Proc. Natl. Acad. Sci. USAVol. 88, pp. 3922-3926, May 1991Genetics

Identification of genes governing filamentous growth and tumorinduction by the plant pathogen Ustilago maydis

(mating type loci/regulatory gene targets/repression/fungal pathogen of corn)

FLORA BANUETTDepartment of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94143-0448

Communicated by Ira Herskowitz, January 2, 1991 (receivedfor review October 1, 1990)

ABSTRACT Two master regulatory loci, a and b, governlife-cycle transitions of the phytopathogenic fungus Ustilagomaydis. Fusion of haploids that differ at both a and b results inproduction of a filamentous dikaryon, which induces tumors inits host, maize. Here I describe identification of genes distinctfrom a and b that play roles in these life-cycle transitions. Thesestudies identify three genes, fuzi, fuz2, and r0%f, that arenecessary for filament formation. fuzi is also necessary fornormal size and distribution of tumors and for teliosporeformation; fuz2 is also necessary for teliospore germination.Mutations in the rtn gene, which are recessive, bypass therequirement of different b alleles for tumor formation. Thisobservation indicates that rfl codes for a negative regulator oftumor induction. Thefuzl, fuz2, and rfl genes may be targetsfor the a and b loci.

Ustilago maydis, a basidiomycetous fungus that inducestumors on corn plants, is characterized by its dimorphism:haploid cells exhibit yeast-like growth on a variety of labo-ratory media and are not pathogenic, whereas dikaryons (theproduct of mating of two compatible haploids-i.e., havingdifferent a and b alleles) are filamentous, grow poorly if at allon laboratory media, and are pathogenic. The filamentousdikaryon differentiates within tumors, where karyogamy andspore formation occur and where these spores (teliospores)acquire competence to undergo meiosis (1, 2). The develop-mental program responsible for producing these two formsand for completing the above steps in the life cycle isgoverned by two master regulatory loci, a and b (the matingtype loci), known from classical genetic studies (3-6).The b locus, with 25 naturally occurring alleles, is the major

pathogenicity determinant (3-8); the presence of different balleles is a prerequisite for tumor induction. In addition, thepresence of different b and different a alleles (the a locus has2 naturally occurring alleles) is necessary for maintenance offilamentous growth (8). The b locus encodes a polypeptidecontaining a homeodomain-related motif (9), suggesting thatit is a regulatory protein. It is proposed to govern expressionof target genes responsible for cell-type specificity (9). The alocus may encode a regulatory protein.

In the budding yeast Saccharomyces cerevisiae, genesdistinct from the mating-type locus (MAT) necessary formating were identified by isolation of mating-defective mu-tants (10, 11). Many of these genes (the STE genes) proved tobe expressed in a cell type-specific manner (for example, ina or a haploid cells but not in a/a diploid cells) and to betargets of the regulatory proteins encoded by MAT (refs. 12and 13; reviewed in ref. 14). Some of these genes code forcomponents of the signaling pathway involved in mating (15,16). Another target gene is RMEI (repressor of meiosis),whose expression in haploids prevents initiation of meiosis

and sporulation. In the a/a diploid, al-a2 represses RMEJexpression, resulting in competence to initiate meiosis andsporulation (17).

I have used a rationale similar to that used in S. cerevisiaeto identify genes in U. maydis distinct from a and b that maybe more directly involved with filament formation, tumorinduction, and teliospore production and function. Thesestudies identify three new genes, fuzi, fuz2, and rtfl, that arenecessary for filament formation and tumor induction andthat may be target genes for the a and b loci.

MATERIALS AND METHODSStrains, Media, Growth Conditions, and Biological Meth-

ods. U. maydis strains are listed in Table 1 (see also Table 5for diploid strains and their construction) and were grown at320C. Media were as described (18). Exponentially growingcells were UV-irradiated as described (19, 20). Conditions forthe fuzz reaction are described in the table legends and in ref.8. Conditions for growth of corn plants (variety B164) and forinoculations are described in the legend to Table 2 (see alsoTable 4). Germination of teliospores and analysis of meioticsegregants are described in ref. 8.DNA Isolation and Nucleotide Sequence Determination. Ge-

nomicDNA from FBf4-lf, isolated as described (21), was usedto construct a library in pUC18. Plasmids containing bi wereidentified as described (9). Nucleotide sequences for bothstrands were determined by using single- and double-strandedtemplates with Sequenase (United States Biochemical) andoligonucleotide primers synthesized at the University of Cal-ifornia, San Francisco, BioMolecular Research Center.

RESULTSIsolation of Fuz- Mutants. An al bi strain (FB1-49) was

mutagenized with UV-irradiation to 8% survival. Colonieswere screened for the Fuz- phenotype after replica matingonto a lawn of an a2 b2 strain (FB2-47) on charcoal nutrientmedium. The wild-type al bi strain forms white fuzzycolonies under these conditions (the Fuz' phenotype; see ref.8) because of formation of dikaryotic filaments. Strains thatcarry identical a or b alleles exhibit a Fuz- phenotype.Thirteen Fuz- mutant candidates were obtained in 11,000colonies. Nine gave an altered fuzz reaction upon retesting(Fig. 1): some produced no filaments, others produced verysparse or morphologically altered filaments. Other screens ofthe same or different strains (a2 b2, a2 bi, or al b2) led to theisolation of a total of 80 Fuz- mutants with fuzz reactionssimilar to those of the original set of mutants.

Location offuz Mutations Relative to a and b. Genetic crosseswere performed with five of the mutants (strains Fuzl-Fuz5;Table 1) to determine if they carry mutations in a or b orelsewhere. Each of the five mutants was independently inoc-ulated with a wild-type a2 b2 strain (FB2). The success of across depends on formation oftumors that produce teliosporescapable of undergoing meiosis. Tumors containing teliosporeswere formed in each case. These teliospores exhibited reducedgermination efficiency (from <2% to 25%; Table 2) compared

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Table 1. List of strains

Strain Genotype Source/ref.FB1 al blFB2 a2 b2 Std. tester strainsFB6a a2 bi (see ref. 8)FB6b al b2FBD-12 al/a2 bJ/b2 pan-+

+/ade-FBD12-3 al/a2 bl/bl Ref. 8FB149 al bJ pan-FB2-47 a2 b2 ade- L

FUV-derived mutant:

FB1-49-2 al bi pan- met- of FB1-49FB6a-91 a2 bi ade- of FB6aFBfa (=Fuzl) al bJ pan-fuzd of FB1-49FBt2 (=Fuz2) al bi pan- fuz2- of FB1-49FBf3 (=Fuz3) al bl pan- rtfl- of FB1-49FBf4 (=Fuz4) al bi pan- rtfl- of FB1-49FBS12-13e a2 bi pan- ade- Meiotic seg.FBS12-4c a2 b2 pan ade- from FBD-12

FBflO-lg,-1i,-5i a2 b2 fuzl-FBflO-3i,-3c al bi fuzl J Seg. from cross 1FBflO-lc,-2c,-2e al b2fuzl-FBflO-5j a2 bl fuzlFBf22-lg al bi fuz2-FBf22-la,-ld,-5f al b2 fuz2-Seg. from cross 2FBf22-5c,-5d a2 bl fuz2-FBf3O-5a,-5i,-6a al bl rtfl-3 Seg. from cross 3FBf3O-11,-9b,-2d a2 bi rtfl-3FBf4-lc,-lf,-lj al bi rtfl-4 Seg. from cross 4FBf4-3c,-3e a2 bi rtfl-4FBS37,40 a2 b2FBS81,8 al bi IFBS13,127 a2biuz seg. from cross 5FBS121,124 al b2

Crosses were: 1, al bl fuzl- x a2 b2 fuzl +; 2, al bl fuz2- x a2b2fuz2+; 3, al bi rtfl-3 x a2 b2 rtf+; 4, al bi rtfl-4 x a2 b2 rtf+;5, a2 bi fuz2- x al b2 fuzl-. Std., standard; Seg., segregant(s).

with >85% germination efficiency for teliospores from wild-type crosses (Table 2). Thus, the mutations in these Fuz-mutants affect teliospore germination.

Meiotic segregants from the above crosses were tested formating type and mutant phenotype (Table 3). Crosses of theFuzl- and Fuz2- mutants to a wild-type strain producedFuz+ segregants of all different mating types. Conversely,Fuz- segregants were observed for all mating types. Theseobservations establish that the Fuzl- and Fuz2- strains carrymutations separable from a and b. Subsequent analysis(described below) shows that these strains carry mutations indifferent genes. It is not possible to determine rigorouslyfrom the data in Table 3 if more than one gene is affected inthese mutants. For simplicity, I assume that each straincarries a single mutation and designate the genes so identifiedas fuzi and fuz2.Crosses of the Fuz3- and Fuz4- mutants to a wild-type

strain yielded Fuz+ segregants, which were primarily of theb2 mating type (85% and 81%, respectively). Conversely,Fuz- segregants were primarily of the bi mating type (97%in each case), indicating tight linkage of the mutation to theb locus. In addition, a small number of recombinants wererecovered: 6 of 68 segregants from the FuzY cross and 6 of58 segregants from the Fuz4- cross. Recovery of suchrecombinants indicates that the mutations are separable fromthe b locus. The mutations in Fuz3- and Fuz4- strains arepresumed to affect the same gene, which is designated rtf7(for regulator of tumor formation).

FIG. 1. Fuzz reaction of wild type and fuz- mutants. Saturatedcultures of wild-type al bJ (FB1) and of mutant strains a! bi rtfl-4(FBf4-f), a! bi fuzl- (FBfIO-3i), and al bi fuz2- (FBf22-5d) (fromleft to right, respectively) were cross-streaked against wild-type a2b2 (FB2) (horizontal line) on charcoal nutrient medium and incubatedfor 48 hr at room temperature.

Cell and Growth Phenotypes of fuzi and fuz2. Both thefuzl- and fuz2- mutations lead to altered cell morphology:mutant cells are approximately twice as long and half as wide

Table 2. Phenotype of Fuz- mutants

TeliosporeFuz Tumor production

Cross phenotype induction (%)1. fuz+ x fUz+ + + + (85)2. fuzl- x fuz1+ +/- + + (16)3. fuzl- x fuzl+-= +/= - (NA)4. fuz2- x fuz2+ +/- + + (17)5. fuz2- x fuz2- - + + (<0.5)6. fuzl- x fuz2- - +/- + (32)7. rtfl-4 x rtf7 +/= + +(2)8. rtfl-3 x rtf1 +/= + + (24)Fuz phenotype was determined by cross-streaking the strains

indicated below on charcoal nutrient medium (8). The reaction wasscored at 16, 24, 48, and 72 hr of incubation at room temperature. Allnine Fuz- segregants from the cross al blfuzl x a2 b2fuzl+ listedin Table 1 were tested with the appropriate tester (cross 2) or amongthemselves (cross 3). All six Fuz- segregants from the cross al bIfuz2- x a2 b2fuz2+ listed in Table 1 were tested with the appropriatetester (cross 4) or among themselves (cross 5). Fuz- strains for cross6 were FBflO-5j (a2 bi) x FBf22-la,-ld,-5f (a] b2); FBflO-lc,-2c (alb2) x FBf22-5c,-5d (a2 bi). All five Fuz- segregants of the cross a!bi rtfl-4 x a2 b2 rtfl+ and all five Fuz- segregants of the cross albi rtfl-3 x a2 b2 rtfl + listed in Table 1 were tested with theappropriate tester strain (crosses 7 and 8, respectively). For cross 1,Fuz+ strains were the standard testers FB1 x FB2 and FB6a x FB6b(Table 1). Tumor formation was assayed after coinoculation of5-day-old corn seedlings, variety B164, with 0.2 ml of a 1:1 mixtureof saturated cultures (2 x 108 cells per ml) of the strains indicatedbelow. Tumor formation was detected as early as 3 days afterinoculation, and its development was followed for 1 month. Cross 3involved 24 independent inoculations with FBfIO-3i (a] bl) xFBf1O-lg,-li,-Si (a2 b2); FBflO-2c (a] b2) x FBfIO-5j (a2 bi). Cross5 involved 8 independent inoculations with FBf22-5d (a2 bl) xFBf22-5a,-5f (al b2). Cross 6 involved 28 independent inoculationswith FBf22-lg (a] bl) x FBflO-lg,-li,-5i (a2 b2); FBf22-5c (a2 bl) XFB10-2c (al b2); and FBf22-la,-5f (al b2) x FBfIO-5j (a2 bi). Forcrosses 2, 4, 7, and 8, the above mutant strains were coinoculatedwith the appropriate tester strains at least twice.fuz+ x fuz+ crosseswere FB1 x FB2 and FB6a x FB6b. Teliospore production wasassessed by direct visual inspection of the tumors (teliospores appearas dark brown specks in the whitish green tumors 8-10 days afterinoculation) and by microscopic observation of tumor slices. Telios-pore germination was examined microscopically on nutrient agarslabs after removal from tumors. Numbers in parentheses indicate %teliospore germination. NA, not applicable; Fuz+, full filamentformation; Fuz+/-, reduced or altered filament formation; Fuz+/=,very reduced filament formation; Fuz-, no filament formation.Tum+, full tumor induction; Tum+/-, reduced tumor size or delayedreaction; Tum+/=, very small tumors and altered distribution.

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Table 3. Segregation offuz- mutations with respect to the a andb loci

Mating type of segregant,Segregants no.

Cross Fuz phenotype No. al bi a2 b2 al b2 a2 biFuzl- x wt + 26 6 8 1 11

- 21 3 5 12 1Fuz2- x wt + 34 4 20 8 2

- 14 2 3 6 3Fuz3- x wt + 33 1 16 12 4

- 35 18 0 1 16Fuz4- x wt + 26 4 11 10 1

- 32 15 0 1 16The Fuz- strains were the original mutants (strains Fuzl-Fuz4),

which were derived from an al bi strain (FB1-49) as described in thetext. They were crossed to a wild-type (wt) a2 b2 strain (FB2), andthe mating type of meiotic segregants was deduced by reaction withstandard testers (8) (Table 1). The Fuz5 mutant gave a complexsegregation pattern (data not shown) and was not characterizedfurther. The greater number ofFuz+ than Fuz- segregants recoveredin the Fuz2- cross has several possible explanations-reducedpenetrance of the mutation, presence of unlinked genetic modifiers,requirement oftwo different mutations for the phenotype, or reducedviability of the Fuz- progeny. Plant inoculations, teliospore germi-nation, and mating reactions were performed as described in thelegend to Table 2 and in ref. 8. The total number of segregantsanalyzed in each cross derives from at least six different teliospores,and the reaction for each segregant was analyzed on two differentoccasions. Fuz phenotype: +, wild-type reaction; -, mutant reac-tion.

as the parental strain. This phenotype cosegregates with theFuz phenotype. Growth rates of the mutants approximatethose of the wild-type parental strain.Tumor Induction. To determine the effect of thefuz - and

fuz2- mutations on tumor induction and teliospore function,plants were coinoculated with sets of fuz- haploids. Thetumors produced byfuz - mutants are very small comparedwith those from control crosses (Table 2, lines 1-3) and arelocalized mainly along the leaf midrib rather than on theentire leaf as observed in control inoculations. No teliosporeswere produced. Thus, thefuzi gene appears to be necessaryfor filament formation, production of full-sized tumors, andformation of teliospores.

Coinoculation of plants withfuz2- haploids leads to tumorinduction similar to that of the controls (Table 2, lines 1, 4,and 5), though tumor formation is delayed 1-2 days comparedwith the normal situation. fuz2- x fuz2- tumors producedteliospores, but these teliospores were incapable of germi-nation (0 of 200 germinated' Table 2). Thus, the fuz2 geneappears to be necessary for filament formation and forgermination of teliospores.The presence of afuz- mutation in both mating partners

causes a more severe defect on filament formation (Table 2,lines 3, 5, and 6) than when only one of the mating partnersis mutant (Table 2, lines 2 and 4).

Allelism Tests offuzl and fuz2. The phenotypes exhibitedbyfuzl- orfuz2- mutants suggest that the mutations are indifferent genes. Crosses were performed between fuzl- andfuz2- strains to test whetherfuzl andfuz2 are allelic. Of 120meiotic segregants from 21 different teliospores from onecross, 30 were Fuz+, 66 were Fuz-, and 24 gave otherreactions (see below). The large fraction of Fuz' segregantsrecovered indicates that the Fuzl- and Fuz2- strains carrymutations in different genes. Backcrosses of the Fuz' seg-regants (Table 1) to a wild-type strain confirmed that they aregeneticallyfuz' (data not shown). The 24 unusual segregants(dual maters and fuzzy constitutives) may be fuzl fuz2-double mutants or may have arisen from meiotic nondisjunc-tion and have not been studied further.

Tumor Induction by rtfl- Mutants. Fuz3- and Fuz4-strains (Table 1) exhibited an unexpected behavior withrespect to tumor formation. Coinoculation of plants withhaploids carrying identical b alleles and an rtfl mutationyielded tumors identical in size and distribution (Table 4,experiment A) to those produced by two haploids carryingdifferent b alleles and a wild-type rtfl gene (Table 4, exper-iment D). These observations indicate that the rtfl - mutationbypasses the requirement for the presence of different balleles. If the rtfl - mutations bypass the need for combina-tions of different b alleles, then a haploid rtfl - strain (whichhas only one type of b allele) might be pathogenic. Indeed,one of the mutants, rtfl-4, appears to be weakly pathogenicin the haploid state: 20%o of inoculations with pure culturesyielded tumors (Table 4, experiment C). rtfl- haploids arenot as pathogenic in single infection as in coinfections. Thisdifference may indicate that mating itself or the presence ofdifferent a alleles is necessary for establishment of a fullypathogenic form.

rtfl Mutations Are Recessive. To determine dominance orrecessiveness of the rtfl- mutations, a set of diploids be-tween rtfl - and rtfl + strains was constructed. Inoculation ofplants with al/a2 bWI/b rtf1-4/rtf1+ or with al/a2 bWI/brtfl -3/rtfl + diploids did not induce tumor formation (Table 5,experiment A). Thus, rtfl-4 and rtfl-3 are both recessive tortfl+. al/a2 bl/b2 rtf/rtflJ diploids resulted in tumorinduction in all inoculations (Table 5, experiment B). Therecessiveness of rtfl - indicates that these mutations result inthe loss or reduction of Rtf function.

rgf Is Distinct from b. The crosses described above indicatethat the rtfl- mutation is close to but separable from the b

Table 4. Tumor production after coinoculation with rtfl- strains% tumor

Exp. Strains Tum' Tum- productionA al bl rtfl-4 + a2 b rtfl-4 25 7 78*

al bi rtfl-3 + a2 bi rtfl-3 12 13 48*B al b] rtfl-4 + a2 b2 rtfl+ 9 0 loot

a] b] rtfl-3 + a2 b2 rtfl 5 0 lootC ax bi rtfl-4 alone 3 12 20t

ax bi rtfl-3 alone 0 9 <10D a] b] rtfl+ + a2 b2 rtfl+ 10 0 100E a] bi rtf+ + a2 b rtfl 0 10 <10Conditions for growth of strains, plant inoculations, assessment of

teliospore formation, and abbreviations are as described in the legendto Table 2. Plants were maintained in a Conviron chamber (14 hr oflight at 280C; 10 hr of dark at 200C). In experiment A, coinoculationswith rtfl- strains (Table 1) were FBf4-lf + FBf4-3c (9) or + FBf4-3e(8); and FBf4-lj + FBf4-3c (7) or + FBf4-3e (4). For rtfl-3 strains,coinoculations were FBf3O-6a + FBf3O-9b (7); FBf3O-S5i + FB3O-2d(8); and FBf3O-5i + FBf3O-9b (5) or + FBf3O-1 (5). In experiment B,coinoculations with rtfl-4 strains were FBf4-lf + FB2 (5); andFBf4-3c + FB6b (4). For rtfl-3 strains, coinoculations wereFBf3O-6a + FB2 (3); and FBf3O-9b + FB6b (2). In experiment C, thertfl-4 strains were FBf4-lf (4), -lj (4), -3c (4), and -3e (3); the rtfl-3strains were FBfO-6a (3), -5i (3), and -9b (3). Numbers in parenthesesare numbers of independent inoculations. ax = a] or a2. In exper-iments D and E, the strains were FB1 + FB2 and FB1 + FB6a,respectively.*The tumors were indistinguishable from those obtained by coinoc-ulation of wild-type strains carrying different b alleles (experimentD) with respect to size, distribution, time course of development,teliospore production, and induction of anthocyanin pigmentation.The fraction of inoculated plants producing tumors was less than inthe controls.tTumor development and distribution were similar to that observedby coinoculation ofwild-type strains (experiment D), but the tumorswere smaller than in control inoculations.tOnly a few very small tumors were observed, and their developmentwas delayed compared with tumors from control inoculations(experiment D). Induction of anthocyanin pigmentation was weak.

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Table 5. Tumor production by rtfl-/rtfl + diploids% tumor

Exp. Strain Tum' Tum- production

A al/a2 bl/bl rtfl-jrtflJ 0 39 <3B al/a2 bl/b2 rtfl-Irtfl' 18 0 100C al/a2 bl/bl rtfl+/rtflI 0 10 <10D al/a2 bl/b2 rtf7l/rtf1' 6 0 100

In experiment A, the rtfl-4/nrtfl diploids are FBD91-f (6), -j (6),FBD13-f (6), and -j (6). The rtfl-3/rtfl diploids are FBD91-305 (6),-306 (2), FBD2-309 (5), and FBD13-306 (2). In experiment B, thertfl-4/rtfl' strains are FBD4-f (3) and -j (6). The rtfl-3/rtfJ'diploids are FBD4-305 (6) and -306 (3). In experiments C and D, thediploid strains are FBD12-3 and FBD-12, respectively. Numbers inparentheses are numbers of independent inoculations. FBD91-f, j,-305, and -306 are diploids (al/a2 bl/bl rtfl-/rtfl adej/+ hygs!hygr) obtained by mating FB91H with FBf4-lf, FBf4-lj, FBt3-6a,and FBf3O-5a, respectively, with selection for hygromycin-resistant(hygr) prototrophs on minimal medium containing 200 .g of hygro-mycin per ml. FBD13-f, j, and -306 are diploids (al/a2 bl/blrtfl -/rtflJ+ ade-/+ pan-I+ hyg5/hyg1) obtained by mating FB13eHwith FBf4-lf, FBf4-lj, and FBf3O-6a, respectively, with selection asfor FBD91-f. FBD2-309 is a diploid (al/a2 bl/bl rtfl -/rtf+ +/met-+/pan-) obtained by mating FB2H with FB13O-9b. FBD4-f, j, -305,and -306 are diploids (al/a2 bl/b2 rtf1-/rtfl+ ade-/+ pan-I+hygs/hygr) obtained by mating FB4cH with FBf4-lf, FBf4-lj, FB30-5a, and FB3O-6a, respectively, with selection as for FBD91-f. Plantinoculations, other conditions for strain manipulations, and abbre-viations are described in the legend to Table 2. FB6a-91 and FB1-49-2(Table 1) are auxotrophic derivatives of FB6a and FB1-49, respec-tively, and were obtained as described in ref. 8. FB-D12 andFB-D12-3 are diploids described in ref. 8 (see Table 1). FBS12-13eand FBS124c are haploid meiotic segregants (Table 1) from FB-D12.FB2H, FB91H, FB13eH, and FB4cH are hygr derivatives of FB1-49-2, FB6a-91, FBS12-13e, and FBS12-4c, respectively, and wereobtained by transformation with plasmid pcM54 (22) by the proce-dure of Tsukuda et al. (22).

locus. The possibility exists that the segregants scored asrecombinants were instead due to unlinked genetic modifiersor to reduced penetrance of the mutation. If so, the rtflmutations might affect the b locus itself. This was directlyexamined by cloning the bi allele ofthe rtfl-4 strain (FBf4-lf)and determining the nucleotide sequence of the bl openreading frame. No changes were detected in the open readingframe or in a 412-base-pair region upstream of the startingATG. The 3' untranslated region has not been sequenced.Although the mutation could reside in this region, theseanalyses together with the recovery of putative recombinantsindicate that the rtfl - mutation is in a gene near but distinctfrom b.

DISCUSSIONThree genes,fuzl,ffuz2, and rtfl, necessary for completion ofsteps in the life cycle of U. maydis have been identified.These genes were identified by isolation of mutants of an albi strain unable to form filaments when mated with an a2 b2strain. fuzi and fuz2 are not linked to a and b, whereas rtflappears to be closely linked to b. Because the two masterregulatory loci, a and b, are necessary for maintenance offilamentous growth, these new genes may represent targetsfor a or b.

Possible Roles offuzi andfuz2. Thefuzi gene is necessaryfor filament formation on nutrient medium, for normal tumorinduction, and for teliospore formation. fuzl- mutants pro-duce only small tumors that are few in number and restrictedto certain leaf areas. These tumors are devoid of teliospores.The defect in tumor formation offuzl- mutants could be dueto inefficient mating, to poor growth of the filamentous formwithin the plant, or to inadequate signaling between U.maydis and its host. Failure of fuzi- mutants to produce

teliospores may be due to a defect in the differentiation ofhyphae within the tumors (1, 2).The fuz2 gene is necessary for filament formation on

nutrient medium and for teliospore germination. fuz2- mu-tants are able to form tumors and teliospores, but theseteliospores are incapable of germination. During teliosporegermination, the thick cell wall of the teliospore breaks downand a short filament (the promycelium) is formed into whichthe diploid nucleus migrates for the ensuing meiotic divisions(23, 24). fuz2 may encode an enzyme necessary for cell-wallbreakdown during germination, or its product may be nec-essary for growth of the promycelium.The altered cell shape offuzi andfuz2 mutants leads to the

suggestion that these genes may code for components of thecytoskeleton. A deficiency in a cytoskeletal componentmight interfere with localization of materials at the growingtip of the bud and the filament or with localization of materialnecessary for teliospore maturation or germination. InSchizophyllum commune, a wood-rotting Basidiomycete,genes distinct from the mating-type factors that are necessaryfor nuclear migration (25) could also be imagined to encodecytoskeletal components (26, 27) and to be the targets of theB mating factor, which governs nuclear migration (28).

Possible Roles for rtf. The results presented here haveshown that recessive mutations in rtfl bypass the need fordifferent b alleles: two rtfl- haploids carrying the same ballele induce tumors that are identical to those obtained bycoinoculation of strains carrying different b alleles. A varietyof hypotheses (Fig. 2) can explain this observation. Inhypothesis I, rtfl is a negative regulator of initiation of tumor

I. NEGATIVE REGULATION OF tum:

In haploids:bl-bl

In dikaryons:

bl-b2 - rtf tiED

TUV

TuM

II. NEGATIVE REGULIATION OF CRYPTIC b (b') ALLELE:

In wildtype haploids:

bi

In mutant haploids:

bl

rtf - | b'

rtf

III. NEGATIVE REGULATION OF b:

In wildtype haploids:

rtf - I bl-bi

In mutant haploids:

rtf bl-bl -

br TU+b' Turn

tum TuM

+Tortuo

FIG. 2. Possible roles for rtfl. Hypotheses to explain the obser-vation that recessive mutations in the rtfl gene bypass the need fordifferent b alleles for tumor induction. In hypothesis 1, rtfl codes fora negative regulator of tumor induction, which is expressed inhaploids and inhibits tumor formation. In dikaryons, bi and b2 forma heteromultimer that represses rtfl and thus leads to tumor forma-tion. In hypothesis 11, rtfl codes for a negative regulator of a crypticb allele (b'). In rtfr- mutant haploids, the b' product interacts withbl to create a heteromultimer that leads to tumor induction. Inhypothesis III, rtfl is a negative regulator of b, which is essential fortumor induction. In rtfl - mutant haploids, b stimulates expression oftum genes. In wild-type dikaryons, either the bl-b2 heteromultimeris insensitive to rtfl, or bl-b2 inhibits rtfl.

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induction. In a bJ/b2 dikaryon, the regulatory protein formedby interaction of different b monomers represses the rtflgene, thus allowing tumor induction by this cell type. Inhaploid cells, rtfl is not repressed; consequently, its productinhibits tumor formation (Fig. 2). This situation is analogousin some respects to control of sporulation in S. cerevisiae, inwhich haploid cells express RME1, an inhibitor of initiationof meiosis and sporulation. In a/a diploids, the al-a2 com-binatorial activity represses RME1, thereby allowing initia-tion of meiosis and sporulation (17). Recessive mutations inRMEJ bypass the requirement for al-a2 (29). Thus, rtfl andRME1 appear to be analogues in that they are both negativeregulators of sexual development of the cell type resultingfrom mating; and both may be subject to negative regulation(by bl-b2 or al-a2 multimer, respectively).

In a second hypothesis, rtfl is a negative regulator of acryptic b allele (designated b') (Fig. 2), or rtfl itself is thecryptic b allele. The recessive rtfl mutation allows expres-sion of b', which has a different allele specificity than bi (theallele present in the mutant). Expression of the b' allele thusresults in the presence of different b alleles in the cell andconsequently in ability to initiate tumor induction. In S.

cerevisiae, recessive mutations in any of the four SIR genesallow expression of silent copies of MAT, resulting in abilityto initiate meiosis and sporulation (30). There is presently noevidence for cryptic b alleles in U. maydis, but there isprecedent for functionally redundant mating type loci in otherBasidiomycetes (28).

In a third hypothesis, rtfl is a negative regulator of b. Inwild-type haploids, rtfl inhibits expression or activity of b,which is essential for tumor induction; consequently, tumorsare not formed. rtfl- mutant haploids have b activity and thusactivate the tum genes. Another postulate of this hypothesisis that a bl-b2 heteromultimer formed in a dikaryon mayeither be insensitive to rtfl or may inhibit rtfl.

In addition to its role in tumor production, rtfl is alsonecessary for filament formation. One possibility is that rtflis a positive regulator of a gene (fll) necessary for establish-ment of filamentous growth and the fil product, like theproducts offuzl andfuz2, needs to be present in both partnersat the time of mating.Although the precise roles offuzl, fuz2, and rtfl cannot be

deduced at present, it seems likely that they shall be ofinterest from the standpoint of both gene regulation and cellbiology. Cloning of the U. maydis fuz genes will make itpossible to determine whether their expression is indeedregulated by a or b. The cloned fuz genes will also provide aprobe for examining other phytopathogenic fungi for fuzhomologues, which might be determinants of pathogenicity.

I thank Ira Herskowitz for discussion of this work and comments

Proc. Nati. Acad. Sci. USA 88 (1991)

on the manuscript; Regine Kahmarn and the members of herlaboratory and the reviewers for comments on the manuscript; andJames DeVay for maize seeds. This work was supported by theWeingart Program in Developmental Genetics at the University ofCalifornia, San Francisco, and by a Research Grant (to Ira Her-skowitz) from the National Institutes of Health (Al 18738).

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