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Summary:

During the early stages of plant colonisation, the oomycete Phytophthora infestans forms expanded intracellular structures termed infection vesicles, from which invasive hyphae emerge. Surrounding these structures and the haustoria that develop in subsequently colonised cells are specialised plant-derived membranes that separate the plant cytoplasm from the pathogen cell wall. In other contexts, within plant membranes, the enzyme phosphoinositide-specific phospholipase C (PLC) is known to release lipid-derived signalling molecules via hydrolysis of phosphoinositides, PI4P and PI(4,5)P₂.

Perk et al. (2025) show that tomato (Solanum lycopersicum) SlPLC2 is a susceptibility factor in the context of Phytophthora infestans infection. SlPLC2 loss-of-function mutants generated using CRISPR-Cas9 were more resistant to infection, as reflected by their reduced disease symptoms and associated pathogen biomass. During infection, SlPLC2 mutants also displayed altered salicylic acid and jasmonic acid responses at the level of transcription, reduced hydrogen peroxide accumulation and callose deposition, and supported the formation of fewer P. infestans-derived infection vesicles. The authors report SlPLC2 localisation at the plasma membrane, including that surrounding infection vesicles, supporting a model where localised lipid modification could contribute to pathogen establishment and the release of second messengers to perturb host immune signalling.

General comments:

The manuscript is generally well-written and a variety of experiments have been presented to demonstrate the role of SlPLC2 as a susceptibility factor during P. infestans infection of tomato, and later Nicotiana benthamiana. The figures are well-presented and the colour-coding of boxplots according to genotype is helpful for interpretation throughout.

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)?

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.

Section- and figure-specific comments:

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.

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.

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.

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.

Lines 309-315: Supplementary figures are referenced incorrectly.

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.

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.

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.

Figure 5:

Panel A is useful for interpretation, but it would also be helpful if labels were added to panel B.

Which statistical test was used for analysis in panel C?

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.

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.

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.

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.

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.

Figure 7:

A suggestion to improve labelling of infection structures: the ‘zoospore’ could be labelled as a ‘cyst’ as it undergoes encystment before germination.

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?

Discussion:

Line 504: This sentence might be incomplete?

Line 659: A suggestion for alternative wording - ‘SlPLC2 could promote the depletion of these lipids from membranes’, as this was not explicitly demonstrated.

Overall, we thought the discussion was thorough and provided some interesting speculation regarding the mechanisms underlying SlPLC2 function during infection.

Competing interests

The authors declare that they have no competing interests.

Use of Artificial Intelligence (AI)

The authors declare that they did not use generative AI to come up with new ideas for their review.