Filamentous Fungi

Fungi are a diverse group of organisms, whose biological activities affect our daily life in many ways. The filamentous fungi are in particular 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.
Filamentous fungi produce a diverse array of secondary metabolites, which serve the pharmaceutical sciences as prolific source of chemical compounds for the development of new drugs.

Growth of filamentous fungi

The vegetative growth of filamentous fungi start 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.

The genetic analysis has produced a deep understanding of both the physiology of Aspergillus and the organisation of the genome (7). This research has advanced the study of eukaryotic cellular physiology and contributed to our understanding of metabolic regulation, development, DNA repair, morphogenesis, and human genetics diseases (6). Furthermore, the recent sequencing of the complete genome of A.nidulans has created a tremendous potential to obtain insight into important aspects of fungal biology such as transcriptional regulation, secondary metabolite production and pathogenicity (8).

Gene targeting

Accurate 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 in 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 below. Different DNA fragments 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 Non Homologous End Joining 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 concept

Several 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 verification of the reporter gene is only made by fluorescence microscopy.

We succeeded in proving that the Plug 'n' Play assembly system is easily applied for constructing fungal vectors. Transformation and expression of the fluorescence proteins GFP and RFP was successfully conducted 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 fungi also showed a pronounced number of unspecific vacuoles. The changes occur due to the DNA fragments were randomly integrated and have inflicted some pathways in the fungi. However, this was not unexpect when using integration by random Non Homologues End Joining.

Device BBa_K678060

Green fluorescence can be observed evenly spread in the hyphae. This correlate with what was expected for the device BBa_K678060 that holds the gene GFP for green fluorescence and with no specific targeting signal.

The constructed pJEJAM 12 plasmid consist of device BBa_K678060 (pgdA,GFP,TrpC,pyrG) , which are cut at the two NotI site before transformation, ensuring linearised DNA fragment for optimal result.

Aspergillus nidulans with device BBa_K678060 - detected with DIC light.	Aspergillus nidulans with device BBa_K678060 - detected with GFP filter.

Aspergillus nidulans with device BBa_K678060 - Shown from front	Aspergillus nidulans with device BBa_K678060 - Shown from Back

Device BBa_K678061

Green fluorescence is can be observed in the hyphae and in clear spots. The occurrence of clear spots was expected for device BBa_K678061, which holds the gene GFP encoding green fluorescence proteins and targeting signal for the peroxiomes. This correlates fine with comparison of device BBa_K678060, which have non-targeting signal.
However, we cannot conclude that the signal is accumulated in the peroxisomes, since they are not dyed. Though, it can be concluded that the GFP signal is targeting to a specific place and accumulated somewhere in the fungi compared to the results from device BBa_K678060.

The pJEJAM 13 plasmid constructed holding device BBa_K678061, are cut at the two NotI site before transformation, ensuring linearised DNA fragment for optimal result.

Aspergillus nidulans with device BBa_K678061 - detected with DIC light.	Aspergillus nidulans with device BBa_K678061 - detected with GFP filter.

Aspergillus nidulans with device BBa_K678061 - Shown from front	Aspergillus nidulans with device BBa_K678061 - Shown from back

Device BBa_K678062

Observed is red fluorescence spread evenly in the hyphae, which correlate with what expected for the device BBa_K678062, that holds the gene for red fluorescence protein RFP with no specific targeting signal.

The constructed pJEJAM 14 plasmid holding device BBa_K678062, are cut at the two NotI site before transformation, ensuring linearised DNA fragment for optimal result.

Aspergillus nidulans with device BBa_K678062 - detected with DIC light.	Aspergillus nidulans with device BBa_K678062 - detected with RFP filter.

Aspergillus nidulans with device - shown from front	Aspergillus nidulans with device - shown from back

Device BBa_K678063

Red fluorescence can be observed in clear spots. The occurrence of clear spots and compared to the results from device BBa_K678062, correlate with what expected for the device BBa_K678063 that holds the gene for red fluorescence protein RFP with the targeting signal for the nucleus. However, we cannot conclude that the signal is accumulated in the nucleus, since they are not dyed. Though, it can be concluded that the RFP signal is targeting to a specific place and accumulated somewhere in the fungi compared to the results from device BBa_K678062.

The constructed pJEJAM15 plasmid, holding device BBa_K678063, are cut at the two NotI site before transformation, ensuring linearised DNA fragment for optimal result.

Aspergillus nidulans with device BBa_K678063 - detected with DIC light.	Aspergillus nidulans with device BBa_K678063 - detected with RFP filter.

Aspergillus nidulans with device BBa_K678063 - Shown from front	Aspergillus nidulans with device BBa_K678063 - Shown from back

Control strain

Wild type Aspergillus nidulans - detected with DIC light.	Wild type Aspergillus nidulans - detected with RFP filter.	Wild type Aspergillus nidulans - detected with GFP filter.

Wild type Aspergillus nidulans - Shown from front	Wild type Aspergillus nidulans - Shown from back

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.

[2] Harris, S.D., 1997. The Duplication Cycle in Asoergillus nidulans. Fungal Genetics and Biology, vol. 22, no. 1, pp.1-12.

[3] Harris, S.D. et al., 2009. Morphology and development in Aspergillus nidulans: A complex puzzle. Fungal Genetics and Biology, vol. 46, no. 1, sup. 1, pp.S82-92.

[4] Timberlake, W.E., 1990. Molecular Genetics of Aspergillus Development. Annual Review of Genetics, vol. 24, pp.5-36.

[5] Nielsen, J.B., 2008. Understanding DNA repair in Aspergillus nidulans - paving the way for efficient gene targeting. Technical University of Denmark.

[6] Galagan, J.E. et al., 2005. Sequencing of Aspergillus nidulans and comparative analysis with A. fumigatus and A. oryzae. Nature, vol. 438, no. 7971, pp.1105-15.

[7] Doonan, J.H., 1992. Cell division in Aspergillus. Journal of Cell Science, vol. 103, no. 3, pp.599-611.

[8] Nielsen, M.L. et al., 2006. Efficient PCR-based gene targeting with a recyclable marker for Aspergillus nidulans. Fungal Genetics and Biology, vol. 43, no. 1, pp.54-64.

[9] Krappmann, S., 2007. Gene targeting in filamentous fungi: the benefits of impaired repair. Fungal Biology Reviews, vol. 21, no. 1, pp.25-29.

[10] Ninomiya, Y., Suzuki, K., Ishii, C. & Inoue, H., 2004. Highly efficient gene replacements in Neurospora strains deficient for nonhomologous end-joining. PNAS, vol. 101, no. 33, pp.12248-53.