Molecular genetics of Ergot-Alkaloidbiosynthesis in Claviceps purpurea
(DFG Tu 50/13; SPP 1152 "Evolution of metabolic diversity"; Link:   http://www.ipb-halle.de/dfg-spp-1152/homepage/index.htm
Ergot alkaloids are toxic ergoline derivatives, which are synthesized by several members of the ascomycetes family Clavicipitaceae, especially by Claviceps spec. The pharmacological relevant peptide alkaloids (ergopeptides) are produced e.g. by Claviceps purpurea, whereas other species synthesize only lysergic acid (LA) and simple LA derivatives (C. paspali) or only precursors of LA, ergoline alkaloids (C. fusiformis). We have recently discribed a cluster of genes involved in peptide alkaloid biosynthesis in C. purpurea; we could identify the genes coding for the non-ribosomal-peptide synthetases (NRPS) involved in the peptide moiety in an ergotamine producing strain. In this project the analysis of cluster genes will be continued . We will focus on functional analysis of NRPS genes, in comparison between C. purpurea strains with different alkaloid spectra, to investigate the basis for diversity of peptide alkaloids within the species C. purpurea ("chemical races"). The next step -based on these informations - will be the analysis of alkaloid genes (presence and function) in different Claviceps species (C. paspali, C. fusiformis) to study the evolution of the biosynthetic pathways of ergot alkaloids within the genus Claviceps.

Early stages of pathogenicity in plant pathogenic fungi
(Trilateral DFG program: cooperation with Tel Aviv and Hebron University)
Plant pathogenic fungi devised various strategies to infect plants. Regardless of the pathogenic life style, the initial stages of infection are common to most fungi: spores germinate on the host surface, undergo a short period of polarized growth and then invade the plant tissue. These early events are crucial for the development of disease and hence are strictly regulated at several levels. We study these early processes in two fungi with different pathogenic life styles: the hemibiotroph Colletotrichum gloeosporioides and the necrotroph Botrytis cinerea. We generated and characterized mutants with defects in various stages of early pathogenic development and use such mutants to determine the role of signal pathways in regulation of early pathogenic development. The results will provide better understanding of these complex interaction systems, and could constitute a basis for the development of alternative disease control systems (definition of targets). We use an integrated approach combining classical (physiology, cytology) and molecular techniques (including array and imaging technology). The 3 partner labs contribute to this research specific expertise and work in close collaboration, including intensive transfer of knowledge and data between the partners, leading to a substantial broadening of the expertise of all partners with special focus on the Hebron lab.

Role of reactive oxygen species (ROS) in the interaction of phytopathogenic fungi and their hosts (DFG project)
ROS play a major role in defense reactions of plants and animals against pathogens. Phytopathogenic fungi face considerable oxidative stress by the so-called "oxidative burst", an early plant defense reaction. We could show that phytopathogenic fungi not only possess effective ROS-detoxification systems, but that they can produce ROS themselves and thus contribute to the ROS status during the interaction. We study two interaction systems showing highly different strategies: Claviceps purpurea, a biotrophic pathogen of rye, obviously has adapted to his host at a high degree, since formation of plant-derived and self-produced ROS is minimized. The necrotrophic grey mould Botrytis cinerea , on the other hand, stimulates the "oxidative burst" and contributes significant amounts of ROS, causing death of the infected tissue. We are analysing in parallel ROS production and detoxification in both systems, focusing on signal chains and ROS-generating systems. We want to test the hypothesis that both fungi use the same system compounds to achieve these different strategies, and we want to prove that the differences in strategy can be recognized mainly at the level of ROS generating and detoxifying systems.

Molecular Interactions of Pathogens with Biotic and Abiotic Surfaces (International Graduate School, GRK 1409; http://zmbe.uni-muenster.de/GRK1409/
Perception and transduction of signals is essential for early infection stages of phytopathogenic fungi. Genes expressed in the early stages (germination of spores on the plant surface, penetration of cuticle, start of colonization) will be identified e.g. by array analyses, using defined mutants of essential signalling pathways. Candidate genes will be functionally analysed by gene replacement and reporter gene analyses. The studies will be performed in close context with the major projects of the group, within one of the two major systems: Claviceps purpurea, the Ergot fungus, on rye, or Botrytis cinerea, the grey mould, on bean. Beyond the whole set of classical molecular genetic techniques, the project will include fungal transformation (gene replacement, reporter gene arrays), protein techniques (western, phosphorylation analyses), array technology and bioinformatics (for B. cinerea a genome sequence is available).

Mey G, Held K, Scheffer J, Tenberge KB, Tudzynski P (2002) CPMK2, an Slt2-homologous MAP-kinase is essential for pathogenesis of Claviceps purpurea on rye: evidence for a second conserved pathogenesis-related MAP-kinase cascade in phytopathogenic fungi. Molec Microbiol 46: 305-318.90.

Correia T, Grammel N, Ortel I, Keller, U, Tudzynski P (2003) Molecular cloning and analysis of the ergopeptine assembly system in the ergot fungus Claviceps purpurea. Chemistry & Biology 10: 1281-1292.

Nathues E, Joshi S, Tenberge KB, von den Driesch M, Oeser B, Bäumer N, Mihlan M, Tudzynski P (2004) CPTF1, a CREB-like transciption factor is involved in the oxidative stress response in the phytopathogen Claviceps purpurea and modulates ROS level in its host Secale cereale. Mol Plant Microbe-Interact 17: 383-393.

Elad Y, Williamson, B, Tudzynski P, Delen N (eds.). Botrytis: biology, pathology and control. Kluwer Academic Press, (2004).

Tudzynski P, Scheffer J (2004) Claviceps purpurea: Molecular aspects of a unique pathogenic lifestyle. Molec. Plant Pathol. 5, 377-388.

Scheffer J, Ziv C, Yarden O, Tudzynski P (2005) The COT homologue CPCOT1 regulates polar growth and branching and is essential for pathogenicity in Claviceps purpurea. Fungal Genetics Biol. 42, 107-118.

Siewers V, Viaud M, Jimez-Teja D, Collado IG, Schulze Gronover C, Pradier J-M, Tudzynski B, Tudzynski P (2005) Functional analysis of the cytochrome P450 monooxygenase gene bcbot1 of Botrytis cinerea: role in botrydial biosynthesis and impact on virulence in different wild strains. Mol Plant Microbe-Interact (in press)

Scheffer J, Chen C, Heidrich P, Dickman MB, Tudzynski P (2005) A CDC42 homologue in C. purpurea is involved in vegetative differentiation and is essential for pathogenicity. Eukary. Cell 4: 1228-1238.

Haarmann T, Ortel I, Tudzynski P, Keller U (2006) the cytochrome P450 monooxygenase CpP450-1 is the bridge between clavine alkaloid and D-lysergic acid amid formation in the ergot alkaloid pathway in Claviceps purpurea. Chem. Biochem. 7: 645-652.

Siewers V, Kokkelink L, Smedsgaard J, Tudzynski P (2006) Identification of an abscisic acid gene cluster in the grey mould botrytis cinerea. Appl. Env. Microbiol. 72: 4619-4626.

Segmüller N, Ellendorf U, Tudzynski B, Tudzynski P (2007) BcSAK1, a stress-activated MAPkinase is involved in vegetative differentiation and pathogenicity in Botrytis cinerea. Eukaryotic Cell, 6: 211-221.

Nathues E, Jörgens C, Lorenz N, Tudzynski P (2007) The fungal histidin kinase CpHK2 has impact on spore germination, oxidative stress and fungicide resistance, and virulence of the ergot fungus Claviceps purpurea. Mol Plant Pathol 8: 653-665.

Lorenz N, Wilson EV, Machado C, Schardl C, Tudzynski P (2007) Comparison of ergot alkaloid biosynthesis gene clusters in Claviceps species indicate loss of late pathway steps in evolution of C. fusiformis. Appl Env Microbiol 73: 7185-7191.