Identification of RNAi-Related Genes and Transcriptome Assembly of Loblolly Pine (Pinus taeda, L.) Seedlings Exposed to Insect-Specific dsRNA
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsRNA interference (RNAi) is a powerful technology that offers new opportunities for pest control through silencing of genes that are essential for the survival of pests. This study sequenced the transcriptome of loblolly pine seedlings treated with dsRNA, annotating and analyzing differential genes. A comparison with the RNAi mechanism of insect pests revealed changes in two key proteins induced by exogenous dsRNA in the seedlings of the experimental group. The RNAi mechanism in the seedlings was found to differ slightly from that of insect pests. Detailed annotation of the transcriptome, enrichment analysis of differential genes in variable pathways, and homologous alignment of proteins in similar species. Understanding the host plant's response to RNAi-mediated pest control is crucial for advancing this technology in pests or pathogenic microorganisms. In a word, the authors provide strong data to support the conclusions drawn.
The following are a few suggestions:
1. In the introduction, the changes of trees and insect pests caused by climate change are over described, slightly redundant and suggested to be reduced.
2. Why choose 150 μg of dsRNA to treat seedlings? Is this dose lethal to pests?
3. Page 5, line 209: BLAST x, line 205: BLAST, please describe it uniformly.
4. In the experimental method section, please add the absolute range of changes in differential gene expression.
5. Page 10, line 341: Genes and Genomes (KEGG) PATHWAY database, should be Genes and Genomes (KEGG) pathway database.
6. The existing literature suggests that exogenous dsRNA can be used to control plant diseases and insect pests through two main mechanisms. The first involves dsRNA is directly absorbed by the plant diseases and insect pests themselves. The second mechanism involves dsRNA is absorbed by plant cells, leading to cross-border RNAi activation through splicing in plant cells, ultimately triggering RNAi against the pests and diseases. The question raised here is whether the mechanism of cross-border RNAi is similar to the two key regulatory pathways of RNAi discussed in this paper.
7. Latin scientific names need to be in italics.
Author Response
Please see attachment
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for Authors
This interestiing contribution addressed the response of seedlings challenged with insect-specific dsRNA as a tool for pest management. However, the sample size is quite low, with only two seedlings per treatment. Despite the lack of detectable RNAi response, other findings reflected the complex impacts of exogenous dsRNA, thus laying the foundation for further studies.
I have a few suggestions that might help improve the MS.
Minor comments,
L99-102. Looking at these lines, it would appear that the study will focus only on loblolly pine; however, several results involve comparative analysis of transcriptomes from other species. What was the purpose of that? Please, indicate.
L101. Some context from previous studies is required about the use of dsSHI.
L127. There should be a reason for using such a small sample size; any practical limitation (for instance, seed availability or germination issues) may prevent the same mistakes in future studies.
L173. Data availability should be confirmed before publication. Currently, project PRJNA993588 does not show any items.
L231-232. Why test the similarity of core RNAi genes across other species?
L241-242. Confirmation of the presence of dsDHI should be provided as supplemental material
L307-309. That is a relevant finding; add supporting data.
Fig. 4, Fig. 5. Improve the quality of both figures; support values are hard to differentiate from the branch.
Table 1. More clarity is needed in the description of the Table. Add' 'this study' since any other transcriptomes may also come from experimental data.
Author Response
Please see attachment.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsThis manuscript reports de-novo assembly of loblolly pine transcriptome in response to treatment by insect-specific dsRNAs, annotated RRPs and compared RRPs in related pine species by phylogenetic analysis, followed by identification of differentially expressed transcripts as compared to control. Overall, this study provides useful information in understanding host plant response to RNAi mediated pest control. The manuscript is mostly well written, but some parts in Method and Result sections need more details for clarification, so that the reader can better understand and evaluate the full analysis undertaken in the study.
I would like to authors to deposit the de-novo assembled transcriptome in GenBank with an accession number for public access and data-sharing for research community. The full statistical analysis is not explained or is left out entirely for DET identification. The methods and result sections have some other gaps that should be addressed.
I have a few suggestions below that I believe would improve the manuscript before publication:
Line 18-24: You described DETs first and then annotated RRPs. But your DETs included two RRPs. It may be more easier for readers to follow if annotation of RRPs was first described.
Line 61: add a reference for RNAi as it first appeared in your text.
Line 65: add a reference for PTGS as it first appeared in your text.
Line 101: add “(RRP)’ in Abstract for readers to follow.
Line 102: Spell dsSHI out in full-length term as it first appeared in the text.
Line 106: “Dicer-like” or “dicer-like”, and “DCL”, keep them consistency through the text.
Line 113: Add an Supplementary table to list dsRNA IDs and sequences, and their target genes of insects.
Line 132: Please clarify what is the final concentration of dsRNA in the water.
Line 147: You only analyzed 4 seedlings, but here you showed 6 seedlings in Figure 1. Please clarify to avoid misleading.
Line 183: Add GenBank registration accession number for your de-novo assembly. This dsRNA-responsive transcriptome needs to be registered in GenBank for data sharing.
Line 194: Here in this subsection of Method, you described annotation first, and detection of DET at next subsection, in conflict to the order of description in Abstract.
Line 222: Please describe what statistic method for DET identification, and what cut-offs of change folds of gene expression levels and p-values were used to filter DETs. Was any method used to correct p-values for DEG identification? Please clarify and describe them in details.
Line 236: Add an supplementary file to show a.a. sequence of “each protein” you used in phylogenetic comparison.
Line 245: Delete “Annotation of RRPs for each pine species was conducted on transcripts assembled by Trinity”, as it was detailed in next subsection.
Line 246-247, Table 1: Register your assembly of loblolly pine transcriptome and add its accession number here for public accession of the dataset of 161,900 transcripts.
Line 257: You annotated RRPs in this subsection. As for other pine species, the assemblies are not registered in GenBank, please add a supplementary file to show sequences of RRPs of in each of related species as reported in this study, with proper GenBank acc numbers if available.
Line 293: You used the Ptaeda2.0 as reference for gene expression study. How many genes of Ptaeda2.0 for 85,443 transcripts to be mapped to?
Line 294: DET counted at 7,131 is a huge number. Can you narrow drown by increasing their change folds ( for example from 2 to 4 ,or 8), and increasing the significance of the p values with proper correction? Add results of those most significant DETs. The same issue for KOG category analysis and KEGG pathway analysis.
Line 294: Please clarify “with an average length of ~52,426 bp”, and confirm this average length because the average transcript length of your transcriptome assembly in Table 1 was only 726-bp. The same confusions are present in line 300 (~56,130 bp) and 301 (46,102 bp).
Line 298: How to define "long" here? It should be described in the methods section. If there are so many longROFs/transcript, a further refining analysis is necessary to pick the top one that make the most sense biologically/bioinformatically. Usually one transcript is translated into one protein (isoform).
Line 306: It is not described how to “only single isoform was considered for the DE count” in the Method section.
Line 307: List their IDs and sequence for all 16 deRRPs in a Supplementary file.
Line 311: please clarify “differential gene expression” vs. “differential transcript expression”
Line 326: Please clarify how many percentage was assigned to either groups of “unknown function or had no significant matches”.
Author Response
Please see attached.
Author Response File: Author Response.pdf
Round 2
Reviewer 3 Report
Comments and Suggestions for Authors For "No specific fold change cut-off was applied for filtering DETs", it would be easy to filter DETs by a cut-off of fold changes as DESeq2 has reports on both "Log 2 fold change" and "Tests of log2 fold change above or below a threshold". Also, "statistic method for DET identification" needs to be clarified M&M section. Thanks for your consideration. Line 222: Please describe what statistic method for DET identification, and what cut-offs of change folds of gene expression levels and p-values were used to filter DETs. Was any method used to correct p-values for DEG identification? Please clarify and describe them in details. The authors recognize the oversight and have elaborated on the details concerning DESeq2 parameters for DET identification. "Counts were normalized using size factor normalization to account for differences in library sizes across samples and differentially expressed transcripts (DETs) were filtered based on an adjusted p-value threshold of 0.05, calculated with the Benjamini Hochberg method to minimize the false discovery rate [64]. No specific fold change cut-off was applied for filtering DETs; instead, all DETs meeting the adjusted p-value threshold were included." [Rev.1: 243-248] Line 246-247, Table 1: Register your assembly of loblolly pine transcriptome and add its accession number here for public accession of the dataset of 161,900 transcripts. As previously stated, the authors will be more than happy to upload loblolly pine assembled transcriptome to GenBank under the BioProject associated with this experiment; however, this process will not be able to be completed during the short review period provided. The authors are currently in the process of preparing these transcriptomes for upload. Line 294: DET counted at 7,131 is a huge number. Can you narrow drown by increasing their change folds ( for example from 2 to 4 ,or 8), and increasing the significance of the p values with proper correction? Add results of those most significant DETs. The same issue for KOG category analysis and KEGG pathway analysis. The authors can conduct further limiting of the DE pool; however, as this will require re-extraction of longORFs and predicted peptide sequences as well as all subsequent analyses, we cannot provide this reanalysis on the short time provided for this revision.
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Round 3
Reviewer 3 Report
Comments and Suggestions for Authors
I have asked the editor to give you time long enough to revise the manuscript and add the GenBank acc before acceptance for publication.
As for "No specific fold change cut-off was applied for filtering DETs", at least you should clarify what default setting for fold change when DETs were analyzed in the M&M section.
Also I do not think that filtering fold change of DETs needs re-extraction of longORFs and predicted peptide sequences.
Author Response
Dear Editor,
We do not yet have the assembled transcriptome accession numbers from GenBank. Our first author (Z. Bragg) has graduated and moved to another institution, and is obtaining authorization and familiarizing themselves with the cluster account there. We estimate 3-4 weeks to get the accession numbers. We are happy to provide these as soon as they're available and have every intention of doing so. Thanks so much. Sincerely,
Lynne Rieske and Zach Bragg
Author Response File: Author Response.pdf
