The use of FLP-mediated recombination for the functional analysis of an effector gene family in the biotrophic smut fungus Ustilago maydis

Ustilago maydis, a dimorphic hemibasidiomycete fungus, is the causative agent of corn smut disease and has become one of the models for the study of biotrophic interactions. The establishment of biotrophic growth critically depends on secreted effector molecules. Among the novel secreted U. maydis effectors some are encoded by gene families which may have redundant functions. Due to the limited number of selectable markers it was not possible to perform sequential gene deletions when this thesis was started, i.e. the functional analysis of effector gene families was not possible. To solve this problem I have established an inducible FLP-mediated marker recycling system in U. maydis. It consists of three main steps: i) the generation of a deletion mutant in which the selectable marker introduced is flanked by directly oriented FRT (FLP recombination targets) sites, ii) the introduction of an inducible FLP gene on an autonomously replicating plasmid and iii) the induction of FLP expression and the subsequent screening for the loss of the selectable marker as well as the FLP donor plasmid. To eliminate possible inter- and intramolecular recombination events between identical FRT sites left in the genome after excision, FRT sequences with different point mutations in the core region were employed. The FLP-mediated selectable marker removal technique was successfully applied to delete a family of 11 effector genes (eff1) using five sequential rounds of recombination. All Eff1 proteins have the same architecture, consisting of an N-terminal signal sequence, a central region predicted to be natively unstructured, and a conserved C-terminal domain, which presumably represents the only folded part of these proteins. I showed that expression of all 11 genes is specifically upregulated during the biotrophic phase. Strains carrying deletions of 9 or all 11 genes displayed a significant reduction in virulence and this phenotype could be partially complemented by the introduction of different members from the gene family, demonstrating redundancy. The combined deletion analysis and complementation studies conducted for members of the eff1 family has revealed that three of the 11 eff1 genes contribute most significantly to virulence, while all the other members of this gene family contribute to virulence only weakly

Electrochemical approach for isolation of chitin from the skeleton of the black coral cirrhipathes sp. (Antipatharia)

The development of novel and effective methods for the isolation of chitin, which remains one of the fundamental aminopolysaccharides within skeletal structures of diverse marine invertebrates, is still relevant. In contrast to numerous studies on chitin extraction from crustaceans, mollusks and sponges, there are only a few reports concerning its isolation from corals, and especially black corals (Antipatharia). In this work, we report the stepwise isolation and identification of chitin from Cirrhipathes sp. (Antipatharia, Antipathidae) for the first time. The proposed method, aiming at the extraction of the chitinous scaffold from the skeleton of black coral species, combined a well-known chemical treatment with in situ electrolysis, using a concentrated Na2SO4 aqueous solution as the electrolyte. This novel method allows the isolation of a-chitin in the form of a microporous membrane-like material. Moreover, the extracted chitinous scaffold, with a well-preserved, unique pore distribution, has been extracted in an astoundingly short time (12 h) compared to the earlier reported attempts at chitin isolation from Antipatharia corals. © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/)

The use of FLP-mediated recombination for the functional analysis of an effector gene family in the biotrophic smut fungus Ustilago maydis

Ustilago maydis, a dimorphic hemibasidiomycete fungus, is the causative agent of corn smut disease and has become one of the models for the study of biotrophic interactions. The establishment of biotrophic growth critically depends on secreted effector molecules. Among the novel secreted U. maydis effectors some are encoded by gene families which may have redundant functions. Due to the limited number of selectable markers it was not possible to perform sequential gene deletions when this thesis was started, i.e. the functional analysis of effector gene families was not possible. To solve this problem I have established an inducible FLP-mediated marker recycling system in U. maydis. It consists of three main steps: i) the generation of a deletion mutant in which the selectable marker introduced is flanked by directly oriented FRT (FLP recombination targets) sites, ii) the introduction of an inducible FLP gene on an autonomously replicating plasmid and iii) the induction of FLP expression and the subsequent screening for the loss of the selectable marker as well as the FLP donor plasmid. To eliminate possible inter- and intramolecular recombination events between identical FRT sites left in the genome after excision, FRT sequences with different point mutations in the core region were employed. The FLP-mediated selectable marker removal technique was successfully applied to delete a family of 11 effector genes (eff1) using five sequential rounds of recombination. All Eff1 proteins have the same architecture, consisting of an N-terminal signal sequence, a central region predicted to be natively unstructured, and a conserved C-terminal domain, which presumably represents the only folded part of these proteins. I showed that expression of all 11 genes is specifically upregulated during the biotrophic phase. Strains carrying deletions of 9 or all 11 genes displayed a significant reduction in virulence and this phenotype could be partially complemented by the introduction of different members from the gene family, demonstrating redundancy. The combined deletion analysis and complementation studies conducted for members of the eff1 family has revealed that three of the 11 eff1 genes contribute most significantly to virulence, while all the other members of this gene family contribute to virulence only weakly

Sequence Capture of Mitochondrial Genome with PCR-Generated Baits Provides New Insights into the Biogeography of the Genus Abies Mill.

Mitochondrial DNA (mtDNA), being maternally inherited in plants of the family Pinaceae, is an important source of phylogeographic information. However, its use is hindered by a low mutation rate and frequent structure rearrangements. In the present study, we tested the method of genomic libraries enrichment with mtDNA via the sequence capture method yielding mtDNA data which were further used to reconstruct the phylogenetic tree of the genus Abies. The baits for hybrid capture were obtained by long-range PCR using primers designed on the basis of the assembly of Abies sibirica Ledeb. mitochondrial genome. Mitochondrial genomes of Picea sitchensis (Bong.) Carr., Larix sibirica Ledeb., and Keteleeria davidiana (Bertrand) Beissn. were used as an outgroup. The resulting phylogenetic tree consists of two sister branches, including the Eurasian and American species, respectively, with some exceptions. The subclade of A. sachalinensis (F. Schmidt) Mast. and A. veitchii Lindl. (Japan and Sakhalin islands) occupies a basal position in the branch of American firs, probably due to the complex history of fir migrations from North America to Eurasia. The tree has high support for majority of clades. For species represented by more than one sample an intraspecific variability was found which is suitable to design mtDNA markers for phylogeographic and population studies

Identification and first insights into the structure of chitin from the endemic freshwater demosponge Ochridaspongia rotunda (Arndt, 1937)

Studies on the identification, properties and function of chitin in sponges (Porifera), which are recognized as the first multicellular organisms on Earth, continue to be of fundamental scientific interest. The occurrence of chitin has so far been reported in 21 marine sponge species and only in two inhabiting fresh water. In this study, we present the discovery of alpha-chitin in the endemic demosponge Ochridaspongia rotunda, found in Lake Ohrid, which dates from the Tertiary. The presence of chitin in this species was confirmed using special staining, a chitinase test, FTIR, Raman and NEXAFS spectroscopy, and electrospray ionization mass spectrometry (ESIMS). In contrast to the case of marine sponges, chitin in O. rotunda has been found only within its holdfast, suggesting a role of chitin in the attachment of the sponge to the hard substratum. Isolated fibrous matter strongly resemble the shape and size of the sponge holdfast with membrane-like structure

3D chitin scaffolds of marine demosponge origin for biomimetic mollusk hemolymph-associated biomineralization ex-vivo

none18siStructure-based tissue engineering requires large-scale 3D cell/tissue manufacture technologies, to produce biologically active scaffolds. Special attention is currently paid to naturally pre-designed scaffolds found in skeletons of marine sponges, which represent a renewable resource of biomaterials. Here, an innovative approach to the production of mineralized scaffolds of natural origin is proposed. For the first time, a method to obtain calcium carbonate deposition ex vivo, using living mollusks hemolymph and a marine-sponge-derived template, is specifically described. For this purpose, the marine sponge Aplysin aarcheri and the terrestrial snail Cornu aspersum were selected as appropriate 3D chitinous scaffold and as hemolymph donor, respectively. The formation of calcium-based phase on the surface of chitinous matrix after its immersion into hemolymph was confirmed by Alizarin Red staining. A direct role of mollusks hemocytes is proposed in the creation of fine-tuned microenvironment necessary for calcification ex vivo. The X-ray diffraction pattern of the sample showed a high CaCO3 amorphous content. Raman spectroscopy evidenced also a crystalline component, with spectra corresponding to biogenic calcite. This study resulted in the development of a new biomimetic product based on ex vivo synthetized ACC and calcite tightly bound to the surface of 3D sponge chitin structure.noneWysokowski M.; Machalowski T.; Petrenko I.; Schimpf C.; Rafaja D.; Galli R.; Zietek J.; Pantovic S.; Voronkina A.; Kovalchuk V.; Ivanenko V.N.; Hoeksema B.W.; Diaz C.; Khrunyk Y.; Stelling A.L.; Giovine M.; Jesionowski T.; Ehrlich H.Wysokowski, M.; Machalowski, T.; Petrenko, I.; Schimpf, C.; Rafaja, D.; Galli, R.; Zietek, J.; Pantovic, S.; Voronkina, A.; Kovalchuk, V.; Ivanenko, V. N.; Hoeksema, B. W.; Diaz, C.; Khrunyk, Y.; Stelling, A. L.; Giovine, M.; Jesionowski, T.; Ehrlich, H