Handbook Part 2 - International Mycological Association

S38PS2 - 0340
Peroxisomal acetyl-CoA is essential for appressorial melanization, and virulence in Magnaporthe
M Ramos-Pamplona, NI Naqvi
Temasek Life Sciences Laboratory, Singapore, Singapore
The long-chain fatty acids undergo beta-oxidation primarily in the peroxisomes and the resultant acetyl-CoA
molecules (and the chain-shortened fatty acids) are exported via the cytosol to the mitochondria for further
breakdown and usage. In a forward genetics approach, we identified a loss-of-function mutation in the Magnaporthe
grisea PEROXIN6 locus. Disruption of PEX6 function led to total nonpathogenicity. Further characterization revealed
that Mgpex6-delete strain lacks functional peroxisomes and is incapable of beta-oxidation of long-chain fatty acids,
and that the peroxisomal acetyl-CoA is essential for the host invasion step of the rice-blast disease. The Mgpex6delete
lacked appressorial melanin and could not elaborate penetration hyphae, and was thus rendered nonpathogenic.
Interestingly, the vegetative hyphae and conidia showed normal pigmentation. A peroxisome-associated carnitine
acetyltransferase (CrAT1) activity was identified as being essential for the appressorial function in Magnaporthe.
CrAT1-minus appressoria showed reduced melanization, but were surprisingly incapable of elaborating penetration
pegs. Exogenous addition of excess glucose during infection stage caused partial remediation of the pathogenicity
defects in the CrAT1delete strain. Moreover, Mgpex6delete and CrAT1delete mycelia showed weakened cell wall
biosynthesis in a glucose-deficient environment leading to appressorial dysfunction in these mutants. Thus, our
characterization of a peroxisome biogenesis mutant and an acetyl-CoA transport mutant suggests that peroxisomal
beta-oxidation contributes metabolites for melanin and cell wall synthesis during appressorium-mediated host
penetration.
These results will be discussed along with our recent data that suggests a possible involvement of Tyrosinases in the
pigmentation of vegetative hyphae in Magnaporthe.
1145-1345
SYMPOSIUM 39 - Biosynthetic Gene Clusters for Fungal Secondary Metabolites
S39IS1 - 0725
The sirodesmin biosynthetic gene cluster of the plant pathogen, Leptosphaeria maculans
BJ Howlett 1, CE Elliott1, EM Fox 1, DM Gardiner 2, AJ Cozijnsen1
1the University of Melbourne, Parkville, Victoria, Australia, 2 CSIRO Plant Industry, St Lucia Queensland, Australia
Genes responsible for the biosynthesis of secondary metabolites are typically clustered in filamentous fungi. We have
cloned a cluster of 18 genes involved in the biosynthesis of an epipolythiodioxopiperazine (ETP) toxin, sirodesmin, from
Leptosphaeria maculans, which causes blackleg disease of canola. We are analyzing regulation of sirodesmin
biosynthesis and its role in disease. Silencing of a Zinc binuclear cluster (Zn2Cys6) gene in the cluster leads to loss of
sirodesmin production and decreased transcription of the biosynthetic enzymes. Screening of random insertional
mutants for loss of sirodesmin production has led to identification of genes outside the cluster that regulate sirodesmin
production. One of these controls biosynthesis of amino acids, which are precursors of sirodesmin.
We are using sirodesmin-deficient mutants to determine the role of sirodesmin in blackleg disease. A mutant in a
peptide synthetase, a key enzyme within the cluster, like the mutants in transcriptional regulators described above,
does not produce sirodesmin. This mutant makes similar sized lesions on cotyledons to those made by the wild type
isolate. However, it colonises stem tissue less effectively than the wild type. A promoter fusion of peptide synthetase
with Green Fluorescent Protein has been used to track sirodesmin biosynthesis during growth in planta. Transcription
of this gene is first detected in hyphae ten days after inoculation of cotyledons. At later stages the gene is transcribed
at high levels in pycnidia and during growth in the stem. These findings implicate sirodesmin as a virulence
determinant in the late stages of infection of canola when stem cankering occurs.
S39IS2 - 0240
Terrequinone biosynthesis in Aspergillus nidulans
Dirk Hoffmeister
Albert-Ludwigs-University, Freiburg, Germany
The asterriquinones are a prominent class of fungal secondary metabolites. As they exhibit valuable pharmacological
activities, such as antidiabetic or antiviral properties, they have potential in drug lead development. LaeA, a global
transcription regulator for secondary metabolism in Aspergilli, was employed for microarray-based screening of the
Aspergillus nidulans genome to identify actively transcribed natural product genes. Follow-up investigations included
gene inactivation, and analytical methods (HPLC, LC/MS, 1D and 2D NMR techniques).
Among the genetic loci found during our screen the genes responsible for biosynthesis of terrequinone A, a member
of the asterriquinone class of compounds, were identified, and confirmed by gene inactivations. This is the first report
for an asterriquinone gene cluster. Based on these results a generic biosynthetic blueprint for fungal quinoid natural
products has emerged. As A. nidulans was not known before to produce asterriquinones, we have demonstrated that
LaeA represents a powerful mining tool even if the compound is unknown from a given species or if
chemical/structural information is unavailable.
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