Effective Antiviral Application of Antisense in Plants by Exploiting Accessible Sites in the Target RNA

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Antisense oligodeoxynucleotides (ASOs) into clinical practice to selectively inhibit or modulate gene expression at the RNA level are being actively and effectively introduced. However, questions remain about the practicality of using antisense technologies due to the difficulty of reliably predicting sites accessible to siRNA and ASO in complex structures. The article by Gruber S. Et al., “Effective Antiviral Application of Antisense in Plant by Exploiting Accessible Sites in the Target RNA” is devoted to assessing the possibility of using ASO technology in plants to trigger the phenomenon of RNA-induced silencing of viral RNA in vitro, for reliable identification sites accessible to Argonaute endonucleases directed by ASOs or siRNAs. The authors of the work believe that optimized ASO oligomers can find application in crop protection. The work convincingly shows that, selected using the eNA-screen method, ASOs effectively reduced the development of symptoms of TBSV disease. However, the authors did not illustrate how viral RNA accumulates in the tissues of infected plants and how the rate of viral RNA replication changes in the absence and presence of ASO. The conclusions are clear. The article is interesting and after explaining the question posed can be published.

Author Response

We thank the reviewer for this very positive evaluation. The following important point was made: "However, the authors did not illustrate how viral RNA accumulates in the tissues of infected plants and how the rate of viral RNA replication changes in the absence and presence of ASO". 

Answer: This is correct, our analysis of the infection experiments mainly relied on the distinction symptomatic/symptom-free. In the applied N. benthamiana/TBSV system, this is very easy to perform (evident symptoms such as systemic necrosis). Moreover, from previous work, we know that in plants that are still symptom-free 3-5 weeks after virus inoculation, viral RNA is generally not detectable in upper, non-inoculated leafs (Gago-Zachert et al., 2019). To explain this, we included an additional paragraph into the Materials an Methods section (lines 576-581 of the revised manuscript) and changed the title of the paragraph (line 565 of the revised manuscript) as follows:

Plant treatment, TBSV challenge and analysis of infection assays.......The analysis of the infection assays was based on the simple distinction of symptomatic/ symptom-free. In the N. benthamiana/TBSV system used, this is very easy to achieve by detecting obvious symptoms such as systemic necrosis. RNA accumulation was not monitored as we knew from previous work that in plants that are still symptom-free 3-5 weeks after inoculation with TBSV, viral RNA is usually not detectable in the upper, non-inoculated leaves [20].

Reviewer 2 Report

Comments and Suggestions for Authors

Very nicely designed set of experiments that demonstrate the similarities between AGO/RISC and RNase H based cleavage mechanisms. The results are clearly presented and the conclusions obtained are very convincing. The main question addressed by the determination of similarities/differences between RNA silencing and RNase H mediated degradation in order to validate the use of antisense DNA for antiviral activity. It's relevant as it shows that plants can achieve the same goal (prevent viral infection) using different mechanisms that have a similar approach (degrade viral RNA). I believe this is the 1st comparison of the efficacy of antisense DNA oligos to siRNAs. I would like to applaud the authors for including the original, uncropped/uncomposed gel images for integrity purposes. Will these be also available as supplementary material or was it only for review purposes?

Author Response

We thank the reviewer for the very positive evaluation of our manuscript. Concerning the kind statement and question "I would like to applaud the authors for including the original, uncropped/uncomposed gel images for integrity purposes. Will these be also available as supplementary material or was it only for review purposes?" is answered by us in the following way:

Actually, this was requested by IJMS during submission, and as far as we understood, this was thought for review purposes only. 

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript titled “Effective Antiviral Application of Antisense in Plants by Exploiting Accessible Sites in the Target RNA” presents a comprehensive exploration of RNA silencing through ASO, providing potential insights into antiviral applications.

The paper demonstrates a well-structured approach to investigating the efficient silencing of target genes, indicating broader implications for the field. 

The results suggest promising avenues for further research on the antiviral potential of antisense technology in plants.

While the title reflects the paper’s focus, minor revisions may be considered to enhance its alignment with the content discussed in the manuscript.

The manuscript could be improved through a careful review of certain sentences (for example lines 35-38) to ensure better clarity for readers.

It is recommended to thoroughly cross-check the entire manuscript to verify the correct placement of main and supplementary figure numbering, ensuring accuracy and coherence.

Comments on the Quality of English Language

Minor editing of English language required

Author Response

We thank the reviewer for the very positive evaluation of our manuscript. Two points were made

(1) "The manuscript could be improved through a careful review of certain sentences (for example lines 35-38) to ensure better clarity for readers."

Answer: We did so. The mentioned sentence and several others were improved. A native speaker improved the English language of the entire text. This is now mentioned in the acknowledgement

(2) "It is recommended to thoroughly cross-check the entire manuscript to verify the correct placement of main and supplementary figure numbering, ensuring accuracy and coherence."

Answer. We thank the reviewer for this valuable comment. In fact, we forgot to mention Supplementary Figure 2 in the manuscript. This has now been done (line 199 of the revised manuscript). Moreover, we adjusted the numbering of the citations and the references to the new format of the manuscript. 

Reviewer 4 Report

Comments and Suggestions for Authors

In my opinion, in this research the application of antisense oligodeoxynucleotides in the RNase H-mediated antisense silencing in plants is interesting. I think the research is well done and well presented in the manuscript. I think it is publishable in the form it has been submitted, and that only the format needs to be checked and, if possible, some additional bibliographical references added.

Comments on the Quality of English Language

In my opinion, only minor revision is required.

Author Response

We thank the reviewer for the very positive evaluation of our manuscript. One point was made: "I think it is publishable in the form it has been submitted, and that only the format needs to be checked and, if possible, some additional bibliographical references added."

Answer: Following also the suggestion of reviewer 3, we adjusted the format. With 55, the number of references is already high. Therefore, we decided to keep it as is.