PREreview of Exploring fern pathosystems and immune receptors to bridge gaps in plant immunity

Review of “Exploring fern pathosystems and immune receptors to bridge gaps in plant immunity”

The manuscript presents a novel investigation into disease resistance in ferns, combining pathogen inoculation experiments across multiple fern species with genome mining for immune receptors, notably RLKs, RLPs, and NLRs. The work addresses a major evolutionary gap in our understanding of plant immunity. However, we suggest improvements in clarity, figure presentation, methodological detail, and logical structure to enhance the message of the paper. More specifically: refining phylogenetic interpretations and clarifying novel findings will substantially improve the paper’s message.

1. Summary

 -              The statement in line 38 “They also encode diverse NLRs” is vague. The authors should specify the nature of this diversity.

 -              The phrase in line 40 “These findings provide insights into disease resistance evolution” should be made more specific, especially since RLKs/RLPs were the primary focus of the comparative analysis. In addition, what are the “promising perspectives for crop protection strategies”?

2. Materials and Methods

 -              Include references for software/tools used such as ImageJ and AlphaFold.

-              Clarify the criteria used to retain the 3,775 NLRs from the initial set of 3,637 proteins. What filters or thresholds were applied?

-              Confirm whether the phylogenetic trees were built using NB-ARC domains. This information is critical for interpreting the NLR classification.

-              Specify the number of infection trials and biological replicates performed per species/pathogen combination.

3. Figures and Data Presentation

 Figure 1:

-              Currently difficult to interpret due to small size and missing axis labels.

-              Figure 1A could be moved to the Supplementary.

-              Heading (of the figure and paragraph) should reflect that only some ferns are susceptible to several pathogens.

-              Start the Results with more compelling data rather than noting microbes that caused no symptoms.

-              Statistical tests appear to lack multiple comparison correction. Kruskal-Wallis followed by Dunn's test or Benjamini-Hochberg correction is recommended.

-              A time-course of infection symptoms would enhance the understanding of phenotypic variability.

Figure 2:

-              Heading should be more specific: "Pteris vittata gametophytes and sporophytes respond differently to some pathogens."

-              The observed generation-specific responses could be a major result. Is there gene expression data to support these phenotypic differences?

-              Some of this data appears to overlap with Figure 1 and may not need separate presentation.

-              Not all pathogens are discussed. e.g., Cubensis is omitted.

Figure 3:

 -              Heading (of the Figure and paragraph) should be qualified with "some pathogens".

-              Include days post-infection (dpi) in images for clarity.

Figure 4:

 -              The transition between RLK/RLP and NLR discussion is abrupt; a better bridge would be nice.

-              Clarify whether the data includes splicing variants and how redundancy was handled.

-              Present RLK family ratios as relative percentages.

-              Arrange species in phylogenetic order.

Figure 5:

 -              Clarify the claim that Cluster III NLRs are related to CC-NLRs from Camellia sinensis; it is unclear where this is shown. Explain why this relationship implies functional diversification.

-              Specify whether RPW8-NLRs in ferns are NRG1 or ADR1 types. This would be the most interesting point regarding RPW8-NLRs

-              EDVID, G10, Cbl-N should be introduced before.

-              DisN-NLRs also seem appear in non-fern lineages.

-              Use species-based color coding to clarify phylogenetic subclades.

-              Explain the identity of the unlabeled "black clade" near TIR-NLRs.

-              Consider comparing NLR with RLK/RLP numbers across species.

4. Misleading Terminology

 The label "Fern genomes encode a novel type of NLRs: disN-NLRs" may be premature. Similar N-terminal disorder may exist in other NLR subgroups. A comparison with other known NLR clades is definitely needed for this claim.

 5. Discussion and Interpretation

-              Figure 7's contribution to the manuscript is unclear. This should be in the results section if retained in the manuscript at all. Statements like “Two well-characterised CC-NLRs were not detected” seem a bit random. The way the data is presented is also not very informative as a potential phylogenomic context is missing (e.g. in which ferns is an ortholog detected).

-              Clarify whether the authors meant “genera” rather than “species” in the sentence about filamentous pathogens.

Minor Points

 -              Ensure consistent use of past tense in the results section. In addition, the last paragraph of the Introduction uses inconsistent tenses.

-              Improve clarity of statistical reporting throughout.

-              Consider editing for conciseness and removing redundant text in the Introduction and Discussion. E.g. The Polypodiales order is mentioned twice in the Introduction; one mention should be removed to avoid redundancy.

-              The general phylogenetic context in the introduction needs clarification for non-specialist readers. A simplified phylogenetic tree of fern orders (including divergence times from seed plants) would add more context.

-              The introduction to NLR subclasses is incomplete. G10 NLRs, which are significant in certain plant lineages, are not mentioned.

-              Discussion of lineage-specific NLR types in bryophytes (Kin-NLRs and Hyd-NLRs) would benefit from a citation to Carella et al., whose work has been central in this area.

Competing interests

The authors declare that they have no competing interests.