A New Strategy of Cross-Protection Based on Attenuated Vaccines: RNA Viruses Are Used as Vectors to Control DNA Viruses
Round 1
Reviewer 1 Report
Overall, the paper is well presented and well structured. The manuscript is clear and relevant to the field of study. The references cited are mostly (64%) non-recent publications (within the last five years). The manuscript has a small number of self-citations. The manuscript results are reproducible as indicated in the methods section; however, they are easy to interpret, understandable, and capture data appropriately and consistently throughout the manuscript. It is suggested that the dates of the RT-PCR reactions and their final reagents and concentrations be reported. The conclusions are consistent with the evidence, and the arguments made in the paper are supported by the listed citations.
In this study, a cross-protection method was investigated to control DNA viral diseases such as Tomato Yellow Leaf Curl Virus and Cucumber Mosaic Virus in tomato plants. The goal was to use RNA virus vaccines for cross-protection to control the disease.The topic is important for research in general because RNA virus-mediated control of DNA virus diseases has not yet been studied. Therefore, the results of this study make an important contribution to the control of virus diseases in plants.
In the methodology, the experiments performed are clearly described; only the PCR reaction conditions should be mentioned. The figures are clear and easy to read and interpret.
The conclusions are consistent with the evidence and arguments presented in the paper and are supported by the citations listed.
Minor comments
I recommend that the manuscript can be accepted for publication.
Minor editing of English language required
Author Response
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Author Response File: 
Author Response.pdf
Reviewer 2 Report
1. First and foremost, as a plant virologist with nearly 40 years of experience, I have never heard plant virus infections referred to as "plant cancers". This statement needs to be removed. When I think of plant cancer, I think more of the galling caused by Agrobacterium species which leads to uncontrolled cell division. In fact, a lot of early research on crown gall was due to its similarities to human cancers and it was hoped that elucidating the mechanism of plant infection would lead to controlling cancer in humans.
2. The authors correctly state the definition of "cross protection" as being the reduction of disease severity by inoculating a milder strain of the pathogenic virus. As such, I cannot accept the use of cross protection in the title when the viruses are not the same. It is more correct to term this as viral induced gene silencing (VIGS), not cross protection.
3. The disease incidence rating scale seems very arbitrary with no clear demarcation between levels. For example, between levels 5 and 7, what would you classify a plant that is stunted to 2/3 of standard height? Do you call it 5 or do you call it 7? Also, why were only odd numbers chosen for indexing purposes. A typical plant disease rating scale (regardless of pathogen) uses a 1-5 scale.
4. It would be clearer if all of the disease rating results were put into 1 master table for ease of comparison between treatments with the various TYLCV inserts rather than inserted below the photos of each group in a rather small font.
5. In lieu of a disease rating scale, have the authors considered using a qPCR/qRT-PCR approach to directly quantify the effects of VIGS on TYLCV (qPCR) and CMV (qRT-PCR) relative infection levels?
6. ln 180: I would not use the term probable as this implies unjustified certainty. Rather, I would use the term possibly as it's more open ended to other interpretations.
7. The gel electrophoresis images seemed tightly cropped with only products between 0.5 and 1 kb being displayed. I would recommend supplying the uncropped images as part of the supplementary material to dispel any doubts about the specificity of the PCR primers utilized in this study.
8. Were the RT-PCR products ever sequenced to ensure that there weren't other spontaneous mutations generated during the infection process?
9. The English needs to be strongly corrected throughout the manuscript as many sentences do not make any sense because they are missing words.
The authors need to submit the manuscript for extensive editing of English. While it is readable, it is not consistent and some terms used are incorrect.
Author Response
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Author Response File: Author Response.pdf
Reviewer 3 Report
This manuscript from Zhu et al details the design and performance of four recombinant viral vaccines against tomato yellow leaf curl virus (TYLCV), which has a wide plant host range and causes significant crop losses. The vaccines were created by inserting three different TYLCV antigens into three available cucumber mosiac virus (CMV) vectors which have been engineered to allow for recombination as well as to create an attenuated viral vector. The group found that the recombinant vaccines were non-pathogenic in plants and protected against TYLCV challenge and TYLCV/CMV co-challenge to varying degrees. The vaccines also stably maintained their recombinant antigens.
As a whole, this paper is straight-forward and well-written. My biggest comments to be addressed prior to acceptance is to expand discussion on the virulence needed for onset of protection in plants (to justify the usage of attenuated viral backbones) and on the time scales of vaccine-mediated protection in plants (since the authors suggest that RNAi by the host is the primary means of anti-viral protection).
I also have the following minor comments to be addressed:
1) In the methods, the authors should explain why they used the four vector/ TYLCV antigen combinations in this paper.
2) Figure 1B would benefit from having at least one of the attenuated backbone CMV viruses (without TYLCV antigen) as proof that the primary means of attenuation is from the viral vector.
3) Lines 180-181 are unclear- the authors should clarify how C1C4 is able to elicit the "SIE" response in 2bPTI ant not 2bPTII, such as any affects on coding frames.
4) In line 187, the authors should clarify the ratio of each vaccine used in the co-vaccination experiment.
5) It appears in figure 2D that the last two lanes have the PCR targets of TYREP and TYR-Rep respectively (since they both seemed to have come from the vaccination experiment) but it isn't entirely clear from their labeling or the figure description- the authors should clarify. The authors should also indicate why their PCR target may have become smaller with co-vaccination.
Author Response
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Author Response File: Author Response.pdf
Reviewer 4 Report
The paper “A new strategy of cross-protection based on attenuated vaccine: RNA viruses are used as vectors to control DNA viruses” is very interesting and the authors described a new approach to cross-protection against DNA viruses. Results obtained in the work are very important for the management of TYLCV, one of the most important viruses for tomato, found in tropical and subtropical regions around the world. I recommend that the manuscript be published after incorporating the observations made, especially those referring to materials and methods.
Comments for author File: Comments.pdf
Author Response
Dear editor,
Thank you for your help and valuable suggestions. We have made revisions based on the comments of the reviewers. We believe that the manuscript is now suitable for publication in Agronomy. Please feel free to contact me for any additional information that could facilitate the revision and publication process of our manuscript.
Sincerely,
Xuefeng Yuan
Department of Plant Pathology, College of Plant Protection,
Shandong Agricultural University
Tai’an, 271000, P. R. China
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
The manuscript has been greatly improved! I only have a few minor corrections in terms of language:
Ln 13 (Abstract): ..threating to agricultural production and food supply. Do you mean ...threatening agricultural production and the food supply?
Ln 78-79: ...recombinant mutants, which were inserted into the identical TYLCV fragment but utilized different CMV vectors. I think this should be ...the identical TYLCV fragments were utilized in different CMV vectors.
Ln 130-131: TaKaRa sells 4 versions of MMLV (https://www.takarabio.com/products/mrna-and-cdna-synthesis/reverse-transcriptases). Can you state which one was used for reproducibility purposes? Also, which polymerase was used in the PCR reactions as I didn't see a specific one mentioned?
Ln 161: ...was performed, and a mixture of wild-type RNA1 and RNA3... I think it should state that the various RNA2 mutants were combined/mixed with wild-type RNA1 and RNA3...
Ln 240: being very nitpicky here but the plural of index is not indexes, rather it's indices.
Much improved but I was still able to find a few grammatical errors.
Author Response
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Author Response File: Author Response.pdf
