The use of FLP‐mediated recombination for the functional analysis of an effector gene family in the biotrophic smut fungus Ustilago maydis

Abstract

Summary In the Ustilago maydis genome, several novel secreted effector proteins are encoded by gene families. Because of the limited number of selectable markers, the ability to carry out sequential gene deletions has limited the analysis of effector gene families that may have redundant functions. Here, we established an inducible FLP‐mediated recombination system in U. maydis that allows repeated rounds of gene deletion using a single selectable marker (HygR). To avoid genome rearrangements via FRT sites remaining in the genome after excision, different mutated FRT sites were introduced. 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. We showed that expression of all 11 genes is up‐regulated during the biotrophic phase. Strains carrying deletions of 9 or all 11 genes showed 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 establishment of the FLP/FRT system in a plant pathogenic fungus paves the way for analyzing multigene families with redundant functions.

Activating Intrinsic Carbohydrate-Active Enzymes of the Smut Fungus Ustilago maydis for the Degradation of Plant Cell Wall Components

“…This strategy might also be a good means to provide flexibility by adapting the activated strains and their ratios depending on the substrate to be degraded. In U. maydis, it is furthermore possible to enable the simultaneous activation of multiple CAZymes in a single strain using resistance marker recycling (47,75). That way, specialized strains for the degradation of different lignocellulose-building blocks could be developed.…”

The Effectors of Smut Fungi

“…Nine of the eff1 genes reside in gene clusters of seven and two genes, respectively. A significant reduction in virulence after seedling infection was observed when all 11 genes were deleted and most of this could be attributed to three of these effector genes, eff1-11, eff1-3, and eff1-4 (Khrunyk et al, 2010;Table 4.1). Three members of the eff1 gene family contain putative nuclear localization motifs (NLS).…”

“…However, um01779 mutants show only a slight reduction in virulence ). In addition, all members of a gene family representing 11 effectors (eff1 family) that are upregulated during biotrophic development have recently been successfully deleted after establishing the FLP/FRT marker recycling system for U. maydis (Khrunyk et al, 2010;Table 4.1). Nine of the eff1 genes reside in gene clusters of seven and two genes, respectively.…”

“…Three members of the eff1 gene family contain putative nuclear localization motifs (NLS). However, based on the fact that these putative motifs do not reside in a conserved sequence context and are not contained in all effectors contributing most significantly to virulence, it seems unlikely that these motifs are relevant for eff1 effector function (Khrunyk et al, 2010).…”

The Corn Smut Fungus Ustilago maydis as an Alternative Expression System for Biopharmaceuticals

“…Due to the limited number of resistance markers available for U. maydis , an efficient resistance-marker recycling system has been invented a few years back that enables multiple gene deletions (Khrunyk et al 2010 ). This marker recycling method is based on a property of the yeast recombinase fl ippase (FLP) to recognize and catalyze recombination between specifi c 34 bp sequences called FRT sites resulting in cleaving off the intervening DNA sequence and leaving one recombination site behind (Hamers-Casterman et al 1993 ).…”