---

Nachwuchsgruppe "Pilzlicher Sekundärmetabolismus und Pathogenität"

Dr. Ralf Horbach

Fungal Secondary Metabolites as Virulence Determinants and Lead Structures in Drug Research

Plant pathogenic fungi cause severe damage on a broad range of crop and ornamental plants, leading to significant yield and quality reduction. A steadily growing demand for food and biofuels requires both increased production of agricultural goods and efforts to reduce losses. Improved pest management strategies may substantially contribute to an increased efficiency in agriculture, however, they require detailed knowledge of the infection biology of phyto-pathogens. The project „Functional analysis of secondary metabolites produced by the maize anthracnose pathogen Colletotrichum graminicola” aims at the understanding of the role of secreted fungal metabolites during the infection process.

Colletotrichum graminicola, the causative of maize anthracnose, produces several secondary metabolites some of which have been previously isolated. Structure elucidation and activity assays revealed hitherto unknown polyketides with antimicrobial and cytotoxic properties. In our present work, we want to study the importance of these compounds for a successful plant infection. The following questions are addressed in our current project: What kind of fungal polyketides can be found in infected host cells particularly during the transition from the biotrophic to the destructive necrotrophic phase? What are the corresponding polyketide synthases (PKSs) and how does the deletion of these genes influence virulence or the ability to compete with other microorganisms. Are there significant differences in the transcription profiles of PKS genes at different time points of the maize infection? Is there an epigenetic component in the control of PKSs expression?

Close examination of the C. graminicola genome revealed the presence of 32 PKS genes and 8 PKS-nonribosomal peptide synthase (NRPS) hybrids. Thus far, targeted gene deletion was confirmed for 8 PKSs and 3 accessory genes belonging to a PKS gene cluster. Corresponding metabolites could be assigned to 3 PKS. Detached leave assays showed that none of the mutants lost the ability to infect host plants completely, however, infection assays with cultivars varying in their susceptibility will be conducted in order to determine putative virulence defects.

In the course of our investigations we identified two adjacent PKSs in the genome of C. graminicola that collectively synthesize the resorcinol lactone monorden as proven by targeted gene deletion. The monorden synthesis cluster contains enzymes, which function in modification and transport of the PKS product, i. e. genes encoding a halogenase, a cyto-chrome P450 reductase to form the epoxide ring and a MFS-transporter which seems to be responsible for carrying monorden across the fungal cell membran. Monorden is known as an effective inhibitor of heat shock protein 90 (hsp90) which, in turn, is a chaperone for plant resistance proteins. This particular function raises questions about the in planta production of monorden and the ability of C. graminicola to inhibit maize hsp90, thereby suppressing plant defense to facilitate host infection. Mutants defective in monorden synthesis were tested in confrontation assays using a panel of fungal maize pathogens which may act as antagonists. Co-cultivation on two different media known to induce the synthesis of monorden did not reveal differences in growth as compared to the wildtype.

The growing pool of mutants with single PKS deletions will be analyzed by means of meta-bolic fingerprinting in cooperation with Prof. Petr Karlovsky (Georg-August-University, Göttingen). Additional compounds produced by C. graminicola in different media or under varying culture conditions will be isolated, and their effects on plant tissue tested. Stage-specific identification of known fungal metabolites during the infection course will be ana-lyzed using Laser Ablation Electrospray-Ionization (LAESI) coupled to mass spectrometry in cooperation with Prof. Akos Vertes (GWU, Washington).

In a previous study, the sfp-phosphopantetheinyl transferase CgPPT1 has been identified as a central activator of all PKS, NRPS and the α-aminoadipate reductase (AAR) required for lysine biosynthesis. Since CgPPT1 is indispensable for fungal pathogenicity the enzyme may represent an excellent target for novel fungicides. In order to enable the screening of com-pound libraries for specific PPTase inhibitors we developed a robust and inexpensive micro-plate assay in cooperation with Prof. L. Wessjohann (IPB, Halle). The assay principle is based on the transfer of a fluorophore labeled phosphopantetheine residue that is covalently attached to a conserved serine of the 100 amino acid acyl carrier protein domain of CgPKS1.

In a second applied project we screen a proprietary fungal strain collection in search for novel secondary metabolites with antimicrobial properties. Among the culture fluid extracts of some 300 fungal strains collected so far approximately ten showed significant antifungal or anti-bacterial effects in agar diffusion assays. Medium-scale fermentation of an Emericellopsis strain designated 2010-119 produced amounts of metabolites sufficient for the isolation of pure compounds and NMR structure elucidation. Bioactive substances were found to be helvolic acid, the peptaiboles heptaibin and emerimicin IV and a yet unknown substance. Inhibitory effects of new compounds will be evaluated using a panel of bacterial and fungal test strains.

Zum Seitenanfang