Genetic Manipulation of the Plant Pathogen <em>Ustilago maydis</em> to Study Fungal Biology and Plant Microbe Interactions

Abstract

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the replacement of the entire gene with a selectable marker via homologous recombination. During homologous recombination, exchange of DNA takes place between sequences with high similarity. Therefore, linear genomic sequences flanking a target gene can be used to specifically direct a selectable marker to the desired integration site. Blunt ends of the deletion construct activate the cell's DNA repair systems and thereby promote integration of the construct either via homologous recombination or by non-homologous-end-joining. In organisms with efficient homologous recombination, the rate of successful gene deletion can reach more than 50% making this strategy a valuable gene disruption system. The smut fungus Ustilago maydis is a eukaryotic model microorganism showing such efficient homologous recombination. Out of its about 6,900 genes, many have been functionally characterized with the help of deletion mutants, and repeated failure of gene replacement attempts points at essential function of the gene. Subsequent characterization of the gene function by tagging with fluorescent markers or mutations of predicted domains also relies on DNA exchange via homologous recombination. Here, we present the U. maydis strain generation strategy in detail using the simplest example, the gene deletion.

A Novel Factor Essential for Unconventional Secretion of Chitinase Cts1

“…All plasmids (pUMa vectors, see below and Table 1 ) generated in this study were obtained using standard molecular biology methods established for U. maydis including Golden Gate cloning ( Brachmann et al, 2004 ; Kämper, 2004 ; Terfrüchte et al, 2014 ; Bösch et al, 2016 ). Oligonucleotides applied for sequencing and cloning are listed in Table 2 .…”

“…Genomic DNA extraction was performed according to published protocols ( Bösch et al, 2016 ), two separate preparations were combined, and the DNA concentration was adjusted to 100 ng/μl using TE-RNase in approximately 500 μl final volume. gDNA quality was verified by PCR reactions using primers specific for the bacterial 16sRNA gene (oRL1124 × oRL1125) and the intrinsic gene uml2 ( umag_01422 ; oRL272 × oRL273).…”

“…To this end, the following primer combinations were used to amplify the indicated genes ( Table 2 ): umag_00493 : oDD691 × oDD729; umag_06269 : oDD692 × oDD730; umag_02631 : oDD693 × oDD731; umag_03776 : oDD694 × oDD732; umag_04298 : oDD695 × oDD733; umag_05386 : oDD696 × oDD734; umag_04385 : oDD697 × oDD735; umag_04494 : oDD698 × oDD736 and umag_11876 : oDD699 × oDD737. gDNA was obtained like described ( Bösch et al, 2016 ). Additional sequencing was conducted for umag_06269 and umag_03776 using progeny #9, #26, #31 and #39 (not shown).…”

Smut infection of perennial hosts: the genome and the transcriptome of the Brassicaceae smut fungus Thecaphora thlaspeos reveal functionally conserved and novel effectors

“…U. maydis growth, infection of maize, and microscopy of maize infection was performed as previously described (Hemetsberger et al ., ; Bösch et al ., ). T. thlaspeos infection of Arabis hirsuta was performed by co‐germination of seeds and spores on soil (Frantzeskakis et al ., ).…”

“…For genomic DNA (gDNA) sequencing of the T. thlaspeos , high‐molecular‐weight gDNA was prepared from pure cultures using phenol extraction (Bösch et al ., ). LF1 gDNA was sequenced by PacBio long‐read sequencing (P6‐C4, Max Planck Genome Centre, Cologne, Germany) and by Illumina short‐read sequencing (2 × 300 bp; Illumina MiSeq, v3 chemistry, Genomics Service Unit at the Biocenter of Ludwig‐Maximilians University, Munich, Germany).…”