VAAM-Jahrestagung 2011 Karlsruhe, 3.–6. April 2011

the autophagosome and its cargo. Localization experiments of SmATG4with a C-terminal GFP fusion showed a cytoplasmatic localization ofSmATG4.FBV017Characterization of a putative α-carbonic anhydrasefrom the filamentous ascomycete Sordaria macrosporaR. Lehneck*, S. PöggelerInstitute for Microbiology and Genetics, Department for Genetics ofEukaryotic Microorganisms, Georg-August-University, Göttingen, GermanyCarbonic Anhydrase (CA) catalyzes the hydration of carbon dioxide tobicarbonate and protons. CAs have evolved in all three domains of life.Based on their amino acid sequence and structure, they can be divided intofive distinct groups (α,β,γ,δ,ξ) which share no sequence similarity and havesupposable evolved independently. All known fungal CAs belong either tothe α- or to the β-class and to date, only β-CAs have been characterized infungi (Elleuche and Pöggeler 2009). Therefore, we investigated in thisstudy, the role of a α-CA in the filamentous ascomycete Sordariamacrospora, termed CAS4. The S. macrospora cas4 gene encodes a putativeprotein of 368 amino acids with a predicted molecular mass of 39.6 kDa.cDNA of the S. macrospora cas4 gene fused to either RGS-His-tag or GSTtagwas heterologously expressed in E. coli. SDS-PAGE and Western-blotanalyses with anti-RGS-His and anti-GST antibodies revealed protein bandswith apparent molecular weights consistent with the calculated molecularweights of the S. macrospora CAS4 protein. CAS4 exhibit a signal peptidefor the endoplasmic reticulum and is therefore predicted to be secreted.Using Western-Blot analysis we were able to show secretion of a Flagtagged-CAS4.To better understand the role of the S. macrospora CAS4, wegenerated a ∆cas4 deletion mutant. The characterization of the ∆cas4 mutantrevealed a reduced vegetative growth rate compared to the wild type. Understress conditions the mutant also shows a slight delay on fruiting bodyproduction. In addition, the expression pattern of cas4 was analyzed bysemi-quantitative RT-PCR.FBV018Characterization, purification and cloning of the O-Methyltransferase of Alternaria alternataA. Neumann* 1 , K. Brzonkalik 1 , C. Syldatk 1Technical Biology, Karlsruhe Institute of Technology (KIT), Karlsruhe,GermanyBlack-moulds of the genus Alternaria contaminate many foodstuffs andagricultural products. In addition to the economical damage these fungi canproduce harmful secondary metabolites, the Alternaria toxins. Some of thesemycotoxins such as alternariol (AOH), alternariolmonomethylether (AME),altenuene (ALT) are polyketides and AOH is produced via the polyketidepathway. AOH is than methylated by the alternariol-o-methyltransferase,transferring a methyl group from SAM to AOH to yield AME. The enzymewas partially purified and characterized, but the sequence is still unknown[1, 2].As Alternaria alternata is not sequenced yet, putative methyltransferaseswere identified by BLAST-analysis in the genome of the close relative A.brassicicola and the sequences were used to clone several SAM dependentmethyltransferases of Alternaria alternata. Three partial and one totalsequence were cloned.With the active expression of the identified genes being not easy, thealternariol-o-methyltransferase of Alternaria alternata was alsocharacterized in crude extracts and partially purified. An SAM dependentactivity-test was developed to identify the enzyme. The products wereanalysed by HPLC. With the N-terminal sequence of the enzyme it shouldbe possible to determine the gene.[1] Gatenbeck and Hermodsson (1965): Enzymic Sythesis of Aromatic Product Alternariol.[2] Stinson and Moreau (1986): Partial Purification and some Properties of an Alternariol-o-Methyltransferase from Alternaria tenuis.FBV019Approaches for directed strain improvement targetingenhanced biosynthesis of gibberellic acid in FusariumfujikuroiS. Albermann*, B. TudzynskiInstitute of Plant Biology and Biotechnology, Molecular Biology andBiotechnology of Fungi, Westphaliam Wilhelms-University, Münster,GermanyThe filamentous fungus Fusarium fujikuroi is known to produce highamounts of different secondary metabolites such as the red pigmentbikaverin, the mycotoxin fusarin C and the phytohormone gibberellic acid(GA). Particularly gibberellins exhibit a great biotechnological impact asapplication of GAs in higher plants induces early flower bud formation andshoot elongation as well as an increased fruit size and enhanced yields ofcrops such as seedless grapes and corn. Therefore, each year about ten tonsof gibberellins are consumed by the agricultural industry as plant growthregulators.Since the knowledge about biosynthesis pathways is accessible to a greaterextent, the rice pathogen F. fujikuroi constitutes a capable species for GAproduction by fermentation. To increase GA yields directed geneticmodifications of pathway genes of competing secondary metabolites wereperformed. These modifications lead to a higher metabolite flux into GAbiosynthesis and thereby enhanced GA production rates compared to thewild type. In addition, regulation of key enzymes of the precursor providingprimary metabolism has to be investigated to circumvent negative feedbackregulation by different intermediates or end products. By understandingenzyme regulation on transcriptional and protein level both gene expressionand enzyme activity will be enhanced significantly. These regulatorymechanisms shall be for example elucidated by identification of positivelyor negatively acting transcription factors.To finally combine all approaches in one strain new selection markers orpossibilities for re-using common markers have to be established for F.fujikuroi.FBV020New insights in the regulation of mycotoxin production inthe plant pathogen F. graminearumJ. Bormann*, P. Ilgen, C. Kröger, B. Hadeler, W. SchäferMolecular Phytopathology and Genetics, Microbiology, Biocenter KleinFlottbek, Hamburg, GermanyThe fungal pathogen Fusarium graminearum is the causal agent of Fusariumhead blight in small grain cereals and of cob rot disease of maize. Thedevastating effect is due to yield losses and mycotoxin contamination.Among the mycotoxins produced by the fungus, the trichothecenedeoxynivalenol (DON) was shown to be important for virulence in wheat.The regulation of DON-production during plant infection and in axenicculture is still not known in detail. Using qRT-PCR and Elisa-based DONmeasurements we analyse the influence of different nitrogen sources andplant substances on a) DON production and b) on genes that play a certainrole in nitrogen signaling. These analyses are accompanied by fluorescencemeasurements using a reporter strain that expresses the green fluorescentprotein GFP under the control of the trichothecene synthase (Tri5) promoter.Using this strain it is possible to directly monitor Tri5 induction underdifferent growth conditions. We show a DON-inducing effect of ammoniumions and plant components. In addition, we started to functionally analysenitrogen signaling regulator proteins like the GATA-transcription factorAreA and the bZIP-transcription factor MeaB in order to assess theirfunction in the regulation of toxin production and virulence.FBV021Molecular analysis of polyketide synthase genes involvedin secondary metabolism of Alternaria alternataD. Saha*, R. FischerKarlsruhe Institute of Technology, Karlsruhe, GermanyFilamentous fungi produce a diverse array of secondary metabolites - smallmolecules that are not necessary for normal growth or development. Classesof fungal secondary metabolites include polyketides, non-ribosomal peptidesterpenes indole terpenes etc. Fungal PKSs are responsible for thebiosynthesis of mycotoxins and other secondary metabolite.spektrum | Tagungsband 2011