Domestication and Genetic Improvement Alter the Symbiotic Microbiome Structure and Function of Tomato Leaf and Fruit Pericarp

Comments 1: [66 In a previous study, Please include the reference]

Response 1: We have included the missing reference for the previous study mentioned on line 66. This reference provides the necessary background and supports the context in which our study was framed.

(Citation:

Lin T, Zhu G, Zhang J, et al. Genomic analyses provide insights into the history of tomato breeding. Nature genetics, 2014, 46(11): 1220-1226.

Zhu G, Wang S, Huang Z, et al. Rewiring of the fruit metabolome in tomato breeding. Cell, 2018, 172(1): 249-261. e12. ).

Comments 2: [176 Please include a reference justifying the use of these reads as bacterial taxa.]

Response 2: We have added a reference that justifies the assignment of specified reads to bacterial and archaeal genomes. This reference discusses the methodology and validation of using such reads for bacterial identification and quantification in metagenomic studies (Citation:

Poore, Gregory D., et al. Microbiome analyses of blood and tissues suggest cancer diagnostic approach." Nature 579.7800 (2020): 567-574.

Roman-Reyna, V., et al., Characterization of the Leaf Microbiome from Whole-Genome Sequencing Data of the 3000 Rice Genomes Project. Rice, 2020. 13(1).).

Comments 3: [198 Syn- echococcus: Please check the scientific name.]

Response 3: We have corrected the scientific name "Syn-echococcus" to "Synechococcus." We apologize for the typographical error and have ensured that the correct nomenclature is used throughout the document.

Comments 4: [201 Why was the composition of the leaf microbiome obtained by metagenome sequencing, while that of the pericarp was obtained by metatranscriptome sequencing? Please discuss.]

Response 4: We have added a discussion explaining why different sequencing approaches were used for the leaf and pericarp microbiomes. The leaf microbiome was analyzed using metagenome sequencing to capture a broad snapshot of the genetic potential, while the pericarp microbiome was analyzed using metatranscriptome sequencing to focus on actively expressed genes, reflecting the dynamic response of the microbiome under fruiting conditions.

Comments 5: [229 Please show the qPCR results of the plants analyzed. Table S2 only shows the primer sequences.]

Response 5: We have updated Table S2 to include the qPCR results (delta Ct and standard deviation) for the plants analyzed. This addition provides quantitative data supporting our findings that the genera we identified are representative members of the tomato leaf microbiome.

Comments 6: [281-283 Please correct the grammar of the paragraph]

Response 6: The grammar of the specified paragraph has been corrected for better clarity and readability. We appreciate your attention to detail in identifying this issue.

[The p-values (P-value ≤ 0.001, Table S4) calculated from PERMANOVA and ANOSIM, based on the Bray-Curtis distance, further demonstrated the significant differences in the leaf and fruit microbiomes among the three tomato clades.]”

 Comments 7: [298 It is a hypothesis, perhaps an experiment can be suggested to answer this question.]

Response 7: Thank you for your comment. We appreciate your suggestion regarding the hypothesis presented in line 298. To clarify, the statement in question is based on established research findings rather than a new hypothesis proposed by our study. We have now added additional references to support this conclusion and provide a more comprehensive background. These references should help to substantiate the basis of our discussion in that section.

(Citation:

Lin T, Zhu G, Zhang J, et al. Genomic analyses provide insights into the history of tomato breeding. Nature genetics, 2014, 46(11): 1220-1226.

Zhu G, Wang S, Huang Z, et al. Rewiring of the fruit metabolome in tomato breeding. Cell, 2018, 172(1): 249-261. e12. ).

Comments 8: [310-311 Please review the meaning of the letters E and F.]

Response 8: Thanks for your comment. We have reviewed and clarified the meaning of the letters E and F in the context they were used, and we have ensured that the meanings are clear.

Comments 9: [368 Please include description of (C).]

Response 9: We have included a detailed description for part (C) of the figure mentioned. Part (C) illustrates the genome-wide association for Capnocytophaga_sp._oral_taxon_878, whose abundance decreased sequentially from PIM to CER to BIG. The left panel features a Manhattan plot, utilizing the tomato leaf dataset, which highlights the major peaks (significant SNPs) distributed across twelve chromosomes, associated with the abundance of Capnocytophaga_sp._oral_taxon_878.

Comments 10: [380 Please write scientific names in italics.]

Response 10: We apologize for the oversight in the formatting of species names. We have carefully reviewed the manuscript and ensured that all species names are now correctly italicized to adhere to scientific naming conventions. This change has been applied throughout the text, including the main body and supplementary materials, to maintain consistency and readability.

4. Response to Comments on the Quality of English Language

Point 1:English is fine, I just suggest reviewing short sentences like the following:

56 Most studies of microbiomes rely solely on rRNA gene sequencing… I suggest: Most microbiome studies rely solely on rRNA gene sequencing, which can introduce biases in the estimation of microbial diversity and abundance.

Response 1: We have revised the sentence you highlighted, along with other similar instances throughout the manuscript, to improve the flow and clarity of the text.We hope that these revisions satisfactorily address your comments and enhance the manuscript. Thank you once again for your thorough review and valuable suggestions.

Comments 1: [Italicize species names.]

Response 1: We apologize for the oversight in the formatting of species names. We have carefully reviewed the manuscript and ensured that all species names are now correctly italicized to adhere to scientific naming conventions. This change has been applied throughout the text, including the main body and supplementary materials, to maintain consistency and readability. 

Comments 2: [Where are the figure and table legends of the supplementary material?]

Response 2: We realize the omission of legends for figures and tables in the supplementary material was an oversight. We have now added detailed legends for each figure and table in the supplementary material. These legends provide a clear description of the data presented, including methodologies and implications, to ensure that readers can fully understand the context and significance of the supplementary information.

We believe these revisions have improved the manuscript and appreciate your help in enhancing the quality of our work. Thank you once again for your thoughtful review and valuable comments.

Comments 1: [A nice in silico based study, but a little descriptive. It should be dicsussed and hypotetized more deeply toward biological background abd relationships of Your findings.]

Response 1: We have expanded the discussion section to include a more detailed analysis of the biological implications of our findings. Specifically, we have added hypotheses on how changes in the symbiotic microbiome might influence tomato plant health and productivity. We also discuss potential mechanisms by which domestication and genetic improvement could alter microbial communities, including changes in plant immune responses that select for different microbial taxa. 

Comments 2: [Formatting of the scienfific names of specie should be checked.]

Response 2: We acknowledge the oversight in the formatting of scientific names and have carefully revised the manuscript to ensure that all scientific names are correctly italicized. This includes genera, species, and higher taxonomic ranks as appropriate.

Comments 3: [Introduction - in silico based analysis and their possibilities must be introduced more deeply.]

Response 3: We have expanded the introduction to include a more comprehensive overview of in silico analysis techniques used in microbiome research. This includes a discussion on the advantages of metagenomic and metatranscriptomic approaches over traditional culture-based methods or rRNA gene sequencing, and how these high-throughput techniques can provide insights into the functional capabilities of microbial communities. We also discuss the relevance of these methods in studying the effects of domestication and genetic improvement on plant microbiomes.

 Response to Comments on the Quality of English Language

Point 1:Only minor correction are needed.

Response 1: Thank you for your feedback regarding the minor corrections needed in our manuscript. We are pleased to inform you that we have carefully reviewed your comments and have made all the necessary revisions. We believe these changes have improved the clarity and overall quality of our manuscript.

We have ensured that each correction was thoroughly implemented and are confident that these adjustments meet your expectations and the standards of the journal. Please find the revised manuscript attached for your review.

We appreciate your guidance and support in refining our work and look forward to any further suggestions you might have. Thank you once again for your constructive feedback.