Simultaneous Detection of Plant- and Fungus-Derived Genes Constitutively Expressed in Single Pseudoidium neolycopersici-Inoculated Type I Trichome Cells of Tomato Leaves via Multiplex RT-PCR and Nested PCR

Round 1

Reviewer 1 Report

Iwasaki et al. have provided a good study to demonstrate that a single trichome cell could be used to monitor the expression of genes of both tomato and the infecting fungus simultaneously. The experiment is well planned and executed and most of the necessary information is provided in the manuscript. However, I suggest authors to add a few lines about the novelty of this manuscript in the abstract. One more point, I understand that authors have worked on tomato and the employed technique is of benefit in this plant species. Can the technique is of benefit in other crops as well? I believe that it can be at least tried in other plant species and may be of benefit. I suggest authors to discuss this in the Discussion part. If possible, it will be good to add a few lines about the same to the abstract. It may increase the readability of the paper and the technique can be seen in a broader context. The figure captions are self-explanatory; however, I would like to suggest adding tomato and maybe cultivar name somewhere in captions of Figure 3-6. For example, leaf type I trichome cells of tomato cv. Moneymaker (MM).

I do believe that the authors should address the mentioned points and enrich the manuscript with crucial information.

Author Response

Dear Reviewer #1,

We would like to express our sincere thanks for the reviewers’ large efforts to improve our manuscript (Ms. ID agriculture-1595525). We attempted to faithfully respond to the reviewers’ comments given to us. According to the reviewers’ request, we indicated changes with line numbers corresponding to line numbering of the revised manuscript (RM) (lines numbers of the final text, without showing track changes). Our responses to the reviewers’ comments were shown below in a ‘point-by-point’ manner. Also, the parts (words, sentences etc.) changed in RM were highlighted in red.  

Reviewer's comments and suggestions to the Authors

Reviewer #1: Iwasaki et al. have provided a good study to demonstrate that a single trichome cell could be used to monitor the expression of genes of both tomato and the infecting fungus simultaneously. The experiment is well planned and executed and most of the necessary information is provided in the manuscript. However, I suggest authors to add a few lines about the novelty of this manuscript in the abstract. One more point, I understand that authors have worked on tomato and the employed technique is of benefit in this plant species. Can the technique is of benefit in other crops as well? I believe that it can be at least tried in other plant species and may be of benefit. I suggest authors to discuss this in the Discussion part. If possible, it will be good to add a few lines about the same to the abstract. It may increase the readability of the paper and the technique can be seen in a broader context. The figure captions are self-explanatory; however, I would like to suggest adding tomato and maybe cultivar name somewhere in captions of Figure 3-6. For example, leaf type I trichome cells of tomato cv. Moneymaker (MM).

I do believe that the authors should address the mentioned points and enrich the manuscript with crucial information.

►Response

Thank you for your kind comments.

We added a few lines about the novelty of this manuscript in ‘Abstract’ section. (lines 42-44 in revised manuscript) 

►Response

We discussed about the novelty pointed out by Reviewer #1 in the ‘Discussion’ section (lines 514-516 in revised manuscript). So, we believe that it will increase the readability of the paper and the technique can be seen in a broader context.

►Response

As pointed out by Reviewer #1, we added cultivar name in captions of Figure 3-6.

‘leaf type I trichome cells of tomato cv. Moneymaker (MM)’

Reviewer 2 Report

In this study, authors have used multiplex RT-PCR and nested PCR to simultaneously detect plant and fungus-derived genes constitutively expressed in single Pseudoidium neolycopersi-ci-inoculated type I trichome cells of tomato leaves. This topic of the manuscript is interesting, and the overall manuscript is written very well. This manuscript covers overall knowledge on this topic. I suggest authors review the whole manuscript carefully, correct all the mistakes, and improve the language. Some shortcomings should be resolved.

Abstract

The authors elaborated the abstract in a good way.

Introduction

The introduction part is well written, but some points must be discussed in detail. The introduction must have a sequence from general to specific, like “In the first paragraph of the introduction discuss the genes, its use in different activities in plants specifically biotic stresses, and effects of fungi on plants by citing recent the following articles.

DOI: 10.1016/j.pmpp.2018.04.002, https://doi.org/10.1016/j.micpath.2020.103966, and https://doi.org/10.3390/agronomy11112124.

In the method section, please add some recent references about PCR i.e., https://doi.org/10.3390/ijms22179175.

Lines 48-51: Rewrite the sentences “Three species………worldwide.

Lines 52-54: Rewrite the sentences. It’s hard to read. Since 1998……….our greenhouses.

Line 54: Mention the names. Five P. neolycopersici isolates

Materials and Methods

This section is well written and explained clearly. References are fully cited, and all the sources are mentioned.

Results

The results section is precise and clear, which can help readers understand quickly. All the sub-sections are discussed appropriately.

Discussion

The discussion part is written well, starting from the general to specific. The authors have justified their results properly.

Conclusion

This section is concluded very well. 

Author Response

Dear Reviewer #2,

We would like to express our sincere thanks for the reviewers’ large efforts to improve our manuscript (Ms. ID agriculture-1595525). We attempted to faithfully respond to the reviewers’ comments given to us. According to the reviewers’ request, we indicated changes with line numbers corresponding to line numbering of the revised manuscript (RM) (lines numbers of the final text, without showing track changes). Our responses to the reviewers’ comments were shown below in a ‘point-by-point’ manner. Also, the parts (words, sentences etc.) changed in RM were highlighted in red.  

Reviewer's comments and suggestions to the Authors

Reviewer #2: In this study, authors have used multiplex RT-PCR and nested PCR to simultaneously detect plant and fungus-derived genes constitutively expressed in single Pseudoidium neolycopersici-inoculated type I trichome cells of tomato leaves. This topic of the manuscript is interesting, and the overall manuscript is written very well. This manuscript covers overall knowledge on this topic. I suggest authors review the whole manuscript carefully, correct all the mistakes, and improve the language. Some shortcomings should be resolved.

►Response

Thank you for your kind comments.

We reviewed the whole manuscript carefully, corrected all the mistakes, and improved the language. So, we believe that some shortcomings, pointed out by Reviewer #2, were resolved.

Abstract

The authors elaborated the abstract in a good way.

►Response

Thank you for your kind comments.

Introduction

The introduction part is well written, but some points must be discussed in detail. The introduction must have a sequence from general to specific, like “In the first paragraph of the introduction discuss the genes, its use in different activities in plants specifically biotic stresses, and effects of fungi on plants by citing recent the following articles.

DOI:10.1016/j.pmpp.2018.04.002, https://doi.org/10.1016/j.micpath.2020.103966, and https://doi.org/10.3390/agronomy11112124.

►Response

Thank you for your kind comments.

We discussed the genes, its use in different activities in plants specifically biotic stresses, and effects of fungi on plants by citing recent articles, pointed out by Reviewer #2, in the first paragraph of the ‘Introduction’ section, due to have a sequence from general to specific. (lines 49-74 in revised manuscript)

In the method section, please add some recent references about PCR i.e., https://doi.org/10.3390/ijms22179175.

►Response

Thank you for your advice.

We added the recent reference (https://doi.org/10.3390/ijms22179175) pointed out by Reviewer #2 in the method section. (line 122 in revised manuscript)

Lines 48-51: Rewrite the sentences “Three species………worldwide.

►Response

We rewrote the sentences pointed out by Reviewer #2.

‘Tomato powdery mildews are caused by three species: Pseudoidium neolycopersici L. Kiss (epiphytic fungus) and Leveillula taurica (Lév.) G. Arnaud (endophytic fungus), two pathogens appeared on host plants in many regions worldwide [12–14], and Golovinomyces lycopersici (Cook & Massee) L. Kiss (epiphytic fungus), reported only from Australia [13,15].’ (lines 76-80 in revised manuscript)

Lines 52-54: Rewrite the sentences. It’s hard to read. Since 1998……….our greenhouses.

►Response

We rewrote the sentences pointed out by Reviewer #2.

‘Powdery mildews caused by P. neolycopersici have occurred regularly on hydroponically cultured tomato (Solanum lycopersicum Mill. cv. Moneymaker) in Japan [16–18].’ (lines 80-82 in revised manuscript)

Line 54: Mention the names. Five P. neolycopersici isolates

►Response

Thank you for your kind comments.

We added the names of five P. neolycopersici isolates in the revised manuscript.

“Five P. neolycopersici isolates (KTP-01, -02, -03, -04 and -05) from infected tomato ….. of powdery mildew fungi [19-22]” (lines 82-85 in revised manuscript)

Materials and Methods

This section is well written and explained clearly. References are fully cited, and all the sources are mentioned.

►Response

Thank you for your kind comments.

Results

The results section is precise and clear, which can help readers understand quickly. All the sub-sections are discussed appropriately.

►Response

Thank you for your kind comments.

Discussion

The discussion part is written well, starting from the general to specific. The authors have justified their results properly.

►Response

Thank you for your kind comments.

Conclusion

This section is concluded very well.

►Response

Thank you for your kind comments.