Transcriptomic Comparison of Soybean Roots Inoculated with Different Rhizobium Strains During Early Symbiosis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Overall Evaluation:

This manuscript presents a comparative study on the symbiotic efficiency and molecular responses of soybean inoculated with two different rhizobial strains: the newly isolated B. ottawaense Bott 59 and the model strain B. diazoefficiens USDA 110. By integrating phenotypic observations with early-stage (3 dpi) root transcriptomic analysis, the authors successfully demonstrate that Bott 59 is a superior strain in terms of nodulation, nitrogenase activity, and biomass accumulation. The study identifies key molecular signatures—specifically the activation of isoflavonoid/flavonoid biosynthesis and more robust hormone signaling (auxin, cytokinin, and JA)—that distinguish the high-efficiency strain. This work provides valuable insights into the specificity of host-rhizobia interactions and offers potential genetic targets for enhancing symbiotic nitrogen fixation in sustainable agriculture. The paper is well-structured and addresses a relevant gap in the field of plant-microbe interactions.

 

Minor comments:

• The superior performance of Bott 59 on the W82 was determined by both the genotype of rhizobium strain (Bott 59) and the genotype of soybean, this should be emphasized and discussed in the discussion section.
• More details are needed in the figure legend of Figure 1F.
• It seems that Bott59 could promote the growth of soybean by compared with USDA110 this should be discussed in the discussion section.
• How about the expression of RIC or NIC peptide genes relative to AON signaling pathway in this study.

Author Response

Dear Reviewer,

We are very grateful for your constructive comments and suggestions for our manuscript entitled "Transcriptomic Comparison of Soybean Roots Inoculated with Different Rhizobium Strains During Early Symbiosis " (ID: plants-4204122). Other changes have been marked in the manuscript using the revision mode (Track Changes). Please see below for our point-by-point responses to the editor's comments and concerns. The specific details of the modifications are shown as follows:

Comment 1: The superior performance of Bott 59 on the W82 was determined by both the genotype of rhizobium strain (Bott 59) and the genotype of soybean, this should be emphasized and discussed in the discussion section.

Response: We sincerely thank the reviewer for this insightful comment. We agree that emphasizing the genotype-by-genotype (G×G) interaction is crucial for interpreting our results, as the superior performance of strain Bott 59 is not an intrinsic property of the strain alone but arises from its specific compatibility with the Williams 82 (W82) host genotype. In response, we have significantly revised the Discussion section to explicitly highlight this G×G dynamic. Specifically:

1. We now clearly state that the high symbiotic efficiency observed is a result of the specific genetic compatibility between Bott 59 and W82, rather than the intrinsic general performance of the rhizobial strain.
2. We clarified that this specificity underscores the importance of selecting compatible strain-cultivar pairs for agricultural applications.

Changes in the manuscript:

We have added the following paragraph to the 3. Discussion section, paragraph 1:

“Phenotypic evaluations at 21 dpi revealed that the Bott 59-inoculated group exhibited higher nodule numbers, nodule fresh weight, and nitrogenase activity compared to the USDA 110 group. These results indicate that strain Bott 59 enhances SNF efficiency. This suggests the potential for a specific genotype-by-genotype (G×G) interaction [58], wherein the genetic compatibility between the Bott 59 genome and the Williams 82 host underlies enhanced symbiotic efficiency. The molecular basis of such specificity often involves precise recognition mechanisms. For instance, the type III effector NopC in the broad-host-range rhizobium Sinorhizobium fredii HH103 affects nodulation across different host genotypes. NopC physically interacts with GmRAC1 to induce the expression of GmNIN2a/2b and GmENOD40, thereby influencing infection thread extension and nodule primordia initiation [59]. We hypothesize that the exceptional compatibility observed in the W82–Bott 59 pair may result from a unique effector repertoire in Bott 59 that either evades W82-mediated immune surveillance or optimally modulate host signaling pathways, thereby inducing the expression of key symbiosis-related genes in the host. This underscores the critical need to dissect the specific G×G molecular determinants governing this mutualism in future studies.”

Comment 2: More details are needed in the figure legend of Figure 1F.

Response: We are grateful for the reviewer's constructive comments. We have revised the figure legend of Figure 1. The additions are indicated below and are also marked in the original text, as follows.

Figure 1. Inoculation with USDA 110 and Bott 59 promotes growth, nodulation, and nitrogen fixation in soybean cv. Williams 82

(A) Growth performance of soybean plants and nodules at various time points post-inoculation under inoculated and non-inoculated conditions. After an initial 10-day cultivation, plant phenotypes are shown at 3, 10, and 21 dpi with or without rhizobia incubation. Nodule development is shown at 21 dpi for USDA 110 and Bott 59 inoculations. Scale bar = 1 cm.

(B-E) Plant height (B), primary root length (C), shoot dry weight (D), and root dry weight (E) at 3, 10, and 21 dpi.

(F) Acetylene reduction activity (ARA) of Williams 82 nodules at 21 dpi with USDA 110 and Bott 59 incubation. The left y-axis represents specific nitrogenase activity per nodule mass (µmol C₂H₄ g⁻¹ h⁻¹), and the right y-axis represents total nitrogenase activity per plant (µmol C₂H₄ plant⁻¹ h⁻¹). The two axes are separated by a vertical dashed line.

(G-H) Nodule number per plant (G) and nodule fresh weight per plant (H) at 10 and 21 dpi. Boxes represent first quartile, median, and third quartile. Whiskers indicate minimum and maximum values; dots represent individual data points (n = 10, except for ARA, where n = 6). Different letters indicate statistically significant differences determined by one-way ANOVA followed by the Student–Newman–Keuls (SNK) post-hoc test for multiple comparisons. All tests were two-tailed, with significance set at P < 0.05. Significant differences between USDA 110 and Bott 59 were determined by a two-tailed Student's t-test (* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001).

 

Comment 3: It seems that Bott59 could promote the growth of soybean by compared with USDA110 this should be discussed in the discussion section.

Response: We appreciate the reviewer’s suggestion to further discuss the growth-promoting effects of Bott 59 compared to the model strain USDA 110. We have expanded the Discussion to explicitly address this comparison. Our revised text now:

1. Details the significant increases in shoot and root biomass observed in the Bott 59-inoculated group relative to USDA 110.
2. Connects these growth improvements directly to the enhanced nodule functionality (number, weight, and ARA) discussed in response to Comment 1, reinforcing the link between efficient G×G matching and systemic plant growth.
3. Integrated recent literature (e.g., Mataboge et al., 2025; Belane et al., 2015) to support the physiological mechanism, explaining that the biomass accumulation is driven by the synergistic assimilation of carbon (via photosynthesis) and nitrogen (via fixation), which is maximized in the compatible W82–Bott 59 combination.

Changes in the manuscript:

The following text has been added to the Discussion section, paragraph 2:

Furthermore, the enhanced SNF efficiency in the Williams 82-Bott 59 combination may be associated with improvements in host growth, with higher shoot and root biomass compared to inoculation with USDA 110. This aligns with the finding that inoculation with compatible, high-efficiency strains significantly boosts shoot biomass, carbon accumulation, and relative symbiotic effectiveness [60]. The correlation we observed between nodule traits (number and weight) and total plant biomass further supports the notion that efficient G×G matching drives systemic physiological improvements. Ultimately, these biomass gains are likely driven by the synergistic assimilation of carbon through photosynthesis and nitrogen through fixation, a process that is maximized only when the specific genetic requirements of both partners are met [60, 61].

 

Comment 4: How about the expression of RIC or NIC peptide genes relative to AON signaling pathway in this study.

Response: We sincerely thank the reviewer for the valuable comments. In response to the reviewer’s comments, we performed expression analysis on gene families related to the AON signaling pathway, including NIC, RIC, and TML. The results showed no significant differences in the expression of NIC family genes under rhizobial infection, which is consistent with our expectations in this study. This study mainly focuses on transcriptomic changes in host genes induced by rhizobial infection, whereas the expression of NIC family genes is primarily regulated by nitrogen treatment.

Expression analysis of the RIC family genes (RIC1a/b, RIC2a/2b) revealed that all RIC family genes were significantly upregulated following inoculation with USDA 110 and Bott 59. The expression patterns of the TML family genes (GmTML1a, GmTML1b, GmTML2) were consistent with those of the RIC family, and their expression was also induced by rhizobial infection. We further verified the expression level of RIC1b (Glyma.13G292300) and TML2 (Glyma.05G077700) using qRT-PCR. As shown in Figure 8, the qRT-PCR results were highly consistent with those from transcriptome analysis. These results indicate that rhizobial infection can activate the AON signaling pathway in soybean, and this activation mechanism is conserved among different rhizobial strains. This conclusion is also in line with findings from related studies in other legume species [22].

Additionally, we have discussed the TML2 and RIC1b genes related to the AON pathway in Part 2.2, paragraph 2. We appreciate the reviewer's insightful comment, which has strengthened the mechanistic connection between our findings and the AON pathway.

Changes in the manuscript:

1. Part 2.2, paragraph 2: Further analysis of these 727 conserved DEGs revealed a series of established key genes involved in SNF, including the master regulator NIN, the early NF signaling component RINRK1, nodule primordium development genes ENOD40 and NPL, and core AON pathway genes RIC and
2. 7. Transcript Level Analysis and mRNA Validation by qRT-PCR.

To verify the dynamic expression changes of DEGs following infection by rhizobial strains Bont 59 and USDA 110, as well as the robustness of the transcriptomic data, we selected four DEGs involved in the flavonoid signaling pathway (IFR, PTS, POD, and IF7GT), genes associated with the hormone transduction signaling pathway (AUX/IAA and A-ARR), and key symbiotic nitrogen fixation genes (NIN1a, ENOD40, RIC1b and TML2) for qRT PCR analysis (Figure 8). The qRT PCR results exhibited expression trends consistent with the transcriptomic analysis, thereby confirming the accuracy of the RNA seq data.

3. The following text has been added to the Discussion, paragraph 3:

“On the other hand, inoculation with different rhizobial strains (USDA 110, Bott 59) also rapidly activates the AON system and induces significant upregulation of negative regulators such as RIC and TML, thereby inhibiting excessive nodulation. These results suggest that soybean employs a sophisticated bidirectional regulatory mechanism in response to rhizobial infection. It not only activates signaling pathways related to symbiotic establishment to ensure effective nodulation, but also utilizes the conserved AON system to restrict excessive nodulation in a timely manner, preventing excessive energy and resource expenditure by the host due to overnodulation. This synergistic “promotion–inhibition” regulatory mode represents an adaptive strategy evolved by legumes over long-term evolution, which not only ensures symbiotic nitrogen fixation efficiency but also maintains the energy balance between host growth and development and the symbiotic process [22].

We sincerely appreciate the reviewer’s valuable feedback, which have significantly improved the quality of our work.

 

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Main Problems Identified in the Manuscript

Lack of independent validation of RNA-seq results. A major methodological weakness is the absence of RT-qPCR validation of selected DEGs. It is standard practice in transcriptomic studies to validate a subset of genes to confirm the reliability of RNA-seq results. No such validation is reported in the manuscript. This omission raises concerns regarding the reliability of key expression patterns highlighted in the study.

Lines 20–22: Limited temporal resolution of transcriptomic analysis - The study design indicates that transcriptomic analysis was conducted only at 3 days post-inoculation. This single time point is insufficient to capture the dynamic transcriptional changes involved in early nodulation and symbiotic signaling. Multiple time points would be necessary to properly describe the temporal regulation of gene expression during symbiosis.

Lines 23–30: The abstract presents strong conclusions regarding strain-specific activation of metabolic pathways (e.g., isoflavonoid and flavonoid biosynthesis) and hormonal signaling. However, these conclusions are based exclusively on transcriptomic data and are not supported by functional validation experiments, such as: RT-qPCR validation of key DEGs, metabolite quantification (e.g., isoflavonoid levels), nodulation assays linked to specific genes. Without some validation, the conclusions remain speculative. This limitation weakens the central claims of the manuscript.

Lines 153–161: The manuscript claims that strain Bott 59 significantly enhances soybean growth and symbiotic performance compared with USDA 110. However, the experimental design does not include sufficient controls or additional strains to support such general conclusions about strain efficiency.

Lines 179–183: RNA-seq analysis is based on a single sampling time point and lacks a clear description of normalization procedures, biological variability evaluation, and batch effect correction. These methodological details are necessary to ensure the reliability of transcriptomic analyses.

Lines 559–562: Each biological replicate consists of pooled root tissues from four plants, which may mask biological variability among samples and potentially bias the transcriptomic analysis. The rationale for pooling samples is not sufficiently discussed.

Lines 616–625: The discussion contains strong mechanistic interpretations regarding transcription factor networks and hormonal signaling pathways. However, these interpretations are speculative and not supported by independent experimental validation such as gene functional assays, mutant analysis, or metabolite quantification.

Minor Issues

1 - Inconsistencies in gene nomenclature: Gene names and formatting are sometimes inconsistent, particularly for soybean gene identifiers (e.g., Glyma IDs). Standardization following accepted plant gene nomenclature guidelines is recommended.

2- Figure clarity: Some figures (particularly pathway heatmaps) are visually dense and difficult to interpret. Improvements in figure labeling, resolution, and annotation would enhance readability.

3: Statistical reporting: Some figures indicate statistical significance but lack clear descriptions of the statistical tests used or adjustments for multiple comparisons.

Comments on the Quality of English Language

The manuscript contains numerous grammatical errors and awkward phrasing throughout the text. Examples include, incorrect article usage, inconsistent tense, typographical errors.

Examples include:

Lines 21–24: Awkward sentence construction and incorrect article usage in the description of the study objectives. The sentence structure should be revised to improve clarity and grammatical correctness.

Lines 76–79: Inconsistent verb tense when describing previous studies and background information. The paragraph alternates between present and past tense.

Lines 154–156: Incorrect article usage and unclear phrasing in the description of plant growth responses following inoculation.

Lines 312–316: Typographical and grammatical inconsistencies in the description of transcriptomic results, making the interpretation of the sentence difficult.

Lines 421–425: Some sentences contain redundant wording and minor grammatical errors that should be revised for clarity.

Lines 612–615: Awkward phrasing and long sentence structure in the discussion section. The sentence could be simplified to improve readability.

Professional English editing is strongly recommended.

 

Author Response

Dear Reviewer,

We are very grateful for your constructive comments and suggestions for our manuscript entitled "Transcriptomic Comparison of Soybean Roots Inoculated with Different Rhizobium Strains During Early Symbiosis " (ID: plants-4204122). Other changes have been marked in the manuscript using the revision mode (Track Changes). Please see below for our point-by-point responses to the editor's comments and concerns. The specific details of the modifications are shown as follows:

Comment 1: Lack of independent validation of RNA-seq results. A major methodological weakness is the absence of RT-qPCR validation of selected DEGs. It is standard practice in transcriptomic studies to validate a subset of genes to confirm the reliability of RNA-seq results. No such validation is reported in the manuscript. This omission raises concerns regarding the reliability of key expression patterns highlighted in the study.

Response 1: We sincerely thank the reviewer for highlighting this critical methodological point. We fully agree that independent qRT-PCR validation is standard practice and essential to confirm the reliability of transcriptomic profiling. In response to this comment, we have now performed independent qRT-PCR validation on a selected of ten DEGs, covering key functional pathways and a broad dynamic range of expression changes. Specifically, we validated four genes involved in the phenylpropanoid, flavonoid, and isoflavonoid biosynthesis pathways (IFR, PTS, POD, IF7GT), two hormone transduction genes (AUX/IAA and A-ARR), and four core symbiotic genes (NIN1a, ENOD40, RIC1b and TML2).

As presented in the revised Section 2.7 (Transcript Level Analysis and mRNA Validation by qRT-PCR.) and Figure 8, the qRT-PCR results exhibited expression trends that are highly consistent with the RNA-seq data across both rhizobial strains (Bont 59 and USDA 110). This independent validation supports the robustness and accuracy of the key expression patterns highlighted in our study.

Changes in the manuscript:

2.7. Transcript Level Analysis and mRNA Validation by qRT-PCR.

To verify the dynamic expression changes of DEGs following infection by rhizobial strains Bont 59 and USDA 110, as well as the robustness of the transcriptomic data, we selected four DEGs involved in the flavonoid signaling pathway (IFR, PTS, POD, and IF7GT), genes associated with the hormone transduction signaling pathway (AUX/IAA and A-ARR), and key symbiotic nitrogen fixation genes (NIN1a, ENOD40, RIC1b and TML2) for qRT PCR analysis (Figure 8). The qRT PCR results exhibited expression trends consistent with the transcriptomic analysis, thereby confirming the accuracy of the RNA seq data.

Comment 2: Lines 20–22: Limited temporal resolution of transcriptomic analysis - The study design indicates that transcriptomic analysis was conducted only at 3 days post-inoculation. This single time point is insufficient to capture the dynamic transcriptional changes involved in early nodulation and symbiotic signaling. Multiple time points would be necessary to properly describe the temporal regulation of gene expression during symbiosis.

Response 2: We sincerely thank the reviewer for this insightful comment regarding the temporal resolution of our transcriptomic analysis. We fully agree that symbiotic establishment is a dynamic process, and capturing gene expression profiles across multiple time points would indeed provide a more comprehensive view of the temporal regulation during nodulation.

However, we would like to clarify the specific rationale behind our experimental design focusing on the 3 dpi point:

1. Rhizobial infection of host roots follows a strict temporal sequence: root hair curling, infection thread formation, and activation of symbiotic signaling primarily occur at the early infection stage of 2-3 days post-inoculation (dpi). This time window represents a critical turning point for divergent interactions between distinct rhizobial strains and host plants, during which conserved symbiotic pathways and strain-specific regulatory pathways are significantly and simultaneously activated. By contrast, regulatory genes at the late infection stage are mainly involved in nodule development, and a large number of early interaction-specific differential signals will be masked. Therefore, sampling at 3 days post-inoculation enables a targeted focus on the initiation of rhizobium-host interactions, facilitating the precise identification of conserved and strain-specific regulatory patterns of host genes, and strengthening the pertinence of our research objectives.
2. The core objective of this study was to perform a horizontal comparison of host regulatory responses induced by different rhizobial strains during the early stages of symbiosis, in order to identify universally co-responsive genes and strain-specific responsive genes, rather than dissecting the temporal regulatory network underlying progressive rhizobial infection. In this experiment, infection conditions, cultivation environments and sampling criteria were strictly unified across all groups. Differences among parallel samples at a single time point are free from the interference of temporal gradients, which eliminates the background deviation caused by developmental discrepancies and ensures the comparability of gene expression profiles among different rhizobial treatments. Adding multiple time points would deviate from the core scientific question.
3. Root development is accompanied by time-dependent fluctuations in endogenous gene expression. Multi-time-point sampling would introduce background variations derived from intrinsic plant growth and development, making it difficult to distinguish rhizobium-specific induced genes from genes altered by natural developmental processes, and thereby increasing interference in subsequent bioinformatic analysis. Late rhizobial colonization and nodule primordium formation trigger extensive non-specific changes in secondary metabolism and cell differentiation, which further obscure the core differential genes underlying strain-specific interactions.

We acknowledge that additional time-series sampling would enable a more comprehensive dissection of host regulatory differences induced by distinct rhizobial strains throughout the entire symbiotic process. In future work, we will analyze multiple developmental stages to further investigate the divergent regulatory mechanisms of different rhizobia in soybean nodule formation and nitrogen fixation. Such content belongs to our future research directions and is not included within the scope and scientific objectives of the present study. We have now explicitly added a paragraph in the 3. Discussion section (the last paragraph) to address this limitation. We emphasize that while our data elucidates the early mechanisms of symbiosis, future studies employing time-series designs are essential for mapping the subsequent transcriptional cascades leading to mature nodule formation.

Changes in the manuscript:

The following text has been added to the 3. Discussion (the last paragraph):

“In this study, we systematically profiled the transcriptional responses of host plants at 3 dpi with diverse rhizobial strains, and identified conserved and strain-specific patterns of transcriptional regulation, including those of genes involved in multiple metabolic pathways. These findings provide essential data support for further dissecting the molecular mechanisms underlying soybean–rhizobium symbiotic nitrogen fixation. However, relying solely on single time-point data makes it difficult to comprehensively depict the continuous dynamic processes of nodule morphogenesis and functional differentiation. Meanwhile, whether such differentially regulated transcriptional changes can be directly translated into distinct alterations in host metabolic metabolites still requires systematic validation and clarification. Collectively, future studies employing diverse rhizobial strains, spanning the full symbiotic continuum from early infection to nodule senescence, and performing integrated multi-omics analyses complemented by metabolomics will provide more comprehensive datasets for systematically elucidating the molecular basis and metabolic regulatory mechanisms underlying strain-specific symbiotic interactions."

 

Comment 3: Lines 23–30: The abstract presents strong conclusions regarding strain-specific activation of metabolic pathways (e.g., isoflavonoid and flavonoid biosynthesis) and hormonal signaling. However, these conclusions are based exclusively on transcriptomic data and are not supported by functional validation experiments, such as: RT-qPCR validation of key DEGs, metabolite quantification (e.g., isoflavonoid levels), nodulation assays linked to specific genes. Without some validation, the conclusions remain speculative. This limitation weakens the central claims of the manuscript.

1. For qRT-PCR Validation: We sincerely appreciate the reviewer for this insightful and critical assessment. As detailed in our response to Comment 1, we have now performed independent qRT-PCR validation on ten key DEGs, including IFR, PTS, POD, IF7GT (phenylpropanoid/flavonoid/isoflavonoid biosynthesis), AUX/IAA and A-ARR (hormone transduction), and NIN1a, ENOD40, RIC1b and TML2 (symbiosis). The qRT-PCR results show high concordance with RNA-seq trends (Figure 8, Section 2.7), confirming that the observed strain-specific transcriptional activation is robust and reproducible.
2. For metabolite analysis: We fully acknowledge and appreciate the reviewer’s valuable comment. Integrated analysis of transcriptomics and metabolomics would facilitate a more comprehensive understanding of the correlation between transcriptional responses and metabolic alterations induced by different rhizobial strains. However, the present study is restricted to transcriptomic data for the two following reasons:
3. This study focuses on systematically dissecting the global responses of soybean to infection by different rhizobial strains at the transcriptomic level. We aim to elucidate the conserved patterns and strain-specific regulatory mechanisms of gene expression, with an emphasis on comparing the similarities and differences in transcriptional regulation and signaling pathways activated by distinct rhizobia. As our research centers on differential regulatory patterns at the transcriptional level, transcriptomic evidence alone is adequate to validate the core scientific hypotheses. Based on functional enrichment analysis, we identified conserved and strain-specific patterns in the transcriptional changes of host genes linked to multiple biological processes, including metabolic homeostasis, plant development, and immune responses, during early symbiotic infection by different rhizobial strains. Therefore, integrated metabolomic analysis is not a prerequisite for addressing the core scientific questions of this study.
4. The time point of 3 days post-inoculation (3 dpi) corresponds to the early symbiotic stage, which is critical for infection thread formation and the initial differentiation of nodule primordia. During this period, soybean initiates rapid transcriptional reprogramming and activates sophisticated signal transduction networks to respond to rhizobial infection and colonization. Nevertheless, biological processes follow a distinct hierarchical temporal sequence, in which transcription, protein translation, and metabolite accumulation are asynchronous. A notable time lag exists from transcript expression to functional protein synthesis, followed by the massive accumulation of downstream metabolites. Accordingly, combined transcriptome and metabolome analysis at the early symbiotic stage may increase the complexity and uncertainty of multi-omics interpretation.

Admittedly, further integration with metabolomic analysis would enable a more comprehensive characterization of the physiological and metabolic changes in host plants induced by rhizobial symbiosis, and help identify key functional genes that precisely regulate critical metabolic pathways, yet it is not indispensable for the present study’s goal of elucidating conserved and strain-specific transcriptomic regulatory networks. We intend to incorporate metabolomics across multiple symbiotic developmental stages in future multi‑omics studies, which lies beyond the scope of the current work.

To ensure scientific rigor and avoid overinterpretation of the dataset, we inferred potential metabolic changes that are explicitly presented as transcriptional predictions in the revised Discussion, while all language implying direct biochemical measurements has been removed or appropriately moderated. In addition, we have discussed the value of performing integrated transcriptomic and metabolomic analyses in future follow-up research.

Changes in the manuscript:

Abstract:

Notably, at the transcript level, Bott 59 specifically induced the expression of genes involved in isoflavonoid and flavonoid biosynthesis, including those encoding I2H and HI4OMT. In addition, Bott 59 was associated with more extensive transcriptional changes in auxin, cytokinin, and jasmonic acid signaling pathways, as well as a broader range of transcription factor genes.

Discussion:

In this study, we systematically profiled the transcriptional responses of host plants at 3 3 dpi with diverse rhizobial strains, and identified conserved and strain-specific patterns of transcriptional regulation, including those of genes involved in multiple metabolic pathways. These findings provide essential data support for further dissecting the molecular mechanisms underlying soybean–rhizobium symbiotic nitrogen fixation. However, relying solely on single time-point data makes it difficult to comprehensively depict the continuous dynamic processes of nodule morphogenesis and functional differentiation. Meanwhile, whether such differentially regulated transcriptional changes can be directly translated into distinct alterations in host metabolic metabolites still requires systematic validation and clarification. Collectively, future studies employing diverse rhizobial strains, spanning the full symbiotic continuum from early infection to nodule senescence, and performing integrated multi-omics analyses complemented by metabolomics will provide more comprehensive datasets for systematically elucidating the molecular basis and metabolic regulatory mechanisms underlying strain-specific symbiotic interactions.

 

Comment 4: Lines 153–161: The manuscript claims that strain Bott 59 significantly enhances soybean growth and symbiotic performance compared with USDA 110. However, the experimental design does not include sufficient controls or additional strains to support such general conclusions about strain efficiency.

Response 4: We greatly appreciate the reviewer's valuable feedback on this point. And we agree that comparing only two strains limits the ability to draw broad, generalizable conclusions regarding the absolute efficiency of strain Bott 59 across diverse genetic backgrounds.

Our primary objective in this study was not to perform large-scale screening of multiple rhizobial strains, but to conduct a comparative transcriptomic analysis between a newly isolated elite strain (Bott 59) and the widely used reference strain (USDA 110) in order to characterize conserved and strain-specific gene regulation. With this focused aim, we only compared the symbiotic characteristics of the two rhizobial strains in host plants and analyzed their differential regulation of host gene expression during the early symbiotic stage.

However, we acknowledge that our original phrasing ("significantly enhances...") might have implied a broader generalization than our experimental design supports. Accordingly, we have taken the following steps:

1. Tempered our conclusions throughout the manuscript. We now explicitly state that the enhanced growth and nitrogen fixation performance of Bott 59 are observed relative to USDA 110 under the tested conditions, rather than making a universal claim about its superiority over all conventional strains.
2. Added a limitation statement in 3. Discussion (the last paragraph), noting that future studies involving a wider panel of rhizobial strains will be necessary to validate the general applicability of these findings.
3. Refined the specific sentence in question to ensure precision.

Changes in the manuscript:

Reviewer's comments on Lines 153–161 have now been revised as follows:

However, by 21 dpi, both Bott 59- and USDA 110- inoculated groups displayed increased plant height, shoot dry weight, and root dry weight compared to the CK group (Figure 1B, D, E). Notably, when comparing the two strains, plants inoculated with Bott 59 exhibited higher shoot and root dry weights than those inoculated with USDA 110 at 21 dpi (Figure 1D, E). Moreover, at both 10 and 21 dpi, both the nodule number and the fresh nodule weight were superior to those in inoculated with Bott 59 than in those inoculated with USDA 110 (Figure 1G and H). Additionally, the nitrogenase activity (ARA) was higher in Bott 59-inoculated plants than in the USDA 110-inoculated plants at 21 dpi (Figure 1F). In summary, compared to the conventional strain USDA 110, inoculation with the strain Bott 59 appears to promote the growth performance and symbiotic nitrogen fixation capacity.

 

Comments 5: Lines 179–183: RNA-seq analysis is based on a single sampling time point and lacks a clear description of normalization procedures, biological variability evaluation, and batch effect correction. These methodological details are necessary to ensure the reliability of transcriptomic analyses.

Response 5: We thank the reviewer for highlighting the need for a clear assessment of biological variability. We agree that rigorous assessment of biological variability and transparent reporting of normalization and batch effect handling are critical for ensuring the reliability of our RNA-seq conclusions. We have taken the following steps to address the concerns regarding data quality and analytical rigor:

1. Assessment of Biological Variability (PCA Analysis): To explicitly evaluate biological reproducibility, we performed a Principal Component Analysis (PCA) on all samples. As shown in the newly added Supplementary Figure S1, the three biological replicates for each treatment group (CK, Bott 59, and USDA 110) cluster tightly together. This confirms high experimental reproducibility and low biological noise within groups. Furthermore, the distinct separation between the control and inoculated groups validates that the observed transcriptional changes are driven by the rhizobial treatments rather than random variation. We have added a description of these results in the Results section (Section 2.2).
2. Clarification of Normalization Procedures. We have updated the Materials and Methods section to explicitly detail our normalization strategy. We clarified that raw count data were normalized using the median of ratios method implemented in the DESeq2 package. This method effectively corrects for differences in library size and RNA composition. The supplementary details are presented in Section 4.3.
3. Batch Effect Correction: Regarding the batch effect concerns, a phased strategy was implemented in the analysis pipeline to address this issue. The specific procedures are detailed below:
   1. Data Preprocessing Stage: Normalization via RSEM

Normalization performed by RSEM effectively mitigated the direct impact of technical biases from the source, ensuring the comparability of expression levels across samples.

1. Differential Analysis Stage: Statistical Correction via DESeq2

Differential expression analysis was conducted using DESeq2. Batch information was explicitly incorporated as a covariate in the design formula of the generalized linear model (design = ~ batch + group). This approach estimates and removes variance attributable to batch effects prior to assessing inter-group significance, thereby ensuring that identified differentially expressed genes are primarily driven by biological conditions.

In summary, the reliability of the differential analysis was safeguarded by combining RSEM-based preprocessing with DESeq2-based statistical modeling. Furthermore, PCA plots comparing samples before and after batch correction are provided in the Supplementary Materials (Figure S1). These plots demonstrate that samples cluster by experimental group rather than by batch after correction, further validating the effectiveness of the approach.

Changes in the manuscript:

Part 2.2

To assess biological variability and sample consistency, Principal Component Analysis (PCA) was performed. As shown in Supplementary Figure S1, the three biological replicates for each treatment group (CK, Bott 59, and USDA 110) cluster tightly together, suggesting high experimental reproducibility and low biological noise. Furthermore, the distinct separation between the control and inoculated groups validates that the observed transcriptional changes are driven by the rhizobial treatments rather than random variation.

Part 4. Materials and Methods 4.3.

Gene expression levels were quantified using RSEM software and are reported in FPKM (Fragments Per Kilobase of transcript per Million mapped reads). DEGs were identified using DESeq2, with potential batch effects statistically corrected by explicitly including the batch variable in the design matrix (design = ~ batch + condition). Genes with a P-value < 0.05 and |log₂FC| > 1 were considered significantly differentially expressed.

 

Comments 6：Lines 559–562: Each biological replicate consists of pooled root tissues from four plants, which may mask biological variability among samples and potentially bias the transcriptomic analysis. The rationale for pooling samples is not sufficiently discussed.

Response 6: We sincerely thank the reviewer for raising this important methodological consideration. This design aims to average out stochastic inter-plant variation through bulk sampling, reduce sampling error, and enhance sample representativeness. Accordingly, the observed transcriptional changes can accurately reflect genuine treatment effects, rather than accidental deviations derived from individual plant outliers.

We have now explicitly detailed this rationale in the revised

Methods section 4.1 (the last paragraph). The updated text reads:

“To minimize transcriptomic fluctuations derived from stochastic plant-to-plant variation and microenvironmental noise, while retaining inherent biological variance across replicates, we established three independent biological replicates, with each sample generated by pooling root tissues from four individual plants.”

We clarify that pooling was employed not to replace biological replication, but to complement it: each of the three replicates represents a distinct pool, preserving inter-replicate biological variance. We appreciate the reviewer’s rigorous feedback, which has significantly improved the methodological rigor of our study.

 

Comments 7：Lines 616–625: The discussion contains strong mechanistic interpretations regarding transcription factor networks and hormonal signaling pathways. However, these interpretations are speculative and not supported by independent experimental validation such as gene functional assays, mutant analysis, or metabolite quantification.

Response 7: We sincerely thank the reviewer for pinpointing this specific passage. We fully agree that the original wording overstepped the evidence by implying direct mechanistic activation without functional or metabolite validation. We have added qRT-PCR validation (Section 2.7. Transcript Level Analysis and mRNA Validation by qRT-PCR). Furthermore, we have completely rewritten Lines 616–625 (original) to accurately reflect the transcript-level nature of our data, replace definitive claims with evidence-aligned inferences, and explicitly acknowledge the need for downstream validation.

The revised text now reads (5. Conclusion, paragraph 3 and 4)

“These data suggest that Bott 59 effectively reinforces the early molecular dialogue with the host, thereby accelerating nodule primordium initiation. Bott 59 elicits more robust auxin, cytokinin, and JA signaling responses while orchestrating a more extensive array of TFs from the MYB, bHLH, and B3 families. This sophisticated regulatory network likely drives the rapid physiological transition of the root system from basal defense to high-efficiency symbiotic development. While these correlations are strong, future functional assays will be essential to definitively dissect the causal mechanisms within this network.

In conclusion, Bott 59 shows great potential for high-efficiency nitrogen fixation. We clarified the molecular mechanisms driving its improved growth performance and pinpointed key regulators, such as HI4OMT and I2H. Collectively, these results provide valuable insights for enhancing soybean nitrogen fixation via breeding and genetic engineering.”

We appreciate the reviewer’s rigorous attention to mechanistic precision, which has substantially improved the scholarly rigor and balance of our discussion.

 

Minor Issues

Comment 8: Inconsistencies in gene nomenclature: Gene names and formatting are sometimes inconsistent, particularly for soybean gene identifiers (e.g., Glyma IDs). Standardization following accepted plant gene nomenclature guidelines is recommended.

Response 8: We sincerely thank the reviewer for this critical observation. We fully acknowledge that soybean gene nomenclature has evolved over time, resulting in varied formats (e.g., differences in prefixes, punctuation, capitalization, and digit counts) across major databases such as EnsemblPlants, Phytozome, and SoyBase, as well as in legacy literature. To address this issue, we adopted a more general representation method: gene identifiers are formatted in italics, i.e., "Glyma.XXGXXXXXX". We then verified every soybean gene identifiers throughout the manuscript, including the abstract, main text, figures, and supplementary materials, to ensure consistency with the chosen standard.

 

Comment 9: Figure clarity: Some figures (particularly pathway heatmaps) are visually dense and difficult to interpret. Improvements in figure labeling, resolution, and annotation would enhance readability.

Response 9: We sincerely thank the reviewer for this constructive comment regarding figure clarity. In response, we have carefully revised the pathway heatmaps in Figures 4 and 6 to reduce visual density and improve interpretability. Specifically, we have:

1. Increased the font sizes for axis labels and legends.
2. Added targeted explanatory annotations to facilitate direct interpretation.
3. These modifications have significantly enhanced readability while preserving essential data. The updated figures are now presented in the revised manuscript (please see the revised Figures 1, 4 and 6). We greatly appreciate this valuable suggestion, which has substantially improved the visual presentation and clarity of our results.

 

Comment 10: Statistical reporting: Some figures indicate statistical significance but lack clear descriptions of the statistical tests used or adjustments for multiple comparisons.

Response 10: We thank the reviewer for this important comment regarding statistical transparency. In the revised manuscript, we have updated the legends of Figure 1 and Figure 8 to explicitly state the statistical tests, multiple comparison adjustment, and test directionality. All tests were two-tailed, and the significance threshold was set at P < 0.05. These details have been clearly added to the corresponding figure legends and are fully consistent with the Statistical Analysis section. Please see the legends for Figures 1 and 8.

 

Comment 11: Comments on the Quality of English Language

The manuscript contains numerous grammatical errors and awkward phrasing throughout the text. Examples include, incorrect article usage, inconsistent tense, typographical errors.

Examples include:

Lines 21–24: Awkward sentence construction and incorrect article usage in the description of the study objectives. The sentence structure should be revised to improve clarity and grammatical correctness.

Lines 76–79: Inconsistent verb tense when describing previous studies and background information. The paragraph alternates between present and past tense.

Lines 154–156: Incorrect article usage and unclear phrasing in the description of plant growth responses following inoculation.

Lines 312–316: Typographical and grammatical inconsistencies in the description of transcriptomic results, making the interpretation of the sentence difficult.

Lines 421–425: Some sentences contain redundant wording and minor grammatical errors that should be revised for clarity.

Lines 612–615: Awkward phrasing and long sentence structure in the discussion section. The sentence could be simplified to improve readability.

Professional English editing is strongly recommended.

Response 11: We sincerely thank the reviewer for the constructive feedback regarding the language quality of our manuscript. We fully acknowledge the grammatical, stylistic, and typographical issues present in the original submission. In response, we have engaged a professional English editing service to carefully proofread and polish the entire text. All identified concerns including incorrect article usage, inconsistent verb tenses, awkward phrasing, and redundant wording, have been systematically corrected. Specifically, we have carefully revised the sentences highlighted by the reviewer (original Lines 21–24, 76–79, 154–156, 312–316, 421–425, and 612–615) to ensure clarity, grammatical accuracy, and logical flow. The detailed revisions for these sections are provided below. A certificate of professional English editing from MDPI has been included in the Supplementary Materials. We believe the revised manuscript now reads clearly and meets the journal’s language standards, and we greatly appreciate this valuable suggestion.
The revised sentences are as follows:

Lines 21–24 (original)：Symbiotic phenotypes were evaluated after inoculation and root transcriptomic analysis was conducted at 3 dpi to assess early molecular responses. Results: At 21 dpi, Bott 59-inoculated plants outperformed plants inoculated with USDA 110 in nodule number, nitrogenase activity, and biomass.

Lines 76–79 (original): Flavonoids are crucial signaling molecules during the establishment of rhizobia-legume symbiosis [23]. Isoflavonoids and flavonoids share the upstream biosynthetic pathway starting from phenylalanine to naringenin chalcone, catalyzed sequentially by phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H), 4-coumarate-CoA ligase (4CL), and chalcone synthase (CHS) [24].

Lines 154–156 (original): Moreover, at both 10 and 21 dpi, the nodule number and the fresh nodule weight were superior to those in inoculated with Bott 59 than in those inoculated with USDA 110 (Figure 1G and H).

Lines 312–316 (original): Transcriptomic data displayed consistent regulatory directions across multiple hormone signaling pathways following inoculation with different strains, confirming that hormonal regulation is a conserved and essential strategy for establishing symbiosis. While the activation of BR and SA signaling represents a highly conserved molecular mechanism, the auxin, cytokinin, and JA pathways showed distinct strain-specific responses, with Bott 59 inducing more extensive transcriptional reprogramming.

Lines 421–425 (original): We sincerely thank the reviewer for this insightful comment Guided by your suggestion, and to ensure consistency with overlapping feedback from the other reviewers, we have comprehensively rewritten and replaced the content at the indicated locations. This revision was undertaken to better integrate the relevant concepts, improve logical coherence, and fully address the shared concerns raised by multiple reviewers regarding the interpretation of the data. The updated text now provides a more precise, well-structured, and scientifically rigorous discussion. Please refer to the revised manuscript (3. Discussion, Paragraphs 1–3). We greatly appreciate your valuable guidance, which has substantially strengthened this part of the manuscript.

Lines 612–615 (original): While both strains share conserved mechanisms for mobilizing the common symbiosis signaling pathway (e.g., NIN), phenylpropanoid biosynthesis pathway, BR, and SA signal transduction pathway, Bott 59 elicits a broader range of gene responses. Notably, transcriptomic profiling indicates a pronounced enrichment of isoflavonoid biosynthesis-associated genes following Bott 59 inoculation, with upregulation of key pathway genes including HI4OMT and I2H.

We sincerely appreciate the reviewer’s valuable feedback, which have significantly improved the quality of our work.

 

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

In this manuscript, the authors present a comparative transcriptomic study between two rhizobia strains at three days’ post-inoculation (3 dpi) in soybean plants. The study is well-grounded; the introduction provides a clear and correct context, and the data analysis is robust. Furthermore, the figures are comprehensive, the results sections are logically structured, and the conclusions are in full alignment with the findings presented.

However, there are two significant concerns that must be addressed to meet the standards of the field:

1. Experimental Validation: There is an absence of experimental validation for key genes via qRT-PCR, which is standard practice to support transcriptomic findings.
2. Data Availability: The current Data Availability Statement is insufficient for a transcriptomic study. According to the journal’s policy and international standards for high-throughput sequencing data, the raw RNA-seq reads (FASTQ files) and processed data must be deposited in a public repository (such as NCBI GEO or SRA). The authors must provide the accession number(s) in the revised manuscript to ensure reproducibility and transparency. Additionally, comprehensive tables containing the list of Differentially Expressed Genes (DEGs) and their corresponding Log₂(FC) values must be made available, either within the chosen repository or as Supplementary Material.

Additionally, there are several minor points regarding formatting, consistency, and figure clarity that should be addressed to improve the manuscript's quality.

Please find my specific suggestions below:

General and Figures

• Line 151: Please define the acronym CK upon its first mention, including within the legend of Fig. 1.
• Fig. 1 (Panel E, 21 dpi): The colors for USDA and Bott should be swapped to remain consistent with the other charts.
• Fig. 1 (Panels F and G): Increase the spacing between these panels to ensure the Y-axis labels are clearly separated and to avoid confusion.
• Fig. 5A: In the panel showing genes related to the TMK1/4 pathway, all entries appear to be the same gene. Please verify if this is correct.

Text Corrections and Formatting

• Line 170: Add a missing space between “activity” and “of”.
• Line 177: Change the section numbering from 3.2 to 2.2.
• Line 184: Since this is the first mention of Log₂(FC), please provide the full term (Fold Change) followed by the abbreviation in parentheses.
• Line 209: Change numbering from 3.3 to 2.3.
• Line 266: Change numbering from 3.4 to 2.4.
• Line 325: Change numbering from 3.5 to 2.5.
• Line 331: There is a discrepancy regarding the gene encoding 4CL: the ID in the text (Glyma.03G028700) does not match the one provided in the table (Glyma.19G075800).
• Line 356: Regarding the phrase "two homologs encoding F3H (Glyma.02G048400)": Since the text mentions two homologs but only provides one ID, I suggest either listing both gene IDs or omitting them entirely, as they are already detailed in the figure table.
• Line 378: Specifying the comparison again here is redundant, as it was established in the previous line for the entire figure. I recommend deleting “from the Bott 59 vs. USDA 110 comparison” and “from the same comparison.”
• Line 379: Replace the comma after “represent Log₂(FC)” with a dot.
• Line 381: Change numbering from 3.6 to 2.6.
• Line 420: Format the "2" in Log₂(FC) as a subscript.
• Line 450: This appears to refer to Figure 6A instead of 7A. Please check.
• Line 543: Delete the redundant phrase “nutrient solution.”

References

• Citations: Several references are missing the journal title (e.g., 6, 11, 13, 18, 20, ...). All citations must include the journal title in its abbreviated form, according to the journal's specific formatting guidelines.

Author Response

Dear Reviewer,

We are very grateful for your constructive comments and suggestions for our manuscript entitled "Transcriptomic Comparison of Soybean Roots Inoculated with Different Rhizobium Strains During Early Symbiosis " (ID: plants-4204122). Other changes have been marked in the manuscript using the revision mode (Track Changes). Please see below for our point-by-point responses to the editor's comments and concerns. The specific details of the modifications are shown as follows:

In this manuscript, the authors present a comparative transcriptomic study between two rhizobia strains at three days’ post-inoculation (3 dpi) in soybean plants. The study is well-grounded; the introduction provides a clear and correct context, and the data analysis is robust. Furthermore, the figures are comprehensive, the results sections are logically structured, and the conclusions are in full alignment with the findings presented.

However, there are two significant concerns that must be addressed to meet the standards of the field:

Comment 1: Experimental Validation: There is an absence of experimental validation for key genes via qRT-PCR, which is standard practice to support transcriptomic findings.

Response 1: We sincerely thank the reviewer for highlighting this critical methodological point. We fully agree that independent qRT-PCR validation is standard practice and essential to confirm the reliability of transcriptomic profiling. In response to this comment, we have now performed independent qRT-PCR validation on a selected of ten DEGs, covering key functional pathways and a broad dynamic range of expression changes. Specifically, we validated four genes involved in the phenylpropanoid, flavonoid, and isoflavonoid biosynthesis pathways (IFR, PTS, POD, IF7GT), two hormone transduction genes (AUX/IAA and A-ARR), and three core symbiotic genes (NIN1a, ENOD40, RIC1b and TML2).

As presented in the revised Section 2.7 (Transcript Level Analysis and mRNA Validation by qRT-PCR.) and Figure 8, the qRT-PCR results exhibited expression trends that are highly consistent with the RNA-seq data across both rhizobial strains (Bont 59 and USDA 110). This independent validation supports the robustness and accuracy of the key expression patterns highlighted in our study.

 

Comment 2: Data Availability: The current Data Availability Statement is insufficient for a transcriptomic study. According to the journal’s policy and international standards for high-throughput sequencing data, the raw RNA-seq reads (FASTQ files) and processed data must be deposited in a public repository (such as CNCB GEO or SRA). The authors must provide the accession number(s) in the revised manuscript to ensure reproducibility and transparency. Additionally, comprehensive tables containing the list of Differentially Expressed Genes (DEGs) and their corresponding Log₂(FC) values must be made available, either within the chosen repository or as Supplementary Material.

Response 2: Response: We sincerely thank the reviewer for this important reminder regarding data transparency and reproducibility. We have deposited all raw RNA-seq reads (FASTQ files) in the CNCB Genome Sequence Archive (GSA). The corresponding BioProject accession number is PRJCA062000 which has been added to 4.7. Data availability statement. (The data is currently undergoing the validation process. We commit to releasing the data immediately upon the acceptance of the manuscript.)

Furthermore, comprehensive supplementary tables containing all Differentially Expressed Genes (DEGs) and Transcription Factors (DETFs), along with their corresponding Log₂(FC) and statistical values (P-value), have been provided as Supplementary Materials. Specifically, we have included:

Table S3: Total DEGs (P-value < 0.05) in soybean roots: Bott 59-inoculated vs. CK.

Table S4: Total DEGs (P-value < 0.05) in soybean roots: USDA 110-inoculated vs. CK. Table S5:

Total DEGs (P-value < 0.05) in soybean roots: Bott 59-inoculated vs. USDA 110-inoculated.

Table S6: Total DETFs (P-value < 0.05) in soybean roots: Bott 59-inoculated vs. CK.

Table S7: Total DETFs (P-value < 0.05) in soybean roots: USDA 110-inoculated vs. CK.

Table S8: Total DETFs (P-value < 0.05) in soybean roots: Bott 59-inoculated vs. USDA 110-inoculated.

Table S9: FPKM values of all expressed genes in soybean roots under CK, USDA 110 inoculated, and Bott 59.

Table S10: KEGG enrichment analysis of Bott 59-inoculated vs. CK.

Table S11: KEGG enrichment analysis of USDA 110-inoculated vs. CK.

Table S12: KEGG enrichment analysis of Bott 59-inoculated vs. USDA 110-inoculated.

Table S13: GO enrichment analysis of Bott 59-inoculated vs. CK.

Table S14: GO enrichment analysis of USDA 110-inoculated vs. CK.

Table S15: GO enrichment analysis of Bott 59-inoculated vs. USDA 110-inoculated.

 

Comments: Additionally, there are several minor points regarding formatting, consistency, and figure clarity that should be addressed to improve the manuscript's quality.

Please find my specific suggestions below:

General and Figures

Line 151: Please define the acronym CK upon its first mention, including within the legend of Fig. 1.

Response: We sincerely thank the reviewer for this helpful suggestion. As requested, we have defined the acronym “CK” at its first appearance in the main text as “control (CK)” (Part 2.1). The full definition has also been added to the legend of Figure 1 for consistency. Thank you again for pointing this out, which has improved the clarity of our manuscript.

Fig. 1 (Panel E, 21 dpi): The colors for USDA and Bott should be swapped to remain consistent with the other charts.

Fig. 1 (Panels F and G): Increase the spacing between these panels to ensure the Y-axis labels are clearly separated and to avoid confusion.

Response: We sincerely thank the reviewer for pointing out this inconsistency. As suggested, we have updated the color scheme in Figure 1. In the revised figure, we have also increased the horizontal spacing between Figure 1F and Figure 1G to ensure clear separation of the Y-axis labels and prevent visual overlap. This adjustment has significantly enhanced the clarity of the composite figure.

 

Fig. 5A: In the panel showing genes related to the TMK1/4 pathway, all entries appear to be the same gene. Please verify if this is correct.

We sincerely thank the reviewer for this careful observation. The repeated appearance of the same gene label in the TMK1/4 pathway panel of Figure 5A was due to a labeling error during figure assembly. We have carefully cross-checked the original dataset and corrected all gene annotations accordingly. The revised Figure 5A now accurately displays the distinct genes associated with the TMK1/4 pathway. This correction has been implemented in the revised manuscript (please see the updated Figure 5). We apologize for any confusion this oversight may have caused and greatly appreciate the reviewer’s rigorous attention to detail.

 

Comment: Text Corrections and Formatting

Line 170: Add a missing space between “activity” and “of”.

Line 177: Change the section numbering from 3.2 to 2.2.

Line 184: Since this is the first mention of Log₂(FC), please provide the full term (Fold Change) followed by the abbreviation in parentheses.

Line 209: Change numbering from 3.3 to 2.3.

Line 266: Change numbering from 3.4 to 2.4.

Line 325: Change numbering from 3.5 to 2.5.

Line 381: Change numbering from 3.6 to 2.6.

Response: We sincerely thank the reviewer for the meticulous proofreading of our manuscript. All the suggested text and formatting corrections have been carefully implemented in the revised version:

Line 170 (original): The legend of Figure 1F has been rewritten.

Line 177 (original): The section numbering has been corrected from "3.2" to "2.2".

Line 184 (original): The first mention has been revised to "|log₂ fold change (FC)|" to provide the full term followed by the abbreviation in parentheses.

Line 209 (original): The section numbering has been corrected from "3.3" to "2.3".

Line 266 (original): The section numbering has been corrected from "3.4" to "2.4".

Line 325 (original): The section numbering has been corrected from "3.5" to "2.5".

Line 381 (original): The section numbering has been corrected from "3.5" to "2.6".

We have also conducted a thorough proofread of the entire manuscript to ensure consistency in formatting, section numbering, and abbreviation usage. All changes are clearly marked in the tracked-changes version and incorporated into the clean revised manuscript.

Comment: Line 331: There is a discrepancy regarding the gene encoding 4CL: the ID in the text (Glyma.03G028700) does not match the one provided in the table (Glyma.19G075800).

Line 356: Regarding the phrase "two homologs encoding F3H (Glyma.02G048400)": Since the text mentions two homologs but only provides one ID, I suggest either listing both gene IDs or omitting them entirely, as they are already detailed in the figure table.

Response: We sincerely thank the reviewer for their meticulous proofreading and helpful suggestions. Both points have been carefully addressed in the revised manuscript:

Line 331 (original): We have corrected the 4CL gene ID in the text to match the table entry (Glyma.19G075800) (Part 2.5).

Line 356 (original): Following the reviewe’s suggestion, we have removed the specific gene ID from the main text to avoid redundancy and potential inconsistency (Part 2.5).

Comment: Line 378: Specifying the comparison again here is redundant, as it was established in the previous line for the entire figure. I recommend deleting “from the Bott 59 vs. USDA 110 comparison” and “from the same comparison.”

Line 379: Replace the comma after “represent Log₂(FC)” with a dot.

Response: We sincerely thank the reviewer for these precise suggestions to improve the conciseness and clarity of the manuscript. As recommended, we have:

Line 378 (original): We have deleted the sentence “The lower left panel shows DEGs in the isoflavonoid pathway from the Bott 59 vs. USDA 110 comparison; the lower right panel shows DEGs in the flavonoid pathway from the same comparison.” to avoid unnecessary repetition (Part 2.5, Legend of Figure 6C).

Line 379 (original): Corrected the punctuation by replacing the comma after “represent Log2(FC)” with a dot (Part 2.5, Legend of Figure 6C).

Line 420: Format the "2" in Log₂(FC) as a subscript.

Response: We thank the reviewer for this suggestion The “2” in “Log₂(FC)” has been formatted as a subscript as requested (Part 2.6, Legend of Figure 7C).

Line 450: This appears to refer to Figure 6A instead of 7A. Please check.

Response: We appreciate the reviewer’s careful reading. The citation has been corrected from “Figure 7A” to “Figure 6A”(3.Discussion, paragraph 5).

Line 543: Delete the redundant phrase “nutrient solution.”

Response: Thank you for pointing this out. The redundant phrase “nutrient solution” has been removed to improve clarity and conciseness (Part 4.1).

References

Citations: Several references are missing the journal title (e.g., 6, 11, 13, 18, 20, ...). All citations must include the journal title in its abbreviated form, according to the journal's specific formatting guidelines.

Response: We have checked and corrected all references. These corrections are clearly marked in the tracked-changes version. We greatly appreciate the reviewer’s rigorous attention to detail, which has significantly improved the accuracy and consistency of our manuscript.

We sincerely appreciate the reviewer’s valuable feedback, which have significantly improved the quality of our work.

 

Author Response File: Author Response.docx

Reviewer 4 Report

Comments and Suggestions for Authors

This study investigates the symbiotic relationship between soybean and an efficient rhizobial strain (B. ottawaense Bott 59), providing valuable candidate genes for early symbiotic responses through comparative transcriptome analysis. However, there are structural numbering, formatting, and grammatical errors in the manuscript that require careful revision.

Lack of qPCR validation for transcriptome data: This study relies on RNA-seq data to characterize gene expression profiles without experimental validation. Using independent experimental methods to verify high-throughput sequencing results is a fundamental standard practice. The authors are advised to perform RT-qPCR analysis on a subset of representative differentially expressed genes (DEGs), particularly core genes emphasized in the discussion section (such as IF7MAT, I2H, and key hormone signaling genes), to confirm the reliability of the transcriptome data.

Non-standard statistical presentation in KEGG analysis (Figure 3): The x-axis of the KEGG pathway enrichment analysis in Figure 3 uses gene count. Simple gene count cannot reflect the statistical significance of pathway enrichment, as basic metabolic pathways containing large numbers of genes often have high count values but may not be specifically enriched. The authors should redraw this figure using statistical significance indicators (such as P-value or FDR) as the x-axis, or adopt a standard bubble plot (where dot size represents gene count and color intensity represents significance).

Formatting standards: The n representing sample size and P representing significance in figures should both be italicized.

Color scheme: Please avoid using red-green color schemes in figures (such as bar/box plots in Figure 1) to accommodate readers with color vision deficiency.

Image clarity: The scatter points in the box plots of Figure 1 lack sufficient clarity; please upload higher resolution images.

Figure legends: Many main figure legends are overly simplistic (e.g., Figure 4). Legends should be sufficiently detailed to enable readers to "understand the figure content by reading the legend alone."

Section duplication: Another "3. Discussion" appears at line 421. This means the article has two Section 3s; the logical hierarchy of the entire Results and Discussion section needs to be reorganized and renumbered.

Inconsistent title capitalization: The title "3.6. Expression patterns of Differentially expressed transcription factors (DETFs)..." (line 381) uses all lowercase in the latter half, while previous titles (such as 3.5) used title case format. Please standardize this.

Species scientific names: The scientific name of Lotus japonicus is misspelled at lines 88 and 115; the correct Latin name should be L. japonicus, not L. japonicas.

Basic spelling: Line 150 "primay root length" should be "primary"; lines 183-184 "Bott 59-innoculated" has an extra 'n' and should be "inoculated."

Line 43-44: "into ammonia (NH3), it can be directly assimilated..." contains a comma splice and should be changed to "...(NH3), which can be..."

Lines 59-60: "promotes... induces... and ultimately trapping..." contains a parallel structure error and should be changed to "traps."

Lines 67-68: "sustained calcium oscillations... is triggered" contains subject-verb disagreement and should be "are triggered."

Line 100: "coordinately governing" should be changed to "govern."

Line 488: "In this study found that" is a mixed sentence structure and should be changed to "This study found that..."

Proper noun capitalization: Line 143 uses capitalized "Williams 82" in the title, but line 147 in the main text uses lowercase "williams 82"; please maintain consistency.

Mixed Chinese and English punctuation: In the Acknowledgments/Funding section, lines 633-634 contain errors with half English parentheses and half Chinese parentheses (e.g., "Grant No.2023C4S02001）"); please uniformly use English half-width parentheses.

Ion symbol formatting: The "+" in "H+-ATPase" at lines 278-279 should be formatted as a superscript.

Text hyphenation artifacts: The text contains numerous hard line break hyphens (such as "nodu-lation" at lines 16-17), likely remnants from PDF export. Please carefully check and remove these before submitting the final manuscript.

Author Response

Dear Reviewer,

We are very grateful for your constructive comments and suggestions for our manuscript entitled "Transcriptomic Comparison of Soybean Roots Inoculated with Different Rhizobium Strains During Early Symbiosis " (ID: plants-4204122). Other changes have been marked in the manuscript using the revision mode (Track Changes). Please see below for our point-by-point responses to the editor's comments and concerns. The specific details of the modifications are shown as follows:

Comment 1: This study investigates the symbiotic relationship between soybean and an efficient rhizobial strain (B. ottawaense Bott 59), providing valuable candidate genes for early symbiotic responses through comparative transcriptome analysis. However, there are structural numbering, formatting, and grammatical errors in the manuscript that require careful revision.

Response 1: We sincerely appreciate your positive assessment of our work and your constructive feedback regarding the manuscript’s structure, numbering, formatting, and language. In response, we have conducted a comprehensive revision of the entire manuscript. All section, figure, table, and equation numbering have been carefully verified and corrected. Furthermore, to ensure linguistic accuracy, academic clarity, and stylistic consistency, the revised manuscript has been professionally edited by a certified academic editing service. A certificate of professional English editing from MDPI has been included in the Supplementary Materials. All modifications have been clearly tracked in the revised version. We believe these revisions have substantially improved the manuscript’s readability, structural coherence, and overall quality. Thank you again for your valuable and meticulous comments.

 

Comment 2: Lack of qPCR validation for transcriptome data: This study relies on RNA-seq data to characterize gene expression profiles without experimental validation. Using independent experimental methods to verify high-throughput sequencing results is a fundamental standard practice. The authors are advised to perform RT-qPCR analysis on a subset of representative differentially expressed genes (DEGs), particularly core genes emphasized in the discussion section (such as IF7MAT, I2H, and key hormone signaling genes), to confirm the reliability of the transcriptome data.

Response 2: We sincerely thank the reviewer for this critical and constructive suggestion. We fully agree that independent experimental validation is essential to confirm the reliability of transcriptomic data. In response, we have performed qRT-PCR on nine representative DEGs that span a broad dynamic range of expression and cover the core functional pathways emphasized in our discussion. Specifically, we validated four genes in the phenylpropanoid/flavonoid/isoflavonoid biosynthesis pathway (IFR, PTS, POD, and IF7GT), two hormone signaling components (AUX/IAA and A-ARR), and three well-established symbiotic nodulation genes (NIN1a, ENOD40, RIC1b and TML2).

Regarding the selection of genes: Although we chose to validate IF7GT instead of IF7MAT/I2H, IF7GT catalyzes a functionally downstream step in the same isoflavonoid metabolic branch and effectively captures the transcriptional dynamics of this pathway, which aligns with the reviewer’s intent to validate core metabolic responses. Additionally, as the reviewer mentioned IF7MAT, during our cross-check of the original data, we identified an error in its value and have since corrected it. All related descriptions of IF7MAT in the text have also been revised accordingly.

As detailed in the revised Part 2.7 and presented in the newly added Figure 8, the qRT-PCR results show expression trends that are highly consistent with the RNA-seq data. All corresponding text, methods, and figures have been updated accordingly in the revised version (Part 2.7 and 4.5).

 

Comment 3: Non-standard statistical presentation in KEGG analysis (Figure 3): The x-axis of the KEGG pathway enrichment analysis in Figure 3 uses gene count. Simple gene count cannot reflect the statistical significance of pathway enrichment, as basic metabolic pathways containing large numbers of genes often have high count values but may not be specifically enriched. The authors should redraw this figure using statistical significance indicators (such as P-value or FDR) as the x-axis, or adopt a standard bubble plot (where dot size represents gene count and color intensity represents significance).

Response 3: We sincerely thank the reviewer for this insightful and methodologically important suggestion. We fully agree that using raw gene counts alone does not adequately reflect the statistical significance of pathway enrichment, as highly represented basal pathways can dominate the x-axis without biological specificity. In response, we have completely redrawn Figure 3 as a standard enrichment bubble plot. In the revised figure, bubble size represents the number of enriched genes, while color intensity reflects the statistical significance (P-value). This format clearly distinguishes truly enriched, biologically relevant pathways from those with high background gene counts. The updated Figure 3 has been replaced in the revised manuscript. We appreciate this constructive comment, which has significantly improved the rigor and interpretability of our KEGG analysis.

 

Comment 4: Formatting standards: The n representing sample size and P representing significance in figures should both be italicized.

Response 4: We thank the reviewer for the comment. After checking the entire manuscript, we have made corrections one by one accordingly.

Comment 5: Color scheme: Please avoid using red-green color schemes in figures (such as bar/box plots in Figure 1) to accommodate readers with color vision deficiency.

Image clarity: The scatter points in the box plots of Figure 1 lack sufficient clarity; please upload higher resolution images.

Response 5: We sincerely thank the reviewer for these important suggestions regarding figure accessibility and presentation quality. In response, we have made the following corrections to Figure 1:

Color scheme: We have changed the color scheme to avoid using red and green.

Image resolution & clarity: Figure 1 has been regenerated and re-uploaded at a higher resolution.

The updated figure has been inserted into the revised manuscript. We appreciate these constructive comments, which have significantly improved the visual clarity, accessibility, and overall publication readiness of our figures.

 

Comment 6: Figure legends: Many main figure legends are overly simplistic (e.g., Figure 4). Legends should be sufficiently detailed to enable readers to "understand the figure content by reading the legend alone.

Response 6: We sincerely thank the reviewer for this important suggestion. We fully agree that figure legends should be sufficiently detailed to allow readers to understand each figure independently, without needing to refer back to the main text. In response, we have thoroughly revised and expanded the legends for Figures 1, 3, and 4. Legend updated now includes: (1) clear descriptions of experimental conditions and treatments and (2) explicit definitions of symbols and colors. These revisions have significantly improved the interpretability and publication readiness of our graphical data.

Comment 7: Section duplication: Another "3. Discussion" appears at line 421. This means the article has two Section 3s; the logical hierarchy of the entire Results and Discussion section needs to be reorganized and renumbered.

Response 7: We sincerely thank the reviewer for carefully identifying this structural oversight. The Results section has been systematically reorganized and sequentially renumbered to ensure a clear, logical hierarchy.

 

Comment 8: Inconsistent title capitalization: The title "3.6. Expression patterns of Differentially expressed transcription factors (DETFs)..." (line 381) uses all lowercase in the latter half, while previous titles (such as 3.5) used title case format. Please standardize this.

Response 8: We appreciate the reviewer’s attention to formatting details. The heading for Section 3.6 has been corrected to standard title case “2.6. Expression patterns of Differentially Expressed Transcription Factors (DETFs) in Early Roots Under Incubation Different Rhizobial Strains”.

 

Comment 9: Species scientific names: The scientific name of Lotus japonicus is misspelled at lines 88 and 115; the correct Latin name should be L. japonicus, not L. japonicas.

Basic spelling: Line 150 "primay root length" should be "primary"; lines 183-184 "Bott 59-innoculated" has an extra 'n' and should be "inoculated."

Mixed Chinese and English punctuation: In the Acknowledgments/Funding section, lines 633-634 contain errors with half English parentheses and half Chinese parentheses (e.g., "Grant No.2023C4S02001）"); please uniformly use English half-width parentheses.

Line 43-44: "into ammonia (NH3), it can be directly assimilated..." contains a comma splice and should be changed to "...(NH3), which can be..."

Response 9: We sincerely thank the reviewer for the meticulous reading and for catching these typographical and formatting oversights. All specified errors have been corrected in the revised manuscript:

(1) “L. japonicas” has been corrected to “L. japonicus”.

(2) “primay” and “innoculated” have been corrected to “primary” and “inoculated,” respectively.

(3) All mixed-width parentheses in the Acknowledgments and Funding sections have been standardized to English half-width parentheses.

(4) The revised sentence now reads: “...into ammonia (NH₃), which can be directly assimilated...”.

 

Comment 10: Lines 59-60: "promotes... induces... and ultimately trapping..." contains a parallel structure error and should be changed to "traps."

Lines 67-68: "sustained calcium oscillations... is triggered" contains subject-verb disagreement and should be "are triggered."

Line 100: "coordinately governing" should be changed to "govern."

Line 488: "In this study found that" is a mixed sentence structure and should be changed to "This study found that..."

Proper noun capitalization: Line 143 uses capitalized "Williams 82" in the title, but line 147 in the main text uses lowercase "williams 82"; please maintain consistency.

Ion symbol formatting: The "+" in "H⁺-ATPase" at lines 278-279 should be formatted as a superscript.

Response: We sincerely thank the reviewer for your careful reading and constructive comments. We have thoroughly revised the manuscript according to all your suggestions. Below is our response:

We have corrected the parallel structure by changing “ultimately trapping” to “and ultimately traps”(1. Introduction, Paragraph 2).

The verb has been revised from “is triggered” to “are triggered” (1. Introduction, Paragraph 2).

We have replaced “coordinately governing” with “govern” to improve grammatical correctness and readability (1. Introduction, Paragraph 4).

The sentence has been revised to “This study found that…” to resolve the mixed construction (3. Discussion, Paragraph 8).

We have standardized the capitalization to “Williams 82” throughout the manuscript, including the title, main text, and all other occurrences.

Response: We appreciate this formatting note. The + in H+-ATPase has been corrected to a superscript (H⁺-ATPase) in accordance with standard biochemical notation (Part 2.4, Paragraph 1).

All modifications have been highlighted in the Tracked Changes version.

Furthermore, to ensure linguistic accuracy, academic clarity, and stylistic consistency, the revised manuscript has been professionally edited by a certified academic editing service. A certificate of professional English editing from MDPI has been included in the Supplementary Materials.

 

Comment 11: Text hyphenation artifacts: The text contains numerous hard line break hyphens (such as "nodu-lation" at lines 16-17), likely remnants from PDF export. Please carefully check and remove these before submitting the final manuscript.

Response: We have thoroughly proofread the manuscript to ensure the final version is now free of such formatting issues.

We sincerely appreciate the reviewer’s valuable feedback, which have significantly improved the quality of our work.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for your careful revision of the manuscript. The changes implemented, particularly the inclusion of qRT-PCR validation and the improvements in statistical analysis and discussion, have significantly strengthened the study. While some limitations remain, they have been appropriately acknowledged and do not detract from the overall quality of the work. I believe the manuscript is now suitable for publication.

Best regards,

Comments on the Quality of English Language

The manuscript contains numerous grammatical errors and awkward phrasing throughout the text. Examples include, incorrect article usage, inconsistent tense, typographical errors.

Examples include:

Lines 21–24: Awkward sentence construction and incorrect article usage in the description of the study objectives. The sentence structure should be revised to improve clarity and grammatical correctness.

Lines 76–79: Inconsistent verb tense when describing previous studies and background information. The paragraph alternates between present and past tense.

Lines 154–156: Incorrect article usage and unclear phrasing in the description of plant growth responses following inoculation.

Lines 312–316: Typographical and grammatical inconsistencies in the description of transcriptomic results, making the interpretation of the sentence difficult.

Lines 421–425: Some sentences contain redundant wording and minor grammatical errors that should be revised for clarity.

Lines 612–615: Awkward phrasing and long sentence structure in the discussion section. The sentence could be simplified to improve readability.

Professional English editing is strongly recommended.

 

Author Response

Dear Reviewer,

We are very grateful for your constructive comments and suggestions for our manuscript entitled "Transcriptomic Comparison of Soybean Roots Inoculated with Different Rhizobium Strains During Early Symbiosis " (ID: plants-4204122). Other changes have been marked in the manuscript using the revision mode (Track Changes). Please see below for our point-by-point responses to the editor's comments and concerns. The specific details of the modifications are shown as follows:

Comment 1: Lines 21-24: Awkward sentence construction and incorrect article usage in the description of the study objectives. The sentence structure should be revised to improve clarity and grammatical correctness.

Response:

Thank you for pointing out this issue. We have revised the sentence structure and corrected the article usage to make the description of the study objectives clearer and more accurate. The revised content can be found in Line 21-23.

Comment 2: Lines 76-79: Inconsistent verb tense when describing previous studies and background information. The paragraph alternates between present and past tense.

Response:

Thank you for your observation. We have standardized the verb tenses in this section according to academic writing conventions to ensure consistency and improve the logical flow of the background information. Past tense is now consistently used to describe previous studies. The modifications are detailed in Lines 84-86.

 

Comment 3: Lines 154-156: Incorrect article usage and unclear phrasing in the description of plant growth responses following inoculation.

Response: Thank you for your comment. We have corrected the article errors and rephrased the sentences to provide a clearer description of the plant growth responses after inoculation, thereby enhancing the accuracy of the data presentation. The modifications are detailed in Lines 164-168.

 

Comment 4: Lines 312-316: Typographical and grammatical inconsistencies in the description of transcriptomic results, making the interpretation of the sentence difficult.

Response: We have corrected these mistakes and refined the sentence structure to ensure the transcriptomic results are presented clearly and professionally, facilitating easier interpretation. The modifications are detailed in Lines 274-283.

 

Comment 5: Lines 421-425: Some sentences contain redundant wording and minor grammatical errors that should be revised for clarity.

Response:

Thank you for your suggestion. We have removed redundant words and corrected the grammatical errors to make the sentences more concise and clear, thereby improving the overall readability of this section. The revised paragraph is now presented in Lines 400-405.

 

Comment 6: Lines 612-615: Awkward phrasing and long sentence structure in the discussion section. The sentence could be simplified to improve readability.

Response:

Thank you for your comment. We have simplified the sentence structure and improved the phrasing in the discussion section to enhance readability and ensure our arguments are conveyed more effectively. The revised content can be found in Lines 563-568.

 

Comment 7: Professional English editing is strongly recommended.

Response:

Thank you for the suggestion. We did have the manuscript edited by MDPI Author Services initially, but we realized that the extensive revisions we made afterward might have introduced some language issues. Therefore, after finishing the revisions, we invited a native English-speaking scholar with expertise in our research field to carefully go through the entire manuscript again. We have now corrected awkward expressions, grammatical errors, and word choice issues throughout the text. We believe the current version meets the journal's standards for academic English writing.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have meticulously addressed all the concerns raised during the first round of revision. The manuscript has significantly improved in terms of technical soundness, data transparency, and biological validation. I am satisfied with the revisions and the additional efforts made by the authors to strengthen their findings.

Experimental Validation: I highly appreciate the inclusion of the qRT-PCR experiments.

Data Availability: The authors have complied with the Open Science standards by uploading the raw RNA-seq data to a public repository (CNCB Genome Sequence Archive (GSA).

Supplementary Information: The addition of the detailed tables for Differentially Expressed Genes (DEGs) provides the necessary depth for readers to explore the genomic data.

Textual Corrections: All minor suggestions, clarifications, and grammatical corrections have been integrated into the final version of the manuscript.

Minor Corrections

Line 459: There is a small typographical error. The word "Bont" should be corrected to "Bott".

In view of the substantial improvements and the rigorous validation provided, I recommend the manuscript for publication once the minor typographical error mentioned above is corrected.

Author Response

Dear Reviewer,

We are very grateful for your constructive comments and suggestions for our manuscript entitled "Transcriptomic Comparison of Soybean Roots Inoculated with Different Rhizobium Strains During Early Symbiosis " (ID: plants-4204122). Other changes have been marked in the manuscript using the revision mode (Track Changes). Please see below for our point-by-point responses to the editor's comments and concerns. The specific details of the modifications are shown as follows:

Comment 1: Line 459: There is a small typographical error. The word "Bont" should be corrected to "Bott”.

Response 1: We thank the reviewer for carefully checking the manuscript. We apologize for this typographical error. The word "Bont" has now been corrected to "Bott" in the revised manuscript. The change is clearly marked at Line 467.

Author Response File: Author Response.docx