Proc. Natl. Acad. Sci. USAVol. 74, No. 9, pp. 3831-3834, September 1977Botany

Occurrence of a major protein associated with fruiting bodydevelopment in Neurospora and related Ascomycetes

(electrophoretic variants/serotypic variants/Sordariaceae)

JUNE BOWMAN NASRALLAH AND ADRIAN M. SRBSection of Genetics, Development, and Physiology, Cornell University, Ithaca, New York 14853

Contributed by Adrian M. Srb, June 20, 1977

ABSTRACT Electrophoretic and immunological analysisof fruiting body (perithecial) extracts demonstrates the occur-rence of a major phase-specific perithecial protein in all Neu-rospora species and in the closely related Gelasinospora cerealisand Sordaria fimicola. The perithecial proteins from thesedifferent species fall into a number of groups with differentelectrophoretic mobilities. They appear to be immunologicallyclosely related but not identical to one another even within thesame genus, with only partial identity exhibited between theheterothallic and pseudohomothallic Neurospora on the onehand and the homothallic Neurospora on the other hand. Inimmunological analysis of fruiting body extracts of the otherAscomycetes, Podospora anserina, Cochliobolus maydis, andAspergillus nidulans, and of ascus extracts of Saccharomycescerevisiae, no crossreaction with the Neurospora perithecialprotein was found.

Biochemical analysis of components of the Neurospora lifecycle has revealed, in N. crassa, N. sitophila, N. tetrasperma,and N. terricola, a specific association between fruiting body(perithecial) development and a major protein species resolv-able on polyacrylamide gels (1, 2). This- protein can be detectedin unfertilized fruiting bodies, but its concentration increasesdrastically in fertilized fruiting bodies until 4 or 5 days afterfertilization, when it constitutes a major fraction of the totalperithecial proteins. Later in maturation, as perithecial contentsare lost in the perithecial exudate and subsequently in sporedischarge, the levels of the major perithecial protein decrease(1, 2). Detailed reports of this type of perithecial protein haveso far been limited to the usual laboratory species, N. crassa andN. tetrasperma. An important biological function in fruitingbody maturation for such a phase-specific protein would beindicated if a comparable protein species were found to occurmore generally among Ascomycetes. All the available Neu-rospora species and members of a number of other Ascomy-cetous genera have now been examined for the presence of sucha protein, as summarized in this report.

MATERIALS AND METHODSStrains of Neurospora. The strains of Neurospora used in

this study were the following: (a) as representatives of theheterothallic species-the standard laboratory wild-type St.Lawrence strains of N. crassa, 74A and 77a; Honduras 3A andla, wild strains of N. crassa isolated in Honduras and obtainedfrom R. H. Stover and S. R. Freiburg; N. intermedia P420 andP405, obtained from the Fungal Genetics Stock Center (FGSC);and N. sitophila 540-34A and 2a from J. R. S. Fincham; (b) asrepresentatives of the pseudohomothallic species-homokar-yotic strains of N. tetrasperma from Borneo, originally isolated

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as strain T-220 by J. H. Warcup, and designated N. tetrasperma(Warcup); N. tetrasperma strains 1270 (85A) and 1271 (85a),derived from an isolate by B. 0. Dodge, obtained from theFGSC, and designated N. tetrasperma (Dodge); N. toroi (FGSCno. 688) obtained from the FGSC; (c) as representatives of thehomothallic species-N. terricola, obtained from S. E.Gochenaur, N. africana (Africana N200), N. dodgei (ATCC15509), N. galapagosensis (Galapagosensis G349), and N.lineolata (ATCC 18966), all obtained from the FGSC.The Neurospora strains were selected to represent established

species of the genus, essentially as classified by Frederick et al.(3). Some named species were omitted, as warranted by morerecent taxonomic work, namely, the exclusion of the stromaticN. phoenix from the genus (4), or by lack of live material, as isthe case for N. erythrea. A strain of N. torcn was included in theserological study although recent work on the crossing behaviorof strains assigned to this species suggests their identity withstrains of N. tetrasperma (5, 6).Other Genera. Members of the other genera tested were

Gelasinospora cerealis, obtained from the FGSC; Podosporaanserina, from D. Marcou; Sordaria fimicola, from L. S. Olive;Aspergillus nidulans (FGSC no. 4), from Etta Kifer; Saccha-romyces cerevsiae, from G. R. Fink; and Cochliobolus maydis,from 0. C. Yoder.

Culture Techniques. Maintenance of stocks and of culturesfor immunological work with vegetative mycelia of Neuro-spora, Gelasinospora, and Sordaria was at 250 on the minimalmedium of Beadle and Tatum (7) supplemented with 2% agarwhen solid medium was required. Vegetative mycelia of thehomothallic strains were produced as submerged cultures inErlenmeyer flasks containing liquid minimal medium andharvested before fruiting bodies formed. Production of proto-perithecia and crosses involving Neurospora and Gelasinosporawere carried out at 250 on Difco cornmeal agar or on the liquidcrossing medium of Westergaard and Mitchell (8) adjusted topH 5.7, as described earlier (1), and supplemented with 2% agarwhere required. Podospora perithecia were produced on Difcocornmeal agar in the light; Sordaria crosses were made on en-riched cornmeal agar (9); Aspergillus crosses were made ac-cording to Pontecorvo et al. (10), and sporulating cultures ofSaccharomyces were produced according to Fowell (11). Co-chilobolus perithecia were provided by 0. C. Yoder.

Harvesting of Material and Biochemical and Immuno-chemical Analysis. Harvesting and extraction of samples on0.1M phosphate buffer (pH 7.0) and electrophoresis on 7.5%polyacrylamide gels were as described earlier (1).The protein antigen for immunological studies was obtained

from perithecial extracts of N. crassa or N. tetrasperma(Dodge) which were subjected to electrophoresis. The proteinwas eluted in 0.1 M phosphate buffer (pH 7.0) from acrylamidegel slices corresponding to the protein band of interest. The gel

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Proc. Natl. Acad. Sci. USA 74 (1977)

FIG. 1. Double diffusion patterns obtained with fruiting bodyextracts. Central wells contained antiserum produced against N.tetrasperma (Dodge) perithecial antigen; peripheral wells containedfruiting body extracts. afri, N. africana; Asp, Aspergillus nidulans;Coch, Cochliobolus maydis; cra, N. crassa; D, Dodge; dod, N. dodgei;gal, N. galapagosensis; Gel, Gelasinospora cerealis; Hon, Honduras;lin, N. lineolata Pod, Podospora anserina; Sac, Saccharomycescerevisiae; sit, N. sitophila; Sor, Sordaria fimicola; terri, N. terricola;tet, N. tetrasperma; tor, N. toroi; W, Warcup.

eluate was dialyzed against the same buffer, and tested forelectrophoretic purity by polyacrylamide gel electrophoresis.Antisera against the antigen were produced in New ZealandWhite rabbits by subcutaneous and intramuscular injection ofa 1:1 mixture of antigen and Freund's complete adjuvant(Difco).Two injection protocols were followed: (i) The rabbits were

injected weekly for 4 weeks and bled 5 weeks after the start ofthe injections. (ii) Two weekly injections were done, and sera

were collected 6 weeks after the first injection. Identical ex-

perimental results were obtained with sera produced by eitherprotocol. To the sera, sodium azide to a final concentration of0.1% (wt/vol) was added as a preservative, and the sera were

frozen and stored at -10° in small aliquots. Immunologicalreactivity was tested by the double diffusion method ofOuchterlony (12), on slides coated with 1% (wt/vol) Difcopurified agar containing 0.1% sodium azide as a preservative.The purity of the sera was demonstrated by the formation ofa single precipitin arc when whole unstained polyacrylamidegels on which the perithecial proteins had been electrophoresedwere placed in agar troughs and analyzed by double diffusion.Antigen was quantitated by the single radial immunodiffusionmethod of Mancini et al. (13), in which the antigen is allowedto diffuse in agar containing undiluted specific antisera in a

ratio of 7:1 (vol/vol). The area, or (diameter)2, of the precipitincircles that develop around the antigen wells is proportional tothe antigen concentration.

RESULTSThe agar gel diffusion test of Ouchterlony with antisera raisedto the N. crassa or N. tetrasperma (Dodge) major perithecialprotein was used to investigate the possible association of a

similar protein with sexual morphogenesis in all the availableNeurospora species and in members of a number of other As-comycetous genera. As shown in Fig. 1, precipitin reactionswere observed for perithecial extracts of all Neurospora species,

ORIGIN_N'--

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Hon gal Iin tern afri dod GelANODE

FIG. 2. Electrophoretic separation of the soluble proteins ofperithecial extracts. Electrophoretic variants of the major perithecialprotein are shown. Abbreviations are given in the legend of Fig. 1.

of Celasinospora cerealis, and of Sordaria fimicola, but not forperithecial extracts of Podospora anserina and Cochilobolusmaydis or for cleistothecial extracts of Aspergillus nidulans orfor ascus extracts of Saccharomyces cerevisiae. Furthermore,on the basis of the immunological reactions obtained with twodifferent antisera, the crossreacting strains can be divided intothree groups, with members of each group exhibiting reactionsof complete identity (end-to-end fusion) with one another andreactions of only partial identity (indicated by spur formation)with members of other groups. Thus, N. crassa, N. crassa(Honduras), N. sitophila, N. intermedia, N. tetrasperma(Dodge and Warcup), and N. toroi fall in one group; N. afri-cana, N. dodgei, N. galapagosensis, N. fineolata, N. terricola,and Gelasinospora cerealis in another; and Sordaria fimicolafalls in still another group.

Polyacrylamide gel electrophoresis of perithecial extracts ofthe various Neurospora species and Gelasinospora cerealhsreveals a major proteinaceous band in each case, with a numberof electrophoretic variants of the protein occurring (Fig. 2). Thecorrelation of this major protein with the crossreacting antigenfor each of the strains tested was demonstrated by immuno-logical analysis of gel slices taken along the length of unstainedgels, inasmuch as only slices corresponding to the major bandreacted with the immune sera. Polyacrylamide gels of peri-thecial extracts from Sordaria fimicola did not reveal such amajor protein upon staining. However, immunological analysisof gel slices showed that reactivity was confined to a region veryclose to the tracking dye, indicating that, under the electro-phoretic conditions used, the Sordarila major perithecial proteinwas not resolved from the front.The functional homology of the crossreacting antigens in the

different species was tested by following the course of devel-opment of the antigens with maturation of the perithecia. First,vegetative mycelia extracts from some of the strains were as-sayed immunochemically (Fig. 3). As was reported earlier forN. crassa and N. tetrasperma (1, 2), all species tested lackedthe perithecial antigen in vegetative mycelia. Then, by thetechnique of single radial immunodiffusion, the changes inconcentration of the antigen in perithecia were measured overa period of 8 days after fertilization for the heterothallic andpseudohomothallic species and 8 days after the first appearanceof recognizable fruiting bodies for homothallic species (Fig. 4).The curve obtained for N. crassa by this method was similarto that obtained previously by electrophoretic protein analysis(1). All species providing a crossreacting protein showed anincrease in antigen concentration from initially undetectablelevels to high levels, followed by a decrease, as illustrated in Fig.5 for four of the strains tested.

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Proc. Natl. Acad. Sci. USA 74 (1977) 3833

0 1 2 3 4 5 6 7 8 0 2 3 4 5 6Days after first appearance of fruiting bodies

FIG. 3. Immunodiffusion pattern of mycelial extracts. Centralwell: antisera produced against N. crassa perithecial antigen. Pe-ripheral wells: 1 and 4, N. crassa perithecial extract; 2, Gelasinosporamycelial extract; 3, Sordaria mycelial extract; 5, N. dodgei mycelialextract; 6, N. africana mycelial extract.

DISCUSSIONThe results presented in this paper demonstrate the generaloccurrence of a protein species associated with sexual mor-

phogenesis in the genus Neurospora and in members of certainclosely related genera of the Sordariaceae. The homology of thevarious perithecial specific protein species identified in Neu-rospora, Gelasinospora, and Sordaria is clearly indicated (a)by their similar behavior on polyacrylamide gels as rapidlymigrating acidic proteinaceous molecules, (b) by their absencein vegetative mycelia, (c) by the observed increase in theirconcentration during fruiting body development, and (d) bytheir close immunochemical relatedness.

Differences among these perithecial proteins do, however,exist. The perithecial proteins from different genera, fromdifferent species within the same genus, and from differentgeographical isolates of the same species have different elec-trophoretic mobilities, with a total of seven variants observed(Table 1). In addition, the patterns of increase of the perithecialantigen during maturation are not identical for all strains tested;

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FIG. 4. Single radial immunodiffusion plate showing changes inantigen concentration associated with fruiting body development ofN. terricola. Numbers indicate days after first appearance of fruitingbodies. Extracts of 25 perithecia were used per well.

FIG. 5. Changes in the levels of the perithecial antigens in peri-thecia collected at different times after the first appearance of fruitingbodies. Maximal reactivity for each strain corresponds to the highestabsolute amount of antigen obtained in the time sequence, with allother values expressed as percentages of that number. (a) N. dodgei;(b) N. lineolata; (c) Gelasinospora cerealis; (d) Sordaria fimicola.

i.e., the maximum concentration attained and the times atwhich that concentration is reached differ. The significanceof the observed differences in pattern of increase is difficult toevaluate, inasmuch as variability is also observed in separateassays of the same strain. Because of asynchronous perithecialdevelopment, absolute values may not be reliable. In any case,the trend in all strains is clearly an increase in antigen concen-tration early in fruiting body maturation.

Immunochemically, differences among the proteins werealso observed. While the serological differences do not correlatewith the electrophoretic differences, they occur in a pattern ofpossible taxonomic significance; that is, by our tests, perithecialproteins from heterothallic and pseudohomothallic Neurospora

Table 1. Immunological relationships and electrophoreticmobilities of the crossreacting perithecial protein from

different species and genera

Electro-Cross- phoreticreaction variant,groups Strains Rf*

I Honduras, N. intermedia 0.73N. tetrasperma (Warcup) 0.77N. crassa, N. sitophila, N. tetrasperma

(Dodge) 0.82II N. galapagosensis 0.77

N. lineolata 0.82N. terricola 0.87N. africana 0.93N. dodgei, Gelasinospora cerealis 0.98

III Sordaria fimicola 0.99

* Perithecial proteins listed as having the same Rf values were shownby electrophoresis of pairwise mixtures of the respective perithecialextracts to exhibit one major band. Perithecial proteins with dif-ferent Rf values were shown in the same way to exhibit two majorbands, each of which was contributed by one of the strains.

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species are serologically indistinguishable but show only partialserological identity with the proteins from homothallic Neu-rospora species and Gelasinospora. The serological criterionmay thus be added to a number of other properties differen-tiating the heterothallic and pseudohomothallic from the ho-mothallic Neurosporas, which, in addition to lacking a matingtype system, generally lack conidiation and have poor mycelialgrowth. The array of distinctions may warrant exclusion of thehomothallics from the genus.

Before a final decision on the taxonomic status of the ho-mothallic Neurosporas is reached, however, the developmentof suitable hybridization techniques (6) is desirable, as are moreextensive serological analyses of varied antigenic componentsin a large number of strains and with a wider range of antiseraraised against the N. crassa and N. tetrasperma perithecialantigen or against perithecial antigens from other relevantspecies.Our failure to detect any crossreacting antigen in the As-

comycetous genera less closely related to Neurospora maysimply indicate phylogenetic divergence and does not excludethe existence of a similar phase-specific protein of analogousfunction, at least in genera that produce fruiting bodies; Elec-trophoretic analysis of the appropriate fruiting body extractsshould indicate whether such a major low molecular weightacidic protein is present.

This work was supported by Grant GM12953 from the NationalInstitute of General Medical Sciences, U.S. Public Health Service.

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in Neurospora," Proc. Natl. Acad. Sci. USA 70,1891-1893.2. Srb, A. M., Nasrallah, J. B. & Basl, M. (1973) "Genetic control of

the development of the sexual reproductive apparatus of Neu-rospora, Brookhaven Symp. Biol. 25, 40-50.

3. Frederick, L., Uecher, F. A. & Benjamin, C. R. (1969) "A newspecies of Neurospora from the soil of West Pakistan," Mycologia61, 1077-1084.

4. Jong, S. C. & Davis, E. E. (1973) "Stromatic Neurosporas," My-cologia 65,458-464.

5. Metzenberg, R. L. & Ahlgren, S. K. (1971) "Structural and reg-ulatory control of aryl sulfatase in Neurospora: The use of in-terspecific differences in structural genes," Genetics 68, 369-381.

6. Perkins, D. D., Turner, B. C. & Barry, E. G. (1976) "Strains ofNeurospora collected from nature," Evolution 30, 281-313.

7. Beadle, G. W. & Tatum, E. L. (1945) "Neurospora II. Methodsof producing and detecting mutations concerned with nutritionalrequirements," Am. J. Bot. 32,678-686.

8. Westergaard, M. & Mitchell, H. K. (1947) "Neurospora V. Asynthetic medium favoring sexual reproduction," Am. J. Bot.34,573-577.

9. Olive, L. S. (1956) "Genetics of Sordaria fimicola. I. Ascosporecolor mutants," Am. J. Bot. 43,97-107.

10. Pontecorvo, G., Roper, J. A., Hemmons, L. M., MacDonald, K.D. & Bufton, A. W. J. (1953) "The genetics of Aspergillus nidu-lans," Adv. Genet. 5, 141-238.

11. Fowell, R. R. (1952) "Sodium acetate agar as a sporulation me-dium for yeast," Nature 170, 578.

12. Ouchterlony, O. (1949) "Antigen-antibody reactions in gels," ActaPathol. Microbiol. Scand. 26,507-515.

13. Mancini, G., Carbonara, A. 0. & Heremans, J. F. (1965) "Im-munochemical quantitation of antigens by single radial immu-nodiffusion," Immunochemistry 2, 235-254.

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