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The B Mating-Type Locus of Ustilago Maydis Contains Variable and Constant Regions

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The B Mating-Type Locus of Ustilago Maydis Contains Variable and Constant Regions Downloaded from genesdev.cshlp.org on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press The b mating-type locus of Ustilago maydis contains variable and constant regions James w. Kronstad t and Sally A. Leong 2 ~Biotechnology Laboratory, Departments of Microbiology and Plant Science, University of British Columbia, Vancouver, British Columbia, Canada; 2Plant Disease Resistance Unit, U.S. Department of AgriculturemAgricultural Research Service, Department of Plant Pathology, University of Wisconsin, Madison, Wisconsin 53706 USA The b locus of the phytopathogenic fungus Ustilago maydis encodes a multiallelic recognition function that controls the ability of the fungus to form a dikaryon and complete the sexual stage of the life cycle. The b locus has at least 25 alleles and any combination of two different alleles, brought together by mating between haploid cells, allows the fungus to cause disease and undergo sexual development within the plant. An open reading frame of 410 amino acids has been shown to specify a polypeptide responsible for the activity of the b l allele, and comparisons of the predicted amino acid sequences for 6 b alleles allowed identification of variable and constant regions within the coding region of the gene. Haploid strains carrying a null mutation at the b locus, created by gene disruption, are viable but fail to interact with formerly compatible strains to give an infectious dikaryon. Analysis of mutants carrying a null allele indicated that the products of different alleles of the b locus combine to form a new regulatory activity and that this activity directly or indirectly turns on the pathway leading to sexual development and pathogenesis. [Key Words: Recognition; plant pathogen; multiallelic gene; dikaryon] Received April 29, 1990; revised version accepted June 8, 1990. The basidiomycete fungus Ustilago maydis causes a 1971). The functions of the a and b mating-type loci disease on corn characterized by the presence of promi- have been demonstrated with diploid strains isolated nent tumors (galls) on the leaves, stems, ears, and tassels from immature gall tissue or from matings between ha- of the plant (Christensen 1963). Three cell types occur ploid strains carrying complementary auxotrophic mu- during the life cycle of the fungus: yeast-like haploid tations (Rowell 1955a; Holliday 1961; Puhalla 1969). cells, dikaryotic cells resulting from mating between ha- Diploid strains heterozygous at both a and b form myce- ploid strains, and diploid teliospores formed by sporula- lial colonies on nutrient medium and induce gall forma- tion of the dikaryon within the plant (Day and Anagnos- tion when injected into com seedlings (Holliday 1961; takis 1971). Haploid cells of U. rnaydis are saprophytic Day et al. 1971). In contrast, strains homozygous at both and nonpathogenic; in contrast, the dikaryotic form of a and b form yeast-like colonies and do not induce gall the fungus is difficult to culture and is generally found development (Holliday 1961; Puhalla 1969; Day et al. only in the infected plant (Day and Anagnostakis 1971). 1971}. Diploids homozygous at a, but carrying any two Infection of the plant, whether initiated by mating or different b alleles, are pathogenic, thus demonstrating directly from germinating teliospores, is required to that the b locus controls the formation of the infectious complete the sexual stage of the life cycle. This is be- dikaryon (Holliday 1961; Puhalla 1968, 1970; Banuett cause the diploid teliospores, which germinate to give and Herskowitz 1989}. It is fascinating, given the mul- meiotic products, can only be formed in association tiallelic nature of the gene (at least 25 alleles}, that each with living plant tissue (Christensen 1963; Hausen and combination of alleles can be distinguished and that the Beiderbeck 1987). state (homozygous or heterozygous) of the b locus influ- Mating between haploid cells is controlled by two ge- ences not only pathogenicity and sexual development netic loci, the a locus with two alleles, al and a2 and the but also cell morphology. b locus with at least 25 alleles (Rowell 1955a, b; Holliday The b locus presents an excellent opportunity to study 1961; Puhalla 1968; Silva 1972; Day 1974). The a locus a mechanism of self versus nonself recognition in- appears to control cell fusion (Rowell 1955a), and the b volving a multiallelic gene in a relatively simple or- locus controls events after fusion, including establish- ganism. U. maydis is attractive experimentally because ment of the infectious dikaryon and sexual development both classical genetic and molecular genetic techniques within the plant (Rowell and DeVay 1954; Day et al. can be applied to the analysis of mating and pathoge- 1384 GENES& DEVELOPMENT4:1384-1395 © 1990 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/90 $1.00 Downloaded from genesdev.cshlp.org on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press Multiallelic recognition nicity (for review, see Froeliger and Kronstad 1990). To bl TP S ROT R S P DDB H begin an analysis of the b locus, we identified clones tl I lll I II l a I II I I II II containing the bl and b2 alleles by complementation p R D S XO SpO and hybridization (Kronstad and Leong 1989). These clones were shown to encode b alleles by their effect on the colony morphology and the pathogenicity of various b2 H TP S ROT R S P DRBD B H haploid and diploid strains. Further proof came from a ! II IIII I II l|ll II II I II I gene replacement experiment in which a strain carrying P R D xo B a b2 allele was transformed with a linear DNA fragment carrying the bl allele and a selectable marker. The transformants resulting from gene replacement events I kb carried the b l allele linked to the selectable marker. In- terestingly, it was also possible to obtain haploid strains Figure 1. Restriction maps of DNA fragments carrying the b l that were both mycelial and pathogenic simply through and b2 alleles. Cleavage sites for nine enzymes are shown on introduction of a different b allele by transformation the 8.5-kb BamHI fragments carrying the bl and b2 alleles. The (Kronstad and Leong 1989). This result serves to confirm cloning of these fragments was described previously (Kronstad further the idea that the b locus is the key regulatory and Leong 1989). The positions of the ORFs for the alleles, as locus controlling the ability of the fungus to cause dis- identified in Figs. 2 and 3, are shown as bars below the maps. ease on the plant. The restriction enzyme designations are as follows: (B) BglII; Schulz et al. (1990)reported recently the cloning and (D) HindIII; (H) BamHI; (S) SalI; (X)XbaI; (O)XhoI; (P)SphI; (T) PstI; (R)EcoRI; (Sp) SspI. sequence analysis of four b alleles, bl, b2, b3, and b4. A single open reading frame (ORF) of 410 amino acids was found for each allele, and sequence comparisons of the similar, especially in the 6-kb region containing iden- alleles revealed a variable amino-terminal portion and a tical restriction sites for the two alleles. This is in con- conserved carboxy-terminal region. The analysis of trast to the situation for mating type genes from other Schulz et al. (1990)also revealed that the b polypeptides fungi, such as Neurospora crassa, where the alleles are contain a motif related to a homeo domain suggesting contained within large regions of nonhomologous DNA that the b polypeptides may be DNA-binding proteins. (Glass et al. 1988; Metzenberg and Glass 1990). In this report, we describe the localization of the b l The location of the bl allele on cloned DNA was de- gene on cloned DNA and the introduction of a stop fined further by Tn5 mutagenesis and subcloning (Fig. codon into the b l allele to identify the correct ORF and 2A, B). The activity of the bl allele was assayed by intro- to demonstrate that a protein product is responsible for duction of plasmids carrying the wild-type allele, sub- b allele activity. Sequence comparisons of six alleles cloned fragments, or fragments with Tn5 insertions into confirm that the alleles are highly homologous but con- a b2/b2 diploid strain (d410) and screening the transfor- tain variable and constant regions. The sequence anal- mants for the mycelial colony morphology expected of a ysis reported here also allows a description of a putative strain carrying two different b alleles. Previous studies nuclear localization sequence in the constant region of (Kronstad and Leong 1989)have shown that the colony the b alleles. In addition, we constructed a null muta- morphology of the strains is an accurate indication of tion of the b l allele and introduced this mutation into their ability to induce galls; that is, mycelial transfor- haploid and diploid strains by gene replacement. Anal- mants are pathogenic when injected into corn seedlings, ysis of strains carrying the null mutation revealed that and yeast-like strains are not. DNA fragments carrying the b locus does not encode an essential gene product the wild-type bl allele generally gave 70-90% mycelial and that the products of different b alleles must act to- transformants in this assay, as did most of the Tn5 in- gether to activate the pathway leading to mycelial sertions throughout the 8.5-kb region containing the bl growth and pathogenicity. allele. An exception was found for the plasmid carrying Tn5 insertion 93; this plasmid yielded a reduced per- Results centage of mycelial transformants (indicating disruption of bl allele function) compared with the plasmid Location of the b i allele on cloned DNA without Tn5. In contrast, Tn5 insertions 45 and 80, Restriction maps of the 8.5-kb BamHI fragments car- which flank 93, did not interfere with the activity of the rying the b l and b2 alleles were constructed to assess b l allele.
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