Key Role of Heat Shock Protein Expression Induced by Ampicillin in Citrus Defense against Huanglongbing: A Transcriptomics Study
Round 1
Reviewer 1 Report
- Grapefruit seedlings were graft-inoculated by phloem-rich stem bark or leaf pieces of HLB-infected lemon.
- Figure 2 requires more labeling
- Authors seem to consider Amp a Hsp stressor but it seems to me to be a stimulator of up regulation of Hsp genes
- TCP should be defined
Comments for author File: 
Comments.pdf
Author Response
Dear editor and reviewers:
Thank you for your letter and the reviewer’s comments on our manuscript entitled " Key Role of Heat Shock Protein Expression Induced by Ampicillin in Citrus Defense against Huanglongbing: A Tran-scriptomics Study " (ID: 1663752). Those comments are very helpful for revising and improving our paper, as well as the important guiding significance to other research. We have studied the comments carefully and made corrections which we hope meet with approval. The main corrections are in the manuscript and the responds to the editors and reviewers’ comments are as follow (the replies are highlighted in red).
Reviewer 1
Grapefruit seedlings were graft-inoculated by phloem-rich stem bark or leaf pieces of HLB-infected lemon.
Response: thank you for your comments, grapefruit seedings were graft-inoculated by phloem-rich stem bark, we have revised it in updated manuscript.
I did not see results of B. subtilis bioassay. Either delete or add to the results section
Response: thank you for your comments, in this manuscript, results of B. subtilis bioassay was inhibitory zone diameter, Amp content in Figure 1B was calculated by the inhibitory zone diameter. We have described these in materials and method section. Because this manuscript displayed accumulated Amp concentrations, we did not provide results of zone diameter, just used these results calculated for Amp concentrations.
Significantly?
Response: thank you for your suggestions, we have revised it.
Figure 2 requires additional labeling especially the lower panel of symptomatic plants.
Response: thank you for your suggestions. Figure 2 have been divided into two figures. The updated Figure 3 can displayed clear HLB-symptom.
Consider replacing with HSP stimulator
Response: thank you for your suggestions. We have revised it.
Regulator?
Response: thank you for your suggestions, we have revised it.
Author Response File: Author Response.pdf
Reviewer 2 Report
Dear Authors,
Congratulations on the manuscript submitted to Agronomy. It is a very interesting and important topic for the citrus industry, and plant pathology in general.
My comments are highlighted in the attached PDF. There are some information missing, specially regarding sample size, and description in the figures.
In a nutshell, please get better resolution pictures. I couldn't see a thing on the DEGs figure, even after zooming the PDF on the computer. A printed version of that figure would be impossible to read.
Also, you deliberately used the word "resistance" to identify a possible phenotype outcome of your research. Please check the correct terminology for it.
Comments for author File: Comments.pdf
Author Response
Dear editor and reviewers:
Thank you for your letter and the reviewer’s comments on our manuscript entitled " Key Role of Heat Shock Protein Expression Induced by Ampicillin in Citrus Defense against Huanglongbing: A Tran-scriptomics Study " (ID: 1663752). Those comments are very helpful for revising and improving our paper, as well as the important guiding significance to other research. We have studied the comments carefully and made corrections which we hope meet with approval. The main corrections are in the manuscript and the responds to the editors and reviewers’ comments are as follow (the replies are highlighted in red).
Reviewer 2
Are you sure you can describe this as "resistance"?
Response: thank you for your suggestions, we have changed “resistance” into “tolerance”.
Please update to the latest season.
Response: thank you for your suggestions, we have updated to the latest season, and also added the reference into updated manuscript.
Please check if resistance is the correct word here.
Response: thank you for your suggestions, we have changed “resistance” into “tolerance”.
Temperature controlled? Humidity controlled? Irrigation schedule? Growing conditions?
Response: thank you for your suggestions, we have provided more details about greenhouse condition.
Is there a specific sponge you used?
Response: Thank you for your suggestions, there is common sponge, we have provided more details about sponge.
How many leaves?
Response: thank you for your suggestions, three leaves, we have added it.
Any pictures of the petri dishes?
Response: thank you for your comments, in this manuscript, results of B. subtilis bioassay was inhibitory zone diameter, Amp content in Figure 1B was calculated by the inhibitory zone diameter. We have described these in materials and method section. Because this manuscript displayed accumulated Amp concentrations, we did not provide results of zone diameter, just used these results calculated for Amp concentrations.
How many?
Response: thank you for your suggestions, three leaves, we have added it.
and three technical repetitions per each biological repetition".
Response: thank you for your suggestions, we have revised it.
You already mentioned it before.
Response: thank you for your comments, we have deleted it.
How many leaves? Symptomatic leaves?
Response: three leaves, the leaves displayed HLB-symptom.
Also grapefruit?
Response: yes, we have provided more details in updated manuscript.
This is not highlighted as a section - edition problem.
Response: thank you for your suggestions, we have revised it.
Statistical design? Number of plants per treatment? State the treatments.
Response: thank you for your comments, we have provided more details.
Please increase the font size of both axis, and make it all black. The gray is not doing you any favors.
Response: thank you for your suggestions, we have revised it.
For what test, and what is your sample size? Error bars mean what?
Response: thank you for your suggestions, we have provided more details.
Same here -> increase font size and change color to black on the axis, the letters and the legend.
Response: thank you for your suggestions, we have revised it.
How there is no statistical difference here?
Response: CLas population is calculated by Scientific notation, we also provided P value which is more than 0.05, in updated manuscript.
Those photos are for which treatment? Please describe the photos in the Figure caption, as it is part of it.
Response: thank you for your suggestions, we have done a updated figure and also revised the figure caption.
Is it necessary to be explained? You can add it into the first phrase of the caption.
Response: thank you for your suggestions, we have added it and revised it.
low-case
Response: we have revised it.
I can't see anything in this figure. Please increase its resolution - it is all pixelated when increased the zoom.
Also, increase font size, or dedicate more space in the manuscript so it is legible to the reader.
Response: we have revised this figure.
So it is not relative expression as it says in the y axis label of Figure 5?
Relative expressions are positive, as you have the 2^-delta delta Ct. Please clarify it in the text or in the Figure caption. This can be very misleading. So what is it shown in the Figure 5? Transcript abundance, gene expression level, or relative expression?
Response: thank you for your suggestions, this is Log2 fold change, we have revised it in updated manuscript.
Which one? Parametric? Non-parametric?
Response: thank you for your suggestions, here is pairwise correlation R2, we have revised it.
Same here -> increase font size and change color to black on the axis, the letters, the legend and error bars.
Response: thank you for your suggestions, we have revised it.
Please explain further what we are seeing here: statistical tests, error bars, sample size.
Also, what is each one of the DEGs?
Response: thank you for your suggestions, we have revised it and provide more details. The DEGs information is in Table S1
For which treatment?
Response: thank you for your suggestions, for Amp treatment, we have revised it.
You are missing a the: "In the plant hormone..."
Response: thank you for your suggestions, we have revised it.
In the RNA processing
Response: thank you for your suggestions, we have revised it.
You said that on line 314 - please move, delete, or modify the description.
Response: thank you for your suggestions, we have deleted it.
Are you sure about using resistance instead of tolerance?
Response: thank you for your suggestions, we have changed resistance to tolerance.
"... in the phloem..."
Response: thank you for your suggestions, we have revised it.
"is localized in"
Response: thank you for your suggestions, we have revised it.
"Foliar spray of gibberellin (GA) in HLB-affected citrus..."
Response: thank you for your suggestions, we have revised it.
You need a comma here.
Response: thank you for your suggestions, we have added it.
I don't think you can say resistance as you still have CLas detection even 240 days after treatment.
Pathologically speaking resistance is when the pathogen DOES NOT multiply inside the host.
Response: thank you for your suggestions. We have changed resistance to tolerance.
Author Response File: Author Response.pdf
Reviewer 3 Report
The authors conducted a systemic analysis on the genes expression profile in citrus which induced by ampicillin by RNA-seq. Furthermore, the results were confirmed by RT-qPCR. It is a novel mechabism for ampicillin on HLB. I learned from the figure 3B, the down-regulated genes in three treatments are 136, however, it was 116 in the manuscript showed in line 226.
Author Response
The authors conducted a systemic analysis on the genes expression profile in citrus which induced by ampicillin by RNA-seq. Furthermore, the results were confirmed by RT-qPCR. It is a novel mechabism for ampicillin on HLB. I learned from the figure 3B, the down-regulated genes in three treatments are 136, however, it was 116 in the manuscript showed in line 226.
Response: thank you for your suggestions, 116 is wrong, we have revised it.
