Agrobacterium tumefaciens-mediated transformation for targeted disruption and over expression of genes in the poplar pathogen Sphaerulina musiva By A. J. Foster 1 , M. J. Morency 1 , A. Séguin 1 and P. Tanguay 1,2 1 Canadian Forest Service, Natural Resources Canada, Laurentian Forestry Centre, 1055 du P.E.P.S., P.O. Box 10380, Stn. Sainte-Foy, Quebec, QC G1V 4C7, Canada; 2 E-mail: philippe.tanguay@NRCan.gc.ca (for correspondence) Summary Sphaerulina musiva causes both leaf spots and cankers on poplar. Leaf spots can lead to defoliation and cankers on branches and primary stems can lead to stem breakage and tree mortality. The recent availability of both the S. musiva and Populus trichocarpa genomes offers a great opportunity to study host–pathogen interactions. To better understand the factors involved in S. musiva pathology, we present a strat- egy for the transformation of this species using Agrobacterium tumefaciens. Binary plasmids were generated with hygromycin B phospho- transferase (hph) flanked by upstream and downstream sequences of polyketide synthase-like (PKS-L1) gene to generate targeted gene disruptants by homologous recombination. Plasmids were also constructed for constitutive expression reporter genes eGFP and mCherry to help with histological characterization of the pathogen during infection. Gene knockouts were identified by PCR and confirmed by sequenc- ing and Southern blotting. No significant differences were observed in melanin production between PKS-L1 disruptants and wild type iso- lates. Colonies expressing reporter genes were identified by fluorescent stereomicroscopy. This method is a promising tool for the characterization of pathogen genes through reverse and forward genetics and for introducing markers for histopathological study. 1 Introduction There are numerous species of poplar with important roles in forest ecology, industry and research, such as Populus tricho- carpa, the first model tree species to have its genome sequenced (Tuskan et al. 2006). Commercially, poplars are used for raw wood material in construction and pulp and paper production and are being studied for their potential use in the gen- eration of biofuels (reviewed in Polle et al. 2013). However, the commercial utilization of poplar is not without difficulty. The fungal pathogen Sphaerulina musiva (Peck) Quaedvlieg, Verkley and Crous (formerly Septoria musiva) causes both leaf spot and stem canker in Populus spp. (Bier 1939; Ostry and McNabb 1985). Within poplar plantations, leaf spots can result in defoliation while stem cankers make the stems and branches more susceptible to wind breakage. Stem canker caused by S. musiva is regarded as the most serious disease of hybrid poplar plantations in North America (Long et al. 1986; Spielman et al. 1986; Royle and Ostry 1995; Feau et al. 2010). The genome of S. musiva was released publicly in 2010 (DOE Joint Genome Institute: http://genome.jgi-psf.org/Sepmu1/Sepmu1.home.html). Relatively little is known about the molecular interactions between poplars and S. musiva during infection, but the public availability of sequenced genomes for both the host and pathogen provides an excellent starting point for future studies utilizing RNAseq platforms. However, a large number of putative virulence factors cannot be functionally annotated (Ohm et al. 2012). Agrobacterium tumefaciens-mediated transformation (ATMT) has successfully been used to generate transfor- mants and gene disruptants in many species of filamentous fungi (reviewed in Michielse et al. 2005) including Mycosphae- rella graminicola, a species related to S. musiva (Zwiers and De Waard 2001). Here, we provide a method for the characterization of S. musiva genes through disruption by homologous recombination and overexpression via ATMT based on the method originally described by De Groot et al. (1998) and modified by other researchers (reviewed in Michielse et al. 2005). Polyketides are a large class of secondary metabolites produced in bacteria, fungi and plants. Polyketides are a broad class of chemicals including mycotoxins like Cochliobolus heterostrophus T-toxin, an important plant virulence factor (Baker et al. 2006) and pigments, such as melanin (Hopwood 1997). Melanin is produced by a wide variety of fungal species and is thought to play a critical role in protecting the fungi from abiotic stresses such as UV irradiation (Wang and Casadevall 1994) and biotic stresses such as enzymatic lysis (Hyakumachi et al. 1987). To validate the efficiency of ATMT to cause disruption in S. musiva, a gene putatively involved in the biosynthesis of 1,8-dihydroxynaphthalene (1,8-DHN) melanin was selected for target disruption. This gene was chosen as it had the poten- tial to block the production of melanin pigments, which could allow for visual confirmation of gene disruption. Targeted disruption of melanin biosynthesis genes in other filamentous fungi resulted in the presence of albino colonies in culture, which were easily differentiated from non-transformed isolates (Kawamura et al. 1997, 1999; Zhang et al. 2003; Sugui et al. 2005). We identified a putative polyketide synthase (S. musiva protein ID 57748; PKS-L1) from the S. musiva genome using BLASTP analysis with C. heterostrophus PKS18 (GenBank accession: AAR90272), which has been identified as a conserved enzyme involved in melanin biosynthesis across a selection of Dothideomycetes genomes (Ohm et al. 2012). However, PKS- L1 shares only a 52% amino acid identity match with C. heterostrophus PKS18 and therefore may not be the gene’s true homologue. PKS-L1 is located on the genome 6.5 kb upstream of a gene (S. musiva protein ID 36082) that annotates to a scytalone dehydratase, which is known to be involved in melanin biosynthesis (Kubo et al. 1996). Received: 22.8.2013; accepted: 31.10.2013; editor: Y. Balci For. Path. 44 (2014) 233–241 doi: 10.1111/efp.12086 © Her Majesty the Queen in Right of Canada 2014 Reproduced with the permission of the Minister of Natural Resources Canada. http://wileyonlinelibrary.com/