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[EN] Current challenges in the study and biotechnological exploitation of filamentous fungi are the optimization of DNA cloning and fungal genetic transformation beyond model fungi, the open exchange of ready-to-use and standardized genetic elements among the research community, and the availability of universal synthetic biology tools and rules. The GoldenBraid (GB) cloning framework is a Golden Gate-based DNA cloning system developed for plant synthetic biology through Agrobacterium tumefaciens-mediated genetic transformation (ATMT). In this study, we develop reagents for the adaptation of GB version 3.0 from plants to filamentous fungi through: (i) the expansion of the GB toolbox with the domestication of fungal-specific genetic elements; (ii) the design of fungal-specific GB structures; and (iii) the ATMT and gene disruption of the plant pathogen Penicillium digitatum as a proof of concept. Genetic elements domesticated into the GB entry vector pUPD2 include promoters, positive and negative selection markers and terminators. Interestingly, some GB elements can be directly exchanged between plants and fungi, as demonstrated with the marker hph for Hyg(R) or the fluorescent protein reporter YFP. The iterative modular assembly of elements generates an endless number of diverse transcriptional units and other higher order combinations in the pDGB3 alpha/pDGB3 Omega destination vectors. Furthermore, the original plant GB syntax was adapted here to incorporate specific GB structures for gene disruption through homologous recombination and dual selection. We therefore have successfully adapted the GB technology for the ATMT of fungi. We propose the name of FungalBraid (FB) for this new branch of the GB technology that provides open, exchangeable and collaborative resources to the fungal research community.

The potato late blight disease, caused by the oomycete Phytophthora infestans, is a major threat for potato production worldwide. To breed potato varieties with durable resistance against P. infestans, it is necessary to combine two or more resistance (R) genes. Single R genes are easily overcome by the rapidly evolving pathogen, whereas the presence of several R genes could probably prevent gain of virulence from a single mutation in the pathogen. The large gene pool available within wild potato species offers sufficient possibilities to identify new and diverse R genes conferring resistance to P. infestans (Rpi). Map-based cloning is the most suitable strategy to isolate such new Rpi genes. The objective of this research was mapping, if possible, followed by cloning of Rpi genes from wild Solanum species. Resistance to P. infestans occurring in four different wild Solanum species were mapped in a major R gene cluster on chromosome 11. Natural stacking of three R genes located on different chromosomes was identified in a natural hybrid. In addition, we initiated studies on another type of defense system that is not based on the typical R genes, namely the response of Solanum to INF1 elicitin.

Een overzicht van de ontwikkelingen van het kloneren van landbouwhuisdieren, met het oog op mogelijke consequenties voor beleid en regelgeving. Er wordt ingegaan op de techniek van dna modificatie, de invloed op veehouderij, fokkerij, praktijk, dierenwelzijn en -gezondheid, nationale veiligheid en regelgeving en de toekomstige ontwikkelingen in de nabije toekomst

Potato late blight, caused by the oomycete pathogen Phytophthora infestans (Mont.) de Bary, is the most disastrous disease on potato worldwide and also the greatest threat to potato production in China. Loss of yield and quality, and the costs of chemical control of potato late blight account for multi-billion US$ annually. Using host resistance is thought to be an economical and efficient approach to control potato late blight. To combat the disease breeders have introduced late blight resistance (R) genes from various Solanum species into the cultivated potato. The proteins encoded by these R genes can recognise specific races of the pathogen. This then triggers a cascade of defence responses ultimately leading to a localized programmed cell death called the hypersensitive response (HR) that arrests growth of the pathogen. However, once the resistant potato cultivars are released into the field, the resistance based on these genes is quickly overcome due to rapid evolution and adaption of P. infestans. In the potato-P. infestans interaction race-specific recognition by R genes is based on the ‘gene-for-gene’ model which predicts that resistance is governed by the (direct or indirect) interaction of an R protein with its corresponding effector, the product of an avirulence (Avr) gene. If either the R gene or the Avr gene is absent or non-functional the interaction is compatible and the host susceptible for disease. Key to a better understanding of the molecular basis of resistance in the potato-P. infestans pathosystem is the unravelling of R protein-effector interactions and, hence, cloning of more R and Avr genes is a prerequisite to study these interactions. This thesis describes the mapping and cloning of Avr genes in P. infestans, and the phenotypic and genotypic diversity in P. infestans field isolates in Northern China. To isolate P. infestans Avr genes a positional cloning strategy was adopted. Chapter 2 presents a molecular-genetic linkage map of P. infest

I introduce the outline of recombinant DNA technology. The most important techniques used in recombinant DNA technology are (1) specific cleavage of DNA by restriction endonucleases, DNA synthesis by DNA polymerase or reverse transcriptase, and DNA ligation by DNA ligase, (2) DNA cloning procedures to utilize plasmids or viruses as a vectorr so that a specific DNA fragment can be amplified in bacteria cells, (3) nucleic acid hybridization to identify the specific sequences and (4) DNA sequencing to determine the nucleotide sequence of cloned DNA fragments.