A Novel Isolate of Bean Common Mosaic Virus Isolated from Crownvetch (Securigera varia L. Lassen)

Round 1

Reviewer 1 Report

Review

In the article titled A Novel Strain of Bean Common Mosaic Virus Isolated from Crownvetch, a new isolate of BCMV found in Securigera varia L. is described, and its molecular characterization is performed through nucleotide sequence and protein analysis including LC-MS/MS Analysis. Overall, the paper can be published with some modifications.

Title: I think that de scientific name of Crownvetch, should be included. On the other hand, the authors indicate that it is a new strain of BCMV, but in the article, they do not explain why they considered it a new strain and not simply a new isolate.

 

Keywords: are correct

Regarding the modifications, I suggest incorporating a recombination analysis since the authors mention the existence of recombinant isolates among different BCMV isolates. However, they do not confirm whether the studied isolate incorporates recombination events.

On the other side, it would be convenient to mark with brackets the different phylogroups in the phylogenetic tree and highlight the isolate under study.

Some minor modifications

Line 37 y 38: change uppercase to lowercase in virus names.

Line 38: bean golden yellow mosaic virus is a Begomovirus, not a potyvirus, maybe you meant bean yellow mosaic virus.

Line 75 I consider that a more detailed explanation of the symptoms observed should be given and if possible, a photo with symptoms should be included.: if not here in M&M.

Line 158: HC-Pro functions should be explained with more details.

HC-Pro is a multifunctional protein that plays a critical role in the life cycle of potyviruses by promoting viral replication, suppressing host defense mechanisms, facilitating viral movement, and inducing disease symptoms.

Line 180: Phaseolus vulgaris (order Fabales, family Fabaceae), Fabales and Fabaceae, should be written with italics.

Line 183: Fabaceae is usually assigned, Fabaceae also in italics.

Figure 4: should explain the origin of the isolates used in the comparison.

Author Response

Odpovede na komentáre recenzenta 1

 

Ďakujem za podnetné komentáre. Našou snahou bolo vyhovieť požiadavkám recenzenta úplne alebo čo najviac.

 

Bod 1: Myslím si, že by mal byť zahrnutý vedecký názov Crownvetch.

Odpoveď 1: Prijaté.

 

Bod 2: Na druhej strane autori uvádzajú, že ide o nový kmeň BCMV, ale v článku nevysvetľujú, prečo ho považovali za nový kmeň a nielen za nový izolát.

Odpoveď 2: Prijaté.

• Ako napísal Kuhn a kol. (Arch Virol 2013; 158(1): 301–311, doi: 10.1007/s00705-012-1454-0) “ Bohužiaľ, väčšina študijných skupín ICTV alebo iných expertných skupín v minulosti neposkytla jasné usmernenia , akceptujúce kmeň a genetické varianty názvov , ako ich navrhovali rôzni výskumníci vo svojich publikáciách, namiesto vytvárania konzistentných nomenklatúrnych schém, ktoré sa vzťahujú aspoň na všetky vírusy jednej rodiny. Status quo je teda taký, že varianty konkrétnych vírusov sú často pomenované podľa rôznych štandardov .
• Tiež v databáze Genbank niektorí autori používajú „kmeň“ a iní používajú „izolovať“ v publikovaných sekvenciách genómu BCMV (https://www.ncbi.nlm.nih.gov/nuccore).
• Van Regenmortel (Infect Genet Evol 2007; 7: 133–144. doi: 10.1016/j.meegid.2006.04.002) definoval variant vírusu ako izolát , ktorého genómová sekvencia sa líši od sekvencie referenčného vírusu.

Preto akceptujeme pripomienku recenzenta a v názve a na jednom mieste v texte rukopisu sme urobili zmenu na „izolovať“. Slovo „kmeň“ sme však ponechali aj v kapitole 1. Úvod , pretože citovaní autori používali tento názov vo svojich publikovaných prácach.

 

Bod 3: Čo sa týka modifikácií, navrhujem začleniť rekombinačnú analýzu, keďže autori uvádzajú existenciu rekombinantných izolátov medzi rôznymi izolátmi BCMV. Nepotvrdzujú však, či študovaný izolát zahŕňa rekombinačné udalosti.

Odpoveď 3: Neuskutočnili sme rekombinantnú analýzu. Zaradenie izolátu BCMV SVK medzi rekombinantné bolo uskutočnené na základe fylogenetickej analýzy. BCMV SVK je extrémne geneticky podobný izolátu BCMV PV 0915, ktorý patrí do rekombinantnej fyloskupiny R1. V tomto zmysle sme upravili text v časti 2.4. Fylogenetické porovnanie BCMV SVK .

However, the essence of our work was not to verify inclusion of the BCMV SVK isolate among some phylogroup, but the genomic and proteomic characterization of the BCMV SVK isolate.

 

Point 4: On the other side, it would be convenient to mark with brackets the different phylogroups in the phylogenetic tree and highlight the isolate under study.

Response 4: Accepted.

Phylogroups (S, C, P, R1-R3) were included in the phylogenetic classification.

 

Point 5: Line 37 y 38 (38-39 now): change uppercase to lowercase in virus names.

Response 5: Accepted.

 

Point 6: Line 38: bean golden yellow mosaic virus is a Begomovirus, not a potyvirus, maybe you meant bean yellow mosaic virus.

Response 6: Accepted.

We had BCMNV in mind. In line 38 we changed BGYMV to BCMNV. At the same time, we also changed the relevant reference [3].

 

Point 7: Line 75 I consider that a more detailed explanation of the symptoms observed should be given and if possible, a photo with symptoms should be included.: if not here in M&M.

Response 7: Accepted.

Figure 8 and symptoms of BCMV infection present on crownvetch plant were included in chapter Materials and Methods (lines 256-258 now).

 

Point 8: Line 158: HC-Pro functions should be explained with more details. HC-Pro is a multifunctional protein that plays a critical role in the life cycle of potyviruses by promoting viral replication, suppressing host defense mechanisms, facilitating viral movement, and inducing disease symptoms.

Response 8: Accepted.

We added some of the functions of the multifunctional HC-Pro protein to lines 163-164. However, our work is not about the Hc-Pro protein, as in the paper of Valli et al. (Mol Plant Pathol. 2018, 19(3): 744–763. doi: 10.1111/mpp.12553) and many others.

We did not want to describe the functions neither of the HC-Pro protein nor all the other proteins. Our interest in this gene was based due to only 2 substitutions discovered in the gene and amino acid sequence of HC-Pro.

 

Point 9: Line 180 (191 now): Phaseolus vulgaris (order Fabales, family Fabaceae), Fabales and Fabaceae, should be written with italics.

Response 9: Accepted.

 

Point 10: Line 183 (195 now): Fabaceae is usually assigned, Fabaceae also in italics.

Response 10: Accepted.

 

Bod 11: Obrázok 4: mal by vysvetliť pôvod izolátov použitých pri porovnaní.

Odpoveď 11: Prijaté.

Názvy jednotlivých izolátov BCMV, uvádzané tu iba kódmi z databázy Genbank, sú na obrázku 7. Sú tam kódy z Genbank, pôvod izolátov (hostiteľ a krajina) a fyloskupiny S, P, C, R1, R2 , R3. Naznačili sme to aj v legende k obrázku 4.

Reviewer 2 Report

The current study is critical where the potential host range of a potyvirus in wild plants is characterized and is frequently associated with viral epidemics in the field. Much scope remains to improve the significance of this study. Moreover, based on the journal where readers are from different research fields, the discussion should be more elaborated which highlights the importance of this study. 

Major comments:

- line 20: " confirmed by western blot and by ...."

- lines 31 to 36: The legumes are also enriched in secondary metabolites which should be highlighted here. Potyviruses are known to influence the secondary metabolites which in turn makes the plants susceptible or resistant to pest attack in field conditions. Thus, due to these intricate interactions, it is important to characterize the host range of a virus. Some papers that can be cited here: doi: 10.3389/fgene.2020.581357; https://doi.org/10.1111/mec.16103; https://doi.org/10.1016/j.foodchem.2020.127786

- Figure 1: The legend should also describe the bands shown in the picture in lane 2 and 4.

- line 84: "oligonucleotide sequences" should be "polypeptide sequence corresponding to the capsid protein"

- Figure 7: Legend should be more descriptive. Was it a maximum likelihood tree. How many bootstraps were used? What are the numbers indicated above the nodes? The phylogroups should be also indicated in the figure that is mentioned in the manuscript.

- line 216 to 223: I would request authors quantify the selection pressure on P1. This can be done in DNASP. The ratio of non-synonymous to synonymous substitution shows the selection pressure. If there is a positive selection pressure that would indicate the role of P1 in host adaptation. 

- lines 225 to 226: I would request authors to cite more relevant papers that highlight the importance of this study in characterizing the host range. There is a recent paper that shows a virus expands its host range from a wild plant to a pepper crop and caused frequent epidemics in Spain. Thus, the current study highlights the importance of crownvetch in disease management of common beans. DOI: https://doi.org/10.1128/jvi.01268-18

 

   

English should be checked by a professional for grammar and correct scientific word usage.

Author Response

Responses to Comments of Reviewer 2

 

Thanks for the stimulating comments. Our effort was to meet the Reviewer's requirements completely or as much as possible.

 

Point 1: line 20: " confirmed by western blot and by ...."

Response 1: Accepted. Changed to western blot.

Both terms - "Western blotting" and “Western blot”, are equally used in scientific papers. Nevertheless, the term “Western blotting” is used in publications disproportionately more often than "Western blott" and is written with a capital “W” rather than a small “w”.

 

Point 2: lines 31 to 36: The legumes are also enriched in secondary metabolites which should be highlighted here. Potyviruses are known to influence the secondary metabolites which in turn makes the plants susceptible or resistant to pest attack in field conditions. Thus, due to these intricate interactions, it is important to characterize the host range of a virus. Some papers that can be cited here: doi: 10.3389/fgene.2020.581357; https://doi.org/10.1111/mec.16103; https://doi.org/10.1016/j.foodchem.2020.127786

Response 2: It is true that legumes also synthesize secondary metabolites, and many of them relate to defence mechanisms during virus attacks. We apologize to the Reviewer, but we do not think that in our manuscript, which is essentially about the molecular characterization of a new BCMV isolate on a new host plant (from the agroecological interface of legume cultivation areas), it is necessary to comment on secondary metabolites, complex interactions, or the characterization of the host range of the virus BCMV. These are well-known facts that have been published many times. One manuscript cannot be about everything.

We read all 3 recommended articles (doi: 10.3389/fgene.2020.581357; https://doi.org/10.1111/mec.16103; https://doi.org/10.1016/j.foodchem.2020.127786). Once again, we apologize to the Reviewer, but we do not think that the topic of secondary metabolites is significantly related to our topic i.e., with the molecular characterization of a new BCMV isolate. Nevertheless, the first paper does not present secondary metabolite-BCMV (or viruses in general) relationships, nor do the second and third articles.

 

Point 3: Figure 1: The legend should also describe the bands shown in the picture in lane 2 and 4.

Response 3: Accepted.

The legend of Figure 1 already contains the information requested by the Reviewer i.e., the description of lines 2 and 4. The legend for this figure is: “Figure 1. Western blotting analysis of BCMV. Lanes: 1 – Protein ladder, 2 – Positive control (BCMV 80 isolate PV 0915), 3 – Negative control, 4 – symptomatic crownvetch plant (from the locality Čachtice), 5 – non-symptomatic crownvetch plant (locality Plavecký Štvrtok)”.

 

Point 4: line 84 (87-89 now): "oligonucleotide sequences" should be "polypeptide sequence corresponding to the capsid protein".

Response 4: Accepted.

This text was really wrong. This is where we really got it wrong (although we were developing antibodies ourselves using oligopeptide sequences). We changed it to the correct form.

However, this sentence is only our guess how the commercial producer (Leibniz Institute, DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany) probably produced the polyclonal antibody against BCMV (DSMZ does not provide any details).

 

Point 5: Figure 7: Legend should be more descriptive. Was it a maximum likelihood tree. How many bootstraps were used? What are the numbers indicated above the nodes? The phylogroups should be also indicated in the figure that is mentioned in the manuscript.

Response 5: Accepted.

The legend now describes specific methods used for construction of the phylogeny tree. Also, the phylogroups are now indicated in the figure.

 

Point 6: line 216 to 223 (229-236 now): I would request authors quantify the selection pressure on P1. This can be done in DNASP. The ratio of non-synonymous to synonymous substitution shows the selection pressure. If there is a positive selection pressure that would indicate the role of P1 in host adaptation.

Response 6: Quantification of selection pressure of the P1 protein of BCMV using DNASP was already performed - by Moradi, Mehrvar (Eur. J. Plant Pathol. 2019, 154, 673–690. https://doi.org/10.1007/s10658-019-01690-6) using a very large set (53) of BCMV isolates. They confirmed very weak selection pressure acting on P1, but some codons in the P1 region were under positive selection.

This request of Reviewer is related to the chapter Discussion in our manuscript. However, in Discussion we do not discuss our results from the selection pressure analysis because we did not do this analysis. In lines 216-223 (229-236 now), we discuss the results from the perspective of previous studies*.

Our comment in these lines is based on logical reasoning about the function of the P1 protein, or it may be a hypothesis for future experimentation.

Many ideas can appear in the Discussion chapter. However, they were not the subjects of the current study, possibly a subsequent one.

We would like to ask the Reviewer not to insist on the need for selection pressure analysis. Again, due to the fact that our work is about the genomic and proteomic analysis of a new isolate of the virus, we do not address other, certainly significant problems and relationships relating to BCMV (phytopathological, genetical, etc.).

*According to Instructions for Authors, defined by the MDPI, the following should be stated in the part Discussion: Authors should discuss the results and how they can be interpreted in perspective of previous studies and of the working hypotheses. The findings and their implications should be discussed in the broadest context possible and limitations of the work highlighted. Future research directions may also be mentioned. This section may be combined with Results.

 

Point 7: lines 225 to 226 (237-245 now): I would request authors to cite more relevant papers that highlight the importance of this study in characterizing the host range. There is a recent paper that shows a virus expands its host range from a wild plant to a pepper crop and caused frequent epidemics in Spain. Thus, the current study highlights the importance of crownvetch in disease management of common beans. DOI: https://doi.org/10.1128/jvi.01268-18

Response 7: Accepted.

We have added new text and 4 new references to this section.

 

Point 8: The current study is critical where the potential host range of a potyvirus in wild plants is characterized and is frequently associated with viral epidemics in the field. Much scope remains to improve the significance of this study. Moreover, based on the journal where readers are from different research fields, the discussion should be more elaborated which highlights the importance of this study.

Response 8: Accepted.

We agree with the Reviewer. We hope that the addition of the text in lines 237-245 has at least partially fulfilled this comment.