Establishment of a Virus-Induced Gene-Silencing (VIGS) System in Passiflora edulis Sims and Its Use in the Functional Analysis of PechlH

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Line 37. Passion fruit also known as passion fruit? It may be convenient to use the common names by which this fruit is known.

In the section of the introduction, it is convenient to explain in more detail what kind of agronomic characteristics or yield components can be improved in passion fruit with the scientific methodology proposed in the paper.

The "Materials and Methods" section is well explained. To enrich this section it may be convenient how they contribute to the validation and reproducibility of the results and what is the advantage of the technique that was used over other.

In the section of "Discussion": How can they transfer technology and what other genetic engineering procedures should be involved in the generation of improved passion fruit varieties?

Author Response

Please refer to the attachment。

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

methods require the addition of the following data:

- please, indicate the sequences to which these gene fragments correspond (from-to);

- please, include the schematic representation of expressed pds and chlCH fragment cDNA inserted in TRV;

- please, include the size of chlCH fragment cloned;

-please, include the accession number of corresponding genes;

- it is necessary to include the developmental stage of plants inoculated, growth conditions of inoculated plants and cultivation condition licencies,

- the SI units require correction, for example mg.L^-1; mmol. dm^-3 etc.

Please, explain the difference between PDS1 and PDS2 fragments cloned to TRV. 

As you know the  VIGS can be significantly improved through the inclusion of small inverted-repeats in plant virus-based vectors, generating a more robust loss-of-function phenotype. What was the reason for the aplification of the pds fragment length of 283 bp?

Author Response

Please refer to the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This article by Zhang et al. has written well. The abstract section needs significant improvements. English writing is not scientific, although authors can get help from MDPI English Language editing services (https://www.mdpi.com/authors/english). The introduction is insufficient, no discussion over the target gene and its importance. The material and Methods section does not have references, which is not common in scientific writing. The section is not well explained. The authors did not keep all the controls, like the plants with only pTRV1+pTRV2 combinations. Statistical analysis results are not shown in the graph. In my opinion, this MS is incomplete and not ready for publication.

Major comments;

1.      Page No. 1, Line No. 17; “several crops” either write several crops or remove the letter a!

2.      Page No. 1, Line No. 22; after 7 days of TRV infection? Similarly. What is 14-16 d?

3.      Page No. 1, Line No. 25; PeCHLH, VIGS passion fruit leaves? The meaning is not clear, re-write.

4.      Page No. 1, Line No. 25-26; “results showed that in passion fruit this gene was also involved in chlorophyll synthesis” Rewrite the sentence!

5.      Page No. 1, Line No. 37; Passion fruit (Passiflora edulis Sims), also known as passion fruit? The authors need to understand what they are writing.

6.      Page No. 2, Line No. 46-47; Remove the gap between the line.

7.      Page No. 2, Line No. 59; PDS gene?

8.      Page No. 2, Line No. 62; “virus silencing technology” there is no such technology exists!

9.      Page No. 2, Line No. 65; observed after 5-6 weeks. After TRV infiltration?  What is GOI gene?

10.  Page No. 2, Line No. 68; what is CsTCS1 gene?

11.  Page No. 2, Line No. 71-72; chloroplast greening?

12.  Page No. 2, Line No. 79; Which species did the authors use? Sometimes they write Passiflora edulis Sims and in materials and methods, it is Passiflora edulis 'Golden. It is very confusing.

13.  Page No. 2, Line No. 87; What kind of comparative bioinformatics analysis?

14.  Page No. 2, Line No. 88; What was the size of PePDS amplicon? Provide the gel image in a supplementary file. How many copies of this gene exist in passion fruits?

15.  Page No. 3, Line No. 94; seamless cloning?

16.  Page No. 3, Line No. 90-100; No references were added in section 2.2.

17.  Page No. 3, Line No. 102; Single colonies of Agrobacterium carrying the target plasmid” Did the authors confirm the construct using PCR and sequencing?

18.  Page No. 3, Line No. 135; Why you cloned PeCHIH? What was the idea behind this cloning?

19.  Page No. 4, Line No. 138; evolutionary tree? Or a phylogenetic tree? And the tree does not have any scale. What was the method used for generating a phylogenetic tree? How many bootstraps did you use? Legends are missing in supplementary files. Why do you have two different clades?

20.  Page No. 5, Line No. 177-191; Figure 2A is not clear. Provide a better picture showing the photobleaching phenotype. Also, provide a picture of the pTRV1+pTRV2 combination. Does TRV infect passion fruits? If yes, what are the symptoms? Why do authors have pTRV2-PePDS1 and pTRV2-PePDS2? And in Figure 2 C they have TRV2-CK. Authors must read the MS carefully before submitting.

21.  Page No. 5, Line No. 177; Figure 2C What is the size of MP and CP amplicon? What was the negative and positive controls in your PCR?

Comments on the Quality of English Language

English writing is not scientific, although authors can get help from MDPI English Language editing services (https://www.mdpi.com/authors/english). 

Author Response

Please refer to the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The MS has been improved significantly. However, the authors can still improve in writing. Important references are missing in the Materials and Methods section. The data analysis part can be a little more elaborated. The authors have not provided a pTRV1+pTRV2 combination image data (control), suggesting this combination might not used! Overall, this is a good attempt to study the transient expression of genes using VIGS analysis in passion fruit. 

Comments on the Quality of English Language

Minor editing is required. Typo errors need to be checked. 

Author Response

Please see the attachment.

Author Response File: Author Response.docx