Transcriptome Analysis Reveals the Involvement of Mitophagy and Peroxisome in the Resistance to QoIs in Corynespora cassiicola

Comments 3: Figure 1 is not a graphical abstract since it does not give an overview of the research article. It could be used in discussion to explain the difference between RI and RII, but not in the introduction. Additionally, please add a title to the figure.

Response 3: Thank you for your suggestion. The original Figure 1 has been moved to the appropriate place in the discussion and the title and comments have been added. Currently listed in Figure 11.

Comments 4: Line 108: what do you mean by "required"? I think it's "added"

Response 4: Thank you for your suggestion. I have revised the word and checked for similar issues throughout the text.

Comments 5: Figure 4: "The up-regulated, down-regulated, and unchanged unigenes are dotted in red, and green, respectively" >> red, green, and blue (third color).

Response 5: Thank you for the suggestion, it was indeed an oversight on our part. I have made an addition to the sentence: “The up-regulated, down-regulated, and unchanged unigenes are dotted in red, green, and blue respectively”.

Comments 6: Figures in general are too small and thus hard to read.

Response 5: Thanks for the suggestion. Have enlarged all figures in the article.

Comments 3: Discuss whether the expression changes observed may compensate for fitness costs of the target mutations. This could provide insight into selective advantages.

Response 3: Thank you for your suggestion. The discussion has been supplemented to target this point, focusing on lines 378-390: “Metabolism-related genes are widely involved in cellular energy metabolism, substance synthesis, catabolism, and transport, which can affect the cell’s ability to adapt to the external environment. It has been demonstrated that polysaccharide metabolites, lipid metabolites, and certain functional genes, such as the R gene, can enhance cellular resistance and can reduce fitness costs caused by resistance such as decreased spore production and reduced spore germination. In our study, we found that metabolism-related genes were enriched focusing on sugar metabolism, lipid metabolism, and amino acid metabolism, and several special genes were found, such as LCB1/2 and FRC1, verifying that cellular autophagy and related metabolism occur. Therefore, it is hypothesized that the changes of related genes within RII strains compensate fitness costs by increasing the expression of metabolism-related genes to promote cellular utilization of resources and energy production to maintain normal physiological function and survival of the cells”.

Comments 4: Elaborate on the proposed model - are there any validated inhibitors of these pathways that could be tested to further support the conclusions?

Response 4: Thank you for your question. In this article, oxidative stress capacity has been measured, validating the pathway to some extent. We are also currently conducting experiments on the production and activity of peroxidase and related proteins.

Comments 5: Carefully proofread the manuscript to fix minor typos, formatting issues, and any unclear wording.

Response 5: Thank you for your advice. We have proofread the text fully for formatting, italics, symbols, spelling, etc., e.g., lines 30 (“is a crucial fungicide” to “are crucial fungicides”), 83 (“Colorectal” to “colorectal”), 161 (“EF-1 alpha” to “EF-1 alpha”), and 235 (supplement of “RⅠ”).

Comments 6: Some sections of the results could be condensed by focusing on the most salient points and removing redundant descriptions.

Response 6: Thanks for the suggestion. The results section has been streamlined or supplemented, focusing on the GO and KEGG enrichment analysis.

For example, the redundant part " which were enriched in 547, 534, 456, and 451 DEGs, respectively " has been deleted.

The sentence " Similarly, two-by-two comparative KEGG analyses were performed for each strain in which peroxisomes, glutamate, glycine, cysteine metabolism, autophagy, and endocytosis were enriched. The highest enrichment factor was all in ribosomes and the highest number of enriched DEGs were in metabolic pathways " has been optimized to “Similarly, two-by-two comparative KEGG analyses were performed for each strain in which the highest enrichment factor was all in ribosomes and the highest number of enriched DEGs were in metabolic pathways”.

Comments 3: on P. 5 list the sensitivity of each fungicide.

Response 3: Thank you for your suggestion. Sensitivity data have been added in lines 183-197 of the text: “The average EC₅₀ values of RII strains were 909.44, 8565.47, 54.69, and 5.55 μg/ml for 4 QoIs (Tri, Kre, Azo, and Pyr), while the average EC₅₀ values of RI strains were 14.49, 1.21, 11.15, and 0.85 μg/ml for 4 QoIs, respectively. Among them, the largest difference in the resistance was observed in Kre, followed by Tri, with the average EC₅₀ of RII strains being 7102.38 and 62.78 times higher than that of RI strains. And the resistance of RII strains to Azo and Pyr was 4.91 and 6.51 times higher than that of RI strains. However, there was no significant difference in resistance between RI and RII strains to the other 7 fungicides (Cya, Tol, Ter, Flu, Dif, Car, Pen), with the average EC₅₀ values were 8529.00, 1.46, 0.37, 0.12, 2.01, 194.90, and 8.88 μg/ml for RII strains and 389.50, 23.69, 0.67, 0.12, 2.88, 341.72, and 0.80 μg/ml for RI strains. Note that due to the extreme resistance of Cya, the EC₅₀ data are only for reference (formulaic extrapolation based on inhibition at low concentrations).

Comments 4: In Fig. 4 it says "up-regulated, down-regulated, and unchanged genes are dotted in red and green, respectively". This lists 3 groups and only 2 colors so meaning is not clear.

Response 4: Thanks for the suggestion, which was indeed an oversight on our part. I have made an addition to the sentence: “The up-regulated, down-regulated, and unchanged unigenes are dotted in red, green, and blue respectively”.

Comments 5: There is no description of statistics used and how multiple tests were analyzed.  Also no indication of regression statistics used to calculate ec50 values (goodness of fit)

Response 5: Thank you for your question and suggestion. EC₅₀ values were calculated by probit regression using SPSS software. The significant analyses of variance were performed by the Duncan test for one-way ANOVA with a significance level was P<0.05.

This has been supplemented in the Materials and Methods section of the article, specifically in lines 124 and 167.

Point 2: On P. 3 the fungicides used are describes as "original drugs". A better term would be "technical grade active ingredient" which I assume is what was meant. However, I am uncertain since no solvent was mentioned. This is an important point. If formulated product was used (not requiring a solvent), the specific formulation must be reported.

Response 2: Thank you for your suggestions. The content has now been changed and solvents added. It is clearly in lines 111-115: “Among them, Car was dissolved with 0.2M HCL and the other fungicides were dissolved with DMSO. The concentrations of active ingredients used were as follows: Kre and Cya (0, 0.1, 1, 10, 100, and 300 μg/mL), Azo and Pyr (0, 0.1, 1, 10, 80, and 240 μg/mL), Ter (0, 0.001, 0.01, 0.1, 1, and 5 μg/mL), Flu (0, 0.005, 0.01, 0.05, 0.1, and 0.5 μg/mL), Dif (0, 0.05, 0.1, 0.5, 3, and 9 μg/mL), Car (0, 10, 50, 200, and 400 μg/mL), Pen (0, 0.1, 0.5, 1, 3, and 10 μg/mL)”.