Genome-Wide Analysis Revealed NBS-LRR Gene Candidates Associated with Bacterial Wilt Resistance in Eggplant (Solanum melongena L.)

Round 1

Reviewer 1 Report

The abstract is not well written and needs to improve. add 2-3 lines of results in the abstract section of the manuscript.

The introduction section is poorly written. Add the citation of the recently published paper

In line no 120, Details of Expasy tool

Improve all figures to at least 300 dpi resolution and describe briefly the legends section.

Check the grammatical mistakes throughout the manuscript.

The discussion section is very poor. Kindly improved

Moderate editing of English language required

Author Response

Reviewer: 1

Comments to the Author

Comments 1: The abstract is not well written and needs to improve. add 2-3 lines of results in the abstract section of the manuscript.

Response 1:

Thank you for your comment. We did not add more results in the abstract section of the manuscript. Since the maximum word requirement for the abstract is about 200 words, and we have stated the most important results in the abstract, which is about 196 words.

 

Comments 2: The introduction section is poorly written. Add the citation of the recently published paper

Response 2:

Good comment! We have cited several recently published papers that related on the NBS genes involved in disease resistance to various species in lines 61-65, also updated references in the References section in lines 471-476, 479-482.

 

Comments 3: In line no 120, Details of Expasy tool

Response 3:

We revised this sentence “The ExPASy tool (https://web.expasy.org/ttools) was used to predict the chemical-physical properties with default parameters” in lines 116-117.

 

Comments 4: Improve all figures to at least 300 dpi resolution and describe briefly the legends section.

Response 4:

Good comment! All figures were improved to at least 300 dpi resolution, and we added brief descriptions in the figure’ legends.

 

Comments 5: Check the grammatical mistakes throughout the manuscript.

Response 5:

Thank you for your comment. The manuscript was checked and corrected for grammatical mistakes according to the comment. Additionally, we submitted our manuscript to MDPI for English editing.

 

Comments 6: The discussion section is very poor. Kindly improved

Response 6:

Thank you for your comment. We added more discussion on results of candidate NBS genes to improve the discussion section according to the comment in lines 370-380.

Author Response File: Author Response.pdf

Reviewer 2 Report

A Well written paper based on a very robust study that increases our understanding not only of the evolution of NBS-LRR genes but also of the genetic basis of bacterial wilt resistance in egg plant. I only have minor suggested revisions.

Line 19: family have been -  family has been

Line 22: wrong bracketing - (231 CNLs (CC-NBS-LRR) - 231 CNLs (CC-NBS-LRR)

Line 26: Evolutionarily – Evolutionary

Line 48: Improper capitalization - Resistance to Powdery MildeW8

Line76: cultivated important – cultivated and important

Line79: members have been - members has been

Line 81/82: In current study - In the current study

Lin3 93: wilt, are provided - wilt, were provided

Line 94: seedlings – the seedlings

Line95: 8 hours dark - 8 hours darkness

Line 204: Change “obviously” to “clearly”

Line 227: we – We

Line 266: Change “obviously” to “clearly”

Line 285: The expressions – the expression

Line 317: but also be greatly significance of - but is also of great significance in

Line 324: change “demonstrated that”  to “reported”

Line 329: Poor bracketing - (231 CNLs (CC-NBS-LRR) -  231 CNLs (CC-NBS-LRR),

Line 353: Revise the last part of the sentence - …………….as well P-loop, GLGL, and kinase-2 were most common and conserve. Suggested revision: …………………with the P-loop, GLGL, and kinase-2 being the most common and conserved.

Linen 379: change – changed

 

Line  380: delete the word “that”

The paper is written in good English. Only minor English language editing is required

Author Response

Reviewer: 2

Comments to the Author

A Well written paper based on a very robust study that increases our understanding not only of the evolution of NBS-LRR genes but also of the genetic basis of bacterial wilt resistance in eggplant. I only have minor suggested revisions.

Thank you very much for the great summary and insightful comments! We have revised our manuscript accordingly as below.

 

Comments 1: Line 19: family have been – family has been

Response 1:

We revised the “family have been” to “family has been” in line 16.

 

Comments 2: Line 22: wrong bracketing – (231 CNLs (CC-NBS-LRR) – 231 CNLs (CC-NBS-LRR)

Response 2:

We removed the redundant bracket in line 19.

 

Comments 3: Line 26: Evolutionarily – Evolutionary

Response 3:

We revised the “Evolutionarily” to “Evolutionary” in line 23.

 

Comments 4: Line 48: Improper capitalization – Resistance to Powdery MildeW8

Response 4:

We revised the “Resistance to Powdery MildeW8” to resistance to powdery mildew8 in line 42.

 

Comments 5: Line76: cultivated important – cultivated and important

Response 5:

We revised the “cultivated important” to “cultivated and important” in line 76.

 

Comments 6: Line79: members have been – members has been

Response 6:

We revised the “members have been” to “members has been” in line 78.

 

Comments 7: Line 81/82: In current study – In the current study

Response 7:

We added “the” between “In” and “current study” in line 81.

 

Comments 8: Line 93: wilt, are provided – wilt, were provided

Response 8:

We revised the “are provided” to “were provided” in line 92.

 

Comments 9: Line 94: seedlings – the seedlings

Response 9:

We revised the “seedlings” to “the seedlings” in line 93.

 

Comments 10: Line95: 8 hours dark – 8 hours darkness

Response 10:

We revised the “8 hours dark” to “8 hours darkness” in line 94.

 

Comments 11: Line 204: Change “obviously” to “clearly”

Response 11:

We deleted the word “obviously” in line 204.

 

Comments 12: Line 227: we – We

Response 12:

We revised the “we” to “We” in line 226.

 

Comments 13: Line 266: Change “obviously” to “clearly”

Response 13:

We changed the “obviously” to “clearly” in line 260.

 

Comments 14: Line 285: The expressions – the expression

Response 14:

We revised the “The expressions” to “the expression” in line 277.

 

Comments 15: Line 317: but also be greatly significance of – but is also of great significance in

Response 15:

Good comment! We revised the “but also be greatly significance of” to “but is also of great significance in” in line 306.

 

Comments 16: Line 324: change “demonstrated that” to “reported”

Response 16:

We revised the “demonstrated that” to “reported” in line 313.

 

Comments 17: Line 329: Poor bracketing – (231 CNLs (CC-NBS-LRR) – 231 CNLs (CC-NBS-LRR),

Response 17:

We removed the redundant bracket in line 318.

 

Comments 18: Line 353: Revise the last part of the sentence - …………….as well P-loop, GLGL, and kinase-2 were most common and conserve. Suggested revision: …………………with the P-loop, GLGL, and kinase-2 being the most common and conserved.

Response 18:

Good comment! We revised the last part of the sentence “as well P-loop, GLGL, and kinase-2 were most common and conserve” to “with the P-loop, GLGL, and Kinase-2 being the most common and conserved” in lines 338-339.

 

Comments 19: Line 379: change – changed

Response 19:

We revised the “change” to “changed” in line 363.

 

Comments 20: Line 380: delete the word “that”

Response 20:

We deleted the word “that” in line 364.

 

Author Response File: Author Response.pdf

Reviewer 3 Report

 This manuscript presents an analysis of NBS genes in eggplant, piecing together computational analysis of previously published data and qPCR assessment of their own conducting.

 

My primary concerns with the manuscript are that it is very difficult to to understand what work has been done as part of this study, and what has been done previously and is being re-presented in this manuscript.  This goes along with a concern that many key methodological details have not been included, contributing to the confusion about what was actually done as part of this study, as well as rendering it difficult to evaluate the quality of the work and the approaches used.  Similarly, many details need to be added to each of the Figure captions (including supplemental).

Another major concern is low image quality of the figures provided.

A few details on specific things that must be fixed in the manuscript (although not exhaustive):

 

Lines 65-66 – remove parentheses around Pseudomonas syringae

Lines 96-97 – Reference 33 does not provide sufficient detail and description of this inoculation method, please provide the details on the inoculation in this section.  Furthermore, no information is provided on the strain of R. solanacearum used or its culture and inoculum preparation, these details are needed.

Line 104 – please refer (here and throughout manuscript) to the genome as ‘GUIQIE-1’ to be consistent with those who published the genome and public databases.

Line 156 – correct typo in “primer3”

Lines 150-156 – no details are provided on reverse transcription reactions (cDNA synthesis)  or on apparatus used for qPCR reactions/analysis. 

Why are the qPCR methods in section 2.6 – so far removed from section 2.1 (which contains RNA isolation methods)?  This should be better organized.

Table 1 needs to be revised to clearly separate the data generated in this study from that coming from other studies.  Please add a column to the table to include references to the other studies.  If somehow all of the data in Table 1 originate from this study, then appropriate methods must be added for the analysis of the additional 10 genomes.

Line 192 – why is this “obvious”?  I recommend changing this word to “Visually”

Figure 1 caption needs more detail.  Figure 1 is too low resolution to read text – please replace with a higher resolution version of the Figure.

Line 204 – remove “Obviously”

Line 213-214 – details like this need to be added to the figure caption.  Figure 2 caption needs more information.

For Table 3, please include in the text which SmNBSs were included and add a column to the table indicating how many SmNBSs contained that specific motif.

Editorial >> Lines 235-241 font change is distracting

Figure 3 – it would be nice to have a higher resolution version of this figure so that all elements of the figure can be read by readers

Figure 3 caption – needs details added.  (including the note from lines 275-276)

Line 266 – Use of “obviously” needs to be justified, changed, or removed – but as is, it is not being used appropriately.

Figure 5 – caption needs additional details.  Color scales for heat maps need units.

Figure 5 - Is there an uninoculated control or only time=0?

Line 287-289 does not fit here, move to the start of section 3.8 or remove.

Figure 6 caption – Use of “symptomatic” here is not suitable.  Reword and add additional details about inoculation, inoculum, RNA, cDNA, qPCR.  Add note to caption.

Data in figure 6 is from two different varieties than all previous data – please comment/discuss in manuscript on how genomic differences between varieties may impact the outcomes and interpretations of data in Figure 6 as compared to the rest of the manuscript.

Lines 316-318 – This sentence does not make sense and must be re-written.

Discussion and Conclusion sections English is poor and should be completely reviewed for English language.

Supplemental figure captions (and also methods section) need additional detail on what was done and how figures were generated (all supplemental figures need this).  Figure legend missing for Figure S4.  All supplemental figure legends should be bigger for readability, and order legend entries either numerically or alphabetically – but not randomly.

Table S3 – It is unclear if the authors re-analyzed the transcriptome data or were able to directly access and download TPM values.  If the former, it appears that a great deal of detail is missing in terms of re-analysis of sequencing data generated previously (if the latter, this should be more explicitly explained).

Several English language errors are present throughout the manuscript.  The errors are especially problematic in the Discussion and Conclusion sections, where several sentences are indecipherable as to what meaning is intended by the authors.

Author Response

Reviewer: 3

Comments to the Author

This manuscript presents an analysis of NBS genes in eggplant, piecing together computational analysis of previously published data and qPCR assessment of their own conducting.

 My primary concerns with the manuscript are that it is very difficult to understand what work has been done as part of this study, and what has been done previously and is being re-presented in this manuscript.  This goes along with a concern that many key methodological details have not been included, contributing to the confusion about what was actually done as part of this study, as well as rendering it difficult to evaluate the quality of the work and the approaches used.  Similarly, many details need to be added to each of the Figure captions (including supplemental).

Another major concern is low image quality of the figures provided.

A few details on specific things that must be fixed in the manuscript (although not exhaustive):

 Thank you very much for your comments, and we believe these comments will help to improve our manuscript a lot! We have clarified the work we have done and the key methodological details, and added more description on Figure captions, then improve the image quality of figures (at least 300 dpi). Meanwhile, we have revised the details on specific things as below.

 

Comments 1: Lines 65-66 - remove parentheses around Pseudomonas syringae

Response 1:

We removed the parentheses around Pseudomonas syringae in line 60.

 

Comments 2: Lines 96-97 - Reference 33 does not provide sufficient detail and description of this inoculation method, please provide the details on the inoculation in this section.  Furthermore, no information is provided on the strain of R. solanacearum used or its culture and inoculum preparation, these details are needed.

Response 2:

Good comment! We completed the details of the cultivating of the R. solanacearum strain, as well as the inoculation method in the materials and methods section 2.1 in lines 96-99, and also cited the reference there.

 

Comments 3: Line 104 - please refer (here and throughout manuscript) to the genome as ‘GUIQIE-1’ to be consistent with those who published the genome and public databases.

Response 3:

Good comment! We revised the genome “GUIQIE” to “GUIQIE-1” throughout manuscript in lines 102, 119, and 141.

 

Comments 4: Line 156 - correct typo in “primer3”

Response 4:

We corrected typo in “primer3” in line 157.

 

Comments 5: Lines 150-156 - no details are provided on reverse transcription reactions (cDNA synthesis) or on apparatus used for qPCR reactions/analysis. 

Response 5:

We added the information of cDNA synthesis and the apparatus of qPCR analysis in the materials and methods section 2.6 in lines 147-149.

 

Comments 6: Why are the qPCR methods in section 2.6 - so far removed from section 2.1 (which contains RNA isolation methods)?  This should be better organized.

Response 6:

Thank you for your comment. We removed the RNA isolation methods to section 2.6, which combined the RNA extract, cDNA synthesis, and qPCR together in line 147-147, and 151-154.  

 

Comments 7: Table 1 needs to be revised to clearly separate the data generated in this study from that coming from other studies.  Please add a column to the table to include references to the other studies.  If somehow all of the data in Table 1 originate from this study, then appropriate methods must be added for the analysis of the additional 10 genomes.

Response 7:

Good comment! We added a column of reference resources from other studies in Table1 to clearly separate the data generated in this study from that coming from other studies.

 

Comments 8: Line 192 - why is this “obvious”?  I recommend changing this word to “Visually”

Response 8:

We revised the “obvious” to “Visually” in line 191.

 

Comments 9: Figure 1 caption needs more detail.  Figure 1 is too low resolution to read text - please replace with a higher resolution version of the Figure.

Response 9:

We added more detailed description in the Figure 1 caption, and the image quality of Figure 1 was improved to at least 300 dpi resolution in lines 198-200.  

 

Comments 10: Line 204 - remove “Obviously”

Response 10:

We removed the word “Obviously” in line 204.

 

Comments 11: Line 213-214 - details like this need to be added to the figure caption.  Figure 2 caption needs more information.

Response 11:

Thank you for your comment. We added more description in the Figure 2 caption in lines 210-212.

 

Comments 12: For Table 3, please include in the text which SmNBSs were included and add a column to the table indicating how many SmNBSs contained that specific motif.

Response 12:

Thank you for your comment. We have added a column in table 3, which is about the number of each motif distribution in SmNBSs (Table 3).

 

Comments 13: Editorial >> Lines 235-241 font change is distracting

Response 13:

The fonts in this section have been standardized in lines 232-236.

 

Comments 14: Figure 3 - it would be nice to have a higher resolution version of this figure so that all elements of the figure can be read by readers

Response 14:

The image quality of Figure 3 was improved to at least 300 dpi resolution.

 

Comments 15: Figure 3 caption - needs details added.  (including the note from lines 275-276)

Response 15:

We added more details in Figure 3 caption in lines 254-255.

 

Comments 16: Line 266 - Use of “obviously” needs to be justified, changed, or removed - but as is, it is not being used appropriately.

Response 16:

We revised the “obviously” to “clearly” in line 260.

 

Comments 17: Figure 5 - caption needs additional details.  Color scales for heat maps need units.

Response 17:

Thank you for your comment. We added the details in Figure 5 caption in lines 281-283. We use the z-scale normalized TPM values (Z-score) to plot heat maps in TBtools. The Z-score represents the distance between the original value and the overall mean in units of the standard deviation of all TPM values for each gene. So that the color scale has no specific units, and the color scale from previous studies also does not use units (Liu et al., 2019; Peng et al. 2022).

 

Liu M, Sun W, Ma Z, Huang L, Wu Q, Tang Z, Bu T, Li C, Chen H. Genome-wide identification of the SPL gene family in Tartary Buckwheat (Fagopyrum tataricum) and expression analysis during fruit development stages. BMC Plant Biol. 2019 Jul 8;19(1):299.

Peng Z, Li W, Gan X, Zhao C, Paudel D, Su W, Lv J, Lin S, Liu Z, Yang X. Genome-Wide Analysis of SAUR Gene Family Identifies a Candidate Associated with Fruit Size in Loquat (Eriobotrya japonica Lindl.). Int J Mol Sci. 2022 Oct 31;23(21):13271.

 

Comments 18: Figure 5 - Is there an uninoculated control or only time=0?

Response18:

The samples at 0 hpi (time=0) were not inoculated with the suspension, which could be considered as uninoculated control.

 

Comments 19: Line 287-289 does not fit here, move to the start of section 3.8 or remove.

Response 19:

The sentence of line 287-289 was moved to the start of section 3.8 in lines 285-286.

 

Comments 20: Figure 6 caption - Use of “symptomatic” here is not suitable.  Reword and add additional details about inoculation, inoculum, RNA, cDNA, qPCR.  Add note to caption.

Response 20:

Thank you for your comment. We modified the “symptomatic” to “phenotype” in Figure 5 caption and add note information to caption in lines 299-302. The additional details about inoculation, inoculum, RNA, cDNA, and qPCR were described in section 2.6 in lines 147-149.

 

Comments 21: Data in figure 6 is from two different varieties than all previous data - please comment/discuss in manuscript on how genomic differences between varieties may impact the outcomes and interpretations of data in Figure 6 as compared to the rest of the manuscript.

Response 21:

Thank you for this insightful comment! The logic of Figure 6 may not be presented well previously.

First, it is true that we used public transcriptome data (from different varieties) to identify the NBS genes differentially expressed upon R. solanacearum infection. Then, we used two different varieties (one resistant and one susceptible) of our own to further validate and compare their expressions upon R. solanacearum infection. We believe the genomic differences among varieties may not impact the results much. Instead, we think using different varieties for validation makes the results more reliable and it may imply that these NBS genes (potential resistance genes) may be more prevalent in the germplasm of eggplants.

 

Comments 22: Lines 316-318 - This sentence does not make sense and must be re-written.

Response 22:

We have re-written this sentence in lines 305-307.

 

Comments 23: Discussion and Conclusion sections English is poor and should be completely reviewed for English language.

Response 23:

We reviewed completely for English language in discussion and conclusion section. Additionally, we submitted our manuscript to MDPI for English editing.

 

Comments 24: Supplemental figure captions (and also methods section) need additional detail on what was done and how figures were generated (all supplemental figures need this).  Figure legend missing for Figure S4.  All supplemental figure legends should be bigger for readability, and order legend entries either numerically or alphabetically - but not randomly.

Response 24:

Very good comment! We added the additional detail of the supplemental figure captions in lines 388-389. The methods of all supplemental figure generated were described in the materials and methods, which are as follows:

1) Figure S1: The conserved domains were described in the section 2.2 in lines 110-114.

2) Figure S2: The exon-intron structures were described in the section 2.3 in lines 118-120;

3) Figure S3: The conserved protein motifs were described in the section 2.4 in lines 126-127;

 

It is too different to present the Figure S4 well due to too many legends, and we deleted it after consideration.

The motif legend order is arranged according to the order of motifs in the sequence, rather than motif numeric order. This kind of motif legend order is consistent with other studies (Ti et al., 2022; Liang et al., 2022).

 

Li D, Tang X, Dong Y, Wang Y, Shi S, Li S, Liu Y, Ge H, Chen H. Comparative genomic investigation of TCP gene family in eggplant (Solanum melongena L.) and expression analysis under divergent treatments. Plant Cell Rep. 2022 Nov;41(11):2213-2228.

Liang S, Xu S, Qu D, Yang L, Wang J, Liu H, Xin W, Zou D, Zheng H. Identification and Functional Analysis of the Caffeic Acid O-Methyltransferase (COMT) Gene Family in Rice (Oryza sativa L.). Int J Mol Sci. 2022 Jul 31;23(15):8491.

 

Comments 25: Table S3 - It is unclear if the authors re-analyzed the transcriptome data or were able to directly access and download TPM values.  If the former, it appears that a great deal of detail is missing in terms of re-analysis of sequencing data generated previously (if the latter, this should be more explicitly explained).

Response 25:

Very good comment! Since the TPM values did not provide directly in the previous study, we only download the raw transcriptome data. In that case, we re-analyzed the transcriptome data to calculate the TPM values and the methods were added in the Materials and Methods section 2.6 in lines 139-143.

 

Comments 26: Comments on the Quality of English Language

Several English language errors are present throughout the manuscript.  The errors are especially problematic in the Discussion and Conclusion sections, where several sentences are indecipherable as to what meaning is intended by the authors.

Response 26:

We carefully checked the manuscript for English language errors (especially in the Discussion and Conclusion) to ensure that the original meaning was correctly indicated. Additionally, we submitted our manuscript to MDPI for English editing.

 

Author Response File: Author Response.pdf

Reviewer 4 Report

In my opinion, the article sent for review is important for the development of modern plant genetics and I confirm what the authors wrote, although the identification and characterization of the NBS-LRR gene family have been extensively described in various species, a comprehensive analysis in eggplant has not been previously documented. The authors' undoubted success is the identification of 269 SmNBS genes in eggplant and analyzed their categorization, chromosomal distribution, gene structure composition, phylogenetic relationship, conserved motifs, gene duplications, and predicted cis-acting regulatory elements (CAREs). Importantly, the results obtained by the authors also have an application aspect, their results provide a comprehensive insight into SmNBSs, which will improve research on eggplant disease resistance and facilitate the breeding of new disease-resistant varieties. This manuscript generally is well-written, and the methodology and conclusions are scientifically sound. I found the paper interesting. The investigations are extensive. The presentation of results is clear and attractive. The conclusions are consistent with the evidence and arguments and address the main questions posed.

Specific comments:

Line 56: Replace ‘ genes in Arabidopsis (Arabidopsis thaliana)’ with ‘genes in Arabidopsis thaliana …

Line 69: ‘The tomato Hero’ please modify/clarify

 

Line 77: The correct version is: ‘Solanaceae family'

Table 1. Please remove 'L.' in species names. We must be consistent when writing Latin names - either we write all Latin names with the abbreviations of the authors of the names, or we omit all abbreviations of surnames.

 

Author Response

Reviewer: 4

Comments to the Author

In my opinion, the article sent for review is important for the development of modern plant genetics and I confirm what the authors wrote, although the identification and characterization of the NBS-LRR gene family have been extensively described in various species, a comprehensive analysis in eggplant has not been previously documented. The authors' undoubted success is the identification of 269 SmNBS genes in eggplant and analyzed their categorization, chromosomal distribution, gene structure composition, phylogenetic relationship, conserved motifs, gene duplications, and predicted cis-acting regulatory elements (CAREs). Importantly, the results obtained by the authors also have an application aspect, their results provide a comprehensive insight into SmNBSs, which will improve research on eggplant disease resistance and facilitate the breeding of new disease-resistant varieties. This manuscript generally is well-written, and the methodology and conclusions are scientifically sound. I found the paper interesting. The investigations are extensive. The presentation of results is clear and attractive. The conclusions are consistent with the evidence and arguments and address the main questions posed.

Thank you very much for the great summary and insightful comments! We have revised our manuscript accordingly as below.

 

Specific comments:

Comments 1: Line 56: Replace ‘ genes in Arabidopsis (Arabidopsis thaliana)’ with ‘genes in Arabidopsis thaliana’

Response 1:

We revised the “genes in Arabidopsis (Arabidopsis thaliana)” to “genes in Arabidopsis thaliana” in line 50.

 

Comments 2: Line 69: ‘The tomato Hero’ please modify/clarify

Response 2:

'The tomato Hero' refers to the Hero gene of tomato. We revised the “The tomato Hero” to “The tomato Hero gene” in line 67.

 

Comments 3: Line 77: The correct version is: ‘Solanaceae family'

Response 3:

We revised the “solanaceous family” to “Solanaceae family” in line 77.

 

Comments 4: Table 1. Please remove 'L.' in species names. We must be consistent when writing Latin names - either we write all Latin names with the abbreviations of the authors of the names, or we omit all abbreviations of surnames.

Response 4:

We removed 'L.' in species names to be consistent the Latin names in Table 1.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors has revised the manuscript accordingly. My recommendation is accept for publication.

Minor editing of English language required