Aspergillus flavus Exploits Maize Kernels Using an “Orphan” Secondary Metabolite Cluster

Round 1

Reviewer 1 Report

The manuscript is well written and interesting, it explores the possible involvment of an "orphan" secondary metabolite cluster in the pathogenic activity of A. flavus on maize kernel. The set of the experiments is well organized and described and also well performed, but the results part need to be revised together with the discussion. Many part of the results fit more better in the discussion, like for example the lines from 151 to 174 of page 5; so I suggest the author to merge the two parts in a unique part: Results and Discussion. In addition the results described in paragraph 2.3 are not congruent with the figures 3a, 3b and 3c. In particular the gene expression of the the genes in the wild type AF3357 are constant and not increase as is written in the results lines 182-189, in neither of the genes tested. While the overexpression of the genes is evident for the strain ZnCys6-OE-GFP which has been modified for the overexpress the Zn factor.  However in -my opinion this experiment did not demonstrate clearly the involvment of these gene in the infection process as in the wild type that is the strain showing the most severe infection damages the expression of the genes is constant and not increase like the the modified Zn strain. Also the ratio between Salycilic acyd and catechol clearly demonstrate the highly infection virulence of the wild type strain respect to the Zn strain, that should result more virulent if this "orphan" cluster is involved in the maize-A.flavus infection. So the sentence in the discussion from line 277 to line 280 is not congruent with the result in my opinion, need to be justified better from the result obtained.

The experiment with the Nyp1 mutant is well conducted and interesting in the results evidencing the possible of this gene in the later stage of the colonization process of the kernel.

A minor point to be checked is in the two paragraph (lines 119-121) in which the authors mentioned "dai 7" in relation to fig 1 that showed the histological progress of the infection till "dai 4"

Finally in my opinion should be very interesting and increase the value of the manuscript to add the analysis of Aflatoxin content in the two experiment performed during the colonizatin of the kernel; and during the histochemical assay. The manuscript after the revision suggested should be very interesting and more attractive to be published in IJMS journal.

Author Response

Thanks for your kind suggestions, please find our reply in the doc attached

Author Response File: Author Response.docx

Reviewer 2 Report

This is an interesting study. The science is sound, however, it is the grammar that needs addressing. The authors vacillate between American and British English often. There are more than 12 pages of suggestions for them to address. Please see attached document or revisions, etc. Best of luck!

Comments for author File: Comments.pdf

Author Response

Thanks for your kind suggestions. We modified the MS according to your precise and kind revision. The modifications have been indicated in the doc attached

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The authors improved the manuscript by answering to the main points evidenced in my revie. I just found to strong the last sentence of the abstract in my opinion about the "centrality of cluster 32 to the onset of infection"; so the statement should be somewhat blunted in my opinion. Finally, it is interesting that the mutant strains seems to produce a reduced amount of aflatoxin, did the authors check if it is statistically different from the other strains? A comment to this result should be added in the manuscript. The manuscript is worthy of publication after this minor integration suggested in my opinion

Author Response

First of all I want personally thanks to your fine and kind revision; it helps us to improve hugely the paper.

Concerning these last few suggestions, we modify the text accordingly. Concerning the abstract we were a bit enthusiastic and we "exceeding" with the final sentence. Now we've changed accordingly.

again, thanks a lot for your patience