Review Reports - Transcriptome Analysis Reveals Key Pathways and Candidate Genes for Resistance to Plasmodiophora brassicae in Radish

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Regarding the manuscript titled "Transcriptome analysis provides key pathways and candidate genes for resistance to Plasmodiophora brassicae in radish"

1.- In the methodology section, 2.1 Plant materials and P. brassica inoculation; the authors have reported their pathogen isolation protocol, inoculation method, and disease index (DI) calculation for P. brassicae preparation in a previous study. However, it is important to describe it in this study so that the reader can better understand the methods used. Without this required information, Table 1 would appear rather vague.

2.- In the methodology section, 2.2 RNA extraction and sequencing; more information is needed on how these activities were carried out.

3.- I would suggest changing the Figure 1 to show a greater visual difference in T6 (resistant). The figure is very dark and requires greater sharpness and clarity to make it appealing to the reader.

4.- Homogenize the word RT-qPCR, in figure 7 it is written as qRT-PCR.

5. In Supplementary Table S2, sequencing data analysis between T14 and T6, the authors describe that the analysis was performed with T30, CORRECT

6. In Supplementary Table S3 and S4, DEGs associated with Ethylene (ET) and JA signaling pathways between T14 and T6, the authors describe that the analysis was performed with T30, CORRECT

Author Response

Reviewer 1

Regarding the manuscript titled’Transcriptome analysis provides key pathways and candidate genes for resistance to Plasmodiophora brassicae in radish’

Comment 1. In the methodology section, 2.1 Plant materials and P. brassica inoculation; the authors have reported their pathogen isolation protocol, inoculation method, and disease index (DI) calculation for P. brassicae preparation in a previous study. However, it is important to describe it in this study so that the reader can better understand the methods used. Without this required information, Table 1 would appear rather vague.

Response: Thank you for your suggestion. A brief description has been added to Section 2.1 as follows lines 114-120: ‘Briefly, the concentration of resting spores was determined using a hemocytometer and adjusted to 1 × 10⁷ spores/mL for inoculation. Ten days after sowing, each seedling was inoculated by injecting 1 mL of the spore suspension into the soil near the root. The disease index (DI) was assessed four weeks post-inoculation based on a 0–4 scale as follows: 0, no galling; 1, a few small galls on lateral roots; 2, moderate galling on lateral roots or main root; 3, numerous galling on the main roots; and 4, club rotten or plant dying.’

Comment 2. In the methodology section, 2.2 RNA extraction and sequencing; more information is needed on how these activities were carried out.

Response: Thank you for your suggestion. We have expanded Section 2.2 ‘We have added a brief description as follows: ‘Total RNAs for transcriptome sequencing were extracted using the Eastep® Super Total RNA Extraction Kit (Promega Co., Ltd., Shanghai, China) following the manufacturer’s protocol. For transcriptome sequencing, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. The purified mRNA was then fragmented and used as a template for first-strand cDNA synthesis with random hexamer primers, followed by second-strand cDNA synthesis. The resulting cDNA fragments were subjected to end-repair, A-tailing, and adapter ligation. After PCR amplification, the final cDNA libraries were constructed. The library construction and sequencing were performed by Annaroad Gene Technology Co., Ltd. (Beijing, China) on an Illumina No-vaSeq 6000 platform, generating 150 bp paired-end reads.’ (lines 134-143)

Comment 3. I would suggest changing the Figure 1 to show a greater visual difference in T6 (resistant). The figure is very dark and requires greater sharpness and clarity to make it appealing to the reader.

Response: Thank you for your suggestion. We have replaced Figure 1 with a higher-resolution version that improves contrast, sharpness, and clarity to better distinguish phenotypic differences, especially in T6 (Figure 1).

Comment 4. Homogenize the word RT-qPCR, in Figure 7 it is written as qRT-PCR.

Response: Thanks for your suggestion. We have standardized the terminology to ‘RT-qPCR’ throughout the manuscript, figures, and supplementary materials for consistency.

Comment 5. In Supplementary Table S2, sequencing data analysis between T14 and T6, the authors describe that the analysis was performed with T30, CORRECT

Response: Thanks for your suggestion. The analysis was performed on the resistant line T6. We have corrected this in Supplementary Table S2.

Comment 6. In Supplementary Tables S3 and S4, DEGs associated with Ethylene (ET) and JA signaling pathways between T14 and T6, the authors describe that the analysis was performed with T30, CORRECT

Response: Thank you for your suggestion. We have corrected the descriptions and headings in Supplementary Tables S3 and S4 to indicate that the DEG analyses were performed between T6 and T14.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The paper of Ma et al. entitled "Transcriptome analysis provides key pathways and candidate genes for resistance to Plasmodiophora brassicae in radish” represents an important contribution in understanding the mechanisms of resistance in radish to this important plant pathogen. Generally, the MS is well written, and transcriptomics analyses have been conducted competently. Some of the references are not that relevant and a few sentences have to be revised for clarity. It is unfortunate that no information related to the growth parameters of lines T6 and T14 are provided, and no physiological or morphological analyses have been conducted. More information on how experiments have been conducted have to be added to the Materials and methods section.

Title.

“Provides” Suggest choosing a different word like, “reveals”.

Abstract

Line 22

Instead of “however” consider “In contrast”

Introduction

Line 38

Avoid sentences like “ particularly in Asian agroecosystems [1]”. Is indeed the production of radish so much specific for Asian agroecosystems? Citation 1 is at least debatable unrelated to the claim! Please indicate the original source of this claim!

Lines 39-40

Revise this information for clarity. Oilseed radish or rapeseed radish can be Brassica napus, not only Raphanus sativus var. oleiformis. There are different subspecies and varieties of radish, Raphanus sativus, not only those listed in the text.

Line 43

References are debatable as are not directly related to the information provided in lines 41-43, especially #3. References 3-5 are more specific for the sentence in lines 43-46.

Lines 48-50.

The sentence has to be revised. Unani is a Perso-Arabic system of medicine. Find the primary source of information. The research in reference 8 is not providing this type of information.

Lines 55 and 347

brassicae belongs to the Phytomyxea (Endomyxa). The term protist is too general to be used in 2025. Please find a better reference.

Line 61

“locales”? What is the meaning of this?

Line 73

For consistency do not use “Cruciferae” but “Brassicaceae”. Cruciferae is the old name of the family.

Line 89

Remove “To address this knowledge gap,”. RNA-Seq was not developed to “address this knowledge gap” in radish!

Lines 100-102

Revised the sentence, it is not clear what the authors want to say: “In this study, a comparative transcriptomic analysis of resistant (T6) and susceptible (T14) radish inbred lines at three time points following inoculation with P. brassicae.”

Line 102

Add “this study” between “pathways” and “aimed”.

Lines 104-106

Revise the sentence. Why using past tense?

“Those findings provided valuable insights into the molecular basis of clubroot resistance in radish and laid a foundation for genetic improvement in radish breeding programs.”

Materials and methods

Line 114

Provide the protocol of inoculation with P. brassicae and provide a reference.

Line 123

Provide more information on the experimental design. How many plants have been used in the experiment? How many biological samples have been sequenced at each time point? Three biological samples, as indicated in figure 8, related to the results of the qPCR experiment?

Discussion section

Lines 363-366

“In contrast, T6 exhibited a more restrained response, charac-363 terized by significant downregulation of genes at early stages (10 DAI), potentially reflect-364 ing resource reallocation from growth to immunity—a hallmark of the ‘growth-defense 365 trade-off’.

Unfortunately, this suggestion lacks experimental support. Why the authors did not record some growth parameters such as size of the plant, fresh and dry weight?

It is surprising that a resistant line exhibits consistent down regulation in the day 10 to day 30 range vs not-inoculated plants. If plants were resistant, it is hard to understand why the down regulation pattern lasts so long. However, the good part is that on day 30 differences between inoculated and not-inoculated plats are reduced, which can be considered as a hallmark of resistance.

Lines 386-388

Revise the sentence (grammar and clarity)

“Hormonal crosstalk modulates resistance outcomes: JA pathway components exhibited striking divergence: JAZ repressors and biosynthesis genes (AOC, OPR) were up-regulated in T6 but suppressed in T14.”

Lines 393-395

Revise the sentence (grammar and clarity)

“ABA and cross-pathway interactions: ABA receptor genes 393 (PYR/PYL/RCAR) showed genotype-specific expression, with Rsa3g000580 (high in T14) 394 and Rsa2g041430 (high in T6), suggesting ABA’s dual role as a defense modulator.”

References

Some references, as previously indicated, are not relevant, and are not the primary sources of information.

Italicize the species names of species, for example, 3, 25.

Comments on the Quality of English Language

Quality is OK. A few sentences have to be revised.

Author Response

Reviewer 2

The paper of Ma et al. entitled ’ Transcriptome analysis provides key pathways and candidate genes for resistance to Plasmodiophora brassicae in radish’ represents an important contribution in understanding the mechanisms of resistance in radish to this important plant pathogen. Generally, the MS is well written, and transcriptomics analyses have been conducted competently. Some of the references are not that relevant and a few sentences have to be revised for clarity. It is unfortunate that no information related to the growth parameters of lines T6 and T14 are provided, and no physiological or morphological analyses have been conducted. More information on how experiments have been conducted have to be added to the Materials and methods section.

Comment 1. Title. ‘Provides’Suggest choosing a different word like, ‘reveals’.

Response: Thank you for your suggestion. We agree that ‘reveals’ conveys our findings more effectively. The revised title is: ‘Transcriptome analysis reveals key pathways and candidate genes for resistance to Plasmodiophora brassicae in radish’

Comment 2. Abstract

Line 22 Instead of ‘however’ consider ‘In contrast’

Response: Thank you for your suggestion. We have revised the sentence as suggested in line 22: ‘In contrast, the resistant line T6 displayed temporally coordinated defense activation marked by rapid induction of core immune mechanisms:’

Comment 3. Introduction

Line 38 Avoid sentences like ‘particularly in Asian agroecosystems [1]’. Is indeed the production of radish so much specific for Asian agroecosystems? Citation 1 is at least debatable unrelated to the claim! Please indicate the original source of this claim!

Response: Thank you for your suggestion. We have replaced the original reference with a more appropriate source: ‘1. Swaamy K: Origin, distribution, genetic diversity, and breeding of radish (Raphanus sativus L.). International Journal of Development Research 2023, 13:61657-61673.’

Comment 4. Lines 39-40: Revise this information for clarity. Oilseed radish or rapeseed radish can be Brassica napus, not only Raphanus sativus var. oleiformis. There are different subspecies and varieties of radish, Raphanus sativus, not only those listed in the text.

Response: Thank you for your suggestion. We have revised the sentence to improve accuracy in lines 39-40: ‘As a versatile crop, various radish types contribute substantially to global vegetable production and agricultural economies.’

Comment 5. Line 43: References are debatable as are not directly related to the information provided in lines 41-43, especially #3. References 3-5 are more specific for the sentence in lines 43-46.

Response: Many thanks. We have re-evaluated the citations and agree that they were not optimally placed. We have restructured the references as follows:

For lines 41-43:

Gaba, J., Bhardwaj, G., Sharma, A., Nayik, G., & Gull, A. Antioxidants in Vegetables and Nuts-Properties and Health Benefits. In.: Springer; 2020,75-103
Goyeneche R, Roura S, Ponce A, Vega-Gálvez A, Quispe-Fuentes I, Uribe E, Di Scala K: Chemical characterization and antioxidant capacity of red radish (Raphanus sativus L.) leaves and roots. Journal of Functional Foods 2015, 16:256-264.

And for lines 43-46:

Baenas N, Piegholdt S, Schloesser A, Moreno DA, García-Viguera C, Rimbach G, Wagner AE: Metabolic activity of radish sprouts derived isothiocyanates in drosophila melanogaster. International Journal of Molecular Sciences 2016, 17(2):251.
Ishida M, Kakizaki T, Morimitsu Y, Ohara T, Hatakeyama K, Yoshiaki H, Kohori J, Nishio T: Novel glucosinolate composition lacking 4-methylthio-3-butenyl glucosinolate in Japanese white radish (Raphanus sativus L.). Theoretical and Applied Genetics 2015, 128(10):2037-2046.
Malik MS, Riley MB, Norsworthy JK, Bridges Jr W: Variation of glucosinolates in wild radish (Raphanus raphanistrum) accessions. Journal of Agricultural and Food Chemistry 2010, 58(22):11626-11632.

Comment 6. Lines 48-50. The sentence has to be revised. Unani is a Perso-Arabic system of medicine. Find the primary source of information. The research in reference 8 is not providing this type of information.

Response: Thank you for pointing out this issue. We agree that the reference provided did not adequately support the broad claim about traditional medicinal uses. After a thorough review, we were unable to locate a suitable primary academic source to substantiate this specific sentence. We agree and removed this sentence from the manuscript to maintain scientific rigor.

Comment 7. Lines 55 and 347: brassicae belongs to the Phytomyxea (Endomyxa). The term protist is too general to be used in 2025. Please find a better reference.

Response: Thank you for your suggestion. We have revised the terminology to ‘plasmodiophorid protist’ for greater specificity in lines 52 and 354: ‘Despite its agronomic and medicinal importance, radish production faces a critical threat from clubroot disease, caused by the obligate biotrophic plasmodiophorid protist P. brassicae’.

‘Clubroot disease, caused by the obligate biotrophic plasmodiophorid protist P. brassicae, poses a significant threat to global Brassicaceae crop production.’

Comment 8. Line 61: ‘locales’? What is the meaning of this?

Response: Thank you for highlighting this potentially unclear word choice. To improve clarity, we have replaced ‘locales’ with ‘locations’ in line 59.

Comment 9. Line 73: For consistency do not use ‘Cruciferae’ but ‘Brassicaceae’. Cruciferae is the old name of the family.

Response: Thank you for noting this inconsistency. We have corrected this throughout the manuscript.

Comment 10. Line 89: Remove ‘To address this knowledge gap,’. RNA-Seq was not developed to ‘address this knowledge gap’ in radish!

Response: Thank you for your suggestion. We have removed this phrase for better clarity and accuracy.

Comment 11. Lines 100-102: Revised the sentence, it is not clear what the authors want to say: ‘In this study, a comparative transcriptomic analysis of resistant (T6) and susceptible (T14) radish inbred lines at three time points following inoculation with P. brassicae.’

Response: Thank you for your suggestion. We have revised the sentence as lines 97-98: ‘In this study, we performed a comparative transcriptomic analysis of resistant (T6) and susceptible (T14) radish inbred lines at three time points following inoculation with P. brassicae.’.

Comment 12. Line 102: Add ‘this study’ between ‘pathways’ and ‘aimed’.

Response: Thank you for your suggestion. The sentence has been revised to line 100: ‘By characterizing global gene expression profiles and enriched pathways, this study aimed to uncover the molecular basis of clubroot resistance in radish...’.

Comment 13. Lines 104-106: Revise the sentence. Why using past tense?

‘Those findings provided valuable insights into the molecular basis of clubroot resistance in radish and laid a foundation for genetic improvement in radish breeding programs.’

Response: Thank you for your suggestion. We have revised lines 102-104 ‘These findings provide valuable insights into the molecular basis of clubroot resistance in radish and lay a foundation for the genetic improvement of radish breeding programs.’

Comment 14. Materials and methods

Line 114: Provide the protocol of inoculation with P. brassicae and provide a reference.

Response: Thank you for your suggestion. We have included a detailed description of the inoculation protocol and disease indexing in Section 2.1 of the Materials and Methods. As follows lines 114-120: ‘Briefly, the concentration of resting spores was determined using a hemocytometer and adjusted to 1 × 10⁷ spores/mL for inoculation. Ten days after sowing, each seedling was inoculated by injecting 1 mL of the spore suspension into the soil near the root. The disease index (DI) was assessed four weeks post-inoculation based on a 0–4 scale as follows: 0, no galling; 1, a few small galls on lateral roots; 2, moderate galling on lateral roots or main root; 3, numerous galling on the main roots; and 4, club rotten or plant dying.’

Comment 15. Line 123: Provide more information on the experimental design. How many plants have been used in the experiment? How many biological samples have been sequenced at each time point? Three biological samples, as indicated in Figure 8, related to the results of the qPCR experiment?

Response: Thank you for your suggestion. We have added the following description to Section 2.1 (lines 126-132): ‘Total of 36 root samples were harvested from both inoculated and mock-treated plants of susceptible and resistant lines at 10, 20, and 30 days after inoculation (DAI). Each sample consisted of three roots pooled from three plants. Three independent biological replicates for each treatment(inoculated and mock-inoculated) and each time point. All samples were immediately immersed in liquid nitrogen and stored at –80°C until further processing for RNA extraction and sequencing or RT-qPCR analysis.’

Comment 16. Discussion section

Lines 363-366: In contrast, T6 exhibited a more restrained response, characterized by significant downregulation of genes at early stages (10 DAI), potentially reflecting resource reallocation from growth to immunity—a hallmark of the ‘growth-defense trade-off’.

Unfortunately, this suggestion lacks experimental support. Why the authors did not record some growth parameters such as size of the plant, fresh and dry weight?

Response: Thank you for your suggestion. Our study did not include physiological growth measurements, which is a limitation. Therefore, we cannot definitively conclude a growth-defense trade-off occurred. Therefore, we cannot definitively conclude a growth-defense trade-off occurred. To address this, we have rephrased the statement to be more speculative and to present it as a plausible hypothesis based on the transcriptomic data, rather than a conclusion. Lines 369-371 ‘This pattern could suggest a potential reallocation of resources from growth toward defense, a strategy often associated with the ‘growth-defense trade-off’ hypothesis in plant immunity. The subsequent reduction in the number of DEGs at 30 DAI in the T6 line may indicate a successful containment of the pathogen, allowing the plant to restore metabolic homeostasis.’

Comment 18. Lines 386-388: Revise the sentence (grammar and clarity)

‘Hormonal crosstalk modulates resistance outcomes: JA pathway components exhibited striking divergence: JAZ repressors and biosynthesis genes (AOC, OPR) were up-regulated in T6 but suppressed in T14.’

Response: Thank you for the suggestion. We have revised the sentence to improve its clarity and flow lines 392-395: ‘Hormonal crosstalk appears to be critical in modulating resistance outcomes. Our analysis revealed a striking divergence in the jasmonic acid (JA) pathway, where key components, including JAZ repressors and biosynthesis genes (AOC, OPR), were up-regulated in the resistant T6 line but suppressed in the susceptible T14 line.’

Comment 19. Lines 393-395: Revise the sentence (grammar and clarity)

‘ABA and cross-pathway interactions: ABA receptor genes 393 (PYR/PYL/RCAR) showed genotype-specific expression, with Rsa3g000580 (high in T14) 394 and Rsa2g041430 (high in T6), suggesting ABA’s dual role as a defense modulator.’

Response: We have rephrased this sentence as suggested to enhance readability. Lines 401-406 ‘Our results also suggest the involvement of abscisic acid (ABA) and cross-pathway interactions. Specifically, ABA receptor genes (PYR/PYL/RCAR) showed genotype-specific expression, with Rsa3g000580 highly expressed in the susceptible T14 line and Rsa2g041430 highly expressed in the resistant T6 line. This finding suggests a potential dual role for ABA as a defense modulator in this plant-pathogen interaction system.’

Comment 20. References

Some references, as previously indicated, are not relevant, and are not the primary sources of information.

Italicize the species names of species, for example, 3, 25.

Response: Thank you for this general comment. We have taken it very seriously and have conducted a thorough review of our entire reference list. We replaced or removed non-primary or irrelevant sources.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors addressed my comments and suggestions.

Author Response

Dear Editor,

Many thanks for your and reviewers positive and constructive comments on this manuscript entitled ‘Transcriptome analysis reveals key pathways and candidate genes for resistance to Plasmodiophora brassicae in radish (Manuscript ID: horticulturae-3674313)’

The comments provided have been instrumental in refining both the quality and coherence of our article. Each author has thoroughly examined the feedback and has conducted the requisite modifications to the manuscript as advised. We have diligently outlined these adjustments and implemented the respective corrections.