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I have examined your revision - thank you for addressing most of the concerns from the reviewers. The described methods should be very useful for gene function studies in Paeonia and other perennial woody plants.
# PeerJ Staff Note - this decision was reviewed and approved by Julin Maloof, a PeerJ Section Editor covering this Section #
Reviewer#1 and #2 suggested number of changes that should make the manuscript acceptable. It is important to address the issue of repeatability and also testing one or two additional genes to show that VIGS is robust in Paeonia ostii. Both reviewers also suggested carefully editing of the manuscript for grammatical and related issues.
This manuscript reported the application of a TRV-based virus-induced gene silencing (VIGS) in the tree peony species P. ostii. Gene of P. ostii phytoene desaturase (PoPDS) was cloned and then used as a marker to test the TRV-based VIGS system in three-year-old P. ostii seedlings. According to this research, the Agrobacterium-mediated seedling vacuum-infiltration was determined as an appropriate way. After TRV-PoPDS inoculation, a typical photobleaching phenotype was shown in the newly-sprouted leaves of tree peony seedlings. Infection of TRV and silencing of PoPDS were confirmed by RT-PCR and qRT-PCR. In addition, the fluorescent tracer study of TRV-GFP in P. ostii seedlings indicate the potential of utilizing TRV-based VIGS system to analyze gene functions in different tissues.
This manuscript conformed to an acceptable format of ‘standard sections’. Sufficient introduction and background were provided. Figures are relevant to the content of the article, but the description need to be improved. Raw data have been made available. In general, this manuscript is worth of publication, but the English written should be improved and there are a number of ways that this manuscript could be stronger.
Following are some general comments for this manuscript and corrections to English grammars errors and typos that will help improve this manuscript:
Please make sure the usage of spaces between numerical values and unit symbols are correct, as suggested by International System of Units. For instance: “10 min”, “94℃”, “48 h”, “1797 bp” and “2-5 μg”.
Line 25: Replace “significance” by “values”;
Line 26: Replace “current limitation for its” by “limitation for its current”
Line 28: Replace “P. ostii” by “Paeonia ostia”
Line 30: Replace “infection” by “inoculation”
Line 30: Replace “agrobacterial” by “Agrobacterium”
Line 36: Replace “of inoculated plants” by “of plants inoculated”
Line 41: Replace “Virus-induced” by “virus-induced”
Line 42: Replace “Green” by “green”
Line 61: Add “ (P. ostii)” behind “Paeonia ostia”
Line 63: Replace “functional analysis” by “function analysis”
Line 67: Replace the sentence “an efficient genetic transformation system is still not established” by “the lack of efficient genetic transformation system”
Line 77: Please carefully check the description of short interfering RNAs. “21-24 nucleotides” or “21-23 nucleotides”?
Line 75: and Line 369 Please check the publish year, 2003 or 2011?
Line 154: Please double check that it was in a “growth chamber” without shaking.
Line 156: The 600 in “OD600” should be subscript.
Line 165: Delete “The”.
Line 181: Delete “in ”
Line 187: Delete “infected with TRV-GFP, ”
Line 193: Please provide the producer or origin of “anti-GFP antibody conjugated to alkaline phosphatase”, because Bedoya et al did not use alkaline phosphatase conjugated GFP antibody in the referred paper (Bedoya et al., 2012).
Line 209: Replace “and not in” by “but not in”
Line 204: The sentence “These two methods acted on the leaf back and whole plant, respectively.” is ambiguous and confusing. Please revise it.
Lines 205-206: Replace “vacuum way” and “syringe way” by “vacuum infiltration” and “syringe infiltration”
Line 207: Add “Furthermore, ” before “the syringe infiltration”
Lines 213-215: Replace the sentences “Using the transcriptome data obtained from developing leaves of tree peony, we PCR-amplified the open reading frame (ORF) nucleotide sequence of phytoene desaturase (PDS) of P. ostii, annotated as PoPDS, which was commonly used as a reporter for silencing” by “Phytoene desaturase (PDS) is commonly used as a visible reporter for silencing. Based on the transcriptome data obtained from developing leaves of tree peony, we PCR-amplified the open reading frame (ORF) nucleotide sequence of P. ostia PDS, annotated as PoPDS.”
Line 215: Replace “PoPDS encodes” by “PoPDS was predicted to encode”
Line 218: Replace “PoPDS amino acids shared” by “amino acid sequence of PoPDS shared”
Line 220: Replace “peptides of PoPDS” by “amino acid sequence of PoPDS”
Line 227: Replace “seemed” by “seems”
Lines 231-233: It is insufficient here to make a conclusion that “PoPDS of tree peony could be silenced by VIGS and TRV infection was systemically established.”. Authors should draw this conclusion at the end of next paragraph.
Lines 235-237: It is inaccurate to say “DNA fragment of TRV1 and TRV2”. Please totally rewrite the sentence “DNA fragments of TRV1 and TRV2 were detected in TRV empty vector- and TRV-PoPDS-infected leaves, but not in the mock control plants”.
Line 238: “PoPDS insert was detected in the leaves agro-infiltrated with TRV-PoPDS”, but in legend for figure 4 “Semi-quantitative RT-PCR analysis of TRV1 and TRV2 accumulation levels in systemically-infected P. ostii leaves”. Agro-infiltrated leaves or systemically-infected leaves, which one is correct?
Line 243: Please be careful with the use of extreme words in academic writing, such as “definitely”.
Line 249 Add “(dpi)” after “5 days post inoculation”
Lines 262-264: The sentence is difficult to read. please rewrite it.
Line 276: Change “seeding” to “seedling”
Line 280: Change “small-scale” to “limited area”
Line 280: Change “infected with TRV” to “infiltrated with TRV ”, because (as shown in the text) you did not verify the virus infection in plants inoculated via syringe-infiltration method.
Line 295: Replace “Because the reproductive buds of triennial tree peony plants didn’t take shape” by “Because no reproductive buds were formed at this stage”.
Line 296: Replace “in the following work” by “in future work”
Line 301: Replace “possibility of VIGS in tree peony” by “possibility of applying VIGS in tree peony”
Line 303-304: Replace “didn’t except some lesions resembling TRV empty vector-treated leaves” by “showed lesions resembling those of TRV empty vector”
Line 320-321 “viral propagation and the silenced systemic response” “viral propagation and/or systemic silencing response”
Lines 324-325: This sentence is grammatically wrong. Please rewrite it.
Line 325: The reference (Quadrana et al., 2011) sited here is inappropriate. Quadrana et al established a VIGS system based on the transgenic plants expressing exogenous GFP and evaluated its impact on metabolism, they did not mention TRV-GFP.
Line 326: Change “In present work” to “In the present work”
Line 327: Change “abundances” to “accumulation levels”
Lines 331-332: Move the sentence “Previous findings proved that TRV virus possesses the ability to move efficiently within the roots of infected plants (Macfarlane & Popovich, 2000).” To line 329, right ahead “We found a higher…”.
Line 333: Add “for the” behind “underlying mechanism”
Line 335: Please explain the meaning of the phrase “TRV-GFP vector was positive to tree peony plants”
Line 365: Replace “Yanlog” by “Yanlong”
Line 399: Delete the empty line
Lines 402-404, 107, and 328 : Please check the initial and last names of the authors, “Ji T” or “Tian J”? “Shuai Z” or “Zhang S”?
Lines 444-445: Please add the name of book, publisher, and page number.
Lines 468-470 and lines 402-404: reference duplicated.
Line 472: delete one “:”
Line 485” Change “pp.01064.02016” to “853-862.”
Lines 500, 503, and 506: Please add the page numbers
Figure 1d: TRV transcripts accumulation levels in vacuum-infiltrated leaves and syringe-infiltrated leaves are different. It would be better to emphasize this point in the text, since it may provide an reason for the choice of vacuum-infiltration method. “18S-26S-ITS” should be “18S-26S ITS”
Figure 3a: Please describe the meaning of the black box, such as “Black box indicates the PoPDS insert”
Figure 4b: “the primers for TRV2-1 were designed outside the multiple cloning sites (MCS)” should be “the primers for TRV2-2…”.
Actually, since Figure 3 has described the details, there is no need to mention again the primers here. I noticed that the size of TRV2-1 products from TRV-PoPDS-infected plants and TRV-infected plants are different. Would you please give an explanation.
Figure 5: There is no need to mark units to all the scale bars. I prefer to delete all the units in figures and describe them in the figure legend, such as “Scale bars equal to 100 μm (a-f) or 75 μm (g-i)”. “Merge” should be “Merged”.
Figure 5 legend: Replace the whole paragraph by “Confocal microscopy image of P. ostii leaves and roots infected with TRV-GFP at 5 days post-infiltration (dpi). Fluorescence was not observed in leaves (a-c) and roots (g-i) of mock-treated plants.” by “ ”. Replace the sentence “The cell outline (a, d, g, j), the dark fluorescence (b, e, h, k), and the combination photographed in bright field (c, f, i, l) are shown.” by “The bright field (a, d, g, j), the GFP channel (b, e, h, k), and the merged images (c, f, i, l) of the bright field and the GFP channel are shown.”.
Figure 6a: To make the figure easy to read, please delete all the “kDa” behind numbers and left one “kDa” on the top of numbers. Please label the blot with “anti GFP” and the stained gel with “Coom.” or “Loading” at their right side, and then provide explanation of any abbreviated label in the figure legend. I think the line beneath “Mock Leaf Root” should be on the top of “Leaf Root”.
The research is relevant and the experimental design is simple but appropriate.
The following additions would be useful:
Please describe how many replicates are made for each assay.
Lines 181-182: Please make an definition or explanation of “Transient assay of GFP”. In addition, the author only mentioned “leaf cells” here, how about the GFP imaging in root cells? Please describe the details.
Line 231: It would be better to display the images of stable phenotype at 5 months as you indicated in the text, or else please add “(data not shown)”.
In the result section: “Validation of TRV-GFP in P. ostii leaves and roots”. I will recommend to add the TRV infected plants as negative control in addition to mock control, due to the potential nonspecific fluorescence from damaged cells caused by virus infection.
Most of the conclusions are well stated and linked to the research performed.
In lines 346-348 of conlusion section, please make sure that the main purpose of the TRV-GFP assay is to reveal “a valid means to a valid means to monitor the viral spread in different tissues”. In my opinion, you try to use this result to prove that TRV is a versatile tool to analyze gene function in different tissues. If so, please rewrite this part.
The authors should carefully correct for all English usage and clarity.
Literature related to whether the tree is host for TRV, not provided. Is this a natural host plant?
Much of the data can be moved to supplement and additional data needed for making inferences like 'silencing protocol established in this species'.
Already known methods are used in this study. Authors should have attempted to use different age of seedlings and shown the best stage for the silencing experiment. Statistics and replicate details should have been better described.
Extending the VIGS protocol in this tree species is useful. But, authors have simply used already known vector and inoculation protocol and standardized to silence PDS gene. There is no novelty here. Authors should generate more data and validate the functional relevance of an important gene and demonstrate VIGS as tool for advancement of research in this species.
Number of replicates and independent experiments are not clear.
In this manuscript, authors attempted to establish TRV-based VIGS in tree peony. They have taken already available VIGS vector and cloned partial fragment of PoPDS and infiltrated the young plants. They have tried both syringe and vacuum methods for agrobacterium inoculation and state that they found the later works better. They have also traced the GFP signals in roots.
The main concern with this study is the evidences are preliminary and need additional data by showing silencing of other genes involved in specific plant process. Overall, the reproducibility of the silencing data could not be perceived from the evidences presented. How many times each experiment was repeated? It should be stated in the legend.
Line #116, three year or four week old, confusing.
I could not understand how the GFP construct was made and how finding GFP signals in root indicate silencing? Authors’ claims in this regard are vague and evidence is too preliminary.
Fig 5, GFP signals shown are very weak, though the western has detected the GFP protein.
Fig 4, what is TRV2-1 and TRV2-2? I can hardly see photo-bleaching in the leaves.
Authors need to show silencing of genes involved a specific plant process and substantiate the silencing. PDS silencing alone is not adequate to conclude that the VIGS system is established.
Table 1, Figure 1-3 should be moved to supplementary.
The lack of effective molecular genetic tools in tree peony limit its genetics, gene function, and molecular mechanism investigations. In the current research, Lihang Xie et al. have attempted to establish a novel approach by Tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) in the tree peony species Paeonia ostii. This is an interesting paper demonstrating TRV-based VIGS technique could be adapted for high-throughput functional characterization of genes in tree peony. In my opinion, it will certainly attract numerous citations in the future. The English in this manuscript is generally correct and intelligible. Also, the scientific background and the research as a whole are sound. Therefore, I suggest accepting this manuscript with some minor revisions.
Please provide detailed information regarding data deposition statement (PoPDS,…) in revised manuscript.
There are still typos and formatting errors in the manuscript. Number and unit should be in between space, for example, “5 min” instead of “5min” in line 133, etc.
Figure 2, sequence IDs should be clearly represented in legend.
Figure 6a, what is the bottom panel? Something like there were no targeted bands in this panel?
The references should be in a consistent format with species name in italics, articles and journal names in similar illustration, etc.
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