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Copyright 0 1986 by the Genetics Society of America
CHARACTERIZATION OF THE HETEROKARYOTIC
AND VEGETATIVE DIPLOID PHASES OF
MAGNAPORTHE GRISEA
MARK S. CRAWFORD,’ FORREST G. CHUMLEY: CAROLYN G. WEAVER
BARBARA VALENT‘,’
AND
Department of Chemistry, University of Colorado, Boulder, Colorado 80309
Manuscript received November 14, 1984
Revised copy accepted August 18, 1986
ABSTRACT
The heterokaryotic and vegetative diploid phases of Magnaporthe grisea, a
fungal pathogen of grasses, have been characterized. Prototrophic heterokaryons
form when complementary auxotrophs are paired on minimal medium. Hyphal
tip cells and conidia (vegetative spores) taken from these heterokaryons are
auxotrophs with phenotypes identical to one or the other of the parents. M.
grisea heterokaryons thus resemble those of other fungi that have completely
septate hyphae with a single nucleus per cell. Heterokaryons have been utilized
for complementation and dominance testing of mutations that affect nutritional
characteristics of the fungus. Heterokaryons growing on minimal medium spontaneously give rise to fast-growing sectors that have the genetic properties expected of unstable heterozygous diploids. In fast-growing sectors, most hyphal
tip cells are unstable prototrophs. The conidia collected from fast-growing sectors include stable and unstable prototrophs, as well as auxotrophs that exhibit
a wide range of phenotypes, including many recombinant classes. Genetic linkage
in meiosis has been detected between two auxotrophic mutations that recombine
in vegetatively growing unstable diploids. The appearance of recombinants suggests that homologous recombination occurs during vegetative growth of M.
grisea. No interstrain barriers to heterokaryosis and diploid formation have been
detected. The mating type of the strains that are paired does not influence the
formation of heterokaryons or diploids.
M
AGNAPORTHE grisea is the name given to the perfect (sexual) state of
what was formerly considered to be two fungal species: Pyricularia oryzae, pathogens of rice (Oryza sativa), and Pyricularia grisea, pathogens of grasses
other than rice (BARR1977; YAEGASHIand UDAGAWA
1978a,b). Rice-infecting
strains are the agents of rice blast disease, which causes serious damage to
crops in many areas of the world. Whereas M . grisea as a species has a very
broad host range, any particular isolate is able to infect only one or a few
’
Present address: Institute of Biological Chemistry, Washington State University, Pullman, Washington
99163.
* Present address: Central Research and Development Department, The DuPont Company, Experimental
Station, E402/2208, Wilmington, Delaware 19898.
Author to whom inquiries should be directed.
’
Genetics 1 1 4 1 1 1 1 - 1 129 December, 1986.
1112
M. S. CRAWFORD E T AL.
species of grasses (ASUYAMA1965; KATO 1978). Hundreds of races are distinguished among rice-infecting strains according to their ability to infect particular cultivars of rice (LATTERELL,MARCHETTI
and GROVE1965; ATKINSet al.
1967; KIYOSAWA1976; YAMADAet al. 1976; Ou 1980). M . grisea thus offers
a unique opportunity to study the genetic basis of host species and cultivar
specificity in a fungal plant pathogen.
Although pathogenic variants and auxotrophic mutants of M. grisea have
been reported (YAEGASHI1978; YAEGASHI
and ASAGA1981; TAGA
et al. 1982;
NAGAKUBO
et al. 1983), this important pathogen is still undeveloped as a subject for rigorous genetic analysis. The work reported here was undertaken to
characterize heterokaryons and diploids of M . grisea in order to utilize these
forms of the fungus in conducting genetic analysis. Either a heterokaryotic or
a diploid form of the fungus is necessary for complementation and dominance
testing of genetic traits of M. grisea, which grows normally as a haploid.
This study presents new results in several areas. We have discovered that
complementary M. grisea auxotrophs form prototrophic heterokaryons in
which conidia and single hyphal tip subcultures are auxotrophs with phenotypes identical to one or the other of the parental strains, as first described
for the plant pathogen Verticillium dahliae (PUHALLAand MAYFIELD1974).
Fast-growing sectors that emerge from heterokaryons are at least transiently
diploid. These diploid cultures of M. grisea are highly unstable; conidia derived
from them include stable prototrophs (which are probably haploid) and auxotrophs, including many that have undergone recombination of markers in the
parents. This observation confirms previous reports (YAMASAKI
and NIIZEKI
1965; GENOVESIand MAGILL1976) that the parasexual cycle (PONTECORVO
1956) is active in M . grisea. Conidia that show properties expected of unstable
diploids have also been detected, although these are rare among the conidia
obtained from diploid mycelia. T w o auxotrophic mutations that are linked
meiotically recombine in the parasexual cycle, suggesting the occurrence of
intrachromosomal mitotic recombination in M. grisea. We have determined
that the mating-type locus in M. grisea does not condition vegetative incompatibility as does the mating-type locus in Neurospora crassa (BEADLEand COONRADT 1944). Finally, no interstrain barriers to the formation of heterokaryons
have been detected in our studies.
MATERIALS AND METHODS
The organism: M. grisea is a filamentous heterothallic Ascomycete, group Pyrenomycetes. It is the perfect state of the plant pathogens, P. oryzae (pathogens of rice), and
P. grisea (pathogens of grasses other than rice). No morphological distinction can be
made between the two groups of pathogens. In a number of laboratories, including our
own, rice-infecting strains have been crossed with strains that infect other grasses (TANAKA, MURATAand KATO 1979; YAEGASHI and ASAGA1981; VALENTet al. 1986; M.
TAGA,personal communication). Progeny from these crosses can be backcrossed to
both parents, yielding viable progeny (VALENT
et al. 1986). Since no basis exists for the
species distinction between strains that infect rice and strains that infect other grasses,
we now refer to all Pyricularia strains as M. grisea.
The asexual spores of M. grisea initiate infections on leaves, nodes and panicles of
susceptible grasses. A single conidium contains three cells, each with a single nucleus.
M . GRISEA HETEROKARYONS AND DIPLOIDS
1113
All three nuclei have a common origin and, thus, are genetically identical (YAMASAKI
and NIIZEKI 1965). On the host plant, germ tubes emerge from one or more of the
cells of a conidium and penetrate directly through the cuticle by means of an appressorium. Hyphae grow intracellularly within the host tissue. Conidiating lesions occur 57 days after initiation of infection. Resistance to this pathogen in plant tissue is often
associated with a hypersensitive response, the death of the first host cells to come in
contact with fungal hyphae (SUZUKI
1965).
M . grisea is normally isolated as a haploid, with six chromosomes per nucleus (YAEGASHI and HEBERT1976; TANAKA,
MURATAand KATO 1979; LEUNC1984). The hyphae are septate containing one nucleus per cell.
Strains: The M.grisea field isolates used to develop the strains described in this study
were SM81-11, SM81-4, WGG-FA40, Ken60-19 (all generously provided by H. YAEGASHI) and 0-42 (collected by H. KATO and B. VALENT,Tochigi Prefecture, Japan).
These isolates originated from lesions either on finger millet (Eleusine coracana) or rice
(Oryza sativa) growing in Japan. Specific strains used in this study are listed in Table
1 . Most of the auxotrophic mutants studied were isolated from parental strains several
generations removed from the original field isolates. Several multiply marked strains
were used to form heterokaryotic and diploid colonies. [Unless otherwise indicated, the
data listed in Tables 5, 6 and 7 are derived from pooling the results of experiments
that involved several closely related strains that contain the same markers (see table
legends)].
Most of the strains given a number preceded by the letters “CP” were isolated
following mutagenesis. Strains designated by a three-part number are single ascospore
progeny of genetic crosses. The first part of the strain number indicates the cross serial
number, the second is the ascus number and the third is the ascospore number. Formerly, the two mating types of M. grisea were referred to as “A” and “a” (KATO,
YAMACUCHIand NISHIHARA1976; YAEGASHIand NISHIHARA1976). In order to use a
consistent nomenclature at all loci and to avoid implying a dominance relationship
between these alleles, we refer to “A” as m a t l - l and “a” as matl-2. We have followed
the conventions for genetic nomenclature suggested by YODER,VALENTand CHUMLEY
(1 986). A three-letter gene designation that recalls the relevant phenotype has been
assigned to all mutations ( i e . , lys). A permanent, unique, serial allele number has been
assigned to each mutation, and it follows the gene designation, separated by a hyphen
( i . e . , lys-1). In many cases, mutations at more than one genetic locus can lead to the
same phenotype. Where such loci have been distinguished through complementation
and recombination tests, a locus number has been assigned. The locus number follows
the gene designation immediately, preceding the allele number, which is still set off by
a hyphen (i.e., lysl-1). All cultures are stored dried and frozen in cellulose filter paper,
et al. 1986).
in silica gel or in infected host tissue (VALENT
Media: All media used in this study were solidified with 1.5%agar unless otherwise
stated.
Minimal: 1 % sucrose, Vogel’s N Salts (VOGEL1964), 1 mg/liter thiamine, 5 pg/liter
biotin.
Minimal plus sorbose: 0.15% sucrose, 3% sorbose, Vogel’s N Salts, 1 mg/liter thiamine, 5 pg/liter biotin.
Complete: 1 % sucrose, 0.6% yeast extract, 0.6% casein enzymatic hydrolysate.
Complete plus sorbose: 0.05% glucose, 0.05% fructose, 3% sorbose, 0.6% yeast extract, 0.6% casein enzymatic hydrolysate.
Misato-Hara medium: 1 % soluble starch, 0.2% yeast extract (TAGA
et al. 1982).
Oatmeal: 50 g of rolled oats are heated in 500 ml of water at 70” for 1 hr, then
are filtered through cheesecloth. The volume of the filtrate is adjusted to 1 liter with
water.
Mutagenesis and detection of auxotrophs by replica printing: Conidia are washed
from a culture growing on oatmeal agar and diluted to 1 X 106/ml in sterile 0.025%
Tween 20. Five ml of this spore suspension are stirred in a 3.5-cm Petri dish while
1114
M. S. CRAWFORD ET AL.
TABLE 1
M. grisea strains
Strain
Mating type
Ken6O- 19
0-42
SM81-11
matl-2
matl-1
matl-1
SM81-4
matl-2
WGG-FA40
matl-1
CP8
CP18
CP20
CP21
CP22
CP28
CP3 1
CP62
CP103
CP125
CP141
CP143
CP167
70-5-7
281-9-2
859-15-4
1951-24-2
3596-5-1
3829- 1-2
3829-1-6
3995-26-1
3995-26-4
3998-4-3
4007-9-1
4007-17-2
4065-5-2
4065-1 1-1
4065-20-1
4069-4-2
4069-5-1
4069-5-4
4069-8-1
4069-8-3
4069-1 1 - 1
4069-1 1-2
4075-7-2
4075-29-1
4085-3-4
4085-9-2
4086-2-2
4089- I - 1
matl-1
matl-1
matl-1
matl-1
matl-1
matl-1
matl-l
matl-1
matl-1
matl-1
matl-1
mall-2
matl-2
mall-I
matl-1
matl-2
mall-1
matl-1
matl-2
matl-1
-
matl-1
mat 1-2
matl-2
matl-2
matl-1
matl-2
matl-1
matl-1
matl-1
matl-1
-
matl-2
matl-2
matl-2
matl-1
matl-2
matl-2
-
Genotype
Rice pathogenic field isolate (H. YAEGASHI)
Rice pathogenic field isolate (B. VALENTand H. KATO)
Finger millet and goosegrass pathogenic
field isolate (H. YAEGASHI)
Finger millet and goosegrass pathogenic
field isolate (H. YAECASHI)
Finger millet and goosegrass pathogenic
field isolate (H. YAEGASHI)
arg-I
ade-8
lys-1
lys-2
lys-3
phe-I
ser-2
buf-4
sor-1 I
arg-6
asn-2
Rice pathogenic laboratory strain
nic-2
Prototrophic laboratory strain
Prototrophic laboratory strain
Goosegrass pathogenic laboratory strain
Goosegrass pathogenic laboratory strain
Prototrophic laboratory strain
arg-1 buf-4
arg-1 buf-4
buf-4 phe-1
arg-1 phe-1
lys-2
arg-1 lys-1
arg-1 buf-4 lys-I
arg-I ser-2
ser-2
arg-1 ser-2
arg-1 phe-1
arg-1 phe-I
arg-1 phe-1
arg-l phe-1
arg-1 phe-1
arg-1 phe-1
arg-1 phe-1
arg-1 lys-I ser-2
lys-I ser-2
phe-1 sor-11
buf-4 phe-1 sor-1 I
ade-8 buf-4
phe-I
.
.
arg-1 ser-2
M . GRZSEA HETEROKARYONS AND DIPLOIDS
1115
TABLE 1-Continued
~
~
Strain
~~
Mating type
4089-2-1
4089-2-4
4089-4-1
4089-8-1
4089-8-5
4089-9-3
4089-10-2
4089-1 2-2
4091-5-8
matl-1
matl-2
matl-2
4 105-1-1
4105-1-2
41 05-1-3
matl-2
matl-I
matl-2
mall-2
-
Genotype
ser-2
ade-8 buf-4 ser-2
ade-8 buf-4
ade-8 buf-4 ser-2
arg-I bu.4 ser-2
ade-8 buf-4
ade-8 buf-4 ser-2
ade-8 buf-4
Goosegrass and weeping lovegrass
pathogenic laboratory strain
phe-1 ser-2 sor-1 I
phe-1 ser-2 sor-I1
are-I ser-2 sor-I 1
Strains of M. grisea used in this study. The phenotypes conferred by mutations are listed in
Table 2. The two mating-type alleles are matl-1 and matl-2. For some strains, mating type has
not been determined (indicated in the table as -).
being irradiated with UV light for 4 min (Ultraviolet Products Mineralight, Model No.
UVG-11, placed 9 cm above the conidial suspension). This treatment kills 93-98% of
the conidia. The irradiated conidia are diluted and spread on complete plus sorbose
medium. Sorbose inhibits radial growth of M . grisea, causing the formation of compact
colonies and allowing up to 200 viable conidia to be plated per dish. After 24 hr, a
piece of sterile cellulose filter paper is placed over the surface of the plate, and the
colonies are allowed to grow into the filter paper for 2 days at room temperature. The
filter paper is then transferred to minimal plus sorbose medium for replica printing.
After 2 days, the prototrophic colonies have grown into the minimal medium and the
filter paper is discarded. Comparison of the replica plates reveals the auxotrophic colonies, which are picked off the complete plus sorbose plate and are tested to determine
the nature of the auxotrophy. All putative auxotrophs are stored immediately in dried,
frozen filter paper (VALENTet al. 1986).
Heterokaryon and diploid formation: When two complementary auxotrophs are
paired on minimal medium, prototrophic heterokaryotic growth appears 1-3 wk after
pairing. No growth occurs when the two strains are separated by a dialysis membrane.
Fast-growing sectors arise spontaneously when these heterokaryons are grown on minimal medium (within 1-3 wk at 25"). The fast-growing sectors have the properties of
unstable diploids, as described below.
A faster method for obtaining heterokaryotic growth involves cocultivating auxotrophic strains on complete medium. After 3 days, cocultivated mycelia are transferred
to minimal medium. Prototrophic growth appears within 2 days. Prototrophic growth
from paired auxotrophs is initially two to three times slower than wild-type growth,
and the colonies are very compact, similar to wild-type growth on sorbose-containing
media.
Genetic crosses: Sexual crosses of prototrophic strains are performed by pairing
strains of opposite mating type on oatmeal agar and allowing the two strains to grow
together at 21" under fluorescent light. Perithecia form at the intersection of growth
between the two strains. Sexual crosses between an auxotroph and a prototroph or
between two noncomplementary auxotrophic strains are performed by placing plugs
from the strains to be mated on Misato-Hara medium and incubating them as above.
Crosses between complementary auxotrophic strains of opposite mating type are accomplished by forming a heterokaryon between the two strains, which is then transferred
to oatmeal medium and incubated as above. Perithecia appear 2-wk later. Tetrads are
1116
M. S . CRAWFORD E T AL.
dissected by hand on 4% water agar (supplemented with the nutrient requirements of
the parental strains) using a finely drawn glass needle and a 50X Wild stereomicroscope.
Germinated ascospores are transferred from the Supplemented water agar to complete
medium supplemented with the nutrient requirements of the parental strains.
Cytology: T h e number of nuclei per cell in conidia was determined by staining with
4 ',6-diamidino-2-phenylindole (DAPI) (RUSSELL,
NEWMAN
and WILLIAMSON
1975). Conidia were collected by washing a culture growing on solid medium with trichloroethane.
T h e spore suspension was evaporated to dryness, and spores were resuspended in 3 ml
of H 2 0 containing 3 mg of DAPI, 100 mM Tris, 100 mM NaCI, 10 mM EDTA, 0.01%
(v/v) Triton X-100, pH 7.0. Nuclei were visualized using incident light from a mercury
lamp. A filter with a high wavelength cutoff at 360 nm was placed between the light
source and the sample, and another filter with a low wavelength cutoff at 436 nm was
placed in the tube of the microscope. Stained nuclei fluoresce bright green. Cross walls
in conidia are easily visualized using illumination from a tungsten lamp.
RESULTS
Isolation of mutants: Strains that showed either interesting pathogenicity
traits or better than average sexual fertility were chosen for mutagenesis.
Twenty-one auxotrophs were isolated from three rice-infecting strains. Two of
these rice-infecting strains were field isolates (Ken6O-19) and 0-42), and the
third (CP143) was a single ascospore progeny of a cross between Ken60-19
and 3596-5-1, a strain developed in our laboratory. The remainder of the
auxotrophs were isolated from finger millet-infecting isolates (SM81-11 and
WGG-FA40), from progeny of crosses between different finger millet-infecting
isolates (281-9-2 and 70-5-7) and from a strain derived from a cross between
a finger millet-infecting isolate and a weeping lovegrass-infecting isolate (409 15-8). The mutants recovered are shown in Table 2.
The yield of auxotrophic colonies following UV mutagenesis (approximately
0.35% of the survivors) is consistent with each of the mutagenized strains being
haploid. Colonies with growth that is not restricted by sorbose (Sor-) arise at
a frequency of about
Colonies with a particular defect in pigmentation
(Buf-) arise at a frequency of about
to
Meiotic analysis of M. grisea mutants: Auxotrophic, sorbose-resistant and
pigmentation-defective mutants were crossed with wild-type strains. Table 3
shows the results of both random ascospore and tetrad analysis including all
asci in which five or more ascospores germinated. Ascospore germination in
these crosses ranged from 20 to 80% depending on the strains involved. In all
cases, segregation in tetrads is consistent with a single-gene defect causing the
phenotype. No aberrant ratios within a tetrad were seen.
Mutant strains were intercrossed to construct multiply marked strains and
to determine linkage relationships. Results of both random ascospore and
tetrad analysis are shown in Table 4. All markers appear unlinked, with the
exception of ade-8 and phe-1, which are separated by a distance of 8.3 map
units. No tetrads were seen that would indicate that any phenotype is the
result of more than one mutation. The segregation patterns presented in Tables 3 and 4 are entirely consistent with M . grisea being haploid.
Heterokaryons: We have observed prototrophic growth resulting from the
pairing of many different complementary auxotrophs, examples of which are
M . GRZSEA HETEROKARYONS AND DIPLOIDS
1117
TABLE 2
Mutant strains of M. grisea
Genotype
adeahsargamatz
buf
CYh
cys-
g1uhishomi1vinoleu1ys-
metnit$heserSOT
tyrVal-
No. of isolates
17
2
10
5
1
76
60
4
1
4
2
3
2
4
6
16
3
1
5
18
1
1
Phenotype
Requires adenine
Requires adenine and histidine
Requires arginine
Requires asparagine
Aminotriazole resistance
Pigmentation block Mycelium buff instead of gray
Cycloheximide resistance
Requires cysteine or methionine
Requires glutamate
Requires histidine
Requires homoserine or methionine and threonine
Requires isoleucine and valine
Requires inositol
Requires leucine
Requires lysine
Requires methionine
Requires nicotinamide
Requires phenylalanine
Requires serine
Sorbose resistance
Requires tyrosine
Requires valine
Auxotrophic, drug-resistant and morphological mutants derived by UV mutagenesis.
Some of the buf - mutants indicated appeared spontaneously.
shown in Table 5 . Such prototrophic growth could result from any one of
several factors: one or both of the auxotrophic strains could have reverted to
prototrophy, the paired strains could be cross-feeding, the colony could be
diploid or the colony could be heterokaryotic. The use of double auxotrophs
in these studies reduces the chances that prototrophic growth is due to reversion of auxotrophic mutations. Reversion to prototrophy is ruled out by the
recovery of all auxotrophic markers originally present in the parental strains
(Table 5 and Table 6). The fact that prototrophic growth does not occur if
the parental strains are separated by a dialysis membrane indicates that the
prototrophic growth is not due to cross-feeding. If prototrophic growth were
the result of the formation of stable heterozygous diploid nuclei, then conidia
harvested from the colony would be expected to be diploid and prototrophic.
However, the prototrophic growth gives rise only to auxotrophic conidia (Table 5). If prototrophic growth were due to the formation, breakdown and
continued reformation of unstable heterozygous diploid nuclei, then some of
the conidia recovered should exhibit recombination of the parental markers
due to mitotic crossing over and/or chromosome loss during reversion to haploidy (parasexual recombination). N o such recombinants are detected (Table
5). Thus, the prototrophic growth resulting from the pairing of two M. grisea
1118
M. S. CRAWFORD E T AL.
TABLE 3
Segregation ratios of alleles at loci controlling morphology and nutritional characteristics
No. of tetrads recovered
Ratio in a tetrad
ade-8
buf-4
lys-I
1:4
2:3
3:2
4: 1
2:4
3:3
4:2
3:4
4:3
4:4
Aberrant
1
1
2
1
3
2
1
1
2
1
6
5
1
1
7
4
11
17
0
1
2
0
0
2
2
2
1
4
3
1
0
55
7
5
17
22:27
21:18
154:118
Total no. of
tetrads
Random ascospores
6
3
1
0
21
110:111
193:225
lys-2
2
1
1
1
1
2
phe-I
1
1
ser-2
1
2
2
1
4
2
3
5
6
sor-11
arg-l
4
2
1
6
3
1
1
10
7
9
0
1
1
1
0
5
5
35
10
41
175:185
81:105
3
0
420:332
The data presented are the pooled results of a large number of crosses in which strains that
carried the mutant allele indicated were mated with strains that carried the wild-type allele. The
strains that were crossed are included in Table 1 . The column headed “Ratio in a tetrad” reports
the results from tetrads in which five or more of the eight ascospores germinated. The ratio of
wild-type to mutant ascospores is presented. Results from random ascospore analysis are given as
the ratio of wild-type to mutant ascospores. The segregation of mating type has been observed
among the progeny of hundreds of crosses; the ratio of the two mating types is always 1 : l .
strains carrying complementary auxotrophies is not due to reversion, crossfeeding o r heterozygous diploidy and must be due to heterokaryosis.
M. grisea heterokaryons can be propagated by mass hyphal transfers taken
behind the leading edge of the colony. However, single hyphal tips from the
leading edge of the heterokaryon are auxotrophic and carry all markers corresponding to one or the other of the parental strains (Table 6). The conidia
derived from heterokaryons are also auxotrophic and are identical to one or
the other of the parental strains (Table 5 ) . Neither prototrophic conidia nor
conidia that are recombinant for the auxotrophies present in the parents have
been recovered from heterokaryons.
Diploids: Fast-growing sectors spontaneously emerge from heterokaryons
after 1- to 3-weeks’ growth on minimal medium (Figure 1). These sectors have
a growth rate similar to wild type, but are morphologically distinct, with a very
sparse appearance. In contrast to hyphal tip cultures from heterokaryons, most
hyphal tip subcultures from fast-growing sectors are prototrophic.
Conidia taken from the fast-growing sectors show a wide range of phenotypes, including classes that exhibit recombination of the parental markers
(Table 7). This observation provides evidence that fast-growing sectors arising
from heterokaryotic cultures are at least transiently diploid. These experiments
were conducted by first purifying prospective diploids through single proto-
1119
M. GRISEA HETEROKARYONS AND DIPLOIDS
TABLE 4
Recombination frequencies between several loci that control morphological and nutritional
characteristics
Second marker
First
marker
arg-1
lys- 1
phe-1
1461261
1:5:9
1331260
5:2:12
sor-1 I
ser-2
lys- 1
18/50
0:0:4
#he-1
bu.4
1471298
3:5:19
931135
2:3:10
521110
3:1:5
9/39
1:0:4
44/86
931151
1:2:10
sor-I1
24/42
0:0:4
ser-2
15/33
2:2:17
ade-8
45/98
1:l:S
8/ 109
16:l:l
45/98
1:2:8
The data presented are the pooled results of a large number of crosses in which strains that
carried the mutations indicated were mated as described in the text. T h e strains that were crossed
are included in Table 1. The fractional number is the fraction of the total progeny examined by
random ascospore analysis that were recombinant for two traits involved. The three-part ratio is
parental ditype:nonparental ditype:tetratype tetrads.
TABLE 5
Nutritional requirements of conidia harvested from heterokaryotic colonies
Strains paired
ade-8 ser-2/arg-1 phe-1
arg-1 phe-1 /lys- 1 ser-2
arg-1 #he-llade-8 buf-4 ser-2
arg-1 ser-2 sor-1 l l a d e - 8 buf-4
Frequency of recovered phenotypes
1918/3162 Ade- Ser18/40 Arg- Phe591274 Arg- Phe221134 Arg- Ser- Sor-
1244/3162 Arg- Phe22/40 Lys- Ser2151274 Ade- Buf- Ser1121134 Ade- Buf-
Strains carrying complementary auxotrophic mutations were paired on complete medium and
allowed to grow together for 2 days. The cocultivated mycelium was then transferred to minimal
medium. Prototrophic growth emerged 2-5 days later. Conidia were harvested from this prototrophic growth, and their nutrient requirements were determined. In this table the ade-8 buf4
ser-2 strains are 4089-2-4, 4089-8-1 and 4089-10-2. These same strains were used for the ade-8
ser-2 pairing listed. Buf- was not scored among conidial colonies for this pairing. The arg-1 #he1 strains are 4069-4-2, 4069-1 1-1 and 4069-1 1-2; the lys-1 ser-2 strain is 4075-29-1; the ade-8 buf4 strain is 4089-4-1; and the arg-1 ser-2 sor-11 strain is 4105-1-3.
trophic hyphal tips taken from fast-growing sectors. T h e hyphal tip cultures
were grown on oatmeal agar, and conidia from these cultures were collected
and plated on complete plus sorbose medium. Auxotrophs were identified by
replica printing. T h e phenotypes of colonies obtained in two different sets of
experiments are shown in Table 7. Monoconidial colonies from fast-growing
sectors of arg-1 phe-lllys-1 ser-2 heterokaryons include many recombinant
classes. Every possible recombinant class except Arg- Ser- and Arg- Lys- Seris represented. T h e nonrecombinants (Arg- Phe- and Lys- Ser-) are not the
major classes. Similarly, monoconidial colonies derived from a fast-growing
sector that emerged from a heterokaryon formed between an arg-1 phe-1 par-
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3
M . GRZSEA HETEROKARYONS AND DIPLOIDS
1121
TABLE 7
Nutritional requirements of conidia harvested from purified diploid
colonies
Strains paired
arg-I phe-lllys-I ser-2
Frequency of recovered phenotypes
1347
+
1 Arg- Phe-
5
10
125
3
149
1
3
47
1
1
2
2
Lys- SerArgPheLysSerArg- LysLys- PhePhe- SerArg- PheArg- LysLys- PheArg- Lys-
SerPheSerPhe- Ser-
1697 Total tested
arg-1 phe-llade-8 buf-4 ser-2
17
8
15
1
1
23
3
1
1
+
AdeBufSerAdeAdeBufPheBuf-
Buf - SerSerBufPhe- Ser-
70 Total tested
Heterokaryons were formed by pairing strains that carried the mutations
shown. From these heterokaryons, fast-growing sectors arose spontaneously.
From each fast sector, a single hyphal tip was transferred to minimal medium
and incubated for several days. The colony growing on minimal medium was
subcultured to oatmeal medium by mass hyphal transfer. After 2 weeks' incubation at 25", conidia were harvested from the oatmeal plates and plated on
complete plus sorbose medium. The resulting colonies were replica-printed to
minimal plus sorbose medium to identify auxotrophs. The nutritional requirements of the auxotrophs were then determined. The 1697 conidia tested in
the first part of the table (derived from arg-1 phe-1 and lys-I ser-2 pairings)
represent data pooled from six independent experiments that involved strain
4075-29-1 as the lys-1 ser-2 parent and strains 3995-26-4, 4069-5-1, 4069-5-4,
4069-8-1, 4069-11-1 or 4069-11-2 as the arg-I phe-1 parent. Note that these
arg-I phe-I strains represent both mating types. The 70 conidia tested in the
second part of the table represent the data from a single experiment involving
strains 4069-1 1-2 (arg-I phe-I) and 4089-8-1 (ade-8 buf-4 ser-2) as parents.
The conidia from diploid cultures that give rise to prototrophic colonies
could be either heterokaryotic, diploid or haploid recombinants generated by
the parasexual cycle. Over 2000 conidia from diploid cultures were examined
using DAPI staining to visualize nuclei. All conidia contained only one nucleus
per cell. Since the three nuclei in a conidium are genetically identical due to
1122
M. S. CRAWFORD E T AL.
their common origin, germ tubes originating from different cells within the
same conidium could not form prototrophic heterokaryons. Prototrophic colonies arising from recombinant haploid conidia would be expected to yield
only prototrophic conidia, whereas colonies arising from heterozygous diploid
conidia should yield some auxotrophic conidia through mitotic crossing over
and/or haploidization. Thirty prototrophic colonies that arose from single conidia isolated from the fast-growing sectors shown in Table 7 were cultured on
oatmeal medium. Conidia were harvested from each of these prototrophic
cultures. The 3000-4000 conidia derived from each of the 30 prototrophic
colonies were all prototrophic. Thus, the 30 colonies contained either haploid
prototrophic or stable diploid mycelia. The latter possibility seems unlikely
since the original diploid culture yielded auxotrophic conidia at a high frequency, indicating that in M. grisea the vegetative diploid stage is inherently
unstable. The conclusion that these prototrophic strains are haploid is supported by the observation that they are stable to treatment with p-fluorophenylalanine o r UV-light, treatments that induce haploidization or homozygosis in
diploids (LHOAS1961; WOODand KAFER 1969).
Isolation of diploid conidia: Numerous attempts to isolate diploid cultures
by collecting conidia from diploid mycelia and plating them on minimal medium have failed to reveal any unstable (heterozygous) prototrophs. This is
consistent with the following visual observations of diploid cultures (Figure 2).
Diploid cultures derived from single prototrophic hyphal tips produce sparse,
rapidly spreading growth with very few conidia. One-wk-old mycelium from
these cultures begins to papillate; that is, islands of heavy growth appear, some
of which conidiate heavily. On minimal medium, stable prototrophs, which are
presumably haploid breakdown products, appear and quickly overgrow the
diploid culture. On complete medium (Figure 2), the papillae include recombinant auxotrophic hyphae as well as prototrophic hyphae. Some papillae from
the diploid in Figure 2 carry the arg-l mutation as well as various combinations
of phe-1, ade-8, ser-2 and buf-4. This is in contrast to the result described in
Table 7 with the same diploid in which no conidia carrying the arg-l mutation
were recovered. This supports the idea that arg-1 was not recovered among
conidia produced by this diploid, due to the poor conidiation of hyphae carrying the arg-l mutation.
Unstable prototrophic conidia that are presumably diploid were isolated following careful visual inspection of mycelium that had not yet begun to papillate. Such mycelium contains very few conidiophores; these are elongated in
comparison to normal haploid conidiophores and contain only one or two
relatively large conidia. Conidia picked from these conidiophores, using a fine
platinum wire loop full of sterile 0.025% Tween 20 solution, produce colonies
that subsequently give rise to conidia with recombinant phenotypes, as shown
in Table 8. Thus, these rare conidia show properties expected of unstable
diploids.
Detection of intrachromosomal mitotic recombination: The arg-1 phe-l/
ade-8 buf-4 ser-2 putative diploid cultures described in Tables 7 and 8 were
formed from parents with the same mating type. Pairing of these strains has
M . GRISEA HETEROKARYONS AND DIPLOIDS
1123
FIGURE2.-Papillation
in a diploid culture. A single hyphal tip was taken from a fast-growing
sector that emerged from a heterokaryon formed on minimal medium between strains 4069-1 1-2
( m a l l - 2 arg-l $he-1) and 4089-8-1 ( m a l l - 2 ade-8 buf-4 ser-2). This tip was subcultured on complete
medium. T h e figure shows this subculture after 14 days’ incubation at room temperature. Note
the sparse, rapidly spreading hyphae (Hy) at the leading edge of the colony and the papillae (P)
that have appeared in the interior. Both Buf- and normally pigmented (Buf+) papillae occur in
approximately equal numbers. T h e significance of these papillae is discussed in the text. T h e bar
is I cm in length.
never produced perithecia, the organs of the sexual cycle. T h e putative diploids contain a pair of auxotrophic mutations that show linkage in meiosis, ade8 and phe-Z (Table 4). Since ade-8 and phe-1 were introduced in different
parents, a reciprocal recombination event between the loci would generate one
homologue with both prototrophic alleles, whereas the other homologue would
carry both the ade-8 and phe-Z mutations. Such a mitotic recombination event,
followed by haploidization, would yield prototrophic haploids and haploids with
both auxotrophic markers. Stable prototrophic progeny have been recovered
from all experiments with the arg-Z phe-Zlade-8 buf-4 ser-2 diploids. However,
the frequency with which stable prototrophs are observed is reduced in comparison to experiments involving the arg-Z phe-ZlZys-Z ser-2 diploids (Table 7).
T h e higher frequency of stable prototrophs recovered from the arg-Z phe-Z/
Zys-Z ser-2 diploids may be due to an arrangement of these markers that does
not require an intrachromosomal mitotic recombination event for recovery of
prototrophic haploids, but merely depends on independent assortment of chromosomes. Progeny containing both ade-8 and phe-Z have been recovered from
diploids 1, 3 and 4 in Table 8. These prototrophic and double auxotrophic
1124
M. S. CRAWFORD ET AL.
TABLE 8
Spontaneous haploidization in diploid cultures derived from single conidia
Colonies identified from
Phenotypes recovered
BufAdeBufAdeAdeBufPheAdeAdeBufPheAde-
+
SerBuf- SerBuf- Phe- SerBufPhe- Ser-
Monoconidial Monoconidial Monoconidial Monoconidial
1
2
3
4
1
2
1
1
1
14
6
14
SerPheSerPhe-
2
2
2
2
3
0
0
5
5
0
1
0
8
16
13
7
14
2
1
1
0
0
0
1
1
5
3
1
4
3
1
2
0
4
10
9
1
0
1
0
2
2
2
3
The nutritional requirements of conidia harvested from four independently cultured prospective diploid conidia derived from pairing 4089-8-1 (matl-2 ade-8 bu.4
rer-2) and 4069-1 1-2 (matl-2 arg-I p h e - l ) are shown. These prospective diploid conidia were isolated by picking single conidia from distinctive conidiophores produced
by diploid mycelium, using a fine platinum wire loop holding a drop of sterile 0.025%
Tween 20 solution. The data were obtained as described in the legend to Table 7.
recombinants suggest that homologous recombination occurs during the vegetative growth of M. grisea diploids.
Normally pigmented gray prototrophic colonies were at least ten times more
abundant than Buf- prototrophic colonies among the recombinants derived
from the arg-1 phe-llade-8 buf-4 ser-2 diploid cultures (Tables 7 and 8). In
some experiments only gray prototrophs were recovered, even though auxotrophs with the B u f phenotype were recovered in high frequency. This asymmetry suggested to us that the buf-4 allele might be carried on the same
chromosome as one of the auxotrophic markers in the Buf- parental strain.
Indeed, data from five independent experiments (Tables 7 and 8) show that
from 77 to 93% of the buf-4 colonies recovered also have the ser-2 mutation.
Thus, these data suggest that buf-4 and ser-2 are located on the same chromosome, and occasional recombinants may be due to mitotic crossing over.
Since the two mutations are not linked meiotically (Table 4), the proof that
they reside on the same chromosome must await further experimentation.
Vegetative incompatibility and complementation: No interstrain barriers to
the formation of heterokaryons or diploids have been detected. Although some
of the auxotrophic strains that could be expected to complement do not, there
is as much apparent incompatibility between auxotrophs derived from a common parent as there is between auxotrophs derived from different parents
(Table 9). The mating type of the paired strains has no effect on heterokaryon
or diploid formation.
The mutants carrying lys-I, lys-2 and lys-3 were independently isolated. Based
1125
M . GRISEA HETEROKARYONS AND DIPLOIDS
TABLE 9
Complementation of auxotrophic mutations
Second mutation
Mating
Origin
tYPe
SM81-11
281-9-2
28 1-9-2
281-9-2
281-9-2
0-42
CP 143
WGG-FA40
Both
Both
Both
matl-1
Both
matl-1
mat 1-2
matl-1
First
mutation
arg-1
lys- I
lys-2
~YS-3
ser-2
asn-2
nic-2
arg-6
~
arg-1
lys-I
lys-2
Iys-3
-
+
+
+
-
-
-
~
~
~
~~~
ser-2
asn-2
nic-2
+
+
+
+
+
+
+
-
NT
NT
NT
NT
NT
NT
-
+
NT
+
-
+
~
+
-
~
arg-6
+
+
+
-
~~~
Strains carrying the auxotrophic mutations indicated were tested for the ability to form prototrophic heterokaryons. Cases where the strains showed complementation are indicated by a “+”
sign. In all cases that showed no complementation (indicated by a =-” sign), at least eight independent trials were conducted. Possible pairings that were not tested for heterokaryosis are indicated by “NT.” All cultures used in these tests originated from either a single ascospore or
conidium. The column headed “Origin” indicates the parental strain from which the auxotrophic
mutant was originally isolated. T h e column headed “Mating-type tested indicates the mating type
of strains that were used in the complementation tests. In those cases where a single mating type
is shown (matl-1 or matl-2), the strain tested was the original mutant, as isolated from the parent
listed under “Origin.” In the cases where “both” is entered, two strains were tested, one being the
original mutant and the other being a strain of the opposite mating type, produced by crossing
the original mutant with a strain closely related to the parent of origin,
on their failure to complement and their similar growth response to intermediates in lysine biosynthesis (K. PARSONS,
unpublished results), we believe they
are alleles of a single gene. Thus, we will now designate them as lysl-1, Zysl-2
and lysl-3. T h e mutants carrying arg-1 and arg-6 differ in their growth response to biosynthetic intermediates (K. PARSONS,unpublished results), and
they complement, indicating that different genes are affected. We will now
designate these mutations as argl-1 and arg2-6.
DISCUSSION
Different modes of heterokaryotic growth have been described in mycelial
fungi. N . crassa forms heterokaryons in which all cells in the mycelium are
and COONRADT
1944; PITTENGER
and ATWOOD1956;
heterokaryotic (BEADLE
DAVIS 1966). Conidia and hyphal tip subcultures from heterokaryons of N .
crassa are themselves heterokaryotic. Heterokaryons of V. dahliae are quite
different. The only binucleate cells are at points of anastomosis 1-2 mm behind
and MAYFIELD1974). Intrahyphal
the growing edge of the colony (PUHALLA
diffusion of small molecules, such as amino acids, from these relatively rare
heterokaryotic cells feeds the growing hyphal tip. Conidia and hyphal tip subcultures from such heterokaryons are homokaryotic and exhibit the phenotype
of one or the other of the parents.
Heterokaryosis and parasexual recombination have been previously reported
in M. grzsea (YAMASAKI
and NIIZEKI1965; GENOVESI
and MACILL 19’76), but
these studies did not discriminate between heterokaryon and diploid formation,
1126
M. S . CRAWFORD E T A L .
and they did not describe M. grisea heterokaryons in detail. The prototrophic
growth that occurs when two M . grisea auxotrophic strains are paired is not
the result of cross-feeding, since complementary auxotrophs separated by a
dialysis membrane fail to grow. Hyphal tip subcultures from M. grisea heterokaryons are auxotrophs with phenotypes identical to the parental strains. A11
M . grisea conidia from heterokaryotic cultures have the growth requirements
of one or the other of the parental strains. All those conidia that we have
examined contained only one nucleus per cell; no heterokaryotic conidia were
detected. These properties of M . grisea heterokaryons resemble those of V.
dahliae heterokaryons; therefore, M . grisea heterokaryotic growth may be fed
by binucleate cells at points of anastomosis behind the leading edge of the
colony. The similarity between M . grisea and V. dahliae heterokaryons is not
surprising since the mycelia produced by these two fungi are similar in having
septate cells with one nucleus per cell. Operationally, the properties we have
observed for M . grisea heterokaryons define the usefulness of these heterokaryons for genetic analysis of the pathogen. As we have already shown (Table
9), M. grisea heterokaryons will be useful for complementation and dominance
testing of mutations that affect nutritional phenotypes. However, straightforward complementation and dominance testing of genes that determine pathogenicity would be impossible using M . grisea heterokaryons, since they produce
only conidia of each parental type, and the normal route of infection is through
conidia.
Fast-growing sectors arising from heterokaryotic colonies are at least transiently diploid, as indicated by the recovery of conidia that show recombination
of parental auxotrophic markers. We have isolated rare, unstable prototrophic
conidia from diploid cultures that segregate the original markers present in
the diploid strain. We have referred to these conidia as diploids because we
are able to obtain from them all markers characteristic of the original parental
strains; however, these conidia may be aneuploid. The formation and behavior
of aneuploids in Aspergallus nidulans was analyzed using diploids marked at
numerous loci representing all eight chromosomes (KAFER 1960, 196 1). Such
highly sophisticated genetic analysis will be required to further describe diploidy and aneuploidy in vegetative cultures of M . grisea.
It appears that the sexual cycle is an unlikely source of variation in nature
for M. grisea strains that infect rice. This is because newly acquired isolates of
M . grisea that infect rice are uniformly female sterile (ITOI et al. 1983; VALENT
et al. 1986), and rice pathogens from a single geographical area appear to be
predominantly of the same mating type. Our results support the suggestion
that the parasexual cycle is an important source of variation for this fungus in
and MAGILL 1976). We have
nature (YAMASAKIand NIIZEKI1965; GENOVESI
been able to detect both haploidization and mitotic recombination in a very
unstable vegetative diploid phase. M . grisea diploids appear to be less stable
than those of A. nidulans, the perfect fungus in which extensive characterizaet al. 1953; KAFER
tion of the parasexual cycle has been achieved (PONTECORVO
1960, 1961; LHOAS1967). T h e degree of instability we have observed may be
M . GRISEA HETEROKARYONS AND DIPLOIDS
1127
more similar to that seen in some imperfect fungi, including the plant pathogen, Verticillium albo-atrum (HASTIE1968).
The suggested importance of the parasexual cycle as a source of variation
of M . grisea in nature is consistent with our failure to detect interstrain barriers
to the formation of heterokaryons or diploids. T h e strains used for this study
were derived from Japanese field isolates that infect either finger millet or
rice. These field isolates were collected over a period of 20 yr, and at diverse
areas within Japan. Our studies have been restricted so far to Japanese strains,
but the lack of vegetative incompatibility is nevertheless an interesting observation, since in other fungi, field isolates from the same area often show as
much incompatibility as isolates from different areas in the world (MYLYK
1976; ANAGNOSTAKIS
and WAGGONER1981).
One measure of the relative fertility of M . grisea strains is the frequency of
viable ascospores produced in a sexual cross (VALENTet al. 1986). T h e crosses
reported here yielded 20-80% viable ascospores depending on the strains involved. We have demonstrated the potential for fertility improvement in M .
grisea strains by inbreeding and selection of highly fertile progeny (VALENTet
al. 1986). Since the auxotrophic mutants reported here were not isolated from
the most fertile strains now available in our laboratory, the potential for genetic
analysis of this fungus is even more favorable than these studies might indicate.
T h e results reported in this paper suggest that mitotic mapping of genes may
be possible in this system.
This work was supported by the Department of Energy (DE-AC02-76ERO-1426and DE-ACO284ER13 160), by The Rockefeller Foundation (RF81042) and by Monsanto Agricultural Products
Company. We should like to acknowledge the support and encouragement given by PETERALBERSHEIM,
in whose laboratory this work was performed. We should also like to thank FRANC=
LATTERELL
for introducing us to Pyricularia and for supplying fungal cultures; many techniques
currently in use in our laboratory were developed by her. We are especially grateful to HAJIME
KATO and HIROSHIYAEGASHIfor sharing strains of Pyricularia and for providing helpful insights
on the Pyricularia as pathogens. HAJIMEKATO generously arranged field trips for B.V. in Ibaraki
and Tochigi Prefectures in Japan during July, 1982, for the purpose of collecting fresh field
isolates of the fungus. HIROSHIYAEGASHIhas generously collected fresh field isolates of the
pathogen at our request. We should also like to thank JILL SKARSTAD
and DOREENLEWANDOWSKI
for expert help in preparing this manuscript.
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Communicating editor: D. BOTSTEIN