PGENETICS-D-24-01497

Systematic exploration of domain assortments in NOD-like receptors uncovers two types of NACHT domains in Sordariales fungi

PLOS Genetics

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PLOS Genetics

Geraldine Butler

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PLOS Genetics

Aimée Dudley

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PLOS Genetics

Anne Goriely

Editor-in-Chief

PLOS Genetics

Journal Requirements:

1) Please ensure that the CRediT author contributions listed for every co-author are completed accurately and in full.

At this stage, the following Authors/Authors require contributions: Lucas Bonometti, Florian Charriat, Noah Hensen, Silvia Miñana-Posada, Hanna Johannesson, and Pierre Gladieux. Please ensure that the full contributions of each author are acknowledged in the "Add/Edit/Remove Authors" section of our submission form.

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5) Thank you for stating that “Genome assemblies, gene models, HMM profiles, orthogroups and NLR sequences generated in this study are available from Zenodo (doi: 10.5281/zenodo.14204294).” We noted that  the data files are restricted to users with access. Please assure that the datasets are publicly accessible.

Reviewers' comments:

Reviewer's Responses to Questions

Reviewer #1: In this study, the authors performed a systematic exploration of NLR diversity in the Sordariales order of Ascomycota fungi. They improved the annotation of the NB domain, and found a high variation in the number of NLR genes among species. They found NLR were organized in clusters based on their definition of clusters in these fungi.

Overall, I found this study highly novel in term of approach and methodology that should be of interest for scientific community interested in the evolution of immune systems and fungi. I haven't detected major methodological flows. The authors tackle putative biais due to differences in genome quality in their dataset and use both automatic and trained annotation pipelines accompanied with manual curation steps.

I have only a few minor remarks:

-L110: it is unclear to me what means "the determinants of NLR"

-the author claims the Sordariales are good systems to study the link between ecology and genome evolution (L128) but conclude in the discussion that there is not enough knowledge on Sordariales lifestyle (L507); This is contradictory and I wonder if the authors could have tried to correlate substrate of origin of strains and their NLR repertoire for examples

-the author conclude the fungi share many similarity in NRL repertoire to animals (L603), but very little information on NLR repertoire in animals is given ; most comparison are done with plants, in particular for clusters. Is there a specific reason for this?

-L145 : a N50 of 14.1 kb seems very low and it seems unlikely that many genes can be annotated in this genome

-L393 and beyond: I failed understanding the main results of this analysis ; L420 the author claims "only a subset of possible combinations was observed in Sordariales" and conclude L426 that the "realized proportion of possible domain assortments were only marginally lower in Sordariales than in Dikarya"; the two sentences seems contradictory to me

Reviewer #2: In their manuscript, ‘Systemic exploration of domain assortments of NOD-like receptors uncovers two type of NACHT domains in Sordariales fungi’, Bonometti et al. characterize in silico the NLR gene repertoire, genomic organization, and diversity in the fungal order of Sordariales. They authors identify and analyze 4613 NLR genes in 82 Sordariales genomes, uncover and report several intriguing findings about fungal NLRs in Sordariales, including the presence of NLR gene clusters, and the presence of two main types of NACHT domains in fungi. These findings, as well as the reported association of number of non-canonical C-terminal domains, which are hypothesized to represent ‘integrated NLR decoys’ in Sordariales, represent exciting contributions to NLR biology in general, and its exploration in fungi in particular. In addition, the presented work is methodologically sound and the manuscript mostly well-written. Below, I have some comments and suggestions that would, in my view, ameliorate the manuscript.

1) Considering the multiple discoveries and the broad scope of the analyses, the title appears to be somehow limiting into presenting the overall impact of the paper. An alternative could perhaps appear to better reflect the whole and also more appealing for a boarder readership.

2) In this context, I think that two other findings ‘NLR clustering’ and ‘Accessory C-ter NLR domains’ could benefit from additional illustrations. For example, cartoons showing some examples of such clusters, NLRs as illustrations. Plus supplementary Tables where one could easily find each such gene from these findings, with species names, and gene ID.

3) Regarding the NLR gene clusters in fungi and their definition/discovery. In my view, the text could be slightly clearer regarding the explanations of their identification (the phrase lines 258-261). Intuitively, one would think that clusters inside the same gene family would depend on the number of gene members and the genome size and gene density. To establish if clustering of gene members exist, one would need to determine if associations between members is more frequently found than what one would expect purely by chance. The description doesn’t provide enough context in my view to make this clear. Are the 1000 randomly-selected gene sets members of similarly sized (as the NLRs) gene families?

What could one expect to find in fungi, if we simply take the plant definition and ‘scale it down’ to account for the average differences in genome sizes, and distance between genes, and average size of the number of genes inside the plant and fungal NLRomes? Wouldn’t another option be to take such parameters from plants and attempt to apply the definition of NLR clusters from there, adapting it for fungi? How would such approach differ from what the authors propose?

4) It feels like the section regarding the identified orthogroups could be presented with a little more details. What are the NLR architectures in the largest orthogroups? Which species are concerned? One could hypothesize that largest orthogroups play important roles and potentially have conserved function in all Sordariales species, so it would be a valuable information for the readers. What percentage of NLRs in Sordariales do not fall in any of the orthogroups? In general, adding some % in this section would be beneficial for the article, especially for future experimental analysis of some of these NLRs.

Some additional minor comments below:

5) The definition of ‘head-to-head NLRs’ and its implication, I think, is only provided in the Discussion section. That could be provided earlier, when the term is initially mentioned.

6) The very first line of the Abstract, the NLRs in fungi are defined as ‘analogs’. Considering the presence of NLRs in bacteria, and their implication in immunity as anti-phage defense systems, and that the fungal NLRs paly similar role in immune defense in the context of allorecognition, it would seem justified to consider NLRs evolutionary related, originating from an ancient NB (NOD) domain, and thus distant homologs.

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Reviewer #1: Yes

Reviewer #2: None

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Reviewer #1: No

Reviewer #2: No

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Dear Dr Gladieux,

We are pleased to inform you that your manuscript entitled "Genomic organization, domain assortments, and nucleotide-binding domain diversity of NLR proteins in Sordariales fungi" has been editorially accepted for publication in PLOS Genetics. Congratulations!

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Benjamin Schwessinger

Academic Editor

PLOS Genetics

Geraldine Butler

Section Editor

PLOS Genetics

Aimée Dudley

Editor-in-Chief

PLOS Genetics

Anne Goriely

Editor-in-Chief

PLOS Genetics

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Comments from the reviewers (if applicable):

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