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Comment by Enzo Ariel Perk
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- CC BY 4.0
Response to Reviewers
We sincerely thank the reviewers for taking the time to carefully read our manuscript and for providing thoughtful and constructive comments. We greatly appreciate the opportunity to consider these observations, and we were pleased to read the reviewer’s perspective on our work. Below, we respond point by point to each comment.
General comment 1: While it is clear that infection progress is inhibited/delayed in the SlPLC2 mutant plants, we believe that evidence for SlPLC2 facilitating vesicle formation, as stated in the title of the paper, could be stronger. Perhaps there could be other explanations for the reduced number of vesicles observed on mutant vs WT leaves reported in Figure 5C (for instance, reduced host penetration)?
Response: We thank the reviewer for this important comment. We agree that the reduction in infection vesicles observed in the SlPLC2 knockout lines should not be interpreted in isolation as definitive proof of a direct mechanistic role in vesicle biogenesis. Our interpretation is instead based on the results related to early infection phenotypes reported in the manuscript. In addition to the reduced number of infection vesicles, the knockout lines showed delayed induction of the biotrophy-associated effector PiAvrblb2, lower PiEF1-α accumulation, and reduced disease progression overall, including lower biomass, lesion size, and sporulation. Besides SlPLC2-GFP is located surrounding these structures (Figure 6). We therefore consider that the title reflects the overall biological interpretation of the study rather than a claim based on Figure 5C alone.
General comment 2: We appreciate the detail given in the Statistical Analysis section of the Materials and Methods. To further aid interpretation of figures, it might be helpful to mention the GLMMs that were applied to each dataset (and their associated fixed/random factors) in figure legends throughout.
Response: We thank the reviewer for this suggestion. We agree that indicating the statistical model more explicitly in the figure legends would facilitate interpretation and improve transparency regarding the fixed and random effects used in each dataset. We simplified this by providing a general explanation in the Materials and Methods section, while indicating only the specific comparison test applied in each figure.
Abstract: Lines 37–38: Wording of the PiAvrblb2 gene expression finding is not very consistent with the data presented. The sentence implies that expression is generally increased, whereas Figure 5D and the accompanying description on lines 369–373 suggest that the peak in expression is delayed. From the abstract alone, it is unclear how increased expression of an effector gene would lead to impaired establishment of infection.
Response: We appreciate this observation and agree that in the abstract should stay a delay in expression and not an increase.
Figure 2: Panel D and Line 239: Are the three independent experiments pooled onto one graph, as in panel B? If so, were the data normalised to take into account variation in control samples between each replicate? It might also be helpful to colour-code the datapoints according to the replicate they were collected from.
Response: No averaging or pooling of replicate means was performed for this panel. Each point corresponds to an independent biological measurement, and the boxplots summarize the distribution of these individual observations obtained across three independent experiments.
Figure 3: Is there a particular reason for the difference in experimental design between panels A/B/D/E and C/F/G (0 dpi or 6 dpi non-infected leaves chosen for comparison with 6 dpi infected leaves)? This makes comparison between panels a little harder.
Response: We thank the reviewer for raising this point. The difference reflects the nature of the two types of readouts. The transcriptional markers were used to establish the temporal dynamics of SA- and JA-associated responses during infection, which required a time-course analysis, and in figure 3 it is only shown the time points where we detect an increase in expression. In contrast, hormone quantification by LC-MS/MS was designed as an endpoint comparison at a biologically informative stage of infection under the same experimental conditions.
We were surprised that SA was not elevated in WT infected vs non-infected samples in panel C, as panel B suggests SA biosynthesis is upregulated during infection. Although, we appreciate that many factors will influence hormone levels in plants and that quantification is challenging.
Response: We agree that this observation is remarkable. The lack of SA at 6 dpi might reflects additional layers of regulation, including metabolic conversion, conjugation, or spatial heterogeneity of infection across the tissue. Thus, transcriptional activation of SA-related responses does not necessarily translate into increased levels of free SA in whole-tissue measurements at this stage.
Lines 309–315: Supplementary figures are referenced incorrectly.
Response: We thank the reviewer for pointing this out. This was an editorial issue, and the supplementary figure references were corrected and reorganized accordingly.
Line 318: Expression profiles of more than one response gene would help to increase confidence in the conclusion that the mutant plants have altered SA and JA responses.
Response: We appreciate this comment. While we did not rely on multiple response-marker genes of the same category for each pathway, our interpretation was based on multiple independent readouts per hormonal pathway, including a response marker, a biosynthetic gene, and hormone quantification.
Overall, we found this figure difficult to integrate conceptually into the final model of the paper, as it is unclear whether SlPLC2 is directly regulating hormone levels and/or responses. However, we acknowledge the discussion of these results (lines 525–537), and think that the suppression of SA levels/responses in the mutant plants could be emphasised more in the results text. Alternatively, some of this data could be moved to the supplement to avoid confusion, especially the MeJA measurements.
Response: We thank the reviewer for this thoughtful comment. We agree that the hormonal data should not be interpreted as demonstrating that SlPLC2 directly regulates hormone levels. Our intention was instead to show that the loss of SlPLC2 is associated with attenuated SA- and JA-related responses during infection. Importantly, in the revised manuscript we discuss the alternative interpretation that the altered hormonal outputs in the knockout lines may result, at least in part, from reduced pathogen colonization and delayed infection progression, rather than from a primary defect in hormone signaling itself. We believe these framing places the hormonal data more appropriately within the overall model of the paper.
Figure 4: Could a masked image be included to clarify the quantification of callose deposition? To an unfamiliar reader, the individual deposits are not very clear in panel C.
Response: We appreciate this suggestion.
Figure 5: Panel A is useful for interpretation, but it would also be helpful if labels were added to panel B.
Response: The interpretation of the structure shown in panel B is supported by the explanatory scheme in panel A and by the figure legend, but we agree that direct labeling would make the panel more accessible.
Which statistical test was used for analysis in panel C?
Response: We thank the reviewers for pointing this out. It was an oversight not to include the statistical specifications. The analysis was performed using a GLMM with Gaussian distribution, with replicate included as a random effect and line as a fixed effect. We quantified the number of vesicles within a defined area.
Figure 6: Use of a plasma membrane marker would greatly aid visualisation of the infection vesicular membranes. For example, in the first row of panel A, SlPLC2-GFP appears mostly cytoplasmic and its localisation differs from the PI4P and PI(4,5)P2 markers below, making its localisation to the membrane difficult to interpret. This also applies to Figure S7.
Response: We thank the reviewer for this important suggestion. In the revised version, we addressed this point experimentally by incorporating RFP-SYP121/PEN1 as an independent plasma membrane marker. This allowed us to show that SlPLC2-GFP is detected at the plasma membrane and at membranes surrounding infection vesicles, thus strengthening the interpretation that SlPLC2 associates with host-derived membranes at the pathogen interface. In addition, we used the PI4P biosensor 2xCherry-PH(OSBP) to examine the colocalization of SlPLC2 with this phosphoinositide. These experiments further supported the presence of SlPLC2 at infection vesicles and reinforced the conclusion that these structures are enriched in PI4P-containing host membranes.
It would be beneficial to include non-infected controls in the main figure, especially considering lines 481–482 in the discussion. Currently, SlPLC2’s specific ‘recruitment to infection vesicles’ is not obvious from panel A alone, although it suggests the protein is in close proximity to these structures.
Response: We would like to emphasize that our main interest was to highlight the role of SlPLC2 at infection vesicles rather than under control conditions. For this reason, the observations made in the absence of infection were included in the supplementary material, while the main figures focus on the pathogen-induced localization patterns that are central to the study.
Some readers might prefer to see the germinating cyst alongside the infection vesicle to clarify which is which, however only the vesicle is shown in panel A.
Response: We thank the reviewer for this suggestion. We agree that providing a broader morphological context could be useful. However, in this study we chose to focus specifically on infection vesicles, where the most direct and biologically relevant role of PLC2 is likely to occur at the epidermal cell–pathogen interface.
Panels B and C seem a little out of place (especially given the title of this figure legend) and could be moved to an earlier location, for example, when discussing the role of SlPLC2 as a susceptibility factor in Figure 2.
Response: We appreciate the reviewer’s perspective. However, we chose to keep these panels together because they form a coherent section linking subcellular localization with functional relevance. In our view, presenting the localization data together with the susceptibility assay in Nicotiana benthamiana helps maintain the internal logic of the section devoted to how SlPLC2 contributes to early colonization and host susceptibility.
Could the title of the legend be adjusted to ‘surrounding P. infestans infection vesicles’? The current title implies SlPLC2-GFP is located inside the infection structures.
Response: We agree with this point.
Figure 7: A suggestion to improve labelling of infection structures: the ‘zoospore’ could be labelled as a ‘cyst’ as it undergoes encystment before germination.
Response: We thank the reviewer for this accurate terminological correction.
SlPLC2 is proposed to play a role during the early stages of infection, yet its elevated expression is only detected 6 dpi in Figure S1 (when infection vesicles have presumably already formed). Could it be that the activation of existing pools of SlPLC2 is most relevant for establishing infection?
Response: We appreciate this important comment. The absence of strong early induction in whole-leaf samples does not exclude an early role for SlPLC2, because P. infestans infection is highly localized at initial stages and early transcriptional changes can be diluted in whole-tissue samples. In the revised analysis, we addressed this limitation by examining leaf discs collected around the infection site, where SlPLC2 was the only family member significantly induced at early time points, whereas SlPLC1 and SlPLC3 were not. This result, together with the reduction in infection vesicles, the delayed PiAvrblb2 induction, and the localization of SlPLC2 at membranes surrounding infection vesicles at 1 dpi, supports an early role for SlPLC2 during infection establishment.
Discussion: Line 504: This sentence might be incomplete?
Response: In our opinion is Ok the sentence.
Line 659: A suggestion for alternative wording - ‘SlPLC2 could promote the depletion of these lipids from membranes’, as this was not explicitly demonstrated.
Response: We appreciate this comment and agree with the reviewer that direct depletion of PI4P and PI(4,5)P2 by SlPLC2 at infection-associated membranes was not measured experimentally in this study. Our discussion was intended to present a mechanistic model based on three elements: the known biochemical activity of PI-PLCs, the presence of PI4P and PI(4,5)P2 at these membranes, and the localization of SlPLC2 at the same interface. We therefore agree that these statements should be interpreted as a proposed model rather than as direct experimental demonstration of local phosphoinositide depletion.
Competing interests
The author of this comment declares that they have no competing interests.