evidence for a ustilago maydis steroid 5 -reductase by for a ustilago maydis steroid 5 -reductase by...

Evidence for a Ustilago maydis Steroid 5�-Reductase byFunctional Expression in Arabidopsis det2-1 Mutants1

Christoph W. Basse*2, Christine Kerschbamer, Markus Brustmann, Thomas Altmann, andRegine Kahmann2

Institut fur Genetik und Mikrobiologie der Universitat Munchen, Maria-Ward-Strasse 1a, D–80638 Munich,Germany (C.W.B., C.K., M.B., R.K.); and Max-Planck-Institut fur Molekulare Pflanzenphysiologie, KarlLiebknecht Strasse 25, D–14476 Golm, Germany (T.A.)

We have identified a gene (udh1) in the basidiomycete Ustilago maydis that is induced during the parasitic interaction withits host plant maize (Zea mays). udh1 encodes a protein with high similarity to mammalian and plant 5�-steroid reductases.Udh1 differs from those of known 5�-steroid reductases by six additional domains, partially predicted to be membrane-spanning. A fusion protein of Udh1 and the green fluorescent protein provided evidence for endoplasmic reticulumlocalization in U. maydis. The function of the Udh1 protein was demonstrated by complementing Arabidopsis det2-1 mutants,which display a dwarf phenotype due to a mutation in the 5�-steroid reductase encoding DET2 gene. det2-1 mutant plantsexpressing either the udh1 or the DET2 gene controlled by the cauliflower mosaic virus 35S promoter differed from wild-typeColumbia plants by accelerated stem growth, flower and seed development and a reduction in size and number of rosetteleaves. The accelerated growth phenotype of udh1 transgenic plants was stably inherited and was favored under reducedlight conditions. Truncation of the N-terminal 70 amino acids of the Udh1 protein abolished the ability to restore growth indet2-1 plants. Our results demonstrate the existence of a 5�-steroid reductase encoding gene in fungi and suggest a commonancestor between fungal, plant, and mammalian proteins.

The basidiomycete Ustilago maydis is a facultativebiotrophic fungus that grows in a yeast form in cul-ture. The pathogenic life cycle is initiated when com-patible, haploid sporidia fuse and form a filamentousdikaryon, which is able to penetrate into host tissue(Snetselaar and Mims, 1993; Mills and Kotze, 1981;for review, see Banuett, 1995). Prerequisites for com-patibility are different a and b loci (for review, seeKahmann et al., 2000). The a locus encodes apheromone-based recognition system required formating (Bolker et al., 1992; Spellig et al., 1994). The blocus encodes the homeodomain proteins bE and bWthat trigger pathogenic development of the dikary-otic hyphae (Kronstad and Leong, 1990; Schulz et al.,1990; Gillissen et al., 1992).

The pathogenic phase is characterized by extensivehyphal growth within the infected plant tissue fol-lowed by karyogamy and differentiation of the dip-loid teliospores (Banuett and Herskowitz, 1996). Theplant reacts to the infection by tumor formation,which is associated with cell enlargement and prolif-eration (Snetselaar and Mims, 1994; I. Potrykus, per-

sonal communication). For more than four decades, ithas been speculated that phytohormones, like auxins,cytokinins, and gibberellins released by U. maydis,may trigger the morphological alterations in the host(Wolf, 1952; Turian and Hamilton, 1960; Mills andvan Staden, 1978; Sokolovskaya and Kuznetsov,1984; Basse et al., 1996). However, up to now there isno convincing evidence, to our knowledge, that thesecompounds are responsible for tumor induction. An-other class of hormones that have a wide distributionthroughout the plant kingdom and that control plantgrowth and development are brassinosteroids (Man-dava, 1988; for review, see Altmann, 1998; Clouseand Sasse, 1998). In Arabidopsis, det2-1 mutants de-fective in brassinolide biosynthesis have been iso-lated (Chory et al., 1991). They are characterized bydramatic dwarfism, dark green leaves as conse-quence of an increased number of chloroplasts in areduced cell volume, reduced male fertility and api-cal dominance, and delayed senescence and flower-ing (Chory et al., 1991; Li et al., 1996; for review, seeAltmann, 1998; Clouse and Sasse, 1998). det2-1 plantsare mutated in the DET2 gene, which encodes asteroid 5�-reductase with high similarities to mam-malian steroid 5�-reductases. To date, DET2 repre-sents the only cloned plant steroid 5�-reductase. Inmammals, this class of enzyme catalyzes theNADPH-dependent conversion of testosterone to di-hydrotestosterone, the potent androgen in male sexdifferentiation (for review, see Russell and Wilson,1994). Detailed metabolic investigations have shownthat the Arabidopsis DET2 protein catalyzes the 5�-

1 This work was supported by the Leibniz program of theDeutsche Forschungsgemeinschaft and through grant no. SFB369.

2 Present address: Max Planck Institute for Terrestrial Microbi-ology, Department of Organismic Interactions, Karl-von-Frisch-Strasse, D–35043 Marburg, Germany.

* Corresponding author; e-mail [email protected];fax 49 – 6421–178 –509.

Article, publication date, and citation information can be foundat www.plantphysiol.org/cgi/doi/10.1104/pp.001016.

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reduction of (24R)-24-methylcholest-4-en-3-one to(24R)-24-methyl-5�-cholestan-3-one, the third step ina four-step process leading to the formation ofcampestanol from campesterol (Li et al., 1996; Fu-jioka et al., 1997; Noguchi et al., 1999). In microor-ganisms, the only gene with similarity to steroid5�-reductases resides in the genome of Schizosaccha-romyces pombe (GenBank accession no. T39193), how-ever, evidence for a function as steroid 5�-reductaseis lacking.

Here, we describe the U. maydis udh1 gene, whichencodes a protein with high similarity to knownsteroid 5�-reductases from mammals and Arabidop-sis and provide evidence for its function by comple-mentation of Arabidopsis mutants homozygousfor det2-1.

RESULTS

Isolation of udh1

A number of U. maydis genes that are stronglyexpressed during the tumor stage of infected maize(Zea mays) plants have been identified by applyingthe method of differential display (Basse et al., 2000,2002). One of these U. maydis genes is udh1 (for U.maydis DET2 homolog). A fragment comprising theudh1 3� portion was used to identify genomic clonesfrom a U. maydis cosmid library. Sequence analysisof a genomic AgeI-HindIII fragment revealed anopen reading frame (ORF) of 1,164 bp encoding apredicted protein of 388 amino acids with an esti-mated molecular mass of 42.3 kD (Fig. 1). The ab-sence of introns in the udh1 ORF sequence and thepresence of a poly(A) site were demonstrated by theisolation of cDNA clones and reverse transcription(RT)-PCR analysis (Fig. 1; see “Materials and Meth-ods”). The deduced amino acid sequence of the udh1gene is most similar to those of type 1 steroid-5�-reductases from mammals (Andersson and Russell,1990; Lopez-Solache et al., 1996), the ArabidopsisDET2 protein (Li et al., 1996), and a putative steroidreductase from S. pombe (GenBank accession no.T39193), with 30% sequence identity to the mamma-lian and plant enzymes and 35% sequence identity tothe predicted S. pombe protein (Fig. 2A). For compar-ison, the Arabidopsis DET2 and rat type 1 steroid-5�-reductase (rS5R1) proteins share 41% sequenceidentity. In a number of positions, the Udh1 sequencematched either the mammalian or the Arabidopsissequence (Fig. 2B), implying a common ancestor pro-tein. In support of a function as steroid-5�-reductase,the Glu-311 residue of the Udh1 protein aligned withan invariant Glu residue of mammalian enzymes thatis absolutely required for activity. This residue is alsoconserved in DET2 but is replaced by Lys-204 in themutant det2-1 protein (Fig. 2A; Li et al., 1996). Fur-thermore, the Gly-52 residue of the Udh1 sequencematched the conserved Gly-34 residue of human type2 steroid-5�-reductase (hSR2) implicated in testoster-

one binding (Thigpen and Russell, 1992). In addition,Udh1 contains five (Arg-266, Pro-295, Gly-297, Asn-307, and Arg-380) of six conserved amino acids thatare part of a cofactor binding domain typical formammalian 5�-reductases (for review, see Russelland Wilson, 1994). The Udh1 amino acid sequenceremarkably differed in length from known 5�-steroidreductases by about an additional 130 amino acids(Fig. 2A). The additional amino acids are inserted insix stretches (I–VI) and comprise positions 1 to 25 (I),80 to 102 (II), 174 to 213 (III), 222 to 234 (IV), 271 to289 (V), and 330 to 348 (VI). Two of these stretches (IIand V) also partially exist in the S. pombe ORF se-quence (Fig. 2A) and emphasize the closer relation-ship between the fungal proteins. The U. maydisamino acid sequence contained eight potentialtransmembrane-spanning domains according to the

Figure 1. Nucleotide sequence and derived amino acid sequence ofudh1. Shown is the DNA sequence of the 1,722-bp genomic AgeI-HindIII fragment (GenBank accession no. AF502086) containing theudh1 gene. The translation start ATG codon is underlined and thestop codon of udh1 is indicated by an asterisk. Primers used forgenerating udh1-eGFP fusions are indicated by arrows. The deletionin �udh1 strains comprised the region from Phe-78 to Ala-358(boxed). The poly(A) site is indicated at position 1,453.

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program TMPRED, of which four aligned with thetransmembrane-spanning domains of rS5R1 pre-dicted by the same program (Fig. 2A). Two of theadditional transmembrane domains in the U. maydisORF sequence overlapped with stretches II and III.Thus, the U. maydis Udh1 seems to form a distinctclass among 5�-steroid reductases.

Expression of udh1

As judged by northern analysis the U. maydis udh1gene was expressed at similar levels during buddinggrowth of haploid sporidia and during filamentousgrowth of either dikaryotic hyphae resulting from across of haploid strains FB1 and FB2 or the solopatho-

Figure 2. Multiple alignment of Udh1 with other 5�-reductase se-quences. A, Udh1, U. maydis Udh1; SP5R, putative S. pombe 5�-reductase; DET2, Arabidopsis DET2; rS5R1, rat type 1 5�-reductase.Amino acids (numbered at the borders) dominating a column areboxed and shaded. Gaps have been inserted to maximize the numberof identities. Predicted transmembrane helices of Udh1 and rS5R1are in boldface and emphasized by bars (gray for Udh1 and black forrS5R1). The highly conserved Glu residue, which is altered in thedet2-1 mutant and corresponds to Glu-311 of Udh1, is marked by adot. Conserved positions within Udh1 stretch V and the respectiveregion of SP5R are boxed. B, Assignment of amino acids from theUdh1 sequence to matching positions from either the rS5R1 (top) orthe DET2 (bottom) amino acid sequence. Amino acids are numberedfrom the N to C terminus.

Figure 3. Expression analysis of udh1. A, U. maydis strains indicatedat the top were grown on CM charcoal for 2 d before RNA isolation.Five to 15 �g of RNA was loaded in each lane. B, Lane 1, RNA (15�g) from strain FB2 grown on CM charcoal for 2 d. Lanes 2 through9, RNA (5 �g; 2 �g in lane 9) prepared from infected maize tissuebetween 2 and 12 d after inoculation with a mixture of strains FB1and FB2. The northern blots were probed with a 32P-labeled udh1cDNA fragment (positions 1,019–1,449 in Fig. 1). A and B, Radio-active signals were quantified (boxed) and standardized (FB2 � 1) bycomparison with signals obtained by hybridization with the consti-tutively expressed U. maydis ppi gene. Staining with methylene bluereflected the amounts of total RNA loaded. The fungal and maizeribosomal RNA bands are indicated.

Rescue of Arabidopsis det2-1 Mutants by the udh1 Gene

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genic, diploid strain FBD11 (Fig. 3A). During biotro-phic growth, udh1 transcripts were detected 3 d afterinoculation, which coincides with an increase of fun-gal mass and the onset of visible tumor development(Fig. 3B). udh1 transcript levels were markedly in-creased 7 and 8 d after inoculation compared withthe barely detectable transcript levels of the consti-tutively expressed ppi gene during biotrophic growth(Fig. 3B). This reflects the small amount of fungalbiomass within infected leaves and emphasizes up-regulation of the udh1 gene during tumor formation,thus, explaining the identification of the udh1 gene bydifferential display analysis at this stage. Standard-ized in comparison with the ppi signal strength, udh1transcript levels were 26-fold increased 7 d postin-oculation compared with those in strain FB2 duringaxenic growth.

Localization of Udh1

Previous studies have indicated that human andrat 5�-steroid reductase isozymes reside in either themembranes of the endoplasmic reticulum (ER) or thenucleus depending on the tissue source of the en-zyme as demonstrated by immunological detection

and sedimentation analysis of enzyme activity (forreview, see Russell and Wilson, 1994). To localizethe U. maydis Udh1 protein, the green fluorescentprotein encoding eGFP gene was translationallyfused to the 3� end of udh1. Two independent U.maydis strains CL13/pugh1#8 and CL13/pugh1#10harboring ectopic insertions of the udh1:eGFP fusionconstruct under control of udh1 promoter sequenceswere analyzed by fluorescence microscopy. EGFPfluorescence mainly resided in the periphery ofnuclei, which were localized by 4,6-diamidino-2-phenylindole (DAPI) staining (Fig. 4). This is indic-ative for localization of the Udh1 protein in mem-branes of either the nucleus or the ER, which iscontinuous with the nuclear membrane. In addition,fluorescent patches were visible at the cell periphery,which are indicative for the typical ER network struc-ture in close vicinity to the plasma membrane (Fig. 4;Pichler et al., 2001; Wedlich-Soldner et al., 2002).

Complementation of Arabidopsis det2-1 Mutants

Previous investigations have demonstrated that theArabidopsis DET2 gene was functional in culturedhuman cells and, conversely, human 5�-steroid type

Figure 4. Localization of Udh1. U. maydisstrains CL13/pugh1#8 (column A), CL13/pugh1#10 (column B) harboring ectopic inser-tions of the udh1-eGFP fusion construct, andstrain CB35 (Basse et al., 2000; column C), ex-pressing the eGFP gene under the constitutivestrong otef promoter, were grown in YEPS me-dium and assayed by differential interferencecontrast (DIC) light microscopy (top row), andepifluorescence to detect eGFP (medium row)and DAPI (bottom row) distribution. The barrepresents 10 �m. Bottom, Schematic depictionof the udh1-eGFP fusion construct. udh1 ORFpromoter and 3� sequences are depicted bygrayed arrows and bars, respectively.

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1 and type 2 reductases could complement Arabidop-sis det2-1 mutants (Li et al., 1997). To investigatewhether udh1 encodes a functional 5�-steroid reduc-tase, the udh1 gene was introduced into the binaryvector pBAR-35S in such a way that expression wasdriven by the constitutive cauliflower mosaic virus(CaMV) 35S promoter (Odell et al., 1985). Homozy-gous Arabidopsis det2-1 and wild-type Columbia(Col-0) plants carrying the resulting plasmid pBUB1were generated by Agrobacterium tumefaciens-mediated transformation and selection for Basta re-sistance. Transgenic lines from two independenttransformation experiments were investigated infour series (Table I). All transgenic lines were testedfor the insertion of the udh1 gene into their genomesby PCR using primers derived from the CaMV 35Spromoter and a primer located in the 3�-untranslatedudh1 sequence and by a subsequent sequence analy-sis of the PCR products (Table I). This analysis con-firmed the transgene in most plants analyzed but alsorevealed its absence in a few homozygous det2-1plants, implying that Basta selection was not com-pletely tight. As expected, these plants showed adwarf phenotype (Table I). To confirm the geneticbackground of all transgenic lines, cleaved amplifiedpolymorphic sequence (CAPS) analysis based on theabsence of the MnlI site at position 610 in the det2-1ORF was employed (Fig. 5; Table I). Whereas PCRproducts amplified from Col-0 DNA contained aMnlI restriction fragment of 152 bp, a 193-bp MnlI

fragment was retrieved from amplified det2-1 DNA(Fig. 5). Three of the investigated descendants frominfiltrated homozygous det2-1 plants that resistedBasta selection were heterozygous (DET2/det2-1) andhave probably emerged from cross-pollination (TableI). In all cases, plants that carried the transgene werehomozygous with respect to the det2-1 allele.

Northern analysis was performed to determinewhether transgenic plants carrying the pBUB1 con-structs expressed the transgene (Fig. 6A). RNA wasextracted from leaves of det2-1/pBUB1#1, 2, 8, and 12plants (see Fig. 7D) and probed with the udh1 gene.RNA preparations from Arabidopsis Col-0 and U.maydis FB1 were included as control to exclude cross-hybridization of the udh1 gene with ArabidopsisRNA and to indicate the length of the U. maydis udh1transcript. Expression of the udh1 transgene was de-tected in all pBUB1 transformants (Fig. 6A, lanes1–4). The udh1 transcript size of pBUB1 transfor-mants was larger than that of the endogenous udh1gene expressed in U. maydis, implying differences intranscription start sites conferred by the differentpromoters.

Transformed plants were compared with homozy-gous det2-1 mutants and wild-type Col-0 plants atdifferent time points. Under the experimental condi-tions employed, Col-0 plants rarely displayed elon-gated shoots or flowers 42 d after sowing but nor-mally required an additional period of at least 10 dfor further development (Fig. 7A; Table I). At the

Table I. Analysis of transgenic Arabidopsis lines

Transgenic lines from two independent transformation experiments were investigated in four series.

LinesAnalyzed

LinesGenotypea Presence of 35S-

udh1/35S-DET2bFlower/Seed

DevelopmentcPhenotyped

Series 1det2-1/pBUB1 4/2e det2-1/det2-1 Yes All (3) after 50 d ACol-0/pBUB1 3 Det2/Det2 Yes All (3) after 50 d BCol-0 6 Det2/Det2 – 1 (0) after 50 d Cdet2-1 3 det2-1/det2-1 – None (0) after 50 d D

Series 2det2-1/pBUB1 9 det2-1/det2-1 Yes 4 (0) after 42 d ACol-0 8 DET2/DET2 – 2 (0) after 42 d Cdet2-1 4 det2-1/det2-1 – None (0) after 42 d D

Series 3det2-1/pBUB1 4/2/1e det2-1/det2-1 Yes All (3) after 48 d ACol-0 6 DET2/DET2 – None (0) after 48 d C

Series 4det2-1/pBUA1 5/7/1e det2-1/det2-1 Yes None (0) after 43 d Ddet2-1/pBDET2 6/1/3e det2-1/det2-1 Yes All (5) after 43 d ACol-0 4 DET2/DET2 – None (0) after 43 d C

a The genotypes of all transformants, all det2-1/DET2 heterozygous and det2-1 homozygous plants,and some wild-type Col-0 plants were confirmed by CAPS (see “Materials and Methods”). b Con-firmed by PCR/sequencing (see “Materials and Methods”). c The number of seed pods developed isindicated in brackets. d A, Accelerated stem elongation, flower and seed development, thin stems,and reduced size and number of rosette leaves. B, Like A, but increased stem thickness and size andnumber of rosette leaves. C, Wild-type Col-0 phenotype. D, det2-1 phenotype. e x/y/z, x is the numberof plants that corresponds to the genotype in column 3 and carries the transgene. y is the number ofheterozygous plants. z is the number of plants that is homozygous for det2-1, escaped Basta selection,and lacked the transgene.




same stage, control det2-1 plants showed a character-istic dwarf phenotype, small dark green leaves, andlacked flowers (Fig. 7B; Table I). All homozygousArabidopsis det2-1 lines harboring the udh1 trans-gene remarkably were no longer dwarfs. Instead,they displayed an elongated main stem and devel-oped flowers even earlier than wild-type Col-0plants, indicating accelerated development due tothe transgene (Fig. 7C; Table I). In addition, theseplants differed from Col-0 plants by a reduced sizeand number of rosette leaves (Fig. 7, A and C). Dur-ing further growth, transgenic plants showed accel-erated seed pod development and additionally dif-

fered from wild-type Col-0 plants by excessivelyelongated stems, which were unable to keep the plantin an upright position (Fig. 7, D and E; Table I). Thephenotype of Col-0 plants carrying pBUB1 resembledthe one of det2-1/pBUB1 transformants with respect

Figure 6. Expression analysis of transgenic Arabidopsis plants. A andB, RNA (0.3–1.8 �g) from the indicated plants (see “Materials andMethods”) and from U. maydis strain FB1 (20 �g) grown on CMcharcoal for 2 d was loaded. Radioactive signals were quantified(boxed) on both membranes and standardized (det2-1/pBUB1#1 �100) by comparison to signals obtained after subsequent hybridiza-tion with the Arabidopsis 32P-labeled actin probe (ACT2). A, RNAfrom wild-type Col-0 plants 62 d after sowing was included ascontrol. The northern blot was probed with the 32P-labeled AgeI-HindIII udh1 fragment. Arrowheads indicate the full-length udh1, thetruncated udh1 (udh1�N70), the U. maydis endogenous udh1, andthe actin transcript. The actin signal in the U. maydis lane is causedby cross-hybridization with the ACT2 probe. The different sizes of theudh1 and udh1�N70 transcripts were also reflected by faster migra-tion of the truncated udh1 transcript. B, RNA from two differentwild-type Col-0 and det2-1 mutant plants 61 d after sowing wasincluded as control. The northern blot was probed with the 32P-labeled DET2 EcoRV fragment. DET2 and ACT2 transcripts are indi-cated by arrowheads. Faintly detectable endogenous DET2 tran-scripts are indicated by asterisks.

Figure 5. Genotypic verification by CAPS analysis. A, Schematicrepresentation of CAPS analysis. Numbers refer to MnlI restrictionsites and respective fragments. The 152-bp MnlI fragment (boxed) isonly contained in wild-type fragments. B, Representative example ofCAPS analysis. PCR products obtained from amplification with prim-ers det3h/det3r were restricted with MnlI and subjected to Southernanalysis using the DET2 EcoRV fragment as probe (see “Materials andMethods”). Minor signals above the expected MnlI bands in thevarious lanes reflect incomplete digests of PCR products.

Basse et al.



to accelerated elongation of the main stem, flower,and seed pod formation. However, main stems weremore stable and the number of rosette leaves was lessseverely reduced than in the det2-1 transformants(Table I; data not shown).

To test whether the phenotype of pBUB1 transfor-mants relied on the Udh1 function or were a conse-quence of overexpression, transgenic det2-1 linesexpressing the DET2 gene under control of theCaMV 35S promoter were generated (see “Materials

Figure 7. Development of Arabidopsis pBUB1, pBDET2, and pBUA1 transformants. A to C, Plants from series 2, 42 d aftersowing. A, Wild-type Col-0. B, Mutant det2-1. C, Mutant det2-1 transgenic for pBUB1. Arrows indicate terminal flowers thatdeveloped in Col-0 and transgenic det2-1 plants. D and E, Plants from series 3, 61 d after sowing. D, Mutant det2-1transgenic for pBUB1 (det2-1/pBUB#1, 2, 8, and 12). Seed pods are indicated by arrows. E, Wild-type Col-0 plants. Seedpods have not yet developed. F to H, Plants from series 4, 43 d after sowing. F, Transformants det2-1/pBDET2#1, 2, 3, 6,7, and 8 are numbered from 5 to 10. det2-1 plants carrying the pBDET2 construct (5–10) display seed pod development(arrows). Homozygous det2-1 (1–3) and heterozygous DET2/det2-1 (4) plants that escaped Basta treatment but lacked thetransgene. G, Control wild-type Col-0 plants. H, pBUA1 transgenic det2-1 plants (2 and 3) are phenotypically indifferentfrom a det2-1 plant (1) that escaped Basta selection but lacked the transgene.




and Methods”). The genomic DET2 sequence wasintroduced into pBAR-35S and the resulting vectorpBDET2 was transformed into homozygous det2-1plants.

Transformants det2-1/pBDET2#1, 2, 3, 7, and 8were analyzed for DET2 expression (Fig. 6B). Thisindicated expression of the transgene in all cases,whereas expression of the endogenous gene wasbarely detectable in wild-type Col-0 or mutant det2-1plants (Fig. 6B, lanes 6–8). The expected position ofthe transgenic DET2 transcript was deduced from thefaint endogenous DET2 signal of Col-0 and det2-1RNA preparations. The presence of transcripts heter-ogeneous in size may reflect the use of differenttranscription start sites in transgenic plants. Stronglyelevated DET2 expression levels like in the det2-1/pBDET2#3 transformant were not reflected by addi-tional phenotypic alterations compared with the det2-1/pBDET2#1 transformant, which expressed thetransgene at a 10-fold reduced level (see below),pointing to saturation in the conversion of the DET2reaction product.

Like det2-1/pBUB1 transformants, all det2-1/pBDET2 transformants displayed long, thin stems,few rosette leaves of reduced size, and acceleratedflower and seed pod development (Fig. 7, F and G;Table I), indicating that overexpression of either theudh1 or the DET2 gene accounted for the morpholog-ical differences compared with wild-type Col-0plants.

The Udh1 amino acid sequence contained anN-terminal extension, which was absent in known5�-steroid reductases (see Fig. 2A). To assess whethera truncated ORF starting from the second in-frame-positioned Met-71 codon resulted in a functional en-zyme, the plasmid pBUA1 was constructed, whichwas derived from pBAR-35S by insertion of the 5�-truncated udh1 ORF. All of the five investigated det2-1/pBUA1 transformants were indistinguishable

from homozygous det2-1 plants (Fig. 7H; Table I).This implicates the functional importance of theN-terminal 70 amino acids for Udh1 activity. Fromthe relatively high expression levels of det2-1/pBUA1#13, 14, and 16 transformants determined bynorthern analysis (Fig. 6A, lanes 6–8), we infer thatthe inability of the truncated udh1 gene to restoregrowth in det2-1 transformants did not reflect theabsence of transgene expression.

Accelerated Growth in the T2 Progeny under DifferentLight Conditions

To further investigate whether a general stress re-sponse from repeated Basta selection promoted theobserved growth difference between wild-type andtransgenic plants, we analyzed the T2 progeny de-rived from selfing of the T1 det2-1/pBUB1#1 plant(series 1). According to Mendelian inheritance, three-fourths of the T2 progeny should carry at least onecopy of the transgene. Seedlings derived in the ab-sence of Basta selection from T1 det2-1/pBUB1 andCol-0 were transferred to individual pots and eitherkept in the greenhouse with a 12-h photoperiodicityor transferred to the phytochamber with a 16-h pho-toperiodicity. Of the investigated T2 progeny fromT1det2-1/pBUB1 plants, 78% displayed rescuedgrowth (Table II), indicative for the presence of thetransgene. As expected, some T2 plants (22%) dis-played a dwarf phenotype reminiscent of det2-1 mu-tant plants. All of these plants lacked the transgene(Table II), demonstrating a strict linkage between itspresence and rescued growth in the T2 progeny.Under long-day conditions development of trans-genic T2 plants was weakly favored compared withF1 Col-0 plants, as illustrated from accelerated stemand flower bud formation and from seed pod devel-opment between 31 and 35 d after sowing (Table II;Fig. 8, D and E). Furthermore, stems from 19 of 22

Table II. Analysis of transgenic Arabidopsis T2 lines under different light conditions

LinesAnalyzed

Linesc GenotypePresence of35S-udh1

Stem and FlowerDevelopment 31/33/40

d after Sowingd

Seed Pods35/40 d

after Sowinge

Series 5a

T2det2-1/pBUB1 25 (10) det2-1/det2-1 Yes 10 (14)/22 (3)/25 (0) 15/25T2det2-1/pBUB1 6 (6) det2-1/det2-1 No 0 (0)/0 (0)/5 (1) 0/0F1Col-0 8 (4) DET2/DET2 No 0 (0)/0 (1)/6 (1) 0/0

Series 6b

T2det2-1/pBUB1 22 (10) det2-1/det2-1 Yes 3 (14)/19 (3)/21 (1) 18/20T2det2-1/pBUB1 7 (7) det2-1/det2-1 No 0 (0)/0 (0)/5 (0) 0/0F1Col-0 10 (6) DET2/DET2 No 0 (3)/5 (3)/10 (0) 2/7a Plants were always kept in the greenhouse with a 12/12-h photo (15,000 lux)/dark period at 22°/15

to 18°C. b Plants were kept in the greenhouse for 17 d as described in (a) and then transferred to aphytochamber maintained with a 16/8-h photo (16,000 lux)/dark period at 22°/18°C. c In brackets,number of lines that were also analyzed with respect to the presence of the transgene by PCR and thegenotype by CAPS (see “Materials and Methods”). These all corresponded to the genotypes indicatedin column 3 and carried or lacked the transgene as indicated in column 4. d Number of floweringplants. In brackets, number of plants with elongated stems (�2 cm) and flower buds. e Number ofplants having seed pods developed.

Basse et al.



T2det2-1/pBUB1 plants exhibited lengths between 10and 19.5 cm compared with stems of the Col-0 vari-ety with only 1 of 10 having elongated to a length of10 cm 35 d after sowing (data not shown). Surpris-ingly, differences became more apparent upon expo-sure to a shorter photoperiod.

Although development of F1 Col-0 plants exposedto a 12-h photoperiod was attenuated compared withgrowth under long-day conditions (Table II; Fig. 8, Band E), development of det2-1/pBUB1 plants wascomparable under both light conditions, as illus-trated by early flowering and seed pod developmentbetween 31 and 35 d after sowing (Table II; Fig. 8, Aand D). This suggests that udh1 overexpression cancompensate for reduced light conditions and demon-strates the accelerated growth phenotype in the ab-sence of Basta selection. In addition, all det2-1/

pBUB1 plants developing under reduced lightconditions differed from Col-0 plants by decreasednumbers of rosette leaves and weakened stem stabil-ity, as already noticed in the T1 plants (Fig. 8A; datanot shown). Northern analysis of transgenic T2plants from series 6 demonstrated udh1 expression inall investigated plants. Although transgene expres-sion levels were severely reduced in the T2det2-1/pBUB1#9 plant, they still exceeded the faintly detect-able endogenous det2-1 expression levels (Fig. 9). TheT2det2-1/pBUB#11 and 12 plants displayed signifi-cantly reduced stem lengths and had not-yet-developed flowers despite udh1 transcript levels ashigh as in the fully developed T2det2-1/pBUB1#4plant (Fig. 8D; Fig. 9), indicating that extensive over-expression was not sufficient to promote acceleratedgrowth in all plants under the chosen conditions.

Figure 8. Development of T2det2-1/pBUB1plants (series 5 and 6) compared with F1Col-0plants. A to C, Plants from series 5, 33 d aftersowing. D to F, Plants from series 6, 33 d aftersowing. All presented T2det2-1/pBUB1 plants,four (B), and six (E) Col-0 plants, respectively,were analyzed with respect to the genotype andthe presence (A and D) or absence (B, C, E, andF) of the transgene. Growth conditions were asspecified in Table II. A and D, T2det2-1/pBUB1.B and E, Wild-type Col-0. C and F, T2det2-1/pBUB1 that displayed a dwarf phenotype andlacked the transgene. Each pot contains twoplants. The numbers in D refer to the plants usedfor northern analysis (see Fig. 9).




Similar results were obtained when udh1 transcriptlevels were determined by northern analysis inT2det2-1/pBUB1 plants that all displayed acceleratedgrowth under a 12-h photoperiodicity (see Fig. 8A).From six analyzed plants, five exhibited comparablelevels of strong overexpression, whereas accumula-tion of udh1 transcripts was 10-fold reduced in oneplant, indicating that moderate levels of overexpres-sion are sufficient for accelerated growth under re-duced light conditions (data not shown). In all cases,udh1 transcript levels significantly exceeded those ofthe endogenous det2-1 gene of T2det2-1/pBUB1plants and the DET2 gene of wild-type Col-0 plantsfrom the same series, respectively (data not shown).

Deletion Analysis of udh1 in U. maydis

To assess the function of the udh1 gene in U. maydis,the respective ORF was deleted in compatible, hap-

loid U. maydis strains (see “Materials and Methods”).Such strains were viable, microscopically indistin-guishable from wild-type cells, and not affected ingrowth (data not shown). Three independent �udh1strain combinations were assessed for pathogenicityin sweet corn (Zea mays var Early Golden Bantam).Tumors comparable in morphology and size withwild-type tumors developed in 47 of 76 plants afterinfection with �udh1 combinations, which compareswith tumor formation (33 of 42) by the respectivewild-type strains FB1 and FB2. Furthermore, the udh1gene was deleted in the haploid strain SG200, whichis solopathogenic due to the presence of a hybrid blocus composed of the compatible bE1 and bW2 genes.Again, the frequency of tumor development wascomparable in plants (sweet corn var Early GoldenBantam) inoculated with either SG200�udh1 (72 of 79plants) or SG200 strain (34 of 35 plants). In addition,the development of teliospores was unaffected by theudh1 deletion (data not shown). A reduction in viru-lence of �udh1 strains may possibly be reflected byattenuated biotrophic growth. We, therefore, inocu-lated mixtures of four independent SG200�udh1 andwild-type SG200 strains and determined their ratio inindividual tumors 10 d postinoculation (sporulationstage). Intensities of strain-specific fragments ampli-fied from �udh1 and wild-type strains were compa-rable when using DNA from preparations of tumors Ato C, whereas the �udh1-specific fragment wasslightly less efficiently amplified from DNA prepara-tion of tumor D (Fig. 10A). This indicated that �udh1mutants were able to proliferate at the same rate aswild-type strains during infection. To assess the pos-sibility of gene redundancy, Southern hybridizationwas performed under non-stringent conditions usingan udh1 fragment, which spanned the deleted ORFregion of the mutant allele (Fig. 10B). Althoughgenomic fragments of the expected sizes were de-tected in restricted chromosomal DNA from wild-typestrain SG200, distinct cross-hybridizing signals werenot detectable in restricted chromosomal DNA fromtwo independently generated SG200�udh1 strains.Under the chosen non-stringent hybridization condi-tions a 2,504-bp BamHI fragment, which overlappedwith only 43 bp of the udh1 probe, showed weakhybridization. This suggests that genes homologous toudh1 are either absent in U. maydis or have becomehighly diverged.

DISCUSSION

Our studies provide molecular evidence for theexistence of a 5�-steroid reductase in U. maydis,which, based on sequence alignment (see Fig. 2, Aand B), likely arose from a common ancestor betweenfungi, plants, and animals. However, the Udh1amino acid sequence is unique with respect to sixadditional protein stretches whose significance re-mains to be determined. Two of them overlapped

Figure 9. Expression of the udh1 gene in differently developed trans-genic Arabidopsis plants. RNA (1 �g) isolated from rosette leaves ofT2det2-1/pBUB1 plants from series 6, 33 d after sowing and from U.maydis strain FB2 (4 �g) grown on CM charcoal for 2 d was loaded(see “Materials and Methods”). Northern blots were probed with the32P-labeled AgeI-HindIII udh1 fragment, the DET2 EcoRV fragment,and the Arabidopsis 32P-labeled actin probe (ACT2) as loading con-trol. The ratio of quantified signals (udh1/ACT2) was calculated(arrowhead). Staining with methylene blue reflected the amounts oftotal RNA loaded. The full-length udh1, endogenous U. maydisudh1, ACT2, and the fungal and maize ribosomal RNA bands areindicated (arrowheads). Bottom, Developmental stage of investigatedplants 35 d after sowing: B, Flower bud; F, flower; S, seed pods. Thenumbers refer to stem lengths in centimeters.

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with predicted transmembrane domains, suggestingthat the Udh1 protein may be more tightly embeddedin the lipid bilayer than mammalian steroid 5�-reductases, which contain four putative transmem-brane domains. Membrane localization of monkeytype 1 and type 2 and human and rat steroid-5�-reductases have been described (Andersson et al.,1989; for review, see Russell and Wilson, 1994; Levyet al., 1995). For the mammalian proteins, evidencewas provided that they reside in either the ER or thenuclear membrane depending on the tissue investi-gated. However, a conclusive discrimination remainsdifficult because the perinuclear localization may re-flect the state of the ER, which is continuous with the

nuclear membrane (for review, see Russell and Wil-son, 1994). Fluorescence microscopy of single U. may-dis cells expressing an Udh1-eGFP fusion proteincorroborates the predicted localization of steroid-5�-reductases in membranes of the ER. Although theUdh1 sequence contains an ER retention element(HDEIL) at position 258, it is unclear whether this isfunctional because these elements are generally po-sitioned at the C-terminal end (for review, see Pel-ham, 1990). As an alternative, the Udh1 protein maybe associated with a protein targeted to the ER.

To determine whether udh1 encodes a functional5�-steroid reductase, transgenic Arabidopsis plantscarrying the udh1 gene under control of the constitu-tive CaMV 35S promoter were generated. In all cases,transformation with pBUB1 rescued the dwarf phe-notype of Arabidopsis det2-1 plants. Intriguingly, alltransformants displayed accelerated stem growthand flower and seed development compared withwild-type Col-0 plants. Because both pBUB1 andpBDET2 transformants promoted the same pheno-type in homozygous det2-1 plants, increased levels of5�-steroid reductase activity, which may encounterelevated levels of brassinosteroids, are responsiblefor accelerated development compared with wild-type Col-0 plants. This is consistent with the lowendogenous DET2 expression levels in wild-typeCol-0 plants (see Fig. 6B). The reduction in size andnumber of rosette leaves and the formation of thin,elongated steps in plants overexpressing 5�-steroidreductase may be the consequence of an imbalancedconcentration of brassinosteroids. The phenotypicdifferences observed in T1 transgenic plants did notresult from repeated Basta selection, because theaccelerated growth phenotype was stably inheritedto the T2 generation in the absence of Basta treat-ment. Under a 16-h photoperiod, individual devel-opmental differences of det2-1/pBUB1 plants werestill maintained in the T2 generation despite similaroverexpression levels, whereas under reduced lightconditions, accelerated growth dominated. The ob-servation that phenotypic differences between det2-1/pBUB1 and wild-type Col-0 plants were stronglyfavored under a 12-h photoperiod compared with a16-h photoperiod of the same light intensity sug-gests that enhanced levels of 5�-steroid reductaseactivity can compensate for low-light conditions,consistent with a role of brassinosteroids in photo-morphogenesis (Clouse, 1997). This conclusionagrees with the recent observation that Arabidopsisantisense lines of the CPD gene, which is implicatedin brassinosteroid biosynthesis, exhibit a more de-layed development compared with wild-type plantsif exposed to low-light conditions (Schluter et al.,2002). All investigated plants expressed transgenelevels that strongly exceeded those of the endoge-nous DET2 gene. This points to saturated levels of5�-steroid reductase activity and may explain why

Figure 10. Analysis of �udh1 strains. A, Detection of SG200 andSG200�udh1 strains in maize tumors. Chromosomal DNA (75 ng)isolated from tumors 10 d after inoculation with mixtures of SG200/SG200�udh1#12 (Tum A), SG200/SG200�udh1#15 (Tum B),SG200/SG200�udh1#20 (Tum C), and SG200/SG200�udh1#21(Tum D) was used as template for PCR with the primer combina-tions a and b (see “Materials and Methods”). Expected sizes of PCRproducts were the 622-bp fragment of the hph gene flanked byudh1 5�-ORF sequences (a) and the 643-bp udh1 fragment (b). Thenumbers above the lanes refer to the PCR cycles performed. Phagelambda (M) DNA (500 ng) digested with PstI was used as sizemarker. The sizes of the respective fragments are indicated inbasepairs. B, Non-stringent Southern analysis of wild-type SG200,SG200�udh1#20, and SG200�udh1#21 strains. ChromosomalDNA isolated from the strains indicated was restricted with eitherBamHI or SalI, and probed with a udh1 fragment spanning thedeletion of the mutant udh1 allele. Attribution of sizes to thehybridizing bands is indicated (arrowheads). The sizes of cross-hybridizing �PstI fragments are marked on the right.




differences in the strength of overexpression werenot reflected by an additional growth phenotype.

To verify the translational start codon of udh1, theDNA sequence encoding the region between Met-1 toMet-71 was deleted. This has indicated that the lessconserved N-terminal portion of the Udh1 protein,which corresponds to the N-terminal 58 amino acidsof the rat rS5R1 protein is crucial for activity. Afour-amino acid segment (VSIV) and the conservedGly residue (matching Gly-52 of Udh1) suggested insubstrate binding of the rat type 1 enzyme and hu-man type 2 enzyme, respectively (Thigpen and Rus-sell, 1992), are contained in this region.

Enzymes of steroid metabolism have been de-scribed in fungi. 17�-hydroxy-steroid-dehydrogenaseactivity was detected in Cochliobolus lunatus and S.pombe (Lanisnik et al., 1992; Dlugonski and Wilman-ska, 1998). 5�-Steroid reductase activity was detectedin Penicillium chrysogenum and Penicillium crustosum,which in culture were able to reduce the 4,5-doublebond in testosterone to give dihydrotestosterone (Ca-beza et al., 1999). Which role these enzymes play infungal growth or development remains to be shown.The finding that U. maydis �udh1 strains are unaf-fected in growth, virulence, and tumor induction ca-pacity rules out the possibility that this enzyme isresponsible for the synthesis of a plant hormone.However, an alternative mechanism for the �4–5 re-duction cannot be excluded. In mammals, steroid 5�-reductases exist as type 1 and type 2 isoforms impli-cated in catabolic and anabolic functions, respectively(Normington and Russell, 1992; for review, see Russelland Wilson, 1994), and the genome of Arabidopsisreveals the presence of a steroid 5�-reductase-like pro-tein (GenBank accession no. T51384). Because theUdh1 sequence was more similar to mammalian type1 enzymes, a possible catabolic function may be en-visaged. However, screening the U. maydis genomesequence (Bayer AG, Leverkusen, Germany) andSouthern analysis provided no evidence for the exis-tence of a second 5�-steroid reductase (data notshown). The S. pombe genome sequence revealed theexistence of only one putative 5�-steroid reductaseencoding gene, whereas a related gene was absent inSaccharomyces cerevisiae. This seems to imply that somebut not all fungi can profit from this enzymatic activ-ity. Taking the elevated udh1 expression during thetumor stage into account, it remains an attractive pos-sibility that U. maydis takes advantage of this enzymeduring completion of its sexual life cycle in the plant.Therefore, it will be challenging to identify the endog-enous substrate for the fungal enzyme.

MATERIALS AND METHODS

Plant Material, Strains, and Growth Conditions

The standard wild-type genotype used was ArabidopsisCol-0. The det2-1 mutation is in the Arabidopsis ecotypeCol-0 background (Chory et al., 1991). The Agrobacterium

tumefaciens strain GV3101pMP90RK (Koncz and Schell,1986), kindly provided by Dr. R. Kunze (Koln, Germany)transformed with the pBAR-b 35S derived vector con-structs (see below) was used to transform ArabidopsisCol-0 and det2-1 plants by the vacuum infiltration method(Bechtold et al., 1993). Seeds were incubated in potting soilfor 2 d at 8°C to induce germination. Five days after main-tenance with a 16-h photoperiod (5,000 lux) at 21°C, seed-lings were subjected to four cycles of Basta (0.01% [v/v]Basta [Aventis CropScience, Strasbourg, France] in 0.01%[v/v] Silwet L-77; Lehle Seeds, Round Rock, TX) selectionin 1-d intervals. Basta-resistant seedlings were transferredto individual soil pots and maintained with a 16-h photo-period at 21°C. To assess the influence of the udh1 trans-gene on development, a T2 progeny was harvested frommature siliques of self-pollinated det2-1/det2-1 plants car-rying the pBUB1 transgene, and a F1Col-0 progeny linewas obtained from self-pollinated Col-0 plants. Seeds werestored under identical conditions and plants were grown asspecified in Table II.

Haploid Ustilago maydis strains FB1 (a1b1), FB2 (a2b2),and the diploid strains FBD11 (a1a2b1b2) have been de-scribed (Banuett, 1995). CL13 (a1bE1bW2) and SG200(a1a2bE1bW2) are solopathogenic haploid strains (Bolker etal., 1995). Cells were grown at 28°C in yeast/peptone/Suc(YEPS; Tsukuda et al., 1988) or complete medium (CM;Holliday, 1974). To test for mating, strains were cospottedon charcoal containing potato dextrose plates and incu-bated at room temperature for 48 h. Plant infections weredone as described (Basse et al., 2000) with the sweet cornvar Early Golden Bantam (Olds Seed, Madison, WI). Esch-erichia coli K12 strain DH5� (Bethesda Research Laborato-ries, Bethesda, MD) and TOP10 (Invitrogen, Karlsruhe,Germany) were used as hosts for plasmid amplification.

DNA and RNA Procedures

U. maydis chromosomal DNA was prepared according toHoffman and Winston (1987). Maize (Zea mays) and Ara-bidopsis chromosomal DNA was isolated with the DNEasyPlant Mini Kit (Qiagen, Hilden, Germany). Transformationof U. maydis followed the protocol of Schulz et al. (1990).RNA from U. maydis grown on solid medium and U.maydis-infected maize tissue was isolated according toSchmitt et al. (1990). Total RNA was extracted from Ara-bidopsis plants with the TRIzol reagent (Invitrogen) or theNucleoSpin RNA II Kit (CLONTECH, Heidelberg). Radio-active labeling of DNA was performed with the megaprimeDNA labeling kit (Amersham-Pharmacia Biotech, Freiburg,Germany). Detection and quantification of the signals wasdone using a STORM PhosphorImager (Molecular Dynam-ics, Sunnyvale, CA) and the ImageQuant software. Nucle-otide sequences were determined by automated sequenc-ing using an ABI Prism 377 DNA sequencer (PerkinElmerLife Sciences, Boston). All PCR products were sequenced.Southern hybridization under non-stringent conditionswas performed at 55°C. Stringent washing steps were omit-ted. All other DNA manipulations followed standard pro-cedures as described by Sambrook et al. (1989). Nucleotide

Basse et al.



sequences were compared using the BLAST program (Alt-schul et al., 1997). Prediction of transmembrane helices weremade with TMPRED (http://www.ch.embnet.org/software/TMPRED_form.html).

Plasmids and DNA Fragments

For subcloning and sequencing, plasmids pUC18,pUC19 (Amersham-Pharmacia Biotech), and pCR2.1-TOPO(Invitrogen) were used. The U. maydis cosmid library hasbeen described (Bolker et al., 1995). Hygromycin (hph) andcarboxin resistance (ip: encoding succinate dehydrogenase)cassettes and the ppi fragment (Bohlmann, 1996) as select-able markers and probes, respectively, for U. maydis havebeen described (Basse et al., 2000). The eGFP gene (CLON-TECH) was excised as a 1,030-bp NcoI-EcoRV fragmentfrom plasmid p123 (Aichinger, 2000). Udh1 was containedin cosmids 9B4, 9B10, and 29C12. A genomic AgeI-HindIIIfragment of 1,722 bp that contained the udh1 gene wasisolated from cosmid 9B4. Plasmid pudh1 contained theend-filled genomic AgeI-HindIII fragment in the SmaI siteof pUC19. A. tumefaciens transformation vector pBAR-b 35S(kindly provided by Dr. R. Kunze, Koln, Germany) is aderivative of pGPTV-BAR (Becker et al., 1992) and containsthe BAR gene as selectable marker and the CaMV 35Spromoter and termination sequences for transgene expres-sion. The udh1 fragment used for non-stringent Southernhybridization spanned positions 491 to 1,324 in Figure 1.

Differential Display and Isolation of udh1cDNA Clones

Differential display was performed as described (Basseet al., 2000). Two individual RNA preparations from leaftumor tissue 6 d after inoculation and mock-infected con-trol leaf tissue were reverse-transcribed using T11CG andT11GG primers. For PCR amplification, the oligo(dT) prim-ers from the first step combined with a 10-mer primer witha defined but arbitrary sequence (5�-SCCATGCACG-3�)were used. The 0.8-kb EcoRI-BamHI fragment spanning the3� portion of udh1 was used as probe to isolate two inde-pendent cDNAs from a cDNA library of FBD11 (Schau-wecker et al., 1995). Both clones terminated approximately100 bp downstream of the predicted translational initiatorATG codon. The remaining 5� end of the cDNA was ob-tained by RT-PCR with DNase-treated RNA isolated fromleaf tumors 6 d after inoculation with strains FB1 and FB2as described (Basse et al., 2000) using the forward primerBspe (5�-GGACTAGTCCATGTCGATCCTCACCTGGATC-3�; positions 251–272 in Fig. 1) and the reverse primer ABsp(5�-TTGATCGAAGGCGAGAAGAGC-3�; positions 566–586in Fig. 1).

eGFP-udh1 Fusion Constructs and Microscopy

For construction of the udh1:eGFP fusion construct pugh1,pudh1 was amplified with the forward primer udh1-2 con-taining a EcoRV restriction site (5�-TGTCGATATCTACCTT-TCCGAAATTACCAC-3�; positions 1,419–1,438 in Fig. 1)

and the reverse primer udh1-1 containing a NcoI restrictionsite (5�-AGGTCCATGGAGACAAAAGGGATGATTGCC-3�;positions 1,394–1,413 in Fig. 1). PCR reactions were per-formed in the presence of Taq precision polymerase (Strat-agene, Amsterdam) using parameters as described (Basse etal., 2000). PCR products were cleaved with EcoRV and NcoIand ligated to the eGFP gene isolated as NcoI-EcoRV frag-ment to yield pug1. The sequence of the amplified udh1 geneshowed one deviation at the wobble position of the Ala-206codon, which, therefore, did not alter the amino acid se-quence. The plasmid pugh1 was generated from pug1 byligating the hph cassette as end-filled NotI fragment into theEcoRV site. The 6,274-bp SspI-SphI fragment was isolatedfrom pugh1 and ectopically integrated into U. maydis strainCL13 (strains CL13/pugh1#8 and CL13/pugh1#10). ForSouthern analysis to confirm ectopic integration, genomicDNA was cleaved with BamHI and probed with the udh1AgeI-HindIII fragment. U. maydis cells grown in YEPS me-dium were fixed with 4% (w/v) formaldehyde and stainedwith 0.5 �g mL�1 DAPI (Sigma, Taufkirchen, Germany) inphosphate-buffered saline (pH 7.2) for 15 min at 60°C andsubsequent washing with phosphate-buffered saline. Sam-ples were observed with differential interference contrastoptics or under fluorescence microscopy (excitation/emis-sion for DAPI: 365 nm/�397 nm; excitation/emission foreGFP: 450–490 nm/515–565 nm) using an Axiophot (ZeissJena, Germany).

A. tumefaciens Transformation Vectors

Plasmids pBUB1 and pBUA1 were derived from Saccha-romyces cerevisiae vectors p426ADH-udh-B1 and p426ADH-udh-A1, which contained the full-length udh1 gene and a5�-truncated udh1 gene fragment, respectively (C. Basse,unpublished data). For construction of p426ADH-udh-B1and p426ADH-udh-A1, the udh1 5� portions were ampli-fied from pudh1 using Pfu polymerase (Stratagene) andthe primer combinations Bspe/ABsp and Aspe610 (5�-GGACTAGTCCATGGAGATTCCTTCGCCCATC-3�; posi-tions 461–482 in Fig. 1)/ABsp, respectively. PrimersAspe610 and Bspe contained SpeI restriction sites. PCRproducts were restricted with SpeI/BamHI and ligated intothe respective sites of p426ADH1#1041 (kindly providedby Dr. R. Kunze, Koln, Germany) to yield p426AuB1 andp426AuA1, respectively. p426ADH-udh-B1 and p426ADH-udh-A1 were obtained by ligating the BamHI fragment,which contained the 3� portion of the udh1 gene (positions534–1,629 in Fig. 1), into the BamHI sites of p426AuB1 andp426AuA1, respectively. The end-filled HindIII-SpeI frag-ments containing the udh1 gene were isolated fromp426ADH-udh-B1 and p426ADH-udh-A1, respectively,and ligated into the SmaI site of pBAR-b 35S. The resultingvectors were termed pBUB1 and pBUA1, respectively. TheDET2 gene was isolated from genomic Col-0 DNA by PCRin the presence of Pfu polymerase, the forward primerudhe5n (5�-AATTGATATCCCCGAAAAATGGAAGAA-ATCG-3�; positions from �8 to �13 in the DET2 ORF), andthe reverse primer udhe3n (5�-AAACGATATCGGAATTA-AACCGGTTACTGG-3�; positions 78–97 downstream of




the DET2 ORF). Both primers contained EcoRV recognitionsites. Sequence analysis of the genomic DET2 clone con-firmed the presence of a short intron with a length of 85 bpinserted at position 397 of the DET2 ORF (Li et al., 1996).However, two alterations to the sequence described by Liet al. (1996) were detected (a C to G transversion and a C toT transition at positions 516 and 592, respectively).Whereas the first substitution affected the wobble positionof the Arg-172 codon, the latter resulted in an Arg-198 toCys conversion. A comparison with the respective regionon chromosome 2 of Arabidopsis (GenBank accession no.AC007661) revealed the same alterations as determined inthe amplified DET2 sequence, which we, therefore, con-sider to be correct. The PCR product was cloned into theSmaI site of pUC18 to yield pUCDET2. The EcoRV fragmentwas reisolated from pUCDET2 for insertion into the SmaIsite of pBAR-b 35S to yield pBDET2.

Genotype and Expression Analysis ofArabidopsis Transformants

The presence of constructs pBUA1 and pBUB1 in A.tumefaciens and Arabidopsis was verified by PCR using theprimer combination C13 (5�-ATCTCCACTGACGTAAGG-GATGACG-3�; binds in the CaMV 35S promoter)/ABspand the combination C13/udh511 (5�-CAGTGTGGTAATT-TCGGAAAGG-3�; positions 1,421–1,442 in Fig. 1), respec-tively. The pBDET2 construct was detected by PCR usingthe primer combination C13/det2r (5�-GCAATGTACCA-CTTGTGACTCG-3�). All PCR products were sequencedfrom both ends. In addition, the PCR product of the det2-1/pBUB1#1 transformant (series 1) was completely se-quenced. This indicated the presence of the complete andcorrect udh1 ORF sequence. Control PCR using the primercombinations C13/udh511 and C13/det2r performed withDNA from Col-0 and det2-1 plants did not result in PCRproducts of the expected size. To confirm the genetic back-ground of analyzed plants, chromosomal ArabidopsisDNA was amplified with the primers det3h (5�-GTCG-GACCGCACTTTGGTACG-3�)/det3r (5�-CGAGTCTTGG-GATACTCTTCC-3�), which primed in the close context ofthe polymorphic MnlI site for subsequent RFLP analysisusing the DET2 EcoRV fragment as probe. The G610 to Atransition of the det2-1 ORF was additionally confirmed bysequence analysis of all PCR products (series 1) cloned intopCR2.1-TOPO. To analyze the genotype of pBDET2 trans-formants, an initial PCR was performed using primers detx(5�-AATTCCATAACCCGAAAAATGG-3�) and dety (5�-GTTGTGCATTGTTGAAGATACC-3�), which both bindadjacent to the DET2 sequence present in pBDET2. Gel-purified PCR products were reamplified using the primercombination det3h/det3r for RFLP analysis. For expressionanalysis, RNA was isolated from leaves of det2-1/pBUB1#1, 2, 8, and 12 and wild-type Col-0 plants (series 3)62 d after sowing; det2-1/pBUA1#13 and 14 plants (series 4)61 after sowing; det2-1/pBUA1#16 (series 4) 75 d aftersowing; and det2-1/pBDET2#1, 2, 3, 7, and 8 plants (series4) and two different wild-type Col-0 and det2-1 mutantplants (series 2) 61 d after sowing; as well as from T2progeny plants (see Fig. 9).

Construction of udh1 Deletion Strains

For construction of the udh1 deletion plasmid, pudh1was amplified with the forward primer udet3 (5�-AAGGC-CTCCTAGAGCTACCAACGGTCAC-3�; positions 1,326–1,346in Fig. 1) and the reverse primer udet1 (5�-AAGGC-CTGATGGGCGAAGGAATCTCC-3�; positions 464–482 inFig. 1), which both contain a StuI restriction site. Thedeletion comprised the complete udh1 ORF except the ini-tial 231 bp at the 5� end and the terminal 91 bp at the 3� end.PCR parameters were as described (Basse et al., 2000). PCRproducts were cleaved with StuI and ligated to the hphcassette as an end-filled BamHI-EcoRV fragment to yieldpudh1ko. To replace the resident udh1 gene with the hphcassette, a 4,434-bp SspI-SphI fragment was isolated frompudh1ko and transformed into U. maydis strains FB1, FB2,and SG200. For Southern analysis, genomic DNA wascleaved with either AgeI/HindIII or BamHI and probedwith the udh1 containing AgeI-HindIII fragment. Becauseudh1 null-mutants were generated only in strains SG200and FB2, FB1 udh1 null-mutants were obtained from thesexual offspring of a cross between FB2�udh1 and FB1.

Detection of U. maydis DNA in Maize TissueInfected with Mixtures of SG200 andSG200�udh1 Strains

Maize plants (var Early Golden Bantam) were inoculatedwith mixtures of SG200 and SG200�udh1 cultures adjustedto a density of 7 to 10 � 107 cells mL�1. Four independentSG200�udh1 strains were used. In each case, DNA wasprepared from a single leaf tumor of an infected maizeplant 10 d after inoculation using the DNEasy plant kit(Qiagen). Chromosomal DNA was used as template forPCR under standard conditions. The following primercombinations were used to amplify fragments from (a) thehph gene flanked by udh1 5� ORF sequences and (b) theudh1 gene: (a) 5�-GCAGGTTCGCGTTCGATAGC-3�/5�-CCATGCAGTCTACGCAGTCG-3�; (b) 5�-CCGTGTTGCA-GCCATTGAGG-3�/5�-GGGAAAGAGACAGGGTGGTG-3�.Primer combinations a and b resulted in comparableamplification efficiencies using identical amounts of chro-mosomal DNA from either SG200�udh1(a) or SG200 (b)strains as template.

Distribution of Materials

Upon request, all novel materials described in this pub-lication will be made available in a timely manner fornon-commercial research purposes, subject to the requisitepermission from any third-party owners of all or parts ofthe material. Obtaining any permission will be the respon-sibility of the requestor.

ACKNOWLEDGMENT

We thank Kathrin Auffarth for technical assistance.

Received November 26, 2001; returned for revision January21, 2002; accepted March 25, 2002.

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evidence for a ustilago maydis steroid 5 -reductase by for a ustilago maydis steroid 5 -reductase by...

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