The Jujube TCP Transcription Factor ZjTCP16 Regulates Plant Growth and Cell Size by Affecting the Expression of Genes Involved in Plant Morphogenesis

Round 1

Reviewer 1 Report

The manuscript by Yang et al. characterized jujube (Ziziphus jujuba) ZjTCP16 which is shown to be induced by the pathogen ‘Candidatus Phytoplasma ziziphi’. They observed that overexpression of ZjTCP16 in Arabidopsis dramatically hampered vegetative and reproductive growth resulting in a dwarf phenotype. They also generated CRISPR/Cas9 mutants of ZjTCP16 in jujube and found the shoot height was higher than control plants. Overall, ZjTCP16 plays an important role in plant growth and regulates several genes that are involved in plant morphogenesis in jujube.

The experimental methodology is correct and detailed presented. Although I can provide my comments and suggestions for the betterment of this draft.  

The authors claimed ‘that ZjTCP16 could have functions in both the host cell membrane and nucleus can  provide respective protein sequences corresponding to the mutations that produce the knockout mutations’ (Figure 1c). In practice to claim the subcellular localization we provide specific fluorescent dyes as control or tacking. Such as nuclear localization DAPI (4',6-diamidino-2-phenylindole) staining is commonly used.

Please explain the individual lane for the fig. 1a.

Please check the sentence Line 199 ‘Figure 3. Overexpression of ZjTCP16 in Arabidopsis induced and dwarfism and reduced leaf size.’

Authors reported that overexpression of ZjTCP16 reduced the siliques size, so they also observed any difference in seed size.

In my opinion, this will be better if the authors can write which one is sgRNA and PAM sequences in fig 5a.

I think the authors already checked the CRISPR/Cas9 of sgRNA off-targets? If yes please provide data/comments.

I also suggest presentation with more elaborate conclusions detailing the obtained results and future prospects of research.

Author Response

Dear editor

We are grateful to your comments and suggestions for improving our manuscript entitled “The jujube TCP transcription factor ZjTCP16 regulates plant growth and cell size by affecting the expression of genes involved in plant morphogenesis” Here, we have interpreted the questions raised by you and made carefully revisions according to your suggestions.

Here below are our point to point response to the reviewers.

Q1: The authors claimed ‘that ZjTCP16 could have functions in both the host cell membrane and nucleus can provide respective protein sequences corresponding to the mutations that produce the knockout mutations’ (Figure 1c). In practice to claim the subcellular localization we provide specific fluorescent dyes as control or tacking. Such as nuclear localization DAPI (4',6-diamidino-2-phenylindole) staining is commonly used.

Reply: Thank you very much for your professional suggestion. In subcellular localization assay, pSAK277-GFP and pSAK277-ZjTCP16-GFP were injected to different tobacco leaves of the same plant. We performed multiple repetitions and confirmed that ZjTCP16 is located on the cell membrane and nucleus. We think the result is accurate, so we did not use specific fluorescent dyes as controls during the subcellular localization.

Q2: Please explain the individual lane for the fig. 1a.

Reply: The explanation of each lane in Figure 1a has been added. (Line172 -Line173)

Q3: Please check the sentence Line 199 ‘Figure 3. Overexpression of ZjTCP16 in Arabidopsis induced and dwarfism and reduced leaf size.

Reply: This section was changed to “Figure 3. Overexpression of ZjTCP16 in Arabidopsis induced phenotype of dwarfism and reduced leaf size.” (Line208 -Line209)

Q4: Authors reported that overexpression of ZjTCP16 reduced the siliques size, so they also observed any difference in seed size.

Reply: In the study, we mainly focused on the effect of ZjTCP16 gene on Arabidopsis and jujube morphogenesis. We will further explore more growth indicators such as seed size, seed vigor and number of seeds in future study.

Q5: In my opinion, this will be better if the authors can write which one is sgRNA and PAM sequences in fig 5a.

Reply: Thank you very much for your professional suggestion. The sequences of sgRNA and PAM has showed in Figure 5.

Q6: I think the authors already checked the CRISPR/Cas9 of sgRNA off-targets? If yes please provide data/comments.

Reply: Yes, we have already checked the CRISPR/Cas9 of sgRNA off-targets. Now this section has been added to Figure S1. (Line125 -Line128) (Line224 -Line226)

Q7: I also suggest presentation with more elaborate conclusions detailing the obtained results and future prospects of research.

Reply: Thank you very much for your professional suggestion. This section has been added. (Line334 -Line337).

Reviewer 2 Report

The manuscript “The jujube TCP transcription factor ZjTCP16 regulates plant growth and cell size by affecting the expression of genes involved in plant morphogenesis” by Yang et al is a good elucidation of the TCP transcription factor ZjTCP16 in the jujube genome. The author cloned and analyzed the TCP protein structure through expression in the model plant Arabidopsis and also in Jujube. The authors also performed the Y1H and Y2H to understand their function at the cellular level. In my opinion, this study is well designed and the findings are also robust. However, the authors should improve the manuscript according to the comments given below. 

I have a question about the main theme of the paper. Authors claimed that The TCP transcription factor ZjTCP16 participates in the plant response to the JWB phytoplasma and may be a target of a JWB phytoplasma effector. However, the authors did not provide clear evidence and link between these hypotheses. Although, the authors stated that ZjTCP16 regulates plant growth and cell size by altering the expression pattern of morphogenesis-related genes in jujube. But where and how does it connect with JWB resistance? If the authors discovered a novel finding they should mention it in the abstract. 

Please modify the first three lines of the abstract in a simple way. For example, JWB is caused by Ca. P. Ziziphus is a destructive disease.
The manuscript is full of grammar mistakes. I would like to suggest the authors get English editing from a native English speaker. 
Line 54-56: Please mention the crop plant of SJP1 and SJP2?
Line 124: What are EV and WT?
Line 171: How old were the leaves and how severe were the symptoms?

The discussion part should be revised and improved. Authors should add more information and compare their findings with the previous similar findings. Authors should write more about effectors, especially the virulence-related factors and their connection with TCP TF. 

The authors should add a separate heading “conclusion” so the reader can get a take-home message. 

Author Response

Dear editor

We are grateful to your comments and suggestions for improving our manuscript entitled “The jujube TCP transcription factor ZjTCP16 regulates plant growth and cell size by affecting the expression of genes involved in plant morphogenesis” Here, we have interpreted the questions raised by you and made carefully revisions according to your suggestions.

Here below are our point to point response to the reviewers.

Q1: I have a question about the main theme of the paper. Authors claimed that The TCP transcription factor ZjTCP16 participates in the plant response to the JWB phytoplasma and may be a target of a JWB phytoplasma effector. However, the authors did not provide clear evidence and link between these hypotheses. Although, the authors stated that ZjTCP16 regulates plant growth and cell size by altering the expression pattern of morphogenesis-related genes in jujube. But where and how does it connect with JWB resistance? If the authors discovered a novel finding they should mention it in the abstract.

Reply: The main theme of this article is the gene function of ZjTCP16. The interaction between ZjTCP16 and JWB effector is not the focus of this article. The abstract has been modified to make the theme clearer.

Q2: Please modify the first three lines of the abstract in a simple way. For example, JWB is caused by Ca. P. Ziziphus is a destructive disease.

Reply: The sentences in the abstract have been replaced.

Q3: The manuscript is full of grammar mistakes. I would like to suggest the authors get English editing from a native English speaker.

Reply: The revised version has submitted to the MDPI language services to improve the language level.

Q4: Line 54-56: Please mention the crop plant of SJP1 and SJP2?

Reply: The revised sentences have been added. (Line56)

Q5: Line 124: What are EV and WT?

Reply: The EV means pSAK277 empty vector and The WT means wild type. The revised sentences have been added. (Line131)

Q6: Line 171: How old were the leaves and how severe were the symptoms?

Reply: The revised sentences have been added. (Line97-Line99)

Q7: The discussion part should be revised and improved. Authors should add more information and compare their findings with the previous similar findings. Authors should write more about effectors, especially the virulence-related factors and their connection with TCP TF.

Reply: The discussion part has been revised. The expression of jujube TCP TFs in response to JWB phytoplasma, the interaction of jujube TCP TFs with phytoplasma effectors were discussed. (Line322 -Line333)

Q8: The authors should add a separate heading “conclusion” so the reader can get a take-home message.

Reply: Thank you very much for your professional suggestion. This section has been added. (Line334 -Line337)