PPATHOGENS-D-26-00373

Chemotaxis to Plant Defense Compounds in Phytopathogens

PLOS Pathogens

Dear Dr. Krell,

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Kind regards,

Nian Wang

Academic Editor

PLOS Pathogens

Shou-Wei Ding

Section Editor

PLOS Pathogens

Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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Reviewers' Comments:

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: The manuscript by Genova et al. investigates the chemotaxis response of the plant pathogen Pectobacterium atrosepticum. The authors identify important plant defense compounds as chemoreceptor ligands for three chemoreceptors and show a role of PacG in plant virulence. This is a well-written manuscript describing carefully composed and conducted experiments. The identification of specific chemoattractants and their corresponding chemoreceptors will contribute to a better understanding of plant pathogen/host interaction, which is an understudied area of research.

Reviewer #2: This is a revised mansucript for which I did not serve as an original reviewer. The authors seem to have addressed the reviewers comments. I have only one conceptual questions and additional editorial comments.

Reviewer #3: The paper by Genova et al. present the findings of studies on three plant pathogen chemoreceptors, terms PacG, PacH, PacI. The authors find the ligands of these chemoreceptors, and determine that they function in chemotaxis, and also are important for plant infection. The manuscript is well written and clear. Overall the work is compelling, impactful, and interesting. I have only a few comments. My only suggestions are slight to change some wording in the introduction about the idea that because chemoreceptors are found in plant pathogen, they must sense plant compounds. While a decent idea, it’s hypothetical so please tone down:

• Line 58 and 72 are too strong. E.g. at 58, change to “suggesting that many of these receptors might sense…”

Reviewer #4: In this manuscript, the authors investigated three chemoreceptors in Pectobacterium. They identified the ligands. They also conducted mutagenesis study. Overall, this study contains some interesting information. There are some major issues that must be addressed though.

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Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: None.

Reviewer #2: Authors provide MIC for eahc of the predicted LBD ligands and also provide semi-quantitative capillary assays for chemotaxis responses usign arange of concentations. Despite the compounds being toxic at high concentrations, the positive chemotaxis responses suggest these are attractants - this makes sense sine they are chemotactically stimulating cells behavioural responses at concentrations far below the MIC for all compounds tested except for vanillin (Fig. S7). For vanillin, a positive chemotaxis response is detected for concentrations >MIC (6mM per Table 1 for PacH and Fig S7). The observation is not addressed despite its incongruence. While it makes senses that a phytopathogen could detect a potentially toxic compound as an attractant at concentrations far below the MIC, for this attractant, it is not clear what would be the advantage to sense a chemattractant at a concentration that can kill the cells. Shouldn't the results suggest that chemotaxis attraction to vanillin/LBD binding to vanillin may not be physiologically relevant? Other issues?

Reviewer #3: None, it's good

Reviewer #4: 1. Statistical analysis was missing for data related to Fig. 4 and Fig. 5B

2. Line 226, the authors stated: To monitor chemotaxis of SCRI1043 to the ligands identified above, we conducted quantitative capillary chemotaxis assays. Initial experiments conducted in standard conditions, involving bacterial cell culture in minimal medium supplemented with glucose as sole carbon source, did not show any taxis to vanillin, salicylate or benzoate (data not shown). Chemotaxis can sometimes be induced by the presence of the cognate ligands in the culture medium [57], so we next monitored chemotaxis to vanillin, salicylate and benzoate in cultures grown in minimal medium supplemented with vanillin or benzoate.

Based on the aforementioned writing, the authors need to clearly state what was included in Fig. 4 figure legends for each experiment.

3. For the virulence assays, please include pictures of infected roots and leaves in supplementary figures.

4. It is required to conduct complementation assay for pacG mutant and pacHIG mutant.

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Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: None.

Reviewer #2: 1. Fig. 1 legend has not been revised from the previous version and include innformation that have been moved to the SI (e.g. reference to red brackets and a panel B)

2. Fig S7- please add labels A, B and C to the individal histogram graphs since the figure caption refers to them.

Reviewer #3: See above

Reviewer #4: 1.Fig. 1. It listed A), but there is no A) in the figure. This figure quality needs to improve.

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Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

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Dear Dr. Krell,

We are pleased to inform you that your manuscript 'Chemotaxis to Plant Defense Compounds in Phytopathogens' has been provisionally accepted for publication in PLOS Pathogens.

Please correct the following in your proof stage:

Please provide the statistical method for Fig. 5B legend.

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Nian Wang

Academic Editor

PLOS Pathogens

Shou-Wei Ding

Section Editor

PLOS Pathogens

Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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Reviewer Comments (if any, and for reference):