Optimization of Hairy Root Transformation and Application of RUBY as a Reporter in Lotus corniculatus

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors describe the use of the chimeric gene RUBY as a report gene in hairy root cultures obtained by A. rhizogenes transformation. Plant cells expressing RUBY can produce the red pigment betalain and can be easily distinguished from not transformed roots.

The manuscript uses A. rhizogenes-mediated transformation to induce hairy roots and develop composite plants, as a model to study the interaction between Lotus corniculatus and rhizobia or mycorrhizal fungi. The authors checked the transformation efficacy on seedlings and young branches.  Researchers found that young branches were transformed with higher frequencies than seedlings and produced composite plants with better growth in vermiculite soils. Moreover, the expression of RUBY and its product did not affect nodules formed in the transgenic hairy roots of composite plants. The manuscript is very similar to the previous one presented from the same authors (Anthocyanin, a novel and user-friendly reporter for convenient, non-destructive, low-cost, directly visual selection of transgenic hairy roots in the study of rhizobia-legume symbiosis).

 

Comments  

I think the title should be changed since is not an optimization of a protocol because the authors only optimize one variable, the transformation of seedlings versus young branches.

The authors suggest that this method is better than the previous reporter system but they did not carry out an experiment in parallel to compare. Moreover, they should explain why they did not compare composite plants with control plants (without A. rizhogenes transformation).

 

Legend to table 1

Letters A and B, represented by different letters, are very significantly different at p = 0.01 according 146 to two-tailed unpaired t tests. The values are presented as the means ± standard deviations of 30 147 independent composite plants.

May be it is better to write are significantly differents

Author Response

 Comments  

I think the title should be changed since is not an optimization of a protocol because the authors only optimize one variable, the transformation of seedlings versus young branches.

Answer: The protocol of hairy root transformation method in Lotus corniculatus were optimized in this study. The hairy root transformation method in L. corniculatus was previously reported by cutting the embryonic roots, coating A. rhizogenes on the hypocotyl, and inducing hairy roots on the culture medium (Boisson-Dernier et al., 2001) which requires a sterile environment and tissue culture to induce composite plants. In this report, the transformation method was improved by using young branches as explants, coating them with A. rhizogenes, and then inserting into vermiculite to produce hairy roots in the natural environment.

 

The authors suggest that this method is better than the previous reporter system but they did not carry out an experiment in parallel to compare. Moreover, they should explain why they did not compare composite plants with control plants (without A. rizhogenes transformation).

Answer: From Lane185 to Lane198, we did not suggest that this RUBY reporter system is better than previous reporter system. We think that RUBY reporter system is an alternative reporter system, and other reporter systems are still defective. For example, AtMYB75 reporter system cannot cause anthocyanin accumulation in some plants.   This study is to optimize transformation method, so the previously reported method is used as a control.  

Legend to table 1

Letters A and B, represented by different letters, are very significantly different at p = 0.01 according 146 to two-tailed unpaired t tests. The values are presented as the means ± standard deviations of 30 147 independent composite plants.

May be it is better to write are significantly differents

Answer: Yes, thank you. The sentence has been modified.

Reviewer 2 Report

Comments and Suggestions for Authors

In the present study, the authors described the possibility of using RUBY as a reporter for the selection of transgenic roots during transformation of Lotus corniculatus, and at the same time optimized the transformation process itself, proposing to use young plant branches instead of seedlings as explants. Although the results of the work may be of practical interest to specialists in this field, there are gaps and inaccuracies in the description of the experimental methodology that must be clarified before publication. The manuscript needs to be revised taking into account the comments and questions below.

Comments:

Line 63: Please indicate how the pDR5::RUBY plasmid has been obtained

Lines 63-64: Primers have identical names (RubyS1),

Line 65: SalI restriction sites are not underlined as it was mentioned in the text

Lines 85, 86 – primers specific to …  were used for PCR analysis

Figure 1. Only the restriction sites used for cloning should be shown on the vector map.

Line 81 - How did the authors wet vermiculite? What medium was used?

Questions:

Which restriction sites were used for cloning – SalI (line 65), “SalI XhoI” both sites -? (as mentioned in M&M, line 66) or XhoI (as it was mentioned in the Results section, line 107)? This should be clarified. If a single restriction enzyme was used for cloning, please indicate how the orientation of the RUBY ​​insert was checked.

Did the authors check whether betalain accumulation affects nitrogenase activity in nodules?

Please indicate the age of the plants from which young branches were taken for transformation. Were these plants in the flowering stage? How many branches can be taken for transformation from one plant? How many internodes were there in such young branches? Please, describe this information in more details, since this is the basis of the proposed method.

Does the use of RUBY as a reporter allow us to detect cases of transgenic root chimerism in composite plants? This issue may be particularly important for functional studies given that it was mentioned that redness of transgenic roots gradually decreased and even disappeared. Please, discuss this issue.

Does betalain accumulation interfere with GUS staining or imaging of fluorescent reporter proteins? What is known about this?

 

Comments on the Quality of English Language

There are grammar errors, typos and cases of incorrect use of English throughout the manuscript. It should be revised.

Author Response

Comments:

Line 63: Please indicate how the pDR5::RUBY plasmid has been obtained

Answer: Yes, the acknowledgment was added in Lane211-212. We thank Dr. Yubing He (Chinese Academy of Agricultural Sciences, China) for providing the pDR5:RUBY vector.

Lines 63-64: Primers have identical names (RubyS1),

Answer: Thank you. The name of primer have been revised.

Line 65: SalI restriction sites are not underlined as it was mentioned in the text

Answer: Yes, SalI restriction sites are underlined.

Lines 85, 86 – primers specific to …  were used for PCR analysis

Answer: Yes, the sentence has been modified.

Figure 1. Only the restriction sites used for cloning should be shown on the vector map.

Answer: Yes, the vector map was modified. There is no XhoⅠ restriction site in the new vector p35SRUBY, because SalⅠ (GTCGAC) and XhoI(CTCGAG) are homotailed enzymes and the RUBY cassette fragment digested with SalⅠ was inserted between XhoⅠ restriction sites of pCAMBIA1305, and then the XhoⅠ restriction sites of new vector p35SRUBY were disappeared.  

Line 81 - How did the authors wet vermiculite? What medium was used?

Answer: The vermiculite was wetted with sterile water. The sentence was modified with ‘The young branches coated with A. rhizogenes were directly planted in sterile vermiculite wetted with sterile water and covered with a highly transparent plastic bag in a growth chamber.’.Homotailed enzyme

Questions:

Which restriction sites were used for cloning – SalI (line 65), “SalI XhoI” both sites -? (as mentioned in M&M, line 66) or XhoI (as it was mentioned in the Results section, line 107)? This should be clarified. If a single restriction enzyme was used for cloning, please indicate how the orientation of the RUBY insert was checked.

Answer:  SalⅠ (GTCGAC) and XhoI(CTCGAG) are homotailed enzyme. In order to express more clearly, we modified the sentence as follows: The PCR fragment was digested with SalⅠ and pCAMBIA1305 binary vector was digested with XhoI, and then those digested DNA fragments were mixed in one-step digestion–ligation reaction with XhoI and T4 ligase. The purpose of adding XhoI in the one-step digestion–ligation reaction is to prevent the self cyclization of the vector digested with XhoI.

Did the authors check whether betalain accumulation affects nitrogenase activity in nodules?

Answer: We don’t do that.

Please indicate the age of the plants from which young branches were taken for transformation. Were these plants in the flowering stage? How many branches can be taken for transformation from one plant? How many internodes were there in such young branches? Please, describe this information in more details, since this is the basis of the proposed method.

Answer: The young branches of L. corniculatus as explants used for hairy root transformation should be the tender branches in the vegetative growth stage, rather than in the flowering stage. Young branches with 1-2 internodes are preferred. Branches with more internodes are older and are not easy to produce hairy roots. As L. corniculatus is a perennial plant, new branches will soon grow after cutting.

Does the use of RUBY as a reporter allow us to detect cases of transgenic root chimerism in composite plants? This issue may be particularly important for functional studies given that it was mentioned that redness of transgenic roots gradually decreased and even disappeared. Please, discuss this issue.

Answer: The article reported by He et al. (2020) showed that RUBY as a reporter is used to detect gene expression and indicate transgenic events in plant genetic transformation. And the article reported by Timoneda et al. (2021) showed that betalain colouration is specifically induced in root tissues and cells where fungal colonisation has occurred. So we think that RUBY as a reporter can detect cases of transgenic root chimerism in composite plants.

The disappearance or reduction of betanin content in transgenic roots may be related to the glycosylation of intermediate metabolites. Glycosylation enhances the half-life of betanidin and isobetanidin when exposed to reactive oxygen species, resulting in a 17-fold increase (Sadowska-Bartosz and Bartosz, 2021). Glycosylation serves as a crucial modification process that significantly impacts the solubility, physicochemical stability, biological longevity, and permeability through biological membranes of plant-derived secondary metabolites. So, In this study, the difference in color between roots and nodules tissues may be related to the degree of glycosylation of intermediate metabolites of betalains synthesis in different organs or developmental stages. And those sentences were added in the discussion.

Does betalain accumulation interfere with GUS staining or imaging of fluorescent reporter proteins? What is known about this?

Answer: In our other research, hairy root transformation was performed with the vector harboring both RUBY and RFP and the results showed that betalain accumulation do not interfere with RFP. As for whether it affects GUS staining, we have not carried out, but we think it should not interfere each other.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

No comments for the authors

Reviewer 2 Report

Comments and Suggestions for Authors The authors responded to the reviewer's questions and comments.

 

Comments on the Quality of English Language

Minor editing of English language required.