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)