Fungal hyphae—and in some cases, spores—are multi-nucleate. During genetic transformation of these spores or mycelia, only one nucleus generally receives the transferred T-DNA generating heterokaryotic colonies. Characterization of genetic changes, such as the effects of gene disruption in the transformants, requires purified homokaryotic lines. Hyphal tip transfer has conventionally been used to isolate homokaryons. We developed an alternative method for purification of fungal homokaryons from transformed heterokaryotic lines of Sclerotinia sclerotiorum. Ultrasound pulses were used to generate bi-septate, unicellular hyphal fragments that regenerate under selection to produce homokaryotic lines that can be easily identified using colony PCR. This technique facilitates the purification of transformed lines, which allows for routine genome manipulation, and should be adaptable for other filamentous fungi.

Introduction

Functional characterization of genes requires the development of mutant lines that disrupt or precisely alter the function of the resultant protein. In this context, simple and reliable genetic transformation systems are required. Traditionally, fungal spheroplasts or protoplasts were transformed by electroporation (1,2) or other means such as polyethylene glycol (3). However, protoplasts can be difficult to produce in some species and the activity of cell wall–degrading enzyme varies between lots and thereby requires ongoing optimization (4,5). As such, methods for the direct transformation of intact fungal mycelia or spores, such as biolistic transformation (6,7), are more desirable, de Groot and colleagues (8) reported the first use of Agrobacterium tumefaciens, historically used for plant transformation, as a vehicle for the introduction of DNA into fungi. This method is 100–1000 times more efficient than conventional transformation approaches and has been used with many filamentous fungal species (9,10).

Transformation of filamentous fungi often results in the uptake of the introduced DNA by only one of the nuclei, which leads to the formation of heterokaryons—cells in which two or more types of nuclei coexist. In addition, the plasmid DNA can integrate into the chromosome by either homologous or ectopic recombination (11). To perform reliable genetic analyses, homokaryotic clones must be isolated. In Neurospora crassa, this was achieved through multiple rounds of single spore isolation (12). The hypha of ascomycetes usually contains a single nucleus near the tip (13,14) and therefore hyphal tip transfer can be used for purification of genetic clones (15). Three to five rounds of hyphal tip transfer are usually required to isolate pure homokaryons and only trained individuals with excellent motor skills can perform this technique.

Sclerotinia sclerotiorum is an important plant pathogen that can infect over 400 different hosts, including important crops (16). Protocols for transformation of ascospores and mycelia using A. tumefaciens have been established (17,18); however, these tissues are multi-nucleate and methods to isolate homokaryons are lacking. The carpogenic production of homokaryotic ascospores requires 4–40 weeks based on the isolate (19). Here we provide an alternative technique for homokaryon isolation that uses ultrasound pulses to generate bi-septate, unicellular structures. Clones derived from mycelial fragments having a single nucleus are then identified using colony polymerase chain reaction (PCR).

Materials and methods

Fungal transformation and detection of homo- and heterokaryons

Sclerotinia sclerotiorum (Lib.) de Bary strain 1980 (No. 18683, ATCC, Manassas, VA, USA) was propagated in minimal salt–glucose (1% w/v) (MS-glu) broth as per Li et al. (20). Mycelia were transformed using A. tumefaciens strain AGL1 harboring the pCB301 binary vector (21) containing a MAP kinase gene (Smk3, SS1G_05445.1; www.broad.mit.edu/annotation/genome/sclerotinia_sclerotiorum), which was disrupted with a transposon carrying a hygromycin resistance cassette (hph). The transformation was carried out as per Liberti et al. (18). Briefly, mycelia grown in MS-glu media for 4 days in the dark were blended in a Waring blender (Model no. 33BL73; Torrington, CT, USA) for 10 s, pelleted by centrifugation and resuspended in 2 mL induction medium (IM) per 0.5 g mycelial wet weight. A. tumefaciens AGL-1 containing the transformation construct pCB301-Smk3υ (Figure 1) was grown in minimal media for 1 day at 27°C with aeration. This culture was used to inoculate IM containing kanamycin (50 µM) and acetosyringone (200 µM) and grown to an OD₆₀₀ of 0.3. Blended mycelia was diluted 1/100 in IM and mixed with an equal volume of A. tumefaciens, spread onto a cellophane membrane overlaid onto co-cultivation medium and incubated in the dark at 20°C for 3 days. Filters were transferred to MS-glu agar medium containing hygromycin (100 µg/mL), cefotaxime (200 µM), and moxalactum (100 µM) and incubated at 20°C for 3 days. Fungal colonies that were resistant to hygromycin after three rounds of subculturing were tested by PCR. Plugs containing colonies were transferred to minimal salt–glucose–hygromycin (MS-glu-hyg; 100 µg/mL) liquid medium and incubated as above for 3 days. Nucleic acids were extracted by transferring a 500 µL wet volume of mycelia to a 1.5 mL microcentrifuge tube and centrifuging at 13000 rpm for 2 min. The supernatant was removed and the tissue ground for 15 s using a pestle. Subsequently, 400 µL extraction buffer (200 mM Tris-HCl pH 8.0, 250 mM NaCl, 25 mM EDTA, 0.5% SDS) was added and the solution mixed with a vortex for 5 s and then centrifuged at 13000 rpm for 1 min. Three hundred microliters of the supernatant were transferred to a tube containing 300 µL isopropanol. The DNA precipitated at room temperature for 2 min followed by centrifugation for 5 min at 13000 rpm. The pellet was washed once with 75% ethanol, air-dried for 5 min, and dissolved in 50 µL ddH₂0. PCR was used to test for (i) homologous integration of the Smk3υ construct into the Smk3 gene and (ii) whether the resultant colony contained homo- or heterokaryotic mycelia. Homologous integration was examined using two sets of primers complementary to the hph cassette and either the 5′ or 3′ untranslated region (UTR) of the Smk3 gene, both of which were outside of the region used to construct pCB301-Smk3υ. The first combination consisted of Pmod Seq R (5′-GAGCCAATATGCGA-GAACACCCGAGAA-3′) and Smk3 5′ (5′-CCTCAACCTCAACCAACCTCAA-3′), while the second consisted of Pmod Seq F (5′-GCCAACGAC-TACGCACTAGCCAAC-3′) and Smk3 3′ (5′-TCGGGAATCTTGTATCATACGCTTA-3′). The presence of non-recombinant nuclei containing the wild-type Smk3 gene was evaluated using primers complementary to the 5′ UTR and 3′ UTR, again outside of the region used to construct pCB301-Smk3υ. The product amplified from the 5′ flanking region was sequenced to confirm that the construct had integrated properly. Amplification of a region of the SsNep1 gene (encoding the necrosis and ethylene-inducing peptide 1) (SS1G_03080.1; www.broad.mit.edu/annotation/genome/sclerotinia_sclerotiorum) using the SsNep1 F (5′-GGAATTCCATATGCTCCA GTCGAGGG-3′) and SsNepl R (5′-CC GCTCGAGTTAGATTTGTGC-CTCTG-3′) primers served as control for the PCR.