An Ammonium Transporter Gene Contributes to the Aggressiveness of the Dutch Elm Disease Pathogen Ophiostoma novo-ulmi

Round 1

Reviewer 1 Report

This study reports the characterization of the genomic region co-97 segregating with the pat1 locus and presents experimental evidence that candidate se-98 quences for pat1 include a gene encoding a high-affinity ammonium transporter. They investigated amtA contributing to Y-M transition and aggressiveness in O. novo-ulmi.

The following suggestions are provided to help the authors further improve this study.

1. The image clarity is insufficient, for example, in Figure 2.
2. In Figure 3A and 3B, the result of gene relative expression is not correctty presented. 
3. In Figure 4, it's better to present the growth phenotypes of the wild-type and CRISPR-Cas9 mutant.
4. In Figure 5, it's better to present the growth phenotypes of the wild-type and CRISPR-Cas9 mutant on medium supplied with various sources of carbon and nitrogen.
5. In Figure 6, it's better to present the pathogenicity phenotypes of the wild-type and CRISPR-Cas9 mutant.

Author Response

The Figures in this manuscript requires major revisions. The following suggestions are provided to help the authors further improve this study.

1. The image clarity is insufficient, for example, in Figure 2.

Following comments from Reviewers 1 and 4, the original Figure 2 has now been split in two: the new Figure 2 shows the alignment of amt-encoding genes, whereas the new Figure 3 shows results of phylogenetic (Fig. 3A) and TMHMM (Fig. 3B) analyses. This provided more room for a proper display of the phylogenetic tree (Fig. 3A) and also resulted in replacing the long caption for the original Fig. 2 with shorter captions for new Figures 2 (L337-345) and 3 (L365-375).

2. In Figure 3A and 3B, the result of gene relative expression is not correctly presented. 

Thank you for picking up this mistake which escaped our attention in our original submission. We have corrected the labeling of the Y axis in the new Figures 4A and 4B (“2 ^-DDCT” replaced with “Normalized number of molecules”). We also modified the figure caption accordingly: “Ct values were converted into corrected molecule numbers and normalized with the actin control gene” (L391-393 and 395-396).

3. In Figure 4, it's better to present the growth phenotypes of the wild-type and CRISPR-Cas9 mutant.
4. In Figure 5, it's better to present the growth phenotypes of the wild-type and CRISPR-Cas9 mutant on medium supplied with various sources of carbon and nitrogen.
5. In Figure 6, it's better to present the pathogenicity phenotypes of the wild-type and CRISPR-Cas9 mutant.

 We are not sure what is meant by the reviewer regarding these three figures, as they illustrate phenotypic differences between wild-type strain H327 and CRISPR-Cas9 mutants derived from it. Nevertheless, we made minor corrections to the figure captions (L442-448 and L486-494) in order to correct mistakes in our original submission and address comments made by other reviewers. We hope the new captions facilitate interpretation of results shown in these figures.

Reviewer 2 Report

Authors found that an ammonium transporter gene, identified through chromosomal analysis, contributes to the aggressiveness of the fungus Ophiostoma novo-ulmi, which causes Dutch elm disease. This article is within the scope of the “Journal of Fungi". The article's title is appropriate to the material presented. The research methods and approaches used are consistent with the article's objectives. The experiments and data adequately address the problem. The discussion and interpretation of the data are consistent with the presented material. The article is well written. It is difficult to find any critical comments or inaccuracies. Thus, the paper merits for publication in “Journal of Fungi".

Point 1: The abstract should be a total of about 200 words maximum.

Point 2: In the Introduction section, authors should more clearly indicate the novelty of their work.

Point 3: Give a name for table S4. Also give a name for the first column.

Comments for author File: Comments.pdf

Author Response

Some minor revisions are needed before acceptance. Precisely:

Point 1: The abstract should be a total of about 200 words maximum.  

We reduced the length of the Abstract from 249 words to 202 words (L17-33).

Point 2: In the Introduction section, authors should more clearly indicate the novelty of their work.

 We added the following statement at the end of the Introduction (L92-94): “Although amt genes have been implicated in aggressiveness for a number of plant pathogenic fungi [31–33], they have not been studied in DED fungi.”

Point 3: Give a name for table S4. Also give a name for the first column. 

Based on the suggestion from Reviewer 3, we moved the original Table S4 to the body of the manuscript (new Table 1, L437-440). We added a title for Table 1 and a label for the first column. We added a new Table S4 entitled “Expression in vitro and in planta of Ophiostoma novo-ulmi H327 amt genes measured in previous transcriptomic analyses”.

Reviewer 3 Report

The article “An ammonium transporter gene identified by chromosome walking contributes to the aggressiveness of the Dutch elm disease fungus Ophiostoma novo-ulmi ” is devoted to the important and acute theme of the role of ammonium uptake in aggressiveness of plant pathogens.Authors used modern methods, including CRISPR-Cas  Authors provide and analyze a lot of information on this theme, but minor edition should be made.

Title: In my opinion, it should be more concretize, such as, for example “An ammonium transporter gene ONUg0282 contributes to the aggressiveness of the new dangerous Dutch elm disease fungus Ophiostoma novo-ulmi”

Lines 97-100 It is important to clearly articulate the purpose of the investigation rather than talk about the results obtained.

I think authors should set up Table S4 in the manuscript and replace Figure 6B with Figure S3 (fig 6A may be added to Fig S3 in the text of the manuscript).

Authors must specify full systematic names of species then they are mentioned for the first time (for example A. brasilense Tarrand, Krieg & Döbereiner, 1978)

Why did Ulmus americana x U. parvifolia hybrid clone 2213 was used for pathogenicity tests? Why weren’t elm leaves tested?

Technical comments:

The text contains different fonts, please, correct.

Line 152 - italic

Lines 186-187 - is it paragraph title?

In some places the country and city of manufacturers aren’t described (Qiagen for example).

Line 267 - reference

Fig. 6 - please, check names of pathogens

Author Response

Reviewer 3

Title: In my opinion, it should be more concretize, such as, for example “An ammonium transporter gene ONUg0282 contributes to the aggressiveness of the new dangerous Dutch elm disease fungus Ophiostoma novo-ulmi”

Thank you for the suggestion. We shortened the title which now reads “An ammonium transporter gene contributes to the aggressiveness of the Dutch elm disease pathogen Ophiostoma novo-ulmi”.

Lines 97-100 It is important to clearly articulate the purpose of the investigation rather than talk about the results obtained.

As indicated in our answer to Point 2 raised by Reviewer 2, we added a short statement about the link between amt genes and aggressiveness in plant pathogens, and the fact that this topic has not been investigated in the Dutch elm disease fungi (L92-94).

I think authors should set up Table S4 in the manuscript and replace Figure 6B with Figure S3 (fig 6A may be added to Fig S3 in the text of the manuscript).

 We followed the reviewer’s suggestion. The original Table S4 is now Table 1 (L437-440), whereas the new Figure 7 includes results from all inoculation trials with CRISPR-Cas9 mutants. A new caption was written for Figure7 (L486-494).

Authors must specify full systematic names of species then they are mentioned for the first time (for example A. brasilense Tarrand, Krieg & Döbereiner, 1978)  

We checked Journal of Fungi guidelines, as well as several recently published articles and did not see evidence for this, although we are aware this is the norm in several other publications. We will be happy to provide full systematic names if the Journal of Fungi Editor confirms this is required.

Why did Ulmus americana x U. parvifolia hybrid clone 2213 was used for pathogenicity tests? Why weren’t elm leaves tested? 

Ulmus americana is highly susceptible to Dutch elm disease, whereas the U. americana x U. parvifolia hybrid clone 2213 is moderately resistant (L260-261). Because hybrid clone 2213 was no longer available, we conducted subsequent experiments on U. americana saplings. 

The standard procedure to test isolates of DED fungi is to inoculate the main stem of elm saplings (in the case of large trees, branch inoculations can be conducted). Wilting of leaves is due to disfunction of vessel elements in the xylem due to infection of DED fungi.

Detailed comments

Technical comments:

The text contains different fonts, please, correct.  Done.

Line 152 – italic.  This has been corrected (L147).

Lines 186-187 - is it paragraph title?  

It is not but we agree this may confuse readers. We amalgamated this portion of text with the next paragraph (L181-198).

In some places the country and city of manufacturers aren’t described (Qiagen for example).  Line 267 – Check reference.   Fig. 6 - please, check names of pathogens. 

Thank you for pointing out these omissions or inconsistencies. Corrections have been made for the three issues raised by the reviewer.

Reviewer 4 Report

1.Evidence for narrowing the pat1 region is insufficient; the justification for selecting only ONUg0282 (amtA) is incomplete

Chromosome walking led to a ~154 kb interval containing 58 predicted genes, yet the manuscript focuses entirely on ONUg0282 (amtA).Neither the main text nor the supplementary materials provide:a systematic assessment of all candidate genes,exclusion criteria for the other genes,nor additional fine-mapping or co-segregation evidence.This makes the selection of amtA appear premature and under-justified.

Required revision:

Provide either (i) a rationale for dismissing the remaining pat1-interval genes (annotation, expression, predicted function), or (ii) clarify that amtA is one strong candidate among many rather than the sole prioritized one.

2.The authors acknowledge in the Discussion that “available data do not permit to conclude that ONUg0282 is the pat1 locus.”Nevertheless, the narrative and Title imply strong association.

Required revision:

Explicitly revise claims throughout the manuscript to avoid suggesting that amtA is pat1. It should be framed as “a gene within the pat1-linked region contributing to aggressiveness.”

3.ΔamtA mutants show enhanced hyphal growth under starvation, but the manuscript attributes this to possible MAPK/PKA dysregulation without providing molecular evidence.Supplementary data do not include:Transcriptomics,Pathway activation assays,Quantitative Y–M switching metrics

Required revision:

Rephrase the signaling-related interpretation to hypothesized mechanisms unless direct evidence is added.

4.Supplementary Table S3 reports WT in planta expression of amt genes, but:ΔamtA in planta expression is not measured,Compensatory expression of amtB–amtE in the knockout lines is not assessed.This is critical for interpreting the functional significance of amtA during host colonization.

Required revision:

Either provide qPCR data for mutant vs. WT during infection or explicitly acknowledge the limitation and adjust conclusions accordingly.

5.The manuscript reports inconsistent results between apple fruit and elm seedlings, and across inoculum dosages. However:Statistical analyses do not include mixed-effects or GLM models,Supplementary figures show variability that is not formally tested,Host × dose interaction remains unquantified

Required revision:

Include appropriate statistical modeling or clarify limitations in interpreting dose responses.

6.The Title refers to “chromosome 2 walking”, while figures and text place the interval on chromosome I (reference genome).This inconsistency must be corrected or explicitly explained.

1.Introduction

Consider expanding the background on fungal ammonium transceptors and nitrogen availability in xylem environments to contextualize the role of Amt/Mep proteins during infection.

2.Methods

Chromosome walking would benefit from a schematic summarizing contigs, gaps, and alignment to the reference genome.

CRISPR section should summarize sgRNA target sites in the main text (currently dispersed in Supplementary).

Provide a concise table distinguishing ΔamtA-3 vs ΔamtA-35 alleles and predicted structural consequences.

Microscopy analysis should include quantitative metrics (hyphal growth rate, branch density), not only qualitative descriptions.

3.Results

Figure 4 colony zones should be quantified with statistics.

Supplemental phenotyping (e.g., methylammonium sensitivity) should be clearly referenced in the main text.

Divergent results between apple and elm hosts require a more explicit discussion of tissue nitrogen context.

4.Discussion

Reduce over-interpretation related to signaling pathways unless additional data are added.

Expand discussion about functional redundancy among the five Amt/Mep genes, especially since amtB shows high in planta expression in Supplementary Table S3.

5.Figures / Tables

Figure 2B phylogeny contains text too small to be readable.

Figure 5 panels are dense; consider splitting into two figures.

Supplementary tables should explicitly link gene IDs to locus tags in the genome (to avoid confusion between ONUg IDs and UniProt/PHI-base annotations).

6.Language

Several long paragraphs in the Results and Discussion should be split for clarity.

Opening sentences of major sections should better summarize key findings to improve narrative flow.

Author Response

Major comments

1.Evidence for narrowing the pat1 region is insufficient; the justification for selecting only ONUg0282 (amtA) is incomplete. Chromosome walking led to a ~154 kb interval containing 58 predicted genes, yet the manuscript focuses entirely on ONUg0282 (amtA). Neither the main text nor the supplementary materials provide:a systematic assessment of all candidate genes, exclusion criteria for the other genes, nor additional fine-mapping or co-segregation evidence. This makes the selection of amtA appear premature and under-justified.

Required revision: Provide either (i) a rationale for dismissing the remaining pat1-interval genes (annotation, expression, predicted function), or (ii) clarify that amtA is one strong candidate among many rather than the sole prioritized one.

We added the statement about ONUg0282 being “one of several strong candidates for pat1” in the Results section (L311).  Please note that we also emphasize that other candidate genes for remain to be investigated before one can conclude unequivocally about the identity of pat1 (L681-683).

2.The authors acknowledge in the Discussion that “available data do not permit to conclude that ONUg0282 is the pat1 locus.” Nevertheless, the narrative and Title imply strong association.

Required revision: Explicitly revise claims throughout the manuscript to avoid suggesting that amtA is pat1. It should be framed as “a gene within the pat1-linked region contributing to aggressiveness.” 

We thank the reviewer for the suggestion. We modified relevant portions of the manuscript (e.g., L90, L311). In addition, in response to comments from Reviewers 3 and 4, “identified by chromosome walking” was dropped from the title. This shortens the title and avoids suggesting that amtA is the sole candidate for pat1.

3.ΔamtA mutants show enhanced hyphal growth under starvation, but the manuscript attributes this to possible MAPK/PKA dysregulation without providing molecular evidence. Supplementary data do not include: Transcriptomics, Pathway activation assays, Quantitative Y–M switching metrics 

Required revision: Rephrase the signaling-related interpretation to hypothesized mechanisms unless direct evidence is added.

We drastically shortened the signaling-related interpretation (L668-677) and emphasized that microscopy results shown in the original Fig. 5 (new Figure 6) were qualitative results (L225 and L450) from preliminary experiments.

4. Supplementary Table S3 reports WT in planta expression of amt genes, but: ΔamtA in planta expression is not measured, Compensatory expression of amtB–amtE in the knockout lines is not assessed. This is critical for interpreting the functional significance of amtA during host colonization.

Required revision: Either provide qPCR data for mutant vs. WT during infection or explicitly acknowledge the limitation and adjust conclusions accordingly.

We do not yet have qPCR data for CRISPR-Cas9 mutants during infection of elm saplings. We have indicated in the text (L333-335 and L571-573) that in planta gene expression data presented in Table S3 and new Table S4 are from previously published work on wild-type O. novo-ulmi H327. We have modified Table S3 (L3) accordingly. The title of new Table S4 also makes it clear that transcriptomic data for O. novo-ulmi amt genes are only available for WT strain H327.

5.The manuscript reports inconsistent results between apple fruit and elm seedlings, and across inoculum dosages. However: Statistical analyses do not include mixed-effects or GLM models, Supplementary figures show variability that is not formally tested, Host × dose interaction remains unquantified

Required revision: Include appropriate statistical modeling or clarify limitations in interpreting dose responses.

We deleted the portion of text about dose response in the interaction between CRISPR-Cas9 mutants and elm saplings (L482-484).

6.The Title refers to “chromosome 2 walking”, while figures and text place the interval on chromosome I (reference genome). This inconsistency must be corrected or explicitly explained.

The pat1 gene region is indeed located on chromosome I. The “2” in “chromosome 2 walking” was part of the line numbering system in the JoF template. In any instance, as indicated above, we have deleted “identified by chromosome walking” from the Title.

Detailed comments

1.Introduction

Consider expanding the background on fungal ammonium transceptors and nitrogen availability in xylem environments to contextualize the role of Amt/Mep proteins during infection.

We thank the referee for the suggestion. We added information on this topic in the Discussion (L641-649), as we thought it would fit better there. We feel that presenting fungal ammonium transceptors in the Introduction might give readers the impression that we had an initial interest in these proteins. Our original goal was to find candidate genes for pat1. Once we found that the list of candidates included the amtA gene, we decided to investigate the latter and realized that it encoded an ammonium transceptor.

2.Methods

Chromosome walking would benefit from a schematic summarizing contigs, gaps, and alignment to the reference genome. 

We toned down section on chromosome walking and, therefore, did not add the schematic.

CRISPR section should summarize sgRNA target sites in the main text (currently dispersed in Supplementary).

Sequences of sGRNA target sites for genes ONUg0282 (amtA) and ONUg7434 (ade7) are now provided in the CRISPR-Cas9 subsection of Materials and Methods (L189-190).

Provide a concise table distinguishing ΔamtA-3 vs ΔamtA-35 alleles and predicted structural consequences.

Nucleotides deleted by CRISPR-Cas9 in the two mutants are now indicated in Table 1 that is included in the Results section (L437-440).

Microscopy analysis should include quantitative metrics (hyphal growth rate, branch density), not only qualitative descriptions.

We have modified the text to emphasize that the microscopy analysis results are preliminary data (L450). Even though we cannot provide results from statistical due to lack of replication, we would like to retain the microscopy analysis in the manuscript.

3.Results

Figure 4 colony zones should be quantified with statistics.

We modified the caption of original Fig 4B (new Fig 5B ) so that readers understand that colony zones shown in panel A were quantified with statistics shown in panel B (L446-447).

Supplemental phenotyping (e.g., methylammonium sensitivity) should be clearly referenced in the main text.

Thank you for the suggestion. This has been taken care of by transferring original Table S4 into the main text (new Table 1, L437-440).

Divergent results between apple and elm hosts require a more explicit discussion of tissue nitrogen context.

We added information on Golden Delicious apple tissue nitrogen context in the Discussion (L646-649).

4.Discussion

Reduce over-interpretation related to signaling pathways unless additional data are added.

We toned down our interpretation related to signaling pathways (see also our response to Major Comment #3).

Expand discussion about functional redundancy among the five Amt/Mep genes, especially since amtB shows high in planta expression in Supplementary Table S3. 

We added a short discussion on functional redundancy versus functional diversity (L588-600) and emphasized that a more definitive discussion on Amt/Mep genes in DED fungi must await results from thorough functional studies for all five genes.

5.Figures / Tables

Figure 2B phylogeny contains text too small to be readable.

 As indicated above, the original Figure 2 was split into two separate figures. Figure 2B is now Figure 3A and should be easier to interpret by readers. Figure captions for new Figures 2 and 3 were rewritten accordingly (L337-345 and L365-375).

Figure 5 panels are dense; consider splitting into two figures.

We modified the spatial arrangement of the 16 photographs that make up Figure5 (Figure 6 in revised ms, L470-474).

Supplementary tables should explicitly link gene IDs to locus tags in the genome (to avoid confusion between ONUg IDs and UniProt/PHI-base annotations).

Thank you for the suggestion. We added gene IDs in revised Tables S3 and S4.

6.Language

Several long paragraphs in the Results and Discussion should be split for clarity. Opening sentences of major sections should better summarize key findings to improve narrative flow. 

We tried our best to split the longer paragraphs in the Results and Discussion sections. We altered the beginning of major sections. We also shortened several sentences that were long.

Reviewer 5 Report

Dear Authors,

This manuscript described a gene ONUg0282 encoding a high-affinity ammonium transporter, as a major contributor to the high aggressiveness of the Dutch elm fungal pathogen Ophiostoma novo-ulmi by chromosome walking method. The predicted ONUg0282 gene product showed structural features of Mep2-type transceptors. The manuscript was well written. Some issues should be concerned.

Specific comments and suggestions

1. Title, The phrase “disease fungus” as well as in the text should be revised as “fungal pathogen” or “pathogen”.

2. line 487, Figure 6. The photos of symptoms should be provided.

3. Supplementary Materials, Figure S3. It was better to provide the photos of leaf symptoms inoculated with the fungal strains.

 

Author Response

Specific comments and suggestions

1. Title, The phrase “disease fungus” as well as in the text should be revised as “fungal pathogen” or “pathogen”.

We changed expressions “Dutch elm disease fungus”, “DED fungus” and “DED fungi” for “Dutch elm disease pathogen”, “DED pathogen” and “DED pathogens” in the Title and elsewhere in the text, except in a few instances where “pathogen” was already mentioned in the same sentence.

2. line 487, Figure 6. The photos of symptoms should be provided. 3. Supplementary Materials, Figure S3. It was better to provide the photos of leaf symptoms inoculated with the fungal strains.

We included a photograph (new Figure S3) of elm saplings that include one individual showing external DED symptoms and one healthy individual that was injected with sterile distilled water (non-inoculated control).

Round 2

Reviewer 1 Report

All the suggestions have been revised.

All suggested changes have been implemented.

Author Response

Thank you.