Virus–Host Interactions and Genetic Exchange in Mixed Infections of Tomato Yellow Leaf Curl Virus (TYLCV), Tomato Leaf Curl New Delhi Virus (ToLCNDV), and Tomato Chlorosis Virus (ToCV)
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript titled ‘Virus-host interactions and genetic exchange in mixed infections of tomato yellow leaf curl virus (TYLCV) and tomato leaf curl New Delhi virus (ToLCNDV)’is interesting and well-developed, addressing significant questions about the role of viral mixed infection in different host plants.
Overall, I would consider the manuscript well worth of publication, as it sheds light on the effects of genetic exchange in pathogenicity and contributes to a better understanding of mixed infections between TYLCV and ToLCNDV in different tomato lines. The data well support the conclusions.
However, several aspects require the author’s attention to improve clarity and impact.
Moreover, English language and style should be reviewed in several parts throughout the manuscript.
Please see below my comments:
Line 6 -13: The affiliation of the first and the third authors is very similar except for ‘Área de Genética, Facultad de Ciencias’. Please, correct.
Line 17-18: Avoid using the same adjective in one sentence.
Line 32-35: Keywords should be insert following the alphabetical order.
Line 49-51: A sentence about the difference (geographical distribution, genome organization, genetic diversity….) between Old World and New World begomoviruses should be added to better clarify their diversity.
Line 47: References must be inserted in progressive order. This mistake is also repeated elsewhere in the manuscript.
Line 71: Add ‘s’ to restrict.
Line 76: Choose one type of brackets to use for reference numbers in the manuscript.
Line 95: Change ‘inform’ with ‘informing’.
Line 98-127: The use of first person is not recommended in a manuscript.
Line 102: Specify if Nicotiana benthamiana plants, used as controls in the experiments, are susceptible to both viruses.
Line 108: Correct ‘DNAB’.
Line 118-120: The sentence should be rephrased as follows: ‘Two infectious clones were used for Agrobacterium tumefaciens-mediated inoculation: the Istraelian TYLCV-IL isolate (ES-Alm-Pep-99; GenBank Acc. N. AJ489258) [61]; and the Spanish ToLCNDV-ES isolate (ES-Al-661-Sq-13; DNA-A GenBank Acc. N. KF749223 and DNA-B GenBank Acc. N. KF749226) [58]’ since is not clear the provenance of the clones in materials and methods section.
Line 118-120: Specify better the reason why two different inoculation methods were used (Agrobacterium and vector mediated) for different viruses and not a comparison between them for all viruses taken into account.
Line 122: In which proportion the infectious clones are present in the culture?
Line 123: Correct the subscript of MgCl2.
Line 125: Indicate which is the final optical density of the suspension.
Line 127: Before starting with ToCV vector-mediated inoculations, explain better the procedure of inoculations for TYLCV-IL e ToLCNVD-Es, if single and multiple and why.
Line 143: A total DNA extraction with a purification step was not performed, so please, change the title of paragraph. It could be ‘Symptoms evaluation and virus detection by tissue blot hybridization’
Line 144: Delete ‘the presence of’.
Line 146: Add ‘moreover’ at the beginning of the sentence.
Line 163: Insert also the short form ‘dpi’.
Line 165: Which is the concentration of the extract?
Line 166: Insert the manufacturer of the EvaGreen Master Mix
Line 170-175: Change ‘included’ with ‘were’.
Line 170-177: Specify if the primer pairs are custom-designed or not. In the first case, the program used should be indicated, otherwise the respective reference.
Line 180: Change ‘quantification’ with ‘standard curve’.
Line 183: Add a sentence about the preparation of the nucleic acids used for sequencing.
Line 199-201: Change the sentence as follows: ‘TYLCV-IL (GenBank AJ489258), ToLCNDV-ES DNA-A (GenBank KF749223), and ToLCNDV-ES DNA-B (GenBank 200 KF749226) were used as reference sequences.’
Line 210: Explain better what you mean with the word ‘concatenating’.
Line 215: Insert ‘s’ of plural.
Line 220: Change ‘corresponding’ with ‘cloning’.
Line: 223: Delete ‘or’.
Line 222-227 and 240-244: These two sections should be moved to the Results section as outcomes of the work.
Line 248: Change ‘TYLCD’ with ‘TYLCV’.
Line 258-265: This part could be moved in Materials and Methods section to better explain the inoculation procedure.
Line 269: Put firstly Figure 1 then Table 1.
Line 271: Change with ‘between non-infected and infected condition’.
Line 286: Why only some plants are present in the figure 1? Please, correct the image by inserting all the plants used for this study.
Line 287: The strain TYLCV-IL is Israelian or Spanish? Please clarify this issue here and in the Material and Methods section.
Table 1: Change ‘interaction between’ with ‘Detection of’
Table 1: Specify the correspondence of each column with the inoculation method (if sequential or simultaneous).
Table 1: Distinguish from tomato lines N. benthamiana in the table as susceptible control.
Line 298: Where is letter ‘a’ in the table? Please correct.
Line 302: Change ‘squash’ with tissue.
Line 308: Add ‘in addition to what is observed in N. benthamiana plants’
Line 308: Co-inoculation or simultaneous inoculation?
Line 309: Delete ‘so’
Line 323: Delete ‘or antagonistic’ because TLCNDV does not efficiently infect if there is also TYLCV.
Line 334: Add ‘with ToLCNDV-ES’.
Line 354: Delete ‘in’.
Table 2: Bring the same correction as suggested for Table 1.
Line 363: Change ‘resistant’ with -susceptible.
Line 365: Change’squash’ with ‘tissue’.
Line 382: Add the software version.
Line 383-388: Correct properly the figure 2a by marking these three cases, because in particular for N. benthamiana, is reported TYLCV-IL/ToLCNDV-ES-Nb" instead of "TYLCV-IL+ToLCNDV-ES-Nb" e "TYLCV-IL/ToLCNDV-ES-Nb".
Line 424-425: Where is the figure corresponding to this passage?
Figure 3: The resolution of this image is low. Please, modify the picture.
Line 541: The clone names are on the left while the size is on the right. Please, correct.
Reference: The reference section is too long, please, reduce.
Comments on the Quality of English Language
English language and style should be reviewed in several parts throughout the manuscript.
Author Response
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Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsDear Authors,
Thank you for the opportunity to review your manuscript titled “Virus-host interactions and genetic exchange in mixed infections of tomato yellow leaf curl virus (TYLCV) and tomato leaf curl New Delhi virus (ToLCNDV)”. Your study presents an important and timely contribution to the field of plant virology, particularly in understanding the dynamics of mixed begomovirus infections in tomato and the potential for genetic exchange through defective DNA molecules. The experimental design is thorough, and the use of both molecular and high-throughput sequencing techniques strengthens your findings. To help improve the clarity and impact of your manuscript, I have highlighted several suggestions for minor revision directly in the annotated PDF file.
Comments for author File: Comments.pdf
Author Response
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Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsThis article evaluates the impact of mixed infection of ToLCNDV with TYLCV-IL or ToCV in tomato and N. benthamiana plants.
Susceptible and resistant tomato plants were single or mixed inoculated with these viruses and infection and disease development were recorded. The occurrence of genetic exchange between TYLCV and ToLCNDV was analyzed by HTS from RCA products in a mixed of sampling collected at 90 and 110 days post inoculation.
Results show that ToLCNDV can infect susceptible tomato plants but at a very low rate, with A or A and B components. However the replication of TOLCNDV-B component could not be assisted by TYLCV. ToLCNDV did not infect TY-1 or Ty-2 resistant plants. Sequence analyses did not reveal recombination events between A component and TYLCV in tomato but two events were detected in N. benthamiana among defectives genomes
ToLCNDV infection is not enhanced by ToCV in tomato .
Authors conclude that mixed infection of TYLCV and ToLCNDV do not lead to pathogenicity modifications, nor to the emergence of recombinants genomes.
The purpose is relevant because thee is a diversity among ToLCNDV isolates in the mediterranean basin and different results were obtained in a previous study with an italian isolate. The experimental design sounds correct and most of the results look interesting. In general, the document is well written but I have some concerns for many things in the methods and results parts:
Abstract: There is no mention of ToCV in the title. Why ?
Part 2 :
The number of plant tested in the different experiments should be given in part 2
Title of 2.3 : I suggest « Symptoms evaluation and virus detection »
The symptom scale is adapted from Friedman but we do not know how. Is it justify to have six levels ? pictures could help understanding this, at least in the suppl data
How did the mixed inoculum for TYLCV, ToLCVA and B component was prepared ?
How did they perform the agroinoculation of ToLCNDV and vector inoculation of ToCV ? at the same time or sequentially ? it is written under table 3 and not in the method part;
Sequential methods doesn’t provide any information in the paper. I would suggest to delete or put it in supplementary data. At least lines 258-263 should be in part 2 or in supplementary file
2.5 : a HTS analysis would allow a better description of the diversity ofrecombinants ; while the analyse performed here shows only the dominant genomes
100ng of total DNA seems to be a huge amount for PCR. Is it correct ?
Part 3 Results
Are you sure that agroinoculation of ToLCNDV at the five to six leaf growth stage is efficient in tomato ? there is no positive control on tomato and I understand why, but you should discuss that
Line 268 : the result is that you could not detect TYLCV in ty2 resistant plants then you can suppose that TYLCV failed to infect ; not the opposite
Line 269 : Why « notably » since you switch to susceptible plants ? and for me 9/10 rondeno plants were infected with TYLCV.
Table 1, 2 and 3 are very difficult to read. Many things are useless in these table, or maybe I did not understand : how can you claim that you do not detect ToLCNDV (A+B or A or B) in coinfected plants with a TYLCV Probe ? same thing for TYLCV detection in coinfected plants with ToLCNDV A or B probe
Why do you present 3 colums of results under probes A and B for ToLCNDV ES treatment in table 1? there is no sequential inoculation there…
table 2 and 3 : I suppose that you used a common probe for A and B component. it should be written in the legend; what is "-" in table 2? I suppose that you did only one assay on Moneymaker. but you claim 2 assays line 331
Lin 307 is in contradition with 271-272 ?
Line 316: You cannot conclude « no functional complementation » if TYLCV did not infect resistant plants
3.3 line 350: it is not easy to understand the pretreatment with virus free B tabaci because youd did not explain how you coinoculated both viruses with different methods
Line 384 specify TY+To coinfected sample
Line 401: It is not clear if you speak about rate of infection or virus accumulation
Line 405: There is no statistic there, is it significant ?
Figure 2 a: can you add the same figure for TYLCV-mld and TYLCSaV in the suppl part
I was very surprised to see that defective molecules detected in N.benthamiana exhibit simple structures, with just a deletion of an entire part of the genome of TYLCV. In the lirtterature, defectives of monopartite begomovirus genomes are often composed of copy past in the right or the inverted orientation of different part of the genomes. Is it linked to the detection method used here?
Line 550 : you cannot say that if TYLCV did not infect resistant plants
Line 560 I don't agree with the sentence because it is known that Indian isolate of ToLCNDV can infect tomato plants with only A components (Jyothsna et al 2013, Sivalingam et al, 2012)
Line 564 there is no statistics on figure 2b and we do not know how many plants were analyzed
Line 615 : We do not know if the number of plants analyzed allows to conclude about rare events.
Line 656 : please specify that this is for the B component
Author Response
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Author Response File: Author Response.pdf
