Review Reports - Genetic Background-Dependent Expression of Lr34, Lr67 and the Lr46-Associated Candidate Gene Lr46-Glu2 in Wheat (Triticum aestivum L.) Highlights Contrasting Transcriptional Responses Following Puccinia triticina Infection

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

General comments

The manuscript by Spychała et al. is well-designed and addresses a crucial topic in wheat breading, and pathology about how adult plant resistance (APR) genes behave when introgressed into different elite winter wheat backgrounds. This study is clear, logically structured, and is based on the authors’ prior research and provides useful novel data on expression dynamics and genetic background effects. The experimental approach is combines RT-qRT-PCR, ddPCR, and marker-assisted selection across BC₂F₁ populations. Although the findings are interesting and relevant to wheat breeding, the manuscript needs major revisions for clarity, scientific rigor (particularly phenotype linking and statistics), interpretation, and some structural tightening before it is accepted for publication.

Major Concerns

The authors said the plants were inoculated with Pt, yet the results have no disease severity, infection type, pustule density, latency period or any resistance phenotype. This is an important gap for a resistance-gene study. Gene expression alone cannot prove functional resistance; therefore, the authors must add at least one figure/table showing phenotypic responses for the same genotypes/time points or a separate adult-plant assessment. Correlate expression patterns with resistance levels if possible. if phenotype data exist but not shown, include them, but if not collected, explain why and temper claims about resistance.
The author claims about interaction as it reads “expression and interaction of Lr34, Lr46 and Lr67” and “reveal distinct modes.” However, the data primarily show individual gene expression in different backgrounds, not clear epistatic interactions or synergistic effects between the three loci. The authors must revise the title, probably to: Genetic background-dependent expression of Lr34, Lr46, and Lr67 in wheat reveals distinct modes of adult plant resistance. Or else, keep interaction only if you add data on combined vs single-locus effects.
Clarify the genetic and experimental design limitations. I think BC₂F₁ hybrids are heterogenous, however, the authors treat BC₂F₁ individuals as if they are genetically uniform, but they are segregating for background genes. This probably contributes to the observed expression variation. Therefore, the authors must explicitly state this limitation and consider discussing how it may increase background-dependent effects.
The authors state that miRNA levels did not consistently reflect mRNA expression, but no direct correlation analysis was presented. Therefore, authors must add a simple correlation table or scatterplot for instance, miRNA-5384-3p vs Lr46-Glu2 expression across time points and genotypes. This would strengthen the claim of post-translational regulation.
Address the absence of non-inoculated controls in expression Figures 1 and 2 because they only show inoculated samples over time. However, the methods describe two treatments – inoculated and non-inoculated, so present non-inoculated controls to distinguish infection-induced changes from developmental or circadian shifts. This is very important for Lr34 and Lr46, which show high expression at 0 hpi.
The conclusion that APR loci do not function as isolated genetic determinants is well supported. However, the claim that the patterns are preserved across distinct experimental systems rely on comparing to a single previous study by the same group (ref 19). Although this is reasonable, it should be framed as consistent with prior observations in spring wheat, not general conservation across all systems. Please tone down over-generalization.

Minor concerns

In the methods and materials, specify the Pt isolate used (race/virulence profile) if possible and confirm if vernalization and growth conditions are appropriate for winter types. Were all genotypes fully vernalized because winter wheat lines may differ in vernalization requirements which could possibly affect APR expression.
Figure 1 panels: Panels (b) and (c) are both Lr46 but split across two panels without clear differentiation. Furthermore, the figure 1 y- and x-axis lack descriptive labels, please revise this.
The manuscript requires thorough proofreading and all genes must be italicized.
Some sentences are overly long/awkward, please check and revise.
Avoid repetitive phrasing, for example, “strong effects of genetic background” is mentioned multiple times. Also, in the discussion opening; “The present study provides new insights into the functional behaviour….” is okay, but later, “A central observation emerging from this study is ………” repeats the same idea. Please revise this.
Although supplementary tables mark significant difference, the main text should briefly summarize statistical outcomes, such as, expression differed significantly between backgrounds at 12 hpi (P < 0.05). And also, the normalization method must be clarified, did the authors use the ΔΔCt method?

Comments on the Quality of English Language

The manuscript requires thorough proofreading and all genes must be italicized.
Some sentences are overly long/awkward, please check and revise.

Author Response

Major Concerns

The authors said the plants were inoculated with Pt, yet the results have no disease severity, infection type, pustule density, latency period or any resistance phenotype. This is an important gap for a resistance-gene study. Gene expression alone cannot prove functional resistance; therefore, the authors must add at least one figure/table showing phenotypic responses for the same genotypes/time points or a separate adult-plant assessment. Correlate expression patterns with resistance levels if possible. if phenotype data exist but not shown, include them, but if not collected, explain why and temper claims about resistance.

The aim of this study was to analyse the expression profiles of APR-associated loci and related miRNAs following inoculation with Puccinia triticina in different genetic backgrounds. The experimental design was focused on intensive sampling for RNA and miRNA analyses at five time points after inoculation. As a result, no separate phenotypic evaluation of disease severity, infection type or latency period was performed on the analysed plants. We agree that expression data alone do not provide direct evidence of resistance effectiveness. To clarify the scope of the study, we added a statement at the beginning of the Discussion indicating that the results should be interpreted in the context of gene regulation and temporal expression dynamics rather than as a direct assessment of resistance levels.

The author claims about interaction as it reads “expression and interaction of Lr34, Lr46 and Lr67” and “reveal distinct modes.” However, the data primarily show individual gene expression in different backgrounds, not clear epistatic interactions or synergistic effects between the three loci. The authors must revise the title, probably to: Genetic background-dependent expression of Lr34, Lr46, and Lr67 in wheat reveals distinct modes of adult plant resistance. Or else, keep interaction only if you add data on combined vs single-locus effects.

We agree that the term “interaction” may overstate the scope of the presented data. The study primarily investigates the expression patterns of Lr34, Lr46 and Lr67 in different genetic backgrounds rather than direct epistatic interactions or synergistic effects among these loci. Following the Reviewer's suggestion, we revised the title to: “Genetic background-dependent expression of Lr34, Lr46, and Lr67 in wheat reveals distinct modes of adult plant resistance”. Moreover, corresponding wording has also been adjusted throughout the manuscript where appropriate.

Clarify the genetic and experimental design limitations. I think BC₂F₁ hybrids are heterogenous, however, the authors treat BC₂F₁ individuals as if they are genetically uniform, but they are segregating for background genes. This probably contributes to the observed expression variation. Therefore, the authors must explicitly state this limitation and consider discussing how it may increase background-dependent effects.

The analysed BC₂F₁ plants were selected using gene-linked molecular markers confirming the presence of the target APR loci (Lr34, Lr46 and/or Lr67). Therefore, the expression analyses were performed on individuals carrying the respective resistance loci. We agree, however, that BC₂F₁ populations are not genetically fixed and that segregation of other genomic regions may contribute to variation in gene expression. This aspect is particularly relevant when interpreting background-dependent expression patterns. We have therefore added a statement to the Discussion acknowledging that residual genetic segregation outside the analysed loci may have contributed to the observed variability among genotypes.

The authors state that miRNA levels did not consistently reflect mRNA expression, but no direct correlation analysis was presented. Therefore, authors must add a simple correlation table or scatterplot for instance, miRNA-5384-3p vs Lr46-Glu2 expression across time points and genotypes. This would strengthen the claim of post-translational regulation.

Following this recommendation, we performed a correlation analysis between tae-miR5384-3p abundance and expression of the candidate gene Lr46-Glu2 across all analyzed genotypes and sampling time points (Figure S1). No significant correlation was detected (Pearson’s r = −0.070, P = 0.713). This result has been added to the revised manuscript and supports the conclusion that miRNA abundance did not consistently reflect Lr46-Glu2 transcript levels. We have also revised the text to avoid overinterpretation and to clarify that potential miRNA-mediated regulation requires further functional validation.

Address the absence of non-inoculated controls in expression Figures 1 and 2 because they only show inoculated samples over time. However, the methods describe two treatments – inoculated and non-inoculated, so present non-inoculated controls to distinguish infection-induced changes from developmental or circadian shifts. This is very important for Lr34 and Lr46, which show high expression at 0 hpi.

We thank the Reviewer for this important comment. We agree that time-matched non-inoculated controls would allow a clearer separation of infection-related responses from temporal, developmental or circadian variation. In the present experiment, however, non-inoculated controls were not collected at each post-inoculation time point. The 0 hpi samples were used as the pre-inoculation baseline for each genotype. We have revised the Materials and Methods section to clarify the experimental design and removed wording suggesting the presence of two full treatments across all time points. The Results and Discussion have also been moderated accordingly, and expression changes are now described as temporal responses following inoculation rather than as definitive infection-induced effects. This limitation has been explicitly acknowledged in the Discussion.

The conclusion that APR loci do not function as isolated genetic determinants is well supported. However, the claim that the patterns are preserved across distinct experimental systems rely on comparing to a single previous study by the same group (ref 19). Although this is reasonable, it should be framed as consistent with prior observations in spring wheat, not general conservation across all systems. Please tone down over-generalization.

We thank the Reviewer for this comment. We agree that the original wording was too wide. Our interpretation was based primarily on comparison with previous observations obtained in spring wheat rather than on evidence from multiple experimental systems. The relevant statements have therefore been revised to indicate that the observed expression patterns are consistent with our previous findings in spring wheat, rather than suggesting general conservation across different systems.

Minor concerns

In the methods and materials, specify the Pt isolate used (race/virulence profile) if possible and confirm if vernalization and growth conditions are appropriate for winter types. Were all genotypes fully vernalized because winter wheat lines may differ in vernalization requirements which could possibly affect APR expression.

The objective of the study was to analyse the expression of APR-associated loci, which confer race non-specific resistance. Therefore, plants were inoculated with a mixed urediniospore population collected from naturally infected wheat fields during the experimental season rather than with a single P. triticina isolate. This approach was intended to provide a broad infection stimulus representative of field conditions and to avoid focusing on race-specific host–pathogen interactions. We have clarified this point in the Materials and Methods section. Regarding vernalization, all winter wheat genotypes were subjected to the same vernalization regime for eight weeks before transfer to growth conditions promoting adult plant development. The analysed material reached the adult plant stage prior to inoculation. A statement confirming this has been added to the Materials and Methods section.

Figure 1 panels: Panels (b) and (c) are both Lr46 but split across two panels without clear differentiation. Furthermore, the figure 1 y- and x-axis lack descriptive labels, please revise this.

Figure 1 has been revised to improve clarity. Descriptive x- and y-axis labels have been added to all panels. The x-axis now indicates time after inoculation (hpi), and the y-axis indicates relative expression level calculated using the 2^-ΔΔCt method. Panels (b) and (c), both presenting Lr46-Glu2 expression, have also been clearly differentiated: panel (b) shows TX-derived genotypes, whereas panel (c) shows Purdue-derived genotypes. The figure caption has been revised accordingly.

The manuscript requires thorough proofreading, and all genes must be italicized. Some sentences are overly long/awkward, please check and revise. Avoid repetitive phrasing, for example, “strong effects of genetic background” is mentioned multiple times. Also, in the discussion opening, “The present study provides new insights into the functional behaviour….” is okay, but later, “A central observation emerging from this study is ………” repeats the same idea. Please revise this.

The manuscript has been carefully proofread and revised for language, style and consistency. Gene names have been checked and italicized where appropriate. In addition, several repetitive statements and overly long sentences have been shortened or rephrased to improve readability and avoid redundancy.

Although supplementary tables mark significant difference, the main text should briefly summarize statistical outcomes, such as, expression differed significantly between backgrounds at 12 hpi (P < 0.05). And also, the normalization method must be clarified, did the authors use the ΔΔCt method?

To improve clarity, we added brief references to the main statistically significant differences in the Results section while retaining the complete statistical information in the Supplementary Tables. The revised text now indicates the key expression changes that were statistically significant (P < 0.05). In Methodes section we clarified that relative gene expression was calculated using the 2^-ΔΔCt method. Expression values were normalized against two reference genes (TUBβ and ARF), and the non-inoculated samples collected at 0 hpi were used as calibrators.

Comments on the Quality of English Language

The manuscript requires thorough proofreading and all genes must be italicized.
Some sentences are overly long/awkward, please check and revise.

Corrected and improved.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Prof. Editor

The manuscript entitled “Genetic background-dependent expression and interaction of Lr34, Lr46 and Lr67 in wheat (Triticum aestivum L.) reveal distinct modes of adult plant resistance” investigates the expression dynamics of three important adult plant resistance (APR) loci following their introgression into elite winter wheat backgrounds. The topic is relevant to wheat breeding and durable disease resistance research, and the manuscript presents a substantial amount of gene expression and miRNA data collected under controlled infection conditions.

The study provides evidence that expression patterns of APR-associated genes vary among genetic backgrounds and suggests that different APR loci may exhibit distinct temporal expression profiles following infection. In particular, the observed differences among recipient backgrounds are interesting and may have practical implications for resistance breeding programs.

However, while the dataset is potentially valuable, several aspects of the manuscript require clarification and revision before publication.

My main concern is that some of the biological conclusions are stronger than the evidence presented. The authors conclude that Lr34 and Lr67 function primarily as constitutive defense components, whereas Lr46 acts as an inducible early-response component. Although the expression profiles are consistent with such an interpretation, the study measures transcript abundance only. No phenotypic disease assessments, pathogen biomass measurements, protein accumulation analyses, or functional validation experiments were performed. Therefore, these conclusions should be presented more cautiously as interpretations supported by expression data rather than demonstrated biological mechanisms.

A second concern relates to the treatment of the Lr46 locus. The manuscript correctly states that the causal gene underlying Lr46 has not yet been conclusively identified. Nevertheless, several sections discuss Lr46-Glu2 almost as if it were the confirmed resistance gene. The distinction between the Lr46 locus and the candidate gene Lr46-Glu2 should be maintained consistently throughout the manuscript.

The statistical analysis section also requires additional detail. The experimental design includes multiple genotypes, multiple time points, and biological replicates, yet the statistical procedures are described only briefly. The authors should clearly explain how comparisons were performed, how biological and technical replicates were handled, and whether genotype-by-time interactions were evaluated. Additional justification for the statistical approach would strengthen confidence in the conclusions.

The miRNA section would also benefit from deeper analysis. While the authors report variation in miRNA abundance across genotypes and sampling times, the biological significance of these patterns remains unclear. Correlation analyses between miRNA abundance and corresponding target-gene expression could considerably strengthen this part of the manuscript and help support the proposed regulatory relationships.

From a presentation standpoint, Figure 1 should be improved. The current figure is difficult to interpret because of overlapping expression profiles, small labels, and the absence of visible measures of variability. Similarly, Figure 2 would benefit from clearer statistical annotations and improved readability.

I also encourage the authors to include a short limitations paragraph in the Discussion. Such a section should acknowledge that the study focuses primarily on transcriptional responses and does not directly evaluate resistance phenotypes, protein accumulation, or causal miRNA–target interactions. This would help place the findings into the appropriate biological context.

Despite these concerns, I believe the manuscript contains useful data and addresses an important question regarding the influence of genetic background on APR-associated gene expression. The study does not require extensive new experimentation, but the interpretation of the results should be more conservative and the methodological description should be expanded.

Section-Specific Comments

Abstract

The abstract is generally well written but contains several conclusions that are stronger than warranted by the data.

The statement that APR loci function as components of a background-dependent regulatory system should be moderated because regulatory interactions were not directly investigated.

Similarly, claims regarding breeding deployment should be expressed more cautiously given the absence of phenotypic validation.

Introduction

The Introduction provides a useful overview of APR biology.

However, the novelty of the study should be more clearly articulated.

Several cited publications from the same research group have already investigated expression of Lr34, Lr46-associated genes, Lr67, and related miRNAs.

The authors should better explain how the present study advances beyond previous work.

Results

The Results section contains extensive descriptions of expression values but relatively little biological interpretation.

In several places, the narrative becomes a list of numerical observations rather than a synthesis of biologically meaningful trends.

The authors should focus more on major patterns and less on exhaustive reporting of individual values.

The exceptionally strong induction of Lr46-Glu2 in the TX × Symetria background deserves further discussion.

Likewise, the biological significance of the unusually high baseline expression of Lr34 in some backgrounds should be considered.

Discussion

Many conclusions are reasonable but are often presented as demonstrated mechanisms rather than interpretations.

The authors should reduce speculative statements and focus on conclusions directly supported by the data.

A dedicated limitations section should be included.

Such a section should acknowledge that:

resistance phenotypes were not evaluated,
• protein abundance was not measured,
• causal miRNA–target interactions were not validated,
• mechanistic regulatory pathways were not investigated.

Materials and Methods

Several methodological details require clarification.

The authors should specify:

the number of technical replicates used in RT-qPCR analyses;
• whether biological replicates consisted of independent plants;
• infection verification procedures;
• statistical software and versions used;
• treatment of technical and biological replication during analysis.

Additional information regarding the five Puccinia triticina isolates would also be useful.

Figures

Figure 1 requires substantial improvement.

The current figure is difficult to interpret due to overlapping profiles and limited visualization of variability.

Error bars should be included and readability improved.

Figure 2 would benefit from statistical annotations and clearer presentation.

Minor Comments

Use consistent notation for BC₂F₁ throughout the manuscript.
Standardize genotype abbreviations.
Report exact P-values whenever possible.
Clarify marker validation procedures, especially for csLV46G22.

Comments for author File: Comments.pdf

Author Response

We thank the Reviewer for this comment and agree that transcript abundance alone does not demonstrate biological function. The conclusions have therefore been revised to more clearly distinguish between direct observations and their biological interpretation. Throughout the Discussion, sentences and statements regarding the roles of Lr34, Lr46 and Lr67 have been rephrased as interpretations supported by expression data and existing literature rather than as demonstrated mechanisms. We also emphasize that further studies integrating phenotypic, molecular and functional analyses will be required to validate these proposed roles.

We thank the Reviewer for this important observation. We agree that the distinction between the Lr46 resistance locus and the candidate gene Lr46-Glu2 should be maintained consistently throughout the manuscript. The text has therefore been revised to clarify that Lr46-Glu2 represents a candidate gene located within the Lr46 genomic region and that its expression profile should not be interpreted as direct evidence of the molecular identity of the Lr46 resistance gene.

The Statistical Analysis section has been expanded to provide additional details regarding the experimental design and data analysis. We now clarify the number of biological and technical replicates, the treatment of replicate data, and the statistical procedures used for comparing expression levels across time points. The revised Methods section also specifies that comparisons were performed relative to the corresponding 0 hpi control within each genotype. We acknowledge that “genotype-by-time” interactions were not formally evaluated and have clarified the scope of the applied statistical analyses.

We agree that correlation analyses between miRNA abundance and target-gene expression could provide additional insight into potential regulatory relationships. However, the primary objective of this study was to characterize temporal expression patterns of APR-associated genes and selected miRNAs across different genetic backgrounds. Given the genotype-specific and time-dependent nature of the observed responses, as well as the limited number of biological observations available for robust correlation analyses, we considered such analyses to be beyond the scope of the present study. The Discussion has been revised to clarify that the observed associations between miRNAs and target genes should be regarded as putative and that direct regulatory relationships remain to be experimentally validated.

Figure 1 has been revised to improve readability. The panels have been reformatted, axis labels have been added, and the distinction between the Lr46-Glu2 panels has been clarified. In the revised version, panel (b) shows TX-derived genotypes, whereas panel (c) shows Purdue-derived genotypes. To avoid further overcrowding of the figure, detailed statistical information and variability measures for the gene expression data are provided in Supplementary Tables S1–S3. Figure 2 has also been checked for readability. The detailed numerical data and statistical information supporting the miRNA expression profiles are provided in Supplementary Tables S4–S6.

A short paragraph has been added to the Discussion to acknowledge the main limitations of the study. In particular, we clarify that the work focused on transcriptional responses and did not include phenotypic assessment of disease resistance, protein-level analyses, or experimental validation of miRNA–target interactions. These aspects should be addressed in future studies to further elucidate the biological significance of the observed expression patterns.

Section-Specific Comments

Abstract

The abstract is generally well written but contains several conclusions that are stronger than warranted by the data. The statement that APR loci function as components of a background-dependent regulatory system should be moderated because regulatory interactions were not directly investigated. Similarly, claims regarding breeding deployment should be expressed more cautiously given the absence of phenotypic validation.

We agree that some statements in the Abstract were stronger than warranted by the scope of the data. The Abstract has been revised to more clearly distinguish observations based on expression profiles from broader biological interpretations.

Introduction

The Introduction provides a useful overview of APR biology. However, the novelty of the study should be more clearly articulated. Several cited publications from the same research group have already investigated expression of Lr34, Lr46-associated genes, Lr67, and related miRNAs. The authors should better explain how the present study advances beyond previous work.

Previous studies, including our own, focused primarily on individual APR-associated genes, candidate genes within the Lr46 region, or miRNA-mediated regulation in specific genetic backgrounds. The present study extends these observations by simultaneously analysing the expression of Lr34, Lr46-Glu2 candidate and Lr67 following introgression into multiple elite winter wheat cultivars. In current version, particular emphasis was placed on the influence of genetic background on APR-associated expression patterns.

Results

The Results section contains extensive descriptions of expression values but relatively little biological interpretation. In several places, the narrative becomes a list of numerical observations rather than a synthesis of biologically meaningful trends. The authors should focus more on major patterns and less on exhaustive reporting of individual values. The exceptionally strong induction of Lr46-Glu2 in the TX × Symetria background deserves further discussion. Likewise, the biological significance of the unusually high baseline expression of Lr34 in some backgrounds should be considered.

The Results section was intentionally structured to present the experimental observations in a descriptive manner, while biological interpretation and mechanistic considerations were reserved for the Discussion section. Nevertheless, we expanded the Discussion to address your two notable observations: the particularly strong induction of the candidate gene Lr46-Glu2 in the TX × Symetria background and the unusually high baseline expression of Lr34 observed in selected genetic backgrounds. These aspects are now discussed in the context of genotype-dependent regulation of APR-associated loci.

Discussion

Many conclusions are reasonable but are often presented as demonstrated mechanisms rather than interpretations. The authors should reduce speculative statements and focus on conclusions directly supported by the data. A dedicated limitations section should be included. Such a section should acknowledge that:

resistance phenotypes were not evaluated,
• protein abundance was not measured,
• causal miRNA–target interactions were not validated,
• mechanistic regulatory pathways were not investigated.

We agree that the limitations of the study should be stated more explicitly. The Discussion has been revised to include a dedicated paragraph outlining the main limitations of the work. In particular, we acknowledge that the study focused on transcriptional responses and did not include direct phenotypic assessment of resistance, protein-level analyses, validation of miRNA–target interactions, or investigation of the underlying regulatory pathways. We have also revised several statements throughout the Discussion to ensure that biological interpretations are presented as hypotheses consistent with the observed expression patterns rather than as demonstrated mechanisms.

Materials and Methods

Several methodological details require clarification.

The authors should specify:

the number of technical replicates used in RT-qPCR analyses;
• whether biological replicates consisted of independent plants;
• infection verification procedures;
• statistical software and versions used;
• treatment of technical and biological replication during analysis.

Additional information regarding the five Puccinia triticina isolates would also be useful.

The Materials and Methods section has been expanded to provide additional information regarding the experimental design and data analysis. We clarified the number and nature of biological replicates, the handling of replicate data during statistical analyses, the RT-qPCR normalisation procedure, and the statistical software used. We also added further details regarding the inoculation procedure and the origin of the Puccinia triticina inoculum used in this study.

Figures

Figure 1 requires substantial improvement. The current figure is difficult to interpret due to overlapping profiles and limited visualization of variability. Error bars should be included and readability improved. Figure 2 would benefit from statistical annotations and clearer presentation.

Figure 1 has been substantially revised to improve readability. The panels have been reformatted, descriptive x- and y-axis labels have been added, and the distinction between the Lr46-Glu2 panels has been clarified. In the revised version, panel (b) presents TX-derived genotypes, whereas panel (c) presents Purdue-derived genotypes. Detailed variability measures and statistical results supporting Figure 1 are provided in Supplementary Tables S1–S3. Figure 2 has also been checked and revised for clearer presentation. The numerical data supporting the miRNA expression profiles are provided in Supplementary Tables S4–S6. In addition, the relationship between tae-miR5384-3p abundance and Lr46-Glu2 expression was further assessed by correlation analysis and is now presented in Supplementary Table S7 / Figure S1.

Minor Comments

Use consistent notation for BC₂F₁ throughout the manuscript.
Standardize genotype abbreviations.
Report exact P-values whenever possible.
Clarify marker validation procedures, especially for csLV46G22.

The manuscript has been carefully revised to ensure consistency in the notation of BC₂F₁ material and genotype abbreviations throughout the text, tables and figures. Exact P-values have been reported where available. We also clarified the marker validation procedures described in Section 4.2. Marker analyses were used to classify genotypes according to the presence or absence of the target resistance loci, and the resulting marker profiles are summarised in Table 1. Only marker-positive materials were included in subsequent expression analyses. With regard to the csLV46G22 marker, we have clarified its role in the selection of plant material for expression studies. However, additional technical details cannot be provided, as the marker has not yet been formally published by its developer and is therefore not available for public disclosure.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript is devoted to the complex analysis of three known adult plant resistance (APR) loci in winter wheat (Triticum aestivum) hybrids and is an obvious continuation of the work of the same authors, published a year earlier in IJMS and dedicated to spring wheat. Two genes (Lr34 and Lr67) are relatively well-studied while the third locus (Lr46) has been investigated primarily on a phenotypic basis. Based on recent research, a gene TraesCS1B02G454200.1 (Lr46-Glu2) has been selected for analysis of Lr46. The authors examined the expression levels of mentioned genes (480 plant samples) before and after inoculation with a fungal pathogen Puccinia triticina at several time points. As a result, it was demonstrated that APR depends significantly on the genetic environment and includes both constitutive and inducible components. This work is interesting from a fundamental point of view, and also has a clear perspective for use in wheat breeding. The reliability of the data obtained is beyond doubt.

The text is written logically and consistently; the English language does not cause any complaints.

Remarks:

Section 4.2: “DNA quality and concentration were suitable for downstream PCR analyses”. In my opinion, this statement is insufficient. Please provide more details on how "quality control" was conducted.

Section 4.2: PCR and restriction analysis of key genetic markers is not presented. I guess this data would be appropriate in the Supplementary section.

Section 4.4. “Primers were designed based on miRNA sequences complementary to Lr34 and the candidate gene associated with Lr46 (Glu2) and their sequences were described in the research team’s previous work [18, 28]”. Primers for Lr67 detection are not specified here. The text goes on to say that (in the context of ddPCR) “primers targeted Lr34, Lr67, and the candidate gene for Lr46 (Glu2) were designed and their sequences were described in the research team’s previous work [18, 28]”. Please clarify.

Editorial notes:

Section headings 3.1., 3.2., 3.3. and so on, up to 4.5.: Not enough space(s)

Section 4.4.: The word “loop” with a hyphen seems out of place in the text

Author Response

The Materials and Methods section has been revised to provide additional details regarding DNA quality assessment, including the methods used to determine DNA concentration, purity and suitability for downstream PCR analyses.

Section 4.2: PCR and restriction analysis of key genetic markers is not presented. I guess this data would be appropriate in the Supplementary section.

The marker analyses were performed solely to verify the presence or absence of the target APR-associated loci in parental lines and BC₂F₁ materials. The resulting genotyping outcomes are summarised in Table 1, which was subsequently used for the selection of material included in the expression analyses. As the PCR and restriction assays employed well-established, previously published marker systems and did not constitute a primary objective of the study, we elected not to include representative gel images in the manuscript.

We thank the Reviewer for identifying this inconsistency. The corresponding section has been revised to clearly distinguish between primers used for RT-qPCR analysis of APR-associated genes (Lr34, Lr67 and the candidate gene Lr46-Glu2) and stem-loop primers used for miRNA expression analyses by ddPCR.

Editorial notes:

Section headings 3.1., 3.2., 3.3. and so on, up to 4.5.: Not enough space(s). Section 4.4.: The word “loop” with a hyphen seems out of place in the text

We thank the Reviewer for noting typographical issues. The text has been corrected accordingly.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

May the authors kindly resubmit a manuscript with marked changes because its difficult to see what changes were made in the manuscript

Comments on the Quality of English Language

May the authors kindly resubmit a manuscript with marked changes because its difficult to see what changes were made in the manuscript

Author Response

We have resubmitted the revised manuscript (2nd revision) with all changes clearly marked using the Track Changes function to facilitate the review process.

Reviewer 2 Report

Comments and Suggestions for Authors

I appreciate the considerable effort invested in revising the manuscript. The clarification of Lr46-Glu2 as a candidate gene, the expanded methodological description, and the improved contextualization relative to previous studies have strengthened the manuscript.

Nevertheless, several points require additional attention before publication.

Major Remaining Issues

Conclusions remain stronger than supported by the data

Although the Discussion has been revised, several statements still imply biological functions that have not been directly demonstrated. Examples include statements suggesting that:

Lr34 and Lr67 contribute to constitutive defence layers.
Lr46 acts as an inducible early-response component.
APR loci operate as components of a background-dependent regulatory system.

The expression data are certainly consistent with these interpretations; however, the study does not include phenotypic resistance measurements, pathogen quantification, protein-level analyses, or functional validation experiments.

I recommend consistently using more cautious wording throughout the Abstract, Discussion, and Conclusions. For example:

"The observed expression patterns are consistent with a potential constitutive defence role for Lr34 and Lr67..."

rather than direct functional statements.

Dedicated limitations section is not clearly visible

The response letter indicates that a dedicated paragraph describing study limitations was added. However, such a section is not readily identifiable within the Discussion.

I recommend adding a clearly labeled paragraph near the end of the Discussion acknowledging that:

resistance phenotypes were not evaluated;
pathogen biomass was not quantified;
protein accumulation was not measured;
causal miRNA-target interactions were not validated;
regulatory mechanisms were inferred from expression patterns only.

A clearly identifiable limitations section would improve transparency and interpretation.

Correlation analysis requires clarification

The response letter states that a correlation analysis between tae-miR5384-3p abundance and Lr46-Glu2 expression was performed and presented in Supplementary Table S7 and Figure S1.

However, these supplementary materials are not clearly referenced within the manuscript and do not appear in the supplementary materials list currently provided.

Please ensure that:

Supplementary Table S7 and Figure S1 are included in the submission;
the corresponding analysis is cited in the Results or Discussion section;
the biological interpretation of the analysis is briefly discussed.

Figure 1 presentation

Figure 1 is improved compared with the previous version; however, error bars are still absent despite being requested during the previous review round.

If variability measures are available, I recommend including error bars directly within the figure. If the authors prefer to present variability only in supplementary tables, this should be explicitly justified in the figure legend or Methods section.

Minor Comments

Ensure consistent formatting of BC₂F₁ throughout the manuscript.
Carefully check spacing and formatting in subsection headings (e.g., "3.1", "3.2", etc.).
Ensure that all supplementary materials referenced in the response letter are included in the final submission package.
Verify that all statements regarding Lr46-Glu2 consistently describe it as a candidate gene associated with the Lr46 locus.

Author Response

We thank the Reviewer for the careful evaluation of the revised manuscript and for identifying the remaining points requiring clarification. We agree that the transcriptional data should be clearly distinguished from functional evidence of resistance. We have therefore further revised the manuscript to use more cautious terminology throughout the Highlight, Abstract, Discussion and concluding paragraph. We have also added a clearly labelled Study limitations subsection, clarified and fully documented the correlation analysis, included the corresponding supplementary material, and added variability measures to Figure 1. The terminology referring to Lr46-Glu2 has also been standardised throughout the manuscript to clearly identify it as a candidate gene associated with the Lr46 locus.

Nevertheless, several points require additional attention before publication.
Major Remaining Issues
1. Conclusions remain stronger than supported by the data
Although the Discussion has been revised, several statements still imply biological functions that have not been directly demonstrated. Examples include statements suggesting that:
* Lr34 and Lr67 contribute to constitutive defence layers.
* Lr46 acts as an inducible early-response component.
* APR loci operate as components of a background-dependent regulatory system.
The expression data are certainly consistent with these interpretations; however, the study does not include phenotypic resistance measurements, pathogen quantification, protein-level analyses, or functional validation experiments.
I recommend consistently using more cautious wording throughout the Abstract, Discussion, and Conclusions. For example:
"The observed expression patterns are consistent with a potential constitutive defence role for Lr34 and Lr67..."
rather than direct functional statements.

We have further revised the manuscript to clearly distinguish the observed transcriptional patterns from inferred biological functions. Statements describing Lr34 and Lr67 as constitutive defence components, Lr46 as an inducible early-response component, or APR loci as components of a defined regulatory system have been removed or reformulated.
Throughout the revised manuscript, we now describe Lr34 and Lr67 as showing predominantly baseline-associated expression, whereas the Lr46-associated candidate gene Lr46-Glu2 displayed a more infection-responsive transcriptional profile in several genetic backgrounds. We explicitly state that these patterns are consistent with different transcriptional behaviours but do not establish distinct functional roles in resistance.
The Highlight, Abstract, relevant parts of the Discussion, and the concluding paragraph have been revised accordingly.

2. Dedicated limitations section is not clearly visible
The response letter indicates that a dedicated paragraph describing study limitations was added. However, such a section is not readily identifiable within the Discussion.
I recommend adding a clearly labeled paragraph near the end of the Discussion acknowledging that:
* resistance phenotypes were not evaluated;
* pathogen biomass was not quantified;
* protein accumulation was not measured;
* causal miRNA-target interactions were not validated;
* regulatory mechanisms were inferred from expression patterns only.
A clearly identifiable limitations section would improve transparency and interpretation.

Although limitations were discussed in the previous revision, they were not presented under a clearly identifiable heading. We have therefore added a dedicated subsection entitled “Study limitations” near the end of the Discussion.
This subsection now explicitly states that resistance phenotypes were not quantitatively evaluated, pathogen biomass was not quantified, protein accumulation was not measured, causal miRNA–target interactions were not experimentally validated, and regulatory mechanisms were inferred from expression patterns only. We also retained the limitation associated with the use of segregating BC₂F₁ material.

3. Correlation analysis requires clarification
The response letter states that a correlation analysis between tae-miR5384-3p abundance and Lr46-Glu2 expression was performed and presented in Supplementary Table S7 and Figure S1.
However, these supplementary materials are not clearly referenced within the manuscript and do not appear in the supplementary materials list currently provided.
Please ensure that:
* Supplementary Table S7 and Figure S1 are included in the submission;
* the corresponding analysis is cited in the Results or Discussion section;
* the biological interpretation of the analysis is briefly discussed.

We thank the Reviewer for pointing out this inconsistency. The correlation analysis has now been expanded and is presented in Supplementary Table S7, including the overall dataset, donor-derived groups, and individual genotypes. Because the graphical representation did not add substantive information beyond the numerical results and could suggest a stronger pattern than supported by the data, Figure S1 has been removed from the revised submission. The manuscript has been updated accordingly, and all references to Figure S1 have been deleted.
The Statistical analyses subsection has been expanded to describe the Pearson correlation analysis between tae-miR5384-3p abundance and Lr46-Glu2 expression. The Results section now reports the correlation coefficient and statistical significance and refers directly to Table S7 The biological interpretation has also been clarified in the Discussion.
No significant relationship was detected across the six BC₂F₁ genotypes and five sampling time points (Pearson’s r = −0.070, P = 0.713), indicating that tae-miR5384-3p abundance did not consistently correspond to variation in Lr46-Glu2 transcript levels. We therefore emphasise that a causal miRNA–target relationship cannot be inferred from these expression data alone.

4. Figure 1 presentation
Figure 1 is improved compared with the previous version; however, error bars are still absent despite being requested during the previous review round.
If variability measures are available, I recommend including error bars directly within the figure. If the authors prefer to present variability only in supplementary tables, this should be explicitly justified in the figure legend or Methods section.

We thank the Reviewer for this comment. We carefully reconsidered the presentation of Figure 1 and initially evaluated versions of the expression plots including error bars. However, because variability was relatively high for several genotype × time-point combinations, the resulting error bars substantially reduced the readability of the figure and obscured the temporal expression patterns that Figure 1 is intended to illustrate.
We therefore decided to replace the line plots with heatmaps. The purpose of Figure 1 is to provide a clear visual overview of differences in the magnitude and temporal dynamics of expression among genotypes rather than to serve as a graphical presentation of the statistical analysis. In our view, the heatmap format allows these expression trends to be compared more clearly across genes, genetic backgrounds and sampling time points without implying a level of graphical precision that is better conveyed by the numerical data.
Importantly, no information on biological variability or statistical testing has been omitted. Mean expression values, standard deviations from biological replicates, and the results of the statistical comparisons are reported in full in Supplementary Tables S1–S3. Thus, the revised presentation separates the two purposes: Figure 1 provides a concise visual representation of the temporal expression patterns, whereas Tables S1–S3 provide the complete quantitative information, including variability and statistical significance.
The Figure 1 legend has been revised to make this distinction explicit.

We have made all corrections mentioned as minor comments

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

I would like to thank the authors for satisfactorily addressing my concerns. The manuscript can now be accepted for publication. However, the authors must italicize "Puccinia triticina" in Figure 1 caption on their final revision before publication.