Application of the NanoString nCounter System as an Alternative Method to Investigate Molecular Mechanisms Involved in Host Plant Responses to Plasmodiophora brassicae
Round 1
Reviewer 1 Report
Comment on
The submitted paper by Qinqin Zhou, Leonardo Galindo-González, Sheau-Fang Hwang and Stephen E. Strelkov to Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW is emphasising to employ the NanoString nCounter System as an alternative method to investigate molecular mechanisms involved in 3 host plant responses to the soil borne, obligate parasite pathogen protist Plasmodiophora brassicae, a fungus resembling slime mold within the class Phytomyxea (plasmodiophorids), Cercozoa of the super-group Rhizaria. P brassicae is an important disease causing protist of canola (Brassica napus) and other crucifers. For studying changes in gene expression in the hosts of P. brassicae the submitted paper compared qPCR, RNA-sequencing and NanoString data and came to the conclusion: the NanoString platform kit is a robust tool that should become a common alternative for identifying resistance/susceptibility plant markers challenged by different P. brassicae pathotypes.
The by the NanoString nCounter System to identifying wealth of host genes focussing paper emphasises the NanoString nCounter System kit advantage by having little soil borne P brassicae influences on the plant-protist-soil interactivities in the methodical consideration. For me as soil microbiologist interested in getting better insights in the the plant-protist-soil interactivities is the submitted, well-written paper only very partially in the focus of my interest and I recommend publishing or declining, the more because the paper majorly confirms already conveyed information.
Author Response
REPLY: We thank Reviewer 1 for their overall positive assessment of this paper. To the best of our knowledge, the suitability of the NanoString system has not been evaluated previously for the clubroot pathosystem (or other soilborne plant diseases). Therefore, this paper represents a novel contribution, which not only shows the utility of this tool for these systems, but also presents a detailed comparison with other leading technologies in terms of operational requirements, time and cost.
Reviewer 2 Report
In this paper the authors show the value of using the Ncounter system to profile panels of selected genes to characterise transcriptional responses to clubroot infection in Brassica napus. They demonstrate the reliability of data collected this way and highlight several advantages. While the central finding that gene expression changes determined by RNA-Seq or Ncounter are well correlated is not particularly novel this platform has been little used in the study of plant pathogen interactions and so will be of interest to researchers in the field contemplating adopting this approach. As well as evaluating the data quality the authors detail their approach to selecting genes which may discriminate responses in clubroot resistant and susceptible varieties which will be of interest to other clubroot researchers. Furthermore, they highlight the advantages of Ncounter in terms of time and budget when compared with using qPCR to validate changes in gene expression.
The paper is very clear and well written. Use of the Ncounter system requires an investment in the preparation of probes, for those considering using the system this paper will be a useful source of information.
One section that could be expanded slightly would be the consideration of the use of reference genes. The authors suggest that at least 3 should be used, it might be helpful to cite a publication which addresses the appropriate / minimum number of reference genes. There are a variety of statistical tools to compare and rank the stability of reference genes used for qPCR. I think it would be interesting to compare the stability and performance of the four references used in the Ncounter and qPCR analyses to show that they have similar stability since all the other analyses are based on this foundation.
One advantage of the Ncounter system is that cruder RNA extractions can be accurately profiled. I wonder if the authors have ever compared RNA extractions without RNAEasy cleanup as this would feed into the cost considerations and higher throughput screening.
FAD7 is referred to as a regulator of SA JA pathways, as a metabolic enzyme surely the regulators of FAD7 expression and turnover are the regulators. Furthermore, in Brassica napus there is no functional characterisation of its influence on clubroot disease, I think it might be clearer to describe it as a potential marker of host responses rather than a "key regulator".
Minor points:
Line 137 "BSMT1" is repeated
Line159/160 "indicated upregulation in the susceptible cultivar and downregulation in the susceptible cultivar"
Figure 1 - fonts on the charts are very small / hard to read in print
Table 2 - "Design and synthase Codesets"
Author Response
REVIEWER COMMMENT: One section that could be expanded slightly would be the consideration of the use of reference genes. The authors suggest that at least 3 should be used, it might be helpful to cite a publication which addresses the appropriate / minimum number of reference genes. There are a variety of statistical tools to compare and rank the stability of reference genes used for qPCR. I think it would be interesting to compare the stability and performance of the four references used in the Ncounter and qPCR analyses to show that they have similar stability since all the other analyses are based on this foundation.
AUTHOR REPLY: We thank Reviewer 2 for this suggestion. We agree with their comment, and therefore we now present stability tests for the four reference genes (please see Lines 110-120 and 396-402 of the revised manuscript showing changes, as well as the new supplementary files Fig. S1 and Tables S2 and S3)
REVIEWER COMMENT: One advantage of the Ncounter system is that cruder RNA extractions can be accurately profiled. I wonder if the authors have ever compared RNA extractions without RNAEasy cleanup as this would feed into the cost considerations and higher throughput screening.
AUTHOR REPLY: We thank the reviewer for this point. We have now included some discussion on the capacity of the NanoString system to generate reliable data from highly degraded samples (Lines 263-269), but note that the RNA samples still need to be pure, with 260/280 and 260/230 OD ratios close to 2.0, regardless of the extraction method used.
REVIEWER COMMENT: FAD7 is referred to as a regulator of SA JA pathways, as a metabolic enzyme surely the regulators of FAD7 expression and turnover are the regulators. Furthermore, in Brassica napus there is no functional characterisation of its influence on clubroot disease, I think it might be clearer to describe it as a potential marker of host responses rather than a "key regulator".
AUTHOR REPLY: Good point. We have revised the text accordingly as per this suggestion (Line 183).
MINOR POINTS FROM REVIEWER (& AUTHOR REPLIES):
Line 137 "BSMT1" is repeated
REPLY: We deleted the superfluous “BSMT1” (Line 150).
Line159/160 “indicated upregulation in the susceptible cultivar and downregulation in the susceptible cultivar”
REPLY: We revised the sentence to “indicated upregulation in the susceptible cultivar and downregulation in the resistant cultivar” to match the data interpretation and results (Line 173-174).
Figure 1 - fonts on the charts are very small / hard to read in print
REPLY: We increased the fonts accordingly.
