The Defense Mechanism of PpCAD4 in Physcomitrium patens Against Botrytis cinerea

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The paper is well done and carfully written, revealing novel details of the PpCAD-4 overexpression. I'd suggest to improve Fig. 1, as the resistence promoting is not clear from it. Text claims the difference between 1F pictures of WT and PpCAD-4 line plants after 12h per inoculation. I don't see big difference between these images.  The greater difference is at 24hpi, but nearby picture of the effect of 24 hours (1G) shows that the plants at this stage do not differ significantly.  Most Supplementary Material data address to 6 and 12 hpi, but not 24 hpi, thus a doubt on is raised on the efficience of resistence. Otherwise the text can be publised as it is.

Author Response

Dear Reviewer:

We would like to express our special thanks for your careful editing manuscript entitled “The defense mechanism of PpCAD4 in Physcomitrium patens against Botrytis cinerea” (ID: plants-4101724). The comment is precious and helpful, enabling us to improve our paper. We have made modifications. We would love to thank you for allowing us to resubmit a revised copy of the manuscript.

The paper is well done and carfully written, revealing novel details of the PpCAD-4 overexpression. I'd suggest to improve Fig. 1, as the resistence promoting is not clear from it. Text claims the difference between 1F pictures of WT and PpCAD-4 line plants after 12h per inoculation. I don't see big difference between these images. The greater difference is at 24hpi, but nearby picture of the effect of 24 hours (1G) shows that the plants at this stage do not differ significantly. Most Supplementary Material data address to 6 and 12 hpi, but not 24 hpi, thus a doubt on is raised on the efficience of resistence. Otherwise the text can be publised as it is

Response: We sincerely thank your positive feedback on our work and for the thoughtful suggestion regarding Figure 1. The point about the efficiency and consistency of resistance is well-taken. We agree that the original image alone may not clearly demonstrate the phenotypic differences described in the text. We have added micrographs for the 6 hpi and replaced more representative images at 24 hpi. The magnified images of specific lesion areas showed in the box are provided to highlight the differences in resistance between WT and PpCAD4-OE plants at 12 hpi. The significant brown maceration regions appeared at the tips of the phyllid in the WT, whereas only discernible symptoms of brown spots were observed in PpCAD4-OE at the same period (Figure 1).

Figure 1. Disease symptoms observed in B. cinerea-infected gametophores of wild-type and PpCAD4-OE lines at various hours post inoculation (hpi). The box showed the magnified images of brown maceration areas of P. patens at 12 hpi.

 

Finally, many thanks for your careful review. We really appreciate your time in reviewing our manuscript. 

 

Best regards,

Sincerely yours,

Huiqing

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript reports the role of PpCAD4, a cinnamyl alcohol dehydrogenase family member, in the defense response of the moss Physcomitrium patens against the necrotrophic pathogen Botrytis cinerea, which aims to place PpCAD4 in an evolutionary context of early land plant defense. Using PpCAD4 overexpression lines, the authors integrate phenotypic measurements, transcriptomics, metabolomics, transcriptional asays (Y1H and dual-Luc), and protein–protein interaction analyses (Y2H, BiFC) to propose a multi-layered defense mechanism involving phenylpropanoid accumulation and ROS modulation via a PpCAT2-PpE3-PpCAD4 module.
The study is interesting and important, particularly for understanding how phenylpropanoid-related enzymes diversified prior to the emergence of vascular lignin-based defenses. The dataset is extensive and potentially valuable. However, substantial revision is required before the manuscript can be accepted for publication.
A major concern is the entire study relies on PpCAD4 overexpression (OE) lines, which limits mechanistic interpretation. Without knockout, knockdown, or CRISPR mutant provided, it is impossible to conclude whether PpCAD4 is required for defense, rather than merely sufficient when overexpressed. Specially, nearly 4,000 genes were differentially expressed between WT and OE suggest strong pleiotropic effects of PpCAD4 overexpression unrelated to pathogen defense. At least the author should clearly acknowledge the limitation and significantly temper causal claims throughout the manuscript. The infection phenotype shows delayed disease progression in OE lines at early time points (6-12 hpi), but not at later stage (48 hpi).
Given that B. cinerea is a typical necrotrophic pathogen and that jasmonic acid (JA) and ethylene (ET) signaling pathways are widely recognized as central regulators of plant defense against necrotrophs, which often interacting antagonistically or synergistically with salicylic acid (SA) signaling (Li et al. 2019; PMID: 30720746 and DOI: 10.3390/ijms20030671). The comprehensive review could be appropriately incorporated into the Introduction section, which would help readers better understand the biological relevance of the hormone-related pathways identified in the transcriptomic analysis, as well as the observed ROS-associated defense responses.
Lines 149-153: At 12 hpi, the authors report “716 upregulated” and “6,525 downregulated” genes, which exceeds the total number of total differentially expressed genes (“1,358”).
The PpE3-PpCAT2-PpCAD4 module seems much speculative. While in vitro and transient assays show interactions, no evidence of ubiquitination or protein degradation is provided.

Author Response

Dear Reviewer:

We would like to express our special thanks for your careful editing of our manuscript entitled “The defense mechanism of PpCAD4 in Physcomitrium patens against Botrytis cinerea” (ID: plants-4101724). Those comments are precious and helpful, enabling us to improve our paper. We have studied the comments carefully and made corresponding modifications, which we sincerely hope meet the requirements for publication in plants. We would love to thank you for allowing us to resubmit a revised copy of the manuscript, entitled according to the reviewer’s suggestion. Revisions in the text are shown using the red highlight for corrections. Here, Point-by-point responses are listed below.

Reviewer: The manuscript reports the role of PpCAD4, a cinnamyl alcohol dehydrogenase family member, in the defense response of the moss Physcomitrium patens against the necrotrophic pathogen Botrytis cinerea, which aims to place PpCAD4 in an evolutionary context of early land plant defense. Using PpCAD4 overexpression lines, the authors integrate phenotypic measurements, transcriptomics, metabolomics, transcriptional asays (Y1H and dual-Luc), and protein–protein interaction analyses (Y2H, BiFC) to propose a multi-layered defense mechanism involving phenylpropanoid accumulation and ROS modulation via a PpCAT2-PpE3-PpCAD4 module. The study is interesting and important, particularly for understanding how phenylpropanoid-related enzymes diversified prior to the emergence of vascular lignin-based defenses. The dataset is extensive and potentially valuable. However, substantial revision is required before the manuscript can be accepted for publication

Response: We are grateful to the reviewer for their time and insightful comments, which have been most helpful in improving our manuscript. In response, we have undertaken extensive revisions to the text and figures. We believe these changes have significantly strengthened the study. Our point-by-point responses to each comment are detailed below, and all modifications in the manuscript have been highlighted.

 

1. A major concern is the entire study relies on PpCAD4 overexpression (OE) lines, which limits mechanistic interpretation. Without knockout, knockdown, or CRISPR mutant provided, it is impossible to conclude whether PpCAD4 is required for defense, rather than merely sufficient when overexpressed. Specially, nearly 4,000 genes were differentially expressed between WT and OE suggest strong pleiotropic effects of PpCAD4 overexpression unrelated to pathogen defense. At least the author should clearly acknowledge the limitations and significantly temper causal claims throughout the manuscript.

Response：We thank the reviewer for raising this critical point, which we fully acknowledge as a key limitation of our study. We agree that the reliance solely on overexpression (OE) lines restricts definitive claims about the necessity of PpCAD4 in defense. The phenotypic response to B. cinerea infection was not pronounced in the PpCAD4 knockout line, which may be attributed to functional redundancy or compensatory mechanisms among other members of the CAD gene family, which comprises four homologs in P. patens. The observation of nearly 4,000 differentially expressed genes indeed highlights the broad transcriptional changes induced by overexpression, some of which may be pleiotropic. We have significantly tempered all causal claims. This revision has been applied in the Abstract, Results, and Discussion sections.

 

2. The infection phenotype shows delayed disease progression in OE lines at early time points (6-12 hpi), but not at later stages (48 hpi).

Response：We agree with the reviewer that the enhanced resistance in PpCAD4-OE lines is most pronounced at early time points (6-12 hpi) and diminishes by 48 hpi. We believe this pattern is biologically meaningful and common in plant-pathogen interactions. Botrytis cinerea, as a robust necrotroph, employs a potent arsenal of cell-wall-degrading enzymes and toxins. We interpret our data to suggest that the constitutive defense priming conferred by PpCAD4 overexpression (e.g., via early phenylpropanoid accumulation and ROS modulation) is highly effective in delaying initial infection establishment. However, this may not be sufficient to completely block a determined pathogen over an extended period, especially once the necrotrophic phase is fully established.

 

3. Given that B. Cinerea is a typical necrotrophic pathogen and that jasmonic acid (JA) and ethylene (ET) signaling pathways are widely recognized as central regulators of plant defense against necrotrophs, which often interact antagonistically or synergistically with salicylic acid (SA) signaling (Li et al. 2019; PMID: 30720746 and DOI: 10.3390/ijms20030671). The comprehensive review could be appropriately incorporated into the Introduction section, which would help readers better understand the biological relevance of the hormone-related pathways identified in the transcriptomic analysis, as well as the observed ROS-associated defense responses.

Response：This is an excellent point. We appreciate the reviewer's suggestion to cite the key reviews (Li et al., 2019) and have now incorporated a concise overview of the established roles of JA/ET signaling in defense against necrotrophs, including their complex interplay with SA signaling, into the Introduction section (lines). This addition provides essential context for readers to better interpret the hormone-related pathways identified in our transcriptomic analysis and links them logically to the observed ROS-associated defense responses, which are often downstream of these hormone signals.

 

4. Lines 149-153: At 12 hpi, the authors report “716 upregulated” and “6,525 downregulated” genes, which exceeds the total number of differentially expressed genes (“1,358”).

Response：We thank the reviewer for their meticulous attention to detail in identifying this discrepancy. The reviewer is absolutely correct. There was an error in the original manuscript. The number of downregulated genes at 12 hpi was incorrectly stated as “6,525” due to a stray digit. The correct numbers are 716 upregulated and 652 downregulated genes, which sum to the correct total of 1,368 differentially expressed genes. We sincerely apologize for this error. We have meticulously rechecked all the transcriptomic data and corrected the text accordingly.

 

5. The PpE3-PpCAT2-PpCAD4 module seems very speculative. While in vitro and transient assays show interactions, no evidence of ubiquitination or protein degradation is provided.

Response：Thanks for your comment. We agree with the reviewer that the proposed PpE3-PpCAT2-PpCAD4 regulatory module, while supported by our protein-protein interaction data (Y2H, BiFC), requires future validation through functional ubiquitination assays. We have revised the manuscript throughout to reframe our conclusion. We now present this as a testable working model or hypothesis. We appreciate this suggestion, as it has helped us present our findings with greater precision and scholarly rigor.

 

Finally, many thanks for your careful review. We really appreciate your time in reviewing our manuscript. Our deepest gratitude goes to you for your careful work and thoughtful suggestions that have helped improve this paper substantially. All the authors think this manuscript is considerable to publish in plants. We sincerely appreciate your consideration of our manuscript and look forward to receiving comments from the reviewers. Our deepest gratitude goes to you for your reply.

 

Best regards,

Sincerely yours,

Huiqing

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have adequately addressed the majority of the reviewer comments. The revised manuscript shows significant improvement with clarity and scientific validity. I recommend acceptance for publication pending minor language and editorial corrections.