Universität zu Köln

Hempel, Anna Ines (2023). Development of a Modular Recombinant Fungal toolkit for gene expression in Ustilago maydis. PhD thesis, Universität zu Köln.

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Abstract

Summary In recent years, the development of tools for manipulating gene expression has significantly advanced. These tools have been applied to model organisms, including mammalian cells, plants, bacteria, and fungi, to unravel complex cellular mechanisms and natural interactions. This study focuses on the model organism Ustilago maydis, a biotrophic fungal plant pathogen that produces corn smut disease in maize (Zea mays). U. maydis is amenable to reverse genetic engineering, is easy to manipulate in laboratory conditions, has a sequenced genome and well-defined interactions with its host maize. One of the most relevant fields of research carried out in U. maydis is the study of its effector genes and their functions in virulence. Various techniques have been used in U. maydis to functionally characterize its effector genes. However, there are still limitations due to their functional redundancy, or the indispensability of certain genes for the development of the disease, making knockout approaches ineffective. In this study, new genetic tools to enhance recombinant gene expression in U. maydis are introduced. This dissertation describes a novel methodology called “Modular Recombinant Fungal toolkit for gene expression”, or “MoRFunG toolkit”, which leverages the Golden Gate and Molecular Cloning systems. As a proof of concept, tailor-made transcript units were constructed and tested in U. maydis, to show the capacity of the technique to complement large genomic deletions. Additionally, steps towards standardization of the method were taken, in order to establish a group of promoters available to be used for differential gene expression during the infection process. Furthermore, experimental development was carried out to incorporate this system in the routinely cloning genetic locations already established and well-defined in U. maydis. Moreover, novel optogenetic tools were evaluated for potential implementation in U. maydis as a methodology to control the expression of effector genes. Thusly, this study summarizes a series of experiments that serve to introduce a new high throughput cloning methodology, allowing systematic, user-friendly, and time-saving options for the manipulation of gene expression in the U. maydis system.

Item Type:	Thesis (PhD thesis)
Translated abstract:	Abstract Language UNSPECIFIED English
Creators:	Creators Email ORCID ORCID Put Code Hempel, Anna Ines air.hempel@gmail.com UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-718207
Date:	2023
Language:	English
Faculty:	Faculty of Mathematics and Natural Sciences
Divisions:	Ehemalige Fakultäten, Institute, Seminare > Faculty of Mathematics and Natural Sciences
Subjects:	Life sciences
Uncontrolled Keywords:	Keywords Language Ustilago maydis English gene expression English MoRFunG toolkit UNSPECIFIED
Date of oral exam:	6 December 2023
Referee:	Name Academic Title Hammerschmidt, Matthias Prof. Dr
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/71820