Technological Advancements Development of resources for the analysis of gene function in Pucciniomycotina red yeasts Giuseppe Ianiri a,b , Sandra A.I. Wright b , Raffaello Castoria c , Alexander Idnurm a,⇑ a Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri-Kansas City, MO 64110, USA b Dipartimento di Scienze Animali, Vegetali e dell’Ambiente, Facoltà di Agraria, Università del Molise, Via Francesco De Sanctis, 86100 Campobasso, Italy c Dipartimento di Scienze e Tecnologie Agro-Alimentari Ambientali e Microbiologiche, Facoltà di Agraria, Università del Molise, Via Francesco De Sanctis, 86100 Campobasso, Italy article info Article history: Received 25 November 2010 Accepted 8 March 2011 Available online 12 March 2011 Keywords: a-Isopropylmalate synthase Biocontrol Essential gene Sporidiobolus Urediniomycetes abstract The Pucciniomycotina is an important subphylum of basidiomycete fungi but with limited tools to analyze gene functions. Transformation protocols were established for a Sporobolomyces species (strain IAM 13481), the first Pucciniomycotina species with a completed draft genome sequence, to enable assessment of gene function through phenotypic characterization of mutant strains. Transformation markers were the URA3 and URA5 genes that enable selection and counter-selection based on uracil auxotrophy and resis- tance to 5-fluoroorotic acid. The wild type copies of these genes were cloned into plasmids that were used for transformation of Sporobolomyces sp. by both biolistic and Agrobacterium-mediated approaches. These resources have been deposited to be available from the Fungal Genetics Stock Center. To show that these techniques could be used to elucidate gene functions, the LEU1 gene was targeted for specific homologous replacement, and also demonstrating that this gene is required for the biosynthesis of leucine in basidio- mycete fungi. T-DNA insertional mutants were isolated and further characterized, revealing insertions in genes that encode the homologs of Chs7, Erg3, Kre6, Kex1, Pik1, Sad1, Ssu1 and Tlg1. Phenotypic analysis of these mutants reveals both conserved and divergent functions compared with other fungi. Some of these strains exhibit reduced resistance to detergents, the antifungal agent fluconazole or sodium sulfite, or lower recovery from heat stress. While there are current experimental limitations for Sporobolomyces sp. such as the lack of Mendelian genetics for conventional mating, these findings demonstrate the facile nature of at least one Pucciniomycotina species for genetic manipulation and the potential to develop these organisms into new models for understanding gene function and evolution in the fungi. Ó 2011 Elsevier Inc. All rights reserved. 1. Introduction The Pucciniomycotina is one of the three subphyla within the basidiomycete fungi and comprises about a third of all described basidiomycete species (Aime et al., 2006; Hibbett et al., 2007). These organisms include the plant pathogenic rust-causing fungi that are of such importance to agriculture so to have influenced its history (Ainsworth, 1981). For example, the rusts cause micro- bial diseases that have been deified (the Roman fertility gods Robi- gus/Robigo), were the first illustrated plant pathogens under a microscope in Robert Hooke’s 1665 Micrographia (rose rust Phragmidium mucronatum), are responsible for multiple nations’ preference for tea rather than coffee (coffee rust Hemileia vastatrix), and led to the development of the concept of the gene-for-gene system in plant–pathogen interactions (flax rust Melampsora lini). Currently new strains of Puccinia graminis f. sp. tritici (stem rust) and Puccinia striiformis (stripe rust) have emerged that threaten wheat crops internationally (Stone, 2010). The rust fungi are diffi- cult to cultivate in vitro, i.e., outside their plant hosts, making the analysis of gene functions within them a technical challenge. An- other disadvantage is that the requirement to maintain them on specific plant host cultivars selects for specific genetic races, which may not be a desirable genetic background in which to carry out a complete genetic analysis. In contrast, other members of the Pucci- niomycotina can be cultivated easily, such as the ‘‘red’’ basidiomy- cete yeasts. These organisms are found world wide on all continents, and from polar to tropical climates and growing on a wide diversity of substrates (Aime et al., 2006). Furthermore, they encompass yeast strains such as Rhodosporidium kratochvilovae LS11 (formerly classified as Rhodotorula glutinis) that has been uti- lized as a biocontrol agent of fungal pathogens of plants and fruits (Castoria et al., 2005, 2008; Lima et al., 1998). There are surprisingly few reports of gene function studies in the Pucciniomycotina fungi. Genetic transformation was described for one red yeast species, Rhodosporidium toruloides (Tully and Gil- 1087-1845/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.fgb.2011.03.003 ⇑ Corresponding author. Address: School of Biological Sciences, University of Missouri-Kansas City, 5100 Rockhill Road, Kansas City, MO 64110, USA. Fax: +1 816 235 1503. E-mail address: idnurma@umkc.edu (A. Idnurm). Fungal Genetics and Biology 48 (2011) 685–695 Contents lists available at ScienceDirect Fungal Genetics and Biology journal homepage: www.elsevier.com/locate/yfgbi