Proc. Nati. Acad. Sci. USAVol. 85, pp. 865-869, February 1988Genetics

Gene transfer system for the phytopathogenic fungusUstilago maydis

(DNA transformation/hygromycin B/corn smut)

JUN WANG*, DAVID W. HOLDEN*, AND SALLY A. LEONGtttPlant Disease Resistance Unit, U.S. Department of Agriculture-Agricultural Research Service, and *Department of Plant Pathology, University of Wisconsin,Madison, WI 53706

Communicated by Ira Herskowitz, October 13, 1987 (received for review June 14, 1987)

ABSTRACT A selectable marker for transformation wasconstructed by transcriptional fusion of a Ustiago maydis heatshock gene promoter with the hygromycin B phosphotransfer-ase gene of Escherichia coli. U. maydis was transformed tohygromycin B resistance by polyethylene glycol-induced fusionof spheroplasts following exposure to plasmid DNA that car-ried the marker gene. Transformation frequencies of 50 and1000 transformants per ,ug of DNA per 2 x 107 spheroplastswere obtained for circular and linear vector DNA, respec-tively. In the majority of transformants, the vector wasintegrated at a single chromosomal site, in either single copy ortandem duplication, as determined by Southern hybridizationanalysis of electrophoretically separated chromosomes and ofrestriction-endonuclease-cleaved DNA. The predominant form(82%) of vector integration was by nonhomologous recombi-nation; the remainder carried the plasmid at the homologousheat shock gene locus. No evidence for gene conversion or genereplacement was obtained in 28 transformants. Hygromycin Bphosphotransferase activity and resistance to hygromycin Bwere roughly correlated with the copy number of the inte-grated vector at the homologous location. Transforming DNAwas stably maintained during mitosis and meiosis. This trans-formation procedure and associated vector should permit thecloning of genes by direct complementation in U. maydis.

Research on the basidiomycete Ustilago maydis, a pathogenof corn, has deepened our understanding of the genetics andbiochemistry ofDNA replication, repair, and recombination(1, 2). This fungus can be cultured with ease on simple,chemically defined medium, and the yeast-like, uninucleatehaploid stage of growth facilitates genetic analysis. U.maydis infects corn and forms smut galls, plant neoplasticstructures. Due to the rapid expansion of knowledge of themolecular biology and genetics of corn, U. maydis shouldprove to be valuable for molecular studies of host-parasiteinteractions. Studies on this fungus may also provide modelsfor research on other basidiomycete phytopathogens includ-ing the cereal smuts and rusts. These fungi are causativeagents of many severe plant diseases that can result insignificant crop losses (3).The long-term goal of our work is to elucidate the struc-

ture, regulation, and role of genes involved in phytopatho-genic and saprophytic growth of U. maydis. Molecular-genetic analysis of these genes and others requires thedevelopment of a gene-transfer system. Genetic transforma-tion in U. maydis based on kanamycin resistance has beenreported by Banks (4); however, only low-frequency, unsta-ble transformants were obtained. Here we report the estab-lishment of an efficient and reproducible DNA transforma-tion system for U. maydis based on resistance to hygromy-cin B (HygB), an antibiotic that has been successfully

utilized as a selective agent in a variety of organisms rangingfrom yeast to mouse cells (5-9).

MATERIALS AND METHODSStrains and Media. Escherichia coli HB101 (F- hsdS20-

(rB ,mB ) recA13 ara-14 proA2 leu-6 lacY gal K2 rpsL20(Str)xyl-S mtl-i supE44 g-) was employed. U. maydis strainsobtained from R. Holliday (National Institute for MedicalResearch, Mill Hill, London) were 518(a2b2), 521(albl),227(adel-1, metl-2, narl-6, rec2-1, a2b2), and 288(panl-1,inol-3, narl-i, recl-i, aibi). Other U. maydis strains werenatural isolates collected in Wisconsin. Minimal, complete,and plate-mating media used in fungal studies were asdescribed (1) except that Casamino acids were used at aconcentration of 1% in the plate-mating medium.

Chemicals and Reagents. HygB was a gift of Eli Lilly andwas later purchased from Calbiochem. Novozym 234 wasobtained from Novo Laboratories (Wilton, CT). Dimethylsulfoxide was from EM Science (Cherry Hill, NJ). D-Sorbitol, PEG 3350, and other chemicals were from Sigma.DNA Isolation and Manipulation. DNA was isolated from

the fungus by the method of Specht et al. (10). For analysisof large numbers of transformants, a protocol for rapid DNAisolation from spheroplasts (11) was adopted. DNA hybrid-ization was carried out as described by Amasino (12) exceptthat PEG and NaCl were omitted from the hybridizationbuffer. Orthogonal-field-alternation gel electrophoresis(OFAGE) was performed essentially according to themethod of Carle and Olson (13).

Preparation of Spheroplasts. Haploid cells of U. maydiswere incubated at 28°C in a 250-ml Erlenmeyer flask con-taining 40 ml of Holliday's complete medium until thedensity had reached 2 x 107 cells per ml (OD6. 1.0-1.2).These early logarithmic-phase cells, consisting mostly ofactively budding cells, were harvested by centrifugation at1100 x g for 8 min in a table-top centrifuge and resuspendedin 20 ml of a freshly prepared solution of 5 mM EDTA, pH8.0/25 mM 2-mercaptoethanol. After gentle shaking at roomtemperature for 20 min, cells were collected by centrifuga-tion and resuspended by brief Vortex mixing in 10 ml ofbuffer I (1 M sorbitol/50 mM sodium citrate, pH 5.8). Afilter-sterilized stock solution of Novozym 234 (lot PPM86,100 mg/ml in buffer I) was added to the suspension to give afinal enzyme concentration of 0.25%. Spheroplast formationwas monitored with a phase-contrast microscope. Within2-5 min at room temperature, 20-30% of the cells wereconverted to spheroplasts. At this time spheroplasts werecollected by centrifugation at 140 x g for 8 min in a table-topcentrifuge. Spheroplasts continued to form during centrifu-gation. The pellet, containing mostly spheroplasts, was

Abbreviations: HygB, hygromycin B; OFAGE, orthogonal-field-alternation gel electrophoresis.tTo whom reprint requests should be addressed.

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washed twice with 10 ml of buffer 11 (25 mM CaC12/25 mMTris1HCl, pH 7.5/1 M sorbitol). Spheroplasts were diluted to108 cells per ml in buffer II. Between 5 x 10' and 7 x 108spheroplasts were routinely obtained from a 40-ml culturewith this protocol, corresponding to a yield of >60%.DNA Transformation. Spheroplasts (2 x 107 spheroplasts

in 200 1l of buffer II) were incubated with 0.5-1 ,ug ofplasmid DNA at room temperature (20'C-260C) for 20 min ina mixture containing 2 1L of 2-mercaptoethanol (final con-centration 110 mM) and 50 A1 of 66% (wt/vol) PEG 3350 in25 mM CaCl2/25 mM Tris1HCl, pH 7.5. Then, a total of 2.5ml of 66% (wt/vol) PEG 3350 in 25 mM CaCl2/25 mMTris-HCI, pH 7.5, was added in a stepwise manner asfollows. First one drop was added, then 0.5 ml was added,and finally the remainder of the solution was added andgently shaken to mix, with an intermission of 1 min betweenadditions. After an additional 15 min of incubation at roomtemperature, the spheroplast-DNA suspension was dilutedto a volume of 35 ml with buffer II by the sequential additionof 1 ml, of 5 ml, and followed by the remainder of the buffer.The spheroplasts were collected by centrifugation as beforeand resuspended in 0.2 ml of 2 x liquid complete mediumcontaining 1 M sorbitol and shaken gently (100 rpm) for 3 hrat room temperature. No evidence for cell division wasobtained during this period. The spheroplasts were spreadwith a glass rod onto the surface of a two-layer selectivemedium plate containing a 12-ml lower layer (completemedium in 1 M sorbitol and HygB at 700 ,ug/ml) and a 12-mltop layer (complete medium in 1 M sorbitol). The top layerwas poured immediately before the transformed sphero-plasts were spread. Any residual liquid on the surface of theplate was removed with a sterile cotton swab before spread-ing the protoplasts. The plate was incubated at 28°C, andresistant colonies appeared within 3-4 days. The regenera-tion frequency of spheroplasts after the above treatment was20-30% on antibiotic-free medium. Less than 0.01% of thespheroplast population was observed to regenerate if thespheroplast suspension was pretreated with water.HygB Phosphotransferase Assay. Fungal cells were first

made into spheroplasts, as described above, and then dis-rupted by sonication in ice-cold 10 mM Tris-HCl/10 mMMgCl2/25 mM NH4Cl/0.6 mM 2-mercaptoethanol, pH 7.8.The S30 supernatant, obtained after centrifuging the soni-cated cells at 30,000 x g for 45 min, was assayed for enzymeactivity by the method of Haas and Dowding (14) with HygBas substrate. Enzyme activity was linear over 10 min with upto 0.5 ,g of protein per ml under these assay conditions.

Genetic and Cytological Procedures. U. maydis diploidstrains were constructed as described (1). Pathogenicitytests and genetic crosses were carried out on the maizecultivar Golden Bantam according to standard methods (1,15). To analyze the random segregants of meiosis, thefollowing procedure was adopted: diploid teliospores frommaize smut galls were rinsed briefly with ether to killvegetative cells and then with water before suspending in asolution of 0.4% tetracycline. Teliospores were spread oncomplete medium plates and incubated at 28°C for 24-30 hr.The resulting haploid basidiospores were collected with amoistened cotton swab and suspended in water. This solu-tion was then spread on complete medium plates. After 2-3days of growth, colonies were replica-plated onto varioustest media plates.Mithromycin staining (16) was used to detect fungal nu-

clei.

RESULTSCharacteristics of Vector pHLL. A derivative of pUC12,

pHL1 contains a 3-.3-kilobase (kb) HindIII insert that carriesthe bacterial HygB phosphotransferase gene (5) in transcrip-tional fusion with a U. maydis hsp7O gene promoter (Fig. 1).

pSL7

Bg

X 5.9kb Hligation PL

pSL1 1

BRI B

Baum fagment from pLG90containing hygB gen coding

sequence

Bg

BaI31

Bg Linker

1 ligationDTtX-J6 kb~ HP11PL X j P(5.0kbHPL-~~P~AuY// Ligation Bg8 Bg

pHL1 pDWH10

FIG. 1. Construction of transformation vector pHL1. pSL7consists of a 4.6-kb HindIII fragment of U. maydis genomic DNAinserted into the HindIII site of pUC12. This insert contains amember of the hsp7O gene family of this fungus (D.W.H. andS.A.L., unpublished data). The two internal Bg1 II fragments ofpSL7 were deleted to give rise to pSL11. pDHW10, a deletionderivative of pSL11, was obtained by linearization at the unique BgIII site, treatment with BAL-31 nuclease, and ligation to Bgl IIlinkers. The endpoint of this deletion is located -25 base pairsdownstream of the transcription initiation site of this hsp7O gene(D.W.H. and S.A.L., unpublished data). pHL1 was constructed byinsertion of the 1.05-kb BamHI fragment, carrying the HygB phos-photransferase gene coding sequence of pLG90 (5), into the Bgl IIsite of pDHW10. Among the unique restriction sites in pHL1 are SstI and BamHI (in the polylinker) and Xho I (in the U. maydis insert).A detailed restriction map of pSL7 will be presented elsewhere(D.W.H. and S.A.L., unpublished data). U. maydis DNA sequenceson the 5' and 3' side of the HygB phosphotransferase gene codingsequence are 1.25 kb and 1.0 kb long, respectively. H, HindIII; R1,EcoRI; X, Xho I; Bg, Bgl II; and PL, polylinker. pUC12 is indicatedby the thinnest line; the HygB phosphotransferase gene codingsequence is represented by a medium-thick line; and U. maydisDNA is represented by the thickest line.

This plasmid confers ampicillin but not HygB resistance in E.coli, and HygB resistance in U. maydis. HygB is particularlyuseful as a selection for Ustilago as it completely inhibitsgrowth of this fungus at 150 ,g/ml, spontaneous resistance islow, and background growth is marginal (unpublished obser-vations). In contrast, U. maydis is highly resistant to therelated drugs G418, neomycin, and kanamycin.

Transformation of U. maydis. Unless specified otherwise,the transformation experiments were carried out with U.maydis 518, a prototrophic, wild-type strain. All of thefungal strains we have examined have shown similar sensi-tivity to HygB. The frequency of spontaneous resistance tothe antibiotic at a concentration of 250 jg/ml, a level lowerthan the one used in the selective medium for transformationexperiments, was <10' for whole cells and spheroplasts.U. maydis isolates originating from a variety of sources havebeen transformed successfully with pHL1. Utilizing thestandard transformation protocol, we have routinely ob-tained >500 stable transformants per jug of linearized pHL1DNA per 2 x 107 spheroplasts. The numbers of transfor-mants obtained were proportional to the amount of plasmidused when 0.1-1 ,ug of DNA was added.To assess the role played by the hsp7O gene promoter in

the transformation vehicle, two additional vectors, pHL2and pHL1-B, were constructed. pHL2 bears the same fea-

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tures as pHL1 except that the orientation of the BamHIinsert, which contains the HygB resistance gene, was in-verted relative to the hsp7O gene promoter. pHL2 failed totransform U. maydis to HygB resistance. Inversion of theHindIII fragment of pHL1 gave rise to plasmid pHL1-B,with the E. coli lac promoter of the vector directed awayfrom the HygB phosphotransferase gene. pHL1-B was func-tionally equivalent to pHL1 in transformation experiments.The protocol we employed for U. maydis DNA transfor-

mation shares some basic similarities with other fungal genetransfer systems. Three factors contributed greatly to theefficiency of transformation. First was the use of earlylogarithmic-phase cells to prepare spheroplasts. Sinceyounger or older cells could be converted into spheroplastsand regenerated at the same frequency as early logarithmic-phase cells, this difference in transformation frequency mustbe due to intrinsic physiological properties of early logarith-mic-phase cells. Second, linearization of pHL1, either at theXho I site (on the 5' side of the heat shock promoter) or inthe pUC12 polylinker sequence, increased transformationfrequency by up to 20-fold. This is in marked contrast toresults obtained with other filamentous fungi, where linear-ization of the plasmid usually has little or no effect ontransformation frequency (17). In Saccharomyces cerevisiaeimproved transformation has also been achieved with linearDNA and has been attributed to the recombinogenic natureof free ends of DNA (18). Third, the genetic background ofthe recipient strains also influenced the frequency of trans-formation. Compared to strain 518, strain 227 gave a trans-formation frequency that was lower by a factor of 50-100,whereas strain 288 consistently gave a 2- to 3-fold increase ofHygB-resistant colonies, perhaps because the red mutationexhibits increased spontaneous mitotic recombination (19).

Fate of Transforming DNA. Twenty-eight HygB-resistantcolonies arising on selective medium after transformationwith linear pHL1 were purified by three rounds of single-colony isolation under continuous drug selection. All of thecolonies contained uninucleate cells as revealed by mi-thromycin staining. No heterokaryons were detected. Totalcellular DNA was isolated from each of the 28 colonies, andSouthern transfers of undigested, HindIII- or EcoRI-digested DNA were hybridized with two probes: (i) theBamHI fragment of pLG90 containing the HygB phospho-transferase gene coding sequence was used to detect thepresence of the antibiotic resistance determinant, and (ii)

intact pHL1 was employed to determine the location as wellas copy number of the integrated plasmid.The 28 transformants displayed several common features.

(i) They all hybridized strongly with the BamHI fragment;DNA from untransformed recipient strains failed to hybrid-ize. (ii) Southern blots of undigested, sheared DNA failed toshow hybridization to bands other than sheared DNA, evenafter an extended period of exposure (data not shown). Thisargues against the presence of free monomeric plasmid in thetransformants. The integration pattern of selected U. maydistransformants is illustrated in Figs. 2 and 3. Two types ofintegration events were found: integration at the residentchromosomal site of the hsp7O gene by way of a singlehomologous recombination or integration elsewhere in thegenome, corresponding respectively to type I and type IIintegration in Aspergillus nidulans as described by Yelton etal. (20). No gene replacement or type III integration events(20) were detected.

It is evident from Fig. 3 that integration of the pHL1vector into the hsp7O locus by homologous recombinationdisrupted a large (13.2-kb) EcoRI fragment and gave rise totwo new EcoRI fragments of 7.2 kb and 7.4 kb. This type ofintegration is represented by transformant 2-4 (Fig. 2). Uponprobing with pHL1 (Fig. 2D, lane 3), the only other EcoRIfragment that hybridized was 4.6 kb long. This correspondsto the larger EcoRI fragment of the vector. When subjectedto HindIII digestion (Fig. 2B, lane 2), three fragments of 3.3kb, 2.7 kb, and 4.5 kb corresponding to vector fragments andthe recombinant HindIII genomic fragment carrying thehsp7O gene were revealed as expected. These data as well asthe intensity of hybridization signals suggests that a singlecopy ofpHL1 had integrated at the hsp7O locus in transform-ant 2-4. Transformant 4-1 (Fig. 2D, lane 4) also lacked thelarge 13.2-kb EcoRI fragment and possessed the same newEcoRI fragments characteristic of homologous recombina-tion. In addition, a third EcoRI fiagment of 1.4 kb, corre-sponding to the smaller EcoRI fragment of pHL1, wasobserved. These data can be best explained by a tandemintegration of two copies of the plasmid. The same hybrid-ization pattern of HindIII fragments displayed by transform-ants 2-4 and 4-1 when probed with the BamHI fragment orpHL1 (Fig. 2 A, lanes 2 and 3, and B, lanes 2 and 3) alsosupported this interpretation.Homologous integration was also suggested by hybridiza-

tion of the BamHI fragment probe to intact chromosomes

1 2 3 4 5 6 2 3 4 5 6 2 3 4 5 6 1 2 3 4 5 6

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FIG. 2. Southern hybridization analysis of selected HygB-resistant transformants. Approximately 5 ,ug of total genomic DNA or 2.5 ng ofpHL1 was digested with HindIII (A and B) or EcoRI (C and D) and electrophoresed in 0.5% agarose gels by using phage A DNA digested withHindIII as a size marker. Southern transfers of gels were hybridized with a radiolabeled 1.05-kb BamHI fragment encoding the HygBphosphotransferase gene from pLG90 (A and C) or pHL1 (B and D) and autoradiographed. In A and B, the lanes contain DNA as follows: 1,strain 518; 2, transformant 2-4; 3, transformant 4-1; 4, transformant 4-2; 5, transformant 4-5; and 6, pHL1. In C and D, the lanes contain DNAas follows: 1, pHL1; 2, strain 518; 3, transformant 2-4; 4, transformant 4-1; 5, transformant 4-2; and 6, transformant 4-5.

A

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H H XHXI I1 I

X HH X RIIl I I I I

>IfK%

H H XHX RI H11 I

RIH X-. I

X HHI I I

7.4kb 4.6 kb 7.2kb-01.4n <-

FIG. 3. Integration of pHL1 by homologous recombination into the U. maydis genome. The predicted structure of the hsp7O locus afterintegration of the vector by a single crossover at the Xho I site in pHL1. Abbreviations are as in Fig. 1. The 4.6-kb HindIII fragment thatencodes the hsp7O gene is located in the 13.2-kb EcoRI fragment of U. maydis genomic DNA and is indicated by the hatched box; thecorresponding homologous fungal DNA in pHL1 is indicated by the thickest line; the HygB phosphotransferase gene coding sequence indicatedby a medium-thick line; other fungal DNA is indicated by a thin line; and pUC12 is indicated by the thinnest line.

separated by OFAGE (Fig. 4). The probe hybridized tochromosome cluster XII-XIII, where the hsp7O gene used toconstruct the vector is known to be located (T. Kinscherfand S.A.L., unpublished data).The majority of the transformants possessed pHL1 at

locations elsewhere in the genome as represented by trans-formants 4-2 and 4-5 (Fig. 2). Unlike 2-4 or 4-1, transform-ants 4-2 and 4-5 retained the large 13.2-kb EcoRI fragment.In the case of 4-2 (Fig. 2 A, lane 4, B, lane 4, C, lane 5, andD, lane 5), the 3.3-kb HindIII fragment and 1.4-kb EcoRIfragment have been replaced by two new fragments suggest-ing the insertion of a single copy ofthe vector. For transform-ant 4-5 (Fig. 2B, lane 5), however, all three HindIII frag-

2 3 4

FIG. 4. Southern hybridization analysis of the electrophoretickaryotype of transformants. OFAGE was carried out essentially asdescribed by Carle and Olson (13) but with 2 x 109 protoplastsembedded in 5 ml of 2.5% low-melting agarose in 1 M sorbitol/0.5 MEDTA, pH 7.4. Chromosomal DNA was liberated from protoplastsby incubation of the agarose sheet in proteinase K at 1 mg/ml and1% N-laurylsarcosine, sodium salt, in 0.5 M EDTA (pH 7.4) at 50°Cfor 12 hr. Southern transfer was probed with the 1.05-kb BamHIfragment containing the coding region of the HygB phosphotrans-ferase gene of pLG90 and then autoradiographed. The lanes containthe chromosome complement as follows: 1, strain 518; 2, transform-ant 2-4; 3, transformant 4-1; and 4, transformant 4-2. (Left) Ethidiumbromide-stained gel. (Right) Autoradiogram of Southern transfer.

ments (2.7 kb, 3.3 kb, and 4.5 kb) were retained in additionto four new HindIII fragments, which implies vector inser-tion at two locations. Data for EcoRI digestion of transfor-mant 4-5 (Fig. 2 C, lane 6, and D, lane 6) DNA also supportthis interpretation.

In summary, 5 of 28 transformants examined by Southernanalysis resulted from homologous integration (type I integra-tion). Of the 5 transformants, one involved a single-copyinsertion, whereas 4 were the result of tandem insertion of thevector. The remaining 23 were generated by illegitimate recom-bination of pHL1 into the host genome (type II integration).Included in this group were single-copy insertions (3 transfor-mants) and tandem integration of the vector with or withoutadditional rearrangements or deletions (10 transformants ineach case). No evidence for type III integration (double-homologous recombination or gene conversion) was obtained.HygB Phosphotransferase Activity. HygB phosphotransfer-

ase activity was readily detected in transformed fungal cells,and the enzyme levels were roughly correlated with both thelevels of resistance to the drug and vector copy numberfortype I transformants (Table 1). No enzyme activity wasfound in the recipient strain.

Mitotic and Meiotic Stability of Transformants. Of the 28transformants subjected to Southern analysis, 3 with type Iintegration and 3 with type II integration were examinedfurther for stability of the transforming DNA. Purified singlecolonies of each transformant were grown in liquid complete

Table 1. HygB phosphotransferase activity of U. maydistransformants

Vector,no. of Specific activity, pmol/min

Extract source copies per ug of proteinStrain 518 0 <0.01 + 0.00Transformant 2-4 1 0.73 + 0.04Transformant 4-1 2 1.60 + 0.05Transformant 5-10 Multiple 2.50 + 0.08

All transformants carried the pHL1 vector at the homologoushsp7O locus. Enzyme activities were determined in triplicate and areexpressed as means + SEM.

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medium without HygB for -30 generations before spreadingon complete medium plates free of antibiotic. The resultingcolonies were transferred onto selective and nonselectivemedium to test for sensitivity to HygB. For each sample, 200colonies were scored. All of these retained the HygB-resistant phenotype.The meiotic stability of transformed U. maydis cells was

investigated by crossing four transformants of strains 518,2-1, 24, 4-1, and 4-2 with strain 521, a wild-type U. maydisisolate. Two hundred basidiospore segregants from eachcross were examined, and half of these were found to beresistant to HygB (data not shown). From each cross sixHygB-resistant segregants as well as six HygB-sensitivemeiotic segregants were subjected to Southern analysis. Nohybridization of the BamHI fragment probe was found in anyof the antibiotic sensitive segregants (data not shown).Moreover, the plasmid integration patterns of the HygB-resistant segregants remained unchanged when compared tooriginal parental transformants (data not shown).

Transformation of Diploid U. maydis. Utilizing the same

transformation procedure, we have also introduced pHL1into a diploid U. maydis strain D-2, which was constructedby fusing haploid strain 227 with 288. A low frequency of 10transformants per pug of plasmid DNA was obtained. Thetransformants were uninucleate as demonstrated by mithro-mycin straining, maintained the ability to grow on minimalmedium, and were solopathogenic-that is, they were ableto elicit corn galls after inoculation of host plants. Theteliospores recovered from these galls were germinated oncomplete medium and resulting basidiospore segregantswere examined for HygB resistance. Two hundred segre-

gants were checked for each of 4 transformants, and a 1:1numerical ratio of HygB-resistant/HygB-sensitive cells wasfound (data not shown).

DISCUSSIONWe have established an efficient and reliable DNA transfor-mation procedure for the phytopathogenic fungus U. maydisthat uses the drug HygB as a selective agent. The vectorpHL1 contains a transcriptional fusion of a U. maydis hsp7Ogene promoter and a structural gene encoding the HygB-resistance determinant. Several factors contributed to thehigh transformation frequencies that we obtained. Amongthese were the use of early logarithmic-phase cells in thepreparation of spheroplasts, the inclusion of 2-mercaptoeth-anol in the transformation mixture, and linearization of theplasmid vector DNA.hsp7O gene promoters from other eukaryotes have been

used in construction of selectable markers (6, 21, 22). Yeastand Drosophila hsp7O promoters have also been fused withthe E. coli B-galactosidase gene (23, 24). In both cases, a

substantial basal-level expression of the gene was observedunder normal growth conditions. Results from S1-protectionanalysis show that the U. maydis hsp7O gene employed inthe development of the selection marker is expressed atnormal growth temperatures (D.W.H. and S.A.L., unpub-lished data). Since pHL2 failed to transform the fungus toHygB resistance and pHL1B was as effective as pHL1, we

conclude that it is the hsp7O promoter fused to the HygBphosphotransferase gene that drives the expression of theantibiotic resistance determinant in transformants.

In all of the transformants exhibiting type I integration, thecrossover event always occurred within the U. maydis DNAsequence on the 5' side of the HygB phosphotransferasegene in the vector. Evidently, the linearization of pHL1 (atan Xho I site) promoted recombination at this region, or theinsertion of a bacterial DNA sequence somehow interferedwith homologous exchange between the DNA sequence onthe 3' side of the insert and its counterpart on the fungal

chromosome. This would also explain our failure to detect asingle case of type III integration among the transformantsanalyzed, since this type of integration requires a doublecrossover or gene conversion at the hsp7O locus. Anotherexplanation for the failure to see replacement of the wild-type hsp7O gene by the disrupted hsp7O gene (carrying HygBphosphotransferase gene within it) is that this hsp7O gene isessential for growth. Disruption of a cognate hsp7O gene ofS. cerevisiae is a lethal event (25). Transformation of pHL1into diploid U. maydis cells would make it possible toinvestigate such recessive lethal mutations.The integration pattern observed here in U. maydis, in

which pHL1 was integrated predominantly at chromosomalsites other than the hsp7O locus, resembles that found inNeurospora crassa and A. nidulans but differs from S.cerevisiae where all transformants are the result of homolo-gous recombination (17). This phenomenon may be markerdependent as has been shown in A. nidulans (26).

U. maydis transformants were stable mitotically and meiot-ically, whether they carried single or multiple copies of thevector. The data obtained for meiotic passage of vectorsuggest that it segregates as a single Mendelian trait whenintegrated at one location. No evidence for autonomousreplication of the transforming plasmid was obtained. Con-sidering the ease with which large numbers of U. maydisspheroplasts can be generated, it should now be feasible toclone genes directly by complementation in this fungus withthe methods described here.We thank Lucille Strassman and Steve Vicen for help in prepara-

tion of the manuscript and Thomas Kinscherf for preparation of theOFAGE gel. This work was supported by Grant 1 R01 GM33716 fromthe National Institutes of Health, the U.S. Department of Agricul-ture, and the Graduate School of the University of Wisconsin.1. Holliday, R. (1974) in Handbook of Genetics, ed. King, R. C. (Plenum,

New York), Vol. 1, pp. 575-595.2. Kmiec, E. B. & Holloman, W. K. (1986) Cell 44, 545-554.3. Saari, E. E. & Prescott, J. M. (1985) in The Cereal Rusts, eds. Roelfs,

A. P. & Bushnell, B. (Academic, Orlando, FL), Vol. 2, pp. 259-298.4. Banks, G. R. (1983) Curr. Genet. 7, 73-77.5. Gritz, L. & Davies, J. (1983) Gene 25, 179-188.6. Kaster, K. R., Burgett, S. G. & Ingolia, T. D. (1984) Curr. Genet. 8,

353-358.7. van den Elzen, P. J. M., Townsend, J., Lee, K. Y. & Bedbrook, J. R.

(1985) Plant Mol. Biol. 5, 299-302.8. Santerre, R. F., Allen, N. E., Hobbs, J. N., Jr., Nagaraja, R. &

Schmidt, R. J. (1984) Gene 30, 147-156.9. Turgeon, B. G., Garber, R. C. & Yoder, 0. C. (1987) Mol. Cell. Biol. 7,

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Genetics: Wang et al.

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