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Proof of concept - Fungi
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Filamentous Fungi
Fungi are a diverse group of organisms, whose biological activities affect our daily life in many ways. The filamentous fungi particularly are of great importance in production of medicine, in the industry, in agriculture, and in basic biological research. Some of the filamentous fungal species are pathogenic to humans, whereas others have great value in the production of antibiotics such as penicillin. The fungi are therefore importance for industrial as well as medical production. Growth of filamentous fungiThe vegetative growth of filamentous fungi starts with the germination of a spore when the conditions are right . The spore germination leads to formation of hyphae (1). A fungal hypha is a long tubular modular structure composed of individual cells (2). Hyphae extend only at their tips and are typically divided into individual cellular compartments by the formation of septa (3).
Filamentous fungi grow by the polar extension of hyphae and multiply by branching (4). The branched hyphae forms a network of interconnected cells called a mycelium (1). The mycelium forms a radially symmetric colony that expands over large area until growth is limited by for example lack of nutrients (1,5). The fungal mycelium appears to be a formless collection of corresponding vegetative cells. However, the various cells within the mycelium interact to form an ordered network with different hyphae or cells playing distinct roles in the acquirement of nutrients from the environment and development (1). Aspergillus nidulans
The filamentous fungus Aspergillus nidulans is a widely recognised model organism (3). A. nidulans possesses today, in contrast to most other aspergilla, a well characterized sexual cycle and a well-developed genetics system (6). Furthermore, in A.nidulans the parasexual cycle has been extensively utilized. Parasexual genetic involves examination of recombination in the absence of sexual reproduction. Gene targetingAccurate manipulation of genes is a key approach in fungal molecular biology (9). The method of gene targeting facilitates precise genome manipulations which are called site directed alterations. They are performed by use of deletions, replacements and insertion of the target locus. Gene targeting is achieved by transforming fungi with a suitable linear DNA fragment that contains sequences that are identical to the target site in the genome, see figure. Different DNA fragment are constructed depending on deletion, replacement or insertion. The fungi can integrates linear DNA fragment in its genome by using its repair system of DNA double stranded breaks.
Two mechanisms of DNA double strand break repair ensure that the introduced piece of DNA is pasted into the fungal genome, to be replicated stably: Homologous Recombination and Non Homologous End Joining, also called illegitimate recombination (9). Homologous recombination involves interaction between homologous sequences, whereas NHEJ involves ligation of the strand ends independently of DNA homology (10). Precise genome manipulation can often be tedious and time-consuming, because fungi appear to favour Non Homologous End Joining over Homologous recombination resulting in low gene targeting efficiencies (9). Proof of conceptSeveral separate plasmid was constructed by use of the Plug 'n' Play assembly system to verify the systems function in filamentous fungi. To perform a successful transformation in fungi the backbone plasmid for the fungal devices BBa_K678046 have two NotI restriction sites flanking the device. Hereby, the device can be cut and a linearised DNA fragment can be transformated into the fungi. The devices for proof of concept in fungi are not design to be inserted at a specific site at the fungal genome. Therefore, a fungal strain with intact pathway for non homologous recombination was used. Therefore, the device can integrate at any site in the fungi and thereby also at site of essential genes. All the fungal devices for proof of concept were constructed with the same strong constitutive promoter PgpdA, the TtrpC terminator, and the marker cassette of pyrG. Different fluorescence reporter genes was used for creating a reporter system, were different compartments of the fungi could be targeted. The compartment attempted to target was the nucleus, peroxisomes, and the mitochondria. All transformation was performed in the laboratory Aspergillus nidulans stain: argB2, pyrG89, veA1 by random non-homologues-end-joining integration, since we only want to verify the reporter gene by fluorescence microscopy. We succeeded in proving that the Plug 'n' Play assembly system is easily applied for constructing fungal vectors. Transformation and expression in fungi succeed by expressing the two fluorescence proteins GFP and RFP in A. nidulans not localized to any compartment. Furthermore, expression of fluorescence proteins GFP and RFP in A. nidulans localized to the nucleus and the peroxiomes succeeded. However, the transformation of the fungi with the mitochondrial targeting signal did not succeed. The obtained transformants with integration of the four different devices changed pronounced morphology compared with the wild type control strain. The strains was visible sick and had slow growth. The change occur due to the DNA fragment were randomly integrated and have inflicted some pathways in the fungi. However, this was to expect when using integration by random non-homologues-end-joining. The idea was to target the fungi with a fluorescence protein to specific compartments and in general to express the fluorescence proteins. The results are displayed in the following section. pJEJAM12 BBa_K678060The results shows fluorescence spread evenly in the hyphae which correlate with that in device BBa_K678060, the gene of interest, only consists of a green fluorescence protein GFP with no specific target.
pJEJAM13 BBa_K678061The results shows fluorescence spread evenly in the hyphae and with spots of fluorescence. Device BBa_K678061 consists of GFP with a target signal to the peroxisomes which can explain the spots compared to the results from device BBa_K678060. We cannot conclude that the signal is accumulated in the peroxisomes since they are not dyed, though can it be concluded that the GFP signal is target a specific place and accumulated somewhere in the fungi compared to the results from device BBa_K678060.
pJEJAM14 BBa_K678062The results shows fluorescence spread evenly in the hyphae which correlate with that in device BBa_K678061, the gene of interest, only consists of a green fluorescence protein RFP with no specific target.
pJEJAM15 BBa_K678063The results show spots of fluorescence as in Device BBa_K678061 . Device BBa_K678063 consists of RFP with a target signal to the nucleus which can explain the spots compared to the results from device BBa_K678060 and the spores with a single nuclei contains a lot of RFP signal. We cannot conclude that the signal is accumulated in the nucleus since they are not dyed, though can it be concluded that the RFP signal is target a specific place and accumulated somewhere.
Control strainThe control strain shows no auto-fluorescence.
References
[1] Adams, T.H., Wieser, J.K. & Yu, J.-H., 1998. Asexual Sporulation in Aspergilus nidulans. Microbiology and Molecular Biology Reveiws, vol. 62, no. 1, pp.35-54.
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