Fig 1.
Organization of the different ETP biosynthesis gene clusters and structure of gliotoxin and sirodesmin.
(A) Shown is the ETP gene cluster in C. purpurea in comparison to the gliotoxin and sirodesmin producing gene clusters from A. fumigatus and L. maculans. Orientation of the arrows indicates the direction of transcription. Genes in black are common ETP moiety genes present in all three clusters. For gene designations see Table 1. (B) Structure of gliotoxin and sirodesmin PL with the bolded characteristic diketopiperazine moiety with an internal disulfide bridge.
Table 1.
Predicted functions and homologs of the ETP gene cluster in C. purpurea.
Fig 2.
Gene expression and co-regulation of the ETP cluster genes.
(A) In planta gene expression of tcpG. Shown are three independent experiments in the C. purpurea wild-type strain in planta 10, 15 and 20 days post-infection (dpi) and in axenic culture. Expression levels were normalized against the housekeeping genes encoding β-tubulin, γ-actin, and glyceraldehyde-3-phosphate dehydrogenase. (B) Co-regulation of the ETP cluster genes. The wild-type as well as three independent OE::tcpZ mutants were grown for seven days in liquid Mantle media and northern blot analysis was performed as described in methods.
Fig 3.
Comparison of the HPLC-MS metabolite profile (total ion chromatograms) between the wild type of Claviceps purpurea strain 20.1 (A), OE::tcpZ (B) and OE::tcpZ/OE::tcpN (C). (D) Extracted ion signals of compounds 1a/b, 2a/b and 3 of OE::tcpZ.
Fig 4.
Newly identified secondary metabolites as products of an ETP gene cluster in Claviceps purpurea.
(A) Compounds 1–3 were produced by OE::tcpZ strain in planta and in axenic culture. (B) Selected 2D- NMR data for 2. Red arrows indicate the specific couplings of H-7a/b and the blue arrows those of S-CH3. (C) HRMSn fragmentation of 1a as [M+H]+ illustrating the rapid cleavage of the thiomethyl group and dimethylallyl group.
Fig 5.
Identified structures of the OE::tcpZ/OE::tcpN strain.
(A) 4-8 occur in axenic culture. (B) HRMSn analysis of 4a as [M+H]+ clarifies the characteristic fragmentation and underlines the differences concerning the N-4 substitution. This characteristic fragmentation allows structure elucidation of other unknown intermediates based on HRMSn.
Fig 6.
Phylogenetic analysis of TcpC and its homologs from other fungal ETP clusters.
Amino acid sequences were obtained from GenBank database (GliC EDP49542.1, GliF AAW03300.1, SirE AAS92549.1, SirB XP_003842422.1, SirC AAS92547.1, ataF XM_001212649.1 ataTC XM_001212652.1). Sequences were aligned with MUSCLE (v3.8.31) and the phylogenetic tree was constructed with the maximum likelihood method using Phylogeny.fr [83]. Branch support values are indicated.
Fig 7.
The characteristic MS/MS spectra of the identified clapurines as end-products of the ETP gene cluster in C. purpurea.
(A) The clapurines are so far unknown ETPs and were characterized in the OE::tcpZ/OE::sirC strain. The HRMSn experiments show a neutral loss of the sulfur groups and the common cleavage of the dimethylallyl group for dithioclapurine (B), trithioclapurine (C) and tetrathioclapurine (D) as [M+H]+.
Fig 8.
Proposed biochemical pathway for the formation of diketopiperazine metabolites 1-8 and the clapurines.
Compounds 12–15 are hypothesized intermediates.
Fig 9.
Tentative biochemical pathway for the diastereomers 1a/b and 2a/b.
Compounds 16 and 18 are hypothesized intermediates.