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 e...
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|Summary:||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.|