Molecular Mutation of the Coat Protein (CP) Gene in Alfalfa Mosaic Virus (AMV) and White Clover Mosaic Virus (WCMV) Combined Infection and the Role of the WCMV CP Gene When Infected with AMV
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsDear Authors
The revision of manuscript number: ID: agronomy-3713339 Type of manuscript: (Molecular Mutation of the Coat Protein (CP) Gene in Alfalfa Mosaic Virus (AMV) and White Clover Mosaic Virus (WCMV) Combined Infection and the Role of WCMV CP Gene When Infected with AMV) has been submitted.
Alfalfa mosaic virus (AMV) is one of the most widely distributed viruses worldwide. Manuscript (Molecular Mutation of the Coat Protein (CP) Gene in Alfalfa Mosaic Virus (AMV) and White Clover Mosaic Virus (WCMV) Combined Infection and the Role of WCMV CP Gene When Infected with AMV) analyzed the CP sequences of two viruses after the co-infection of AMV and WCMV in Nicotiana benthamiana, and found that the CP sequences of the two viruses mutated after co-infection with AMV and WCMV compared with the sequences during single infection with both viruses. The research is interesting and thoroughly conducted. The manuscript is also well written, though a few minor changes should be considered to improve its presentation. Some details are included in the manuscript, and other minor suggestions are included below.
Kind regards,
-In Abstract and introduction:
-Lines 37 and 42: use italic format to cite the name of virus family.
-Line 43: The authors the authors refer that ´This species primarily infects leguminous plants,…… and causes leaf blight ……´
-The virus species does not cause the infection or induce symptoms, it is the virus that causes the infection and causes the disease. Use species only in the context of taxonomic classification as defined to in the ICTV.
Comments:
In the introduction, the production of alfalfa and the significance of this crop will be included. It may encompass general information about virus complexes that infect alfalfa, with over 30 viruses reported to affect this crop.
Materials and Methods
The methodology was correctly conducted.
In the 2.1 section, line 107: Purified AMV and WCMV viruses were stored at −80 °C (Gansu Agricultural Uni-107 versity, Lanzhou, China) at a concentration of 300 pg·mL−1.
-Mention which method of purification was used?
Results
The results of the study provide a basis for in-depth research on the pathogenic mechanism of viral CPs variants in co-infection. Some figures and tables could be included in the supplementary material, such as figures 4 and 5, tables 2-4.
Discussion
According to the authors, in line 486-487: `The highest antagonism inhibition rate on AMV infection occurred when the WCMV CP: AMV was 2:1. These results lay the foundation for further studies on the synergistic and antagonism of these two viruses. Therefore, this study provides a basis to develop more effective disease control strategies´.
-In addition to the significance of this finding, it should be considered that AMV is seed-borne, which favors its dispersal in cultivated areas. Under natural conditions, the interaction discussed would occur frequently, can its results be addressed in effective disease control strategies? This could be addressed, such as avoiding or reducing the incidence or severity of this viral complex.
-The headings in sections 4.2 and 4.3 could be removed, keeping the discussion in block form.
Comments for author File: 
Comments.pdf
Author Response
Dear editors and reviewers,
We sincerely thank you for taking the time to review this manuscript. We have fully revised our manuscript according to the comments as well as added new analyses to further state our work. The revised parts have been marked in red font in the manuscript. Please find the detailed responses below and the corresponding revisions in the submitted files.
Reviewer 1
Comments 1: Lines 37 and 42: use italic format to cite the name of virus family.
Response 1: Thank you for pointing this out. We agree with this comment. The names of the virus families have been cited in italic format (51line, 56 line).
Comments 2: Line 43: The authors the authors refer that ´This species primarily infects leguminous plants,…… and causes leaf blight ……´
The virus species does not cause the infection or induce symptoms, it is the virus that causes the infection and causes the disease. Use species only in the context of taxonomic classification as defined to in the ICTV.
Response 2: Thank you for pointing this out. We agree with this comment. Has been revised to “this primarily infects leguminous plants” (56-57 line).
Comments 3: In the introduction, the production of alfalfa and the significance of this crop will be included. It may encompass general information about virus complexes that infect alfalfa, with over 30 viruses reported to affect this crop
Response 3: Thank you for pointing this out. We agree with this comment. In the introduction, the
production and significance of alfalfa have been supplemented. The details are as follows: alfalfa (Medicago sativa), a globally cultivated perennial leguminous forage grass renowned as the "King of Forages," is rich in minerals and vitamins. In China, driven by the ongoing implementation of the "Revitalize the Dairy Industry with Alfalfa Initiative" and the "Grain-to-Feed" policy, the alfalfa cultivation area has continuously expanded, reaching approximately 6.5 million mu (about 433,000 hectares). However, with increasing cultivation years and frequency of mechanical harvesting, the accumulation and occurrence of viruses have risen annually, leading to significant reductions in both the yield and quality of alfalfa. It is reported that over 50 different viruses infect alfalfa, with alfalfa mosaic virus (AMV) and medicago sativa alphapartitivirus 2 (MsAPV 2) being the predominant pathogens; the incidence rates were 91.7% and over 74.4%, respectively. As the province with the largest alfalfa cultivation area in China, Gansu Province experiences widespread alfalfa viral diseases, with an average disease incidence of 32.15% and a disease index of 16.27. These diseases pose a serious threat to the alfalfa industry, causing substantial economic losses (32-45 line).
Comments 4: In the 2.1 section, line 107: Purified AMV and WCMV were stored at −80 °C (Gansu Agricultural University, Lanzhou, China) at a concentration of 300 pg·mL−1.
Mention which method of purification was used?
Response 4: Thank you for pointing this out. We agree with this comment. In the Materials and Methods section, the purification methods for AMV and WCMV have been supplemented. The details are as follows: naturally infected alfalfa leaves collected from fields in Lanzhou, Gansu Province, served as the virus source for inoculation onto Vigna unguiculata by friction inoculation. According to the symptoms of AMV infection in V. unguiculata, it produces spots, while WCMV infection in V. unguiculata produces systematic mosaic leaves. Moreover, AMV can produce spots 48 h after inoculation, and the time of WCMV infection to produce systematic mosaic leaves is about 72 h, so the two viruses can be preliminarily separated by collecting single-spot and new leaves at different times. The two separate viruses were inoculated with cowpea three times by the same method; AMV was obtained by the single-spot separation method, and WCMV was obtained by collecting new leaves of V. unguiculata, and then the two viruses were inoculated with N. benthamiana at the four-leaf stage, labeled, and placed in an artificial intelligence assisted climate chamber (Hangzhou, China) for virus propagation, and the number of diseased leaves was collected after 15 days post inoculation (dpi), and AMV and WCMV were purified according to the following methods.
The purification of AMV referred to the method of Jin et al [39]. Diseased leaves were collected and washed, and 50 g of tissue was weighed, and potassium phosphate buffer (pH 7.5, 0.5 M) containing 0.01 M EDTA, 1% Triton X-100, and 1% mercaptoethanol was added at 1:2 (w/v). After grinding and homogenizing in a frozen mortar, 5% chloroform-n-butanol (equal volume mixing) was added, and after 40 min of standstill, low-temperature high-speed refrigerated centrifuge (5430R, Eppendorf, Germany) was carried out, the supernatant was taken, and 6% PEG6000 and NaCl were added and stirred for 4 h. After centrifugation, the precipitation was dissolved in 0.01 M EDTA, 1% Triton X-100 potassium phosphate buffer (pH 7.4, 0.02 M), and after multiple centrifugations, the precipitation was suspended in phosphate buffer (pH 7.0, 0.02 M), divided into 2 mL sterile centrifuge tubes, labeled, and stored in a -80°C freezer (Gansu Agricultural University, Lanzhou, China) for later use.
The purification of WCMV referred to the method of Wetter et al [40]. Diseased leaves were collected and washed, and 50 g of tissue was weighed, and phosphate buffer (pH 7.8, 0.05 M) containing 0.2 EDTA and 0.2% Na2SO3 was added at 1:4 (w/v). After grinding and homogenizing in a frozen mortar, the supernatant was taken after low-temperature high-speed refrigerated centrifuge (5430R, Eppendorf, Germany), the supernatant was adjusted to pH 5.0 by acetic acid, the supernatant was taken by centrifugation, 4% PEG6000 and 0.3 M NaCl were added and allowed to stand for 2 h, and the precipitation was suspended in boron acid borax buffer (pH 8.6, 0.13 M). After several centrifugations, the precipitation was suspended in phosphate buffer (pH 7.0, 0.02 M), aliquoted into 2 mL sterile centrifuge tubes, labeled, and stored in a -80°C freezer (Gansu Agricultural University, Lanzhou, China) for later use (121-157 line).
Comments 5: The results of the study provide a basis for in-depth research on the pathogenic mechanism of viral CPs variants in co-infection. Some figures and tables could be included in the supplementary material, such as figures 4 and 5, tables 2-4.
Response 5: Thank you for pointing this out. We agree with this comment. Figures 4 and 5, tables 2-4 have been included in the supplementary materials.
Comments 6: According to the authors, in line 486-487: The highest antagonism inhibition rate on AMV infection occurred when the WCMV CP: AMV was 2:1. These results lay the foundation for further studies on the synergistic and antagonism of these two viruses. Therefore, this study provides a basis to develop more effective disease control strategies´.
In addition to the significance of this finding, it should be considered that AMV is seed-borne, which favors its dispersal in cultivated areas. Under natural conditions, the interaction discussed would occur frequently, can its results be addressed in effective disease control strategies? This could be addressed, such as avoiding or reducing the incidence or severity of this viral complex.
The headings in sections 4.2 and 4.3 could be removed, keeping the discussion in block form.
Response 6: Thank you for pointing this out. We agree with this comment. In addition to the significance of this finding. Furthermore, the antagonistic effect can be utilized. A preparation made with WCMV CP as the effective component can be applied in the field to effectively prevent and control the occurrence and severity of virus diseases caused by co-infection (532-535 line).
The sections 4.2 and 4.3 have been removed.
We tried our best to improve the manuscript. We appreciate the editors and reviewers earnest work and hope the correction will meet with approval. If there are still any problems, we will continue to revise and clarify. Once again, thank you very much for your comments and suggestions.
Author Response File: Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsAuthors of this manuscript compared the molecular characteristics and protein structures of coat protein (CP) of alfalfa mosaic virus (AMV, Alfamovirus) and white clover mosaic virus (WCMV, Potexvirus) when they were singly and co-inoculated to Nicotiana benthamiana. Symptom appearances were also compared in this study. While the findings are interesting, I found that some of the reported results were difficult to believe. Especially on the mutations experienced by isolates in co-infections. Materials and Methods section related to isolates used in the experiment, treatment, and sequence analysis should be explained in detail. The Authors need to address them adequately in the text. Below are my comments and suggestions for the manuscript.
Line 97 Experiments were conducted between 8/2022 and 6/2024.
Line 102 ‘in an artificial intelligence assisted climate chamber’.
Lines 110-112 The sentence can be revised into ‘The leaves were thoroughly rinsed with sterile water until clean then a small amount of carborundum was delicately sprinkled onto the leaves’.
Line 108-114 These sentences here might be better to be written as the first paragraph of ‘Virus Inoculation’ subsection. Meanwhile, a brief explanation how to purify the AMV and WCMV isolates, and their original host and location can be written in the ‘Plant and Virus Materials’ subsection. Were the two viruses isolates from single or mixed infection? Was the original host(s) free from other viruses, and how to confirm it needs to be explained as well.
Line 117 Please mention concentrations of AMV and WCMV inoculated singly, respectively.
Lines 136-138 Please explain here what was the need to use different primers to target genes Co-AMV CP, Si-AMV CP, Co-WCMV CP, and Si-WCMV CP. Can Co- and Si- of a virus be targeted with the same primer? Authors supposedly do not know any mutation at the CP up to this point.
Lines 228-229 The sentence ‘The AMV CP, and WCMV CP encode 221 and 207 amino acids that were each composed of 20 amino acids (Figure 3)’ is likely a mistake, please revise it.
Lines 231-252 How many replicates of each treatment was sequenced? Were all the replicates behaving exactly the same in term of mutations? How do you analyze the data when replicates showed different sequences? These should be explained in M&M or Results sections. Moreover, 7.69% nucleotide and 9.05% amino acid mutations for AMV, and 3.37% nucleotide and 5.77% amino acid mutations for WCMV seem too high after a single pass to N. benthamiana. The authors need to clearly mention the original CP sequences of AMV and WCMV isolates before inoculated to N. benthamiana. They also should register these sequences in NCBI GenBank and provide the accession numbers in the manuscript.
Comments on the Quality of English LanguageThe quality of English Language is fine but a few minor grammatical and writing errors are found throughout the text. These should be corrected to help the readers clearly understand the sentences.
Author Response
Dear editors and reviewers,
We sincerely thank you for taking the time to review this manuscript. We have fully revised our manuscript according to the comments as well as added new analyses to further state our work. The revised parts have been marked in red font in the manuscript. Please find the detailed responses below and the corresponding revisions in the submitted files.
Reviewer 2
Comments 1: Line 97 Experiments were conducted between 8/2022 and 6/2024.
Response 1: Thank you for pointing this out. We agree with this comment. It has been revised to experiments were conducted between 8/2022 and 6/2024 (111 line).
Comments 2: Line 102 ‘in an artificial intelligence assisted climate chamber’.
Response 2: Thank you for pointing this out. We agree with this comment. It has been revised to an artificial intelligence assisted climate chamber (116 line).
Comments 3: Lines 110-112 The sentence can be revised into ‘The leaves were thoroughly rinsed with sterile water until clean then a small amount of carborundum was delicately sprinkled onto the leaves’.
Response 3: Thank you for pointing this out. We agree with this comment. It has been revised to the leaves were thoroughly rinsed with sterile water until clean then a small amount of carborundum was delicately sprinkled onto the leaves (162-164 line).
Comments 4: Line 108-114 These sentences here might be better to be written as the first paragraph of ‘Virus Inoculation’ subsection. Meanwhile, a brief explanation how to purify the AMV and WCMV isolates, and their original host and location can be written in the ‘Plant and Virus Materials’ subsection. Were the two viruses isolates from single or mixed infection? Was the original host(s) free from other viruses, and how to confirm it needs to be explained as well.
Response 4: Thank you for pointing this out. We agree with this comment. The sentences about inoculation with the Nicotiana benthamiana virus have been placed in the first paragraph of the “Virus Inoculation” section (161-168 line).
In the Plant and Virus Materials section, the purification methods for AMV and WCMV have been supplemented. The details are as follows: naturally infected alfalfa leaves collected from fields in Lanzhou, Gansu Province, served as the virus source for inoculation onto Vigna unguiculata by friction inoculation. According to the symptoms of AMV infection in V. unguiculata, it produces spots, while WCMV infection in V. unguiculata produces systematic mosaic leaves. Moreover, AMV can produce spots 48 h after inoculation, and the time of WCMV infection to produce systematic mosaic leaves is about 72 h, so the two viruses can be preliminarily separated by collecting single-spot and new leaves at different times. The two separate viruses were inoculated with cowpea three times by the same method; AMV was obtained by the single-spot separation method, and WCMV was obtained by collecting new leaves of V. unguiculata, and then the two viruses were inoculated with N. benthamiana at the four-leaf stage, labeled, and placed in an artificial intelligence assisted climate chamber (Hangzhou, China) for virus propagation, and the number of diseased leaves was collected after 15 days post inoculation (dpi), and AMV and WCMV were purified according to the following methods.
The purification of AMV referred to the method of Jin et al [39]. Diseased leaves were collected and washed, and 50 g of tissue was weighed, and potassium phosphate buffer (pH 7.5, 0.5 M) containing 0.01 M EDTA, 1% Triton X-100, and 1% mercaptoethanol was added at 1:2 (w/v). After grinding and homogenizing in a frozen mortar, 5% chloroform-n-butanol (equal volume mixing) was added, and after 40 min of standstill, low-temperature high-speed refrigerated centrifuge (5430R, Eppendorf, Germany) was carried out, the supernatant was taken, and 6% PEG6000 and NaCl were added and stirred for 4 h. After centrifugation, the precipitation was dissolved in 0.01 M EDTA, 1% Triton X-100 potassium phosphate buffer (pH 7.4, 0.02 M), and after multiple centrifugations, the precipitation was suspended in phosphate buffer (pH 7.0, 0.02 M), divided into 2 mL sterile centrifuge tubes, labeled, and stored in a -80°C freezer (Gansu Agricultural University, Lanzhou, China) for later use.
The purification of WCMV referred to the method of Wetter et al [40]. Diseased leaves were collected and washed, and 50 g of tissue was weighed, and phosphate buffer (pH 7.8, 0.05 M) containing 0.2 EDTA and 0.2% Na2SO3 was added at 1:4 (w/v). After grinding and homogenizing in a frozen mortar, the supernatant was taken after low-temperature high-speed refrigerated centrifuge (5430R, Eppendorf, Germany), the supernatant was adjusted to pH 5.0 by acetic acid, the supernatant was taken by centrifugation, 4% PEG6000 and 0.3 M NaCl were added and allowed to stand for 2 h, and the precipitation was suspended in boron acid borax buffer (pH 8.6, 0.13 M). After several centrifugations, the precipitation was suspended in phosphate buffer (pH 7.0, 0.02 M), aliquoted into 2 mL sterile centrifuge tubes, labeled, and stored in a -80°C freezer (Gansu Agricultural University, Lanzhou, China) for later use (121-157 line).
The two viruses were originally isolated from co-infected alfalfa, which was isolated by cowpea and purified separately by propagation by N. benthamiana. In this study, purified AMV and WCMV were used for single inoculation of N. benthamiana as the control, and the mixture of AMV and WCMV 3:1 was inoculated into N. benthamiana as the treatment, so that after repeated inoculation for 10 generations, disease samples were collected, the total RNA of N. benthamiana in AMV and WCMV co-infection and single infection was extracted, the RNA quality was detected, the CP of the two viruses was used as primers for PCR amplification, and the products were detected by 1% gel electrophoresis and then recovered by glue and sent to Beijing Qingke Technology and Biotechnology Co., Ltd. (China) for sequencing. The mutations of CP of the two viruses in co-infection were compared.
Comments 5: Line 117 Please mention concentrations of AMV and WCMV inoculated singly, respectively.
Response 5: Thank you for pointing this out. We agree with this comment. The concentrations of AMV and WCMV inoculated singly were 60 pg·mL−1 and 100 pg·mL−1, respectively (170 line).
Comments 6: Lines 136-138 Please explain here what was the need to use different primers to target genes Co-AMV CP, Si-AMV CP, Co-WCMV CP, and Si-WCMV CP. Can Co- and Si- of a virus be targeted with the same primer? Authors supposedly do not know any mutation at the CP up to this point.
Response 6: Thank you for pointing this out. The same primers were used for both AMV and WCMV co-infection and single infection. The presence of both Co- and Si- primers is intended to facilitate a clearer distinction between Co- and Si-. Duplicate primers have been removed (Table 1).
Comments 7: Lines 228-229 The sentence ‘The AMV CP, and WCMV CP encode 221 and 207 amino acids that were each composed of 20 amino acids (Figure 3)’ is likely a mistake, please revise it.
Response 7: Thank you for pointing this out. We agree with this comment. It has been revised to the AMV CP and WCMV CP encode 221 and 207 amino acids respectively, and both are composed of 20 different amino acids (289-290 line).
Comments 8: Lines 231-252 How many replicates of each treatment was sequenced? Were all the replicates behaving exactly the same in term of mutations? How do you analyze the data when replicates showed different sequences? These should be explained in M&M or Results sections. Moreover, 7.66% nucleotide and 9.05% amino acid mutations for AMV, and 3.37% nucleotide and 5.77% amino acid mutations for WCMV seem too high after a single pass to N. benthamiana. The authors need to clearly mention the original CP sequences of AMV and WCMV isolates before inoculated to N. benthamiana. They also should register these sequences in NCBI GenBank and provide the accession numbers in the manuscript.
Response 8: Thank you for pointing this out. We agree with this comment. In the Virus Inoculation section, the experiment process has been supplemented. The details are as follows: Each treatment inoculated 10 seedlings, repeated three times. The seedlings were labeled and placed in the climate chamber to observe the symptoms of disease daily. Samples were collected at 15 dpi, the total RNA of the plant was extracted, 10 parts of RNA from each treatment were mixed together, and finally there were 3 parts of RNA for each treatment, and the CP of the two viruses was used as primers for PCR amplification, and the product was detected by 1% gel electrophoresis, and the size of the 3 repeated CP bases was found to be basically the same through verification. Then we collected these three strips into one sterile centrifuge tube and sent them to Beijing Qingke Science and Technology Biological Co., Ltd. (China) for sequencing and compared the sequences of AMV and WCMV co-infection and single infection and counted the mutation rate and amino acid mutation rate of the CP base of the two viruses in the co-infection (172-183 line).
Regarding the base and nucleotide mutations of CP in the cases of AMV and WCMV co-infection and single infection, we conducted the following procedure: The purified AMV and WCMV were mixed in a ratio of 3:1 and then inoculated onto N. benthamiana. After 15 dpi, the diseased leaves were collected, ground into a grinding solution, and inoculated onto N. benthamiana again. This process was repeated for 10 generations (166-168 line).
The original CP sequences of the AMV and WCMV isolates have been uploaded in the form of attachments and have been registered in NCBI GenBank. The reference accession numbers for AMV CP and WCMV CP are OL7062601 and X166361, respectively (159 line).
We tried our best to improve the manuscript. We appreciate the editors and reviewers earnest work and hope the correction will meet with approval. If there are still any problems, we will continue to revise and clarify. Once again, thank you very much for your comments and suggestions.
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsAuthors had significantly improved their manuscript, including by incorporating all my corrections. Therefore, I believe the draft is ready for publication in 'Agronomy'.
