Heterokaryons of Gibberella zeae formed following hyphal anastomosis or protoplast fusion

EXPERIMENTALMYCOLOGY 11, 339-353 (1987)

Heterokaryons of Gibberella zeae Formed following I Anastomosis or Protoplast Fusion1r2

GERARD ADAMS,* NANCYJOHNSON,* JOHN E LESLIE,? ANEI L. PATRICK

“Department of Botany and Plant Pathology, Michigan State University, East Lansing, Michigan 48824, and I’Department of Plant Pathology, Throckmorton Hall, Kansas State University, Manhattan, Kansas 66556

Accepted for publication July 16. 1987

ADAMS, G., JOHNSON, N., LESLIE, J. E, AND HART, L. P. 1987. Heterokaryons of Gibberella zeae formed following hyphal anastomosis or protoplast fusion Experimental Mycology 11, 339-353. Heterokaryosis in Gibberella zeae varies depending upon the compatibility of the fused strains and the manner in which the strains are fused. Analysis of nutritionally complementing auxotrophic markers in pairings via hyphal anastamoses revealed a nuclear distribution in which heterokaryosis was restricted to anastamosed cells; apical cells remained auxotrophic but both types of nuclei could be recovered from multinucleate macroconidia. A comparable pairing via protoplast fusion yielded, initially, a fusion product in which apical cells were prototrophic and the conidia contained only one nuclear type. Analysis of subcultures of this fusion product, however, revealed a change. As in the heterokaryon generated by hyphal anastomoses, apical cells were auxotrophic and both types of nuclei could be found in the conidia. Heterokaryons could not be established by pairing vegetatively incompatible hyphal cells but the fusion of protoplasts from incompatible cells yielded a slow-growing, prototrophic colony in which conidia resolved only one nuclear type. This nuclear type was different from either of the parental types, and all conidia were capable of growth on minimal medium. The complementing nuclear types were not recovered. Protoplast fusion between vegetatively incompatible strains (G. zeae Types A and B) apparently produced a heteroploid. 0 1987 Academic Press. hc.

INDEX DESCRIPTORS: Gibberella zeae; Fusarium graminearum; Fusarium roseum; protoplast fusions; heterokaryosis; heteroploid; aneuploid; vegetative compatibility.

Gibberella zeae (Schw.) Petch (anamorph: Fusarium graminearum Schwabe) is an important plant pathogen with a worldwide distribution. It causes head and kernel blight (scab) of small grains (Hagler et al., 1984), stalk and ear rot of maize (Hart et al., 1982), and root rots and seed- ling diseases (Cook, 1968). This fungus also produces numerous mycotoxins as secondary metabolites on infested grain (Marasas et al., 1984). For example, some strains of G. zeae produce zearalenone, a

i Publication No. 12247 from the Michigan Agricul- ture Experiment Station; contribution No. 87-291-J from the Kansas Agricultural Experiment Station, Manhattan.

2 This project was supported in part by BRSG Grant 2-SO7 RR07049-15 awarded by the Biomedical Re- search Support Grant Program, Division of Research Resources, National Institutes of Health.

compound used as the starting material for the synthesis of zearanol, a growth- promoting substance in cattle ( Hidy et al., 1977). Another my duced by some strains of G. zeae, deoxynivalenol, causes feed refusal in both cattle and pigs (Forsyth et al., 1977).

Within the G. zeae species, t logically distinct types can be dis and are known as Types A and al., 1982). These two types can coexist in the same maize field and may r netically isolated populations. lates are pathogenic on maize ears; form a sexual stage readily in culture, grow relatively rapidly, and produce deoxy~~~~~e~o~ and low levels of zearalenone. Type lates are not pathogenic on maize form a sexual stage rarely, if ever, in culture, grow relatively slowly,

339 0147-5975187 $3.00 Copyright Q 1987 by Academx Press. inc. All rights of reproduction in any form reserved.

340 ADAMS ET AL.

duce deoxynivalenol, and produce large quantities of zearalenone. We have studied heterokaryons formed when the parental strains were exclusively Type A or exclusively Type B, and when one strain was Type A and the other strain was Type B.

A heterokaryon is a multinucleate cell containing genetically distinct nuclei in a common cytoplasm; such cells are often created by hyphal fusions between compatible strains. Heterokaryosis provides an opportunity for genetic recombination via the parasexual cycle in sterile, homothallic, or imperfect fungi. Heterokaryons selected by fusion of complementing auxotrophs on minimal medium have been used in the analysis of many genticahy important traits (cf. Burnett, 1975; Fincham et al., 1979 for references).

Two kinds of heterokaryons resulting from hyphal fusions have been described in ascomycetous fungi. One kind was described in studies of Neurospora crassa Shear & Dodge (Beadle and Coonradt, 1944), Aspergillus nidulans (Pontecorvo, 1953), and Penicillium cyclopum (Rees and Jinks, 1952). These heterokaryons are best described as a thallus of growing multinucleate cells containing genetically distinct nuclei; these nuclei are capable of mi- grating into the apical cells of the mycelium. A second type of heterokaryon has been described in Gibberella fujikuroi (Sarv.) Wr. (anamorph: Fusarium moniliforme Sheldon) (Burnett, 1975), and in Verticillium dahliae (Puhalla and Mayfield, 1974). The heterokaryons formed by these fungi are confined to the anastamosed cells. These heterokaryotic cells nourish the linked auxotrophic hyphae by producing nutritional products which are translocated throughout the colony. Thus in this second type of heterokaryon, the heterokaryotic cells are neither actively growing nor dividing. The nuclei in these cells neither migrate throughout the colony nor segregate from one another into the actively growing hyphal tips. The potential

for asexual genetic recombination following hyphal fusion is greatly hindered by the lack of growth of the heterokaryotic cells per se and by the lack of nuclear mi- gration throughout all of the cells of the heterokaryon. The cells resulting from the fusion of protoplasts of fungi which form this type of heterokaryon have not been previously described in detail. The ma- jority of ascomycetes and deuteromycetes are thought to form this latter type of heterokaryon (Burnett, 1975).

Conventionally, heterokaryons are selected by hyphal fusion of complementing auxotrophs on minimal medium. Hetero- karyons formed by hyphal fusion can occur only between vegetatively (heterokaryon) compatible strains. Research on the genetic systems controlling vegetative compatibility in fungi indicates that these compatibility systems are present in many fungal species and are controlled by 10 or more loci (Anagnostakis, 1977, 1982; Brasier, 1983; Croft and Jinks, 1977; Leach and Yoder, 1983; Perkins et al., 1982; Puhalla and Hummel, 1983; Puhalla and Spieth, 1985). For two strains to be vegetatively compatible, they must have identical alleles at all of these loci. Protoplast fusion can bypass the incompatibility caused by these loci in some fungi (Dales and Croft, 1977), although it seems unlikely that such techniques will bypass these loci in all fungi since, for example, the microinjection of cellular contents from an incompatible strain into a N. crassa hypha is sufficient to trigger the incompatibility reaction (Wilson et al., 1961).

The objectives of this study were (1) to determine the type of heterokaryons formed by G. zeae following either hyphal anastamosis or protoplast fusion and (2) to determine if these heterokaryons are stable and capable of self-perpetuation. Nuclear ratios in heterokaryons formed by hyphal fusion and by protoplast fusion were esti- mated. Heterokaryons were formed between strains of the same vegetative com-

HETEROKARYOSIS IN Gibbevella zeae 341

patibility group as well as from strains of different compatibility groups.

MATERIALS AND METHODS

Fungal strains. Two strains of G. zeae and their derivatives were used for these studies: U-5373, a strain from the Fusarium Research Center that was morphologically Type A (Hart et al., 1984), and ATCC 20273, a strain from the American Type Culture Collection that was morphologically Type B (Leslie, 1983). The Type B strain only rarely forms perithecia on car- nation leaves, whereas the Type A strain fruits readily. Auxotrophs which were derived from the two primary strains are des- ignated with the prefix A or B to indicate the strain from which the mutant was derived. For example, Alys- is an auxotroph requiring lysine that originated in the Type A strain. Heterokaryons are similarly desig- nated; for example, Alys-IBnnu- desig- nates a heterokaryon in which the A nucleus is marked with lys- and the B nucleus is marked with nnu- (nitrate nonutilizing mutant). The mutants in the Type B strain have been described in more detaiB elsewhere (Leslie, 1983, 1987; Kinzel and Leslie, 1985).

Mutagenesis. Auxotrophic strains of ATGC 20273 were induced by ultraviolet light and recovered following filtration en- richment (Leslie, 1983).

Auxotrophic mutants of strain U-5373 were also induced with ultraviolet light. A suspension containing l-5 X IO4 macroconidia per milliliter of water was made, and 0.1 ml of the suspension was spread on the surface of a plate containing a complete or appropriately supplemented minimal medium plus sorbose (recipe below). The surface of the plate was dried by evapora- tion in a laminar flow hood and the plate was exposed to 254-nm ultraviolet radia- tion 357 kW/cm2 for 3 min. This dose gave 99.5% kill. Following mutagenesis, the exposed plates were incubated in the dark to prevent photoreactivation. Auxotrophic

colonies were identified by replica plating with Kimwipes (Kimberly-Clark Co ~ 1 Neenah, WI) to a minimal me -I- sorbose (recipe below). Auxotro ultures were reisolated from a single rna~ro~~~~.~ dium. The phenotypes of thes were identified via auxanograp 1949).

Media. Minimal medium ( modification of Neuvospo crossing medium (West Mitchell, 1947) and consiste crose, 1 g of KNO,, 0.5 7H,O, 0.5 g of K2HP0,, 0.39 g of

0.11 g of CaCl, * 2H,O, 0.1 g of NaC 1 ml of a trace element solu media. The trace element sohrtion contained 160 mg of CuS04 * 7H,0, 56 MnCi,, 40 mg of FeCl, s 6H@, 36 boric acid, and 16 mg of Na, in 100 ml of distilled water. dium with sorbose (SMM) containe of sorbose and 2 g of sucrose per liter instead of 20 g of sucrose. S~pp~~rne~t~d minimal medium (SPM) was ~~~irna~ medium plus 200 mg/hter of the amino aci quired for growth by the auxotrop plete medium (CM) was modified from that of Leslie (1983) and contamed IO g of glucose, 2 g of NZ Amine (Type A, Sheffield Products, Norwich, NY), 1 g of yeast extract (Difco), 1 g of beef extract (Difco), 2.5 g of NaCl, 1 ml of the trace ele- ments solution, and I ml of a vitamin stock solution per liter of media. The vita stock solution contained 400 inositol, 200 mg calcium pa~totb~~at~, 0 mg Qf

e, 200 mg of nicotimc aci 100 mg of thiamine-HC1, 5 flavin, 50 mg of pyridoxine- p-aminobenzoic acid, 20 mg of biotin, and

3 Abbreviations used: MM, minimal medium; SMM, minimal medium with sorbose; SPM, supplemented minimal medium; CM, complete medium; SCM, complete medium with sorbose; CMC, carboxymethyl cellulose medium with sorbose; MCMC. minimal CMC; PPM, protoplasting medium; PEaS7 po:y- ethylene glycol; PRM, protoplast regeneration medium.

342 ADAMS ET AL.

0.8 mg of folic acid in 100 ml of distilled water (Beadle and Tatum, 1945). Complete medium with sorbose (SCM) contained 20 g of sorbose in place of the 10 g of glucose. Twenty grams of Bacto-agar (Difco) per liter was added to solidify all media not stated to be liquid. Macroconidia were produced in a liquid carboxymethyl cellulose medium (CMC) containing 15 g of the sodium salt of a low viscosity carboxymethyl cellulose (Sigma), 1 g of NH4N0,, 1 g of KH2P04, 1.0 g of NZ Amine, and 0.5 g of MgSO, . 7H,O per liter of medium (Capel- lini and Peterson, 1967). Minimal CMC (MCMC) was CMC without the NZ Amine. Macroconidia were produced by inocu- lating 100 ml of sterile CMC or MCMC in a 500-ml Erlenmeyer flask with 0.1 g of a sterile soil stock culture. These flasks were incubated at 25°C on a reciprocal shaker at 64 t-pm in diffuse room light for 5 days.

Microscope studies. When self-anastomosis or anastomosis between strains was examined, the cultures were grown on a single thickness of cellophane dialysis tubing (VWR Scientific, San Francisco, CA) overlaying 2% water agar. The number of nuclei per cell in both hyphae and spores was observed in bright field microscopy following staining with haematoxylin (Raju and Newmeyer, 1977).

Protoplast formation and regeneration. Mycelia for protoplast production were grown in six 500-ml flasks containing 50 ml of liquid MM. Each flask was inoculated with 1 x IO5 macroconidia and incubated on a reciprocal shaker at 64 rpm for 28 h at 25°C. The mycelium (approximately 1.4 g fresh wt or, following 12 h at 60°C 0.1 g dry wt) was harvested by filtering the contents of the flask through a IOO-pm nylon mesh screen and then washed three times with sterile distilled water. Macroconidia passed through the mesh while mycelia were retained.

The mycelia from which protoplasts were to be generated were transferred to a 50-ml sterile Erlenmeyer flask containing

12 ml of filter-sterilized protoplasting medium (PPM). This medium (Miller, 1980) contained 10 ml of distilled water, 2.0 g of sucrose, 1 ml of 1.0 M sodium phosphate buffer, pH 7.0, 16.0 mg of r.,-cysteine, 0.3 pg of chloramphenicol, 0.5 kg of strepto- mycin sulfate, and enzymes. Enzymes tested singly and in combinations for their ability to release protoplasts from G. zeae hyphae were Novozyme 234 (NOVO Labo- ratory, Inc., Wilton, CN); cellulase-Tv concentrate (Miles Laboratories, Inc., Elk- hart, IN); chitinase from Serratia marces- tens (Sigma Chemical Co., St. Louis, MO); chintinase from Streptomyces griseus (Sigma); and Type H2 B-glucuronidase (Sigma). The experiment was repeated three times. After stationary incubation at 33°C for 3- 12 h, the PPM broth was filtered through a 15-pm nylon mesh (Tetko, Inc., Elmsford, NY) to separate the protoplasts from the hyphal debris. The filtrate was centrifuged at 8008 for 10 min and the su- pernatant was discarded. The pellet was resuspended in 1 ml of 0.6 M KCl, gently layered over 1 ml of 0.8 M sucrose in a 15 x 75-mm centrifuge tube, and then recen- trifuged (Yabuki et al., 1984). The pellet was resuspended in 1 ml of 0.8 M sucrose. The protoplasts were counted in a hemacy- tometer and 0.25 ml of the appropriate dilu- tion in 0.8 M sucrose was plated onto agar regeneration media. Protoplasts were plated on a variety of media including PRM (Acha et al., 1966), MM, SMM, SPM, CM, SCM, and potato dextrose agar, and the protoplasts were allowed to regenerate into colonies during incubation at 25°C in diffuse light.

Heterokaryon formation. Two techniques were used to form heterokaryons between complementary auxotrophic strains paired on minimal medium. In one method, a 3-mm2 block was cut from a culture of an auxotrophic strain on SPM and placed on MM with aerial hyphae up. A similar block of a second auxotrophic strain was placed with aerial hyphae down

HETEROKARYOSIS IN Gibberellcr zene 343

on top of the first block. Mycelia which grew prototrophically after successive transfers on MM were considered to be heterokaryotic if the additional criteria dis- cussed below were met.

In the second method, heterokaryons were formed following the fusion of protoplasts of complementary auxotrophic strains. Protoplasts of the two strains were mixed together in 2 ml of 0.6 M KCl. This mixture was overlaid on 1 ml of 0.8 M sucrose in a 1.5 x 75-mm centrifuge tube and centrifuged for 10 min at 800g (Yabuki et

al., 1984). The pellet was resuspended in 1 ml of cold (4°C) protoplast fusion medium -an aqueous solution of 25% polyethylene glycol (molecular weight, 3350; Sigma) an 1.32% CaCl, * 2H,O for 30 min (Ferenczy et al., 1975). The protoplasts were col- lected by centrifugation and resuspended in 1 ml of 0.8 M sucrose, and 0.1 ml of the suspension was placed onto each MM plate.

Meterokaryon analysis. The following experiments were performed to confirm plasmogamy and to eliminate cross-feeding as a cause for the prototrophic growth of colonies of paired complementary auxotrophs. Macroconidia from each of two complementing auxotrophic strains were harvested separately from CMC and washed twice with distilled water. A drop of a mac- roconidial suspension, containing approximately 500 macroconidia, from one auxotrophic strain was placed on the surface of the MM plate and dried in a laminar flow hood. These macroconidia were covered with a Nucleopore filter (0.2-pm pore, 47-mm diameter; Nucleopore Corporation, Pleasanton, CA) which had been surface sterilized in 70% ethanol and then dried. A similar drop of a macroconidia suspension from a second auxotroph was placed on the filter on top of the first drop. Control experiments included (1) pairing an auxotro- pkic strain with its prototrophic parent, (2) pairing complementary auxotrophs on MM witkout tke interposed Nucleopore filter,

and (3) placing the auxotrophic strain under the nucleopore filter and a drop PPd of the nutrient required by the a opkic strain on the filter’s surface. These tests ali- lowed us to identify combinations of pa auxotrophs that grew prQtotropkica~ly to cross-feeding and to make ~re~~rni~a~y determinations of vegetative (ketero- karyon) compatibility.

To determine the distribution of component nuclei witk a keterokaryotic co (35 to 45mm diameter), contiguous li blocks were cut along a colony t-a Three radii per colony, replicated times, were sampled. Tkese agar b were placed on MM and SPM and incubated for 7 days at 25°C.

MM. This procedure was repeated twice, successively, under 60 x rnag~~fic~t~Q~. The nuclear content of macroco~~d~2 from heterokaryotic colonies was dete plating dilutions of spores onto S and CM. Germination and growl on the media were quantified.

RESULTS

Microscope studies. G. zeae isolates do not form microconidia (Nelson et ai., 1983); macroconidia observed in our study were composed of an average of five or six cells. In the parental strains, the terminal cells of the macroconidia were generally uninucleate (sometimes binucleate) and remaining cells were generally binucle (sometimes trinucleate). Cells in Ike macroconidia formed by colonies Ilr- and B4rrg-IAtYy-9-) d protoplast fusion of vegetatively incompatible strains were usually u~~n~c~eate and only occasionally binucleate. Hypkae com-

344 ADAMS ET AL.

monly contained four nuclei per cell and less frequently two nuclei per cell. Self- anastomosis of hyphae occurred frequently. Hyphal anastomosis occurred readily in pairings between strains of Types A and B, but lysis of the cytoplasmic contents occurred in the fused cell. Based on this reaction, these two strains were terms vegetatively incompatible.

Formation of protoplasts. Several combinations of enzymes were effective in di- gesting cell walls of G. zeae and releasing protoplasts (Table 1). The results were consistent in three replications of the experiment. Over a period of 2 years different lots of each of the tested enzymes were used in uncontrolled experiments. Regardless of lot number, the enzymes proved consistent in effectiveness within half an order of magnitude in number of protoplasts re- leased. The most effective combinations of enzymes were chitinase plus P-glucuronidase and Novozyme 234 plus P-glucuronidase. The two chitinases were approximately equally effective. The cell walls of isolates of Type A were more resistant to

the Novozyme 234 than were isolates of Type B. Although Novozyme 234 alone was effective in producing a relatively high yield of protoplasts from Type B strains, the addition of P-glucuronidase to the No- vozyme 234 was required for the release of a significant number of protoplasts from the hyphae of the Type A strains. When grown in liquid shake culture, Type A strains also produced numerous spores with cell walls that were resistant to en: zyme degradation. These spores were re- moved by rinsing the mycelial mats three times with sterile water prior to enzyme treatment. Pretreatment of the hyphae with dithiothreitol or P-mercaptoethanol resulted in less hyphal debris following incubation in wall-degrading enzymes, but the resulting protoplasts were unstable.

Protoplast regeneration. Regeneration of colonies from protoplasts of G. zeae was previously reported to require up to 6 weeks on PRM (0.2% agar) (Leslie, 1983). Here, protoplasts regenerated in 3 to -8 days when 0.1 to 0.25 ml of a protoplast suspension in 0.8 M sucrose was spread

TABLE 1 Enzyme Combinations and Their Effectiveness in Initiating Release of Protoplasts from Mycelium of G. zeae

Enzyme

Novozyme 234 B-glucuronidase H-2

Chitinase from Serratiu B-glucuronidase H-2

Chitinase from Streptomyces

B-glucuronidase H-2

Cellulase TV concentrate P-glucuronidase H-2

Novozyme 234 Type A strain Type B strain

Novozyme 234 Chitinase from Streptomyces

Cellulase TV concentrate Chitinase from Streptomyces

Amount (per 20 ml PPM)

10 mg 0.75 ml

10 mg 0.75 ml

10 mg 0.75 ml

10 mg 0.75 ml

10 mg 10 mg

10 mg 10 mg

10 mg 10 mg

Protoplasts Relative amount (per ml) of hyphal debris

1 x 105 +

1 x 105 +

4 x 104 +

4 x 104 ++

1 x 102 +++ 1 x 104 +

1 x 103 +++

0 ++++

Note. Treatment was for 20 h at 25°C.

HETEROKARYOSIS IN Gibberella zeae

over 2.0% agar medium in a 9-cm petri dish, rather than the 20-45 days on PRM (0.2% agar) (Table 2). If 0.8 M mannitol was used to suspend the protoplasts instead of 0.8 M sucrose, then the number of regenerated protoplasts decreased by approximately 20%.

The percentage of protoplasts regener- ating to form colonies following fusion could be determined most precisely when one of the components was a Type B strain carrying nnu- (Bnnu-). Protoplasts of strains carrying this mutation would support limited growth (approximately 1 mm linear growth) prior to lysis on MM. Thus, a count of regenerated protoplasts of

nnu- could be compared with regener- ating fusion products on each SMM plate. Approximately 10% of the Bnnu- protoplasts regenerated on SMM following treatment with polyethylene glycol (PEG) plus CaCl, . 2H,O. Colonies resulting from fusion between protoplasts of Bnnu- and Alys- regenerated on SMM at a frequency of O.3-0.6% of the background count of Bnnu- . Therefore, approximately O.Ol-0.03% of the protoplasts in the fusion mixture could regenerate into colonies on SMM as viable fusion products.

Complementation tests and vegetative compatibility. The prototrophic parental strains grew on MM when inoculated under or on top of the nucleopore filter. Auxo- trophs inoculated either on top of or under- neath the filter grew only if supplementai nutrients were placed in a drop of liquid on

TABLE 2 Regeneration of Protoplasts of G. zeae Suspended in

0.8 M Sucrose on Agar Media

Regeneration

Medium Days

PRM (0.2% agar) 20-45 PRM (2.0% agar) 5 PDA (2.0% agar) 3 SCM (2.0% agar) 3 SMM (2.0% agar) 8

Percentage

10.2 i 3.1 10.1 f 3.4 10.0 * 1.5 4.8 k 3.9 9.5 i 3.0

top of the filter. Of the auxotrophs teste Bnnu- and A@- were capable of cross- feeding. Other auxotrophic strains cross feed complementary auxo strains when any combination of two strains were spearated by e interposed nucleopore filter. Spores a non-cross- feeding auxotrophic strain, whe with spores from a complementary non- cross-feeding auxotrophic strain, grew into a prototrophic colony on MM only both of the auxotrophic strain rived from the same parental phae of auxotrophic strains deri Type A strain did not form a he with hyphae of a Type I3 strain reg of the auxotrophic mutants carried component strains. This lack of karyon formation was presumed to be due to genetically determined barriers to karyon formation such as vegetat heterokaryon, incompatibility loci (cf. Perkins et al., 1982). All combinations of paired complementary auxotrophic strains derived from a single arent could for heterokaryotic, prototrophic colonies t could grow on MM.

Growth rate and colony ~~~~hol~gy~ Heterokaryons of complementing au trophs grew noticeably more slowly MM than did wild-type homokar~o~s~ ex- cept Alys-/Atry- (Table 3). ~eter~ka~ formed by pairing hyphae or spores of etatively compatible au had irregular colony m with different colors ( nies derived from fus colony margins and color were regular at first, When these colonies were tured, however, they gave rise to with irregular margins and patchy color.

Heterokaryons formed by fusing protoplasts of complementary ~~x~t~~~~~ of vegetatively incomp tremely slowly on markedly slower grow was evident on

as minute cushions of

346 ADAMS ET AL.

TABLE 3 Growth Rate of Heterokaryotic Protoplast Fusion

Products, Sectors of Fusion Products, Heterokaryons from Hyphal Fusions, Auxotrophs, and Wild-Type

Strains of G. zeae on MM and SPM

Daily growth rate (mm) at 25°C

Strain MM SPM

Wild types and auxotrophs U-5373 (Type A) 17.8 IT 0.5 9.0 + 0.5 ATCC-20273 (Type B) 7.6 i 0.4 7.8 i 0.4 Tiyptophane- 12.8 2 1.5 Lysine- 17.8 r 2.4 ATCC-48064 Arginine- 5.4 f 0.5 ATCC-48066 Histidine- 6.2 k 0.6 ATCC-48065 AdenineZ- 5.8 -t- 0.4 ATCC-48067 Nitrate

nonutilizing- 8.8 i 0.8 Heterokaryons from hyphal pairings

Alys-IAtty- 17.5 * 3.4 Bnnu-/Burg- 3.7 f 0.2 BnnulBade2- 3.1 Z!I 0.2 Bnnu-IBhis- 3.3 t 0.3 Burg-IBhis- 4.0 -c 0.4 Burg-IBadeZ- 3.4 * 0.1 Bade2-IBhis- 2.8 i 0.4

Heterokaryons from protoplast fusions Barg-IAlys- 2.7 t 1.1 Bnnu-IAlys- 2.8 k 0.9 Barg-IAtry- 2.4 k 0.7 Alys-IAtry 3.6 * 0.5

Sectors from protoplast fusion products” Barg-IAlys-Sdr- 6.0 r 0.6 Barg-lAlys-lcr- 5.5 * 0.5 Barg-IAlys-lcp- 5.5 + 0.4 Barg-IAlys-7- 4.9 5 0.5 Burg-IAlysZb- 5.3 -+ 0.7 Barg-IAtry-llr- 3.9 t 0.2 Barg-IAtty-9- 3.1 r 0.3 Barg-IAtry-lip- 4.5 i 0.5 Bnnu-IAlys-lObp- 4.3 i 0.3

a Growth rates following 6 months of selection for rapid growth and wild-type coloration and mycelial density.

growth with thin, widely spaced peripheral hyphae (called “fried-egg” morphology) (Fig. lC), after approximately 12 days. When transferred to MM, the fusion products grew as thin hyaline colonies that were not clearly visible unless light reflected off the surface hyphae at an oblique angle. Some fusion products retained this characteristic

slow, thin colony growth pattern, while others formed sectors of faster and denser growth. From these colonies we selected sectors with coloration and mycelial density similar to that of the wild type on MM. After 6 months of selection (two to six transfers) for faster growing sectors, the most vigorous sectors closely approached the growth rate of the wild type of Type B (Table 3), even though they retained the fried-egg appearance on MM.

Heterokaryon analysis. The results from sampling contiguous blocks along the radii of heterokaryotic colonies formed following hyphal fusion of vegetatively compatible strains were similar to those of Pu- halla and Spieth (1983) with heterokaryons of G. fujikuroi formed by hyphal anastomosis. The hyphae at the periphery of the heterokaryotic colonies were usually composed of one auxotrophic nucleus for an average of 3 mm (ranging from 2 to 8 mm) behind the growing front. Generally, colonies resulting from subcultures taken more than 3 mm from the periphery were prototrophic. From these data we concluded that hyphal tips of heterokaryons formed following hyphal fusion were auxotrophic.

The distribution of nuclear types among conidia is listed in Table 4 for heterokaryons formed between vegetatively compatible strains by either hyphal fusion or protoplast fusion. Generally, nuclear ratios were not 1:l in heterokaryons formed by hyphal fusion of vegetatively compatible auxotrophic strains, but the disparity never exceeded 1:4. Conidia from heterokaryons formed by protoplast fusion of vegetatively compatible strains initially contained nuclei from only one of the parental strains and hyphae at the colony periphery were prototrophic. After the colony was subcultured, however, the subordinant component was represented in the conidia in proportions similar to those found for heterokaryons formed by hyphal fusion, and hyphal tips were auxotrophic. Thus, neither individual hyphae nor conidia from the heterokaryons

HETEROKARYOSIS IN Gibber& zeae 347

FIG. 1. A comparison of the distinct cultural characteristics of colonies of 6. zeae growing on minimal medium. (A) The wild-type colony. (B) The mosaic or patchy growth of a heterokaryotic colony derived from hyphal fusion between vegetatively compatible strains. (C) The growth of a colony derived from protoplast fusion between vegetatively incompatible strains described as “fried- egg” appearance. The protoplast fusion product has been selected over 6 months for rapid growth rate, wild-type coloration, and mycelial density. Visible several millimeters ahead of the flocculent colony growth extend characteristic widely spaced, sparsely branching. nonanastomosing, single strands of hyphae.

derived from vegetatively compatible strains were capable of perpetuating the prototrophic colony.

Hyphal tips of colonies derived from protoplast fusion of vegetatively incompatible strains were prototrophic. If these colonies were transferred to CM and then subcultured on MM, they remained prototrophic. Fast-growing sectors from these fusion products formed peripheral hyphae which often grew 5 mm on MM without anastomosing. The lack of anastomosis was not similar to wild-type or heterokaryon growth patterns. When these fast- growing sectors were reisolated from hyphal tips to MM three successive times, all of the sectors remained viable prototrophs. Other than with these peripheral hyphae, anastomosis was frequently observed between hyphae in these colonies, and adja- cent conidia were occasionally observed to anastomose with one another at several points (a fusion at each cell).

The distribution of nuclear types of conidia of heterokaryons formed by protoplast fusion of vegetatively incompatible strains is listed in Table 5. Conidia from

these heterokaryons initially apnea germinate and grow only on mented with the requirement of one parental strains, while not growing on supplemented with the other requirement or on the unsupplemented MM. Thus, the initial interpretation was that conidia from these heterokaryons contained nuclei from only one of the parental strains, the dominant nuclear component. In all but one case the dominant nuclear component was Type A. However, after conidia were incubated an additional 2-3 weeks, comdia grew protofrophically on MM and on supplemented with the single r~q~~rerne~t of the other parental strain (the ~~b~rd~~a~t nuclear component). The m the prototrophic growth on mented with the requirement of the subordinant nucleus was not the same as that of either auxotrophic parent. Instead, the morphology was of the fried egg type and resembled the growth of the fusion product on MM. Furthermore, growth on the medium supplemented with the req~~~~~rn~~t of the subordinant component was sometimes slower and more restricted than the

348 ADAMS ET AL.

TABLE 4 Resolution of Heterokaryons of Gibber& zeae Formed by Hyphal and Protoplast Fusion of Vegetatively

Compatible Strains

Proportion of conidia growing on

Heterokaryon Component differential media (No. counted)

formation X Y MM+X” MM + yb MM CM Ratio of X: Y

Hyphal fusion Type B His -

Arg-

Arg-

Type A

Protoplast fusion Type A

Lys -

TV-

After one transfer

After two transfers

Arg- 0.43 0.50

(185) (215) Ade2- 0.23 0.74

(101) (322) His - 0.24 0.80

(121) (401) Ade2 - 0.22 0.79

(97) (342) NfU- 0.33 0.69

(139) (287) TV- 0.22 0.64

(101) (298)

Lys - 0

(0) 0.24

(128) 0.30

(502)

0.98 W3 0.85 (460) 0.71

(1198)

0

(0)

(:I 0

(0)

(419) 1

(464)

1 (372)

(539)

(lQs3)

1:1.16

1:3.22

1:3.33

1:3.59

1:2.10

1:2.91

0:l

1:3.54

1:2.37

a MM + X, minimal medium plus the nutrient required by the component X. * MM + Y, minimal medium plus the nutrient required by the component Y.

growth on MM. Growth from the conidia or hyphal tips transferred to MM plus the nutrient required by the dominant auxotroph resembled wild-type growth. The proportion of conidia growing on MM was approximately equal to the proportion of conidia growing on MM supplemented with the requirement of the dominant nucleus (Table 5). These data suggest that most of these conidia were capable of prototrophic growth and that they did not contain only the subordinant nuclear component. Thus, both individual hyphae and conidia from colonies derived from the fusion of protoplasts of vegetatively incompatible strains were capable of perpetuating the prototrophic colony.

In contrast, neither individual hyphae nor conidia from the heterokaryons derived (by protoplast fusion or by hyphal fusion) from vegetatively compatible strains were

capable of perpetuating the prototrophic colony.

DISCUSSION

Methods of fusion protoplasts of fungi have been used for several years, yet there has been little evaluation and discussion of the nature of the heterokaryon produced. Perhaps this lack of information is a reflec- tion of the paucity of data concerning the nature of the heterokaryon (in Ascomy- cetes) produced by the more conventional method of forcing haploid auxotrophic strains to complement one another following hyphal anastomosis. In this study we made heterokaryons via both hyphal anastomosis and protoplast fusion. Micro- scopic examination revealed that hyphal anastomosis occurred between strains of Type A and Type B of G. zeae, but that successful heterokaryon formation was

HETEROKARYOSIS IN Gibberella zeae

TABLE 5 Resolution of Heterokaryons of G. zeae Formed by Protoplast Fusion of Type A and B Strains

Auxotrophic Proportion of conidia growing on differential media (number counted)

components After 1 week After 3 weeks -

Heterokaryon Type B Type A MMB” MMAb MM CM MMB” MMA” MM CM

AT-9 Arg- Try- 0 1.14

(178) 0.92

(378) 0.79

W-3 0.95

(680) 1.03

(4046) 1.02

(782) 0.74

(1956) 1.04

(146)

1 0 (156)

AT-10 Arg- TrY- 0 0 (412) 1

AT-l ir Arg- TV- 0 0 (468) 1

AT-l lp Arg- TUY- 0 0 (714)

BE-Sb Arg- Lys- 0 cl (39iO)

AL-lcr Arg- Lys- 0 0 (7166) 1

AL-5dr Arg- Lys- 0 0 (2626) 1

GL-lb Nnu Lys- 0 0 (140) 0.97 1

GL-1 lb Nm- Lys - (130) 0 0 (134) 1.01 1

CL-1 Nnu- Lys- 0 (718) 0 (710) 0.75 1

CL-1% Nm- Lys- 0 (348) 0 (466)

a MMB, minimal medium plus the nutrient required by the Type B component. b MMA, minimal medium plus the nutrient required by the Type A component.

0.74 (11‘5) 0.78 (3201 0.71 (330) 0.85 (604) 1.07

(4200) 0.89 i6W 0.78

(2048) 0.87 (122) 0.97 (130) 1.28 (912) 0.97 (452)

1.14 (178) 0.92 (378) 0.79 (372) 0.95 (680) 1.03

(40463 I .02 (782) 0.75

(1956) 1.04 (1461 1.28 (172) 1.01 (718) 0.15 (348)

0.73 1 (114) 1156) 0.52 1 (214) (412) 0.73 (340) i468) 0.96 1. (684) (714) 1.13 1

(4440) (3920) 0.39 1 (300) (766) 0.48 1

(1268) !2626) 0.51 i

(80) (ICSO! 0.94 1 (134) (126) 1,Oi I (718) (710) 0.90 I (420) 1456)

prevented, presumabiy by genetically determined vegetative (heterokaryon) incompatibility factors. If the components of the heterokaryon were vegetatively compatible, the heterokaryons formed by either hyphal anastomosis or protoplast fusion were very similar. If the components were vegetatively incompatible, however, then no viable heterokaryon formed following hyphal anastomosis and the nature of the heterokaryon formed following protoplast fusion differed from that formed following the fusion of protoplasts from vegetatively compatible strains. Although there have been published discussions on the potential of protoplast fusions in overcoming the barrier of vegetative compatibility systems or species differences (cf. Dales and Croft, 1977; Anne and Peberdy, 198.5; Tamaki,

1986), we know of no previous demonstra- tion that the heterokaryon resulting from protoplast fusion is fundamentally different from the heterokaryon formed f~~~owi~g hyphal anastomosis.

Heterokaryosis following hyphaI fusion in G. zene resembled that in 9;. ~u~~~u~~i (Puhalla and Spieth, 1983). In both cases, heterokaryosis was apparently confine the anastomosed cells which then nour- ished the rest of the colony. The apical cehs remained homokaryotic and the conidia were auxotrophic.

The heterokaryotic cells eit grow into heterokaryotic hyphae cell” form of heterokaryosis) or ~~~~~ps growth began but, having no mechanism to assure synchronized nuclear division sec- toring occurred. Resolution of the

350 ADAMS ET AL.

karyon by conidiation gave nuclear ratios that describe a stable, but moderately unbalanced, heterokaryon. The two different nuclei were apparently growing in separate hyphae with hyphal anastomosis restor- ing the heterokaryotic cells in a regular manner. No spores which were capable of prototrophic growth on MM were recovered from these heterokaryons.

With this “single-cell” form of heterokaryosis following hyphal anastomosis, it was difficult to predict the type of cell that would result from protoplast fusion. We have demonstrated that the prototrophic heterokaryon resulting from protoplast fusion between vegetatively compatible auxotrophic strains is self-perpetuating but changes nuclear condition. The initial colony segregates only one nuclear type into the conidia and has prototrophic hyphal tips, but after several transfers on MM both nuclear components that can be recovered from the conidia and hyphal tips are auxotrophic. Two explanations are pos- sible for these data: (1) The initial heterokaryon is grossly unbalanced and the subordinate nuclei are so uncommon that they are only rarely segregated into the conidia. Upon subculture the nuclear ratios ap- proach equality through differential repli- cation of the subordinate nucleus and the heterokaryon becomes similar to the heterokaryon formed following hyphal anastomosis. (2) The fusion product breaks down due to nonsynchronous nuclear division into a colony in which the auxotrophs separate and prototrophic growth is maintained by the restoration of heterokaryotic cells by hyphal anastomosis. Upon subculture the heterokaryon becomes similar to the heterokaryon formed following hyphal anastomosis.

The heterokaryon formed following protoplast fusion of vegetatively compatible strains can be clearly distinguished from the heterokaryon formed following protoplast fusion of vegetatively incompatible strains. The prototrophic heterokaryons

formed following protoplast fusion of vegetatively incompatible auxotrophic strains had an unusual morphology (fried egg), grew slowly, contained distinctive hyphae which did not anastomose with other hyphae at the colony perimeter, and could be perpetuated by subcultures of single macroconidia or hyphal tips. We conclude that the successful synthesis of this stable self- perpetuating product is limited to protoplast fusions between vegetatively incompatible strains since the heterokaryons formed by vegetatively compatible strains all eventually break down into their components. At first, the nature of the vegetatively incompatible heterokaryotic product appeared to be similar to the heterokaryon of Neurospora (Beadle and Coonradt, 1944), since it was both stable and self- perpetuating. The vegetatively incompatible Gibberella fusion product, unlike the Neurospora type of heterokaryon, could resolve only one nuclear type through seg- regation of nuclei into conidia. The conidia did not express the phenotype of either parent. The conidia grew rapidly when supplemented with the nutrient required by one of the auxotrophic nuclear components of the heterokaryon (dominant component). Colonies derived from these conidia and hyphal tips also grew on MM and on MM plus the nutrient required by the subordinant auxotrophic nucleus. This latter type of growth was not the wild type, however. Instead, it was very slow and thin and resulted in a colony with the fried-egg morphology.

We attribute the prototrophic growth of hyphae and conidia from the protoplast fusion product to nutritional complementation in a diploid or heteroploid formed by the two vegetatively incompatible auxotrophic strains. Presumably nuclear fusion occurred during or immediately following fusion of the protoplasts. A similar conclu- sion was reached by Ferenczy et al. (1977) following their studies of protoplast fusion between strains from two different species

HETEROKARYOSIS IN Gibbereiln zeae 351

of Aspergillus. Both aneuploidy and heteroploidy may be important mechanisms of variation in the fungi (Tolmsoff, 1983) and are a frequent result of fusion between cells of different species of either plants or an- imals (Pontecorvo, 1975).

Formation of aneuploids or heteroploids would require karyogamy as well as plasmogamy. Successful protoplast fusion of incompatible strains in 6. zeae may require fusion of the nuclear envelopes. Sectors of more rapid growth and wild-type colony characteristics spontaneously formed from the diploid or heteroploid colonies of 6. zeae. Such instability might suggest that the sectors within the resulting colony could have different chromosome numbers. Stable sectors could be simple haploids or disomics, while the unstable sectors would have a more complex chromosome composition.

We conclude that the product of protoplast fusion between vegetatively incompatible strains of G. zeae is not a stable and self-perpetuating heterokaryon, but instead of heteroploid. We exclude the possibility that the fusion products are diploids based on the extreme variability evident in indi-

ual fusion products in pathogenicity and toxin formation (unpublished data). How- ever, to verify that the nuclei have a more complex chromosome composition, a de- tailed genetic analysis using identified polymorphic chromosome loci will be required. Polymorphic loci should be electrophoreti- tally detectable in the Type A and B strains because they are homothallic species and, presumably, genetically isolated.

Although we termed the Type A and B strains used in our study “vegetatively incompatible,” the exact taxonomic relation- ship between these strains is far from clear. Indeed, it has long been recognized that the species concept in the Fusarium roseum group, to which F. graminearum belongs, is incompletely formed (Messiaen and Cas- sini, 1981; Nelson et al., 1983). Although

yphae of Type A did anastomose with

Type B strains, the two types may re sent sibling species or different biol cal species. Studies characterizing t products of protoplast fusion between ling species, between vegetatively incompatible and vegetatively compatible strains of the same species, and between dif~ere~t species, such as Fusarium ~~~rn~r~rn (W. 6. Smith) Sacc., Fusarium crookwel- lense Burgess, Nelson, and Toussoun~ and Fusarium sambucinum Fuckel, coul crease our genetic understanding of the complex taxonomic relationships of the B;. roseum group. For example, perhaps protoplast fusions between different biological. species within one taxonomic species always form diploids or heteroploids, rather than heterokaryons.

Our results demonstrate the value of fused protoplasts in bypassing natural bia- logical barriers such as species sexual sterility, mating factors, tive (heterokaryon) incompatibility factors. Once these heteroploids are better c terized, they should be useful in studying the effets of gene dosage and genetic inter- actions on the expression of economically important traits such as virulence and the production of secondary ~etabol~t~s such as mycotoxins.

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Heterokaryons of Gibberella zeae formed following hyphal anastomosis or protoplast fusion

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