Copyright 0 1990 by the Genetics Society of America Genetic Analysis of Suppressors of the veAl Mutation in Aspergillus nidulans Jeffrey L. Mooney,' Daniel E. Hassett' and Lawrence N. Yager Department of Biology, Temple University, Philadelphia, Pennsylvania 19122 Manuscript receivedJune 27, 1990 Accepted for publication August 20, 1990 ABSTRACT Light-dependent conidiationin the filamentous ascomycete,Aspergillus nidulans, is contingent on the allelic state of the velvet (veA) gene. Light dependence is abolished by a mutation in this gene (veAl),which allows conidiation to occur in the absence of light.We have isolated and characterized six extragenic suppressorsof veAl that restore the light-dependent conidiation phenotype.Alleles of four genes, defined by complementation tests, were subjected to extensive genetic and phenotypic analysis. The results of light-dark shifting experiments and the phenotypesof double mutant combi- nations are consistent with the possibility that the expression of the light-dependent phenotype is regulated by specific interactions of the suppressor gene products with the velvet gene product and with each other. ONIDIATION (asexual sporulation) in the fila- the activity of a negativeregulator, which controls the C mentous ascomycete, Aspergillus nidulans, is a expression of certainconidiation-specific genes (Moo- complex process involving the induction and coordi- NEY and YAGER1990). nateregulation of many genes. The expressionof Analysis of extragenicsuppressors is a powerful these genes leads to the formation of multicellular method in dissecting complex gene interactions (e.g., differentiated structures called conidiophores which, JARVIK and BOTSTEIN1975). The underlying hypoth- in turn, produce pigmented, haploid conidia at pre- esis of this strategy is that if two gene products inter- cisely scheduled times (AXELROD1972; CLUTTERBUCK act, a deleterious mutation in one gene product can 1969; MARTINELLIand CLUTTERBUCK197 1; TIMBER- besuppressed by acompensating mutation in the LAKE 1980).In wild-type strains,conidiation is in- interacting gene product. Accordingly, to help eluci- duced by exposure of mycelia to red light,but is date the function of the velvet gene and to identify suppressed by animmediate shift to far red light, other genes involved in the light response, we have reminiscent of the phytochrome-mediated responses isolated and characterized six extragenic suppressors of higher plants (MOONEY and YAGER 1990). How- of the veAl mutation that restore the light-dependent ever, light-mediated conidiation is contingent on the conidiation phenotype. allelic state of the velvet (veA)gene. Specifically, strains that are wild-type (veA+)at this locus display the light- MATERIALS AND METHODS dependent conidiation phenotype, while strains bear- Aspergillus strains and genetic techniques:Strains used ing a mutationat this locus, designated veAl, conidiate in this studyare listed in Table 1. Genotypes are designated regardless of the presence or absence of light. as in CLUTTERBUCK(1984). Standardgenetic techniques The veAl mutation shows a wide range of pleio- were employed throughout this study (PONTECORVOet al. 1953; CLUTTERBUCK1977; KAFER 1977). Mitotic haploidi- tropic effects. veAI strains fail to produce theprofuse zation of heterozygous diploidswas performed as described aerial hyphae normally present in wild-type colonies, in MCCULLYand FORBES(1965), substituting benlate as the showing vigorous conidiation instead (KAFER 1965). haploidizing agent (HASTIE1970). CHAMPEet al. (1981) demonstrated that veAl strains Mediaand growth conditions: The completegrowth are acleistothecial at high temperature(42"), and medium and general culture techniques described by YA- GER, KURTZ and CHAMPE(1 982) and BUTNICKet al. (1984) noted that a 36,000 M.W. protein, which is absent in were used throughout this study. Surface grown colonies the veAl mutant, accumulates in veA+ strains. Al- requiringillumination were maintained inincubators though the nature and functionof the velvet gene are equipped with GE 20 W Broad Spectrum fluorescent light unknown, we have suggested that the veA gene prod- bulbspositioned 20 cm fromthe agar surface (average uct may function as a negative regulatoror may affect illumination between 10 and 13 W m-'). Dark conditions were obtainedby loosely wrapping single platesin aluminum foil. All incubations were performed at 32". Submerged ' Present address: Departmentof Genetics, Universityof Georgia, Athens, Georgia 30602. growth rates were determinedas described in MOONEYand ' Present address: Departmentof Immunology and Medical Microbiology, YAGER (1990). The growth of fungi in submerged culture University of Florida College of Medicine, Gainesville, Florida32610. mayobey either a cube-root or exponential relationship Cknetics 126 869-874 (December, 1990) 870 J. L. Mooney, D. E. Hassett and L. N. Yager TABLE 1 TABLE 2 Aspergillus nidulans strains Phenotypes of suppressor alleles in a veA+ background Designation Genotype Designation Phenotype Allele TU 1 veAlpyroA4; suAlveAl F TU 31 yA2,pabaAl; suAlveA1; veAl suBlveAl F TU 32 pabaA1;suAlveAl; veAl suClveAl CA TU 44 pabaA1;suDlveAl;veAl suC2veAI CA TU 47 yA2,pabaAI; suDlveAl; veAl suC3veAI CA TU 48 pabaA1;suC3veA1; veAI suDlveAI CA TU 49 yA2,pabaAl; suC3veAI; veAl The symbols represent the phenotypes of colonies possessing the TU 50 pabaAl;suBlveAl; veAl indicated suppressor in the wild-type veA+ background. F, colonies TU 51 yA2,pabaAl; suBlveAl; veAl are fluffy with profuse aerial hyphae. These colonies remain acon- TU 52 pabaAl;suCIveAI; veAI idial and acleistothecial irrespective of illumination. CA, colonies TU 53 yA2,pabaAl; suClveAI; veA1 are conidial in the light, but aconidial in the dark. TU 54 pabaAl;suC2veAI; veAl TU 55 pabaA1;yA2, suC2veAI; veAI 064 (yA2, pabaA1; veA1)before further analysis. WIM 064" yA2,pabaAl; veA1 Light-dark shifting experiments:Light-dark shifting ex- WlM 126" yA2,pabaA1; veA+ periments were performed as described in MOONEY and FGSC 283b adE20,suAladE20, yA2; YAGER(1 990). Colonies were induced after 36-hr incubation acrAI; galAl; pyroA4; fac- at 32", then shifted from light to dark and vice versa as AN?; sB?; nicB8; riboB2 determined by the experiment. Conidial yields were deter- FGSC 495b lysB5,nicA2, pA2; veAl mined 24 hr afterinduction by harvesting five coloniesfrom each plate and assaying for viable conidia asdescribed above. All strains were derived in this laboratory except as noted. The strains listed are those used as parent and/or tester strains. All other strains are derived from these by standard genetic methods. RESULTS a Obtained from S. P. CHAMPE,Waksman lnstitute of Microbi- ology. Isolation suppressor mutants: Suppressor mu- ' Obtained from the Fungal Genetics Stock Center. of tantswere isolated on the basis of their ability to (COCKERand GREENSHIELDS1977). Using the described producenormal developmental structures when growth conditions cube-root growth was observed and sub- grown in the light, but only vegetativemycelia when merged growth rates were presented as mg dry weight"'/ grown in the dark. In general, this phenotypewas not hr (EMERSON1950; MARSHALLand ALEXANDER1960). Ra- dial colonial growth rates, which obey a linear growth rela- temperature sensitive, since mutants grown at 22", tionship, were determined as described in YAGER, KURTZ 27 O, 32 O and 37 O all produced normal developmental and CHAMPE(1 982). structures andviable conidia when grown in the light Colonies do not normally conidiate in submerged culture, but only vegetative mycelia when grown in the dark. but do if they have acquired competence and are subse- However, all of the suppressor mutants wereaconidial quently exposed to an air interface (AXELROD,GEALT and PASTUSHOK1973). The transfer of mycelia from liquid to in the light and in the dark at 42". In comparison, surface culture, termed induction, is used to initiate and veA+ strains were conidial at 42", although light de- synchronize asexual development (TIMBERLAKE1980). Un- pendency was lost and very fewconidia were pro- less otherwise indicated, induction was performed 36 hr duced. after inoculation of spores into liquid medium, which is well To demonstrate that thesix suppressors behaved as after the time of competence. All conidial yield determina- tions were performed as described in YAGER,KURTZ and single Mendelian alleles, each was meiotically crossed CHAMPE(1982). The time of conidiophore vesicle appear- to either TU 1 or WIM 064. Analysis ofapproxi- ance was scored by microscopic examination using the mately 125 progeny from each cross showed that all method of AXELROD,GEALT and PASTUSHOK(1973). The suppressor mutations segregated from their respective time of competence was determined by the methods of wild-type alleles in a 1:1 ratio. All suppressor muta- AXELROD,GEALT andPASTUSHOK (1973) and BUTNICKet al. (1 984). The initiation of sexualdevelopment was assayed tions also assorted independently from yA2, an un- using the chromogenic staining technique of CHAMPEet al. linked spore color mutation (P > 0.05 for all crosses; (198 l), and the formation of mature cleistothecia and asco- x* test). spores was monitored by microscopic examination and plat- Interactions of the suppressor mutations with the ing of viable spores. wild-type velvet allele: Each suppressor mutant was Isolation of suppressors: Approximately 5 X 1O4 conidia meioticallycrossed to a veA+ strain. The resulting of strain TU 1 (pyroA4; veA1) were suspended in 10 ml of water