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Recombinant b alleles of Ustilago maydis Yee, Arthur Raymond 1991

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RECOMBINANT b ALLELES OF USTILAGO MAYDISbyARTHUR RAYMOND YEEB.Sc. (Agriculture) University of British ColumbiaA THESIS SUBMITTED IN PARTIAL FULFILMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMaster of ScienceinTHE FACULTY OF GRADUATE STUDIESDEPARTMENT OF PLANT SCIENCEWe accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIADecember 1991© Arthur Raymond Yee, 1991In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature) Department of Plant ScienceThe University of British ColumbiaVancouver, CanadaDate^31 December 1991DE-6 (2/88)ABSTRACTThe fungal plant pathogen Ustilago maydis has a multiallelic sexualincompatibility system governed by an estimated 25 alleles at the b locus. Haploidstrains must have different b alleles in order to be sexually compatible and pathogenic.Existing sequence data from several b alleles show a variable domain in the first 110codons and a constant domain through the rest of the open reading frame. Todelineate the region responsible for allelic specificity, a series of recombinant allelescontaining the variable region of bl and the constant region of b2 were constructedusing an in vivo recombination strategy. A haploid strain of U. maydis, carrying the b2allele, was transformed with linear DNA containing progressive 3' end deletions of thebl variable region. Homologous integration of the transforming DNA resulted inreplacement of the resident b2 variable region with part or all of the bl variable region,producing a bl-b2 recombinant. A map of alleles containing recombination pointsthroughout the variable region delineated a 48 codon domain responsible for allelicspecificity. Recombination between bl and b2 within this region generated alleles withdifferent specificities from either bl or b2. Analysis of nucleotide sequences ofrecombinant alleles derived from specific plasmids showed that homologous integrationcan occur anywhere from the end of the linear transforming DNA to about 100 by fromthe end. A comparison of amino acid sequences of recombinant alleles withrecombination points near the left and right borders of the specificity domain showedthat nonconservative amino acid substitutions can alter allelic specificity of the b locus.These results suggest that new alleles of the multiallelic b locus may have evolved by acombination of meiotic crossing over and mutation within the specificity region.llTABLE OF CONTENTSAbstract^ iiTable of Contents^ iiiList of FiguresList of Tables^ viAcknowledgements viiIntroduction^ 1The Disease 1The Pathogen 4Life Cycle and Sexual Incompatibility System^ 4Molecular Studies of the b Locus^ 7DNA Mediated Transformation of Ustilago maydis 10Strategy for in vivo Construction of Recombinant b Alleles^ 11Materials and Methods^ 14Strains and Media 14DNA Procedures 14Construction of Plasmids 15Transformation of U. maydis^ 20Screening of Transformants by Replica Mating^ 21PCR of Genomic DNA from Transformants 24DNA Sequencing^ 26Pathogenicity Tests and Genetic Crosses 28Results^ 30Mating Tests of Transformants^ 30PCR Screen for Homologous Integration^ 34Nucleotide Sequence of Recombinant Alleles ^ 35Analysis of Amino Acid Sequence of Recombinant Alleles^38The Null Class Transformants^ 42Pathogenicity Tests and Genetic Crosses^ 42Discussion^ 46Conclusions 51Bibliography^ 52Appendix A. Media 55Appendix B. Transformation of Ustilago maydis^ 57iiiTable of ContentsAppendix C. Small Scale Preparation of Ustilago DNA.^ 61Appendix D. Abbreviations^ 63ivLIST OF FIGURESFigure 1. Disease symptoms of Ustilago maydis on corn.^ 3Figure 2. Simplified life cycle of Ustilago maydis. 5Figure 3. Mating or incompatibility tests for Ustilago maydis.^ 6Figure 4. Amino acid sequences for several b alleles. 9Figure 5. Strategy for in vivo construction of recombinant b alleles.^ 12Figure 6. Plasmids pUCblhyg and pAR.^ 16Figure 7. Linear maps of pAR plasmids used for transformation ^ 18Figure 8. Location of Sphl linker sites on pAR plasmids.^ 19Figure 9. Replica mating plates.^ 23Figure 10. Location of primers used for PCR and sequencing.^ 25Figure 11. Mating type classes of transformants.^ 31Figure 12. Agarose gel from PCR screen for homologous integration^ 34Figure 13. Nucleotide sequence of recombinant alleles produced from pAR21 ^37Figure 14. Map of recombinant b alleles ^38Figure 15. Amino acid sequences of the left border region. 40Figure 16. Amino acid sequences of the right border region^ 41LIST OF TABLESTable 1. Ustilago maydis strains used in this study^ 15Table 2. Sequences for oligonucleotide primers. 25Table 3. Proportion of transformants belonging to each mating type class as afunction of the plasmid used for transformation.^ 33Table 4. Summary of the recombinant b allele transformants^ 36Table 5. Pathogenicity data for transformants^ 43Table 6. Genetic analysis of a cross between transformant t4-6 (a2bx70) and tester521 (albl)^ 44viACKNOWLEDGEMENTSI would like to thank my supervisor, Dr. J. W. Kronstad, for the opportunityto train in his laboratory, and for his guidance and support during my studies. Iwould also like to thank the members of my thesis committee, Dr. R. J. Copemanfor his sound advice and Dr. N. L. Glass for her insightful questions.Thanks is also given to members of Dr. Kronstad's laboratory for theirwillingness to share their knowledge and experience, to Dr. J. E. Carlson for the useof his computer, and to members of his laboratory for their helpfulness.A very special thanks to my wife Lorraine, for her patience and support.viiINTRODUCTIONThe fungal plant pathogen, Ustilago maydis, is the causative agent of the cornsmut disease. U. maydis has a multiallelic sexual incompatibility system governed byan estimated 25 alleles at a locus called b. Haploid strains must possess different balleles in order to be sexually compatible and pathogenic. The genetics of U. maydisand the b locus have been well characterized and more recently several alleles ofthe b locus have been cloned and sequenced. The organism is easily cultured andmanipulated in the laboratory because it is facultatively biotrophic, grows relativelyquickly, and has a unicellular yeast-like stage. It is also capable of undergoingefficient DNA transformation and targeted gene replacement. Thus, U. maydis andthe b locus provide a convenient system for studying multiallelic recognition in arelatively simple eukaryotic organism.THE DISEASEThe corn smut disease is characterized by prominent galls (tumors) on stems,leaves, ears, and tassels of corn (Zea mays). Dark, sooty, masses of teliosporesdevelop within the tumors, and these diploid spores germinate to give haploidbasidiospores. Associated disease symptoms include chlorosis andanthocyanescence on stems and leaves, especially in the area surrounding the tumor.Figure 1 illustrates some disease symptoms and Christensen (1963) presents a goodreview of this disease. Crop damage from corn smut is highly variable. Yield lossesmay average about 2% with resistant varieties (Agrios 1988) while susceptiblevarieties, such as those of sweet corn, may suffer considerable damage.1IntroductionInfection of the corn plant occurs during the growing season, with theinoculum coming from overwintering teliospores in the soil or plant residue (Agrios1988). The teliospores can germinate on the corn plant or on plant residue to givehaploid sporidia which grow saprophytically. Compatible haploid strains will fuse toform a dikaryon which can infect and grow parasitically within the meristematicregions of the plant. The parasitic growth leads to tumor formation and theproduction of diploid teliospores. These spores act as survival propagules, allowingthe fungus to overwinter in soil or plant residue.Control of the disease is presently achieved by planting resistant cornvarieties, and to a limited extent, by sanitation. Resistance to corn smut ismultigenic and is associated with field corn varieties (Christensen 1963), but novarieties are completely resistant.2IntroductionFigure 1. Disease symptoms of Ustilago maydis on corn.A. A four week old corn plant showing symptoms of chlorosis, anthocyanescence,and a basal stem gall (arrow). B. Close-up of the basal stem gall containingteliospores.3IntroductionTHE PATHOGENU. maydis is a basidiomycete which belongs to the taxonomic orderUstilaginales, the smut fungi. The smut fungi share many characteristics, such as theproduction of dark teliospores within the host, and a life cycle involving asaprophytic haploid stage and an obligately parasitic dikaryotic stage.Life Cycle and Sexual Incompatibility SystemThe life cycle of U. maydis is illustrated in Figure 2 (adapted from Froeligerand Kronstad 1990). Starting from the diploid teliospore stage, the fungusundergoes meiosis at germination to produce a tetrad of haploid basidiospores.These grow as yeast-like sporidia and are saprophytic and nonpathogenic. Haploidlines can mate and they are compatible if they carry different alleles at 2 locidesignated a and b. A compatible mating will produce a mycelial, pathogenicdikaryon. A pathogenic dikaryon can infect a plant, grow parasitically with themeristematic tissue, form galls, and produce diploid teliospores. The production ofteliospores from plant tissue in culture has been reported (Hausen and Beiderbeck1987) but teliospore production ex planta has not been accomplished to date. Thus,for Ustilago, sexual compatibility and pathogenicity are intimately associated.The two sexual incompatiblity or mating type loci are unlinked and havedifferent functions. The a locus has two alleles, al and a2 (Rowell and DeVay1954), and controls the ability of haploid cells to fuse (Rowell 1955). The b locus ismultiallelic (Rowell and DeVay 1954), with an estimated 25 alleles (Puhalla 1970).It controls developmental events involved in pathogenicity and teliospore formationfollowing cell fusion (Froeliger and Kronstad 1990).4Diploidteliospore(2n)Germination& meiosisIntroductionClassical genetic studies of U. maydis have been facilitated in part becausethe fungus can be easily cultured in the laboratory as unicellular, yeast-like haploidsor diploids. Also, sexual incompatibility tests can be easily carried out by a plateassay on a rich medium containing activated charcoal (Day and Anagnostakis 1971;Holliday 1974). These mating tests give yeast-like (fuz-) growth for incompatiblecombinations and mycelial (fuz+ ) growth for compatible combinations (Figure 3).Dikaryotic^Haploidhyphae basidiospores(n+n) (n)Fusion ofcompatiblehaploidsFigure 2. Simplified life cycle of Ustilago maydis.The diploid teliospore undergoes meiosis during germination to produce a tetrad ofhaploid basidiospores. These grow as yeast-like sporidia and can mate with othersporidial lines that carry different a and b alleles to form an infectious, mycelialdikaryon. Infection of corn eventually results in tumor formation and theproduction of diploid teliospores. Karyogamy (fusion of nuclei) occurs just beforeteliospore formation.5IntroductionCompatible521 x 518albl x a2b2Incompatible521 x 032albl xa2b1Figure 3. Mating or incompatibility tests for Ustilago maydis.Overlapping spots of haploid cultures are placed on charcoal mating plates. Thefirst spot is allowed to dry before the second is applied. The two compatible strains,521 and 518, which possess different a and b alleles, show the white mycelialreaction (fuz+) in the overlap area. The two incompatible strains, 521 and 032,which have different a alleles but the same b alleles, show yeast-like growth (fuz-).6IntroductionMolecular Studies of the b LocusSeveral alleles of the b locus have been recently cloned and sequenced. Thefirst isolation of a b allele was reported by Kronstad and Leong (1989). The strategyinvolved transformation of a diploid, b2/b2 strain (which has a yeast-likephenotype) with a cosmid library containing DNA from a haploid bl strain. Thetransformants were screened for the mycelial phenotype expected of a straincarrying different b alleles. The bl allele resided on a 8.5 kilobase (kb) BamHIrestriction fragment. The b2 allele was isolated by using the bl allele to probe agenomic library from a b2 strain.The bl coding region was further defined by the use of Tn5 mutagenesis andsubcloning (Kronstad and Leong 1990). The smallest fragment that gave high bactivity in transformation experiments was a 1.7 Kb BglII-SalI fragment. This regionof DNA was sequenced from both bl and b2 alleles and a 1230 basepair (410 aminoacid) open reading frame (ORF) 1 was found to be common to both alleles. In vitromutagenesis experiments indicated that the 410 amino acid ORF encodes apolypeptide responsible for b activity. Similar work on the cloning of the b2 allelehas been reported by Schulz et al. (1990).Several other b alleles were isolated by DNA hybridization with a b alleleprobe (Schulz et al. 1990) or polymerase chain reaction (PCR) amplification of theb ORF region (Kronstad and Leong 1990). A comparison of sequences betweenalleles showed a variable amino terminus including codons 1 to 110 (about 60%conservation) and a conserved region covering codons 111 to 410 (about 90%1 A list of abbreviations is given in Appendix D.7Introductionconserved). Within the variable region, there were two hypervariable regions, frompositions 47 to 60 and 70 to 75, that displayed most of the variability between alleles(Figure 4).The above results suggested that allelic specificity was determined by thevariable region. Dahl et al. (1991) confirmed this by generating hybrid orrecombinant b alleles using suitable restriction sites between b2 and b3, and thenintroducing these alleles into haploid U. maydis strains by DNA mediatedtransformation. Transformants that had undergone targeted (homologous) genereplacement at the b locus carried a single recombinant b allele. If the N-terminalregion up to amino acid position 115 was derived from b2 and the rest of the ORFderived from b3, the allele showed b2 specificity. If the N-terminal region up toposition 56 was derived from b2 and the rest of the ORF derived from b3, the alleleshowed b3 specificity. Thus, the specificity domain for b2 versus b3 was localized to60 amino acids between positions 56 and 115.8^20^ 40^ 60. .^.bl- MS S DPNFS LI S FLECLNE I EHEFLRDKGENYPVLVRKLRELQQKI PNDIANLPRDPET I Qb2-^NY^T V^L R^Q RR T NV S SY G HbH-^R KL SK V HR Q^T KHV K HE MbJ- T E R II T HV D AH SK EbK-^R KL SK^ V HR^Q^T KHVT NbL- T SQ^V P Q T GHV S LH80^ 100^ 120.^. .^. .bl- Q I HQTTHRIRAVAQAFIRFDQKFVS LCSEVVHGTS KVMQEFNVVS PDVGCRNLS EDLPAYb2-^KVA K^I S^H DA ED ALKKADAS V^DbH- AA DI I D T^A^GEbJ-^A KVAVKT I E A VbK- IA LEVAVKV LHI R^R D ED AL V A A EYbL-^A EVAVKV HI^T G^ V G A. Figure 4. Amino acid sequences for several b alleles.Predicted amino acid sequences for the variable regions of bl, b2, bH, bJ, bK and bL usingthe one letter code (Appendix D). Data are from Kronstad and Leong (1990). Thecomplete bl sequence up to codon 120 is shown, but amino acids for the other alleles areshown only where they differ from bl.9DNA Mediated Transformation of Ustilago maydisDNA mediated transformation of U. maydis was first reported by Banks(1983) but transformants were found at a low frequency and they were unstable. Aprotocol for efficient stable transformation of U. maydis was developed by Wang etal. (1988). This protocol involved, as the first step, the production of protoplastsfrom early log phase cultures using Novozyme 234 (CalBiochem Corporation, SanDiego, CA), a commercial mixture of chitinases and cellulases. An integrativetransformation vector containing a hygromycin B resistance gene was incubated withthe protoplasts in the presence of calcium chloride and polyethylene glycol. Thecells were plated on medium with hygromycin to select for transformants. A similarprotocol for U. maydis transformation was developed by Tsukuda et al. (1988). Inthis report, they described the development of a high frequency transformationvector containing a Ustilago autonomously replicating sequence (UARS).Subsequent experiments demonstrated that targeted gene replacement canoccur in U. maydis at a high frequency of about 70% for the pyr6 gene (Kronstad etal. 1989) and 25% to 50% for the leul gene (Fotheringham and Holloman 1989).Targeted gene replacement occurs when the transforming DNA integrates into thechromosome at a site of homology and replaces the resident DNA sequences. Thispowerful technique allows the analysis of specific changes in a particular gene.The effect of conformation of the transforming DNA on the frequency ofhomologous versus nonhomologous integration was studied by Fotheringham andHolloman (1990), using the leul gene. They showed that DNA integration into theU. maydis genome occurred primarily at nonhomologous sites when the10transforming DNA was circular but at the homologous site if the DNA had beenlinearized within the U. maydis sequence. Also, transformation using linearizedDNA gave a 100-fold higher frequency than with circular DNA. It appears thatDNA ends are highly recombinogenic and promote integration into the U. maydisgenome by homologous recombination. This phenomenon has been previouslydemonstrated for Saccharomyces cerevisiae (Orr-Weaver et al. 1981).The ability to carry out both homologous and nonhomologous recombinationplaces U. maydis intermediate between S. cerevisiae, in which recombination isentirely homologous, and mammalian systems, in which recombination is largelynonhomologous. It is possible that U. maydis has recombination machinery thatpossesses characteristics in common with both systems (Fotheringham andHolloman 1990).STRATEGY FOR IN VIVO CONSTRUCTION OF RECOMBINANT b ALLELESThe ability to do targeted gene replacement in U. maydis and the partialhomology existing between different b alleles set the stage for a novel in situ and invivo strategy to construct recombinant b alleles. To identify the determinants ofallelic specificity, we constructed a series recombinant alleles consisting of the blvariable region and the b2 constant region. A DNA fragment carrying the blvariable region and a selectable marker was transformed into a haploid b2 strain(Figure 5).11Transforming linear DNAwith La variable regionand HygB resistance gene HygB blResident b2 alleleb2Partial gene replacement givesbl -b2 recombinant allelerr17.7n7.77!!!ZT!T.M!!!!rr,Z7!!17!!!!!!!T!r!!TTIHygB^bl -b2Figure 5. Strategy for in vivo construction of recombinant b alleles.The x's represent homologous recombination events into the U. maydis genome.12The transformants were screened for altered mating type and integration atthe b locus. Transformants with these properties were found to contain bl-b2recombinant alleles. By using portions of the bl variable region produced byprogressive deletions from the 3' end, recombination points were obtained thatspanned the entire variable region. This strategy had the advantages of (1)construction and introduction of recombinant alleles into U. maydis in a single step,and (2) production of a series of recombination points that differed from each otherby only a few amino acid positions.Other researchers have also employed in vivo construction of chimeric orrecombinant genes. Pompon and Nicolas (1989) used the recombination machineryof yeast to study the shuffling of functional domains between mouse and rabbit P-450 cytochromes. This work was an extension of the experiments done by Ma et al.(1987) with the in vivo construction of plasmids by homologous recombination inyeast.Our starting hypothesis was that transformation with linear DNA containingmost or all of the bl variable region would give transformants with bl specificity andthat transformation with linear DNA that contained a small amount of the blvariable region would give transformants with b2 specificity. In between theseextremes, we expected a transition region where recombination would delineate asharp boundary, give null alleles, or create a different allelic specificity. Theultimate goal was to generate a fine structure map of the region responsible forallelic specificity.13MATERIALS AND METHODSSTRAINS AND MEDIAEscherichia coli strain DH5a [F-, end Al, hsd R17(ric",mk ), sup E44, thr,rec Al, 080dlac ZM15] from Bethesda Research Laboratories was used for DNAcloning work and was grown in LB medium (Sambrook et al. 1989). U. maydisstrains are given in Table 1. They are all wild type, prototrophic strains obtainedfrom R. Holliday (Commonwealth Scientific and Industrial Research Organization,Laboratory of Molecular Biology, Sydney, Australia). U. maydis cultures weregrown in potato dextrose broth or agar (PDB or PDA; Difco Laboratories) orcomplete medium (CM; Holliday 1974) and mating reactions were carried out ondouble complete charcoal mating plates (Holliday 1974). Media recipes are given inAppendix A.DNA PROCEDURESProtocols used for DNA manipulations and for small scale, boiling-lysis,plasmid preparations are from Sambrook et al. (1989). Small scale, alkaline-lysis,plasmid preparations were done by the "ten minute" method of Zhou et al. (1990).Restriction and DNA modifying enzymes were obtained from Bethesda ResearchLaboratories, Boehringer Mannheim, and Pharmacia. All centrifugations ofmicrofuge tubes were done in an Eppendorf Centrifuge 5415.14Materials and MethodsTable 1. Ustilago maydis strains used in this study.Strain^Genotype^Source518^a2b2^R. Holliday521 a1 b1 R. Holliday031^a1 b2^R. Holliday032 a2b1 R. HollidayAll strains are wild type prototrophs.CONSTRUCTION OF PLASMIDSThe plasmids used for transformation originated from pUCblhyg (Figure 6).Plasmid pUCb lhyg consists of the 8.5 kb BamHI fragment of pb 1 carrying the openreading frame of the bl allele (Kronstad and Leong 1989) cloned into pUC9(Bethesda Research Laboratories, Bethesda, MD). A hsp70 promotor/hygromycinresistance gene (Gritz and Davies 1983; Wang et al. 1988) was inserted into theBglII site about 400 base pairs (bp) upstream of the bl open reading frame. PlasmidpUCblhyg was cut with SphI and recircularized with T4 ligase. This resulted indeletion of the 3' and downstream sequences of the bl allele to give plasmid pAR(Figure 6).15mHISsplBgIIIpAR9.50 Kbb1hsp70Hyghsp70BamHI-Bg111SsplSphlBamHIgillBamHIpUCbl hyg13.40 Kbhsp70HygBhsp70SphlSphlSphlBamHI-Bg111SsplMaterials and MethodsFigure 6. Plasmids pUCblhyg and pAR.The vector (pUC9) is represented by a solid thin line. U. maydis sequencesupstream and downstream of the bl ORF are represented by the light grey shadingand the bl ORF is in solid black. The plasmid pUCblhyg was digested with Sphl todelete the 3' and downstream sequences of the bl ORF and recircularized with T4ligase to obtain plasmid pAR.16Materials and MethodsPlasmid pAR was then cut with SphI and digested with the Klenow fragmentof DNA Polymerase I. The Klenow fragment removes the 3' overhang created bythe SphI restriction enzyme. Progressive deletions extending in both directions fromthe SphI site were produced using the Pharmacia nested deletion kit, by digestionwith Exonuclease III for times ranging from 0 to 20 min, followed by S1 digestion.SphI linkers were ligated to the deleted ends following the protocol in Sambrook etal. (1989) and then digested with SphI. Excess linkers were removed byGENECLEAN and the plasmids were recircularized with T4 ligase. The resultingligation products were used to directly transform E. coli using the standard heatshock protocol (Sambrook et al. 1989).The resulting clones were screened for the presence of a SphI linker sites, atpositions distributed through the variable region, by small scale plasmidpreparations and restriction mapping of the SphI site. A set of 16 plasmids wereselected and designated pAR11 to pAR26 (Figures 7). Large scale preparations ofthese plasmids were produced by CsCl-ethidium bromide gradient centrifugation(Sambrook et al. 1989). The pAR plasmids were later sequenced to determine theexact location of the SphI linker site (Figure 8).17If^4^Endpointk • codon% in b ORF PAR 283pUC9^hsp70/HYgB^hipAR11pAR12pAR13pAR14pAR15pAR16pAR17pAR19pAR20pAR21pAR22pAR23pAR24pAR25pAR26pAR274888M2618181^ Met9 1551391221091079490807562555044392914IRM4SENSItitIMESSMOM=MtKM5Mk&AIIMM*811Figure 7. Linear maps of pAR plasmids used for transformation.Each plasmid is linearized at the single SphI linker site. The vector sequences arerepresented by a thin solid line and sequences upstream and downstream of bl aregiven by light grey shading.18Codon 0Variable^ConstantRegion Region110Endpointcodonin b ORFpAR 283pAR11 155•pAR12 139pAR13 122pAR14 109pAR15 107pAR16 94pAR17 90pAR19 80pAR20 75pAR21 OMMIIIMMOMMMM 62pAR22 55pAR23 50pAR24 44pAR25 39pAR26 Am= 29pAR27 14Figure 8. Location of SphI linker sites on pAR plasmids.A close-up of the linear maps given in Figure 7 showing only the bl ORF section(solid black). The deletion endpoints indicate the positions of the SphI linkers. Theplasmid pAR has not undergone deletion.19Materials and MethodsTRANSFORMATION OF U. MAYDISThe transformations with linearized plasmids were organized in two differentways. The first was to transform with the individual plasmids, such as pAR11 andpAR21, so that recombinant alleles derived from a specific plasmid could beanalysed. The second way was to transform with a pool of 3 or 4 pAR plasmids.This approach made it easier to handle a large number of transformants withouthaving to transform and screen on an individual plasmid basis. The plasmids werepooled as follows: pAR12 to pAR15 (endpoint codons 139 to 107) were grouped inpool a, pAR16 to pAR20 (endpoint codons 94 to 75) were grouped in pool b,pAR21 to pAR24 (endpoint codons 62 to 44) were grouped into pool c, and pAR25to pAR27 (endpoint codons 39 to 14) were grouped in pool d. A comparison ofamino acid differences between bl and b2 (Figure 4) suggested that the specificityregion was probably located between codons 38 and 108; these groupings weremade on this assumption.Transformation of U. maydis was accomplished by a protoplast-polyethyleneglycol-CaC12 procedure (Appendix B). Protoplasts of U. maydis were prepared withNovozyme 234 (CalBiochem Corporation, San Diego, CA) using the protocol ofWang et al. 1988. The protoplasts were stored frozen at -70°C in Buffer II (1 Msorbitol, 25 mM Tris pH 7.5, 50 mM CaCl2 ) with 13-mercaptoethanol, dimethylsulfoxide (DMSO), and polyethylene glycol (PEG) 3350 (Sigma Chemical Co. StLouis, MO).Transformation of U. maydis protoplasts was performed in 1.5 ml microfugetubes with a procedure adapted from Wang et al. (1988) and Specht et al. (1988).The transformation protocol involved incubation of protoplasts with DNA in the20Materials and Methodspresence of PEG and CaC12. A hygromycin resistance gene was used for selectionof transformants. Transforming linear DNA was prepared from pAR plasmids bydigestion with BamHI and SphI, phenol-chloroform and chloroform extraction, andethanol precipitation. Each transformation tube gave 50 to 200 stable transformantsfrom 1 to 5 ug of DNA.SCREENING OF TRANSFORMANTS BY REPLICA MATINGSingle colony transformants were transferred by toothpick, onto master PDAplates with 200 ug/ml hygromycin B (CalBiochem Corporation, San Diego, CA) in a8 by 6 grid pattern that matched a prong type, replica plating device. This replicaplating device had a prong diameter of 1.5 mm and a 9 mm prong spacing thatmatched the well spacing of standard 96 microwell plates. The master plates wereput in closed plastic bags (to keep the colonies moist) and incubated for 1 to 2 daysat 30°C. The transformants were then transferred to charcoal mating plates usingthe prong replica plater and incubated for 1 day at 30 °C to allow 1-3 mm colonies toform. Then a drop of log phase liquid culture containing the tester strains 521(albl) or 031 (alb2) was placed on each colony using a multi-channel pipettor andthe plates were allowed to dry in a laminar flow hood. The plates were then tapedwith Parafilm (American National Can, Greenwich, CT) and incubated at roomtemperature. Formation of white aerial mycelium indicative of a compatible matingreaction was visible within 24 hr and the colonies were scored on day 2. Mycelialphenotype was found to be stronger when incubated at room temperature ratherthan at 30°C. Figure 9 shows the results from a typical set of replica matings.Selected transformants were streaked for single colonies on PDA plates with21Materials and Methods200 ug/ml hygromycin B and then inoculated into 18 x 150 mm test tubes containing5 ml of PDB. These were grown without hygromycin selection for about 24 hrs at30°C until early stationary phase. Previous reports with integrative transformationin U. maydis have shown that transformants were mitotically and meiotically stable(Wang et al. 1988; Kronstad et al. 1989). This liquid culture was used for retestingthe mating type of each transformant, for small scale preparations of total genomicDNA, and for storage of the strain at -70°C.Retesting of mating type was done by spotting about 10 ul of early stationaryphase cultures containing tester strains 521 or 031 onto charcoal mating plates,allowing these spots to dry in the laminar flow hood, and then spotting about 10 ulof liquid culture from the transformant on top of the tester. After drying, the plateswere taped with Parafilm and incubated at room temperature for 2 days.Small scale preparation of total genomic DNA from U. maydis was done byan adaptation of the yeast protocol (Elder et. al. 1983). This protocol involvesvortexing with glass beads and phenol to break up the cells and it is described inAppendix C. Cultures were stored at -70 °C in 7% DMSO.22•• • it, ik, i^.4. • •* . • is • 0 • • 41,*^* G. a . ,t. •• 1 & 4A 0'W031 (alb2) 1tester91725334181624324048Materials and Methods521 (albl)tester1917253341'`• • • • • • • e• • • a • • • •• • • - • • • •• • •• • • •• • • • • • • •• • • • • • • •81624324048Figure 9. Replica mating plates.Haploid transformants to be tested for mating type (originally a2b2) weretransferred to charcoal mating plates using a prong type replica plater.Transformant colonies were incubated for a day at 30PC and then spotted withtester culture (either al bl or alb2). Mating plates were taped with Parafilm andincubated at room temperature for 2 days. White aerial mycelium is indicative of acompatible mating reaction produced by different b alleles. Transformants 14, 16,27, 37, 42, 43, and 48 show a compatible reaction with bl and b2; transformant 20 iscompatible with b2 only, and transformant 26 fails to give a reaction to either tester.All other transformants are compatible with bl only.23Materials and MethodsPCR OF GENOMIC DNA FROM TRANSFORMANTSPCR reactions were performed with a Perkin Elmer Cetus DNA ThermalCycler Model 4800 in 0.5 ml GeneAmp tubes. Cycling parameters were an initial 5min denaturation at 94°C, then 30 cycles of 1 min at 94°C, 1 min at 55°C, and 3 minat 74°C, and a final extension step of 10 min at 74°C. When required, PCRproducts were stored at 4°C overnight or -20°C for longer periods. Reactions werecarried out in volumes of 20 to 100 ul containing 10 mM Tris-HC1 pH 8.3, 50 mMKC1, 2.0 mM MgC12 , 0.001% gelatin, 100 uM each of dATP, dCTP, dGTP, dTTP,0.2 uM of each primer, 2.5 units of Taq DNA Polymerase (Perkin Elmer Cetus) per100 ul reaction, and 0.2 to 1 ug of genomic U. maydis DNA from small scalepreparations per 100 ul reaction. PCR products were electrophoresed on 0.7 %agarose gels and visualized by ethidium bromide staining (Sambrook et al. 1989).Detection of homologous integration in transformants was done by the PCRtechnique of Frohman and Martin (1990). One PCR primer (primer Hph) waslocated within the hygromycin B resistance gene (Gritz and Davies 1983) and theother primer (number 7-1 or 6B) was located at the 3' end of the bl allele sequence.Primers 7-1 and 6B are outside of the sequences present in the transforming blDNA. A PCR product was produced only in transformants in which the hygromycingene and the bl sequences had integrated at the b2 locus. PCR primer sequencesare given in Table 2 and primer locations are given in Figure 10.24Materials and MethodsTable 2. Sequences for oligonucleotide primers.Primer^Sequence (5' to 3')Hph^ACGGATCCGAGGGCAAAGGAATAGAG2 AAGGATCCTCTCAGCAACATCATCAC3^ AGACTACACACAGGATTA7-1 GGGATTGTAGTGAGACAT6B^TGGGATCCTGATTGCGAACAACAGCABamHI recognition sites (GGATCC) plus 2 additional "clamp" nucleotides havebeen added to the 5' ends of primers Hph, 2, and 6B.SspI^SphIHph^2^3453MIMMWMMEMMINMF SWENNSESI^ MEMEMMORM hsp70 7-1 6BHygB bORFFigure 10. Location of primers used for PCR and sequencing.The map shows the arrangement of hsp70/Hy0 sequences after integration at the blocus. Left and right ends of the map are contiguous with hsp70 promotor and bORF downstream sequences, respectively. The arrows point to the 3' end of primer.25Materials and MethodsDNA SEQUENCINGPCR products were sequenced either after cloning into the vector pGEM-3Zf(+ ) (Promega, Madison, WI, USA) or directly sequenced with a methodadapted from Casanova et al. (1990). Sequencing was carried out by the dideoxytermination method (Sanger et al. 1977) using 35S-dATP and the Pharmacia T7Sequencing kit. Sequencing primers used were numbers 2 and 3 in Figure 10 andTable 2.Cloning of PCR products was facilitated by BamHI restriction sites on the 5'ends of the PCR primers. The PCR products were first purified usingGENECLEAN (Bio 101 Inc, La Jolla, CA), digested with BamHI, purified withGENECLEAN again, and then ligated into linearized (BamHI) anddephosphorylated pGEM-3Zf(+). E. coli transformation was done using thestandard heat shock protocol (Sambrook et al. 1989). Clones were screened usingthe small scale, boiling lysis, plasmid isolation procedure.Double stranded plasmid templates were prepared for sequencing from smallscale plasmid preparations by an alkaline denaturation method adapted fromToneguzzo et al. (1988). For each template, 3 to 5 ug of plasmid (about 0.1 pmole),10 pmoles of sequencing primer, and 2 ul of 2 N sodium hydroxide in 2 mM EDTAwere mixed and brought to a total volume of 22 ul with sterile distilled water. Thismixture was incubated on a heat block at 85 °C for 5 min, then transferred to ice.Subsequently 3 ul of 3 M sodium acetate pH 5.2 and 75 ul 100% ethanol were addedand the tube was vortexed for 2 sec. After incubation at -20°C for 20 min, the tubewas centrifuged for 10 min at 14,000 rpm (16,000 x g) and 4°C, the DNA waswashed with 70% ethanol, recentrifuged, and the supernant was aspirated out. The26Materials and MethodsDNA was dried under vacuum for 5 min and dissolved in 12 ul of sterile distilledwater. For sequencing reactions, this 12 ul of denatured DNA was mixed with 2 ulof annealing buffer from the Pharmacia T7 sequencing kit, incubated at 37 °C for 20min and allowed to cool to room temperature for 10 min. The subsequent labellingand termination reactions were performed as outlined in the Pharmacia protocol.Direct sequencing of double stranded PCR product was done with a methodadapted from Casanova et al. (1990) using the Pharmacia T7 sequencing kit. ThePCR product was first purified with GENECLEAN to remove primers anddeoxynucleotide triphosphates, eluted in 12 ul of 10 mM Tris pH 8.0 and 1 mMEDTA buffer (TE), and quantified by electrophoresis of 1 ul of the GENECLEANproduct on an agarose gel. About 0.5 pmoles (0.15 ug) of PCR product, 10 pmolesof sequencing primer, and 2 ul of Pharmacia annealing buffer were mixed withsterile distilled water to a total volume of 14 ul. This mixture was heated on a heatblock at 100°C for 3 min, then immediately snap-cooled in a dry ice-ethanol bath tofreeze the sample.The annealing mix was removed from the dry ice-ethanol bath and 6 ul oflabelling mix was added as it thawed. The tube was centrifuged for 2 sec andlabelling proceeded for 45 sec at room temperature. Then 4.5 ul of the labelledtemplate was added to each termination mix and incubated for 2 min at 37 °C. Thereactions were terminated with 5 ul of stop mix and stored at -20 °C untilelectrophoresis.Electrophoresis of sequencing reactions was done with 6% polyacrylamide-50% urea gels on a BRL Model S2 sequencing apparatus (75 Watts). Prior toelectrophoresis, reactions products were denatured at 80 °C for 2 min and kept on27Materials and Methodsice until loaded. The buffer gradient technique of Sheen and Seed (1988) was used.PATHOGENICITY TESTS AND GENETIC CROSSESThe pathogenicity testing and genetic crosses were done by injecting U.maydis suspensions into 1 week old corn seedings ("Golden Bantam", BuckerfieldsSeeds, Vancouver, B.C.). Each haploid transformant was injected as a mixture witheither strain 521 (albl) or 031 (alb2) to determine if the pathogenicty testscorrelated with the incompatibility tests. Fungal cells were prepared for injection bywashing 2 ml of a late log or early stationary phase culture with sterile distilledwater and diluting each strain to 106 cells per ml in sterile distilled water. Injectionwas done with 0.1 to 0.5 ml of cell suspension injected just above the soil line using a3 ml syringe and 26 gauge needle. Liquid was injected until it came out the top ofthe seedling.Plants were grown in a greenhouse from September to November 1991.Supplemental sodium lights were used in November with a photoperiod of 16 hrs.The soil mixture contained 2 parts sterile soil to 1 part peat with Osmocote fertilizer(Grace-Sierra, Milpitas, CA) at approximately 10 grams per liter of soil mixture.Symptoms appeared about 1 week after injection and teliospores were collectedafter 2 to 3 weeks.Products of meiosis were obtained by germinating the teliospores andisolating random haploid progeny. Fresh or dry tumor tissue containing teliosporeswas ground with a mortar and pestle in about 10 ml of 1.5% (w/v) copper sulfatepentahydrate and filtered though cheesecloth into a 50 ml Falcon tube. Theteliospores were incubated overnight at room temperature in copper sulfate28Materials and Methodssolution, then washed twice in sterile distilled water, and taken up in 1 to 10 ml ofwater, depending on the yield of teliospores. One hundred and fifty ul of thisteliospore suspension was plated on PDA and incubated for 2 days until colonieswere visible.The colonies were transferred, using a sterile cotton swab, to 5 ml of PDBand suspended by vortexing. This suspension was diluted and plated onto PDA togive about 100 colonies per plate. Plates were incubated at 30 °C for 2 days.Individual colonies were transferred by toothpick to PDA in a 8 by 6 grid patternand tested for mating type by using the replica mating procedure described abovewith the testers strains 521 (alb1), 518 (a2b2), 031 (alb2), and 032 (a2b1). Theprogeny were also tested for hygromycin resistance by replica transfer to PDAcontaining 200 ug/ml hygromycin B.29RESULTSMATING TESTS OF TRANSFORMANTSApproximately 1700 hygromycin resistant transformants were tested for theirincompatibility reaction by replica mating with bl and b2 testers. Within this group,there were 192 transformants from plasmid pAR11, and 384 transformants fromeach of the 4 plasmid pools. In addition to these 1700 transformants, there wereabout 100 transformants derived from plasmid pAR21; these were screened foraltered mating type, by cross-streaking of cultures from agar plates, during anexploratory experiment. With respect to mating type, three classes of transform antswere found. Class I transformants were unaltered from the original b2 specificity,class H transformants had changed to a different specificity that was neither bl norb2, and class III transformants had switched from a b2 to a bl specificity (Figure 11).A minor class of transformants, designated as the null class, gave no mating reactionwith either tester.30Results Class Class^ClassI^II III(a2b2) (a2bx) (a2b1)521 (a1b1)031 (alb2)• •• •Figure 11. Mating type classes of transformants.Separate liquid cultures of transformant (originally a2b2) and tester (either albl oralb2) were spotted together on charcoal mating plates. Plates were sealed withParafilm and incubated at room temperature for 2 days. White aerial myceliumindicates a compatible reaction produced by strains containing different a and balleles. There were 3 main classes of transformants: class I - specificity wasunchanged from original b2, compatible reaction with bl only; class II - specificitywas different from bl or b2, compatible reaction with both bl and b2; class III -specificity has changed from b2 to bl, compatible reaction with b2 only.31ResultsThe proportion of transformants belonging to each class depended upon theplasmid used for transformation. These results are summarized in Table 3. Themost frequent class of tranformants was class I (unaltered from original b2specificity). This class comprised about 80% of the transformants for plasmidscontaining more than the first 39 codons of the bl ORF and comprised 100% of thetransformants for plasmids that contain the first 39 codons or less. Class Itransformants represent those in which the transforming DNA has integrated at anonhomologous position (not at the b locus) or in which the transforming DNA hasintegrated at the b locus but without enough bl sequence to alter the mating type.This suggests that the frequency of nonhomologous integration under theseconditions is about 80% and that the first 39 amino acids of the bl variable regiondo not affect allelic specificity.Plasmids that contained endpoints from codons 44 to 94 gave mostly class Iand II transformants. This shows that recombination in the intermediate regionbetween codons 39 and 107 results in alleles that are different from the two parentalleles. Plasmids that contained the first 107 or more codons from bl gavetransformants belonging mostly to class I or III. The class III transformants haveswitched from b2 to bl specificity. These results show that the variable region doesdetermine allelic specificity but they do not accurately map the borders of thespecificity region.32ResultsTable 3. Proportion of transformants belonging to each mating type class as afunction of the plasmid used for transformation.Plasmid Endpoint Class Class Class Nullcodon I II IIIpAR11 155 85% 0% 15% 0%pAR12 topAR15(pool a)pAR16 to139 to10794 to80%81%4%15%15%2%1%2%pAR20(pool b)pAR21 to7562 to39/4880%7/4819%1/480%1/481%pAR24(pool c)pAR25 to4439 to 100% 0% 0%5/3800%pAR27(pool d)14 48/48 0/48 0/48 0/48Class I transformants have the original b2 specificity, class II transformants have aspecificity different from bl or b2, and class III transformants have a bl specificity.Actual numbers of transformants in each class, recorded from subsamples, are givenfor some of the plasmids. All other percentages are estimates based on observationstaken during the replica mating screen.33ResultsPCR SCREEN FOR HOMOLOGOUS INTEGRATIONOut of 54 class I transformants screened by PCR, 9 showed homologousintegration at the b locus, giving a frequency of 17%. These class I transformantswere obtained from plasmid pools c and d. An agarose gel demonstrating theseresults is shown in Figure 12.9:1aN4-3—2—1-Figure 12. Agarose gel from PCR screen for homologous integration.Electrophoresis of PCR products amplified from genomic DNA of U. maydistransformants using primers Hph and 6B (see Figure 10). Ten ul of PCR reactionwas electrophoresed on a 0.7% agarose gel and then stained with ethidium bromide.DNA kilobase (kb) markers were electrophoresed in the far left lane. Samples 34,37, and 40 show a fragment approximately 3 kb in size that is expected fromtransformants that have undergone homologous integration at the b locus.34ResultsNUCLEOTIDE SEQUENCE OF RECOMBINANT ALLELESThe positions of recombination points in transformants showing homologousintegration were determined by PCR amplification of the b variable region andDNA sequencing of the product (Table 4). The recombination point was defined asthe first nucleotide position or amino acid position, depending upon the context ofthe discussion, that switches from bl to b2 sequence.All nucleotide recombination events analysed were found to be in-frame andin-register. That is, the recombination events resulted in a switch from thenucleotide sequence of the bl allele to the corresponding sequence of the b2 allele,without any deletions or insertions. Also, all transforming DNA fragmentscontained SphI linker sequences at their ends and in no cases were SphI sequencesfound in the recombinant alleles.Recombinant alleles resulting from transformation by an individual plasmidwere analysed and these results showed that recombination can occur anywherefrom the end of the plasmid to 100 nucleotides from the end. Figure 13 shows tworecombinant alleles resulting from transformation with pAR21. Plasmid pAR21contains bl sequences up to nucleotide + 184 (codon 62). It produced one allele(bx70) with bl nucleotide sequences up to position + 185 and another allele (bx51)with bl sequences up to nucleotide + 151, which is about 30 nucleotides from theend of the plasmid. Nucleotide position + 1 is the first nucleotide of the initiationcodon. The plasmid pAR11 contains bl sequences up to nucleotide +464 (codon155) and it produced alleles (Table 4) with recombination points ranging fromnucleotide +467, which is right at the end of the bl sequence, to nucleotide +354,about 100 nucleotides from the end of the bl sequence.35ResultsTable 4. Summary of the recombinant b allele transformants.Mating reactions were tested by crossing the transformants with tester strains 521(a1b1) and 031 (alb2).Transformant AlleledesignationRecombination pointCodon^NucleotideMating reaction^521^031^(bl) (b2)t11-2 bx156 156 +467 +t11-2 bx142 142 +424 +t11-7 bx128 128 +354 +to-1 bx112 112 +333 +to-3 bx142 142 +424 +to-5 bx79 79 +235 + +to-23 bx107 107 +320 +to-28 bx128 128 +342 +tb-1 bx70 70 +186 + +tb-5 bx79 79 +235 + +tb-7 bx87 87 +259 + +tb-11 bx48 48 +142 + +tb-14 bx90 90 +269 +tb-16 bx70 70 +209 + +tb-21 bx92 92 +274 +tb-23 bx70 70 + 198 + +t3-1 bx79 79 + 186 + +t4-6 bx70 70 +198 + +t4-8 bx5 1 51 + 152 + +tc-1 bx49 49 + 145 + +tc-3 bx5 1 51 + 152 + +tc-7 bx60 60 +180 + -+td-12 bx28 28 +82 +td-27 bx39 39 +114 +td-35 bx28 28 +82 +The mating reaction ratings: (-) incompatible; (+) compatible.Recombination points were defined as the first nucleotide or codon that switchesfrom bl to b2 sequence. Some recombinant alleles (e.g. bx70) are represented bymore than one nucleotide recombination point if recombination has occurred in aarea where the nucleotide sequence differs between bl and b2 but the amino acidsequence does not.36Resultsnt^+160^ +180Spb.TpAR2 1^CTACCCCGCGATCCCGAAACGATCCAGCAAACGCATQ*bx70^CTACCCCGCGATCCCGAAACGATCCAGCAAATACACCAGACTACTCACAGGATTAAAGTCGCTGCC*b2^CTATCCTACGATCCGGGCACGATCCATCAAATACACCAGACTACTCACAGGATTAAAGTCGCTGCCnt^ +140^ +160^ +180^. SphIpAR2 1^CAACAAAAGATACCCAACGACATTOCAAACCTACCCCGCOATCCCGAAACGATCCAOCKIUWW•ATQ*bx51^CAACAAAAGATACCCAACGACATTGCAAGCCTATCCTACGATCCGGGAACGATCCATCAA*b2^CGACGAAAGACACCCAACAACGTTGCAAGCCTATCCTACGATCCGGGAACGATCCATCAAFigure 13. Nucleotide sequence of recombinant alleles produced from pAR21.The nucleotide recombination point (*) of the two alleles bx51 and bx70 is definedas the first nucleotide position that switches from bl to b2 sequence. The blsequence of plasmid pAR21 is aligned above the recombinant allele sequence andthe b2 sequence is aligned below. Plasmid pAR21 contains bl sequences up toposition +184, followed by the Sphl restriction site sequence.37ResultsANALYSIS OF AMINO ACID SEQUENCES OF RECOMBINANT ALLELESA map of the codon positions of 16 recombination points (Figure 14)revealed a 48 amino acid region responsible for allelic specificity. This regionoccurred between codons 39 and 87. Recombination between bl and b2 within thisregion generated alleles different from either bl or b2 (class II transformants). Inall cases, the recombination events resulted in a switch from the sequence of oneallele to that of the other without the introduction of an additional or incorrectamino acid.Class IIRecombinants here have aspecificity differentClass I^ from bl or b2^ Class IIIRecombinants here haveb2 specificity218^319Recombinants here havebd specificity1I^ I^I I49 90^1124S  51 600^70 79^Si i 92 107i^125^142^156‘^I I^I^1^II^I 1 I 1 1Cotton 0^ Specificity Region 160Variable Region ^Figure 14. Map of recombinant b alleles.Map shows the codon positions of 16 recombination points.38ResultsThe predicted amino acid sequences for recombinant alleles at the left andright borders of this specificity domain are shown in Figures 15 and 16. The leftborder region (Figure 15) was defined by recombinant allele bx39, which has b2specificity, and bx48, which has a specificity different from bl or b2. A comparisonof the amino acid acid sequences of these two alleles reveals that they differ atcodons 39, 42, 43, and 45. At each position except 45, a positively charged aminoacid (arginine) has been substituted for an uncharged one (glutamine). The rightborder region (Figure 16) is defined by the recombinant alleles bx79, which has aspecificity different from bl or b2, and bx87, which has a bl specificity. These twoalleles differ at codons 79 (isoleucine for phenylalanine) and 82 (serine for lysine).Isoleucine and phenylalanine are both uncharged and nonpolar but phenylalaninedoes have an aromatic side chain. Serine is uncharged and polar while lysine ispositively charged. These results show that relatively few nonconservative aminoacid changes at the border regions can change allelic specificity.39ResultsCodon 30^ 40^ 50^ 60* * * •bl^NYPVLVRICLIIIILQQKIPNDIANLPRDPICTIQ++ +bx39^NYPVLVRICLQICLRRICTPNNV A S L S Y D P G T I Hbx48^NYPVLVRICLRKLQQXIPNNV A S L S Y D P G T I H++^+b2^NRPVLVRICLQIILRRKTPNNVASLSTDPGTIRQ - Glutamine: uncharged, polarR - Arginino: basic (positively charged)T - Threonins: uncharged, polarI - Isoleucino: uncharged, nonpolarFigure 15. Amino acid sequences of the left border region.Amino acids are shown using the one letter code (Appendix D). Plus symbolsindicate predicted amino acid differences between alleles bx39 and bx48. Theamino acid sequences of bl and b2 are given above and below the recombinantallele sequences, respectively.40ResultsCodon 70^ 80^ 90^ 100* * * *b 1^RAVAQATIRXDQXXV3LCSEVVEGTSXVMQZ+^+bx79^RAVAQATIRIDQWSLHSDAVEDTSKALXXbx87^RAVAQATIRTDQXTVSLXSDAVEDTSKALXX+^+b2^XVAAXATIRIDWVSLESDAVIDTSKALXXI - Isoleucine: uncharged, nonpolarP - Phenylalanino: uncharged, nonpolarS - Serino: uncharged, polarK - Lysine: basic (positively charged)Figure 16. Amino acid sequences of the right border region.Amino acids are shown using the one letter code (Appendix D). Plus symbolsindicate predicted amino acid differences between alleles bx79 and bx87. Theamino acid sequences of bl and b2 are given above and below the recombinantallele sequences, respectively.41ResultsTHE NULL CLASS TRANSFORMANTSAs mentioned earlier, the replica mating screen identified a minor class oftransformants that gave null mating reactions to both testers. They occurred at afrequency of 0% to 2%. Investigation of 2 out of 8 of these null class transformantsshowed that they were slow growing strains that gave delayed, compatible (fuz + )mating reactions with both bl and b2 testers when retested using liquid culturemating tests. The PCR screen for homologous integration was negative for thesetwo transformants. A possible explanation for these results is that a gene whichaffects growth and mating reaction has been disrupted in these transformants byintegration of transforming DNA.PATHOGENICITY TESTS AND GENETIC CROSSESSelected transformants were tested for pathogenicity by injecting them intocorn seedlings in a mixture with bl or b2 tester strains. The pathogenicity data forthe transformants tested were consistent with the mating tests (Table 5).Compatible combinations of transformant and tester were pathogenic andincompatible combinations were nonpathogenic.42ResultsTable 5. Pathogenicity data for transformants.Transformant AlleledesignationMating Reaction521^031(b 1) (b2)Pathogenicity521^031(bl) (b2)t11-1 bx156 + nd ndt11-2 bx142 + 0 3t11-7 bx128 + nd ndto-1 bx112 + nd ndto-3 bx142 + nd ndto-5 bx79 + nd ndta-23 bx107 + 0 4ta-28 bx128 + nd ndtb-1 bx70 + + 4 3tb-5 bx79 + + 3 2tb-7 bx87 + 0 3tb-11 bx48 + + 4 3tb-14 bx90 + 0 4tb-16 bx70 + + nd ndtb-21 bx92 + 0 3tb-23 bx70 + + 4 4t3-1 bx79 + + 4 4t4-6 bx70 + + 4 3t4-8 bx51 + + 4 4tc-1 bx49 + + 4 4tc-3 bx51 + + nd ndtc-7 bx60 + + 4 4td-12 bx28 + 4 0td-27 bx39 + nd ndtd-35 bx28 + nd ndnd - not doneIncompatibility and pathogenicity was tested by crossing the transformants withtester strains 521 (albl) and 031 (alb2). The mating reaction ratings are: (-)incompatible; (+) compatible. The pathogenicity ratings from 0 to 5 were adaptedfrom Kronstad and Leong (1989): 0 - no symptoms; 1 - anthocyanescence onleaves; 2 - small leaf galls present; 3 - small stem galls present; 4 - large stem gallspresent; 5 - plants dead.43ResultsTeliospores obtained from the pathogenicity tests were germinated and theproducts of meiosis were subjected to genetic analysis. The objectives were todetermine (1) whether the b alleles would segregate independently of the a alleles,(2) whether the recombinant b allele would segregate 1:1 with the tester b alleles,and (3) whether the hygromycin marker would be linked to the recombinant allele.The genetic data for a representative transformant t4-6 (a2bx70) are shown in Table6.Table 6. Genetic analysis of a cross between transformant t4-6 (a2bx70) and tester521 (alb1).Transformant x tester^ ProgenyGenotype^Numbers(a2bx70 Hygr) x (al b1 Hye) albl Hygs^9a2b1 Hygs^11albx70 Hygr^8> 12a1bx70 Hygs^4a2bx70 Hygr^1114a2bx70 Hye 3 46No progeny were found of the genotype a1b1 Hygr or a2b1 Hygr44ResultsThe null hypothesis is that there is no linkage between a and b, and weshould expect a 1:1:1:1 ratio between the progeny classes albl, a2b1, albx70, anda2bx70. A Chi-square (Suzuki et al. 1989) calculated for the above data gives avalue of 1.13. At 3 degrees of freedom and a Chi-square equal to 1.13, theprobability of the null hypothesis being correct is greater than 50%. Thus weconclude that there is no linkage between the a and b from the above data.The bl and bx70 alleles show a 1:1 segregation which confirms that they arealleles at the same locus. Also, the data show linkage between the hygromycinresistance marker and the recombinant bx70 allele. The bx70 Hygs progeny areprobably due to inadequate transfer of these colonies to the hygromycin plate. Theresulting poor growth would cause them to be scored as Hyg s. The fact that therewere no Hygr bl progeny is good evidence that the hygromycin marker was notsegregating from the b locus. Genetic data from two other crosses involving allelesbx51 x b2 and bx79 x bl gave the same results as the cross bx70 x bl, namely, therecombinant allele and tester allele showed a 1:1 segregation and the Hyg r markerwas linked to the recombinant allele.45DISCUSSIONThe in vivo recombination strategy described in this work was successful inproducing a series of bl-b2 recombinant alleles. The analysis of these recombinantalleles provided insight into recombination in U. maydis, into the organization of thespecificity region of the b gene, and possibly, into the mechanism by which newalleles might have arisen. These results also contribute to attempts to developmodels for multiallelic recognition and the function of the b locus.In terms of recombination, it was found that homologous integrationoccurred at a frequency of about 15% to 20%. This is lower than the frequencies of25 to 70% obtained from other targeted gene replacement experiments with othergenes in U. maydis (Kronstad et al. 1989; Fotheringham and Holloman 1990). Itmay be that homologous integration at the b locus does not occur as frequently asintegration of other genes at their homologous sites. The frequency of homologousintegration inAspergillus nidulans has been shown to vary depending on the locusunder investigation (Tilburn et al. 1983). The degree of homology between thetransforming DNA and the target genomic sequences probably affects the frequencyof homologous integration. It is possible that the sequence differences between thebl and b2 variable regions or presence of SphI linker sequences at the end of eachtransforming fragment may have reduced the frequency of homologous integration.The analysis of recombinant alleles derived from specific plasmids showed thathomologous integration can occur anywhere from the end of the linear transformingDNA to about 100 by from the end.46DiscussionThe mating tests with transformants containing recombinant alleles andsubsequent nucleotide sequence analysis of the bl-b2 hybrid alleles allowedconstruction of a fine structure map of the specificity region. The results haveidentified a 48 amino acid region, between codons 39 and 87, that is responsible forallelic specificity. This is in close agreement with Dahl et al. (1991), who reportedthat the specificity domain involving the b2 and b3 alleles was located betweenpositions 56 and 115. The remarkable finding here is that recombination within thespecificity region generated alleles that were different from either of the two parentalleles. A comparison of recombinant alleles (having different specificities) thatdefined the amino terminal and carboxy terminal boundaries of the specificityregion indicated that very few amino acid differences can alter specificity.Given the demonstrated ability to generate new alleles by recombination invivo, it is tempting to speculate that a similar mechanism may have been responsiblefor the development of this multiallelic recognition system. That is, new alleles mayhave evolved by reciprocal crossing-over events between b sequences duringmeiosis. Past searches among meiotic progeny for this type of recombination havebeen unsuccessful (Day et al. 1971; Puhalla 1970). The results presented hereindicate that this failure may be due to the small size of the region (about 144 bp)where recombination would have to occur. The analysis of the borders of the regionof specificity also suggests that mutation could contribute to the generation of newalleles. This idea could be tested using in vitro mutagenesis to introduce amino acidchanges at key codons in the border regions or in the hypervariable regions. Thesealtered alleles could be introduced into U. maydis by transformation and genereplacement and the resulting strains could be tested for altered b allele specificity.47DiscussionMany different models for the molecular mechanism of multiallelicincompatibility have been proposed (Kuhn and Parag 1972; Ullrich 1978) but recentmolecular work with the b locus supports a model involving dimerization of bproteins and then binding of the protein complex to DNA to activate transcriptionof developmental genes. The b proteins are thought to be transcription factorsbecause of the presence of a homeodomain sequence in the constant region (Schulzet al. 1990).Recent evidence from this laboratory (Bakkeren et al., manuscript inpreparation) and from the laboratory of R. Kahmann (personal communication)shows the presence of another gene immediately upstream (about 200 bp) of the bORF. It is thought that the product of this newly discovered gene, designated bW,interacts with the b polypeptide and that the multimeric complex acts to regulatedevelopmental processes. The predicted protein product of bW also possessesvariable, constant, and homeodomain regions and it is likely that the specificitydomain of b interacts with an analogous region in the bW product.In our model, control of transcription could be achieved by the differentialactivity of specific combinations of b and bW proteins. One possibility is that b andbW proteins occur in nature as paired nonfunctional combinations (e.g. bl andbW1). Thus, the combination of b and bW proteins other than the nonfunctionalcombination (e.g. bl and bW2) results in activation of genes involved in the sexualcycle and pathogenicity. An alternate possibility is that specific combinations of band bW proteins, such as bl and bW1, interact with a cytoplasmic anchoring function(Kronstad and Leong 1990) that prevents nuclear localization and thus prevents48Discussiontranscription of developmental genes. A possible nuclear localization signal isfound in the constant region of the b ORF (Kronstad and Leong 1990).The question of whether the b alleles with recombination points within thespecificity region have different specificities from each other is difficult to answer atthis time. If the b protein interacts with the bW protein (and not with other bproteins), then it is not possible to test for differences in specificity between therecombinant b alleles by testing them against each other. They must be testedagainst alleles of bW. One way this could be done is to cross strains carryingdifferent recombinant b alleles with a bank of strains carrying wild type bW allelesand to look for a difference in the pattern of compatible versus incompatiblereactions. A preliminary experiment testing transformants with alleles bx51, bx70,bx79, and bx107 with a set of eight U. maydis tester strains having uncharacterizedbW alleles did not give any differences between alleles bx51, bx70, and bx79. Thebx51, bx70, and bx79 alleles gave compatible reactions with some of the bW allelesand incompatible reactions with others (i.e., the recombinant alleles are notconstitutive) but the pattern of reactions was the same with all three alleles. Thebx107 allele did give a different pattern of mating reactions compared to the other 3recombinant alleles but this was expected because the recombination point forbx107 is outside of the specificity domain and it has a bl specificity (rather than aspecificity different from bl or b2). The set of 8 U. maydis testers may have beentoo small to differentiate the recombinant alleles.Another way to test whether the recombinant b alleles are different fromeach other would be to test them against a series of recombinant bW alleles. A49Discussionseries of recombinant alleles between bW1 and bW2 could be constructed as wasdone for bl and b2. Then, the series of recombinant b alleles could be tested in allcombinations against the series of recombinant bW alleles. Recombinant b allelesthat were different from each other would give a different pattern of matingreactions. Also, the analysis of incompatible combinations of recombinant b andbW alleles would allow an alignment of the b and bW domains that interact witheach other.The results from pathogenicity tests and genetic analysis confirm severalaspects of this work. First, the pathogenicity of transformant-tester combinationswas consistent with the mating tests. Second, the recombinant bx70 allele was stablemeiotically and it segregated as expected, i.e. independently of a and 1:1 with thetester bl allele. Finally, the Hygr marker co-segregated with the recombinant bx70allele. This evidence, in combination with the PCR test for homologous integration,supports the premise that the transforming DNA had integrated at the b locus toproduce a recombinant b allele and that the observed results were not due tointegration at other locations.50CONCLUSIONSThe in vivo generation of a series of recombinant alleles between hi and b2identified a 48 amino acid region responsible for allelic specificity. This region waslocated between codon positions 39 to 87 within the b ORF. Recombination withinthis region produced alleles that were different than either bl or b2. Analysis ofnucleotide sequences of recombinant alleles derived from specific plasmids showedthat homologous integration can occur anywhere from the end of the lineartransforming DNA to about 100 by from the end. A comparison of amino acidsequences of recombinant alleles at the left and right borders of this specificitydomain show that nonconservative amino acid substitutions can alter allelicspecificity of the b locus. These results suggest that the multiallelic recognitionsystem of U. maydis could have evolved through a combination of meiotic cross-overand mutation occurring within this specificity domain.51BIBLIOGRAPHYAgrios, G. N. 1988. Plant pathology. Third edition. Academic Press.Banks, G. R. 1983. Transformation of Ustilago maydis by a plasmid containing yeast2-micron DNA. Curr. Genet. 7:73-77.Casanova, J. L., Pannetier, C., Jaulin, C., and Kourilsky, P. 1990. Optimalconditions for directly sequencing double-stranded PCR products withSequenase. Nucl. Acids Res. 18:4028.Christensen, J. J. 1963. Corn smut caused by Ustilago maydis. Am. Phytopathol. Soc.,Saint Paul, MN. Monograph 2.Dahl, M., Bolker, M., Gillissen, B., Schauwecker, B., Schroeer, B., and Kahmann, R.1991. The b locus of Ustilago maydis: Molecular analysis of allele specificity.In: Hemmecke, H. and Verma, D. P. S., eds. Advances in molecular geneticsof plant-microbe interactions. Kluwer Academic Publishers, TheNetherlands. Vol. 1:264-271.Day, P. R. and Anagnostakis, S. L. 1971. Corn smut dikaryon in culture. Nature NewBiol. 231:19-20.Day, P. R., Anagnostakis, S. L. and Puhalla, J. E. 1971. Pathogenicity resulting frommutation at the b locus of Ustilago maydis. Proc. Natl. Acad. Sci. USA.68:533-535.Elder, R. T., Loh, E. Y. and Davis, R. W. 1983. RNA from the yeast transposableelement Tyl has both ends in the direct repeats, a structure similar toretrovirus RNA. Proc. Natl. Acad. Sci. USA. 80:2432-2436.Fotheringham, S. and Holloman, W. K. 1989. Cloning and disruption of Ustilagomaydis genes. Mol. Cell. Biol. 9:4052-4055.Fotheringham, S. and Holloman, W. K. 1990. Pathways of transformation in Ustilagomaydis determined by DNA conformation. Genetics 124:833-843.Froeliger, E. H. and Kronstad, J. W. 1990. Mating and pathogenesis in Ustilagomaydis. In: Raper, C. A. and Johnson, D. I. eds. Seminars in developmentalbiology. W. B. Saunders Company, London. Vol. 1:185-193.Frohman, M. A. and Martin, G. R. 1990. Detection of homologous recombinantsIn: Innis, M. A., Gelfand, D. H., Sninsky, J. J., and White, T. J. eds.. PCRprotocols: A guide to methods and applications. Academic Press, Inc. SanDiego, CA. p. 228-236.52BibliographyGritz, L. and Davies, J. 1983. Plasmid-encoded hygromycin B resistance: thesequence of hygromycin B phosphotransferase gene and its expression inEscherichia colt and Saccharomyces cerevisiae. Gene 25:179-188.Hausen, G. and Beiderbeck, R. 1987. The development of an in vitro host pathogensystem consisting of maize seedling segments infected with Ustilago maydis. J.Phytopathol. 118:97-102.Holliday, R. 1974. Ustilago maydis. In: King, R. C. ed. Handbook of Genetics.Plenum, New York. Vol. 1. p. 575-595.Kronstad, J. W. and Leong, S. A. 1989. Isolation of two alleles of the b locus ofUstilago maydis. Proc. Natl. Acad. Sci. USA 86:978-982.Kronstad, J. W. and Leong, S. A. 1990. The b mating-type locus of Ustilago maydiscontains variable and constant regions. Genes Dev. 4:1384-1395.Kronstad, J. W., Wang, J., Covert, S. F., Holden, D. W., McKnight, G. L. and Leong,S. A. 1989. Isolation of metabolic genes and demonstration of genedisruption in the phytopathogenic fungus Ustilago maydis. Gene 79:97-106.Kuhn, J. and Par% Y. 1972. Protein subunit aggregation model for self-incompatibitiy in higher fungi. J. Theor. 1% iol. 35:77-91.Ma, H., Kunes, S., Schatz, P. J., and Bolstein, D. 1987. Plasmid construction byhomologous recombination in yeast. Gene 58:201-216.Orr-Weaver, T. L., Szostak, J. W., and Rothstein, R. J. 1981. Yeast transformation:A model system for the study of recombination. Proc. Natl. Acad. Sci. USA.78:6354-058.Pompon, D. and Nicolas, A. 1989. Protein engineering by cDNA recombination inyeasts: shuffling of mammalian cytochrome P450 functions. Gene 83:15-24.Puhalla, J. E. 1968. Compatibility reactions on solid medium and interstraininhibition in Ustilago maydis. Genetics 60:461-474.Puhalla, J. E. 1970. Genetic studies of the b incompatibility locus of Ustilago maydis.Genet. Res. Camb. 16:229-232.Rowell, J. B. and DeVay, J. F. 1954. Genetics of Ustilago zeae in relation to basicproblems of its pathogenicity. Phytopathol. 44:356-362.Rowell, J. B. 1955. Functional role of compatibility factors and an in vitro test forsexual compatibility with haploid lines of Ustilago zeae. Phytopathol. 45:370-374.53BibliographySambrook, J., Fritsch, E. F. and Maniatis, T. 1989. Molecular cloning: A laboratorymanual. Second edition. Cold Spring Harbor Laboratory Press. New York.Sanger, F., Nicklen, S. and Coulson, A. R. 1977. DNA sequencing with chainterminating inhibitors. Proc. Natl. Acad. Sci. USA. 74:5463-5467.Schulz, B., Banuett, F., Dahl, M., Schlesinger, R., Schafer, W., Martin,T.,Herskowitz, I. and Kahmann, R. 1990. The b alleles of U. maydis, whosecombinations program pathogenic development, code for polypeptidescontaining a homeodomain-related motif. Cell 60:295-306.Sheen, J. Y. and Seed, B. 1988. Electrolyte gradient gels for DNA sequencing.BioTechniques 6:942-944.Specht, C. A., Munoz-Rivas, A., Novotny, C. P. and Ullrich, R. C. 1988.Transformation of Schizophyllum commume: An analysis of parameters forimproving transformation frequencies. Exp. Mycol. 12: 357-366.Suzuki, D. T., Griffiths, A. J. F., Miller, J. H. and Lewontin, R. C. 1989. Anintroduction to genetic analysis. W. H. Freeman and Co. New York, NY.Tilbum, J., Scazzocchio, C., Taylor, G. G., Zabicky-Zissman, J. H., Lockington, R.A. and Davies, R. W. 1983. Transformation by integration in Aspegillusnidulans. Gene 26:205-221.Toneguzzo, F., Glynn, S., Levi, E., Mjolsness, S. and Hayday, A. 1988. Use of achemically modified T7 DNA polymerase for manual and automatedsequencing of supercoiled DNA. BioTechniques 6:460-469.Tsukuda, T., Carleton, S., Fotheringham, S. and Holloman, W. K. 1988. Isolationand characterization of an autonomously replicating sequence from Ustilagomaydis. Mol. Cell. Biol. 8:3703-3709.Ullrich, R. C. 1978. On the regulation of gene expression: Incompatibility inSchizophyllum. Genetics 88:709-722.Wang, J., Holden, D. W. and Leon*, S. A.. Gene transfer system for thephytopathogenic fungus Ustilago maydis. Proc. Natl. Acad. Sci. USA. 85:865-869.Zhou, C., Yang, Y. and Jong, A. Y. 1990. Mini-prep in 10 minutes. Biotechniques8:172-173.54APPENDIX A. MEDIALB (Luria-Bertani)For 1 literdistilled water^ 950 mlNaC1^ 10 gbacto-tryptone 10 gbacto-yeast extract 5 gPDB (Potato Dextrose Broth)For 1 literdistilled water^ 1 literDifco potato dextrose broth powder^24 gPDA (Potato Dextrose Agar)distilled waterDifco potato dextrose agar powderAgar (supplemental)For 1 liter1L39 g5gCM (Complete medium)For 1 literCasamino acids^ 5 gAmmonium nitrate 1.5 gYeast extract 10 gSalt solution (see below)^ 62 mlDissolve in 850 ml H2O, adjust pH to 7.0 and bring volume to 1 liter. Autoclave 15to 20 min on liquid cycle.55Appendix A. MediaCharcoal Mating MediumCasamino acidsAmmonium nitrateYeast extractSalt solution (see below)For 1 liter10g3g20 g125 mlDissolve in 800 ml H2O, adjust pH to 7.0 and bring volume to 1 liter.Distribute 500 mis into each of two 1 liter flasks and add 5 grams of activatedcharcoal and 10 grams of agar to each flask. Mix by swirling, cover with foil andautoclave 20 min on liquid cycle. Then add 10 ml of sterile 50% glucose to eachflask. Mix well before and during the pouring of plates.Petri plates should be allowed to solidify quickly by pouring them thin (20 ml or lessper plate) and by not putting them in stacks. This will result in more consistentmating reactions because the charcoal is not given time to settle to the bottom ofthe agar.Ustilago maydis Salt SolutionFor 1 literKH2PO4^ 16 gNa2SO4 4 gKC1 8 gMgSO4 • 7H20 2 gCaC12 .2H20^ 1 gTrace elements solution^ 8 mlUstilago maydis Trace Element SolutionFor 500 mlH3B03^ 30 mgNhiC12 • 4H20 70 mgZnC12 200 mgNa2Mo04 • 2H20^ 20 mgFeC13 • 6H20 50 mgCuSU4 • 5H20 200 mg56APPENDIX B. TRANSFORMATION OF USTILAGO MAYDISA. PREPARATION OF COMPETENT SPHEROPLASTS1. Inoculate 5 ml of PDB with U. maydis cells from a fresh culture. Incubate inan orbital shaker at 30°C and 250 rpm overnight until an O.D.600 of about 2is reached.2. Inoculate 100 ml of complete medium with 1% glucose in a 2 liter Fernbachflask with 30 ul of U. maydis culture (O.D.600 about 2). This inoculationshould be done late in the day. Incubate overnight at,30°C and 250 rpm for18 to 20 hours until 0.D.600 is 0.9 to 1.0 (about 5x10 ' cells/ml for strain521). The cells must be in early log phase and actively budding in order toachieve high competency.3. Pour the 100 ml culture into two 50 ml Falcon tubes. Centrifuge for 5 min atabout 3500 rpm (2000 x g; about setting 90 on IEC Centra 4B table topcentrifuge); decant.4. Resuspend cells in 20 ml of fresh 5 mM EDTA and 25 mM B-mercaptoethanol by vortexing. For 40 ml of this solution, mix 40 nil of steriledistilled H2O, 400 ul 0.5 M EDTA pH 8, and 70 ul B-mercaptoethanol.5. Incubate at room temperature on orbital shaker at about 60 rpm for 20 min.6. Centrifuge the cells again at 3500 rpm (2000 x g) for 5 min and resuspend byvortexing in 10 ml of buffer I (1 M Sorbitol, 50 mM sodium citrate, pH 5.8).Then add 250 ul of filter sterilized Novozyme 234 (100mg/m1 in buffer I) tothe suspension (final concentration of 2.5mg/m1) and mix. Take a 5 ulsample to microscopically monitor spheroplasting.7. Incubate the cells at room temperature on an orbital shaker at about 60 rpmwhile protoplasting is monitored with the microscope. Within 5 to 10minutes, the ends of the rod shaped cells of U. maydis should begin toballoon out. When this happens place cells in centrifuge and centrifuge at600 rpm (60 x g) for 10 minutes. Centrifugation is done at a slow speed sothe cells can be resuspended easily. For strain 518; centrifuge the cells whenabout 25% of them show balloon ends. Protoplasting continues during thecentrifugation so that > 90% of the cells have become protoplasted at theend of the centrifugation.8. Decant the supernatant; resuspend the pellet in the residual supernatant byshaking tube gently back and forth. This should break up pellet and form athick suspension. Then add 10 ml of buffer I. Centrifuge the cells again at600 rpm (60 x g) for 10 min.57Appendix B. Transformation of Ustilago maydis9. Decant and resuspend the cells in the residual supernatant as before. Thenadd 10 ml buffer II (1 M Sorbitol, 25 mM Tris-HC1 pH 7.5, 50 mM CaC12).If the suspension is really clumpy, then resuspension can be done by ,ent -lepipeting up and down with a disposable 10 mlpipet (reusable glass pipetsmay carry some residual soap or contaminants); do not overtreat the cells.Recentrifuge at 600 rpm (60 x g) for 10 min.10. Decant and resuspend the cells in the residual supernatant as before. Thenadd 2 ml buffer II. If the suspension is clumpy, then resuspend by gentlepipetting with a Gilson P1000. Perform a cell count wiSh a hemacytometeron a 1/10 dilution in buffer II. Dilute the cells to 2x10° cells/nil with bufferH.11. For each ml of protoplasts (2 x 108 cells/nil) add:10 ul B-mercaptoethanol50 ul DMSO250 ul 50% PEG with 25 mM Tris 7.5 and 50 mM CaC12.This protoplast mixture can be stored at 0°C for up to 24 hr without loss oftransformation frequency or stored at -70°C with minimal loss of transformationfrequency. Aliquot the final protoplast mixture into 1 ml portions.B. DNA ADDMON.1. Thaw the protoplast mixture on ice (if frozen). In a separate microfuge tube,mix 2 ul of heparin (15 ug/ul), 1 to 5 ug of transforming DNA and steriledistilled water to a final volume of 15 ul. Place the tube on ice for 5 min.2. Then add 125 ul of the protoplast mixture (2x10 7 cells) to the DNA mixture;incubate on ice 10 min.3. Add 100 ul of room temperature 50% PEG in 25 mM Tris 7.5 and 50 mMCaC12. Mix by inversion. Incubate at room temperature for 20 min.4. Add 1 ml of buffer II. Centrifuge at 3500 rpm (1000 x g) for 10 min. Gentlyaspirate the supernatant.5. Add 1 ml buffer H again. Centrifuge at 2000 rpm (325 x g) for 5 min.Aspirate the supernatant.6. Add 200 ul double complete medium with 1% glucose and 1 M sorbitol. Aregeneration incubation is not required.7.^Spread the entire 200 ul on one transformation plate consisting of 10 ml ofdouble complete agar with 1 M sorbitol and 500 ug/ml of hygromycin Boverlain by 10 ml of the same medium without hygromycin.58Appendix B. Transformation of Ustilago maydis8.^Transformants are visible within 3-4 days. Transfer the transformants ontoPDA containing 150 ug/ml Hyg B to test for stability. The transformants canbe stored on PDA at 4 C for 1-2 months.COMMENTSThe 50% PEG in 25 mM Tris pH 7.5 and 50 mM CaC12 solution should be fresh.For 10 ml, add to a 25 ml graduated cylinder;5 g PEG 3350 or 40004 ml (11120Cover with foil, autoclave, cool to 65°C; then add:500 ul 1 M CaCl2250 ul 1 M Tris pH 7.5Make to 10 ml with sdH2O, mix by pipetting, cool to room temperature.The 50% PEG in 25 mM Tris and 50 mM CaC12 should be made the same day, orpossibly the previous day, and it is critical that it be autoclaved without the Tris andCaC12. For best results, buffers I and II should be less than 2 weeks old.The appearance of the cells in the hemacytometer will give an indication of howwell the protoplasting went. If many unprotoplasted cells are visible and protoplastslook bumpy and rough, then protoplasting did not go far enough. The cells will bedumpier and hard to resuspend; transformation frequencies will probably be lower.If the protoplasts look round and smooth, then protoplasting went well. Some olderlots of Novozyme 234 do not protoplast well.A small amount of osmotic shock to the protoplasts improves the transformationfrequencies. A DNA-Heparin volume of 12 to 55 ul works well when using 125 ul ofprotoplast mix. Transformation frequencies drop if DNA-heparin is added in only 5ul of solution for 125 ul of protoplasts. The DNA should not be diluted with anosmotic buffer; use sterile distilled water or TE buffer.The transforming DNA must be relatively clean for successful transformation withintegrative vectors; miniprep plasmid DNA from E. coli will give poor results .DNA should be of PEG precipitation or CsCI gradient quality. DNA that has beenrestriction digested must be phenol-chloroform/chloroform/ethanol precipitated orpurified with GENECLEAN before being used for transformation. Miniprepplasmid DNA will work if using a vector with an autonomously replicating sequence(ARS) and should give 10-100 transformants/ug DNA. Boiling lysis mini reps workbetter than alkaline-SDS lysis because the SDS is damaging to the protoplasts.59Appendix B. Transformation of Ustilago maydisThe buffer II has been modified from the original protocol by increasing the CaC12concentration from 25 to 50 mM. Other fungal transformation protocols haveshown that this is close to optimal for protoplast-PEG-CaC12 transformation(Specht et al. 1988; Judelson and Michelmore 1991).Fifty to two hundred stable transformants can be obtained from 1-5 ug of linearintegrative vector using this protocol.REFERENCESThis protocol has been adapted from Wang et al. 1988 and Specht et al. 1988.Judelson, H. S. and Michelmore, R. W. 1991. Transient expression of genes in theoomycete Phytophthora infestans using Bremia lactucae regulatory sequences.Curr. Genet. 19:453-459..Specht, C. A., Munoz-Rivas, A., Novotny, C. P. and Ullrich, R. C. 1988.Transformation of Schizophyllum commume: An analysis of parameters forimproving transformation frequencies. Exp. Mycol. 12: 357-366.Wang, J., Holden, D. W. and Leong, S. A.. Gene transfer system for thephytopathogenic fungus Ustilago maydis. Proc. Natl. Acad. Sci. USA. 85:865-869.60APPENDIX C. SMALL SCALE PREPARATION OF USTILAGO DNA.1. Inoculate 5 ml of PDB in a 18 x 150 mm test tube with U. maydis and growovernight until a high density is reached; 0.D.600 of 2.0 to 2.5.2. Transfer 1 ml of culture to a 1.5 ml eppendorf microfuge tube, and centrifugefor 30 sec at 14,000 rpm (16.000 x g).3. Aspirate off the supernatant.4. Add about 0.3 g of acid washed glass beads. A scoop made from anEppendorf microfuge tube cap can be used for this. Be careful not to getglass beads near the rim of the microfuge tube or else the tube will leakduring the following vortexing.6. Add 500 ul of lysis buffer. (0.5 M NaCl, 0.2 M Tris pH 7.5, 1% SDS, and 0.01M EDTA )For 10 ml of lysis buffer, mix together 5.7 ml dH2O, 1 ml 5 M NaCl, 2 ml 1 MTris pH 7.5, 1 ml 10% SDS, 0.2 ml 0.5 M EDTA pH 8.0.7. Add 250 ul of Phenol:Chloroform:Isopropanol (PCI - 24:24:1), and vortex for3 min on a S/P vortex mixer set to high (setting 10). If the Genie 2 vortexwith multi-sample head is used, then a 10 min vortex on high (setting 8) willgive equivalent yield. Gloves should be worn when handling PCI and thisshould be done in the fume hood.8. Centrifuge the tubes for 3 min at 14,000 rpm (16,000 x g) to separate thephases. Then transfer 450 ul of the upper aqueous phase to a new tube,being careful to avoid the cell guts at the interface.9. Add 250 ul of PCI; vortex 5 to 10 sec; then centrifuge for 30 sec at 14,000rpm (16,000 x g). Transfer 400 ul of the upper aqueous phase to a new tube.10. Add 0.6 vol of isopropanol or 2 vol of 100% ethanol. Vortex 2-5 sec. Storeat -20°C for 15 mm or more.11. Centrifuge for 5 min at room temperature; a white pellet should be visible;aspirate off the supernatant.12. Wash pellet with about 1 ml of 70% EtOH; aspirate off the supernatant;centrifuge for 5 min before aspirating if the pellet is loose. Dry the pelletunder vacuum for 5 mM.13. Dissolve the pellet in 100 ul of TE buffer (10 mM Tris pH 8 and 1 mMEDTA pH 8).61Appendix C. Small Scale Preparation of Ustilago DNA.COMMENTSThis protocol yields both DNA and RNA. The DNA will digest with restrictionenzymes and will amplify with PCR. Yield of total DNA is usually 20 ug or more.It is recommended to inoculate U. maydis from a fresh culture; inoculation fromcultures stored at 4°C for a few weeks will frequently fail.Occasionally, a microfuge tube will leak during vortexing. Leakage is usually theresult of glass beads trapped between the cap and the tube or faulty microfugetubes.The long vortex times are necessary to break apart most of the cells. Vortex timesof less than 2 min will result in lower, inconsistent yields of DNA (see Hoffman andWinston 1987).REFERENCESThis protocol has been adapted from the protocol for yeast DNA (Elder et al. 1983).Elder, R. T., Loh, E. Y. and Davis, R. W. 1983. RNA from the yeast transposableelement Tyl has both ends in the direct repeats, a structure similar toretrovirus RNA. Proc. Natl. Acad. Sci. USA. 80:2432-2436.Hoffman, C. S. and Winston, F. 1987. A ten minute miniprep from yeast efficientlyreleases autonomous plasmids for transformation of Escherichia coll. Gene57:267-272.62APPENDIX D. ABBREVIATIONSby - base pairDMSO - dimethyl sulfoxideEDTA - ethylenediaminetetra-acetic acidkb - kilobaseORF - open reading framePCR - polymerase chain reactionPEG - polyethylene glycolTE - 10 mM Tris pH 8.0 and 1 mM EDTA pH 8.0 bufferAmino AcidThree-LetterSymbolOne-LetterSymbolAlanine Ala AArginine Arg RAsparagine Asn NAspartic acid Asp DAsn and/or Asp Asx BCysteine Cys CGiutamine Gln QGlutamic acid Glu EGin and/or Glu Glx ZGlycine Gly GHistidine His HAmino AcidThree-LetterSymbolOne-LetterSymbollsoleucine Ile ILeucine Leu LLysine Lys KMethionine Met MPhenylalanine Phe FProline Pro PSerine Ser SThreonine Thr TTryptophan Trp WTyrosine Tyr YValine Val V

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