PONE-D-19-25109

Development of a minigenome cassette for Lettuce Necrotic Yellows Virus: a first step in rescuing a plant cytorhabdovirus

PLOS ONE

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The MS was reviewed by three experts in the field and the majority opined that the research findings presented are worthy of publication in PLoS One, pending minor revisions. I agree with the majority recommendation and I encourage you to address the reviewers' comments and submit a revised version.

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Reviewers' comments:

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #2: N/A

Reviewer #3: Yes

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Reviewer #1: Yes

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Reviewer #3: Yes

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Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

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5. Review Comments to the Author

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Reviewer #1: The paper does not have any major scientific flaws but does not provide any interesting information either. The work presented has been done before alas not with LNYV. Is this good enough to warranty publication in PLoSOne? Not in my opinion. It just lacks originality.

Reviewer #2: The paper by Ibrahim and co-authors describes the set up of a replicating minigenome cassette based on genomic components of a cytorhabdovirus, lettuce necrotic yellows virus.

The paper is well written, with a well thought experimental plan, and the main conclusion seem to be well supported by the experiments carried out.

It needs to be improved a little bit, with further explanations that would make this paper useful for repeating experiments and future planning of the same work by other laboratories.

Here are my suggestions for improving it:

Line 19: maybe better “membrane-bound”?

Line 32: plasmids

Line 59-60: are you sure N. glutinosa is the choice plant species for studying virus biology and pathogenesis? The same can be said for N. benthamiana.

Line 140: N. glutinosa in Italics

In general, I think you need to specifically show in a figure (figure 2) a panel that shows the HHz-ribozyme sequence, with the initial viral sequence, and the putative cleaving site. This is important to show, and it would be better to show the secondary structure. No need to do so for the HDz)

Having said that, what you show in Fig.2.B is some evidence of cleaving (but not of the exact cleaving). Furthermore, cleaving is predicted based on RNA sizes (difficult to estimate in agarose gel, likely non-denaturing, even if not specifically stated in the Fig. 2 legend). So I would extensively use the word “putative” to describe the band assigned to the fragments predicted.

Line 174-175: it would be better to specifically mention the exact nt start and end of the 5’ and 3’ UTR referred to a GenBank reference viral sequence.

Line 251: specify better in which sense they were “unsuccessful”

Line 259-262: as I said above, you should have used northern blots with specific probes to identify better each of the three bands. I think this part of the discussion needs to be tuned down, or erased.

278-281: I am a little surprised about this. Probably working with appropriate filters and positive controls, you should have been able to distinguish dsRED from Autofluorescence. And even if I am not specifically asking for it, it would have been good to also have a GFP construct. Visual aspect of infection are also important, fur future steps when you will add putative movement proteins.

The arrows in Fig 1 are too big and misleading in some way. They need to be on the sides of the intron sites…. Not across the intron side.

Fig 4 should show some loading control (Coomassie or poinceu red).

Reviewer #3: Nice ms, describes difficult work that was well done, and of significant interest both to plant virologists and to plant molecular biotechnologists. I have very few concerns, and those are mainly use of taxonomic terms (see annotated ms). The English was excellent, the work was well described, and the results good enough to merit publication.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Edward P Rybicki

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Submitted filename: PONE-D-19-25109_reviewer edit.pdf

Development of a minigenome cassette for Lettuce necrotic yellows virus: a first step in rescuing a plant cytorhabdovirus

PONE-D-19-25109R1

Dear Dr. Drake,

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With kind regards,

Hanu R Pappu

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: