Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The Manuscript ID: biomolecules-3873443 addresses surfactin structural variants that differentially modulate plant immune responses. It deals with the mechanism of the related small molecule signaling substances that regulate the grey mold pathogen (Botrytis cinerea) behavior, symptoms, and development as important tool for developing environmentally friendly biological control agents. This is especially important as the relevant and searched cyclic lipopeptides (CLPs) is generated by beneficial rhizobacteria such as Bacillus and Pseudomonas species. The study tried to unravel the complexities involved in the molecular mechanisms underlying the immune-modulatory effects and the role of CLP structural diversity. It could demonstrate that specific structural features of surfactin-type CLPs critically influence their ability to trigger early immune responses in plants, including reactive oxygen species (ROS) bursts, nitric oxide (NO) production, calcium fluxes, and ultimately systemic resistance. It indicated that contrasting immune effects of CLPs may stem from the ecological lifestyles of their microbial producers, shedding light on the evolutionary basis of plant-microbe interactions. While root treatment with surfactin (Srf) and lichenysin significantly reduced leaf infection by the grey mold pathogen, no disease reduction compared to controls could be observed with pumilacidin. There is thus a strong correlation between the potential of a definite CLP to trigger early immune events and its efficiency as elicitor of systemic resistance. They also tested a myb72-2 knockout mutant and data showed that the ISR elicitor potential of the CLP is fully conserved in this mutant compared to the wild-type. It indicates that activation of MYB72 by Srf does not lead to resistance to B. cinerea. However, it might be involved in defense against other plant pathogens. Eventually, the obtained results underscore the importance of CLP structural variation in shaping plant defense responses and highlight the potential for structure-informed design of next-generation biosourced small molecules with broad-spectrum efficacy as plant protectants.

The subject is worth publication and the authors did a good job. Yet, further insights might improve the study and specific improvements should be considered:

• The authors are full aware of the fact that the exact pathway of Induced Systemic Resistance (ISR) remains to be determined, though the ability of Srf to stimulate reactive nitrogen species (RNS) can act in concert with ROS as redox regulators during plant immune responses and elevated nitric oxide (NO) levels typically correlate with enhanced ROS production. So, their synergistic interaction can boost defense responses, where ROS promotes NO generation, while NO modulates antioxidant defenses to prevent oxidative damage and sustain cellular redox homeostasis. Their study was based on ISR that was presumably induced by the CLPs generated by beneficial rhizobacteria against Botrytis cinerea on the tested Arabidopsis So, the introduction should have included uses of jasmonic acid (JA) and ethylene (ET) signaling, that enhances physical or chemical barriers, but without pathogenesis-related (PR) protein accumulation for such ISR array.
• In connection with the aforementioned point, in such cases they would discuss also in “Results and Discussion” section the assumption that their studied ISR basically encompasses ROS to induce resistance against the pests/pathogens. It is the oxidation burst by which plants generally reacts early to biotic stresses. Hence, it is better to mention also the complementary part against such pests/pathogens; that is, Systemic Acquired Resistance (SAR) usually based on salicylic acid (SA)-responsive genes. It is important to discuss that both ISR and SAR signaling can commonly crosstalk either antagonistically or synergistically, based on their combinations in the plant. Interestingly, the negative interaction between SAR and ISR signaling can be employed by pathogens to boost plant susceptibility.
• Scientific names when reported for the first time in the text should be written in full with Authority and systematics; e.g. Botrytis cinerea (Helotiales: Sclerotiniaceae)
• Please, do not start a sentence by a number; that is “Two-week-old Arabidopsis seedling roots (from 27 mg seeds) were collected….” instead of “2-week-old Arabidopsis seedling roots (from 27 mg seeds) were collected….”
• It is better to write the used Arabidopsis species (lines 493 myb 72-2, pMYB72:GFP-GUS and pBGLU42:GFP-GUS). Now, there are 12 species of Arabidopsis that are formally accepted.

Therefore, I would suggest accepting it after minor revision.

 

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Ning Ding, Hansong Dong, Romain Thomas, Guillaume Gilliard, Jelena Pršić, and Marc Ongena

Surfactin Structural Variants Differentially Modulate Plant Immune Responses

The work is devoted to the influence of cyclic lipopeptides synthesized by the bacteria Bacillus licheniformis and B. pumilus on the formation of reactive oxygen and nitrogen species, on plant immunity and systemic resistance to pathogens. A large volume of experiments has been carried out, the results are presented compactly, the illustrations carry a lot of information. The work will be of interest to many specialists.

Several comments on the text of the article.

1) It is useful to add the species names of the microorganisms Bacillus licheniformis and B. pumilus, reactive nitrogen species, and reactive oxygen species to the keywords.

2) line 254 (Fig. 1) “Blue or green color highlights the difference of Liche or Pumi, compared to Srf in the cyclic peptide” - blue and especially green letters on the diagram are poorly visible, the font color should be changed.

Author Response

Please see attached file

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This study investigates the impact of minor modifications in the molecular structure of cyclic lipopeptides produced by bacteria from the genus Bacillus, on their capacity to stimulate plant immune responses. Specifically, we compare the immunostimulatory activity of canonical surfactin with two naturally occurring structural variants, lichenisin and pumilacidin. The work combines biochemical analysis, cell biology techniques to measure early immune responses, and phytopathological testing to establish the structure-function relationships of these biological control agents. Overall, the work is well done and the manuscript is written clearly and logically. However, while reading it, I had a few questions.

1.    Why were pure standards of these specific compounds not used for quantification of Liche and Pumi, or a calibration curve not constructed for each of them? How can the authors ensure that the comparison of potency is performed at equal molar concentrations? The mass spectrometer with ionization detection used in the work has a different response factor for different compounds. Even minor changes in the structure of the molecule (such as replacing Glu with Gln) can change its ability to ionize. Therefore, the same concentrations of Srf and Liche may give different signal intensities in the mass spectrum (and vice versa). This could lead to the possibility that the differences in the effects of these lipopeptides on plants are not due to structural features, but rather due to different actual concentrations used in the experiment. Lines 291-294 -- Did the authors even conduct a qualitative experiment, such as titrating Pumi (1, 10, 50 μM) to see if it was active at higher, perhaps realistic, concentrations? 

2.    You write the composition of surfactins homologues (Lines 101-102). What are the homologous compositions (length and branching of fatty acid chains) of the Liche and Pumi preparations? Could you indicate these homologues in the Materials and Methods section?

3.    The authors suggest that the amino acid substitution, and therefore the high hydrophobicity, makes Pumi inactive. Is it possible that high hydrophobicity has led to aggregating in an aqueous solution and being unavailable to plants in the same form as Srf/Liche; Being insoluble at a concentration of 10 µM. Have critical micellar concentrations and aggregation states been controlled for all three preparations in working solutions?

4.    Since the authors' hypothesis posits that the immune activation mechanism is determined by the detergent properties of the lipopeptides (lines 87-88; 259-262; 445-448), a key question arises regarding Pumi's lack of activity. Could it be that Pumi is not a "poor elicitor", but rather a "too effective detergent"? Its higher hydrophobicity might cause such strong or irreversible membrane damage, leading to rapid loss of cell viability and necrosis, rather than a controlled immune response. Could this cytotoxic effect mask its potential eliciting activity in the assays used? Have the authors assessed the viability of the protoplasts after treatment with different CLPs?

Figure 2 needs a better resolution.
Figure 5: Is it possible to enlarge the photographs of the leaves? The changes in the leaves caused by the treatments are not clearly visible.

Author Response

Please see attached file

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

In principle, I am pleased with the responses to my questions. However, I would like to draw your attention to the fact that the authors have not yet corrected Figures 2 and 5. If the authors are satisfied with the diagrams and photos, which are of such low quality, then I won't object.