Cloning, Bioinformatics Analysis and Physiological Function of the Pine Wood Nematode Bxadh2 Gene

Round 1

Reviewer 1 Report

 

In the present study, the Authors explored the role of the Bxadh2 gene, encoding an alcohol dehydrogenase protein from the cytochrome P450s family, in the survival rate and fecundity of the pine wood nematode.  The authors aimed to accomplish this by:

 

1) Identifying putative Bxadh2 gene sequences through in silico PWN transcriptome analysis

2) Designing primers for said Bxadh2 gene

 3) Amplifying sequences from putative Bxadh2 gene from cDNA obtained from total RNA extracted from PWN culture 

4) Creating recombinant Bxadh2-T7 DNA fragments that can be targeted by T7 primers through cloning

5) Designing and using T7-promoter-oligos that can be used to obtain siRNA complementary to the Bxadh2 sequence

6) Confirming the adequacy of the siRNa by RT-PCR targeting the Bxadh2-originated mRNA in PWN cultures exposed and not exposed to the siRNA.

7) Evaluating the role of the Bxadh2 gene in the PWN survival rate and fecundity by exposing live PWN to the siRNA in immersion assays and assessing those parameters at defined time points.

7) Characterizing basic physicochemical properties of the Bxadh2 gene through bioinformatic tools.

The study is well designed and it could produce a valuable insight into the role of the Bxadh2 gene after major corrections. Overall presentation of background, goals and methodology is somewhat confusing, not backed by the references presented and needs further details and rewriting.

Detailed comments below:

2. Materials and methods

2.1. PWN culture

Reference 34 is not verifiable

“It was isolated by the Bellman funnel method” – the name of the method in the reference cited is the Baerman funnel method

“centrifuged at 3000 r/min for 3 min” – detail if rcf(x g) or rpm. Centrifugation speed should preferably be presented in rcf (x g) for reprodutibility purposes.

“The PWN solution was collected in sterile 15 mL tubes, centrifuged at 3000 r/min for 3 min, the supernatant was discarded, treated with 0.1 % streptomycin sulphate for 15 min, rinsed and centrifuged three times with sterile water, and the PWN mixed into a suspension with a concentration of 5000 nematodes/mL and stored at 4 ℃ for backup.” Sentence too long and hard to understand, consider rephrasing

2.2. Cloning of the PWN Bxadh2 gene coding region

“…concentration and purity from each group of samples were tested using Nanodrop 2000” Not tested, assessed.

“RNA was then reverse transcribed into cDNA and stored at -20°C for backup. “ Details of the reaction are missing, including the enzyme, dNTPs, thermal cycler, equipment, etc 

“The primers of Bxadh2 gene were designed based on the transcriptome data, and the cDNA of Bxadh2 gene was amplified by PCR.”  Reference the software used for primer design. The reference for the transcriptome data used is missing, so this cannot be reproduced.

Synthetic cDNA was used as the template for PCR amplification. The reaction conditions were as follows: 95℃, 30s; 95℃, 15s; 58℃, 15s; 72℃, 90s; 33 cycles, 72℃ extension for 5min. Details of the reaction are missing, including the enzyme, dNTPs, thermal cycler, equipment, tc  

 

“Colony PCR was performed using Green Taq Mix (Vazyme, Nanjing) and T7 primers (Forward Primer: 5'-TAATAC-GACTCACTATAGGG-3', Reverse Primer: 5'-GCTAGTTATTGCTCAGCGG-3'). The PCR reaction conditions were 95℃, 30s; 95℃, 15s; 58℃, 15s; 72℃, 90s; 33 cycles, extended for 5min at 72℃. Details of the reaction are missing, including the enzyme, dNTPs, thermal cycler, equipment 

 

2.4. PWN Bxadh2 gene siRNA synthesis

“RNAi primers were designed for the PWN Bxadh2 gene using the online design web-site BLOCK-iT™ RNAi Designer. Four single-strand Oligo DNA with T7 promoter sequences were designed for each species.” In 2.1 PWN culture only one species and strain of PWN is mentioned. Correct if otherwise.

“According to the description of In vitro Transcription T7 Kit (Code No.6140, TaKaRa), the DNA sequences downstream of the T7 promoter were transcribed in vitro, and numerous single-stranded RNA were synthesized. “  Should read  “Single Stranded RNa was synthesized using In vitro Transcription T7 Kit (Code No.6140, TaKaRa) according to the manufacturer’s instructions.”

“After passing detection by ultraviolet spectrophotometer, the RNA was stored at -80 ℃ for reserve.” If referring to a RNA quantification method by spectophotometry, please detail so.

2.5. Functional verification of the Bxadh2 gene after PWN RNAi

2.5.1. Detection efficiency of interference by RT-qPCR Do you mean “Detection of interference efficiency?”

“Around 3000 PWN were immersed in Bxadh2 siRNA and control NC siRNA at a concentration of 1000 ng/μL for 48 h at 20 °C and 180 r/min.” Replace ‘around’ with ‘approximately’. What does NC stand for?

2.5.2. Determination of PWN viability after RNA interference

“10 μL of the suspension was aspirated every 3 days to determine PWN survival rate. PWN survival rate = (number of live worms/total number of nematodes) x 100% for 3 replicates.” Was=were; PWN survival rate was calculated as…

 

 

3. Results and analysis

3.3. PWN Bxadh2 gene function studies

3.3.1. PWN Bxadh2 gene RNAi efficiency Do you mean interference efficiency?

The gene expression of PWN treated with NC siRNA solution was compared with that treated with Bxadh2 siRNA. Gene expression of PWN treated with Bxadh2 siRNA de-creased to 44%, and the difference was significant (Figure 9), indicating that interference was effective. PWN treated with interference could be used for subsequent experimental studies.   Please detail how was relative expression calculated.

3.3.3. PWN fecundity changes following RNA interference

After incubating the PWN on grey grape spores for 5 d, the PWN were collected using the Bellman funnel method and their fecundity calculated. Please correct the name of the method for collecting PWN, as above.

Nematode numbers in the NC siRNA control and Bxadh2 siRNA-treated groups were 8683 and 6217, respectively (Figure 11), indicating that when the expression of the Bxadh2 gene of PWN is suppressed, its reproduction is significantly reduced.

It would be interesting, if possible, to have more detail on this, for example regarding number of juveniles vs number of adults.

 

4. Discussion

Wang studied PWN BxAK1 gene function by RNAi technology and found that down-regulation of the gene expression led to a decrease in reproductive capacity and increased nematode mortality [21]. Kang performed RNAi on the PWN BxVap-1 gene and found that it was closely related to PWN migration ability [38].  This references do not refer to the studies mentioned in the sentence

 cDNA sequence is not referenced, shown or deposited anywhere, so the results can not be verified.

 

The use of the English lexicon and grammar is poor and needs to be reviewed throughout the whole manuscript. Examples from the Introduction:

 

“PWD is characterized by a hidden site…” this statement is not clear, please reformulate

 

“It is also known as pine tree "cancer" [4] and is thought to have spread from North America [5], and Japan was the first country where PWD occurred [6, 7].” Sentences with ‘and….and….’ are unclear, consider rewriting.

 

“affects a variety of pines” should read “affects a variety of pine species”

 

“pathogenesis, the pathogenesis is not clear at present” consider using “pathogenesis whose mechanisms are not clear at present”

Author Response

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Author Response File: Author Response.docx

Reviewer 2 Report

This study investigated the effects of Bxadh2 gene on the survival rate and fecundity of PWN, which provided important theoretical basis for biological control of PWD. These results can provide some guidance for the control of pine wood nematode disease in the future. And I quite approve of this work, but this paper is not up to the standard of being published yet, which requires a great of work by the authors to modify, detailed modification suggestions are as follows:

 

Abstract:

1. Line2: Latin should be italicized.

 

Introduction:

The introduction of this paper is badly written and it is very difficult for the reader to understand what the authors did. The association between “CYP450”, “Alcohol dehydrogenase” and “GD2” does not seem to be well explained and the reader is unable to understand the significance of the study

 

1. Paragraph 1, line6: Japan is the first country in Asia to have PWD, but not the first to have it. The expression here seems to be inaccurate.

2. Paragraph 2, line18: “CYP450” did not appear in the previous article

3. Paragraph 2, line18-21: Lack of references to support.

4. Paragraph 4: Why is the effect of Bacillus cereus strain GD2 on PWN gene expression suddenly studied? The explanation in this section is not strongly correlated, and it is difficult to understand the purpose of doing this study.

 

Materials and Methods:

 

2.1. PWN culture

1. Line 1: What is the virulent PWN strain?

 

2.2. Cloning of the PWN Bxadh2 gene coding region

1. Paragraph 2, Line 5: By what method and what reagents were the recombinant plasmids constructed? Please explain.

2.5. Functional verification of the Bxadh2 gene after PWN RNAi

1. Are the mixed PWN used in the RNAi experiment? It is suggested to supplement RNAi experiments for different instars of nematodes.

 

2.5.1. Detection efficiency of interference by RT-qPCR

1. The optimum culture temperature of nematodes is 25-28℃. Why are nematodes used for RT-qPCR treated at 20℃ and 180 r/min.

2. What exactly is a control NC siRNA? Does GPF processing need to be added?

 

Results:

3.1. Constitutive anatomical traits

 

1. Line 4: Not all the high-susceptible group showed lower CfC and CsX than the low-susceptible group. This seems to be an inaccurate statement.

2. Figure 9: It is suggested that different colors should be used to distinguish column drawings of different treatments, and the presentation of significance analysis should be standardized and unified.

The authors have their work reviewed by a proper translation/reviewing service before submission. Overall, the language is appropriate.

Author Response

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Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The study benefited from the authors corrections but still needs some corrections in order to match the journals’ quality standards. Some english language editing also needed . Please see below, comments in yellow.

“PWD is thought to be spread from North America [4], and Japan was the first country in Asia…” should read “thought to have spread…”

 Unreferenced sentence: “Mamiya found that parenchyma cells and lipid cells in infected pine trees were affected and even died before the nematodes reached the affected area.”

 “So PWN detoxification is also a key factor in its Development” Do you mean in disease development?

 Unverifiable references: Chen Y X, Yan Z M, Tan J J. Identification of the endophytic bacterium GD2 in Pinus sylvestris and its effect on the occurrence 411 of pine nematode disease [J]. Phytosanitary, 2021, (5): 35.

 Chen studied the effect of Bacillus cereus strain GD2 on PWN gene expression using transcriptome sequencing, and taking p - value < 0.05 and | log2FC | > = 0.585 as the standard, selected 143 differentially expressed genes [26]. This belongs in methodology and not in introduction, consider including a section in Methods detailing how you chose the Bxadh2 gene because this is actually part of your methodology and quite interesting

 AA3 was an insect strain isolated from infected pine trees of Pinus hwangshanensis in Anhui province. It sounds as you are working with an insect… consider rewriting as “strain isolated from an insect”

About 5000 pieces of AA3 PWN suspension were stored at 4℃ for later use. Pieces should be aliquotes

 RNA was then reverse transcribed into cDNA and the reaction system was: Anchored Oligo (dT)18 Primer, 0.5 μg, EasyScript RT/RI Enzyme Mix, 1 μL; gDNA Remover, 1 μL; 2×ES Reaction Mix, 10 μL; total RNA, 1 μL; and finally 6 μL of Rnase-free Warter to 20 μL, mix well, then 42°C, 30 min; 85°C, 5s. Stored at -20°C for backup.  “…and stored at -20°C for 112 backup”

. E. coli receptor cells.  Italicize E. coli  and detail strain.

After being qualified by agarose gel electrophoresis, the RNA was stored 160 at -80 ℃ for reserve. Qualified? You mean integrity verified by agarose gel electrophoresis?

 150 nematodes (of mixed age) Should be mixed stage culture

The sequencing results showed that the recombinant vector contained the Bxadh2 gene and the gene sequence was consistent with the reference sequence.  Sequence should be deposited in GenBank and have an Acc Nr, or at least provided in the supplementary files

 the difference was analyzed for significance by SPSS 24 The name of the statistical test should be mentioned, not the software

We successfully cloned the Bxadh2 gene using the PCR technique, analyzed it by raw letter, and found that it has some relationship with cytochrome P450. Please rephrase “analyzed it by raw letter” . Please detail “some relationship”

 

 

 

 

 

 

 

 

see above

Author Response

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Reviewer 2 Report

After reading the author's reply, the author answered every question seriously and made a lot of modifications to the paper. I appreciate this work very much and agree to publish this paper.

Author Response

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