Bioinformatics Analysis and Functional Characterization of the CFEM Proteins of Metarhizium anisopliae

Round 1

Reviewer 1 Report

The paper is devoted to the bioinformatics and experimental analysis of a specific fungal protein family CFEM with some important functions involved in invasion and host adaptation in plant parasitic fungi. As it is well established now that the plant and insect pathogenic fungi have a common origin, it is of particular interest to study properties of these proteins in entomopathogenic fungi. Examination of protein structure, localization and functions is essential for understanding their roles and may be helpful for improvement of insect killing by the fungi applied as biopesticides. In particular, many entomopathogenic fungi, just like other related fungal taxa, are considered as effective endophytes with a range of positive effects incurred to plants.

The paper perfectly fits the JoF scope and represent a sound study using a series of modern approaches, from bioinformatics used for protein search and prediction to genetic manipulations for expression, localization and interactions with plants.

There are however several flaws which need to be addressed before the paper can be accepted.

1) In Figure 5, nuclei staining is lacking which do not convince in subcellular localization. Meanwhile, the authors do not discuss possible reasons and consequences of nuclear localization of MaCFEM88, as opposed to the other proteins.

2) In Figure 7, two parts of the figure could be adjusted so the rows of pictures of the same protein correspond to each other

3) In Figure 8, it is not always clear what is shown on the leaves and the conclusions are not always convincing. For example, in MaCFEM81, it seems there is a slight necrosis in left CFEM spot and no such reaction in the right one. In MaCFEM85, the necrosis tends to form only on the one side of the spot (the upper side). Is it possible that the reactions are affected by neighbor spots. Why not performing this test using separate leaves? The use of repetitions and quantitative image evaluation tools would be appropriate here.

4) Grammar and style need improvement.

 

Author Response

On behalf of my co-authors, I pay my sincere thanks to the reviewer for positive and constructive comments. All of the suggestions made by the reviewers have been addressed and detailed below. Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

The Manuscript entitled (Bioinformatics analysis and functional characterization of the CFEM proteins of Metarhizium anisopliae) introduced good knowledge about the

 Cloning and identification of 13 CFEM genes from M. anisopliae that might be used to illustrate the roles of MaCFEM proteins during M. anisopliea-plant interactions with well descriptions of methods and results.

 

I suggest that the MS in this form can be considered as minor revision

 

Comments to authors:

-        Line 9: (is) instead of (was )

-        Line 32: (Fungi) instead of (Fungus )

-        Line 41: delete (more)

-        Lines 78,84,87: (M. anisopliae ) instead of (Metarhizium anisopliae)

-        Line 83: write the species in complete form, Nicotiana benthamiana, then, abbreviate it in the remaining MS as in line 96.

-        Line 212: in (Metarhizium anisopliae ) instead of (M. anisopliea)

-        Figure 6: it should be with higher resolution because the letters on bars are not obvious.

-        Lines 288,292,296: add df, F and P values, they are important to confirm the significance

-        Line 362: Write (S. turcica) in complete

-        Lines 403-407: add reference(s).

 

Author Response

On behalf of my co-authors, I pay my thanks to the reviewer for positive and constructive comments. All of the suggestions in subsequent paras have been incorporated to the best of my knowledge. Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors implied some effort to improve the manuscript. However, some issues need to be corrected.

1)     staining of nuclei is not convincing. If the authors cannot provide direct proof using specific staining (DNA dye), they may only SUPPOSE that those intensively stained dots are the nuclei. The supposed structures should be clearly indicated on the images of the Figure 5, including nuclei and cytosolic bodies (whatever that means).

2)     Authors claim that the manuscript has been corrected both “by a native English expert and an English professional service” but this work doesn’t seem to be thoroughly done. For example, in the phrase “Metarhizium spp., as …. fungi, is one of …. fungi” (Lines 33-34), the use of the predicate in singular form is not appropriate. The use of preposition doen’t seem to be conventional in the phrase “incubated onto potato sucrose medium” (Line 93). “Plant … was growth” (Line 99) makes no sense. In Lines 134 and 138, the first phrases don’t seem to be complete. “Plasm membrane” in Lines 480-481 seems to be a typo.

3)     Style of the manuscript needs to be reconsidered. For example, “increased resistance … by systemic …resistance” (Line 48-49) is an unnecessary tautology. The use of present perfect in Lines 50-51 is not logical as it describes a permanent process in generalized terms. The full term should precede its abbreviation (CFEMs in Line 65). It is not clear what is meant by “after … cloning, … genes were cloned” (Line 81). Why the personal name is given in lower case? (Line 91). I doubt authors used abbreviated generic epithet for “M. anisopliae” (Line 109) while doing search in Pfam database. The phrase “sequence of these CFEM proteins” (Lines 109 and 111) implies one sequence codes for multiple proteins. Logical connection is lacking in “proteins phylogenetic tree” (Line 122). The use of terms “collections” in regard to samples of mycelium is not appropriate (Line 140). Expression or localization “in the whole cell” sounds odd. First, there is no evidence that the protein is present in each and every cell organelle (as implied by the “whole cell”). Second, protein localization somewhere doesn’t necessarily means it is expressed in the very place. I hardly can imagine “expression …. in cell membrane” (Line 278).

Author Response

On behalf of the authors, I appreciate the reviewer for positive and constructive comments. we have asked an expert for providing linguistic assistance once again. And tried our best to revise and improve the manuscript. Please see the detail in the attachment.

Author Response File: Author Response.pdf