Quantitative Distribution and Transmission of Tea Plant Necrotic Ring Blotch Virus in Camellia sinensis
, Yao Chen 1, Fumei Zhao 2, Changqing Ding 1, Kexin Zhang 1, Lu Wang 1, Yajun Yang 1,*, Xinyuan Hao 1,* and Xinchao Wang 1
Round 1
Reviewer 1 Report
Dear authors,
in your manuscript “Quantitative distribution and transmission of tea plant necrotic ring blotch virus in camellia sinensis” you described an RT-qPCR method for the detection and quantification of PNRBV in tea trees and gave information on its transmissibility and diffusion in plant. There are in fact few pieces of information in literature on this virus, in particular on biology and transmission. Thus, the data you collected in your experiments are really useful to improve the knowledges on PNRBV.
I only have some comments reported below.
Line 45: In this part it is more correct to use the term motifs instead of proteins. In fact in RNA1 there is only 1 ORF that encodes for a protein which contains those motifs. The same for RNA2. See https://doi.org/10.1128/mra.00323-22.
Line 48: for a putative movement protein.
Line 57: there is no logical correlation between the two phrases in line 57. I suggest changing them: BNRBV, that belongs to Blunervirus genus such as TPNRBV, does not move…
59: nevertheless instead of however
69: the diagnostic methods that you are mentioning in this paragraph are general for all plant viruses and not specifically reported for TPNRBV. Only PCR detection is available. It is better to clarify it.
In fig 1 please add some more details of symptoms or add an healthy control, otherwise it is difficult to understand what is a symptom and what is not, in particular when it is not so clear (for example in roots or stems). If some of these parts of plant do not have symptoms, their pictures are not useful for the article, and should be removed.
In fig 2: In square C there are not clear differences between inoculated and uninoculated plants. If some differences are present please highlight them, otherwise they are useless and should be removed.
107: change for 36 days with after 36 days
110: change “;” with a simple comma.
111: delete “;”
119: “five drops”? please report the precise volume
Paragraph 2.2: do you have some references on this inoculation methods? In my experience it is used for agrobacterium inoculum, but not for mechanical virus inoculation.
Line 190-193: All these data are reported clearly in fig3. You can delete them here and write “the standard curve… are reported in fig 3”
Line 209: what did you use as negative control?
Table 1: in some descriptions you used “fomed” instead of “formed”. Please check and correct the mistakes.
Paragraph 3.4
In my opinion it would be better to indicate the cultivar you used for seeds and cuttings collection (you didn’t mention it here). I suppose they are from the cv BY1 infected, as you reported in material and methods, but if it is specified here too, the data will be more understandable.
I have some question about the inoculation: did you check the presence of the virus in the supernatant preparation or in the leaves after inoculation? Just to confirm the presence of the virus, since it is not possible to verify infectivity.
Did you try cla mechanical inoculation?
Line 248: on the basis of your results, the virus is more “not mechanically transmissible” than “easily transmitted”
Line 271 and 274: However is repeated in these two period, please correct them. I suggest changing the second one in a simple “but”
Line 310: remove “efficiently”. The term “not efficiently” implies that a small percentage of inoculated material became infected, but you didn’t obtain any infections.
Author Response
Response to Reviewer 1 Comments
Thank you for your constructive suggestions. We have made careful modifications as follows:
Point 1: Line 45: In this part it is more correct to use the term motifs instead of proteins. In fact in RNA1 there is only 1 ORF that encodes for a protein which contains those motifs. The same for RNA2. See https://doi.org/10.1128/mra.00323-22.
Response 1: The original sentence was changed to ‘RNA1 possesses a single ORF, which contains methyltransferase, cysteine-protease, and helicase. RNA2 possesses a single ORF, which contains helicase-RNA polymerase. All enzymes encoded by RNA1 and RNA2 are involved in viral replication and transcription. RNA3 posseses four ORFs encoding four putative proteins with unknown functions and molecular weights 14, 29, 22, and 22 kDa. RNA4 possesses a single ORF encoding a putative movement protein’.
Point 2: Line 48: for a putative movement protein.
Response 2: The sentence was corrected to ‘RNA4 possesses a single ORF encoding a putative movement protein’.
Point 3: Line 57: there is no logical correlation between the two phrases in line 57. I suggest changing them: BNRBV, that belongs to Blunervirus genus such as TPNRBV, does not move.
Response 3: The original sentence was changed to ‘BNRBV does not move systemically in diseased blueberry and is not transmitted through vegetative propagation’.
Point 4: 59: nevertheless instead of however.
Response 4: The word of ‘however’ was altered to ‘nevertheless’.
Point 5: 69: the diagnostic methods that you are mentioning in this paragraph are general for all plant viruses and not specifically reported for TPNRBV. Only PCR detection is available. It is better to clarify it.
Response 5: According to the suggestion of reviewer 3, the sentence was changed to ‘So far, only PCR methods have been applied for detecting TPNRBV and have shown some inconsistencies in detection, sensitivity, and quantification compared with RT-qPCR [7]. Thus, an improved RT-qPCR method is needed for TPNRBV diagnosis’.
Point 6: In fig 1 please add some more details of symptoms or add an healthy control, otherwise it is difficult to understand what is a symptom and what is not, in particular when it is not so clear (for example in roots or stems). If some of these parts of plant do not have symptoms, their pictures are not useful for the article, and should be removed.
Response 6: The pictures of asymptomatic tissues were removed.
Point 7: In fig 2: In square C there are not clear differences between inoculated and uninoculated plants. If some differences are present please highlight them, otherwise they are useless and should be removed.
Response 7: Figure 2c was deleted.
Point 8: 107: change for 36 days with after 36 days.
Response 8: The phrase of ‘for 36 days’ was corrected to ‘after 36 days’.
Point 9: 110: change “;” with a simple comma.
Response 9: ‘;’ was changed to a simple comma.
Point 10: 111: delete “;”.
Response 10: ‘;’ in line 111 was deleted.
Point 11: 119: “five drops”? please report the precise volume
Response 11: ‘five drops of ’ was changed to ‘40 μL’.
Point 12: Paragraph 2.2: do you have some references on this inoculation methods? In my experience it is used for agrobacterium inoculum, but not for mechanical virus inoculation.
Response 12: The inoculation methods referred to section 3.2 (Extraction and crude purification) of the reference (DOI:10.1385/0-89603-385-6:171).
Point 13: Line 190-193: All these data are reported clearly in fig3. You can delete them here and write “the standard curve… are reported in fig 3”
Response 13: The sentence was changed to ‘The standard curve equation, regression coefficient (r2), and amplification efficiencies (E) of pTPNRBV1, pTPNRBV2, pTPNRBV3, and pTPNRBV4 were reported in Figure 3’.
Point 14: Line 209: what did you use as negative control?
Response 14: The water was used as negative control in previous experiment. According to the suggestions of reviewer 3, This part of the experiment was reworked. The plasmids were diluted with cDNA from TPNRBV-free sample. Thus, both of the cDNA from TPNRBV-free sample (N1) and water (N2) were used as negative control. The note was added to Figure 3.
Point 15: Table 1: in some descriptions you used “fomed” instead of “formed”. Please check and correct the mistakes.
Response 15: ‘fomed’ was corrected to ‘formed’.
Point 16: Paragraph 3.4
In my opinion it would be better to indicate the cultivar you used for seeds and cuttings collection (you didn’t mention it here). I suppose they are from the cv BY1 infected, as you reported in material and methods, but if it is specified here too, the data will be more understandable.
Response 16: The original sentence was changed to ‘Analysis of the transmission of TPNRBV through progeny showed that newly germinated seeds and older mature seeds from TPNRBV-infected ‘BY 1’ were TPNRBV-negative. In addition, leaves of two-year-old cuttings from TPNRBV-infected ‘BY 1’ tea trees were asymptomatic and TPNRBV-negative’.
Point 17: I have some question about the inoculation: did you check the presence of the virus in the supernatant preparation or in the leaves after inoculation? Just to confirm the presence of the virus, since it is not possible to verify infectivity.
Response 17: We detected the concentrations of the four segments of TPNRBV in supernatant via RT-qPCR. The supernatant was TPNRBV-positive and the concentrations of the four segments were 5.32 × 104, 8.82 × 104, 7.79 × 105 and 4.34 × 107 copies/μL, respectively. These data were added to section 3.4.
Point 18: Line 248: on the basis of your results, the virus is more “not mechanically transmissible” than “easily transmitted”
Response 18: Taking the suggestion of reviewer 3 into account, this sentence ‘Thus, TPNRBV is not easily transmitted to tea trees by mechanical infiltration’ was deleted.
Point 19: Line 271 and 274: However is repeated in these two period, please correct them. I suggest changing the second one in a simple “but”.
Response 19: The second ‘However’ was changed to ‘But’.
Point 20: Line 310: remove “efficiently”. The term “not efficiently” implies that a small percentage of inoculated material became infected, but you didn’t obtain any infections.
Response 20: ‘efficiently’ was removed.
Author Response File: 
Author Response.docx
Reviewer 2 Report
The manuscript submitted by Ren et al. focuses on (1) development of reliable qPCR-based detection system of tea plant necrotic ring blotch virus and (2) its distribution and transmission in tea plants. There are several drawbacks that should be addressed before resubmission:
-no internal plant control was employed. Hence, how were comparisons between different samples done without normalisation? I may assume that approximation was done using the same amount of RNA input for cDNA synthesis, but that would not be correct, even taken into account that spectrophotometry-based quantification is very rough. Some dye-based quantification (Qubit or similar) should be used. The normalization would require incorporation of another control that might be plant mRNA(s). This also will remove any doubts about whether negative results were not false-negative.
- reported ranges of viral load (Tables 1 & 2) do not fall into a range of standard curve
- traditional approach of standards generation involves in vitro transcription of RNA targets and their cDNA synthesis, i.e. the same pathway (cDNA synthesis -> PCR) the real samples took. Alternatively custom synthesised RNA or DNA oligos could be used with known concentration. Importantly, if pDNAs were just water diluted then it affects the numbers. Use either salmon DNA or spike pDNA into cDNA from virus-free sample.
- there is confused part about pDNA preparation. The size of pCAMBIA1300 is almost 9kb. How all sizes were calculated? Figure S1 and the text (l.181-182) mention sizes from ~2 to ~6 kb. Were these sizes of inserts?
- it is not clear how plasmids were quantified
- the formula is incorrect (l.172-173), the mol weight value of DNA bp is missing
- there are some contradictions between text and Figure S3. For example, pTPNRBV4 had 1.29 × 1010 copies/μL, was diluted and qPCR-amplified and the system claimed to have detection limit of 1.29 × 10 copies/μL. The Figure S3 shows the highest dilution 10-8 (i.e. theoretical 1.29 x 102). How is that possible?
Author Response
Response to Reviewer 2 Comments
Thank you for your valuable and constructive suggestions. We have made careful modifications according to your comments, which will make our data more reliable. Relevant revisions are shown as follows:
Point 1: -no internal plant control was employed. Hence, how were comparisons between different samples done without normalization? I may assume that approximation was done using the same amount of RNA input for cDNA synthesis, but that would not be correct, even taken into account that spectrophotometry-based quantification is very rough. Some dye-based quantification (Qubit or similar) should be used. The normalization would require incorporation of another control that might be plant mRNA(s). This also will remove any doubts about whether negative results were not false-negative.
Response 1: Due to the lack of Qubit, the RNA concentration was determined using NanoDrop ND-2000 spectrophotometer. The normalized RNA was verified by 1 % gel electrophoresis to assess its quantity and quality. To accurately quantify the virus content, CsPTB and CsEF1 of tea plant were used as internal control that was amplified in paralleled with four segments of TPNRBV. The Ct value of all samples were normalized according to the Ct of CsPTB and CsEF1. Thus we got more accurate data. And we found that these data showed similar patterns to the previous data. Relevant description was added to section 2.7.
Point 2: - reported ranges of viral load (Tables 1 & 2) do not fall into a range of standard curve
Response 2: The standard curve was recreated according to the new Ct of plasmids. Viral loads of all samples were recalculated and fall into a range of new standard curve.
Point 3: - traditional approach of standards generation involves in vitro transcription of RNA targets and their cDNA synthesis, i.e. the same pathway (cDNA synthesis -> PCR) the real samples took. Alternatively custom synthesised RNA or DNA oligos could be used with known concentration. Importantly, if pDNAs were just water diluted then it affects the numbers. Use either salmon DNA or spike pDNA into cDNA from virus-free sample.
Response 3: All pDNA were serially diluted using cDNA from virus-free sample and used for RT-qPCR analysis. The standard curve was redrawn based on these new Ct value.
Point 4:- there is confused part about pDNA preparation. The size of pCAMBIA1300 is almost 9kb. How all sizes were calculated? Figure S1 and the text (l.181-182) mention sizes from ~2 to ~6 kb. Were these sizes of inserts?
Point 5: - it is not clear how plasmids were quantified
Response 4 and 5: The accurate describe was added to 2.4 of materials and methods. The size of the four segments of TPNRBV are 5.9 kb, 4.1 kb, 2.7 kb and 2.3 kb, respectively. The full-length cDNA of these four segments were individually inserted into pCAMBIA1300 vector (9.3 kb) to obtain pTPNRBV1, pTPNRBV2, pTPNRBV3 and pTPNRBV4. These recombinant vectors were used for the calculation of copy number.
Point 6: - the formula is incorrect (l.172-173), the mol weight value of DNA bp is missing
Response 6: Thanks for your reminder. The formula in section 2.7 was corrected to ‘numbers of copies = (Avogadro’s number 6.02 × 1023) × (amount of plasmid in nanograms) / [length of DNA in base pairs (bp) × 660 × 1 × 109]’.
Point 7: - there are some contradictions between text and Figure S3. For example, pTPNRBV4 had 1.29 × 1010 copies/μL, was diluted and qPCR-amplified and the system claimed to have detection limit of 1.29 × 10 copies/μL. The Figure S3 shows the highest dilution 10-8 (i.e. theoretical 1.29 x 102). How is that possible?
Response 7: I am sorry for the mistake due to my carelessness. We checked all the data and made related revisions.
Reviewer 3 Report
Dear colleagues,
The work submitted by Ran et al. is manily about the developing of a detection method for TPNRBV, studying the distribution of the different virus segments in tee trees, and virus transmission via seeds, cuttings and mechanical inoculation and in fields with where TPNRBV-infected trees were found. Overall the work needs some clarifications, including;
The authors need to indicate how they selected the regions for primers design.
The discussion needs some work as it is based on speculations.
I would also suggest that the authors add a figure to represent their experimental design.
Other comment can be found in the attached PDF.
Best regards
Comments for author File: 
Comments.pdf
Author Response
Response to Reviewer 3 Comments
Thank you for your constructive suggestions. We have made careful modifications as follows:
Point 1: What is the role of endophytes in preventing virus infection? I am not sure which reference has this information.
Response 1: Some literatures describe that endophytes are active against viruses as follows:
The article (DOI:10.1155/2018/1470305) describes that ‘Accumulating evidence has shown that endophytic actinomycetes, especially those from medicinal plants, are a promising source of novel metabolites with antimicrobial, antiviral, anticancer, and anti-inflammatory properties’.
The article ( DOI: 10.1007/s10482-015-0502-7 ) describes that ‘In recent years, many of novel antibiotics synthesized by endophytic actinobacteria recovered from medicinal plants found to be active against bacteria, fungi and viruses’.
However, there is no direct report on the antiviral properties of endophytes in tea plants. Thus, the description of the role of endophytes against viruses in our manuscript was deleted.
Point 2: The third paragraph of introduction: Please add references here.
Response 2: The references were added.
Point 3: The forth paragraph in introduction: (1)Suggestion: move the second sentence as third and start with Only polymerase chain reaction (PCR) ... (2) Add reference
Response 3: Modification in the third paragraph of introduction. (1) The second and third sentence was changed to ‘So far, only PCR methods have been applied for detecting TPNRBV and have shown some inconsistencies in detection, sensitivity, and quantification compared with RT-qPCR’. (2) The reference was added.
Point 4: 2.1 Are these replicates from the same plant or different plants?
Response 4: These replicates were from different plants.
Point 5: 2.2 Which homogenizer?
Response 5: The homogenizer is DFT-200A homogenizer (Wenling Linda Machinery Co., Ltd, Zhejiang, China).
Point 6: The authors need to indicate how they selected the regions for primers design.
Response 6: We reported the primers information used for the detection of four segments of TPNRBV via RT-PCR method in our previous study (Hao et al., 2018). Based on the research, we redesigned primers within the four amplicons used for RT-qPCR analysis. Relevant description was added to section 2.5 as follows: ‘We previously designed primers used for the detection of four segments of TPNRBV via RT-PCR [7]. Based on the research, three pairs of primers within the four amplicons were redesigned to detect each segment and to select the most specific pairs for PCR and RT-qPCR analysis’.
Point 7: Section 3.2: How many repetitions did you perform?
Response 7: Three repetitions were performed. And the figure showed the information for one repetition.
Point 8: Table 1: Are these technical replicates or biological replicates?
Response 8: Three biological and two technical replicates were evaluated in each sample. The explanation was added to the note under the table 1 and 2.
Point 9 : The first paragraph of Section 3.4: What kind of analysis? PCR or RTqPCR?
Response 9: ‘Analysis’ was changed to ‘RT-qPCR analysis’.
Point 10: The second paragraph of Section 3.4: by which method?
Response 10: (1) ‘were not detected’ was changed to ‘were not detected via RT-qPCR’.
Point 11: The third paragraph of discussion: By checking the protein sequence of AYE53921 from segment 3, it appears that it is the coat protein (SP24 pfam16504). Please check CDD: NCBI's conserved domain database.
Response 11: Thank you for such professional and accurate comments. The sequence was rechecked using CDD: NCBI's conserved domain database. And we also referred relevant literature (Doi: 10.1128/JVI.02595-13). This part of discussion was rewrote as follows: ‘Notably, in symptomatic mature leaves, the four segments of TPNRBV were detected, but in some asymptomatic tissues, only a part of segments of TPNRBV were detected. Other proteins may be required for TPNRBV movement or spread. Thus, how TPNRBV moves in diseased plants requires further analysis’.
Point 12: Conclusion: Not easily! based on your experiment it was not transmitted via seeds or mechanically. Maybe, change the sentence to: TPNRBV transmission was not successful mechanically and via seeds.
Response 12: The sentence was changed to ‘In addition, TPNRBV transmission was not successful mechanically and via seeds’.
Point 13: Acknowledge: If you don't want to acknowledge any one remove this part.
Response 13: This part was deleted.
Author Response File: Author Response.docx
Round 2
Reviewer 2 Report
The authors addressed comments raised during the first review. However, some points must be clarified:
- the fact that pDNAs were serially diluted with plant cDNA was not mentioned.
- it is not clear what are "CsPTB and CsEF1". There is no description, primers, etc.
-the main issue is the calculation part (lines 182-190). Provide reference to the applied method. From what is indicated, all reference Cts were averaged, then samples's reference Cts were divided by the obtained value. What is the purpose of that? Coefficient of difference (or likely term) from average?
-please provide raw Ct values as either excel or tsv/csv file.
Author Response
Please see the attachment.
Author Response File: Author Response.docx
