Pyrroloquinoline Quinone Treatment Induces Rice Resistance to Sheath Blight through Jasmonic Acid Pathway
Round 1
Reviewer 1 Report
Bioactive inducer is becoming an effective way to enhance rice resistance to diseases. This manuscript showed the PQQ-induced rice resistance to sheath blight (ShB) caused by Rhizoctonia solani through JA pathway. The transcriptome analysis of leaf sheath of G and L two rice varieties identified several differentially expressed genes related to plant-pathogen interaction, ribosome, plant hormone signaling transduction, MAPK signaling pathway and phenylpropanoid biosynthesis which provides valuable information for understanding the mechanism of PQQ-induced rice resistance to ShB. The contents of the MS are substantial and experimental results abundant. In general, the manuscript provides valuable information for green prevention and control of ShB in the field.
Here are some minor points:
1. For the title, why not consider saying more specific like “Pyrroloquinoline Quinone Treatment Induces Rice Resistance to Sheath Blight through jasmonic acid pathway”.
2. In both Abstract and conclusion parts, jasmonic acid was found to might play key role in PQQ-induced rice resistance to ShB, and during transcriptome analysis, it only declared differentially expressed genes also including plant hormone signaling transduction. Did it also contain JA-related genes? If so, it should be clearly reflected.
3. Abstract part, Line 13 and 14, I suggest the author add one sentence to reflect the limitations of current research in order to lead to his own research
Author Response
We would especially like to thank you for the constructive suggestions. In the revised manuscript, we have carefully corrected the mistakes that you highlighted.
Q1. For the title, why not consider saying more specific like “Pyrroloquinoline Quinone Treatment Induces Rice Resistance to Sheath Blight through jasmonic acid pathway”.
Response: Thanks for your advice. We have revised the title.
Q2. In both Abstract and conclusion parts, jasmonic acid was found to might play key role in PQQ-induced rice resistance to ShB, and during transcriptome analysis, it only declared differentially expressed genes also including plant hormone signaling transduction. Did it also contain JA-related genes? If so, it should be clearly reflected.
Response: Thanks for your comments. We have added the relative results including the JA-related genes in both Abstract and Conclusion.
Q3. Abstract part, Line 13 and 14, I suggest the author add one sentence to reflect the limitations of current research in order to lead to his own research.
Response: Thanks for your suggestion. We have added a sentence to reflect the limitations of current research in order to lead to our research.
Reviewer 2 Report
The present research work "Mechanism of PQQ-Induced Rice Resistance to Shealth Blight" is overall well presented and sound in its content. However, there are some ambiguities which need to be improved in some parts and numerous flaws in English language.
Ambiguities:
Lines 17-19, and 340-343: It is misleading to mention only disease symptom ratings of GP+ and LP-. This way it is not clear whether the positive effect is due to the varietal resistance or due to the PQQ treatment. Indicate here the difference between P+ and P- for both varieties G and L, according to Fig. 1.
Lines 26+27: please explain abbreviations (KEGG, MAPK)
Line 159: you mention QC of RNA with agarose gel electrophoresis, but the outcome is not mentioned in the results.
Line 177: please indicate software and the parameter settings employed for the development of the primers.
Line 180ff: how was the specifity of the qPCR results been validated? How can you be sure that you have not produced non-specific amplification products with the Sybr qPCR? Have you treated the RNA with DNAse or made control qPCR runs with RNA (without revese transcriptase)?
Line 208: please explain "DS" in the legend.
Line 231: biological replicates not repeats
Lines 255-256: Increase the font size of description of the pathways in Fig. 4 for better readability.
Line 257: explain KEGG and DEG in the legend.
Line 324: explain "frequent application" and "abuse" of fungicides. You might better say "excessive use of fungicides"
English errors: here below just some examples, please let the manuscript be revised by an English native.
Line 17: did not appear or have not appeared
Line 200: did occur or have occured
Line 205: dehydrated
Line 206: some of the leaves
Lines 201-207: improve language and readability
Line 232: the replicates clustered together
Line 322: (the species) R. solani has developped
Line 324: instead of "few of chemical", some chemical fungicides
Line 326: "replace with" instead of "change to"
Line 363: "As it is well known that" or "JA and SA are well known as important..."
Line 404: not changed
Author Response
We would especially like to thank you for the constructive suggestions. In the revised manuscript, we have carefully corrected the mistakes that you highlighted.
Ambiguities:
Q1. Lines 17-19, and 340-343: It is misleading to mention only disease symptom ratings of GP+ and LP-. This way it is not clear whether the positive effect is due to the varietal resistance or due to the PQQ treatment. Indicate here the difference between P+ and P- for both varieties G and L, according to Fig. 1.
Response: Thanks for your comment. We have revised these results according to Figure 1.
Q2. Lines 26+27: please explain abbreviations (KEGG, MAPK).
Response: Thanks for your advice. We have added their full name.
Q3. Line 159: you mention QC of RNA with agarose gel electrophoresis, but the outcome is not mentioned in the results.
Response: Thanks for your comment. The ojective of quantity and quality determinations of total RNA was only to meet the requirements of the subsequent experiments including RNA-seq and qRT-PCR. Thus, the outcome is not mentioned in the results.
Q4. Line 177: please indicate software and the parameter settings employed for the development of the primers.
Response: Thanks for your comment. We have added the software information and the parameter settings for the development of the primers.
Q5. Line 180ff: how was the specifity of the qPCR results been validated? How can you be sure that you have not produced non-specific amplification products with the Sybr qPCR? Have you treated the RNA with DNAse or made control qPCR runs with RNA (without revese transcriptase)?
Response: Thanks for your suggestions. Dissociation curve analysis of amplified products was performed at 95 °C for 15 s and 60 °C for 1 min, added as a final step to confirm that the PCR product was specific and unique. Total RNA was treated with RQ1 RNase-free DNase (Promega, Madison, WI, USA) to avoid potential genomic DNA contamination.
Q6. Line 208: please explain "DS" in the legend.
Response: Thanks for your advice. We have described the meaning of “DS” in the legend of Figure 1.
Q7. Line 231: biological replicates not repeats
Response: Thanks. We have revised it.
Q8. Lines 255-256: Increase the font size of description of the pathways in Fig. 4 for better readability.
Response: Thanks for your advice. We have revised it.
Q9. Line 257: explain KEGG and DEG in the legend.
Response: Thanks. We have explained them in the legend.
Q10. Line 324: explain "frequent application" and "abuse" of fungicides. You might better say "excessive use of fungicides".
Response: Thanks for your suggestion. We have revised it.
English errors: here below just some examples, please let the manuscript be revised by an English native.
Response: Thanks for your comments. We have revised and corrected them carefully.
Line 17: did not appear or have not appeared
Response: We have revised it.
Line 200: did occur or have occurred
Response: We have revised it.
Line 205: dehydrated
Response: We have revised it.
Line 206: some of the leaves
Response: We have revised it.
Lines 201-207: improve language and readability
Response: Thanks for your advice. We have rewritten these sentences carefully.
Line 232: the replicates clustered together
Response: We have revised it.
Line 322: (the species) R. solani has developped
Response: We have revised it.
Line 324: instead of "few of chemical", some chemical fungicides
Response: We have revised it.
Line 326: "replace with" instead of "change to"
Response: We have revised it.
Line 363: "As it is well known that" or "JA and SA are well known as important..."
Response: We have revised it.
Line 404: not changed
Response: We have revised it.
Reviewer 3 Report
The manuscript reported a study on investigating the mechanism of pyrroloquinoline quinone (PQQ) induced rice resistance to sheath blight. Totally, the manuscript was well organized and easy to follow. However, what I am mainly concerned is the inoculation method because the inoculation method directly affects the result and conclusion. The authors claimed that they used sclerotia with same size to inoculate rice. I would like to know how the authors obtained so many sclerotia with same size. was the age of sclerotia considered? Different age of sclerotia may have various effects on inoculation. In addition, how to put the sclerotia on the leaf sheath of first leaf of such small plants (only 3-4 leaf old)? Overall, if no appropriate inoculation method
Other concerns
1. For figure1, it is better to add a clear image to show disease spot.
2. Since sclerotia need a certain time to recover before germination for inoculation, it is better to show when did the hyphal grow after inoculation. This is important for you to determine why the timepoints of 12, 24, 48hai was chose for RNAseq experiment.
Author Response
We would especially like to thank you for the constructive suggestions. In the revised manuscript, we have carefully corrected the mistakes that you highlighted.
Comments and Suggestions for Authors
The manuscript reported a study on investigating the mechanism of pyrroloquinoline quinone (PQQ) induced rice resistance to sheath blight. Totally, the manuscript was well organized and easy to follow. However, what I am mainly concerned is the inoculation method because the inoculation method directly affects the result and conclusion. The authors claimed that they used sclerotia with same size to inoculate rice. I would like to know how the authors obtained so many sclerotia with same size. was the age of sclerotia considered? Different age of sclerotia may have various effects on inoculation. In addition, how to put the sclerotia on the leaf sheath of first leaf of such small plants (only 3-4 leaf old)? Overall, if no appropriate inoculation method.
Response: Thanks for your comments. In fact, the sclerotia of R. solani with the same size and age were used to inoculate. Additionally, we have drawn a schematic diagram to explain how to perform the operation process of the inoculation.
Other concerns
1. For figure1, it is better to add a clear image to show disease spot.
Response: Thanks. We have added a clear image to show diseas spot in the revised Figure 1a.
2. Since sclerotia need a certain time to recover before germination for inoculation, it is better to show when did the hyphal grow after inoculation. This is important for you to determine why the timepoints of 12, 24, 48 hai was chose for RNAseq experiment.
Response: Thanks for your comment. The ShB sclerotia recover to germinate the hyphal about 8 hours after inoculation.
Round 2
Reviewer 3 Report
The authors have addressed all my concerns and the manuscript could be accepted.
