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

during development of the symbiotic interaction (1). Hydrophobins havebeen shown to play an important role in this interaction. At the same time,the hydrophobin TtHyd1 is specifically expressed in the Hartig’net in acompatible interaction of Tricholoma with pine (2). To investigate itsfunction, heterologous expression in Schizophyllum commune wasperformed, where we know 13 hydrophobins of this class from the genomesequence. So far, a databank was generated and used to search for motifs,new hydrophobins and a phylogenetic tree was calculated based onhydrophobin protein and mRNA sequences. For the characterization ofhydrophobins from Tricholoma vaccinum, an overexpression will be thefuture goal to investigate in which stage of the symbiotic interactionhydrophobins are produced and what kind of role they play with respect tofunction of the symbiotic tissue.[1] Cloning symbiosis-related cDNAs from eucalypt ectomycorrhiza by PCR-assisted differentialscreening.,Tagu et al., 1993.[2] Spezifische Genexpression in der Ektomykorrhizabildung durch den Pilz Tricholoma terreum,Mankel, 2000.SIP004Kunitz-type protease inhibitors are involved in arbusculedevelopment in mycorrhizal symbiosisS. Rech*, N. RequenaBotanical Institute, Karlsruhe Institute of Technology (KIT), Karlsruhe,GermanyThe arbuscular mycorrhiza (AM) is the most widespread symbiosis and it isformed between plants and fungi of the Glomeromycota Phylum. Toestablish the plant-fungal interface, fungal hyphae invade the host rootthrough the epidermal layer and continue growing toward the cortex, wherethey form highly branched structures - called arbuscules. This involvesinvagination of the plant plasma membrane around developing arbuscules toform the mutual interaction zone. Arbuscule development is a dynamicprocess. After several days of maturity arbuscules collapse and die, whereasan invaded plant cell can host successive arbuscules. TC106 encodes asecreted Medicago truncatula (barrel medic) protease inhibitor (Kunitztype)specifically induced upon contact with AM fungi. Deregulation ofTC106 expression level mediated by RNAi silencing and constitutiveoverexpression revealed abberrant mycorrhizal phenotypes showingdisproportionally high numbers of crippled arbuscules. A non-directedyeast-two-hybrid screen identified a secreted cysteine protease as a potentialinteraction partner of TC106. Furthermore, direct yeast-two-hybridinteraction tests showed an interaction of TC100 - also encoding a secretedKunitz protease inhibitor - with a recently described mycorrhiza specificinduced subtilase. Proteolytic cleavage of peptide bonds is crucial tonavigate development and regulatory processes. In this study, allinvestigated proteins contain a secretion signal peptide. This would suggestthe plant-fungal interface as the potential locus of protein-proteininteraction. We hypothezise that distinct members of the Kunitz proteaseinhibitor family are key players in the plant controlled part of arbusculedevelopment. The protease inhibitors would fine-tune proteolytic activityrequired for arbuscule turnover to prepare the invaded plant cell for the nextgeneration of arbuscules.SIP005Molecular basis of symbiosis in phototrophic consortiaP. Henke* 1 , J. Overmann 21 Microbial Ecology and Biodiversity Science, Germany German Collectionof Microorganisms and Cell Cultures (DSMZ), Braunschweig, Germany2 German Collection of Microorganisms and Cell Cultures (DSMZ),Braunschweig, GermanyThe phototrophic consortium Chlorochromatium aggregatum is amorphologically defined multicellular assemblage consisting of a centralmotile chemotrophic Betaproteobacterium that is associated with ~20 cellsof the green sulfur bacterial epibiont Chlorobium chlorochromatii. Theepibionts are connected with each other and the central bacterium throughhair-like ultrastructures. The attachment site of the epibiont to the centralbacterium is characterized by the absence of chlorosomes and a singlecontact layer. The central bacterium is flagellated and extends periplasmictubules to the outer membrane of the epibionts. This highly structuredassociation is culturable making it a model system for understanding themolecular basis of symbiosis between different types of bacteria. Previouswork has described four putative symbiosis genes (Cag1919, Cag1920,Cag0614, Cag0616) of the epibiont which were recovered by suppressionsubstractive hybridization and bioinformatic approches. These four genesare constitutively transcribed and do not occur in genomes of non-symbioticrelatives of the epibiont. Cag1919 contains a haemolysin-type Ca 2+ -bindingregion with several RTX repeats. RTX-type toxins so far have been found inGram-negative bacterial pathogens and Cag1919 may have been recruitedby the epibiont via lateral gene transfer. To facilitate localization of theproteins, Cag1919 was cloned in its entirety into the vector pQE60.Interestingly, expression of Cag1919 was deleterious to E. coli strainscausing the formation of extremely long, filamented cells. Expression of therecombinant protein was achieved in E.coli strain XL1-Blue. RecombinantCag1919 is used to produce antibodies for immunogold labelling andtyramide signal amplification to establish the location of the protein in the C.chlorochromatii in the free-living and symbiotic states. Efforts to identifyand express suitable fragments of the giant Cag0614 and Cag0616 geneproducts will also be described.SIP006The symbiotic gut microbiota of termites andcockroaches: Are there evolutionary patterns in thedictyopteran lineage?T. Köhler*, A. BruneDepartment of Biogeochemistry, Max Planck Institute for TerrestrialMicrobiology, Marburg, GermanyPrevious studies on the intestinal microbiota of termites have identifiednumerous clusters of bacteria that seem to occur exclusively in termite guts.Some of these termite-specific clusters are also affiliated with sequencesoriginating from their closest relatives, the cockroaches. However, themicrobial diversity in cockroach guts has not been studied in any detail, andit is therefore not clear whether this exclusiveness reflects properties of theparticular niches in the dictyopteran gut (e.g., habitat preferences or dietrelatedfactors) or whether these are even evolutionary patterns (i.e.,elements of the gut microbiota are cospeciating with their dictyopteranhosts). To address these points, we investigated the diversity of the bacterialgut microbiota in numerous representatives of the dictyopteran lineage using454 pyrosequencing. Total DNA was extracted from the microbe-packedhindguts of 35 insect species, comprising 16 termites, 15 cockroaches, 1mantid, and 3 insect species outside the Dictyoptera. The 16S rRNA geneswere sequenced after PCR amplification with a modified primer set targetingthe V3-V4 region (ca. 450 bp) to exploit the full capacity of the Titaniumtechnology. The resulting sequences (3,000-15,000 per species) wereprocessed using a pipeline combining Naïve Bayesian classification with amanually curated reference database. OTU assignment, statistical andphylogenetic analyses are being performed using MOTHUR, R, and ARBsoftware. Preliminary results indicate that the gut microbiota of termites andcockroaches contains phylotypes typical of the gut environment in general,and particular lineages are apparently cospeciating with their dictyopteranhost. The presence of bacterial groups occurring exclusively in soil-feedingor fungus-feeding species suggests a participation of these groups in thedegradation of the respective diet.SIP007Will not be presented!SIP008Development of lineage-specific gut microbialcommunities during termite evolutionT. Köhler, A. Mikaelyan*, K. Paul, A. BruneDepartment of Biogeochemistry, Max Planck Institute for TerrestrialMicrobiology, Marburg, GermanyTermites feed on diets consisting of lignocellulosic or humic substrates.They are divided into two groups - the more primitive lower termites, whichpossess cellulolytic gut flagellates, and the evolutionarily advanced highertermites, which lack such flagellates and have developed novel strategies todigest their respective diets. While the lower termites feed almostexclusively on wood, higher termites (family Termitidae), which make upabout 80% of all termite species, comprise several feeding guilds of funguscultivating,soil-feeding, and secondarily wood-feeding forms. Weconducted a comprehensive comparative analysis of the bacterial gutmicrobiota in representatives of all subfamilies and feeding guilds,combining 454 pyrosequencing of the V3-V4 region with additional Sangerspektrum | Tagungsband 2011