Genome-Wide Characterization and Expression Analysis of CBP60 Gene Family in Citrullus lanatus in Response to Fusarium oxysporum Infection and Aphid Infestation
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsKindly add the separate section of statistics analysis in the materials and methods
How many biological and technical replicates were used in RT-PCR
How many reference genes were used to calculate delta ct values
The authors should functionally validate one of the genes in the model plant Arabidopsis by using CRISPR technology: knockout or overexpression”.
The authors should compare their expression data with the already published RNA seq data
Kindly rewrite the discussion and thoroughly explain in the discussion about the role of CPB60 gene family and their potential applications in plant immunity
The final summary of this research article is that the authors identified the CPB60 gene family in watermelon and study their gene expression in response to Fusarium oxysporum and aphid infestations. The study lacks the novelty and functional characterization in model plant. Based on gene expression we cannot speculate their broader role in plant immunity
Author Response
Dear Editors and Reviewers,
We appreciate the opportunity to revise our manuscript. We would like to thank the reviewers for their insightful comments and constructive suggestions, which have significantly improved the quality of our manuscript. We have addressed all the comments point-by-point. All changes in the manuscript are highlighted in [Color/Track Changes]. In addition, the entire manuscript has been thoroughly reviewed and refined by a native English speaker to ensure accuracy, clarity, and linguistic rigor.
Comment 1. Kindly add the separate section of statistics analysis in the materials and methods
Response: Thank you for this valuable suggestion. In the revised manuscript, we have added a dedicated subsection entitled “2.8 Statistical Analysis” within the Materials and Methods section. The new text reads as follows: “Statistical analyses were performed using R software. All experimental data are presented as the mean ± standard deviation (SD). For the fusarium wilt treatment, differences in gene expression between the control group and the F. oxysporum inoculated group at each time point were evaluated using Student’s t-test to determine statistical significance (p < 0.05, p < 0.01). For the aphid infestation treatment, one-way analysis of variance (ANOVA) followed by Tukey’s Honest Significant Difference (HSD) test was applied for multiple comparisons among groups to assess differences in gene expression. A p-value < 0.05 was considered statistically significant, and statistically distinct groups are indicated by different lowercase letters in the figures.”
Comment 2. How many biological and technical replicates were used in RT-PCR
Response: Thank you for this suggestion. We have clarified the number of biological and technical replicates in the revised manuscript. Lines 163–164: "At each time point, samples from three individual plants were pooled to form one biological replicate, and three independent biological replicates were prepared." - Lines 183–185: "Root samples were collected... with three biological replicates per time point and three seedlings per replicate." - Line 197–198: "Three technical replicates were performed per biological replicate."
Comment 3. How many reference genes were used to calculate delta ct values
Response: Thank you for this comment. The revised version as line 196-199 “Gene-specific primers (Table S3) were designed using Primer3, and actin (ClG42_01g0162900.10) was used as the internal reference gene. And three technical replicates were performed per biological replicate. Relative expression levels were calculated using the 2−ΔΔCt method.”
Comment 4. The authors should functionally validate one of the genes in the model plant Arabidopsis by using CRISPR technology: knockout or overexpression.
Response: We appreciate this valuable suggestion. While functional validation (knockout/overexpression) in Arabidopsis or watermelon would provide deeper mechanistic insights, we believe it is beyond the scope of this study, which aims to provide a comprehensive genome-wide identification and expression framework for the ClaCBP60 family in watermelon. To address this, we have added a "Limitations and Future Perspectives" section (Section 4.4) in the Discussion. We emphasize that candidates such as ClaCBP60_01 and ClaCBP60_16 are high-priority targets for subsequent CRISPR-based functional studies to confirm their roles in SA-mediated immunity.
Comment 5. The authors should compare their expression data with the already published RNA seq data
Response: Thank you for this valuable suggestion. We examined the currently available RNA-seq datasets; however, none of the published transcriptomic studies matched the specific experimental conditions or stress treatments used in our work. Therefore, a direct comparison was not feasible. We have noted this limitation in the revised manuscript. Farthemore we will use RNA‑seq to enable a more comprehensive comparative analysis and functional validation in future studies.
Comment 6. Kindly rewrite the discussion and thoroughly explain in the discussion about the role of CPB60 gene family and their potential applications in plant immunity
Response: Thank you for this valuable suggestion. In the revised manuscript, we have substantially rewritten and expanded the Discussion section to provide a more comprehensive explanation of the roles of the CBP60 gene family in plant immunity, as well as their potential applications in crop improvement. In particular, “4.3 Regulatory Roles of ClaCBP60s in Fon and Aphid Defense”, where we discuss how ClaCBP60 genes may function as calcium‑signal decoders, integrate SA‑ and JA‑related defense pathways, and contribute to vascular immunity.
Comment 7. The final summary of this research article is that the authors identified the CPB60 gene family in watermelon and study their gene expression in response to Fusarium oxysporum and aphid infestations. The study lacks the novelty and functional characterization in model plant. Based on gene expression we cannot speculate their broader role in plant immunity
Response: Thanks for your suggestion. To address the concern of novelty, we have highlighted that this is the first genome-wide characterization of CBP60 genes in watermelon, identifying unique lineage-specific clustering and dual responsiveness to fungal and insect stress. We have also toned down speculative language throughout the manuscript, clarifying that our expression and in silico data provide foundational hypotheses that set the stage for future functional confirmation.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe manuscript describes the analysis of the CBP60 gene family in watermelon. Evolutionary features were described and experimental data about watermelon interaction with F. oxysporum infection and aphid infestation were presented using RT-qPCR. The topic is interesting, but improvements are required. Detailed comments appear below.
Introduction section. It would be useful to add information on encoded domains in CBP60 genes that have been previously described. Localization of CBP60s in the cell is also important. Targets for CBP60s reported in other models should be useful too.
Lines 113-118. Specify whether the PF02882 domain corresponds to CaMBD.
Lines 119-123. Complete the passages about the number of organisms analyzed.
Lines 249-151. Specify the term “functionally categorized”.
Line 153-154. Specify whether 'ym-t' corresponds to a specific ecotype material.
Methods. Specified the statistical analysis performed. There is missing information on that in the present version of the manuscript. Defining the species of aphid used is required. There is data on the identification of F. oxysporum, but no data is provided. Adding information such as morphology and conidial features in the supplementary information would be useful.
Section 3.1. It is necessary to ensure consistency between the genomes analyzed and the data provided in the method section.
Table 1. Chromosome number data should be presented as numbers without accompanying letters. Add the unit for the Molecular Weight column.
Lines 223-227. Double check the protein member number of CBP60s within the subfamilies.
Lines 227-229, 252-255, 367-370. The elaboration of speculative propositions should be in the discussion section. Conclusions that are not highly supported are also included in the discussion section.
Line 237-240. The phylogeny is based on amino acid sequences instead of nucleotides. So, correction is required for the term gene family.
Lines 247-249. Defining which motif constitutes the Calmodulin binding domain should be useful. Does it correspond to the motifs 1, 2, 4, 5, and 8 together in the Calmodulin binding domain?
Lines 357-358. Double-check that the exon number described is consistent with the figures.
Lines 263-269. Correction of the scale for the panel b is required. N-terminal and C-terminal must be indicated when describing amino acids instead of DNA.
Lines 294-296. Correction must be made as mentioned ‘illustrating species-specific patterns of gene expansion’ but the data displayed are based on the use of genome data.
Lines 340-342. Congruence of data with the provided description is required. In the figure below, the expression profile of eleven genes is displayed instead of sixteen genes.
Typo lines 14, 313. a space between text and references must be present, for instance, 383.392, 400,408.
Table S1. Completeness of information in the columns. Does the fifth column correspond to the chromosome number?
Table S2. Consistence in the gene names used with the names used in the manuscript is required.
Author Response
Dear Editors and Reviewers,
We appreciate the opportunity to revise our manuscript. We would like to thank the reviewers for their insightful comments and constructive suggestions, which have significantly improved the quality of our manuscript. We have addressed all the comments point-by-point. All changes in the manuscript are highlighted in [Color/Track Changes]. In addition, the entire manuscript has been thoroughly reviewed and refined by a native English speaker to ensure accuracy, clarity, and linguistic rigor. The figures have also been carefully revised to enhance their precision and readability.
The manuscript describes the analysis of the CBP60 gene family in watermelon. Evolutionary features were described and experimental data about watermelon interaction with F. oxysporum infection and aphid infestation were presented using RT-qPCR. The topic is interesting, but improvements are required. Detailed comments appear below.
Comment 1. Introduction section. It would be useful to add information on encoded domains in CBP60 genes that have been previously described. Localization of CBP60s in the cell is also important. Targets for CBP60s reported in other models should be useful too.
Response: Thank you for this helpful suggestion. We have incorporated the requested information into both the “Introduction” and “Materials and Methods” sections. Revised version as lines 61 – 63 “CBP60 proteins are characterized by a conserved calmodulin-binding domain (CaMBD; Pfam PF07887), which enables calcium-dependent regulation, and many members also contain nuclear localization signals…” and line 112-114 “the Hidden Markov Model (HMM) profile corresponding to the calmodulin-binding domain (CaMBD; Pfam PF07887) was retrieved from the Pfam database and used to query the predicted watermelon proteome with...”
Comment 2. Lines 113-118. Specify whether the PF02882 domain corresponds to CaMBD.
Response: Thank you for this suggestion. As clarified in Lines 61–63, the calmodulin‑binding domain corresponds to PF07887. This correction has been made to ensure consistency and accuracy.
Comment 3. Complete the passages about the number of organisms analyzed.
Response: Thank you for this suggestion. We have revised Sections 2.1 and 3.1 to explicitly state that seven cucurbit species were analyzed, including the number of CBP60 members identified in each species (e.g., 23 in C. sativus, 16 in C. maxima). Detailed characteristics are provided in Supplementary Table S1. The revised text reads: “To evaluate evolutionary conservation across the Cucurbitaceae, the identical identification workflow was applied to six additional cucurbit genomes: 23 members in C. sativus, 13 in C. melo, 6 in M. charantia, 11 in L. siceraria, 15 in B. pruriens, and 16 in C. maxima.”
Comment 4. Specify the term “functionally categorized”.
Response: Thank you for this suggestion. Revised version as follows: “The identified elements were classified into functional categories based on their annotated biological roles, including hormone responsiveness (e.g., SA, JA, ABA, and ethylene), stress responsiveness (e.g., drought, low temperature, and pathogen infection), growth and developmental regulation, and light responsiveness. Their distribution patterns across ClaCBP60 promoters were subsequently visualized using TBtools to highlight potential regulatory modules.”
Comment 5. Line 153-154. Specify whether 'ym-t' corresponds to a specific ecotype material.
Response: Thank you for this comment. We have clarified this as follows: “Watermelon seedlings of the inbred line ‘ym‑t’, a cultivar for its pronounced susceptibility to both biotic and abiotic stresses, were cultivated in a climate-controlled chamber…” (Lines 154-156).
Comment 6. Methods. Specified the statistical analysis performed. There is missing information on that in the present version of the manuscript. Defining the species of aphid used is required. There is data on the identification of F. oxysporum, but no data is provided. Adding information such as morphology and conidial features in the supplementary information would be useful.
Response: Thank you for this suggestion. We have added a new subsection, “2.8 Statistical Analysis”. We also added the sentence: “The aphid species was identified as Aphis gossypii, a common phloem-feeding pest of cucurbits.” (lines 158-159 ). Additionally, morphological and conidial features of F. oxysporum have been included in Supplementary Figure S1. Add the sentence as “Initially, classical morphological characteristics, including colony morphology and conidial features, were used for preliminary screening and identification (Supplementary Figure S1).” (Lines 168-170)
Comment 7. Section 3.1. It is necessary to ensure consistency between the genomes analyzed and the data provided in the method section.
Response: Thank you for pointing this out. We have verified and ensured consistency between Sections 2.1 and 3.1. The seven species and their member counts now match Supplementary Table S1. Revised version as “To evaluate evolutionary conservation across the Cucurbitaceae, the identical identification workflow was applied to six additional cucurbit genomes. This analysis identified 23 members in C. sativus, 13 in C. melo, 6 in M. charantia, 11 in L. siceraria, 15 in B. pruriens, and 16 in C. maxima. Detailed physicochemical characteristics of these gene families are provided in Supplementary Table S1.”(Lines 227-231)
Comment 8. Table 1. Chromosome number data should be presented as numbers without accompanying letters. Add the unit for the Molecular Weight column.
Response: Thank you for this suggestion. Table 1 has been revised to present chromosome numbers as numerals only, and “kDa” has been added as the unit for molecular weight.
Comment 9. Lines 223-227. Double check the protein member number of CBP60s within the subfamilies.
Response: Thank you for this correction. The revised text now reads: “Subfamily I emerged as the largest clade, containing 66 members, followed by subfamilies III (16 members) and II (10 members), while subfamily IV was the smallest, with only 8 members.” (Lines 244-246)
Comment 10. Lines 227-229, 252-255, 367-370. The elaboration of speculative propositions should be in the discussion section. Conclusions that are not highly supported are also included in the discussion section.
Response: Thank you for this suggestion. All speculative statements from Lines 227–229, 252–255, and 367–370 have been relocated to the Discussion section.
Comment 11. Line 237-240. The phylogeny is based on amino acid sequences instead of nucleotides. So, correction is required for the term gene family.
Response: Thank you for this comment. Lines 237-240 have been corrected to refer to the “CBP60 protein family.”(Line 254)
Comment 12. Lines 247-249. Defining which motif constitutes the Calmodulin binding domain should be useful. Does it correspond to the motifs 1, 2, 4, 5, and 8 together in the Calmodulin binding domain?
Response: Thank you for this suggestion. We have clarified that Motifs 1, 2, 4, and 5 together constitute the CaMBD. The revised sentence reads: “Notably, the calmodulin binding domain (CaMBD) was mapped to a combination of Motifs 1, 2, 4, and 5, which together form the functional interface for Ca²⁺/CaM interaction.”(Lines 266-269 )
Comment 13. Lines 357-358. Double-check that the exon number described is consistent with the figures.
Response: Thank you. The text has been revised to: “Exon numbers ranged from 6 to 15, with corresponding coding sequence (CDS) lengths varying accordingly.” (Lines 273-274)
Comment 14. Lines 263-269. Correction of the scale for the panel b is required. N-terminal and C-terminal must be indicated when describing amino acids instead of DNA.
Response: Thank you. N‑terminal and C‑terminal labels have been added, and the figure legend now specifies that panel b represents amino acid lengths.
Comment 15. Lines 294-296. Correction must be made as mentioned ‘illustrating species-specific patterns of gene expansion’ but the data displayed are based on the use of genome data.
Response: Thank you for this suggestion. The phrase has been removed from the figure legend, and the Discussion now includes the following clarification: “However, it is essential to note that these patterns are currently inferred from genome assemblies; future functional and evolutionary studies will be required to determine if they correspond to genuine lineage-specific expansion processes.” (Lines 428-431)
Comment 16. Lines 340-342. Congruence of data with the provided description is required. In the figure below, the expression profile of eleven genes is displayed instead of sixteen genes.
Response: Thank you. The text has been revised to: “Of the sixteen genes, eleven of the 16 ClaCBP60 genes displayed high expression at least at one time point relative to mock-inoculated controls (Figure 6).”
Comment 17. Typo lines 14, 313. a space between text and references must be present, for instance, 383.392, 400,408.
Response: Thank you. We have corrected spacing throughout the manuscript.
Comment 18. Table S1. Completeness of information in the columns. Does the fifth column correspond to the chromosome number?
Response: Thank you. The table has been updated to clearly indicate chromosome numbers.
Comment 19. Table S2. Consistence in the gene names used with the names used in the manuscript is required.
Response: Thank you. A “Rename” column has been added to ensure consistency with the nomenclature used in the manuscript.
Reviewer 3 Report
Comments and Suggestions for AuthorsThis manuscript, 'Genome-Wide Characterization and Expression Analysis of CBP60 Gene Family in Citrullus lanatus in Response to Fusarium oxysporum Infection and Aphid Infestation' is very well written, addressing an important gap in cucurbit immunity research.
The introduction can be improved and rearranged for greater clarity. It starts with pathogens and other signaling networks, but I would suggest starting with the plant of interest, its diseases, and linking the protein and signaling hormones.
The objectives are clear and well written.
The labels on Figures are difficult to read and is not clear.
However, the conclusions rely primarily on in silico and transcript-level data, and functional validation is lacking. Future studies incorporating gene knockout and overexpression and phytohormone measurements would be essential to confirm the proposed regulatory roles and mechanistic involvement of these genes in broad-spectrum defense. However, for this manuscript, the gene expression analyses provide supportive evidence for stress responsiveness.
Author Response
This manuscript, 'Genome-Wide Characterization and Expression Analysis of CBP60 Gene Family in Citrullus lanatus in Response to Fusarium oxysporum Infection and Aphid Infestation' is very well written, addressing an important gap in cucurbit immunity research.
Comment 1. The introduction can be improved and rearranged for greater clarity. It starts with pathogens and other signaling networks, but I would suggest starting with the plant of interest, its diseases, and linking the protein and signaling hormones.
Response: Thank you for this suggestion. We have reorganized the Introduction to improve clarity and logical flow. The revised version now begins with watermelon as the focal crop, outlines its major biotic threats (Fusarium wilt and aphid infestation), and subsequently introduces the CBP60 family and its roles in SA‑mediated immunity and vascular defense.
The objectives are clear and well written.
Comment 2. The labels on Figures are difficult to read and is not clear.
Response: Thank you for pointing this out. We have revised the figure labels to enhance readability and visual clarity.
Comment 3. However, the conclusions rely primarily on in silico and transcript-level data, and functional validation is lacking. Future studies incorporating gene knockout and overexpression and phytohormone measurements would be essential to confirm the proposed regulatory roles and mechanistic involvement of these genes in broad-spectrum defense. However, for this manuscript, the gene expression analyses provide supportive evidence for stress responsiveness.
Response: Thank you for this insightful comment. We fully agree that the current conclusions are based primarily on in silico analyses and transcriptl expression data, and that functional validation is essential for establishing the mechanistic roles of ClaCBP60 genes in broad‑spectrum defense. In the revised manuscript, we have explicitly acknowledged this limitation and clarified that our findings provide a foundational framework rather than definitive functional evidence. We have also expanded the Discussion to emphasize that future studies involving CRISPR/Cas‑mediated knockout or overexpression, together with phytohormone quantification and downstream defense‑related assays, will be critical for validating the regulatory functions of key candidates such as ClaCBP60_01 and ClaCBP60_16. Within the scope of this study, however, the gene expression patterns under F. oxysporum infection and aphid infestation offer supportive evidence for their responsiveness to biotic stress.
