The TOR signaling pathway regulates vegetative development, aflatoxin biosynthesis and pathogenicity in Aspergillus flavus

Reviewer #1 (Public Review):

In this manuscript, the authors investigated the roles of the target of rapamycin (TOR) pathway in various pathobiological processes of Aspergillus flavus. They found that rapamycin treatment affects the growth, sporulation, sclerotia, and aflatoxin synthesis of A. flavus. The authors identified four immunophilin genes (FKBP1 -4), among which FKBP3 is involved in both rapamycin and FK506 resistances, with K19 residue being essential for succinylation. The authors identified a single Tor kinase and characterized its function. Subsequently, the authors analyzed a series of downstream effectors of the TOR pathway, including Sch9, TapA, SitA, Ppg1, and Spot7/Nem1, in terms of vegetative growth, sexual development, stress responses, and aflatoxin production.

While the authors provided a large amount of data regarding the genes involved in the TOR pathway, it is highly descriptive and mostly confirmative data, as numerous papers have already shown that the TOR pathway plays essential roles in a myriad of biological processes in multiple fungi. The authors seemed to perform a series of parallel studies in several genes involved in the TOR pathway in other fungi. However, their data are not properly interconnected to understand the TOR signaling pathway in this fungal pathogen. The authors frequently drew premature conclusions from basic phenotypic observations. For instance, based on their finding that sch9 mutant showed high calcium stress sensitivity, they concluded that Sch9 is the element of the calcineurin-CrzA pathway. Furthermore, based on their finding that the sch9 mutant show weak rapamycin sensitivity and increased Hog1 phosphorylation, they concluded that Sch9 is involved in TOR and HOG pathways. To make such conclusions, the authors should provide more detailed mechanistic data.

In the section "Tor kinase plays important roles in A. flavus", some parts of their data are confusing. The authors said they identified a single Tor kinase ortholog, which is orthologous to S. cerevisiae Tor2. And then, they said failed to obtain a null mutant, but constructed a single copy deletion strain delta Tor1+/Tor2-. What does this mean? Does this mean A. flavus diploid strain? So is this heterozygous TOR/tor mutant? Otherwise, does the haploid A. flavus strain they used contain multiple copies of the TOR gene within its genome? What is the real name of A. flavus Tor kinase (Tor1 or Tor2?). "tor1+/tor2-" is the wrong genetic nomenclature. What is the identity of detalTor1+/Tor2-? Please provide detailed information on how all these mutants were generated. A similar issue was found in the analysis of TapA, which is speculated to be essential (what is the deltaTapA1+/TapA2-?). I couldn't find any detailed information even in Materials and Methods. The authors should provide southern blot data to validate all their mutants.

How were the FRB domain deletion mutants constructed? If the FKBP12-rapamycin binding (FRB) domain is specifically deleted in the Tor kinase allele, should it be insensitive and resistant to rapamycin? However, the authors showed that the FRB domain deleted TOR allele was indeed non-functional.

In Figure 4C, the authors should monitor Hog1 phosphorylation patterns under stressed conditions, such as NaCl treatment, and provide quantitative measurements. Similar issues were found in the western blot analysis of Slt2 (Fig. 8D).

For all the deletion mutants generated in this study, the authors should generate complemented strains to validate their data.

Reviewer #2 (Public Review):

In this study, the authors identified the complex TOR, HOG, and CWI signaling networks-involved genes that relatively modulate the development, aflatoxin biosynthesis and pathogenicity of A. flavus by gene deletions combined with phenotypic observation.

They also analyzed the specific regulatory process and proposed that the TOR signaling pathway interacts with other signaling pathways (MAPK, CWI, calcineurin-CrzA pathway) to regulate the responses to various environmental stresses. Notably, they found that FKBP3 is involved in sclerotia and aflatoxin biosynthesis and rapamycin resistance in A. flavus, and that the conserved site K19 of FKBP3 plays a key role in regulating the aflatoxin biosynthesis. In general, there is a heavy workload task carried in this study and the findings are interesting and important for understanding or controlling aflatoxin biosynthesis. However, findings have not been deeply explored and conclusions mostly are based on parallel phenotypic observations. In addition, there are some concerns that exist surrounding the conclusions.

Reviewer #3 (Public Review):

The paper by Li et al. describes the role of the TOR pathway in Aspergillus flavus. The authors tested the effect of rapamycin in WT and different deletion strains. This paper is based on a lot of experiments and work but remains rather descriptive and confirms the results obtained in other fungi. It shows that the TOR pathway is involved in conidiation, aflatoxin production, pathogenicity, and hyphal growth. This is inferred from rapamycin treatment and TOR1/2 deletions. Rapamycin treatment also causes lipid accumulation in hyphae. The phenotypes are not surprising as they have been shown already for several fungi. In addition, one caveat is in my opinion that the strains grow very slowly and this could cause many downstream effects. Several kinases and phosphatases are involved in the TOR pathway. They were known from S. cerevisiae or filamentous fungi. The authors characterized them as well with knock-out approaches.

As for many results, I miss the re-complementation of the created mutants throughout the manuscript. This is standard praxis.

Fig. 1: cultures were grown for 48 h before measuring the transcript level. The authors show that brlA, abaA, and some sexual regulators are less expressed. In my opinion, this does not allow the conclusion that there is a direct control through rapamycin. Since the colonies grow very slowly in the presence of rapamycin, the authors should add rapamycin and follow gene expression after 15, 30, 60, 90 min. The figure legend needs to be more detailed. Which type of cultures were used, liquid, solid medium? Etc.

Why in chapter one Fig. 9 is already cited? Those data should then be included in Fig. 1 for the general phenotype.

The authors wrote that radial growth and conidiation were gradually reduced with increasing rapamycin concentrations. This is not true. There is no gradient! However, it should be tested if there is a gradient if lower concentrations are used. The current data imply that there is a threshold concentration, so either there is 100 % growth or a reduction to 25 %. This looks strange.