. The small heat shock proteins and their clients. Cell Mol. Life Sci. 64, 294–306.

Kraft M., Scheidegger C., Werth S. 2022. Stressed out: the effects of heat stress and parasitism on gene expression of the lichen-forming fungus Lobaria pulmonaria The Lichenologist (2022), 54, 71–83 doi:10.1017/S0024282921000463).

 

Section 3.3 and Figure 4: The authors introduce the gene PcSOD1 without clearly explain what it is or why they are using it as a refence gene….more detail hers is needed  Figure 3: This length of time at –20 °C and at +40 °C should be added to tie figure legend. 

Gene PcSOD1 is a gene encoding superoxide dismutase 1, an antioxidative enzyme, which dismutates superoxide anion radical. This gene was chosen to study because in lichens (as indeed for most organisms) its expression increases in response to most abiotic stresses. This is because stress increases the levels of reactive oxygen species increases, which in turn activates antioxidative defence, including superoxide dismutase.

We have made the timing our treatments more explicit in our figure legend.

Figure 4: the timing of these experiments should be part of the figure legend and discussed in more detail in the text. How long was the lichen thalli held at –20 °C and how long at +40 °C and how long after this was this thallus sampled.   The experiments in Figure 4B should also have some more detail about how long after CuSO₄ exposure and how long after rehydration were these samples taken.

We have made the timing our treatments more explicit in our figure legend.

 

Reviewer 3 Report

Comments and Suggestions for Authors

 Comments to the manuscript “Characterization and Analysis of Expression of the Sterol C-5 Desaturase Gene PcERG3 in the Mycobiont of Lichen Peltigera canina Under Abiotic Stresses” by Swid et al.

General comment
The submitted manuscript analyses in silico the structural characteristics and performs a phylogenetic reconstruction of a sterol C-5 desaturase (ERG3) of the fungal symbiont of the lichen Peltigera canina. The amino acid sequence of the protein showed closed relationships with ERG3 proteins of Peltigeralian and non-Peltigeralian lichens, as well as from ERG3 of free-living fungi. Further RT-qPCR analysis showed that the transcription of the PcERG3 gene increased in response to heat stress and rehydration.  Transcriptional regulation of the genes encoding proteins that respond to heat and oxidative stress are also analyzed. The authors state that the transcriptional regulation observed of PcERG3 in response to temperature fluctuations, dehydration/rehydration cycle, and heavy metal “…suggests that regulating the balance between more and less saturated sterols is key to maintaining membrane functioning during stress.”
 
This manuscript contributes to better understanding on the role of the C-5 desaturase (ERG3) to the response to abiotic stress in lichens. The submitted manuscript is well written, the materials and methods are clearly described and the results and discussion properly presented. Thus, I consider that this work is suitable for its publication in Microbiology Research. I just have a couple of comments regarding the phylogenetic analysis.
 
Specific comments
1. The topological result of the Neighbor-Joining (NJ) tree is based on sequence similarity. Thus, the NJ method is a phenetic one, that is, does not reconstruct phylogenetic relationships between analyzed proteins/genes/species. If you want to infer “…evolutionary relationships between ERG3 in P. canina and other organisms” (lines 192-193), I strongly recommend using the Maximum Likelihood (ML) method, after inferring the best evolutionary model. You can perform all this analysis in the same MEGAX software. The NJ is accepted as fast analysis, and probably you will obtain the same topological order than in the ML reconstruction but always is better to use the correct tool to make phylogenetic inferences. Although it is not mandatory to perform phylogenetic analysis using the ML method for publication of the manuscript, it is strongly recommended.

2. Please add bootstrap values to each bifurcation of the phylogenetic tree of the figure 1. But please consider the previous comment for the phylogenetic analysis. If the bootstrap values are low (<70%) in most of the branches, or in those relevant to finding the phylogenetic relationships of PcERG3, then you should consider performing the analysis using the ML criterion.

Author Response

Reviewer 3

The submitted manuscript analyses in silico the structural characteristics and performs a phylogenetic reconstruction of a sterol C-5 desaturase (ERG3) of the fungal symbiont of the lichen Peltigera canina. The amino acid sequence of the protein showed closed relationships with ERG3 proteins of Peltigeralian and non-Peltigeralian lichens, as well as from ERG3 of free-living fungi. Further RT-qPCR analysis showed that the transcription of the PcERG3 gene increased in response to heat stress and rehydration. Transcriptional regulation of the genes encoding proteins that respond to heat and oxidative stress are also analyzed. The authors state that the transcriptional regulation observed of PcERG3 in response to temperature fluctuations, dehydration/rehydration cycle, and heavy metal “…suggests that regulating the balance between more and less saturated sterols is key to maintaining membrane functioning during stress.”

This manuscript contributes to better understanding on the role of the C-5 desaturase (ERG3) to the response to abiotic stress in lichens. The submitted manuscript is well written, the materials and methods are clearly described and the results and discussion properly presented. Thus, I consider that this work is suitable for its publication in Microbiology Research. I just have a couple of comments regarding the phylogenetic analysis.

 

Specific comments

1. The topological result of the Neighbor-Joining (NJ) tree is based on sequence similarity. Thus, the NJ method is a phenetic one, that is, does not reconstruct phylogenetic relationships between analyzed proteins/genes/species. If you want to infer “…evolutionary relationships between ERG3 in P. canina and other organisms” (lines 192-193), I strongly recommend using the Maximum Likelihood (ML) method, after inferring the best evolutionary model. You can perform all this analysis in the same MEGAX software. The NJ is accepted as fast analysis, and probably you will obtain the same topological order than in the ML reconstruction but always is better to use the correct tool to make phylogenetic inferences. Although it is not mandatory to perform phylogenetic analysis using the ML method for publication of the manuscript, it is strongly recommended.
2. Please add bootstrap values to each bifurcation of the phylogenetic tree of the figure 1. But please consider the previous comment for the phylogenetic analysis. If the bootstrap values are low (<70%) in most of the branches, or in those relevant to finding the phylogenetic relationships of PcERG3, then you should consider performing the analysis using the ML criterion.

We re-did the phylogenetic analysis using the Maximum Likelihood (ML) method, as the reviewer recommended. Figure 1 now shows a phylogenetic tree indicating the relationships between retrieved homologous peptide sequences for PcERG3 constructed in IQ-TREE using the maximum likelihood (ML) method with 2500 replicates of ultrafast bootstraps. Branch support is now marked using numbers. ERG3 from P. canina is indicated in red. The scale represents 1.0 amino acid sequence substitutions per site.

 

We have amended our Methods as follows:

These protein sequences were aligned with the MUSCLE algorithm (Edgar, 2004) with standard parameters and trimmed by the trimAl v.1.2 program (Capella-Gutiérrez et al., 2009) with the «automated1» mode. The phylogenetic tree was constructed using the IQ-TREE program (Nguyen et al., 2015) using a ultrafast bootstrap (Minh et al., 2013). Phylogenetic trees were constructed using the iTOL tool (Letunic & Bork, 2021) and edited in the Inkscape program.

 

References:

Edgar, R.C. Muscle: a Multiple Sequence Alignment Method with Reduced Time and Space Complexity. BMC Bioinformatics 2004, 5, 113, https://doi.org/10.1186/1471-2105-5-113.

Capella-Gutiérrez, S.; Silla-Martínez, J.; M., Gabaldón, T. TrimAl: a Tool for Automated Alignment Trimming in Large-Scale Phylogenetic Analyses. Bioinformatics 2009 8; 25(15),1972–1973, https://doi.org/10.1093/bioinformatics/btp348.

Nguyen, L.-T.; Schmidt, H. A., von Haeseler A., Minh B. Q. IQ-TREE: a Fast and Effective Stochastic Algorithm for Estimating Maximum-Likelihood Phylogenies. Molecular Biology and Evolution 2015, 32(1), 268-74, https://doi.org/10.1093/molbev/msu300 .

Minh, B. Q.; Nguyen, M. A. T.; Haeseler A. Ultrafast Approximation for Phylogenetic Bootstrap. Molecular Biology and Evolution 2013, 15, 30(5), 1188–1195, https://doi.org/10.1093/molbev/mst024.

Letunic, I.; Bork, P. Interactive Tree of Life (iTOL) v6: Recent Updates to the Phylogenetic Tree Display and Annotation Tool. Nucleic Acids Research 2024, 52, W1, W78–W82, https://doi.org/10.1093/nar/gkae268.