Involvement of the Transporter CgTrk1 in Potassium Uptake, Invasive Growth, and Full Virulence in Colletotrichum gloeosporioides
Round 1
Reviewer 1 Report
Comments and Suggestions for Authorsline 181. How similar? even in Figure S1, I cannot infer if identity is high or medium. Please, clarify in the text the number of amino acids, % similarity/identity, etc.
line 287. and 289 type type, delete repeated word
line 320. curial? or crucial?
line 329. reference 38 provides a comprehensive and up-to-date vision of the mechanisms responsible for alkali metal cation transport and their regulation in the model yeast Saccharomyces cerevisiae and to establish, when possible, comparisons with other yeasts and higher plants, is that reference well used in that sentence?
Comments on the Quality of English LanguageNo comments.
Author Response
line 181. How similar? even in Figure S1, I cannot infer if identity is high or medium. Please, clarify in the text the number of amino acids, % similarity/identity, etc.
Response: Thank you for your suggestion. Based on your opinion, we evaluated the similarity of Trk1 and Trk2 using the website servers of ESPript 3 (https://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi). We have updated Figure S1 in the new version. The data showed that the similarity between Trk1 and Trk2 reached 66.42. Additionally, both the Trk1 and Trk2 proteins contain a potassium uptake domain. These data indicate that CgTrk1 and CgTrk2 are highly conserved.
line 287. and 289 type type, delete repeated word
Response: Thank you for your question. We have revised.
line 320. curial? or crucial?
Response: Thank you for your question. We have revised.
line 329. reference 38 provides a comprehensive and up-to-date vision of the mechanisms responsible for alkali metal cation transport and their regulation in the model yeast Saccharomyces cerevisiae and to establish, when possible, comparisons with other yeasts and higher plants, is that reference well used in that sentence?
Response: Thank you for your question. I have provided new reference in updated version. This study reported that the intracellular potassium levels of pathogens played a role in host cell colonization and the expression of type III secretion (T3SS). Furthermore, potassium has been shown to preserve mRNA in the decoding center during the elongation phase of translation and to stabilize tRNA, rRNA, and ribosome subunits. Hence, we propose that deletion of the potassium ion transporter Trk1 in C. gloeosporioides may also impact colonization, virulence factor secretion, and the expression of crucial infection-related genes.
Liu, Y.; Ho, K. K.; Su, J.; Gong, H.; Chang, A. C.; et al. Potassium Transport of Salmonella is Important for Type III Secretion and Pathogenesis. Microbiology. 2013, 159, 1705-1719.
Hayward, J. A.; Mathur, A.; Ngo, C.; Man, S. M. Cytosolic Recognition of Microbes and Pathogens: Inflammasomes in Action. Microbiology and Molecular Biology Reviews, 2018, 82(4), e00015-18.
MacGilvary, N. J.; Kevorkian, Y. L.; Tan, S. Potassium response and homeostasis in Mycobacterium tuberculosis modulates environmental adaptation and is important for host colonization. PLoS Pathogens, 2019,15(2), e1007591.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe study by Zhi et al identifies a high-affinity potassium transporter, CgTrk1, in Colletotrichum gloeosporioides, an economically important pathogen causing anthracnose on crops and trees worldwide. The authors localized CgTrk1 to the cell membrane and showed its role for potassium uptake in C. gloeosporioides, as well as its various aspects of pathogenicity.
The deletion of the CgTRK1 gene leads to decreased potassium uptake, impaired vegetative growth, reduced appressorium development, and diminished virulence. Despite the presence of a homologous protein, CgTrk2, CgTrk1 is demonstrated to play dominant roles in potassium homeostasis, invasive growth, and pathogenicity in C. gloeosporioides.
The study seems well structured and organized with solid experiments. This study seems a continuous (or supplementary) work to their saga including the Involvement of MAPKKK CgMck1, CgSat4 in K+ Uptake, full virulence, cell wall, cation tolerance etc.
The study presents valuable findings regarding the role of CgTrk1 in K+ uptake and pathogenicity, however the study suffers from various weaknesses. Addressing all of them would further enhance the rigor and impact of the research and would subject to different interpretation
The main limitation of this study is merely descriptive. While the study identifies the role of CgTrk1 in K+ uptake and pathogenicity, it lacks a comprehensive understanding of the regulatory mechanisms governing these processes. Further investigation into the (molecular) regulation network of CgTrk1 is suggested. Otherwise, the authors seems to describe one by one process in each paper. Having said that the authors do not delve deeply (or try) into other potential functions or interactions of CgTrk1 within the fungal cell or their previous findings.
Also, seems that the study provides insights into the specific functions of CgTrk1, but it is essential to avoid overgeneralizing the findings to other fungi or organisms without further experimental validation. Throughout the text the authors over speculate their findings without solid data/experiments.
Besides, this reviewer is unclear about the iuncomplete characterization of CgTrk2: Although CgTrk2 is identified as a homologous protein to CgTrk1, its specific role and significance in K+ uptake / pathogenicity / cation uptake are not thoroughly explored in the study. Further characterization of CgTrk2 would provide a more comprehensive understanding (or discard) of K transport in C. gloeosporioides.
Additionally, the study has several weaknesses and pitfalls that must be addressed:
- “Colletotrichum” instead of “Colletrotrichum”
- The graphs and table units must be more clarified for instance Fig 2A; ug/mg of what ?
- L 221-222: This statement “These results indicate that CgTrk1 and CgTrk2 are involved to regulate cell wall integrity” seems too courageous to conclude based on these (incomplete) data.
- Important Fig 1B: is not clear. The subcellular localization of CgTrk1 in appressorium formation is not well depicted. Please provide as a sole new figure the Green fluorescence detected during the stages of germination and appressorium formation. .
- Fig 2 and throughout the text. What various stresses resistance ?? This is very general and this reviewer disagrees the generalization of the “various stress” please be more specific in the figure and whole the text since authors did not text various stresses.
- What about the response to other high osmotic Stress ( NaCl, sorbital, etc)
- Fig 4:Please amend this figure with Trypan blue staining of lesions in picture。
- RT-PCR (gene expression) or metabolite quantification of cell wall biosynthesis-related and potassium-related genes. RT-PCR
- Data on the appressoria formation and the rate percentage on the GelBond membrane are required.
- Does extracellular application of substances (i.e. cAMP) induces appressorium formation in the mutants. This should be tested and discussed.
- A suggestive working model is required, where the authors can explain and/or connect all the players (genes/proteins/metabolites) involved in.
- The discussion section should be expanded to provide deeper insights into the implications of the findings and their significance in the broader context of fungal physiology and pathogenicity.
- The reference list should be amended with additional relevant citations to support the study's findings and contextualize them within the existing literature.
Comments on the Quality of English Language
Extensive editing of English language required
Author Response
Author Response File: 
Author Response.pdf
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsWhile the authors have made some improvements, several critical issues remain unresolved and many of the comments provided in the initial review have not been adequately addressed.
1. The m/s still primarily relies on descriptive content without engaging in deeper analysis or critical discussion. Please move beyond description and provide more substantial insights and discussion on the topic.
2. The writing quality remains a concern. Coherence, and conciseness persist throughout the manuscript. Additionally, there are instances of grammatical errors and awkward phrasing that need to be corrected.
3. The quality/clarity of figures (i.e. subcellular localication) and the inclusion of additional data require significant improvement. Figures lack clarity, proper labeling, and necessary context. Additional data requested in the previous review has not been provided.
4。The suggested model needs to be improved by including further mechanisms and routes. As it stands どえ this figures does not provide any deep insight.
Comments on the Quality of English Language
Suggest that the authors revise the manuscript by a native speaker in the field for improved clarity, coherence, and conciseness.
Author Response
- The m/s still primarily relies on descriptive content without engaging in deeper analysis or critical discussion. Please move beyond description and provide more substantial insights and discussion on the topic.
Response: Thank you for your question. I have presented a new analysis that supports the article’s viewpoint.
The writing quality remains a concern. Coherence, and conciseness persist throughout the manuscript. Additionally, there are instances of grammatical errors and awkward phrasing that need to be corrected.
Response: Thank you for your question. I have revised the manuscript in question and sought assistance from an English-speaking scientist.
The quality/clarity of figures (i.e. subcellular localication) and the inclusion of additional data require significant improvement. Figures lack clarity, proper labeling, and necessary context. Additional data requested in the previous review has not been provided.
Response: Thank you for your question. The figure of localization pattern of the CgTrk1 protein were replaced in new version. The clarity of figures, proper labeling were supplied. In terms of localization pattern of the CgTrk1 protein in appressorium, we found that slightly GFP fluorescence in the cytoplasm was detected in the stages of appressorium formation (followed figure). Thus, we not provided the data in the main manuscript.
The suggested model needs to be improved by including further mechanisms and routes. As it stands this figures does not provide any deep insight.
Response: Thank you for your question. The working model has been revised. we have highlighted the role of kinase Sat4 in phosphorylating CgTrk1, which subsequently facilitates its subcellular localization on the plasma membrane, thereby enabling potassium uptake. In turn, phosphatase Ppz1 dephosphorylated CgTrk1, thereby regulating the activity of CgTrk1. In addition, the legends in question of working model have undergone a process of revision.
