The cross-pathway control system regulates production of the secondary metabolite toxin, sirodesmin PL, in the ascomycete, Leptosphaeria maculans

BMC Microbiology, Jul 2011

Background Sirodesmin PL is a secondary metabolite toxin made by the ascomycetous plant pathogen, Leptosphaeria maculans. The sirodesmin biosynthetic genes are clustered in the genome. The key genes are a non-ribosomal peptide synthetase, sirP, and a pathway-specific transcription factor, sirZ. Little is known about regulation of sirodesmin production. Results Genes involved in regulation of sirodesmin PL in L. maculans have been identified. Two hundred random insertional T-DNA mutants were screened with an antibacterial assay for ones producing low levels of sirodesmin PL. Three such mutants were isolated and each transcribed sirZ at very low levels. One of the affected genes had high sequence similarity to Aspergillus fumigatus cpcA, which regulates the cross-pathway control system in response to amino acid availability. This gene was silenced in L. maculans and the resultant mutant characterised. When amino acid starvation was artificially-induced by addition of 3-aminotriazole for 5 h, transcript levels of sirP and sirZ did not change in the wild type. In contrast, levels of sirP and sirZ transcripts increased in the silenced cpcA mutant. After prolonged amino acid starvation the silenced cpcA mutant produced much higher amounts of sirodesmin PL than the wild type. Conclusions Production of sirodesmin PL in L. maculans is regulated by the cross pathway control gene, cpcA, either directly or indirectly via the pathway-specific transcription factor, sirZ.

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The cross-pathway control system regulates production of the secondary metabolite toxin, sirodesmin PL, in the ascomycete, Leptosphaeria maculans

Candace E Elliott 1 Ellen M Fox 0 1 Renee S Jarvis 1 Barbara J Howlett 1 0 Department of Sustainability and Environment , Gippsland Regional Office, (71 Hotham Street), Traralgon, Victoria (3844) , Australia 1 School of Botany, the University of Melbourne , Victoria, (3010) , Australia - The cross-pathway control system regulates production of the secondary metabolite toxin, sirodesmin PL, in the ascomycete, Leptosphaeria maculans Elliott et al. Open Access The cross-pathway control system regulates production of the secondary metabolite toxin, sirodesmin PL, in the ascomycete, Leptosphaeria maculans Background: Sirodesmin PL is a secondary metabolite toxin made by the ascomycetous plant pathogen, Leptosphaeria maculans. The sirodesmin biosynthetic genes are clustered in the genome. The key genes are a nonribosomal peptide synthetase, sirP, and a pathway-specific transcription factor, sirZ. Little is known about regulation of sirodesmin production. Results: Genes involved in regulation of sirodesmin PL in L. maculans have been identified. Two hundred random insertional T-DNA mutants were screened with an antibacterial assay for ones producing low levels of sirodesmin PL. Three such mutants were isolated and each transcribed sirZ at very low levels. One of the affected genes had high sequence similarity to Aspergillus fumigatus cpcA, which regulates the cross-pathway control system in response to amino acid availability. This gene was silenced in L. maculans and the resultant mutant characterised. When amino acid starvation was artificially-induced by addition of 3-aminotriazole for 5 h, transcript levels of sirP and sirZ did not change in the wild type. In contrast, levels of sirP and sirZ transcripts increased in the silenced cpcA mutant. After prolonged amino acid starvation the silenced cpcA mutant produced much higher amounts of sirodesmin PL than the wild type. Conclusions: Production of sirodesmin PL in L. maculans is regulated by the cross pathway control gene, cpcA, either directly or indirectly via the pathway-specific transcription factor, sirZ. Background Sirodesmin PL is the major phytotoxin produced by the plant pathogen Leptosphaeria maculans (Desm.), the causal agent of blackleg disease of Brassica napus (canola). Sirodesmin PL has antibacterial and antiviral properties [1] and is essential for full virulence of L. maculans on stems of B. napus [2]. This toxin is an epipolythiodioxopiperazine (ETP), a class of secondary metabolites characterised by the presence of a highly reactive disulphide-bridged dioxopiperazine ring synthesised from two amino acids (for review see [3]). The first committed step in the sirodesmin biosynthetic pathway is prenylation of tyrosine [4,5]. As for other fungal secondary metabolites, the genes for the biosynthesis of sirodesmin PL are clustered. The sirodesmin cluster contains 18 genes that are co-ordinately regulated with timing consistent with sirodesmin PL production. Disruption of one of these genes, sirP, which encodes a peptide synthetase, results in an isolate unable to produce sirodesmin PL [6]. Based on comparative genomics, the cluster of genes in Aspergillus fumigatus responsible for the biosynthesis of another ETP, gliotoxin, was then predicted. The pattern of expression of the clustered homologs was consistent with gliotoxin production [7]. The identity of this gene cluster was confirmed via the disruption of peptide synthetase, gliP whereby the resultant mutant was unable to make gliotoxin [8,9]. These ETP gene clusters also encode a Zn(II)2Cys6 transcription factor, namely SirZ for sirodesmin, and GliZ for gliotoxin [7]. Such factors are often found in biosynthetic gene clusters for secondary metabolites and they regulate transcription of the biosynthetic genes and consequently metabolite production. Disruption of A. fumigatus gliZ resulted in a mutant isolate unable to produce gliotoxin [10]. RNAi-mediated silencing of sirZ in L. maculans revealed that sirZ is essential for the transcription of sirodesmin biosynthetic genes and consequently production of sirodesmin PL [11]. In this paper we describe the identification of three genes that regulate sirodesmin PL and are unlinked to the sirodesmin gene cluster. One of these genes is denoted as cpcA (cross pathway control A), and is involved in regulation of amino acid biosynthesis in fungi such as Saccharomyces cerevisiae, Aspergillus nidulans, and A. fumigatus [12-14]. This pathway acts as a metabolic switch to enable the fungus to synthesize amino acids during periods of amino acid limitation. In this paper we describe the effect of starvation on the expression of sirodesmin biosynthetic genes and sirodesmin PL production in L. maculans wild type and cpcA-silenced isolates. Results Identification of genes flanked by T-DNA insertions in sirodesmin-deficient mutants of L. maculans To generate sirodesmin-deficient mutants of L. maculans, wild type isolate IBCN 18 was transformed with plasmid (...truncated)


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Candace E Elliott, Ellen M Fox, Renee S Jarvis, Barbara J Howlett. The cross-pathway control system regulates production of the secondary metabolite toxin, sirodesmin PL, in the ascomycete, Leptosphaeria maculans, BMC Microbiology, 2011, pp. 169, 11, DOI: 10.1186/1471-2180-11-169