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Both reviewers are satisfied with the revision and amended version is acceptable now.
[# PeerJ Staff Note - this decision was reviewed and approved by Robert Winkler, a PeerJ Section Editor covering this Section #]
Clear and unambiguous, professional English used throughout: Ok
Literature references, sufficient field background/context provided: Ok. However, information from the cited literature (Beyaz 2023) is incomplete. It would be good to correct this information in the reference section.
Total pages:388
Page: 161-196
Editors: Dr. Ahmet KAZANKAYA and Dr. Mevlüde Alev ATEŞ
Publisher Name: Iksad Publications (Ankara/Türkiye)
Publication time: July/2023
ISBN: 978-625-367-194-5
online access address: https://www.researchgate.net/publication/373041204_FUNCTIONAL_GENE_SILENCE_TECHNIQUE_IN_PLANTS_VIRUS-INDUCED_GENE_SILENCING_VIGS
Professional article structure, figures, tables. Raw data shared: Ok
Self-contained with relevant results to hypotheses: Ok
Original primary research within Aims and Scope of the journal: Ok
Research question well defined, relevant & meaningful. It is stated how research fills an identified knowledge gap: Ok Line 95-97
Rigorous investigation performed to a high technical & ethical standard: Ok
Methods described with sufficient detail & information to replicate: Ok Line: 155-156
It was also observed that the authors added second housekeeping genes (HmAFD2) besides the Actin gene for validation of target genes in real-time PCR.
Impact and novelty not assessed. Meaningful replication encouraged where rationale & benefit to literature is clearly stated: Ok, Line: 90-97 in introduction
All underlying data have been provided; they are robust, statistically sound, & controlled: Ok
Conclusions are well stated, linked to original research question & limited to supporting results: Ok
It would be good if the references in the manuscript and reference section were reviewed to ensure that they were consistent with the journal format.
The authors addressed well all my comments.
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Please address concerns of all reviewers and amend manuscript accordingly,
**PeerJ Staff Note:** Please ensure that all review, editorial, and staff comments are addressed in a response letter and that any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate.
In the introduction of the manuscript, the justification of the research, hypothesis, research question, and purpose of the research are clearly stated. However, the literature regarding the study is sufficient.
However, information about which gap in the literature this study will fill should be clearly stated in the introduction of the manuscript.
Where and when was research conducted in the material methods section of the manuscript? This should be written.
I did not see a section on the statistical analysis of the data in the hand patch. This must be included in the manuscript. The following questions must be answered:.
According to which trial design was the data collected?
How many repetitions were the treatments made, and how many plants were analyzed in each repetition?
Why did you use only a single housekeeping gene, "HmActin," as a control in the study? The use of a second housekeeping gene for validation will be important for the accuracy of the results of the real-time PCR study.
The discussion part of the manuscript is brief. It would be good to expand it with current literature knowledge (Beyaz R., 2023), "Functional gene silence technique in plants: virus-induced gene silencing (VIGS)".
In the conclusion part of the manuscript, recommendations should be made regarding future logical studies based on this study.
After the above corrections are made, the manuscript can be accepted for publication (Minor revision)
The article aim to use VIGS in the H.mutabilis to increase the use of molecular breeding and functional gene studies.
The experimental design for conducting VIGS is well detailed explained. It can be replicated by following the methodology explained
The experiment has been validated through carrying out QRTPCR for the CLA1 genes which indicates the reduction of the expression. Furthermore for the proof of the work, pictures have been provided.
The article succinctly explains the methodology of carrying out VIGS in the H. mutabilis, it has been written well and explains in detail the methodology. However, the VIGS is a effective way to do functional characterization of the genes, and its being used in several other crops.
The English language is ok.
The literature provided is ok.
The research lacks originality.
10.7717/peerj.7505 (published in Peer J in 2019) has TRV VIGS of CLA1 In Hibiscus hamabo (Wang et al., 2019). The same gene is silenced in different species of the same genus. Cla1 is highly homologous among the Hibiscus genus.
Ok.
The manuscript from Sang et al., describes an attempt to apply a TRV-mediated Virus Induced Gene Silencing (VIGS) protocol in Hibiscus mutabilis - a plant with a potential medicinal interest – in order to serve as a protocol for functional in planta characterization of genes.
The manuscript is descriptive and well-written. The rationale is easy to follow and most experiments described well.
I have some comments that should be addressed by the authors and hopefully will improve the draft and make it more appropriate for publication.
Major comments:
Line 25: “we conducted transcriptome analysis”. I failed to find any relative information in the manuscript. Which dataset was assessed (Public Code in Genbank), how many isoforms were found for CLA1, which one is the best candidate? In lines 207-208 a newly released genome is referenced (Yang et al., 2022), did the authors use this dataset? If yes please describe the pipeline used. If not, you should compare the cDNA isolated with other isoforms identified by this publication.
Line 166: “Characterization of the HmCLA1 gene in H. mutabilis”. In my eyes, this is not a characterization of a the CLA1 gene. I would expect to see genomic coordinates, intron/exon structure, size of the gene and size of the transcribed mRNA, and experiments to see if it is a single-copy gene like a Southern-blot for instance. The title should be modified. Did the authors perform RACE PCR to isolate the CLA1 homologue? Which primers were used and where is the sequence of the cDNA? I only found the sequence of the protein but this is not at all helpful to the readers of this study.
The authors should deposit the sequence identified (even if it is partial) in a public database (i.e. GenBank) and provide the code within the manuscript. The authors provide the sequence of the primers used but not the sequence of the template. How are the readers supposed to see which region of the cDNA is amplified in each case? A figure should be included showing the sequence of the cDNA (not the protein) and highlight where exactly the primers used for generating the pTRV2-HmCLA1 construct hybridize, as well as where the primers used for qPCR hybridize. Also provide within the manuscript the accession number of the Actin gene used for qPCRs.
In addition, the alignment of the CLA1s from other species is not helpful as presented since they only show the amino acid sequences. It would be more helpful to provide also an alignment with the cDNAs in order to see if the region selected to trigger silencing is conserved or not.
Figure 4: In Line 152 the authors mention that “Three biological replicates were analyzed per group”, but in Figure 4 I only see triplicates for the plants Agroinfiltrated with pTRV2-HmCLA1 and only one bar with the plants Agroinfiltrated with EV (Control) and mock plants. Please correct the figure accordingly.
Line 200: “Among the members of the Hibiscus genus, TRV-mediated VIGS systems…” Apart from the case of H. hamado mentioned by the authors, there is a recent publication performing TRV-based VIGS in Hibiscus cannabinus also (Yang et al., Tropical Plant Biol. 16, 146–155 (2023), DOI: 10.1007/s12042-023-09341-1). This work should also be referenced within the manuscript.
no other comment than that reported above
no other comment than that reported above
no other comment than that reported above
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