Diversity of Expression Patterns of Lr34, Lr67, and Candidate Genes towards Lr46 with Analysis of Associated miRNAs in Common Wheat Hybrids in Response to Puccinia triticina Fungus
, Agnieszka Tomkowiak 1,*, Aleksandra Noweiska 1, Roksana Bobrowska 1, Jan Bocianowski 2,*, Aleksandra Sobiech 1 and Michał Tomasz Kwiatek 1,3Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe interaction between a plant and a pathogen is one of the priority areas in agricultural science. Identifying protective mechanisms against Puccinia triticina in one of the most important agricultural wheats is not only important, but also a complex and time-consuming task.
Annotation. Add practical significance to the results obtained.
Introduction. Extensive, lots of interesting data. Move your data (table), since it is unpublished, to the Discussion section. At the end of the Introduction section, indicate in detail the purpose and objective of the study (preferably based on your published data).
Materials and methods. This section is written in detail, perhaps even too detailed. In subsection 2.3. indicate at the very beginning which molecular markers were identified (since it was immediately possible to decide that these were DNA markers (ladder)).
330- decrypt hpi
362- bring unified or hour, or h
491 - there is also tetraploid wheat
Conclusion. The conclusion does not present the results themselves, but only the conclusions.
Author Response
Response to Reviewer 1 Comments
Reviewer #1
Point 1: The interaction between a plant and a pathogen is one of the priority areas in agricultural science. Identifying protective mechanisms against Puccinia triticina in one of the most important agricultural wheats is not only important, but also a complex and time-consuming task.
Response: Thank you very much.
Point 2: Annotation. Add practical significance to the results obtained.
Response: Thank you for your suggestion. The practical significance of our results is completed in the Discussion and Conclusions.
Point 3: Introduction. Extensive, lots of interesting data. Move your data (table), since it is unpublished, to the Discussion section. At the end of the Introduction section, indicate in detail the purpose and objective of the study (preferably based on your published data).
Response: Thank you very much for your suggestion. We have changed the Introduction and Discussion according to your suggestions. In Discussion section we have added more information on our preliminary unpublished studies on the expression of all candidate genes for Lr46/Yr29.
Point 4: Materials and methods. This section is written in detail, perhaps even too detailed. In subsection 2.3. indicate at the very beginning which molecular markers were identified (since it was immediately possible to decide that these were DNA markers (ladder)).
Response: Thank you very much for your precious suggestion. We have changed this fragment considering your suggestion. We have corrected Figure 1 and placed it in section 2.3. Table 1 has been placed in section 2.4. Table 1 has been completed and contains all the genes that were analyzed in our study.
Point 5: 330- decrypt hpi
Response: We have added an explanation of the abbreviation “hpi” – “hours post inoculation”. We previously explained “hpi” in subsection 2.4.
Point 6: 362- bring unified or hour, or h.
Response: Thank you for pointing out our mistake. We have corrected the "h" to "hpi".
Point 7: 491 - there is also tetraploid wheat.
Response: Thank you for your comment. We have changed this fragment considering your suggestion.
Point 8: Conclusion. The conclusion does not present the results themselves, but only the conclusions.
Response: Thank you very much for your suggestion. We have added more information on our preliminary unpublished studies on the expression of all candidates for Lr46/Yr29. In addition, the conclusions have been improved to follow the idea more easily. This section has been updated with the most important results we obtained. Thank you very much for reviewing our manuscript.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThis paper presents interesting results on expression patterns some candidate genes and the analysis of associated miRNAs in wheat in response to rust disease. The quality of work is good and the authors found some novel results.
However, the writing quality of the paper need to be improved. The paper is complex. Also, the paper is wordy. The introduction is unnecessarily long which must be reduced by half. It is written unprofessionally. Also, there should not be any figure or table in this section.
Methodology is also wordy. I suggest concise known methodology and cite original references.
Results are not clear. Please avoid just mentioning the values or range but I suggest compare the entities/treatments very clearly which is easily understandable.
Figures are not professional. Please rebuild the figures. Remove minor gridlines and make the fonts visible. Avoid red colors from bars.
I suggest the authors improving the discussion.
Language quality needs improvement.
Author Response
Response to Reviewer 2 Comments
Reviewer #2
Point 1: This paper presents interesting results on expression patterns some candidate genes and the analysis of associated miRNAs in wheat in response to rust disease. The quality of work is good and the authors found some novel results.
Response: Thank you very much.
Point 2: However, the writing quality of the paper need to be improved. The paper is complex. Also, the paper is wordy. The introduction is unnecessarily long which must be reduced by half. It is written unprofessionally. Also, there should not be any figure or table in this section.
Response: Thank you very much. We have changed the Introduction and Discussion according to your suggestions. We hope that this arrangement will be better for following the aim and implementation of our study. We have respected this valuable comment and moved Table 1 and Figure 1 to the Materials and Methods section.
Point 3: Methodology is also wordy. I suggest concise known methodology and cite original references.
Response: Thank you very much for your comment. We have changed the Methodology according to your suggestions. We have described the methods used in a simpler way.
Point 4: Results are not clear. Please avoid just mentioning the values or range but I suggest compare the entities/treatments very clearly which is easily understandable.
Response: We have taken Reviewer’s comment in full consideration and it will be well reflected by the revised version of manuscript. We have performed additional statistical analysis for better conclusions. The structure of the Results section has been simplified with a division into two subsections. We hope that this arrangement will be better for following the aim and implementation of our study.
Point 5: Figures are not professional. Please rebuild the figures. Remove minor gridlines and make the fonts visible. Avoid red colors from bars.
Response: Thank you for your suggestion. We have improved the quality of the presented figures.
Point 6: I suggest the authors improving the discussion.
Response: Thank you for your important suggestion. The practical significance of our results is completed in the Discussion and Conclusions. We have added more information on our preliminary unpublished studies on the expression of all candidate genes for Lr46/Yr29.
Point 7: Language quality needs improvement.
Response: Thank you very much for your suggestion. We have improved the level of English in each section. We hope that these changes made will increase the comprehensibility of the manuscript.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for Authorscimb-2964082-peer-review-v1
Comments
Thank you for sending me this manuscript by Julia Spychała, et al., to review. Overall, the authors evaluated the expression level for candidates’ genes involved in the rust resistance. They also investigated the expression pattern of miRNA complementary to Lr34 genes and its respective candidates.
In general, the manuscript must be improved in its different sections. Abstract is very confusing and hard to follow the idea. Methods are not well described, and some data are not well documented. Results is very short in some sub section.
There are several major and minor issues that are listed in order, as follow:
1) Abstract must be improved, it is difficult to follow the idea. Please be clearer describing the aims achieved in this manuscript. Clarify what are “the candidate genes”, etc. In general use appropriate words for the context
2) page 1, line 18-23: the paragraph is very confusing, firstly we cannot test a gene against other gene, do authors mean protein, miRNAs? This is for the phrase “we selected four candidate genes against Lr46 out of ten candidates…”
3) page 1, line 21-22: please specify what inoculation the authors are referring to.
4) page 1, line 34: please use a different word rather than “civilization significance”
5) page 2, line 70-71: as the technique was already specify, just write QTL
6) page 2, line 80-81: “…resulting in the pathogen developing more slowly and less rapidly.” More slowly and less rapidly is the same result.
7) page 2, line 86: R genes? I recommend describing these genes first .
8) page 4, line 169: please specify the number of uredospores per ml, or a unit to determine the amounts of spores adding during the treatment.
9) page 4, line 182: Indicate the Tris buffer pH and concentration used for DNA dilution.
10) page 8, line 319: “Another gene with high significant expression was another candidate gene Lr46-RLK3” change to “Another gene with high significant expression was the candidate gene Lr46-RLK3”.
11) page 8, the section 3.3 is very general. Please provide more details.
12) page 9, line 330: “Finally, between 24 hpi and 48 hpi, when we observe an increase in Lr34 gene expression” change to “Finally, between 24 and 48 hpi, when we observe an increase in Lr34 gene expression”.
13) The overall results do not support at high extent the central hypothesis.
14) Please provide higher resolution Fig 1, Fig 4, Fig 5, Fig 6, Fig 7,
15) Please provide the agarose gel mentioned in material and methods.
Comments on the Quality of English Languagethe english style should be improved and the use of adequated words for the context is required
Author Response
Response to Reviewer 3 Comments
Reviewer #3
Point 1: Thank you for sending me this manuscript by Julia Spychała, et al., to review. Overall, the authors evaluated the expression level for candidates’ genes involved in the rust resistance. They also investigated the expression pattern of miRNA complementary to Lr34 genes and its respective candidates.
Response: Thank you very much for reviewing our manuscript.
Point 2: In general, the manuscript must be improved in its different sections. Abstract is very confusing and hard to follow the idea. Methods are not well described, and some data are not well documented. Results is very short in some sub section.
Response: Thank you very much for your suggestion. Yes, we have changed the structure of the Abstract for better understanding. We hope you will find the abstract more logical.
We have described the methods used in a simpler, clearer way. We have performed additional statistical analysis for better conclusions (two-way ANOVA). The structure of the Results section has been simplified with a division into two subsections. We hope that this arrangement will be better for following the aim and implementation of our study.
There are several major and minor issues that are listed in order, as follow:
Point 3: 1) Abstract must be improved, it is difficult to follow the idea. Please be clearer describing the aims achieved in this manuscript. Clarify what are “the candidate genes”, etc. In general use appropriate words for the context
Response: We have taken Reviewer’s comment in full consideration and it will be well reflected by the revised version of manuscript. The abstract has been heavily revised to more easily follow the idea of our study. We have added more information on our preliminary unpublished studies on the expression of all candidates for Lr46/Yr29.
Point 4: 2) page 1, line 18-23: the paragraph is very confusing, firstly we cannot test a gene against other gene, do authors mean protein, miRNAs? This is for the phrase “we selected four candidate genes against Lr46 out of ten candidates…”.
Response: Thank you very much for your valuable suggestion. We have corrected this mistake in the mentioned section.
Point 5: 3) page 1, line 21-22: please specify what inoculation the authors are referring to.
Response: Thank you very much for your comment. This information has been completed in the mentioned section.
Point 6: 4) page 1, line 34: please use a different word rather than “civilization significance”.
Response: Thank you so much for your suggestion. The mentioned phrase has been changed.
Point 7: 5) page 2, line 70-71: as the technique was already specify, just write QTL.
Response: We have corrected this sentence by placing only the abbreviation QTL.
Point 8: 6) page 2, line 80-81: “…resulting in the pathogen developing more slowly and less rapidly.” More slowly and less rapidly is the same result.
Response: Thank you so much for your suggestion. The mentioned phrase has been changed.
Point 9: 7) page 2, line 86: R genes? I recommend describing these genes first.
Response: Thank you for your valuable comment. We have included a short description on R genes, citing relevant references.
Point 10: 8) page 4, line 169: please specify the number of uredospores per ml, or a unit to determine the amounts of spores adding during the treatment.
Response: Thank you very much for your comment. We have included information on uredinospores concentrations in the Materials and methods, 2.2 section.
Point 11: 9) page 4, line 182: Indicate the Tris buffer pH and concentration used for DNA dilution.
Response: Thank you very much for your comment. We checked the protocol and it is an Elution buffer. We apologise for the mistake. Unfortunately, the manufacturer of the mentioned DNA isolation kit did not indicate the pH of this buffer.
Point 12: 10) page 8, line 319: “Another gene with high significant expression was another candidate gene Lr46-RLK3” change to “Another gene with high significant expression was the candidate gene Lr46-RLK3”.
Response: We replaced the sentence "Another gene with high significant expression was another candidate gene Lr46-RLK3." with the sentence "Another gene with high significant expression was the candidate gene Lr46-RLK3.".
Point 13: 11) page 8, the section 3.3 is very general. Please provide more details.
Response: Thank you so much for your suggestion. The Results section has been simplified with a division into two subsections. We hope that this arrangement will be better for following the aim and implementation of our study.
Point 14: 12) page 9, line 330: “Finally, between 24 hpi and 48 hpi, when we observe an increase in Lr34 gene expression” change to “Finally, between 24 and 48 hpi, when we observe an increase in Lr34 gene expression”.
Response: We replaced the sentence "Finally, between 24 hpi and 48 hpi, when we observe an increase in Lr34 gene expression " with the sentence " Finally, between 24 and 48 hours post inoculation (hpi), when we observe an increase in Lr34 gene expression".
Point 15: 13) The overall results do not support at high extent the central hypothesis.
Response: In Discussion section we have added more information on our preliminary unpublished studies on the expression of all candidates for Lr46/Yr29.
Point 16: 14) Please provide higher resolution Fig 1, Fig 4, Fig 5, Fig 6, Fig 7,
Response: Thank you very much for your comment. The quality of the mentioned Figures has been improved.
Point 17: 15) Please provide the agarose gel mentioned in material and methods.
Response: Thank you very much for your precious suggestion. We will send Figures with a description of the molecular markers (csLV34, cfd23, cfd71) to be analysed in separate attachments. We have also included a figure showing the identification of the marker csLV46G22 (E. Lagudah, Unpublished data). However, the interpretation of this result is due to personal correspondence with Evans Lagudah, so please do not distribute this figure as the csLV46G22 marker is still unpublished. Thank you very much for reviewing our manuscript.
csLV34:
cfd23:
cfd71:
csLV46G22:
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsThe revised version is acceptable for publication.
Reviewer 3 Report
Comments and Suggestions for AuthorsI agree with the new version of the manuscript. No futher modification are needed
