GmPBS1, a Hub Gene Interacting with Rhizobial Type-III Effectors NopT and NopP, Regulates Soybean Nodulation

Round 1

Reviewer 1 Report

This paper describes plant responses, including at the transcriptional level, to rhizobial strains mutated in genes encoding two type III effector proteins , NopP and NopT.   The system studied is Ensifer fredii (Sinorhizobium fredii) strain HH103, and Soybean (Glycine max) cultivar Suinong 14.  Differences in nodule number and dry weight in the plants inoculated with the single and double mutants are reported, and a number of genes whose transcription is altered in mutant inoculated plants are identified in an RNA seq experiment. 

Overall this is a useful contribution, though it has flaws.

1) The level of English is not acceptable and needs improvement through substantial editing or a professional editing service. 

2) The mutants are never complemented, thus it cannot be ruled out that the effects detected are due to second site mutations. 

3) The MS is excessively long.  The discussion in particular could be shortened substantially.  No need to repeat material already explained in results. 

4) the vector used to make the mutants is not referenced in the text and the reference is missing from the reference list. 

Some other editorial comments. 

Line 15.  These are not the MAIN factors in establishing the symbiosis.  Nodule formation (except in rare Bradyrhizobia) required nod factor production by NodABC, and appropriate modifications by other Nod proteins depending on host.  Type III effectors do play a role in host range determination in some symbioses, but are completely absent in others.  

Lines 23/24  

Lines 190-194.  Not at all clear how GFP fusion was made and whether it was translational or trancriptional. 

Line 239 - why cDNA - as far as I can tell you did not isolate RNA here, just PCR'd the DNA

 

" The expression of Glyma.19g098600 increased 23 significantly than that of other four genes after soybean was "

 

Should say increased significantly to a much larger extent than that of the other four genes. 

Author Response

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Author Response File: Author Response.docx

Reviewer 2 Report

Li et al., have analyzed the role of type-III effectors of rhizobia such as NopT and NopP in symbiosis development and nodulation of soybean.

Authors have performed the nodulation analysis of soybean root nodules elicited by single mutants of rhizobia effectors NopT and NopP, and by a double mutant NopT/NopP. Authors also compared sequences using the Next Generation RNA Sequencing.

   In connection with the role of the effector AvrPphB, highly homologous to the type-III effector NopT, Li et al. have studied a host plant protein kinase GmPBS1, involved in defense response to flagellin, using a genetic construct with an overexpression of GmPBS1. Due to role of PBS1 a cleavage site for proteases, Li et al have analyzed it’s role in modulation of defense response during the nodule induction and a putative role in symbiosis in relations with effectors NopT and NopP.

According to the nodulation assay the mutations of NopT and NopP produce a multidirectional effects, with NopT shown to have the positive effect for the nodulation trait and NopP has negative effect. Li et al identified the number of differently expressed genes induced by mutations of NopT, NopP and double mutation and performed Gene Ontology and KEGG Pathway Enrichment Analysis with aim to define the functional categories of these genes.

Further Li et al. performed Weighted gene co-expression network analysis (WGCNA) and have identified 5 genes that were specifically expressed in the roots or nodules. The expression levels of these genes have been estimated by PCR analysis and putative key regulatory targets have been selected.

The subcellular localization on tobacco leaves have shown the localization of NopT and PBS1 on plasma membrane and in nuclei.

The nodulation analysis of the nodules elicited by overexpressing under 35S promoter GmPBS1have shown the negative effect for the nodulation.

Authors concluded that GmPBS1 is a hub gene involved in the possible pathways of regulation and co-regulation network of NopP and NopT, triggering gene signaling transduction in soybean.

Li et al., have performed an interesting study, methodological approaches are adequate.  But the text is difficult to read, there is redundant information, for example, in the introduction, which is not related to the topic of the article. I strongly advise Li et al to arrange the correction of the article by an English-speaking editor. Apart of the comment listed below I have left comments in the PDF of the paper (attached)in the form of sticky notes.

Introduction

The Intro is too long and overloaded with unnecessary data and abbreviations. By the same time the Introduction lacks the sound hypothesis and the substantiation of the work.

The role of soybeans as an agricultural crop is well known fact, the description its significance should be shortened, to be described it two or three sentences. The content of oligosaccharides and saponins in soybean milk or other products has no relations to NopT/NopP in root nodules so the text from line 36-44 may be deleted.

Lines 85-112: the descriptions of mechanisms have to be shifted to the Discussion

The last phrase  of Introduction (line 137) only notifies the reader what the authors of the article did: “Therefore, this study explored the involvement of PBS1 in T3SS induced symbiotic nodulation crosstalk in soybean”.

 I think this is insufficient. Authors have to explain why they made this research at the first place. The verbalization of hypothesis, explanation of experimental setting and a putative biological significance has to be added to the Introduction.

Methods

Authors have to give the full names of used programs in Methods like “Weighted gene co-expression network analysis (WGCNA)”, Bimolecular fluorescence complementation (line 212) etc with the explanation of the purpose to use this method and program. Authors have to use a correct names of used products, for example, Kod One, etc.

Wordy descriptions of changes in the Results   should be accompanied by either a table or a diagram.

Line 351: Thе part of the text below has to be presented as a table with full names of the genes:

[(Glyma.16g212300 (GmRj2), Glyma.17g217000 (GmCHIT5), Glyma.07g095800 (GmPR5), Glyma.02g138800 (GmMAPK2) and Glyma.03g236300 (GmR- BOHB), Glyma.16g212300 (GmRJ2), Glyma.02g247600 (GmBIK1)].

Discussion

The fact that NopT changed the effect of NopP on the nodulation, may be explained by a putative co-regulation. The direct regulation of one gene by another has not been shown. Taking under consideration the number of differentially expressed genes, and the number and the functional of the genes, selected for the heat map, I would recommend some changes in the title of the article. The fashionable word “crosstalk” in this case explains nothing. The new title should include the name of plant protein kinase GmPBS1.

In the final part of the Discussion Li et al should draw the conclusion and formulate the biological significance of the study.

The paper of Li et al., may be accepted after the major revision.

Comments for author File: Comments.pdf

Author Response

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Author Response File: Author Response.docx

Reviewer 3 Report

The manuscript is dedicated to the study of two rhizobia proteins, NopT and NopP in root nodulation process. The authors have shown that the proteins lead to changes in gene expression in the plants and have identified one protein directly interacting with NopT. The design of experiment is appropriate. Some minor comments:

1. The text on figures is too small and hardly readable, particularly fig. 5.

2. It is not clear how the bacterial and plant protein can interact directly. Should be at least discussed. It would be better to validate interaction in vivo.

3. The source of plasmids should be mentioned. Please, add some refs.

Author Response

Thank you for your suggestion. We have sorted the detailed modification into the attachment. Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 4 Report

The manuscript  Agronomy- 2255743: „GmPBS1, a hub gene interacting with rhizobial type-III effectors NopT and NopP, regulates soybean nodulation”, presented for peer  review is interesting, but it has some shortcomings, which I will mention below:

Dear authors

In my opinion, the work contains extensive results. I miss discussing and indicating the application of research. Why do we detect and analyze NopT and NopP mechanisms in symbiotic transduction signalling? We know that the type-III effectors such as NopT and NopP secreted by the type-III secretion system of Rhizobium are the important signalling factors for the establishment of symbiosis in soybean. Type III protein secretion systems of nitrogen-fixing rhizobia deliver effector proteins into leguminous host cells to promote or inhibit nodule symbiosis. However, mechanisms underlying effector-triggered inhibition of nodulation remain largely unknown. The question comes up If your findings suggest that legumes face an evolutionary dilemma:

1. of either developing effector-triggered immunity against pathogenic bacteria

2. or establishing symbiosis with suboptimally adapted rhizobia-producing pathogen-like effectors.

These statements could be explored and given what they to your hypothesis, I quote: "We hypothesized that a gene in the host can simultaneously respond to various type-III effectors, and the host signalling network may be regulated and affected by a specific hub gene during the establishment of symbiosis."- Did the hypothesis come true?

I am interested in how the identification of the genes responsible for NopT and NopP and the determination of DEG expression after inoculation with single and double mutants of NopT and NopP will help, for example in plant agronomy. I didn't find this in the discussion and that would be the strength of this article.

I am missing statements, for example, whether this article shows that effector genes can be screened for their ability to improve transformation, or maybe I misunderstand it. Please add something to the discussion or maybe just improve the conclusions.

Please work on the literature because in line 73: Citation error. Please correct the citations and the arrangement of the literature due to the error.

Author Response

Thank you for your suggestion. We have sorted the detailed modification into the attachment. Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 5 Report

This publication presents interesting results of the interaction between GmPBS1 gene and rhizobial type-III effectors NopT and NopP to regulate soybean nodulation. The adopted approach in this study was very interesting since it could improve our understanding of the mechanisms underlying nodules formation.

The manuscript was well introduced, and the authors adopted convincing methods with a discussion of the different obtained results. However, the manuscript needs substantial revisions to be suitable for publication in Agronomy.

General comments

- Comment 1: The English of this manuscript needs moderate improvements.

- Comment 2: The introduction section should be shortened.

- Comment 3: The discussion section should be more developed.

 

Other comments

 

- Abstract

Please shorten the abstract (200 words instead of 264).

Please add “NopT” and “NopP” as keywords.

 

- Introduction

The introduction is too long, please shorten it.

L45-46: Please add a reference.

 

- M&M

L111: italicize “Kan” gene name.  please check throughout the manuscript.

L120: please be more specific on the growth conditions (controlled conditions? relative humidity? light intensity?..)

126-129: please place this subsection at the end of M&M section. The statistical analysis was performed for other parameters not only for nodule numbers. Please specify the statistical analysis perfomed ANOVA? And the degree of significance.

L133: why did you choose 36 hpi?

L169: please change “was” to “were”.

L170: please be more specific on the conditions of growth. The same in L193.

L192: please correct the citation form.

 

- Results

L213: please put the dot at the end of the sentence.

L214: please change the tile of the fig 1 to “Fig. 1(a, b). Insertion of (a) spectinomycin (Spec) (a) and (b) kanamycin B (Kan)-resistance gene fragments downstream of initiation codons of NopT and NopP, respectively and Identification of (c) NopT, (d) NopP and (e) NopT&NopP using PCR; M: Trans 2K Plus DNA marker. 1: HH103 (NopT-LF-F/R), 2: 216 HH103ΩNopT (NopT-LF-F/R), 3: HH103 (NopT-LF-F, Spec-R), and 4: HH103ΩNopT 217 (NopT-LF-F, Spec-R).” Please do the same for the other figures when necessary.

- please place Fig 2 after the description text and provide the citation of the figure in the text. Please check for the other figs.

L231-237: please provide same % values.

Fig 4: Maybe you should remove the X axis legend from fig4 (a and b) and keep it in Fig 4c.

Figs 6 and 7 are not clear.

L346: please remove “however, the specific mechanism of this effect remains to be studied”.

L390: please change “not change significantly” to “not significantly change”.

 

- Discussion

This section should be more developed. Some results should be more discussed.

L406: please change “indicated” to “indicate”.

L406-407: “involved a cross-talking”, cross-talking between what?

L418-419: please change “indicated” to “indicates”.

L433: “hpi” please provide the significance at the first appearance in the text.

L444-445: please provide a reference.

Author Response

Thank you for your suggestion. We have sorted the detailed modification into the attachment. Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The attempt to revise this paper has been minimal and did not provide all the information requested.

The writing is still inadequate and needs to be revised by a professional editing service.  

If the mutants were made in a previous paper (which is suggested in the authors response) this should be properly cited and it should be mentioned that they were complemented (fully complemented for phenotype) in that paper. 

Still no references for vectors like pJQ200Sk which were used in this paper. You need to cite properly and accurately. 

Paper still too long. 

Author Response

Please see the attachment. Thank you.

Author Response File: Author Response.docx

Reviewer 2 Report

Authors have revised the MS, it is sufficiently improved. Several small corrections I marked with a Sticky Notes in PDF (attached). After this correction the paper can be accepted for publication.

Comments for author File: Comments.pdf

Author Response

Please see the attachment.Thank you very much.

Author Response File: Author Response.docx

Reviewer 5 Report

I endorse thepublication of the current form of the manuscript.