Phytotoxin production in Aspergillus terreus is regulated by independent environmental signals

1) The manuscript needs significant editing as it simply does not do justice to the work in its present form and is unlikely to engage the typical eLife reader. The Introduction needs to put the current studies in a more general context, and needs to be completely rewritten. The manuscript is much too long to engage readers through to the end. As noted, the authors did a very thorough set of experiments, but they don't all need to be described in such detail. The authors should describe the significant findings and relegate others to the supplementary information. In particular, much of the material in the iron section could be moved to the supplementary information. Finally there are significant editorial issues with English usage, particularly in the Introduction.

The Introduction has now been completely revised, putting the investigation in a much broader context. We focus now in the Introduction on the potential of secondary metabolites as pharmaceutical compounds without knowledge on the ecological and physiological impact of the metabolite for the producing organism. We hope that the English style is now acceptable.

The Results section of the main manuscript has been shortened by relocating several experimental details and supporting data to the supplemental. This also led to the reduction of total Figure in the main manuscript text that was reduced from 9 to 8 figures. Although comments below required the addition of some information that was not contained in the previous version of the manuscript, the Results section had been shortened by approximately 2 full pages. This included the “iron section”. A complete removal was not possible, because one of the biological functions of terrein is iron reduction and its regulation performs via the iron response regulator HapX. However, most details on the iron acquisition system have been relocated to the supplementary information.

2) The controls found involve AreA mediated response to nitrogen starvation and AreA binding sites in the 5' of terA are characterized. It is shown that the response to methionine is mediated by the A. terreus homolog of the AtfA bZip transcription factor (TF). This is a little more mysterious because previous work with this TF in other fungi indicates a role in osmotic / oxidative stress responses via the SakA MAP kinase. The authors state that SakA is not involved in terrein synthesis. It is not obvious how methionine responsiveness might occur and the authors should indicate this more clearly. Furthermore from yeast studies the Atf1/Sko1 binding site is TGACGTCA – does this motif occur in the TerR promoter?

We also generated a sakA deletion mutant in order to assess SakA/AtfA interactions. However, we did not obtain indications for a stimulating effect of SakA on AtfA. While a SakA mutant revealed an increased sensitivity against osmotic stress, this was not observed for an atfA mutant. The sakA mutant produced pigmented conidia, whereas the atfA mutant did not. The sakA mutant produced terrein under nitrogen starvation, whereas the atfA mutant did not. However, in terms of terrein production, a deletion of sakA allowed terrein production in the presence of some nitrogen sources, which prohibit terrein production in the wild type. This indicates that SakA may act as a repressor on terrein production, but whether this is direct or indirect remains to be elucidated in the future. However, we added some information in the Discussion:

“In several ascomycetes nitrogen starvation response is also mediated by the basic leucine zipper transcription factor AtfA (Hong et al., 2013). In A. nidulans, nitrogen starvation, oxidative or osmotic stresses induce phosphorylation of the MAP kinase SakA that is subsequently transported into the nucleus to activate transcription of target genes by interaction with the constitutively expressed transcription factor AtfA (Lara-Rojas et al., 2011). Interestingly, in A. terreus the interaction between AtfA and SakA has not been solved in detail. While a sakA deletion does not affect terrein production levels under nitrogen limitation, it allows terrein production in the presence of some nitrogen sources, indicating that it may possess a repressor function under some conditions. In addition, a ΔsakA strain still produces pigmented conidia (Figure 4—figure supplement 5) and the sakA mutant, but not the ΔatfA strain showed some increased osmosensitivity (Figure 4—figure supplement 5), which agrees to recent data on ΔatfA and ΔsakA mutants of Penicillium marneffei (Nimmanee et al., 2014). Therefore, it is well conceivable that terrein biosynthesis gene cluster activation via AtfA does not require SakA” and provided an additional supplemental figure (Figure 4—figure supplement 5).

We also analysed all promoters for putative AtfA binding sites and revised the Results section accordingly:

“Therefore, we additionally searched for putative palindromic AtfA/Sko1-binding sites (5′-TKACGTMA-3′) in the promoter regions of the cluster (Proft et al., 2005). Only one hit (TGACGTCA) was identified in the promoter of the structural gene terC. However, if one mismatch is allowed, there is a putative binding site at position −731 relative to the ATG start codon of terR (5′-TGGCGTCA-3′), but it remains speculative whether this binding site is recognised by A. terreus AtfA. Nevertheless, it should be mentioned that even a single half site of the suggested motif could promote transcription factor binding and promoter induction (Proft et al., 2005). Therefore, we conclude that, although direct evidence for AtfA binding at the terR promoter is lacking, both transcription factors seem to regulate terR expression.”

3) The authors comprehensively show that terrein cluster expression responds to Fe starvation and that this is mediated via HapX, a well-studied TF of Fe acquisition genes. HapX acts together with the CAAT binding complex and sites are found upstream of terR. More recently associated HapX binding sites have been found – are these present? They further show that terrein might contribute marginally to iron acquisition in the absence of siderophores and that it is capable of reducing ferric ions, an activity that might be relevant to competition with other organisms in the rhizosphere.

We re-analysed the terR promoter for putative HapX binding sites and added the following text to the Discussion:

“The control of terrein production by HapX depicts an unprecedented example for the activation of a secondary metabolite gene cluster outside the siderophore biosynthesis system. Interestingly, terrein production is only under positive HapX control, but not under negative SreA feedback-control, which is in agreement with a lack of SreA binding sites in the terR promoter (Schrettl et al., 2008), whereas a CCAAT box required for binding of the CCAAT-binding complex (CBC) and its subsequent CBC interaction with HapX (Hortschansky et al., 2007; Gsaller et al., 2014) is present. In the HapX target promoter regions of cycA, sreA, acoA and lysF from Aspergilli a bipartite CBC-HapX-DNA binding motif with consensus 5′-C(C/A)AATCAN_11-12GAT-3′ is present, of which CCAAT is recognised by the CBC complex, while the GAT sequence is bound by HapX (Hortschansky et al., 2015). Interestingly these are all types of promoters that are repressed under iron limitation. In contrast, the cccA promoter, which encodes for a vacuolar iron transporter, is the only known HapX induced promoter known so far with a confirmed in vitro HapX binding site (Gsaller et al., 2014). HapX binds to the GAT motif of a 5′-CCAATN₁₆GATC-3′ sequence, which is also present in the cccA promoter of A. terreus and – with one mismatch – in the terR promoter (position −215). However, further studies will be required to confirm this motif as a HapX binding site.”

4) A further signal is demonstrated – a requirement for glycolytic carbon sources – consistent with plant environments. Perhaps it should be made clear that the mechanism for this is not known.

To make this point a bit more clear, we added the following statement to the Results section:

“However, it should be mentioned that the presence of sugars was always required, since especially hexoses from mono- and disaccharides provoked strong expression under nitrogen limited conditions (Figure 3D), which is in direct contrast to the production of dihydroisoflavipucine in A. terreus that belongs to the class of fruit and root rot toxins. This metabolite is only produced in the strict absence of sugars and requires preferred nitrogen sources such as glutamine or asparagine for induction (Gressler et al., 2011).”