Exogenous Application of IR-Specific dsRNA Inhibits Infection of Cucumber Green Mottle Mosaic Virus in Watermelon

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript "Exogenous application of IR-specific dsRNA inhibits infection of cucumber green mottle mosaic virus in watermelon" presents interesting data on a possible methodology/mechanism to protect plants against virus infection.

Overall, the ms is organized but there are a few areas that can be improved to facilitate the reading. Some of the questions are below. In most cases, the authors might be able to answer them without any further experiments. 

Plasmids L4440

It will be nice to see the fragments that were selected and cloned (location in the genome). The authors only mention their size in bp: MET (1130), IR (1300), and HEL (765).  Perhaps a genomic CGMMV map with the location of the primers used to amplify the fragments.

Figure 6 mentions a schematic representation of virus genome but it was not included in the figure (ORF, regulatory regions, etc.).

 

General Questions.  Answers (and discussion) might help to improve the manuscript.

What was the criterion to select those regions and fragment sizes?

Figure 2

In C and D.  What samples were used for RT PCR and ELISA analyses? 14, 21 dpi? Other?   Text says 21 days.  If so, please include that information in the figure legend to facilitate reading.

Size of GUS-dsRNA?  Compared with the viral fragments used.

 

Additional questions on the Application of dsRNA (total amount vs copy number).

What it means crude extracts? 

Did you quantify the amount of RNA?

Or, did you apply the same amount of dsRNA?

Any idea if the (protective) response is dosage-dependent? In other words, if you inoculate/apply more (or less) dsRNA the protection could be higher, longer (or lower in case of less dsRNA)?

Another way to address that:

Considering that the cloned fragments were different in size, the same amount of total dsRNA used for the treatments would represent different number of dsRNA molecules in each case (copy number)?  Could that be important? 

Yes, why?,  No, why?

 

Systemic movement.

Please provide more information about the systemic leaves used. It means, the next leaf to the inoculated one? Two leaves? 

Was the same leaf position analysed in all time points? At 12 dpi you might get newly developed leaves (not present at the time of inoculation). Could the dsRNA move preferentially to the younger, newly developed leaves?

 

What is the importance of the last base of the vsiRNA?  The las paragraph of the discussion is a bit confusing. Their results show  predominantly 5’A and 3’ U: 
“AGO1 and AGO2 preferentially associate with sRNAs possessing U and 5' termini, respectively, while many AGO proteins participate in the processing of vsiRNA inside the watermelon RISC complex”

Comments on the Quality of English Language

Overall the English language is OK/good. But It is always possible to improve by a extended reviews by a English-native editor.

Author Response

Dear reviewer, we have modified the paper according to your suggestions，thank you for your comments. Best wishes for you.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors

The revision of the manuscript Agronomy-3871140 has been submitted.

 

 

Cucumber green mottle mosaic virus (CGMMV), a member of the genus Tobamovirus, which primarily infects plants in the family Cucurbitaceae, has been disseminated globally via the international seed trade. CGMMV are serious threat in the production of watermelon. Manuscript agronomy-3871140  (Exogenous application of IR-specific dsRNA inhibits infection of cucumber green mottle mosaic virus in watermelon) describes the RNAi technology employs using dsRNAs to target and reduce the expression levels of specific viral genes in plants. The research is interesting, thoroughly conducted, and offers novel data about CGMMV mangement. The manuscript is also well written although a few minor changes should be considered to improve its overall quality. Some recommendations are included in the manuscript, and other minor suggestions were provided below.

 

Kind regards,

1. Introduction:

-In the introduction, the importance of watermelon cultivation could be mentioned, although impact and distribution of the virus were adequately addressed.

-In line 45: Use the original citation or reference for the first CGMMV report in 1935.

2. Materials and Methods

The methodology was correctly conducted. Review line 99. In section 2.5. more details of the implemented techniques could be included.

-Check the style of the units in lines 140 and 141.

3. Results

-The results were adequately presented, and controls were used in all analyses, which is important in these studies. The analyses were very well planned.

-Review some typographical details in lines 163 and 238.

- In section 3.3: The authors verified that the externally applied dsRNA can stably persist in the plant for at least 7 days and is capable of systemic movement. -Based on these results, should a proposed field application frequency be established for disease management? Consider as a recommendation. The use of nanoparticles as carriers and adjuvants would probably have favored the protection period.

4. Discussion

-The discussion is clear and concise. Considering the above in lines 352-357 “Due to the volatility of dsRNA during external application, frequent spraying of dsRNA is essential for sustaining plant resistance to viruses during crop development……”, The period of protection obtained in these trials should be reflected in the conclusions.

4. Conclusion

It is suggested to include the above. If the plants treated with viral-derived dsRNA showed a considerable decrease in both virus titers and vsiRNA levels, the authors should consider their use in the field, specifying the possible frequency, dosage, etc.

 

Comments for author File: Comments.pdf

Author Response

Dear reviewer, we have modified the paper according to your suggestions，thank you for your comments. Best wishes for you.

Author Response File: Author Response.docx