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Home , Genetic analysis

Copyright 0 1990 by the Society of America

Genetic Analysis of Suppressors of the veAl 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 tofar 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 wereused 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 exponentialrelationship

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 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 and irradiated with ultraviolet light at a dosage that phenotypes of each suppressor allele in a veA+ back- produced 10% survival (700 pW/cm2 for 40 sec). A total of ground are given in Table 2. Only suCveA 1; veA+ and 7.5 x lo5 conidia were mutagenized, plated at a density of suDveA 1; veA+ still showed light-dependent conidia- 1.25 x lo2 conidia/plate, and incubated for two to three tion. The other two combinations werealways aconi- daysin the dark. Six colonies that were aconidial in the dark, but conidiated when grown in the light (incandescent dial, irrespective of illumination. illumination at 35 W m-') were selected. These independent Characterization of growthand developmental isolates were backcrossed several times to TU 1 and WIM rates: As shown in Table 3, the suppressor mutant Suppressors of the veAl Mutation 87 1 TABLE 3 tion, none of these diploid strains showed any reduc- Growth properties tion in vegetative growth rate in comparison to a control diploid that was homozygous for ueAI. As Radial growth rate expected, diploids that were homozygous for both the Submerged growth (mm/day) rate (ma drywt"'/ suppressor and ueAI mutations were conidial in the Strain hr)' Light Dark light and aconidial in the dark. On the other hand, WIM 126 (veA+) 0.048 f 0.025 8.7 & 0.4 8.8 f 0.3 diploid strains that were heterozygous for either su- WIM 064 (veAZ) 0.088 f 0.039 8.2 f 0.2 8.2 f 0.8 AlueAl or suBlveAl and veAl displayed reduced veg- TU 31 (suAZveAZ) 0.069 f 0.018" 5.0 f 0.6 4.5 f 1.4 TU 51 (suBZveA1) 0.078 f 0.023" 5.7 f 0.4 6.5 k 0.2 etative growth and conidial yields in comparison to TU 53 (suCZveAI) 0.083 f 0.020" 6.4 f 1.2 6.9 f 0.7 controls, indicating that these suppressors do not ap- TU 55 (suC2veAZ) 0.080 f 0.025" 7.0 f 0.4 7.8 5 0.5b pear to be completely recessive with respect to these TU 49 (suC3veAZ) 0.040 f 0.01 I" 7.0 f 0.4 6.8 f 1.1 phenotypes. The phenotypes of diploids that were TU 47 (suDlveAZ) 0.072 f 0.020" 5.8 f 0.6 6.2 f 0.6 heterozygousfor either suClveAI,suC2veA1, su- All growth rate measurements were performed on complete C3veAI or suDlveAI and veAl were indistinguishable medium. Strain designations are given, with the suppressor allele from a control diploid that was heterozygous only at or allelic state of the velvet gene in parentheses. N = 3 for the submerged growth rate determinations and N = 5 for the radial the velvet locus. growth rate determinations. The significance of differences be- Complementation and linkage analysis: Comple- tween means was determined by calculating t (Student's t test, two- mentation analysis was performed on the six suppres- tailed). Means are f standard deviation. The submerged growth rates of the WIM 126 (veAf) and WIM sors by constructing all possible combinations of het- 064 (veAl)controls and the radial growth rates in light and dark of erozygous diploids. The mutants define four comple- these same controls were not significantly different (P > 0.05 for all comparisons). The growth rates of suppressor mutations were mentationgroups, designated suAveA I, suBveAI, compared to these controls. Unless indicated, the growth rates of suCveA1 and suDveA1. Meiotic crosses involving the the suppressor mutations were significantly different from the ap- noncomplementing suClveAI,suC2ueAI and su- propriate controls (P < 0.05 for all comparisons). a Not significantly different from submerged growth rate con- C3veA I suppressors indicate that they are allelic, with trols (P> 0.05). recombination frequencies of less than 0.1%. Not significantly different from radial growth rate controls (P Chromosomal linkage was determined by mitotic > 0.05). haploidization of heterozygous diploids (Table 4). All suppressors areextragenic to velvet, since none is strainsdid not exhibit significantly different sub- linked to VZZZ, to which veAl maps. merged growth rates fromeither theparental veAl or suAlveA1 is located on chromosome suBIveAl is the wild-type veA+ strains. However, a significant re- ZZ, duction in radial colonial growth rates from the con- located on chromosome V, suClveAI, suC2veA1, and trol strains, regardless of illumination, was observed. suC3veAI are located on chromosome ZZZ, and su- Radial growth rates were not affected by incubation DlveAl is locatedon chromosome V. Although su- on complete medium or on minimal medium supple- BlveAl and suDlveAl map to the same chromosome, mented with the appropriate requirements (data not meiotic analysis indicates that they are not allelic. shown). On the other hand, thesuppressor mutations suBlveAl was localized to the lysB5 region of chro- have no effect on the time of conidiophore vesicle mosome V and was further mapped by a three-point appearance (30 k 0.5 hr) or thetime at which mature cross of strain TU 50 with strain FGSC 495, which conidia form (37.5 - 38.0 hr). indicated 4% linkage from nicA2 and 8% linkage from Since strains containing the veAl mutation produce lysB5 (data not shown). In contrast, suDlveAI did not acleistothecial colonies at 42" (CHAMPEet al. 1981), show linkage toeither of thesemarkers or to su- we tested the suppressor mutants at 42" in the pres- BlveAl. ence and absence of light to see whether they would Double mutant analysis:Double mutants were con- also manifest this phenotype. suAlveAI, suBIveAland structedfrom members of each complementation suDlveAl undergo sexual development at 42", but group to deduce interactions and possible epistatic strains containing the suCveA1 alleles are blocked in relationships. The phenotypes of these double mu- sexual development at this temperature andthus show tants are shown in Table 5. No double mutant re- only partial suppression of this aspect of the ueAl covered had the same phenotype as either single mu- phenotype. tant parent. Double mutants thatsurvived were either Dominance tests: Each suppressor was tested for completely aconidial or always conidial, regardless of dominance by constructing diploids that were heter- the presence or absence of light. A suAlveAI;su- ozygous for the suppressor and homozygous for the DlveAI double mutant could not be constructed, sug- veAl mutation. All diploids were conidial both in the gesting either that this combination is lethal, or that light and in the dark, indicating that, with respect to recovery of this combination is prevented by very the conidiation-control phenotype, all of the suppres- poor growth since, as shown in Table 3, these two sors were recessive to their wild-type alleles. In addi- alleles individually result in the poorest growth. 872 J. L. Mooney, D. E. Hassett and L. N. Yager

TABLE 4 Linkage analysis of suppressor mutations

No. of haploid segregants

suAl suB 1 SUCl SUCPsuDl sue3 Linkage PhenotypicLinkage group marker - + - + - + - 4- - 4- - + I Y+ 11 3341 42 44 18 31 25 20 10 9 24 Y 8 4845 23 35 21 27 31 20 19 9 33 I1 ACR' 19 0 29 43 45 24 38 26 24 21 10 34 ACR 0 81 34 43 34 15 20 30 16 8 8 23 111 GAL' 11 40 38 48 79 1 58 5 8 40 10 29 GAL 41 8 27 38 0 38 0 48 0 24 8 28 IV PYRO+ 13 3446 36 34 18 31 35 21 15 10 23 PYRO 6 47 29 40 45 21 27 21 19 14 8 34 V FAC+ 42 8 65 6 42 29 27 30 22 20 18 4 FAC 11 39 0 80 37 10 31 26 18 9 0 53 VI S+ 11 45 30 53 48 29 27 32 19 16 10 30 S 8 34 35 33 30 10 31 24 21 13 8 27 VI1 NIC+ 52 9 34 43 45 28 24 34 23 21 11 34 NIC 11 37 31 43 33 11 34 22 17 238 7 VI11 RIBO' 13 46 32 51 40 2036 30 16 13 11 37 RIB0 6 3535 33 38 19 28 20 24 16 7 20 Strains TU 32, TU 50, TU 52, TU 54, TU 48, and TU 44 carrying, respectively, the suppressor mutationssuAZueAZ, suBZueAZ, suCZueAl, suC2ueA1, suC3ueAZ and suDlueAZ were used toform heterozygous diploids(homozygous for ueAZ) with strain FGSC 283. The first phenotype listed for each linkage group (-) is that of the suppressor strain (light dependent conidiation). The second phenotype (C)is that of the unsuppressed veAl strain (conidiation occurs regardless of the presence or absence of light).

TABLE 5 TABLE 6 Interactions of suppressors in double mutants Light-dark shifting analysis of suppressor mutations

su- su- Minimum expo- suBlveAlsuClveA1 CPveAl suC3veAl DlveAl sure time re- Critical period of quired to elicit SUAZueA Z F C C C NR responsivenesslight conididtion suBZueA 1 C C C F Strain (hr postinduction) (min) suCZueAZ NR NR C WIM 126 (ueA+) 0-6 15-30 suC2ueA I NR C TU 31 (suAIueAZ) 0-6 15-60 suCjrueA 1 C TU 51 (suBlueAl) 1-6 30-60 The symbols indicate the phenotypes of thetwo suppressors in a TU 53 (suClueAZ) 1-6 30-60 ueA I background. F, colonies are fluffy in appearance with a profuse TU 55 (suC2veAI) 1-6 30-60 aerial hyphae. These colonies remain aconidial and acleistothecial TU 49 (suC3ueAI) 1-6 30-60 irrespective of illumination. C, theueAZ mutation is not suppressed. TU 47 (suDZveAZ) 1.5-6