Diversity and Traits of Multiple Biotic Stressors Elicit Differential Defense Responses in Legumes

Round 1

Reviewer 1 Report

Comments to the authors:

Authors investigated the physical and chemical defense responses of pea (Pisum sativum) with a vector herbivore (pea aphid, Acrythosiphon pisum), a non-vector herbivore (pea leaf weevil, Sitona lineatus), and a virus (Peaenation mosaic virus, PEMV). As a result of different treatments and analyses, the authors found that viruliferous A. pisum (transmitting PEMV) strongly induced callose deposition and certain reactive oxygen species accumulation and programmed cell death in pea. Although weevil reduced the leaf area, but not the photosynthetic efficiency in pea, while viruliferous aphids reduced photosynthetic efficiency. This is an interesting study, which provide new cues to further uncover the mechanism of plant defense responses to insects and pathogens. The MS should be published after revised.

All my questions about this article are as follows： 

1. Abstract section, suggest adding descriptions of key experimental methods.

2. Line18, I don't think “PEMV-infected plants” is accurately described.

3. Keywords, this section is recommended to include photosynthetic efficiency.

4. Introduction section, suggest to add some introduction on reactive oxygen species.

5. Line84, I don't think this paragraph should be part of materials and methods, but rather introduction.

6. Line111, I suggest that the described reverse transcriptase-PCR results be shown.

7. Materials and methods section, the description of the replications is not detailed enough.

8. Line198, 3 - should be followed by the addition of d.

9. The 2 of O₂•-in multiple places in the paper is not angularly labeled.

10. Figure 5 has only one diagram, but the description has four diagrams.

Author Response

Reviewer 1

Authors investigated the physical and chemical defense responses of pea (Pisum sativum) with a vector herbivore (pea aphid, Acrythosiphon pisum), a non-vector herbivore (pea leaf weevil, Sitona lineatus), and a virus (Pea enation mosaic virus, PEMV). As a result of different treatments and analyses, the authors found that viruliferous A. pisum (transmitting PEMV) strongly induced callose deposition and certain reactive oxygen species accumulation and programmed cell death in pea. Although weevil reduced the leaf area, but not the photosynthetic efficiency in pea, while viruliferous aphids reduced photosynthetic efficiency. This is an interesting study, which provide new cues to further uncover the mechanism of plant defense responses to insects and pathogens. The MS should be published after revised.

We thank the anonymous reviewer for the incisive feedback, all the valuable suggestions and comments on our manuscript. We Strongly believe that all these comments and suggestions from the Reviewer helped us improving the manuscript immensely. We are highly grateful to the reviewer for that. 

All my questions about this article are as follows： 

1. Abstract section, suggest adding descriptions of key experimental methods. 

We thank the reviewer for the valuable comment. We have added key experimental methods in the abstract section as suggested by the reviewer.

2. Line18, I don't think “PEMV-infected plants” is accurately described.

Changed as suggested by the reviewer. The revised text on L18-19 is as follows: “High-throughput phenotyping showed that viruliferous aphids reduced plant photosynthetic efficiency, but plants infected with PEMV had increased programmed cell death (trypan blue staining)”.

3. Keywords, this section is recommended to include photosynthetic efficiency.

Keyword “photosynthetic efficiency” has been included as suggested by the reviewer.

4. Introduction section, suggest to add some introduction on reactive oxygen species.

More introduction about ROS has been added to introduction section. We thank the reviewer for the valuable suggestion. We thank the reviewer for the valuable comment. The revised text on L54-61 is as follows: “ROS generation in plants are triggered by various biotic and abiotic stressors. ROS (e.g., H2O2, O2•- etc.) are produced permanently in various cellular compartments including chloroplast, mitochondria, peroxisomes, etc. Overproduction of ROS cause oxidative stress leading to photooxidative damage of various cellular components and ultimately cell death [17]. Hence, plants have evolved various ROS-detoxifying mechanisms including various antioxidant enzymes (e.g., superoxide dismutases, catalases, peroxidases etc.) to harmlessly neutralize damage caused by ROS overproduction [13].”

5. Line84,I don't think this paragraph should be part of materials and methods, but rather introduction. 

We thank the reviewer for the suggestion. This paragraph at L84 has been moved to introduction section as suggested by the reviewer.

6. Line111, I suggest that the described reverse transcriptase-PCR results be shown.

We thank the reviewer for the valuable comment. We have added details RT-PCR in the “Materials and Methods” section the revised manuscript on L128-134 is as follows: “We collected adult aphids from both colonies in microfuge tube, froze in liquid nitrogen and stored samples at −80°C until use. Aphid tissue was ground in sterile conditions with a micro-pestle and liquid nitrogen. We completed RNA extraction using Promega SV total RNA isolation kits (Promega, Madison, WI, United States), cDNA was synthesized from 1 μg of total RNA using Bio-Rad iScript cDNA synthesis kits, and PCR using DreamTaq Green PCR Master Mix (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and PEMV primers for coat protein (see Table 1)”. The purpose of performing RT-PCR was to detect the prevalence of PEMV in infected colonies and making sure that the non-viruliferous aphid colonies remain infection free (PCR negative). The results have been described in detail at L134-139 is as follows: “We sampled 100 aphids from each colony every 2 months since 2019; over 90% of aphids in the PEMV colony were viruliferous and 0% in the non-viruliferous colony had PEMV. While not every aphid in the PEMV colony was viruliferous, the high prevalence of PEMV ensured virus was present in treatments where many aphids were added to plants, while the lack of PEMV in the non-viruliferous colony ensured sham aphids did not transmit PEMV”. 

7. Materials and methods section, the description of the replications is not detailed enough.

We highly appreciate the reviewer for the valuable comment. We have added descripts of sample replications used various measurements in this study in this manuscript on L203-205 is as follows: “The experiment was performed in two separate blocks and there were six biological replicates per treatment in each block at both time points.”

8. Line198, 3 - should be followed by the addition of d.

Changed as suggested by the reviewer.

9. The 2 of O₂•-in multiple places in the paper is not angularly labeled.

We thank the reviewer for pointing out this mistake. This mistake has been corrected throughout the manuscript.

10. Figure 5 has only one diagram, but the description has four diagrams.

We have corrected this mistake in the revised manuscript by incorporating a new figure 5 with four diagrams. We thank the reviewer for pointing out this mistake.

Reviewer 2 Report

Please find comments as below:

1. Line 84: rewrite as ""PEMV is transmitted by aphids in non-persistent manner.

2. Line 111: change to ""Reverse transcription-PCR"

3. Primers for CP is designed for this study or inferred from somewhere.

4. To represent the PCD, more tests like SEM images of organelle or comet assay.

5. In fig 5, there is no Fig5a, 5b, 5c and 5d. Please incorporate. 

Author Response

Reviewer 2

Please find comments as below:

1. Line 84: rewrite as ""PEMV is transmitted by aphids in non-persistent manner.

Changed as suggested.

2. Line 111: change to ""Reverse transcription-PCR"

We thank the reviewer for pointing this out. We have changed this as suggested by the reviewer.

3. Primers for CP is designed for this study or inferred from somewhere.

We have designed the primers by ourselves using coat protein sequences (obtained from NCBI gene bank) of multiple PEMV isolates form their conserved regions so that the primers can also be used for detecting PEMV isolates from filed collected samples for other diagnostic studies.

4. To represent the PCD, more tests like SEM images of organelle or comet assay.

We thank the reviewer for the valuable study. We tried to use Electron microscope to show how various biotic stressors induce PCD and what are effects on various cellular organelle particular vacuole, mitochondria, chloroplast, golgi apparatus etc. But this is beyond the scope due to lack of facilities and expertise. 

5. In fig 5, there is no Fig5a, 5b, 5c and 5d. Please incorporate. 

We have corrected this mistake in the revised manuscript by incorporating a new figure 5 with four diagrams. We thank the reviewer for pointing out this mistake.

Reviewer 3 Report

The manuscript of the article is devoted to a comparative assessment of damage of various levels that occurs in plants under the influence of various insects and viruses.

There are serious comments and questions about the manuscript:

1. What does the phrase “dry peas” mean, line 59?

2. It is obvious that callose is formed with the participation of hydrogen peroxide.

This relationship needed to be discussed and emphasized, which was not done.

3. Programmed cell death (PCD) is assessed according to several parameters, including the very important “DNA ladder” indicator and changes in cell ultrastructure. What is presented in the manuscript may also be the result of necrosis, which is the opposite of ACL. Typically, the external manifestation of PCD is pinpoint necrosis, which is absent in photographs of leaves.

4. There is no clear final conclusion: what is common and what are the fundamental differences between damage caused by insects and viruses. There are a lot of general phrases about nothing.

5. A very poor and primitive discussion of the results obtained.

   Based on the foregoing, the article is not recommended for publication in  the journal "Agriculture".

Author Response

Reviewer 3

There are serious comments and questions about the manuscript:

We thank the anonymous reviewer for the incisive feedback, all the valuable suggestions and comments on our manuscript. We Strongly believe that all these comments and suggestions from the Reviewer helped us improving the manuscript immensely. We are highly grateful to the reviewer for that. 

1. What does the phrase “dry peas” mean, line 59?

Cultivated peas are classified into two types: garden peas (Pisum sativum ssp. hortense), which are identified by the wrinkled nature of their seed and cotyledon, and field peas (Pisum sativum ssp. arvense), also known as dry peas. Dry peas are distinguished by its smooth seed surface (Source: https://www.usapulses.org/resources).

2. It is obvious that callose is formed with the participation of hydrogen peroxide.

This relationship needed to be discussed and emphasized, which was not done.

We thank the reviewer for the valuable comment. This relationship between callose deposition with the participation of hydrogen peroxide has been discussed and emphasized in the revised manuscript. The revised text on L380-383 is as follows: “H2O2 signaling in plants however has been found to be associated with regulation of callose deposition. Virus induced callose deposition in plants require H2O2-mediated transcriptional reprograming to restrict virus transmission [35]”.   

3. Programmed cell death (PCD) is assessed according to several parameters, including the very important “DNA ladder” indicator and changes in cell ultrastructure. What is presented in the manuscript may also be the result of necrosis, which is the opposite of ACL. Typically, the external manifestation of PCD is pinpoint necrosis, which is absent in photographs of leaves.

We thank the reviewer for the valuable comment. We agree with the reviewer that PCD is assessed by investigating several parameters including “DNA ladder” indicator and changes in cell ultrastructure. We initially planned to investigate the changes in various cell organelles including chloroplast, mitochondria, peroxisome and vacuole using SEM, but due to lack of available facility and expertise we were unable to do that. On the contrary, Trypan blue staining is a very standardized histochemical technique for measuring program cell death in response various biotic and abiotic stress responses. In this study, we wanted to see how various combinations of biotic stressors induce program cell death in plants and hence we used trypan blue stating for getting a relative estimate of the amount of induced PCD in pea leaves. As we all know that programmed cell death is a type of plant hypersensitive response, where plants induce rapid cell death within and around the affected area sometimes manifested in the form of necrotic lesions, which can limit proliferation of pathogens or further herbivore feeding. We have clarified this in the revised manuscript and the revised text on L416-420 is as follows: “Programmed cell death is a type of plant hypersensitive response, where plants induce rapid cell death within and around the affected area sometimes manifested in the form of necrotic lesions, which can limit proliferation of pathogens or further herbivore feeding. Whereas necrosis is the result of cell death during disease development [50.”

4. There is no clear final conclusion: what is common and what are the fundamental differences between damage caused by insects and viruses. There are a lot of general phrases about nothing.

We thank the reviewer for the valuable comment. We have revised the conclusion section of the revised manuscript and introduced how the fundamental difference of damage caused by piercing-sap sucking, viruses transmitted by them and chewing non-vector herbivores insects might affects plant responses depending on the trait and complexity of the biotic stressors they are exposed.

1. A very poor and primitive discussion of the results obtained.

We have significantly edited the discussion to provide more and in-depth information regarding the results obtained explained them in the discussion section. We believe the discussion has been improved significantly in the revised manuscript. We thank the reviewer for the valuable comment and help for improving our revised manuscript significantly.

   Based on the foregoing, the article is not recommended for publication in  the journal "Agriculture".

Round 2

Reviewer 2 Report

No revision required.

Author Response

We thank the anonymous reviewer for the incisive feedback, all the valuable suggestions and comments on our manuscript. We Strongly believe that all these comments and suggestions from the Reviewer helped us improving the manuscript immensely. We are highly grateful to the reviewer for that. 

Reviewer 3 Report

The authors made some edits to the manuscript, which undoubtedly brought greater clarity to the text. However, I still have questions about the definition of the term “programmed cell death.” It is well known that apoptosis, that is, programmed cell death, occurs only in the event of the development of incompatible interactions between a resistant host plant variety and an infectious agent caused by exometabolites of the pathogen. The article does not mention the resistance of the pea variety to the virus. An increase in the area of dead cells during co-infection with the virus and the pea budworm also most likely indicates cell death of the necrotic type (lines 268-270). Trypan blue staining is not a marker of apoptosis, since it is a vital dye that reveals any dead cells, regardless of the type of death.

On this basis, we cannot talk about programmed cell death; we can note cell death without discussing the mechanism of death, since there is insufficient evidence of death by apoptosis.

I recommend that authors use these terms more carefully.

Taking this into account, I can say that the section “Effects of biotic stressors on programmed cell death in pea plants” line 258 requires revision taking into account the comments, after which the article can be published in the journal "Agriculture".

Author Response

The authors made some edits to the manuscript, which undoubtedly brought greater clarity to the text. However, I still have questions about the definition of the term “programmed cell death.” It is well known that apoptosis, that is, programmed cell death, occurs only in the event of the development of incompatible interactions between a resistant host plant variety and an infectious agent caused by exometabolites of the pathogen. The article does not mention the resistance of the pea variety to the virus. An increase in the area of dead cells during co-infection with the virus and the pea budworm also most likely indicates cell death of the necrotic type (lines 268-270). Trypan blue staining is not a marker of apoptosis, since it is a vital dye that reveals any dead cells, regardless of the type of death.

On this basis, we cannot talk about programmed cell death; we can note cell death without discussing the mechanism of death, since there is insufficient evidence of death by apoptosis.

I recommend that authors use these terms more carefully.

We thank the anonymous reviewer for the incisive feedback, all the valuable suggestions and comments on our manuscript. We Strongly believe that all these comments and suggestions from the Reviewer helped us improving the manuscript immensely. We are highly grateful to the reviewer for that. 

 We strongly agree with the reviewer that, trypan blue staining is to assess cell viability and it is used as a vital staining technique to selectively stain dead cells blue. Therefore, it does not specifically distinguish between programmed cell death and non-programmed cell death. So, we have rephrased Programmed cell death/PCD with just "cell death” throughout the manuscript as suggested by the reviewer. We are highly grateful to the reviewer for pointing out this very crucial point and help us rectifying in this revised manuscript.

Taking this into account, I can say that the section “Effects of biotic stressors on programmed cell death in pea plants” line 258 requires revision taking into account the comments, after which the article can be published in the journal "Agriculture".

We have changed the heading of this section as suggested by the reviewer. The revised text on L258 is as follows: “Effects of biotic stressors on cell death in pea plants”

We have also changed programmed cell death to cell death everywhere in this section in order to avoid the confusion whether the dead cells were produced through programmed or non-programmed manner for which we have no direct clue. We are highly thankful to the reviewer for pointing this out. The revised text on L259-269 is as follows:

“Similar to the deposition of callose and accumulation of reactive oxygen species, pea leaf samples collected 7 d after the removal of viruliferous pea aphids showed significantly higher levels of cell death compared to control or sham pea aphid treatments, regardless of the presence or absence of pea leaf weevil herbivory. However, effects of viruliferous pea aphids on cell death were not different from any other treatments at 3 d post removal. All other treatments besides viruliferous pea aphids exhibited relatively low levels of cell death induction that did not differ from control treatments (Fig. 4). The extent of dead cells resulting from cell death was lower in all 3 d post addition samples (compared to 7 d post addition), with only pea leaf samples infested by viruliferous pea aphids in the presence of pea leaf weevils showing greater numbers of dead spots compared to other treatments (Fig. 4).”

Author Response File: Author Response.pdf