Reviewer 1's Review (Patrick VanDijck)

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In this paper a novel regulator of sporulation and trehalose biogenesis has been characterized in the filamentous fungus A. nidulans. This is an important finding because this regulator may be a potential target for novel antifungals as a deletion results in a lack of spore viability. This novel regulator, VosA, seems to be conserved among filamentous or dimorphic fungi but a quick check indicated that it is not present in Candida albicans, also a dimorphic fungus but of course, does not produce spores. This may indicate that the VosA protein is more related to spore formation and that the effect on trehalose may be secondary. Interestingly, the VosA also has no homolog in baker's yeast, at least not in the sequenced laboratory strain.

The findings are well-documented and are of good quality and the paper is written very nicely and focussed.

Remarks:

In yeast and in plants it is well-known that the intermediate of the trehalose biosynthesis pathway, trehalose-6-phosphate is an important regulator of glycolysis and plant development respectively. Based on the data presented in Fig. 3, it seems that in the vosA mutant, there may be an imbalance between the expression of tpsA and OrlA. This could result in accumulation of T6P which may cause developmental defects as observed in Arabidopsis. So I think it would be interesting to also measure T6P in these experiments.

Also, I think that there should be more emphasis on the fact that it seems to be TreA that is regulated by VosA (strongly repressed). In S. cerevisiae, upon a heat shock, there is a strong accumulation of trehalose and this occurs despite a strong induction of the trehalase gene. However, this gene is not active and only becomes activated (by phosphorylation) upon lowering the temperature. So it would be interesting to not only measure the expression of trehalase (or the trehalose biosynthesis genes) but also its activity and the activity of the TpsA and OrlA.

As the authors point out, they have no direct evidence for the mechanism of action of VosA. In Fig. 6 they just put an arrow from VosA to trehalose accumulation on the one hand and Conidia maturation on the other hand. If VosA regulates the expression of TpsA, a simple experiment to prove the direct involvement is to replace the promotor of TpsA by a constitutive promotor. When the effect is based on altered transcription, then there should be no difference between presence and absence of VosA. When the effect is posttranscriptionally, or is mediated by T6P, then there may still be an effect.

In summary, this is a great start for a very interesting novel system but as the authors write also, many experiments need to be done in order to understand the mechanism. Some additional experiments may strongly improve the impact of this finding.
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N.B. These are the general comments made by the reviewer when reviewing this paper in light of which the manuscript was revised. Specific points addressed during revision of the paper are not shown.