PHYCI_587572: An RxLR Effector Gene and New Biomarker in A Recombinase Polymerase Amplification Assay for Rapid Detection of Phytophthora cinnamomi
, Aohua Wang 1,†, Xiao Yang 2,3, Xiaowei Yu 1, Wen Tian 4, Yue Xu 1 and Tao Hu 1Round 1
Reviewer 1 Report
Review of PHYCI_587572: an RxLR effector gene and new biomarker in a recombinase polymerase amplification assay for rapid detection of Phytophthora cinnamomi
Summary
This article reports the identification of a unique gene in P. cinnamomi and a method to detect infection by this disease-causing fungus. The ability to decipher P. cinnamomi from other species of Phytophtora is clearly shown though the reason why the other Phytophtora are not a concern is not described clearly. The researchers used an isothermal DNA amplification and claim it is useful in the field but do not describe how the DNA extraction process would work in a field. The gene identified and the assay described appear to be effective.
Comments:
There are 8 subsections of methods and only 3 corresponding subsections of results. Where are your results for the other work you completed? Figure 1 is a Result not a method and needs to be moved to the appropriate section of the paper. In general, I find the results section lacking.
I am not convinced how early identification of P. cinnamomi is helpful. Is there research showing that early ID can help and how that information can be deployed?
Line 70: relies not replies
Line 76: other assays can’t tell the difference between P. cinnamomi and P. parvispora but as the reader, I don’t know why P. parvispora is ok to have. It is the most closely related one but is it non-pathogenic? You have not explained why finding differences between these species is important.
78-79: not a very clear sentence
87: it would be clearer to say “40 samples representing 21 Phytophtora species”. Also, I think you mean “16 other fungal species” not “16 of fungal species.”
116: Isn’t “Hidden HMM” redundant? Hidden (Hidden Markov Model)? Also, what is HMMER?
118: located not locates
125: Amino acid sequence not sequences
187: Is the line disctintly colored or is it clear (no color)? Could you clean up the wording here a little?
191: reaction should be singular
197: I need more details on the NaOH extraction protocol.
215: Are plasmids filtrated or infiltrated? In my experience I have only heard of infiltrating leaves with plasmids and filtrating would be a different process of running them through filters.
257: why is the start of the sentence a different font size?
294: with naked eyes feels like weird wording
296/297 are word for word copies with 315/316. You should reword one of them.
Claiming that this method can be deployed in the field is misleading with the data you have. If DNA extraction is possible in the field, then yes this could be deployed, however, the need for NaOH extraction and purification of DNA seemingly in the lab makes the ability to do the RPA-LFD part in the field moot.
Author Response
Responses to comments of Reviewer 1
This article reports the identification of a unique gene in P. cinnamomi and a method to detect infection by this disease-causing fungus. The ability to decipher P. cinnamomi from other species of Phytophthora is clearly shown though the reason why the other Phytophthora are not a concern is not described clearly.
Response: we carefully revised the manuscript to avoid any confusion. It was not our intention to make the impression that other Phytophthora species are not important, although the goal of this study was to develop a new target and a detection assay specific to P. cinnamomi.
The researchers used an isothermal DNA amplification and claim it is useful in the field but do not describe how the DNA extraction process would work in a field. The gene identified and the assay described appear to be effective.
Response:We have revised the manuscript and added the methods (2.9.Detecting P. cinnamomi in soil samples by RPA-LFD assay ). Details of the NaOH crude DNA extraction method were added to the manuscript. This 5-minutes method could be used for diseased fine roots and leaves under the field condition. For the soils we always use the kits.
There are 8 subsections of methods and only 3 corresponding subsections of results. Where are your results for the other work you completed? Figure 1 is a Result not a method and needs to be moved to the appropriate section of the paper. In general, I find the results section lacking.
Response: Figure 1 has been moved to the Result section.
I am not convinced how early identification of P. cinnamomi is helpful. Is there research showing that early ID can help and how that information can be deployed?
Response: Early detection of P. cinnamomi is essential for successfully implementing control strategies (Kunadiya et al. 2017; 2019). We have revised the manuscript to emphasize the importance of this rapid detection tool.
Line 70: relies not replies
Response: Corrected
Line 76: other assays can’t tell the difference between P. cinnamomi and P. parvispora but as the reader, I don’t know why P. parvispora is ok to have. It is the most closely related one but is it non-pathogenic? You have not explained why finding differences between these species is important.
Response: We carefully revised the manuscript to avoid this confusion. P. parvispora is also an important pathogen. However, the goal of this study is to develop a specific assay for P. cinnamomi.
78-79: not a very clear sentence
Response: Revised as recommended
87: it would be clearer to say “40 samples representing 21 Phytophthora species”. Also, I think you mean “16 other fungal species” not “16 of fungal species.”
Response: We revised to clarity this part. Phytophthora species are oomycetes, not fungi.
116: Isn’t “Hidden HMM” redundant? Hidden (Hidden Markov Model)? Also, what is HMMER?
Response: “Hidden HMM” is redundant. We deleted it. HMMER is used for searching sequence databases for sequence homologs, and for making sequence alignments. It implements methods using probabilistic models called profile hidden Markov models (profile HMMs).
118: located not locates
Response: Corrected
125: Amino acid sequence not sequences
Response: Corrected
187: Is the line disctintly colored or is it clear (no color)? Could you clean up the wording here a little?
Response: Revised as requested. The dipsticks yielded visible control lines, indicating valid tests. Test lines were visible on all dipsticks correlating to positive RPA reactions. A negative detection or NTC in contrast should not result in any signal or color at the position of the test line.
191: reaction should be singular
Response: Corrected
197: I need more details on the NaOH extraction protocol.
Response: More details of the NaOH crude DNA extraction method were added.( 2.8. Detecting P. cinnamomi in artificially inoculated pine needles using RPA-LFD)
215: Are plasmids filtrated or infiltrated? In my experience I have only heard of infiltrating leaves with plasmids and filtrating would be a different process of running them through filters.
Response: Corrected
257: why is the start of the sentence a different font size?
Response: Reduced font size
294: with naked eyes feels like weird wording
Response: Deleted
296/297 are word for word copies with 315/316. You should reword one of them.
Response: One was removed.
Claiming that this method can be deployed in the field is misleading with the data you have. If DNA extraction is possible in the field, then yes this could be deployed, however, the need for NaOH extraction and purification of DNA seemingly in the lab makes the ability to do the RPA-LFD part in the field moot.
Response: NaOH DNA extraction method only requires NaOH and 5 minutes. We have added the methods (2.8. Detecting P. cinnamomi in artificially inoculated pine needles using RPA-LFD ；2.9.Detecting P. cinnamomi in soil samples by RPA-LFD assay ). This 5-minutes method could be used for diseased fine roots and leaves under the field condition. For the soils we always use the kits.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The topic is very interesting and your paper provides some interesting data. However, I am afraid that RPA is an interesting and useful method but in the case of P. cinnamomic cannot be used as an early detection tool, unless you are able to detect the pathogen directly on the host tissues.
General
Development of early detection is really important. Although you briefly describe the technique in the introduction you do not mention what is the initial material used for the analysis. Plant roots? Isolates? Leaves? Something else. Please, provide this information and be precise. I understand that you used P. cinnamomi isolates. Although, I understand that the method works well in the lab and it is quicker that other molecular methods I cannot understand how could it be used as a tool for the early detection of the pathogen. Could this method be applied for host tissues, by omitting the step of the fungal isolation? If yes, then you could claim that it can be used as an early detection tool. I believe that you should revise thoroughly your paper and make clear what was your aim, what you did and why, and then explain what is the value of your research. Reading your manuscript gives me the impression that you did a lot of work but it was not focused to answer certain questions. I believe that it would have been more useful to focus on one or two hosts in order to develop an early detection tool. You claim that the method could be used in the field when you have tested the method only on artificial inoculated pine needles under lab conditions. I believe that you should include some tests of the method in the field as well before you can claim that it can be used as an early detection tool under field conditions.
Spacific
Line 44: what about chestnuts?
Line 128: why did you chose potato virus X (PVX) vectors? Any particular reason? What is the connection with Phytophthora sp?
Line 148: as the above comment. Why did you choose Agrobacterium tumefaciens infiltration? What is the connection with Phytophthora sp?
Line 197: why did you tested only pine needles? Your isolates are coming from a wide range of plants/trees. Why didn’t you include other plant species as well?
Line 315: how can you detect the P. cinnamomi under field conditions since the isolation of the pathogen is a prerequisite for the use of this method?
Author Response
Responses to comments of Reviewer 2
The topic is very interesting and your paper provides some interesting data. However, I am afraid that RPA is an interesting and useful method but in the case of P. cinnamomi cannot be used as an early detection tool, unless you are able to detect the pathogen directly on the host tissues.
Response: As exemplified in this study, the RPA assay could be used on crude DNA extraction samples directly from host tissues(Table 3).
Development of early detection is really important. Although you briefly describe the technique in the introduction you do not mention what is the initial material used for the analysis. Plant roots? Isolates? Leaves? Something else. Please, provide this information and be precise.
Response: We isolated the Phytophthora cinnamomi isolates from many hosts in our privous research, such as the plant roots of Cedrus deodara, Rhododendron simsii and the apples.Phytophthora cinnamomi isolates were obtained from diseased roots of Pinus sp.、Rhododendron simsii and Camellia oleifera from various provinces in China. The roots were surfce disinfected,dried, and placed on Phytophthora selective agar PARP-V8 agar .Surface disinfestation of the diseased roots were accomplished incubation in e70 % ethanol . When using 70 % ethanol, infected roots were given single dips and then dipped in three vials containing autoclaved distilled water for 5-10 min per vial. Following surface disinfestation, roots were surface dried by placing them on filter paper.
I understand that you used P. cinnamomi isolates. Although, I understand that the method works well in the lab and it is quicker that other molecular methods I cannot understand how could it be used as a tool for the early detection of the pathogen. Could this method be applied for host tissues, by omitting the step of the fungal isolation? If yes, then you could claim that it can be used as an early detection tool. I believe that you should revise thoroughly your paper and make clear what was your aim, what you did and why, and then explain what is the value of your research. Reading your manuscript gives me the impression that you did a lot of work but it was not focused to answer certain questions. I believe that it would have been more useful to focus on one or two hosts in order to develop an early detection tool. You claim that the method could be used in the field when you have tested the method only on artificial inoculated pine needles under lab conditions. I believe that you should include some tests of the method in the field as well before you can claim that it can be used as an early detection tool under field conditions.
Response: We added results of testing on naturally infected samples to the manuscript (Table 3). We have done before but the results were not shown in original paper. Details of the NaOH crude DNA extraction method were added to the manuscript. This 5-minutes method could be used under the field condition.
Line 44: what about chestnuts?
Response: chestnut disease has been reviewed.
Line 128: why did you chose potato virus X (PVX) vectors? Any particular reason? What is the connection with Phytophthora sp?
Response: RXLR effectors possess a modular architecture including the N-terminal signal peptide for protein secretion, the following RXLR motif for host translocation, and the diverse C-terminal domain executing virulence activity.A large portion of RXLR effectors have the ability to suppress host PTI and/or ETI.
Based on PVX-based gene expression and functional assay in Nicotiana benthamiana, a high-throughput screenings was carried out to identify the virulence effect of RxLR effectors. Some effectors may block the programmed cell death induced by mouse pro-apoptosis protein Bax or Inf1, while other effectors may elicit plant resistance response.
Line 148: as the above comment. Why did you choose Agrobacterium tumefaciens infiltration? What is the connection with Phytophthora sp?
Response: Revised as requested. Agrobacterium tumefaciens-mediated transformation (agroinfection) was utilized to transiently express P. cinnamomi effector PcAvh87 in the leaves of N. benthamiana to confirm the activity of Suppression of BAX- and elicitin protein INF1-mediated cell death of Nicotiana benthamiana
Line 197: why did you tested only pine needles? Your isolates are coming from a wide range of plants/trees. Why didn’t you include other plant species as well?
Response: We inoculted the hosts (apples) from 1day to 3days. To determine the efficacy of the developed RPA-LFD method, the experimentally infected apple were used to assess the performance. The apples were inoculated with P. cinnamomi P7491(PCN-18-078) mycelium after one day, two days and three days, and visible disease symptom was observed at 1 day on apples (Fig S1). The total genomic DNA of the infection apples were extracted, respectively, and then subjected to RPA-LFD assay. As shown in FigS1, test bands were clearly observed after infection while not observed in healthy apple, suggesting that the developed RPA-LFD assay could successfully detect P. cinnamomi in infected apples.
Tests on naturally infected plants were added to the manuscript(table 3).
Figure S1. Detection of Phytophthora cinnamomi in artificially inoculated apples using the recombinase polymerase amplification-lateral flow dipstick assay. Genomic DNA (10 ng per µL) of P. cinnamomi isolate Pci1 was used as the positive control (PC). Nuclease-free water was used in place of DNA templates in the no template control (NTC). Dipsticks of the first repeat are shown. Identical detection results were observed in the second repeat of the experiment.
Line 315: how can you detect the P. cinnamomi under field conditions since the isolation of the pathogen is a prerequisite for the use of this method?
Response: tests on naturally infected plants were added to the manuscript.
Author Response File: Author Response.pdf
