Production of fungal secondary metabolites is often regulated by pathway-specific transcription factors, acting through global transcription factors that control several physiological processes and respond to environmental cues such as pH, temperature, and nutrition . Given this complexity of regulation, it is not surprising that 1.5% of T-DNA insertional mutants of L. maculans analysed were sirodesmin-deficient. The finding that sirodesmin-deficiency correlated with severely reduced transcript levels of the pathway-specific transcription factor, sirZ, is consistent with studies on the regulation of production of other secondary metabolites. For instance, LaeA a master regulator of secondary metabolism in fungi such as Aspergillus spp. , regulates gliotoxin in A. fumigatus via the pathway-specific transcription factor, gliZ .
Cross pathway control homologs have a complex pattern of regulation. All identified to date are transcriptionally regulated in varying degrees; levels of transcripts increase significantly during amino acid starvation (for example, S. cerevisiae Gcn4p [12, 21]. N. crassa cpc1 , A. nidulans cpcA , A. fumigatus cpcA  and F. fujikuroi cpc1 ). A CPRE element with one different nucleotide to that of the canonical CPRE sequence (5'-TGACTgA-3') is also present in the promoter of sirZ (-610 to -616), which suggests that CpcA may regulate sirZ directly. This element is not present in the promoter region of other genes in the sirodesmin gene cluster. Unfortunately due to the recalcitrance of L. maculans to homologous gene disruption we were unable to mutate the putative CPRE in the promoter of sirZ and test for regulation of sirodesmin PL production via CpcA.
The best studied cross pathway control homolog is S. cerevisiae GCN4. Starvation for any of at least 11 of the proteinogenic amino acids results in elevated transcript levels of targets of Gcn4p. Such targets include enzymes in every amino acid biosynthetic pathway, except that of cysteine, and also in genes encoding vitamin biosynthetic enzymes, peroxisomal proteins, mitochondrial carrier proteins, and autophagy proteins [12, 21]. A comparative study of genes regulated by S. cerevisiae Gcn4p, Candida albicans CaGcn4p and N. crassa Cpc1 revealed regulation of at least 32 orthologous genes conserved amongst all three fungi . These genes mainly comprised amino acid biosynthetic genes including the tryptophan biosynthetic gene trpC [13, 14, 22, 25]. However, aroC, which encodes chorismate mutase, the enzyme at the first branch point of aromatic amino acid biosynthesis, is unresponsive to the cpc-system [14, 18]. As expected, CpcA regulated transcription of trpC in L. maculans but not of aroC in response to amino acid starvation.
The cross pathway control system is also regulated at the translational level, since mutation of upstream uORFs in A. nidulans or S. cerevisiae results in increased translation of cpcA and GCN4 proteins under non-starvation conditions, compared to the wild type strains [13, 26]. In L. maculans the cpcA coding region is preceded by two upstream Open Reading Frames (ORFs), the larger one displaying sequence similarity to an uORF preceding the coding region of cpcA of A. fumigatus and A. nidulans. Thus it is likely that L. maculans cpcA is regulated translationally, as well as transcriptionally.
It is puzzling why the insertion of T-DNA into the 3' UTR of cpcA in mutant GTA7 reduces production of sirodesmin PL but does not appreciably affect levels of cpcA transcript. One explanation is that the T-DNA insertion affects the regulation or increases the stability of the cpcA transcript, resulting in a cross pathway control system that is active in complete media and thus diverts amino acids from sirodesmin production. The importance of the 3' UTR in the regulation of genes is well-documented. For instance, regulatory elements in the 3' UTR control transcript stability of the global nitrogen regulator AreA in A. nidulans . Deletions in 3' UTR of this gene render the transcript insensitive to nitrogen availability. Similarly, the deletion of part of the 3' UTR of cpcA could render the L. maculans isolate insensitive to amino acid levels in the media.
Given that sirodesmin PL is derived from two amino acids, tyrosine and serine, the finding that the transcription of sirodesmin biosynthetic genes, sirP and sirZ, and sirodesmin PL production appears to be regulated by cpcA and by amino acid starvation is not unexpected. It should be noted, however, that integration site effects may have contributed to these phenotypes since the site of insertion of the cpcA-silencing vector in the genome was not determined. It is unclear why the addition of 5 mM 3AT did not have as marked an effect as extreme starvation (absence of carbon and nitrogen) did on the levels of sirodesmin PL in either the wild type or cpcA-silenced isolate, when there was a marked effect on transcript levels of sirP and sirZ with addition of 3AT. This may be due to the significant difference in time periods during which the cultures were treated with 3AT; transcript levels were determined after 5 h, whilst sirodesmin PL levels were measured after eight days, after which time 3AT may have been depleted or degraded. In previous studies using 3AT to induce starvation, the effects on gene transcription were measured after 2 to 8 h [14, 23, 28]. Thus the imidazole glycerol phosphate dehydratase might have been inhibited for only a short period in the L. maculans cultures that were treated for eight days with 3AT. In the wild type culture grown in the absence of carbon and nitrogen, cross pathway control would be active during the entire eight days resulting in reduced levels of sirodesmin PL. In contrast, in the cpcA-silenced isolate grown in the absence of carbon and nitrogen, there is probably insufficient cpcA transcript to downregulate production of sirodesmin PL thereby resulting in an increased level of sirodesmin PL.
Until this report such a link between CpcA and secondary metabolism had only been implicated in two filamentous fungi. In A. nidulans, biosynthesis of penicillin is regulated by CpcA . Penicillin and lysine share a common intermediate, the non-proteinogenic amino acid, α-aminoadipate. Under amino acid starvation conditions, CpcA directs metabolic flux towards lysine biosynthesis instead of penicillin biosynthesis, whilst in nutrient-rich conditions, penicillin is produced. In F. fujikoroi, cpc1 has been implicated in control of production of diterpenoid gibberellins, as deletion of glutamine synthetase leads to down regulation of gibberellin biosynthetic genes and upregulation of cpc1 . However, recent experiments have shown that Cpc1 is not responsible for down-regulation of gibberellin biosynthesis .
Since cpcA regulates sirodesmin PL production, its homolog in A. fumigatus may regulate production of the related molecule, gliotoxin. An A. fumigatus cpcA mutant was attenuated for virulence in pulmonary aspergillosis of neutropenic mice, which had been immunosuppressed with cyclophosphamide and corticosteroids . However, the effect on gliotoxin production was not tested. Several research groups have shown that gliotoxin is not a virulence factor in such neutropenic mice, but is a virulence factor in mice that have retained neutrophil function after immunosuppression by corticosteroids alone (for review see ). In a study of infection of immature dendritic cells by A. fumigatus, gliotoxin biosynthesis genes were downregulated over time. However, this could not be attributed to cross pathway control because cpcA was not differentially expressed .
The following model for regulation of sirodesmin PL production is consistent with all these data. When wild type L. maculans is grown on complete medium, the cross pathway control system is inactive, and amino acid biosynthesis does not occur (or occurs at a low level), but sirodesmin PL is produced. In contrast during starvation, amino acids are diverted from sirodesmin biosynthesis towards amino acid biosynthesis. This effect is mediated either directly or indirectly through the sirodesmin pathway-specific transcription factor, sirZ. Other transcription factors including LaeA and dsp3 may also regulate sirodesmin PL production either directly or indirectly through sirZ as is the case for LaeA with gliZ and gliotoxin .