A Method to Produce vsiRNAs in Plants with Cross-Kingdom Gene Silencing Capacity

Round 1

Reviewer 1 Report

This manuscript examined the production of viral small interfering RNA with cross kingdom gene silencing capacity.

Virus induced gene silencing is widely exploited in control and management of viruses either via host or virus manipulation. In the first step the authors constructed VIGS vector based on Begomovirus,  Euphorbia mosaic virus. This is an important topic for exploration in medicine and this will serve as a good contribution. However, I wish the authors to address some of my worries about this article.

Comments:

1- Can the authors include a schematic sketch for the vector construction?

2- What guided the choice of 432bp fragment from Krt18?

3- Explain clearly and be concise why was the fragment digested with Pst I and EcoR V  in section 2.1.

4- Adjust the y-axis of figure 1f if in percentage!

5- Among the numerous test plants, why N. benthamiana  is the only used in this experiment?

6- Line 57: there is a sting of Chinese characters, is this a sentence on its own?

7- Line 90: change visRNA to vsiRNA

8- Line 98: Is EuMV-YP:Krt18 the same as pEuMV-YP:Krt18? Also applies to line 299.

9- Line 119: space between chII and [23]

10- Line 381and 407: years of publication not bold, check out.

 

Author Response

Dear Reviewer,

We appreciate your comments and we have responded to all of them.
Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

This manuscript by Villanueva-Alonzo et al. describes a method to induce in plants the production of virus-derived small interfering RNAs (vsiRNAs), which could be used to silence genes in mammals, in an example of cross-kingdom gene silencing. The method described here is very interesting, with potential broad use in the medical field. However, more studies would be needed to reach its practical application.

The method is well described, and the results obtained with the pEuMV-YP virus in Nicotiana benthamiana plants seem very promising. Overall, I believe the research merits publication, however, below I have detailed suggestions for improving the manuscript’s clarity and readability.

 

Line 57: some numbers (1.1234) and Chinese characters need to be removed.

 

Line 66: mice mouse; this should be “mouse”

 

Line 108: …enzymes and ligated to…

 

Line 124: It is not clear what “Krt18 vsiRNA cDNA” refers to. Is this cDNA synthesized from the vsiRNA corresponding to the Krt18 gene fragment?

 

Line 125: total RNA – from where?

 

Line 126: typo: vsiRNA (not visRNA)

 

Line 127: How do you know that this vsiRNA was being produced, and why select this specific one for quantification?

 

Line 139: 2.4 sRNA sequencing – this section should be moved below “RNA extraction and sRNA enrichment”.

 

Line 188: our 0 μg?

 

Line 228: percentage of vsiRNA corresponding to plant genes or sequences in the pEuMV-YP:Krt18 vector.

 

Line 229: this should be: percentage of vsiRNAs derived from genes in pEuMV-YP:Krt18.

 

Line 230: How is the color scheme for the heat-map established. Perhaps a scale indicating the colors should be added to this part of the figure.

 

Line 232: GC content of the hotspots (instead of %CG)

 

Line 233: vsiRNA-induced silencing of the Krt18 gene in macrophages...

 

Lines 240-241: The percentages in Figure 1d do not agree with this statement (68 and 32%).

 

Lines 241-242: This is not correct based on Figure 1e and the percentages given in lines 243-244. They are not distributed equally: DNA-B accounted for almost half of the total vsiRNAs (~49%). The DNA-A had a little over 43%.

 

Lines 256-259: It might be worth following up with an RNA-seq analysis of the macrophages from both treatments, to see if other genes besides Krt18 were also silenced. This would have practical implications, as additional vsiRNAs may be also silencing undesired mammalian genes.

 

Lines 268-268: The sentence that starts “Gene silencing intensity…” does not make sense. Please re-word it.

 

Lines 269-270: More information should be provided about how this 432 bp fragment from the mouse Krt18 gene was selected to induce vsiRNAs. Why this fragment? Why this length?

 

Lines 279-280: as mentioned above, the data do not agree with the siRNAs accumulating equally between the A- and B-genomes.

 

Lines 284-285: starting at “the largest vsiRNA accumulation…”, this sentence does not make sense. Perhaps what is meant here is that the largest vsiRNA accumulation corresponds to viral genes with higher expression levels?

 

Lines 295-296: Why was this specific vsiRNA sequence chosen for quantification? Some more information is needed here.

 

Line 308: At the end of the Discussion section, perhaps a discussion should be added here that many vsiRNAs are produced by this method, and an investigation on the possible silencing effects of these additional vsiRNAs on mammalian genes (unintended targets) would be warranted. This could possibly be carried out by RNA-seq analysis of the macrophages described here.

 

Line 312-313: something is missing from the sentence starting at the end of Line 312.

 

 

 

 

 

 

 

 

 

 

 

Author Response

Dear Reviewer,

We appreciate your comments and we have responded to all of them.
Please see the attachment.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Concerns raised during the first round of review has carefully been address by the authors. However, there are still some minor issues to consider. 

Comments:

1- Line 97:

Bear in mind that ICTV rules do not permit the abbreviation of virus species names (italicized, initial capital letter, but no other capital letters unless of a proper name); e.g. ‘Plantago asiatica mosaic virus’ (species name, italics) cannot be abbreviated to ‘PlAMV’. Only the virus common name (Plantago asiatica mosaic virus, all lower case, normal font – initial capital ONLY because it is a genus name not also used as a common name) can be abbreviated as PlAMV. Solution to this is to introduce the virus species name, followed in parentheses by the common name and the abbreviation – thus “Plantago asiatica mosaic virus [species name, italic font] (Plantago asiatica mosaic virus [common name, normal font]; PlAMV)” = “Euphorbia mosaic virus (Euphorbia mosaic virus Yucatan Peninsula; EuMV-YP)” 

2- Line 162:

Fulneček et al. (2004) is not included in the reference list. Is there a special reason for that?

3-Please number the sections in a uniform manner (including or excluding dot ".",) please refer to lines 172, 233, 256, 271.

4- Line 269

What is the significance of "average % GC greater than" in this context? 

5- Figure 2 fits better under material and methods section.

 

Author Response

Dear Reviewer,

We appreciate your comments and we have responded to all of them.
Please see the attachment.

Author Response File: Author Response.docx