Review Reports - Virus-Induced Silencing of a Sequence Coding for Loricrin-like Protein in Phytophthora infestans upon Infection of a Recombinant Vector Based on Tobacco Mosaic Virus

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Author,

I have an honor to review the manuscript entitled “Virus-induced silencing of a sequence coding for loricrin-like 2 protein in Phytophthora infestans upon infection of a recombinant vector based on tobacco mosaic virus” a research article submitted to MDPI Journal, horticulture. Authors of this manuscript used virus-induced gene silencing (VIGS) strategy and silenced a P. infestans ortholog of plant loricrin-like protein, PiLLP, aiming to reduce Phytophthora infestans sexual reproduction identified and slower colony growth and a diminished virulence in the PiLLP-silenced mating. Overall, the experiments are performed well and the results are convincing. Thus, the presented results take up an important topic consistent with the profile of the Journal.

-However, even, manuscript is well organized and well described of the conception, I have some suggestions, which might improve the manuscript to make important to the wider audience.

-This article required firm aim of the study that should be underlined precisely and simultaneously and highlight why this analysis is important to study.

-Some major and other minor comments are as below

-There are many places where grammar can be improved. There are many complex sentences in the result sections those could be simplified for the general readers. I suggest a careful revision by a professional language editor.

I've just noted a few here.

-Few suggestions I have mentioned in the main text pdf file. Please check

Title:

-Title is ok.

-If English is available in affiliation, that should be used

Abstract: -Good organization with results order.

-Keywords: Number is more than enough, you may reduce

-Also, better use alphabetic order

1. Comment in Introduction:

-Just follow the journal instruction, no need indication that you did not cite

-Introduction is well organized, however, needed to be more specific and sequential including some more specific findings referencing recent publications. Rationale with own findings to be elucidated at the end for the wider reader.

2. Materials and Methods

This section is well organized with detailed of the methodology used

3. Results and Discussion

3.1. P. infestans mating types identification

-The authors might have restriction digestion gel picture; therefore, it is better to use side by side of the PCR gel pic

-Wild type, Wild-type, WT these should be uniform throughout the text

Overall: Results are impressive, however, needing comparison between other available and low-cost management system. Otherwise this high valued experiment won’t attract researchers.

More specifically, need improvement of discussion for individual result sections for wider and general readers

-In Figures legend, there should be title of the fig.

-While naming protein it should be no italic, whereas, gene should be italic. Need careful checking for this.

My Recommendation about this article:

Revision needed. This article may be accepted after minor corrections.

Comments for author File: Comments.pdf

Author Response

Response to Reviewer 1 Comments

Dear Author,

The authors wish to thank the reviewer for its positive evaluation and all the very constructive comments to improve the manuscript. Below, a point-by-point response to comments is provided (red text).

-However, even, manuscript is well organized and well described of the conception, I have some suggestions, which might improve the manuscript to make important to the wider audience.

-This article required firm aim of the study that should be underlined precisely and simultaneously and highlight why this analysis is important to study.

We thank the reviewer for the useful comments. Text has been modified/improved as requested. (Lanes 115-141)

-Some major and other minor comments are as below

Text has been revised and modified as requested.

I've just noted a few here.

Few suggestions I have mentioned in the main text pdf file. Please check

Thank you for your suggestions, which have been integrated in the text.

Title:

-Title is ok.

-If English is available in affiliation, that should be used.

English in each affiliation has been used.

Abstract: -Good organization with results order.

-Keywords: Number is more than enough, you may reduce

Keywords number has been reduced, as suggested.

-Also, better use alphabetic order

Keywords alphabetic order has been used.

Comment in Introduction:

-Just follow the journal instruction, no need indication that you did not cite

We would like to thank the reviewer for the comments. We agree with the reviewer that the rationale should be clarified and text has been modified. To the best of our knowledge, recent publications have been added; these are few, therefore some old ones need to be cited. The Rationale and our findings have been added at the end of Introduction (lines 115-141).

Materials and Methods

This section is well organized with detailed of the methodology used

Results and Discussion

3.1. P. infestans mating types identification

-The authors might have restriction digestion gel picture; therefore, it is better to use side by side of the PCR gel pic

As suggested, Figure 1 has been integrated by gel electrophoresis of PCR products obtained for wild type A1 and A2 mating type with S1a/S1b (panel a), PHYB-1/PHYB-2 (panel b), and W16–1/W16–2 primers pairs, followed by HaeIII digestion (panel c).

Wild type, Wild-type, WT these should be uniform throughout the text

Thanks for your comment. Wild type for the first citation, then WT has been used throughout the text.

Overall: Results are impressive, however, needing comparison between other available and low-cost management system. Otherwise this high valued experiment won’t attract researchers.

Thank you for your suggestion, based on it we add a paragraph (please see lines 443-453 of the revised manuscript) explaining attractiveness of this research.

More specifically, need improvement of discussion for individual result sections for wider and general readers.

Thank you for your suggestions. We opted for a Results and discussion format. However, whenever possible, critical evaluation of the results has been improved.

In Figures legend, there should be title of the fig.

Title of the figures has been added as requested.

While naming protein it should be no italic, whereas, gene should be italic. Need careful checking for this.

Checked

We wish to thank you for the useful comments and we hope that all the explanations reported above are good enough to proceed with the positive evaluation of our manuscript for publication in Horticulture.

With many thanks for your attention and time.

Reviewer 2 Report

The manuscript submitted by Labarile et al. describes that TMV induces VIGS in P. infestans. Further, the VIGS of P. infestans reduces tomato infection symptoms. The findings in the submitted manuscript are interesting. However, there are several concerns as follows that need to be addressed and corrected.

First, as the authors also state in their conclusion, it is most important to determine whether suppression of P. infestans by VIGS occurs in plants. I strongly suggest to the authors to conduct the experiment.

An important control seems to be missing from this study. To verify that TMV infection itself does not affect the infectivity of P. infestans, TMV-GFP-1056 must be used as a control in the experiments in Figures 6 and 7.

The name of two P. infestans types need to be unified in the manuscript, sometimes 96.9.5.1 and sometimes A1, etc.

Results 3.1. All A1 and A2 genotyping data should be shown.

Lines 262-276, and Fig. 2. My understanding is that in the TMV-PiLL-1056 vector, the original GFP sequence has been replaced by a PiLLP sequence. Why is GFP still present in the pTMV-PiLLP-1056 plasmid in Figure S1?

Lines 284-298. I think these statements should be moved to the introduction.

Fig. 3. Personally, I would prefer to use the term "lane" instead of "track". It is necessary to explain why sgRNA1 to 3 signals were detected in the plant inoculated with mock.

Lanes 349-351. The 241 bp PCR product result should be shown.

Lane 352. It is not clear to me what the ‘a 3.5-fold and a 7-fold reduced expression’ means. The method of calculation should be shown.

Minor comments

Fig. 1. Arrow cannot be seen.

Fig S1 should be cited in the manuscript.

Author Response

The manuscript submitted by Labarile et al. describes that TMV induces VIGS in P. infestans. Further, the VIGS of P. infestans reduces tomato infection symptoms. The findings in the submitted manuscript are interesting. However, there are several concerns as follows that need to be addressed and corrected.

The authors wish to thank the reviewer for its evaluation and all the very constructive comments to improve the manuscript. We tried to made the revisions according to your comments and suggestions in order to improve the quality and the clarity of the data presented.

First, as the authors also state in their conclusion, it is most important to determine whether suppression of P. infestans by VIGS occurs in plants. I strongly suggest to the authors to conduct the experiment.

Thank you for your suggestion. There are two ways to obtain the evidence requested: i) knockout the LLP by a CRISR/Cas approach and then infect them with P. infestans, whose PiLLP could be silenced by an HIGS mechanism; ii) infect simultaneously tomato plants with P. infestans and TMV-PiLLP-1056. Then is expected that recombinant virus or vsiRNAs produced by the plant against it would probably be acquired by P. infestans to get its PiLLP silenced. Both the processes are interesting but quite long and complex and to our opinion need a dedicated study. On the basis of our previous works, we opted for an in vitro infection of both the A1 and A2 mating types of P. infestans using purified preparations of TMV-PiLLP-1056. The approach achieved part of its goal silencing partially the A1 mating type. We tested A1-PiLLP infectivity on detached tomato leaves, which is an approach routinely used in tests of pathogenicity, observing a reduction in the size of lesions induced. This reduction was statistically significant as shown in the panel (b) of Figure 7 added to this revised version of the manuscript. Unfortunately, we were unable to obtain a viable culture of the A2 mating type after infection with TMV-PiLLP-1056, therefore we could not test its infectivity in detached tomato leaflets.

An important control seems to be missing from this study. To verify that TMV infection itself does not affect the infectivity of P. infestans, TMV-GFP-1056 must be used as a control in the experiments in Figures 6 and 7.

In two recent papers (ref. 26 and 27 in the text) we provided exhaustive evidence that, like in plants, TMV-GFP-1056 can infect, express and induce RNAi in the A1 mating type of P. infestans without altering its infectivity. Therefore, in this instance, we omitted this approach.

The name of two P. infestans types need to be unified in the manuscript, sometimes 96.9.5.1 and sometimes A1, etc.

Thank you for your suggestions. P. infestans A1 and A2 mating type and sometime A1 or A2 is now used in the MS.

Results 3.1. All A1 and A2 genotyping data should be shown.

We agree with the reviewer. Figure 1 has been integrated by gel electrophoresis of PCR products obtained for wild type A1 and A2 mating type with S1a/S1b (panel a), PHYB-1/PHYB-2 (panel b), and W16–1/W16–2 primers pairs, followed by HaeIII digestion (panel c).

Thanks for noting that. That was a mistake in drawing the Figure S1. Indeed, that fragment shows a duplication of the TMVCP promoter to drive the transcription of PiLLP fragment ligated downstream. Figure S1 has been modified accordingly.

Lines 284-298. I think these statements should be moved to the introduction.

We agree with the reviewer and as suggested, we moved description of TMV-GFP-1056 vector at the end of introduction (lines 115-141).

Fig. 3. Personally, I would prefer to use the term "lane" instead of "track". It is necessary to explain why sgRNA1 to 3 signals were detected in the plant inoculated with mock.

That was a mistake. Figure legend has been amended since lane 2 represents RNA preparations extracted from mock-inoculated tobacco plant.

Lanes 349-351. The 241 bp PCR product result should be shown.

We integrated Figure 6 to show the 241bp product as requested.

Lane 352. It is not clear to me what the ‘a 3.5-fold and a 7-fold reduced expression’ means. The method of calculation should be shown.

Text has been modified to better explain the qPCR and the gene expression normalization method used.

Minor comments

Fig. 1. Arrow cannot be seen.

Arrows have been added to Figure 1 to point with size PCR products and some marker bands.

Fig S1 should be cited in the manuscript.

Figure S1 was cited in the M&M in the “2.2. TMV-PiLLP-1056 vector construction” section of the manuscript. However, we cited the Figure S1 in other parts of this revised version of the manuscript.

With many thanks for your attention and time.

Reviewer 3 Report

In the manuscript on Virus-induced silencing of a sequence coding for loricrin-like protein in Phytophthora infestans upon infection of a recombinant vector based on tobacco mosaic virus, the data shown in this paper is not properly and solid, and the author did not explain why they did the tobacco plant inoculation and the infection of infestians separately, what’s the logical beneath this? The new creativity is limited. The English writing is not good and needs to be improved.

There are some main issues I pointed out here:

In the introduction part, you did not indicate your aim and the results by using the TMV-PiLLP-1056, you need to make a simple conclusion about what this recombinant vector can bring.
In the method part, the”2.3 infection in plants” should be “ plant inoculation”. You did not indicate the part of the plant stage and which leaves you used to do the inoculation in the tomato. What is “Gel Red”, please indicate it
In the results part: where is the data of” According to S1a/S1b PCR assay, isolate 96.9.5.1 yielded a product of about 1.25 kbp con- 243 firming that it was the A1 mating type, whereas no amplification was observed from 244 13_A2 (Blue 13) isolate.”?
And in figure 1, where is the arrow? Please label the size number of the marker.
In figure 2, the plant inoculated with mock should be done as another control and show the picture of mock-inoculated leaves
The second paragraph of 3.2, did not indicate on which day you collected the samples for checking the TMV-PiLLP-1056 expression after inoculation. you should collect the samples on the same day when checking the phenotype.
In figure 3, the gel figure should include the marker and the size label. And I cannot see the bands in sgRNA1 and sgRNA2, the data now you showed here is not convincible.
In figure 4, please do not label the number upon the probe dots
If you didn’t have any evidence or reference, please do not write this hypothesis of “probably as 335 consequence of the TMGMV CP that replaced the authentic TMV CP in the recombinant 336 vector.”
In figure 7, the leaf in A1 TMV-PiLLP looks like not in the same stage/condition as A1 WT and A2 WT, so the phenotype cannot the truly confirmed, and you need to inoculate amount of leaves to calculate the size of the lesions and showing the blot data.

Author Response

Response to Reviewer 3 Comments

In the manuscript on Virus-induced silencing of a sequence coding for loricrin-like protein in Phytophthora infestans upon infection of a recombinant vector based on tobacco mosaic virus, the data shown in this paper is not properly and solid, and the author did not explain why they did the tobacco plant inoculation and the infection of infestans separately, what’s the logical beneath this? The new creativity is limited. The English writing is not good and needs to be improved.

The authors wish to thank the reviewer for its evaluation and all the very constructive comments to improve the manuscript.

In our opinion, tobacco was used to biologically test the successful transformation of the original TMV-GFP-1056 into TMV-PiLLP-1056 by the GFP screening and other test, including sequencing, served for its purification to prepare the inoculum. On the other side, P. infestans was infected in vitro by a purified preparation of TMV-PiLLP-1056 whereas we tested its infectivity/pathogenicity in tomato, which is one of its economically important hosts. We think that English has now been improved throughout the manuscript.

There are some main issues I pointed out here:

In the introduction part, you did not indicate your aim and the results by using the TMV-PiLLP-1056, you need to make a simple conclusion about what this recombinant vector can bring.

We agree with the reviewer and at the end of the Introduction we added a quite long paragraph describing the strategy of the approach and anticipating some of the results achieved. We think that the novelty of the approach (as stated in the text) relies in the use of a recombinant plant virus vector to target and silence specific effector genes in an oomycete.

In the method part, the”2.3 infection in plants” should be “plant inoculation”.

Thanks for your suggestion. Plant infection has been replaced with plant inoculation.

You did not indicate the part of the plant stage and which leaves you used to do the inoculation in the tomato.

In the M&M “2.5 Pathogenicity test” section, it has been specified that tomato plants were four week-old at the time the leaflets were collected.

What is “Gel Red”, please indicate it

It has been specified that GelRed^® has been used for staining nucleic acid bands separated by agarose gel electrophoresis.

In the results part: where is the data of” According to S1a/S1b PCR assay, isolate 96.9.5.1 yielded a product of about 1.25 kbp confirming that it was the A1 mating type, whereas no amplification was observed from 13_A2 (Blue 13) isolate.”?

Figure 1 has been integrated by gel electrophoresis of PCR products obtained for wild type A1 and A2 mating type with S1a/S1b (panel a), PHYB-1/PHYB-2 (panel b), and W16–1/W16–2 primers pairs, followed by HaeIII digestion (panel c).

And in figure 1, where is the arrow? Please label the size number of the marker.

Figure 1 has been modified by adding arrows pointing, with size, PCR products, and some marker bands.

In figure 2, the plant inoculated with mock should be done as another control and show the picture of mock-inoculated leaves

We integrated Figure 2 by adding panels d) and e) showing mock-inoculated controls exposed or not to UV.

The second paragraph of 3.2, did not indicate on which day you collected the samples for checking the TMV-PiLLP-1056 expression after inoculation. you should collect the samples on the same day when checking the phenotype.

Text has been modified according to suggestions.

In figure 3, the gel figure should include the marker and the size label. And I cannot see the bands in sgRNA1 and sgRNA2, the data now you showed here is not convincible.

Figure 3 has been modified adding arrows pointing the viral genomic RNA with estimated size of 6.39 kb and the tobacco leaf 25S and 18S rRNA of 3.7 and 1.9 kb in size, respectively. In our opinion, bands of sgRNAs 1 and 2 are visible although the relative abundance of the three sgRNA might be variable. Since it is a hybridization signal, overloading the gel will make the hybridization signal of sgRNA3 to mask the signal of the other two.

In figure 4, please do not label the number upon the probe dots

Thanks for the suggestion. Figure 4 has been integrated with a scheme to show the preparation used for each dot.

If you didn’t have any evidence or reference, please do not write this hypothesis of “probably as consequence of the TMGMV CP that replaced the authentic TMV CP in the recombinant vector.”

Well, on the basis of the experience of the electron microscopist Dr. Angelo De Stradis and of own experience as plant virologists that could be an explanation, since swollen particles cannot be seen in normal TMV preparation. Therefore, we would prefer to maintain that hypothesis.

In figure 7, the leaf in A1 TMV-PiLLP looks like not in the same stage/condition as A1 WT and A2 WT, so the phenotype cannot the truly confirmed, and you need to inoculate amount of leaves to calculate the size of the lesions and showing the blot data.

We integrated Fig. 7 with panel b to support the evidence that the reduction of lesion area observed with A1-TMV, compared to wild type A1 was statistically significant as shown in the panel b of Fig. 7 added to this revised version of the manuscript.

With many thanks for your attention and time.

Round 2

Reviewer 2 Report

Most of my concerns have been remedied in the revised manuscript.

Author Response

Thank you for your positive evaluation of our responses. We revised the manuscript to further improve English

Reviewer 3 Report

The authors have responded to my questions appropriately, except the figure 3.b, it would be better to show another repeat of this experiment with clear bands shown here.

And in the first question, you explained why you did the tobacco plant inoculation and the infection of infestans, I understand your purpose for these experiments, what I mean you should describe and explain this in your manuscript for the transfer role of the different arrangement of your chapter(like between 3.2 and 3.3) that can maker readers more understand the aim and meaning you design the experiments like this.

Author Response

Thank you for the positive evaluation of our responses to most of your questions

With specific reference to figure 3b we tried to explain in the text (lines 331-335) that the different hybridization signal among the three subgenomic RNAs might be due to the fact that the three sequences are under the control of different promoters; thus, the relative abundance of the each sgRNA may vary according to the efficiency of its transcription, which was not evaluated. We hope this explanation may be sufficient to dissipate any doubt about the results of this test.

Furthermore, here you can see a repeat of this experiment.

lane 1= 2 mg of total RNA preparation extracted from mock-inoculated tobacco plant used as negative control; lane 2= 2 mg of total RNA preparation extracted from tobacco plant infected by TMV-PiLLP-1056 at 14 dpi; lane 3= 0.2 mg of RNA preparation extracted from purified preparation of TMV-PiLLP-1056 used as control. Arrows indicate the positions of tobacco leaf 25S and 18S rRNAs of 3.7 and 1.9 kb, respectively (a), and TMV genomic RNA (vgRNA) and subgenomic RNA1, RNA2, RNA3 (sgRNAs) RNAs (b).

Probably we misunderstood your question. However, now we split in two sentences the rationale of using tobacco (lines 298-301) and detached tomato leaves (lines 418-420) as suggested.