FEMS MicrobiologyLetters 2 (1977) 203-205
© Copyright Federation of European MicrobiologicalSocieties
Published by Elsevier/North-HollandBiomedicalPress
FUSION OF RHODOSPORIDIUM (RHODOTORULA) PROTOPLASTS
M. SIPICZKI and L. FERENCZY
Department of Microbiology, Attila J6zsef University, H.6701, Szeged, P.O. Box 428, Hungary
Received 1 August 1977
1. Introduction
CCY 62-2-4 a were provided by Dr. A. Kockov~Kratochvilov~ (Bratislava, CSSR), and the haploid
auxotrophic strains 716 leul ~ and 756 phenl met1
a by Dr. F. B6ttcher (Greifswald, GDR).
Protoplast formation was performed with snail
enzyme [4] in 1 M sorbitol. The method ofprotoplast fusion was similar to that used for Schizosaccharomyces pombe [2]. The protoplasts of the strains
716 and 756, 107 of each, were mixed, centrifuged for
10 min at 2500 ×g, the supematant was removed,
and the sedimented protoplasts were overlayered with
35% polyethylene glycol (PEG, mol. wt. 4000) in 0.1
M CaC12 and gently shaken several times. After incubation at 30°C for 30 min the protoplasts were mixed
with minimal agar medium and plated in a thin overlay onto minimal agar medium [2], both layers being
stabilized osmotically with 0.8 M mannitol. In order
to establish the total number of colony-forming units,
samples were also applied to stabilized minimal medium supplemented with 0.5% peptone. Control experiments were performed as described previously [2].
3-day-old cells grown by shaking in liquid medium
(0.5% yeast extract, 0.5% peptone, 1% glucose, pH
6.5) were used for the determination of the DNA
contents and the sizes of the cells of the mutant
strains and the fusion products. DNA was extracted
by the Bostock procedure [7] and estimated with
diphenylamine [8] using chicken erythrocyte DNA as
standard.
For the analysis of mitotic segregation the cells
were transferred to minimal agar plates supplemented
with 0.5% peptone and 3-(p-fluorphenyl)-DL-alanine
at concentrations of 0, 10, 30, 100 and 300/ag/ml.
After 5 days the cells were collected, spread on nutritionally rich medium (0.5% yeast extract, 0.5% peptone, 1% glucose, 2% agar). The nutritional require-
The life cycle ofRhodosporidium toruloides
(Rhodotorula glutinis) was discovered by Banno [1 ].
The haploid types, previously known as Rhodotorula
yeasts, are yeast-like. Under appropriate conditions,
cells of opposite mating types, A and ct, conjugate
and develop dikaryotic mycelia, which form diploid
resting spores (teliospores). After meiosis four haploid sporidia are produced by each spore. The sporidia propagate by budding, resulting in haploid clones
of either A or a mating type. The cells of the clones
of opposite mating types can conjugate and the cycle
is repeated. Self-sporulating uninucleate diploid sporidia are also produced with low frequency, and form
directly dikaryotic mycelia with resting spores. Stable
diploids not exhibiting spore formation and segregation into haploid sporidia have not been found to
date. Nevertheless, we assumed that stable diploids of
Rhodosporidium can be produced by fusing haploid
protoplasts of identical mating type, similarly to
other species [2,3].
Protoplasts of Rhodosporidium species can easily
be produced with snail enzyme [4,5] and they regenerate readily into normal yeast cells [5]. As regards
yeast protoplast fusion, successful experiments were
recently carried out with auxotrophic Schizosaccharomyces pombe [2] and Saccharomyces cerevisiae
strains [3,6]. We present here results of protoplast
fusion of Rhodosporidium toruloides auxotrophic
mutants belonging to the a mating type.
2. Materials and Methods
The haploid wild-type Rhodosporidium tomloides (Rhodotorula glutinis) strains CCY 62-2-3 A and
203
204
ments o f the colonies were d e t e r m i n e d b y replica
plating.
TABLE 2
Cell size of parental cells, fusion cells, and segregant ceils
Cell volume (V) was calculated for each cell individually
according to the equation
3. Results and Discussion
4 a (b) 2
v=g,,~.
3.1. Fusion of protoplasts o f auxotrophic strains
where a is the long axis and b the short axis of a cell.
The fusion e x p e r i m e n t s were carried o u t w i t h
strains 716 and 756, b o t h being a u x o t r o p h s and a as
regards mating type. The appearance o f colonies on
m i n i m a l plates c o u l d therefore be a t t r i b u t e d to protoplast fusion and c o m p l e m e n t a t i o n . The f r e q u e n c y o f
c o m p l e m e n t a t i o n is 0.14% w h e n the n u m b e r o f colonies appearing o n m i n i m a l m e d i u m is c o m p a r e d to
that o f colonies growing on s u p p l e m e n t e d m e d i u m ,
and 0.02% w h e n the c o m p a r i s o n is m a d e to the original n u m b e r o f protoplast pairs. The considerable difference is due to the aggregation o f protoplasts during
PEG-treatment.
B a c k - m u t a t i o n frequencies o f o n l y 5.8 • 10 - 8 were
o b t a i n e d for b o t h strains and there was n o significant
difference b e t w e e n the n u m b e r s o f revertants f r o m
intact cells or protoplasts.
Strain
number
Length of
cells (/am)
Width of
cells (tzm)
716
756
F1
F2
F3
F4
HI-1
H2-1
7.37 ± 0.50
7.69 ± 0.35
9.90 ± 0.59
9.64 -+ 0.57
9.35 ± 0.63
9.39 ± 0.45
8.37 ± 0.42
8.00+_0.35
3.87 ± 0.25
4.44 ± 0.14
5.27 ± 0.17
4.71 +- 0.16
4.44 ± 0.20
4.25 ± 0.24
4.83 ± 0.21
5.56±0.21
Volume of cells
(um 3)
65.59 ± 12.62
72.50 ± 6.13
130.66 ± 12.23
105.81 ± 10.31
95.31 ± 12.29
86.85 ± 10.99
92.47 ± 9.69
113.69-+10.13
HI-I and H2-1 are leu- segregants fof F1 and F2, respectively.
D N A as the haploid cells participating in the fusion
( 0 . I 0 1 -+ 0.016 pg/cell for strain 716 and 0.093 +
0.009 pg/cell for strain 756), and the D N A c o n t e n t s
o f the segregants varied b e t w e e n the haploid and
diploid values.
Ten p r o t o t r o p h i c clones, p r e s u m e d to be fusion
products, were e x a m i n e d for segregation o f the auxotrophic markers present in the parental strains. The
s p o n t a n e o u s segregation f r e q u e n c y was as low as
3.2. Genetic and cytological investigations or fusion
products
The fusion cells were yeast-like, m u l t i p l y i n g b y
budding, stable w i t h o u t f o r m a t i o n o f m y c e l i a and
sporidia, larger, and c o n t a i n e d nearly twice as m u c h
TABLE 1
Mitotic segregation of fusion clones, induced with 3-(p-fluorophenyl)-DL-alanine
Fusion
clones
Total
colonies
analysed
Prototrophic
colonies
Auxotrophic
colonies
leu-
F
F
F
F
F
F
F
F
F
F
602
315
676
325
340
477
336
506
744
890
445
43
203
94
211
313
164
298
258
749
157
272
473
231
129
164
172
208
486
141
122
272
473
230
129
172
208
486
38
1
2
3
4
5
6
7
8
9
10
phen-
-
met-
35
161
88
leu -
met-
leu -
phen -
phen-
met-
m
m
r
r
m
1
lell -
phenmet
m
m
m
m
m
1
2
m
m
15
205
0.25%. It could be increased significantly by 3-(p-fluorophenyl)-DL-alanine, with the highest value at 30
/ag/ml (Table 1). The appearance of segregants proves
that the fusion products contained an increased number of chromosomes. However, the reason for the
preferential segregation of the three expected markers
is not known. It is most probably caused by incomplete mitotic nondisjunction resulting in various
aneuploids. This is supported by the sizes (Table 2)
and the DNA contents of the cells. The low frequency of segregants requiring phenylalanine can be
attributed to the selective inhibitory effect of
3-(p-fluorophenyl)-DL-alanine.
Attempts to cross the fusion isolates with strains
CCY 62-2-3 A and CCY 62-2-4 a resulted in mycelia
and teliospores with the A strain only. This means
that the fusion products remain of ~ mating type and
are capable of yielding zygotes.
In conclusion, it is possible to carry out fusion of
protoplasts of Rhodosporidium toruloides haploid
strains otherwise incapable of conjugation, as in the
case of Schizosaccharomyces pombe [2]. The stable
fusion products segregate mitotically when treated
with 3-(p-fluorophenyl)-DL-alanine. The results show
the possibilities of hybridization and segregation
analysis with avoidance of the heterokaryotic mycelial phase.
Further characterization of the fusion products is
in progress.
Acknowledgement
The authors wish to thank Dr. A. Kockov~-Kratochvflowi and Dr.F. B6ttcher for Rhodosporidium
strains, and Miss M. Romv~ry for expert technical
assistance.
References
[1] Banno, I. (1967) J. Gen. Appl. Microbiol. 13,167-196.
[2] Sipiczki, M. and Ferenczy, L. (1977) Mot. Gen. Genet.
151, 77-81.
[3] Ferenczy, L. and Mar~z, A. (1977) Nature, in press.
[41 Von Hedenstr6m, M. and HSfer, M. (1974) Arch. Microbiol. 98, 51-57.
[51 Svoboda, A. (1967) in: Symposium tiber Hefe-Protoplasten (Mtiller, R., ed.),'pp. 31-36, Akademie-Verlag,
Berlin.
[6] Svoboda, A. (1977) Arch. Microbiol., in press.
[7] Bostock, G.J. (1970) Exp. Cell Res. 60, 16-26.
[8] Giles, K.W. and Myers, A. (1965) Nature, 206, 93.