Transcriptome Analysis of Berries of Spine Grape (Vitis davidii Föex) Infected by Colletotrichum viniferum during Symptom Development
Round 1
Reviewer 1 Report
In the current study, the authors perform a transcriptome analysis of spine grape to identify pathways and genes associated with Colletotrichum viniferum infection and find the disease-resistance mechanism. Furthermore, the authors reveal that phenylalanine-pathway-related transcription factors and genes are involved in disease-resistant via CA+2 deregulation. The data presented in the manuscript is thoughtfully designed, organized, and delivered. Furthermore, the manuscript is well written and clearly explained. In addition, this study could add to the existing knowledge in understating the mechanism behind the disease-resistant grape against Colletotrichum viniferum.
I have only a few minor suggestions for strengthening the manuscript.
Lines 30 to 33 are hard to read; consider re-writing to make it easier to understand.
Line 66, the authors could consider adding the exact location of the grape planted site.
Line 108, "total RNA was extracted from samples in the same way that it was extracted from" instead of from it should be "for."
A total of 11 figures are too much, so consider combining figures 5, 6, and 7 and move figure 10 into the supplementary file.
Figure 11, consider improving the quality of this figure; hard to read, so change the fonts to Arial and do not make them bold. For example, in lines 16 to 19, this sentence contains a series of three words "(1, 3 and 7 days)," but it did not separate with a comma. Also, there should be a comma before the word which in the same sentence. These mistakes are present throughout the manuscript; please consider revising carefully.
References in the reference list are also not formatted according to guidelines; for example, reference numbers 6, 7, and 29. Therefore, consider checking all the references carefully and correcting them.
Below are the links to a few related references. The authors could consider citing them.
https://link.springer.com/article/10.1007/s11104-015-2436-2
https://sfamjournals.onlinelibrary.wiley.com/doi/abs/10.1111/jam.15019
Author Response
Comments and Suggestions for Authors
In the current study, the authors perform a transcriptome analysis of spine grape to identify pathways and genes associated with Colletotrichum viniferum infection and find the disease-resistance mechanism. Furthermore, the authors reveal that phenylalanine-pathway-related transcription factors and genes are involved in disease-resistant via CA+2 deregulation. The data presented in the manuscript is thoughtfully designed, organized, and delivered. Furthermore, the manuscript is well written and clearly explained. In addition, this study could add to the existing knowledge in understating the mechanism behind the disease-resistant grape against Colletotrichum viniferum.
I have only a few minor suggestions for strengthening the manuscript.
Lines 30 to 33 are hard to read; consider re-writing to make it easier to understand.
Response: Thanks a lot for the reviewer`s suggestion. The sentence was rewritten as ‘Our results offer comprehensive transcriptomic data about molecular responses in C. viniferum-infected grape, these data will aid in understanding of processes underlying plant responses to C. viniferum.’
Line 66, the authors could consider adding the exact location of the grape planted site.
Response: Thanks a lot for the reviewer`s suggestion. The exact location of the grape planted site was 27°04′55.61″N/119°31′29.11″E. And we revised the sentence as ‘…grapevines were planted in XiTa village, MuYun town, FuAn city (27°04′55.61″N/119°31′29.11″E, Fujian province, China).’
Line 108, "total RNA was extracted from samples in the same way that it was extracted from" instead of from it should be "for."
Response: Thanks a lot for the reviewer`s suggestion. ‘from’ was corrected as ‘for’.
A total of 11 figures are too much, so consider combining figures 5, 6, and 7 and move figure 10 into the supplementary file.
Response: Thanks a lot for the reviewer`s suggestion. We combined figures 5, 6, and 7 as a single figure 5 in revised manuscript, consequently, we re-numbered the figures.
For figure 10 (Figure 8 in revised manuscript), we improved the quality of this figure, and we would like to retain this figure in main text as it is very important to show the consistency between selected genes expression levels and the transcriptomic data.
Figure 11, consider improving the quality of this figure; hard to read, so change the fonts to Arial and do not make them bold. For example, in lines 16 to 19, this sentence contains a series of three words "(1, 3 and 7 days)," but it did not separate with a comma. Also, there should be a comma before the word which in the same sentence. These mistakes are present throughout the manuscript; please consider revising carefully.
Response: Thanks a lot for the reviewer`s suggestion. Figure 11 (Figure 9 in revised manuscript), was revised according to reviewer 1`s suggestion. ‘(1, 3 and 7 days)’ was rewritten as ‘1 dpi, 3 dpi and 7 dpi’ throughout the manuscript, all the changes can be found in tracked version of manuscript.
References in the reference list are also not formatted according to guidelines; for example, reference numbers 6, 7, and 29. Therefore, consider checking all the references carefully and correcting them.
Response: Thanks a lot for the reviewer`s suggestion. We revised format of all the reference including 6, 7, and 29.
Below are the links to a few related references. The authors could consider citing them.
https://link.springer.com/article/10.1007/s11104-015-2436-2
https://sfamjournals.onlinelibrary.wiley.com/doi/abs/10.1111/jam.15019
Response: Thanks a lot for the reviewer`s suggestion. We cited https://sfamjournals.onlinelibrary.wiley.com/doi/abs/10.1111/jam.15019 in ‘Discussion’ (ref. 47 in revised version).
Reviewer 2 Report
The following manuscript titled "Transcriptome analysis of berries of spine grape (Vitis davidii Föex) infected by Colletotrichum viniferum during symptom development” provides a detailed insight on the defense mechanism of V.davidii against a grape ripe rot. However, major changes are required to make it acceptable for the scientific community.
Introduction: This portion lacks basic information to describe your manuscript. It is advisable to write more about grape ripe rot losses (calculated data, statistics) etc. In the second paragraph, provide information regarding up till now studies about controlling grape ripe rot either through breeding or biotechnological tools.
Line 66: V. davidii is grown in china as wild or on a cultivated level?
Results:
Figure 1: disease incidence symptoms on berries are not clear, use clear pictures. While in B) use arrows to indicate lesions growth on berries at different dpi
It is better to display pictures of susceptible berries along with resistant berries to differentiate between the level of tolerance in V. davidii and other cultivated grapes.
In the whole manuscript, V.davidii word is used again and again. It is already understood that V.davidii was the only research material used for the study. Thus avoid using it repeatedly.
Why only up to 7 days post inoculation were studied? Are Colletotrichum viniferum spores completely inhabit berries at 7 days or do they need more days to show symptoms? Clarify it.
Figure 4: If it is possible in the KEGG pathway, indicate upregulated and downregulated genes in accordance with the different pathways.
DEGs involved in pathogen resistance genes: Figure 7: in the result description, it is mentioned that gene VIT-19s0093g00510 was downregulated at 7 dpi but in the figure, it is first upregulated at 3 dpi and then decrease at 7dpi. Elaborate them
Discussion:
Line 343: It is written that the degradation of pectin enhances disease resistance in plants. But according to my knowledge pectin in the cell wall are the first defense barrier against disease invasion/penetration. Thus this system seems to confuse readers. If your statement is correct then further describe it.
Author Response
The following manuscript titled "Transcriptome analysis of berries of spine grape (Vitis davidii Föex) infected by Colletotrichum viniferum during symptom development” provides a detailed insight on the defense mechanism of V.davidii against a grape ripe rot. However, major changes are required to make it acceptable for the scientific community.
Introduction: This portion lacks basic information to describe your manuscript. It is advisable to write more about grape ripe rot losses (calculated data, statistics) etc. In the second paragraph, provide information regarding up till now studies about controlling grape ripe rot either through breeding or biotechnological tools.
Response: Thanks a lot for the reviewer`s suggestion. We revised the ‘Introduction’ according to the reviewer`s comment, in L42 ‘Potentially, it causes losses with severities up to 67% or 37% in the Mid-Atlantic US and Northeast China, respectively [3]’ was added.
In L54 ‘Two hundred and thirty-five Vitis and six Muscadinia grapevine cultivars and selections were evaluated for ripe rot resistance and fifity cultivars or selections were classified as highly resistant [8]. Accordingly, it was recently reported that a grape ripe rot resistance locus (Cgr1) has been identified from V. amurensis [9]’ was added.
Line 66: V. davidii is grown in china as wild or on a cultivated level?
Response: Thanks a lot for the reviewer`s comment. Vitis davidii are wild grapes in China. Due to its pleasant flavour and disease resistance, grape breeders selected some excellent accessions for wine and fresh fruit especially in south China. We believe there will be grape cultivars from V. davidii in future.
Results:
Figure 1: disease incidence symptoms on berries are not clear, use clear pictures. While in B) use arrows to indicate lesions growth on berries at different dpi
Response: Thanks a lot for the reviewer`s suggestion. For ‘disease incidence symptoms on berries are not clear…’, full developed disease symptoms need about 11-14d in C. viniferum-infected berries. And the incidence symptom was small even at 7 dpi (and why choose these time points see below), we enlarged Figure 1 to make it clear. The symptoms were showed in B, and arrows to indicate lesions were added in revised Figure 1 according to reviewer`s suggestion.
It is better to display pictures of susceptible berries along with resistant berries to differentiate between the level of tolerance in V. davidii and other cultivated grapes.
Response: Thanks a lot for the reviewer`s suggestion. Actually, we inoculated resistant berries (V. davidii) and susceptible berries (Vitis vinifera × Vitis labrusca, cv. Kyoho) in the same time, therefore we would like to supply a picture of susceptible berries in Supplementary Data (numbered as Figure S1 in revised manuscript), and the description of this picture was added in main text 3.1 accordingly.
In the whole manuscript, V.davidii word is used again and again. It is already understood that V.davidii was the only research material used for the study. Thus avoid using it repeatedly.
Response: Thanks a lot for the reviewer`s suggestion. We checked and deleted many ‘V.davidii ’ words in revised manuscript to avoid using it repeatedly. For example, ‘Uninfected Vitis davidii Föex berries’ are revised as ‘Uninfected berries’, ‘infected V. davidii samples’ are revised as ‘infected samples.’
Why only up to 7 days post inoculation were studied? Are Colletotrichum viniferum spores completely inhabit berries at 7 days or do they need more days to show symptoms? Clarify it.
Response: Thanks a lot for the reviewer`s comment. We would like to clarify it here, C. viniferum spores need more days to show symptoms. In general, first sign of infection appeared as small, faintly visible ripe rot colonies 3-4 d after inoculation, and full developed disease symptom need about 11-14 d in C. viniferum-infected berries (Shiraishi et al. 2007; Oo et al. 2017). In this study, at 7 dpi disease symptoms were clearly visible, many molecular changes (e.g., DEGs) were taken place (Supplementary Data 5). Also, it was easy to extract grape berry RNA before 7 dpi for further analysis. After 7 dpi, RNA of grape berries is easily to degrade during extracting in preliminary experiment. Therefore, we choose 1 dpi, 3 dpi and 7 dpi to analysis the molecular processes during infection.
Shiraishi, M.; Koide, M.; Itamura, H.; Yamada. M.; Mitani, N.; Ueno, T.; Nakaune, R. Nakano M. Screening for resistance to ripe rot caused by Colletotrichum acutatum in grape germplasm. Vitis. 2007, 46 (4):196-201.
Oo, M.M.; Oh, S. Identification and characterization of new record of grape ripe rot disease caused by Colletotrichum viniferum in Korea. Mycobiology. 2017, 45 (4): 421-425.
Figure 4: If it is possible in the KEGG pathway, indicate upregulated and downregulated genes in accordance with the different pathways.
Response: Thanks a lot for the reviewer`s suggestion. We would like to make an explanation here, KEGG pathway here help us to understand the potential pathways involved in C. viniferum infection. It would be hard to combine upregulated and downregulated genes in accordance with the KEGG pathway in one picture, and the upregulated and downregulated genes in accordance with the different pathways can be found in (Supplementary Data 5 and 7).
DEGs involved in pathogen resistance genes: Figure 7: in the result description, it is mentioned that gene VIT-19s0093g00510 was downregulated at 7 dpi but in the figure, it is first upregulated at 3 dpi and then decrease at 7dpi. Elaborate them
Response: Thanks a lot for the reviewer`s comment, and the sentence was revised as ‘The DEGs (VIT_00s0698g00010 and VIT_04s0044g00020) that belonged to peroxisome were down-regulated during C. viniferum infection, while for VIT_19s0093g00510, it was first up-regulated at 3 dpi and then decrease at 7 dpi’ in 3.7.
Discussion:
Line 343: It is written that the degradation of pectin enhances disease resistance in plants. But according to my knowledge pectin in the cell wall are the first defense barrier against disease invasion/penetration. Thus this system seems to confuse readers. If your statement is correct then further describe it.
Response: Thanks a lot for the reviewer`s suggestion. Indeed, the reviewer is correct, many studies confirmed that pectin plays role to overcome fungal pathogen, and we revised the sentence as ‘…in order to prevent the degradation of pectin, hence enhancing plant disease resistance.’(L332)
Reviewer 3 Report
Colletotrichum viniferum, a phytopathogenic plant fungus, causes huge losses in Vitis species. The authors describe a transcriptome analysis of berries from a C.-viniferum-tolerant species, Vitis davidii, after 1, 3, and 7 days inoculation time of berries with C. viniferum and comparison to non-infected V. davidii berries. The annotated genome of Vitis vinifera was used as reference. C. viniferum triggered several signaling pathways, activated WRKY-, ERF-, and MYP-like transcription factors regulating expression of genes involved in metabolism, plant-pathogen interaction, and PR proteins.
The manuscript is well written. I have only one question and a few typos have to be corrected (see below).
Lines 139--141: This difference of mapping ratios between infected and non-infected samples (76 vs 87%) seems quite large, but is this significant given the three replicates?
Typos:
Most information on first page, left column is missing.
Line 22: "7dpi" => "7 dpi"
Line 70: What is "PDA"?
Line 84: "co. ltd" => "Co. Ltd."
Line 108: "extracted from RNA-seq" => "extracted for RNA-seq"
Otherwise I don't understand this sentence.
Line 130: "Uninfected berries at 0 d (CK) and Vitis davidii Föex berries" => "Uninfected Vitis davidii Föex berries at 0 d (CK) and berries"
Line 178: "dpi ." => "dpi."
Line 237: "MLO-like" => "Mildew Locus O (MLO)-like"
Line 252: "encoded" => "encoding"
Line 299: "About two genes, including MYB4 and ERF113," => "Two genes, MYB4 and ERF113,"
Lines 331f: What is meant by the numbers in brackets? To me, this is not obvious from Figure 3.
Line 333: "categories were activated to participate" => "categories participate"
Figures 10, 11: Increase font size of labels; these are hard to read. Avoid the use of graphics in JPEG format.
Ref. 52: Remove the underlining from the DOI.
Author Response
Colletotrichum viniferum, a phytopathogenic plant fungus, causes huge losses in Vitis species. The authors describe a transcriptome analysis of berries from a C.-viniferum-tolerant species, Vitis davidii, after 1, 3, and 7 days inoculation time of berries with C. viniferum and comparison to non-infected V. davidii berries. The annotated genome of Vitis vinifera was used as reference. C. viniferum triggered several signaling pathways, activated WRKY-, ERF-, and MYP-like transcription factors regulating expression of genes involved in metabolism, plant-pathogen interaction, and PR proteins.
The manuscript is well written. I have only one question and a few typos have to be corrected (see below).
Lines 139--141: This difference of mapping ratios between infected and non-infected samples (76 vs 87%) seems quite large, but is this significant given the three replicates?
Response: Thanks a lot for the reviewer`s comment. We would like to clarify that the lowest mapping ratio was 75.81 % (infected samples at 7 dpi), while the highest mapping ratio was 87.13% (infected samples at 3 dpi), but not ‘infected and non-infected samples (76 vs 87%)’.
Indeed, the difference of mapping ratios between the samples (76 vs 87%) are quite large, especially 75.81 % (infected samples at 7 dpi). We would like to make an explanation here, in RNA-seq, the mapping rate should above 70%, our data was 75.81%, so it was acceptable for further analysis.
And there may be two factors that lead to the big difference compared to other mapping rate. No.1, the grape we used belongs to wild species, the reference genome comes from Vitis vinifera cv Pinot Noir, there will be a different between the two grapes. No.2, as the pathogen are accumulated at 7 dpi, the mapping ratios are become lower, and the lower mapping ratio may also due to the individual difference.
Typos:
Most information on first page, left column is missing.
Response: Thanks a lot for the reviewer`s suggestion, we supplied the citation in left column and the other information should be provided by editors if this manuscript could be accepted.
Line 22: "7dpi" => "7 dpi"
Response: Thanks a lot for the reviewer`s suggestion, the word has been revised according to the suggestion.
Line 70: What is "PDA"?
Response: Thanks a lot for the reviewer`s suggestion, the word has been revised as ‘potato dextrose agar medium (PDA)’ according to the suggestion.
Line 84: "co. ltd" => "Co. Ltd."
Response: Thanks a lot for the reviewer`s suggestion, the word has been revised according to the suggestion.
Line 108: "extracted from RNA-seq" => "extracted for RNA-seq"
Otherwise I don't understand this sentence.
Response: Thanks a lot for the reviewer`s suggestion, the sentence has been revised according to the suggestion.
Line 130: "Uninfected berries at 0 d (CK) and Vitis davidii Föex berries" => "Uninfected Vitis davidii Föex berries at 0 d (CK) and berries"
Response: Thanks a lot for the reviewer`s suggestion, the sentence has been revised according to the suggestion.
Line 178: "dpi ." => "dpi."
Response: Thanks a lot for the reviewer`s suggestion, the word has been revised according to the suggestion.
Line 237: "MLO-like" => "Mildew Locus O (MLO)-like"
Response: Thanks a lot for the reviewer`s suggestion, the word has been revised according to the suggestion.
Line 252: "encoded" => "encoding"
Response: Thanks a lot for the reviewer`s suggestion, the word has been revised according to the suggestion.
Line 299: "About two genes, including MYB4 and ERF113," => "Two genes, MYB4 and ERF113,"
Response: Thanks a lot for the reviewer`s suggestion, the sentence has been revised according to the suggestion.
Lines 331f: What is meant by the numbers in brackets? To me, this is not obvious from Figure 3.
Response: Thanks a lot for the reviewer`s comments, it means up/down-regulated genes, and we revised the sentence as ‘these DEGs were enriched in 42 (28 up-regulated genes /14 down-regulated genes) terms, including 5 (2 up-regulated genes /3 down-regulated) biological process, 2 (2 up-regulated genes /0 down-regulated) cellular components and 35 (24 up-regulated genes /11 down-regulated) molecular functions (Figure 3).
Line 333: "categories were activated to participate" => "categories participate"
Response: Thanks a lot for the reviewer`s suggestion, the sentence has been revised according to the suggestion.
Figures 10, 11: Increase font size of labels; these are hard to read. Avoid the use of graphics in JPEG format.
Response: Thanks a lot for the reviewer`s suggestion, we revised Figures 10 and 11 (Figure 8 and 9 in revised manuscript) according to the suggestion. Size of labels were increased, and graphics are in TIF format now.
Ref. 52: Remove the underlining from the DOI.
Response: Thanks a lot for the reviewer`s suggestion, the reference has been revised according to the suggestion.
Round 2
Reviewer 2 Report
I appreciate the efforts put in by the authors to address all my concerns effectively. I feel the MS is now suitable for its possible acceptance.
Good Luck!
Author Response
We appreciate for the reviewer`s approval of our manuscript and thanks for your work.
