Putative LysM Effectors Contribute to Fungal Lifestyle

Round 1

Reviewer 1 Report

The authors have carried out a mainly bioinformatic study. In this study they have found 4 putative LysM effectors in the genome of Pochonia chlamydosporia (Pc123). Using different bioinformatic tools and software they have analyzed several characteristics of these four effectors and have found that these effectors are different compared to those present in phytopathogenic fungi. In this way, they have managed to establish a relationship between LysM effectors and the fungal lifestyle.

I would have liked to see some more experiment determining the presence of these effectors not only at the expression level but also at the protein level. In this way, the authors could be concluded not only that they are expressing themselves but that they are also carrying out a function in some specific location of the plant for example. In addition, some analysis of the behavior of the plant could also have been done after inoculating it with the fungus. Since although it is described that they overcome plant defences, in the same experiment that you use to analyze the expression of the effectors, it would be necessary to study at least the expression of genes related to plant defence hormones (JA-, SA-, and ET-related genes).

I any case, I believe that this is a good work that lays the foundations to allow the development of deeper molecular work in the study of the LysM effectors of endophytic and phytopathogenic fungi. Furthermore, how its characterization can help to understand the behavior of fungi in different environments or even to predict how they will behave in future environmental environments.

Author Response

Reviewer 1,

Many thanks for your comments on this article and the experimental ideas to extend this work. Certainly, this is a first article to other studies we are currently conducting in our laboratory. The main goal of this paper is to carry out modelling, phylogenetic and evolutionary analyses of putative LysM effectors. Protein detection and localization is not the topic of this paper. We will tackle this in future works.

In the work of Larriba et al., 2015, barley microarrays of Pochonia chlamydosporia 123 colonizing barley were performed. In this system, it was observed that genes belonging to plant hormone biosynthesis pathways were mostly up-regulated. P. chlamydosporia induced plant biosynthesis and transport hormone proteins -mainly associated with salicylic (e.g. Salicylic acid-binding protein 2) and jasmonic (e.g. 12-Oxophytodienoic acid reductase) acids metabolism. Arrays on barley colonised by P. chlamydosporia show that all members of HSP families are up-regulated in colonized roots.

Since this looks like very interesting information, text has been added to the paper to clarify these points (lines 277-280, 294-296). New information has been highlighted in yellow.

Author Response File: Author Response.pdf

Reviewer 2 Report

This manuscript reports an interesting observation, that there seem to be a different set of LysM proteins in endophytes compared to phytopathogens. At first glance this appears to be backed up by the data in Fig2, however on refelction I question whether this is correct. From this data I could equally argue that invertebrate pathogens have a different set of LysM effectors to others. If the title of the paper is to be upheld, then I think the datasets need to include more of the widely-accepted model endophytes to show this is a broad-reaching conclusion rather than perhaps being an artefact of the species sampled.  Include species such as Epichloe, Claviceps and others widely recognised as true endophytes. Include entomopathogens that aren't endophytes.

Do comment on why  protein RZR70225.1 was not regarded as a LysM given its close relationship to LysM1.

In fig 2, clade II, A.oryzae is indicated as being endophyte, it isn't an endophyte, its other (saprophyte).

For Fig 3, I suspect the range of plant pathogens analysed is also introducing bias, where are the mildews, rusts smuts etc?

section 2.5, I would have expected to see far more growth conditions being assessed before declaring that the LsyM gene are constitutively expressed. All  that can be said is they are expressed in Banana roots. What happens to these during nematode colonisation?

I note that the supp figures don't tally with the figure legends - something is wrong with the numbering.

Author Response

Reviewer 2,

We appreciate the comments you have made on our manuscript. They have been important for us to set new sights. First, please note that all the changes that have been made to the text are highlighted in yellow.

In our revised analysis, we have included 27 new phytopathogenic and endophytic fungi following the suggestion of reviewer 2. No proteins qualifying as LysM effectors for these fungi have been found in all organisms selected (see supplementary table 1). The information has been added to clarify it in lines 119-121. More text is highlighted in yellow because we had to make some text modifications according to the new phylogenetic tree. Supplementary Table 1, Figure 2 and Table 1 have also been modified.

Do comment on why protein RZR70225.1 was not regarded as a LysM given its close relationship to LysM1.

Because of the absence of signal peptide. Our criteria to study LysM effectors fit necessary the presence of signal peptide to include these genes as putative LysM effectors since they must be secreted in order to carry out their function. Corresponding explanations have been added to the text (lines 72-74).

In fig 2, clade II, A.oryzae is indicated as being endophyte, it isn't an endophyte, its other (saprophyte).

This has been cited in the discussion (lines 269-270). It is not its main lifestyle, but it can adopt an endophytic lifestyle, as it has been shown in Sun et al., 2018 https://pubmed.ncbi.nlm.nih.gov/29948125/

For Fig 3, I suspect the range of plant pathogens analysed is also introducing bias, where are the mildews, rusts smuts etc?

We have considered new organisms among mildews, rusts, and smuts (mainly Peronospora, Puccinia and Ustilago). Almost none of the selected microorganisms had proteins that accomplish all considerations for being LysM effectors (Supplementary Table 1). Of course, they have some LysM motifs in some proteins, but they show more domains than LysM or do not have signal peptide. For the avoidance of doubt on this point, information has been added in the text from line 118, as it was commented before. These new organisms are now included in this study.

section 2.5, I would have expected to see far more growth conditions being assessed before declaring that the LsyM gene are constitutively expressed. All that can be said is they are expressed in Banana roots. What happens to these during nematode colonisation?

We have expression data during colonization of nematode eggs in an RNA-seq recently published in Environmental Microbiology (https://0-sfamjournals-onlinelibrary-wiley-com.brum.beds.ac.uk/doi/full/10.1111/1462-2920.15408) and we have seen that putative LysM effectors have a basal expression when they colonize root-knot nematodes eggs (Suarez-Fernandez et al., 2021).  This has been added to the text in lines 261-263.

I note that the supp figures don't tally with the figure legends - something is wrong with the numbering.

Thank you very much for notifying us of this error of which we had not been aware. Tables, figures and legend numbers have been revised and modified.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

the inclusion of the attitional species is welcomed and the corrections made are appropriate, although I can find you many more papers that call Aspergillus oryzae a saprophyte than an endophyte!

I do however still have a major problem with the conclusions and title. I still maintain that you could just as easily have identified these species as invertebrate pathogens rather than as endophytes, ie those that can attack chitinaceous hosts. It is telling that the true endophyte Epichloe does not fit your pattern of novel LysM effectors, whilst Cordyceps militaris, an insect pathogen, does fit with these classes of LysM.

Yes, you have identified an interesting class of LysM, however I think that your title is extremely misleading.

Author Response

Reviewer 2, 

we have taken your comments into account and modified the title so that it is not so misleading. We have decided to re-write it so as not to conclude that this work only includes endophytes and pathogens.