Bialaphos resistance as a dominant selectable marker in Neurospora crassa

Current Genetics

Curr Genet (1989) 16:369-372

© Springer-Verlag 1989

Bialaphos resistance as a dominant selectable marker in Neurospora crassa Javier Avalos*, Robert E Geever, and Mary E. Case Department of Genetics, University of Georgia, Athens, GA 30602, USA

Summary. Conidia of Neurospora crassa are sensitive to the herbicide bialaphos at concentrations of 160 rag/ 1 in Westergaard's or Fries' minimal media. Plasmid pJA4 was constructed by inserting a truncated bar gene from Streptomyces hygroscopieus fused to the his-3 promoter from N. crassa into pUC19. The bar gene in plasmid pJA4 confers resistance to bialaphos when transformants are selected on a medium containing bialaphos. The bar gene can be used as an additional dominant selectable marker for transformation of fungi. Key words: Bialaphos - Neurospora crassa - transformation

Introduction Transformation in Neurospora crassa generally requires donor DNA containing a selectable marker to complement an appropriate auxotrophic mutant (Case et al. 1979). Dominant selectable markers are desirable for transformation of genes with no mutant phenotype, or for introduction of genes into a wild-type strain. At the present time, only the [3-tubulin gene (Bml) from N. crassa, conferring benomyl resistance, has been used successfully for transformation as a dominant selectable marker in N. crassa (Orbach et al. 1986). Bialaphos is a new, potent, nonselective herbicide produced by Streptomyces hygroscopicus. This compound is a unique tripeptide composed of two Lalanine residues and an analogue of glutamic acid known as phosphinothricin (PPT; Thompson et al.

* Present address: Department of Genetics, Faculty of Biology, University of Seville, E-41012 Sevilla 12, Spain Offprint requests to: M. E. Case

1987). Following cleavage of the two alanines from PPT, this substanie is a potent inhibitor of glutamine synthetase in plants. Inhibition of glutamine synthetase by PPT results in a rapid accumulation of ammonia, which leads to death in plant cells (Tachibana 1986). PPT can be chemically synthesized (trade name Basta) while bialaphos is produced by fermentation with S. hygroscopicus (trade name Herbiace). The gene encoding resistance to bialaphos (bar gene) in S. hygroscopicus has been recently cloned and characterized in E. coli (Thompson et al. 1987) and has been introduced into tobacco, tomato, and potato plants (DeBlock et al. 1987). The transgenic plants showed a complete resistance to high doses of bialaphos. This paper reports the construction of plasmid pJA4 containing the truncated bar gene from S. hygroscopicus fused to the his-3 promotor from N. crassa and demonstrates the ability of this plasmid to confer bialaphos resistance when transformed into N. crassa.

Materials and methods Strains and media. Wild type 74-OR23-1A (74A) and a met- 7 strain (a

methionine-requiring strain allele 4894, Fungal Genetics Stock Center number 4088) were used in the transformation experiments. All transformants were crossed to a pan-2 strain (allele B23-1a, pantothenic acid-requiring) to obtain F1 homokaryotic isolates. Conidial growth tests for bialaphos resistance were made on the following synthetic media; Fries', Westergaard's, or Vogel's medium N (Davis and de Serres 1970) supplemented with various concentrations of bialaphos. Bialaphos was a gift from Dr. Kozo Nagaoka, Pharmaceutical Research Laboratories, Meiki Seika Kaisha Ltd, Kohoku-ku, Yokohama, 222, Japan. Plating with benomyl followed the procedure described by Orbach et al. (1986). Benomyl was a gift from E.I. du Pont, De Nemours, Wilmington, DE. Plasmid. The plasmid pBG 195 (Thompson et al. 1987) containing the bar gene was a gift from Dr. Charles J. Thompson, Institut Pasteur, Paris, France. pJA4 was derived from pBG195 by inserting the his-3 promoter region (Legerton and Yanofsky 1985) 5' to the bar gene, as

370

Table2. Sensitivity of wild type spheroplasts to different concentra-

(Spll/Asp 718)

tions ofbialaphos on Westergaard's minimal medium. Innocula were 0.1 ml containing about 10 7 spheroplasts added to a 15 ml top layer

t

APr

Bar/~ pJA4

4.2 kb

/ ~

(Nco I / Bsp Itl)

Bialaphos concentration (mg/1)

Growth

0 10 20 40 80 160

+++ +++ ++ + 0 0

Table3. Transformation

frequencies obtained with the bar gene, pJA4, and the Bml gene, BT3, in wild type 74A and the met-7 strain Xba I Recovery conditions

Plasmid

Transformants/gg DNA

Fig.1. Dominant

selection marker plasmid, pJA4. The plasmid consists of the N. crassa his-3 promoter and 5'-leader (a 0.9 kb XbaI/ NcoI fragment, hatched) from pNH60 (Legerton and Yanofsky 1985) constructed as a translational fusion to the coding region of S. hydroscopicus bar gene (solid) and modified from pBG195 (Thompson et al. 1987). The latter entailed an intermediate construct, pJA3 (not shown), in Which a BspHI site (TCATGA) was created, by standard restriction cleavage and ligation methods, to replace the unusual GTG initiation codon of bar without altering the second codon. The integrity of the junction was confirmed by DNA sequencing. Restriction sites at fragment juctions an in the pUC19 polylinker are indicated. Ap r = ampicillin resistance

Table 1. Number of viable conidia after a one week incubation at 25 ° C on three different media at three different concentrations of bialaphos. Initial innocula were 5 X l0 s conidia Minimal Media

Fries' Vogel's Westergaard's

74A

met-7

Fries' + bialaphos Fries' -4-bialaphos

None pJA4

0 60-110

0 660-1,920

Fries' + benomyl Fries' + benomyl

None pBT3

0 820-900

0 2,000-6,000

Westergaard's + bialaphos Westergaard's ÷ bialaphos

None pJA4

0 110-180

0 540- 700

Westergaard's + benomyl Westergaard's ÷ benomyl

None pBT3

0 800-880

0 2,240-6,000

1 ~tg plasmid DNA used in each experiment. Transformation frequencies indicate range obtained in different experiments

bacterial transformation experiments were carried out following the calcium chloride procedure of Daggert and Erlich (1979).

Bialaphos mg/1 0

40

80

120

> 1,000 > 1,000 > 1,000

40 15 3

7 16 0

7 11 0

indicated in Fig. 1. The plasmid pBT3, which confers benomyl resistance, was obtained from Dr. Marc Orbach (Orbach et al. 1986). Transformation. Transformation of wild type 74A or the met-7 strain was performed as previously described (Case 1982) except that Novozyme 234 (Nova Industries, Danbury CT) (Volmer and Yanofsky 1986) replaced glusulase in generating spheroplasts. The met-7 transformants were selected on methione-supplemented medium. Plasmid pBT3, which confers resistance to benomyl, was used as a control for spheroplast transformation. One gg of circular plasmid DNA was used in each experiment. Spheroplasts were added to 3% agar containing 1 M sorbitol and 15 ml/plate overplated onto a 15 ml bottom layer containing 320mg/1 bialaphos (final concentration 160 mg/l). Transformation with benomyl followed the plating procedure described by Orbach et al. 1986. For plasmid construction,

Southern blot analyses. DNA was isolated by the procedure of Yelton et al. (1984) from the N. crassa transformants grown on Fries' medium with or without 160 mg/1 bialaphos. DNA were digested with Eco RI, run on an 0.8 % agarose gel, and blotted to a nitran filter following the procedure of Southern (1975). There are no EcoRI sites within the bar gene (Thompson et al. 1987). The blot was probed with a 32p-labeled AhaII/PvuII 479 bp internal bar gene fragment isolated from pBG195.

Results and discussion Initial tests were required to determine the sensitivity of wild type 74A to bialaphos. Conidia were plated on three different minimal media, Fries', Vogel's and Westergaard's, with and without three different conc e n t r a t i o n s o f b i a l a p h o s (40,80, a n d 120 m g / 1 , T a b l e 1). Resistant colonies were counted after a one week i n c u b a t i o n a t 2 5 ° C . T h e s e r e s u l t s i n d i c a t e d t h a t N. crassa is s e n s i t i v e t o c o n c e n t r a t i o n s o f 4 0 m g / 1 , a n d greater, of bialaphos. However, resistant colonies were

371

Fig.2. Southern blot analyses of N. crassa DNA isolated from heterokaryotic and homokaryotic transformants. The N. crassa DNAs weredigestedwithEcoRI and probed with a 3Z-labeledAhaII/ Pvu I1479bp bar genefragmentfrom pJA4. Heterokaryotictransformants; 40, lane 2; 27, lane 5; 13, lane 8; and 11, lane 10. Homokaryotic isolates of the same transformants; 40-1, lane 1, 27-1, lanes 3 and 4; 13-1 and 13-2, lanes 6 and 7; and 11-1, lane 9

still obtained and the number of colonies in the higher concentrations of bialaphos depended on the minimal medium used. Resistant colonies were observed at all three concentrations of bialaphos with Fries' and Vogel's minimal media. However, no colonies were observed on Westergaard's minimal medium in bialaphos concentrations over 40 mg/1. Conidial plating of the m e t - 7 strain showed a similar sensitivity as wild type on the highest concentration of bialaphos on Fries' and Westergaard's media. Table 2 indicates the sensitivity of spheroplasts overlayered onto Westergaard's medium either alone or supplemented with various concentrations ofbialaphos ranging from 10 to 160 mg/1. From these results, medium supplemented with 160 rag/1 bialaphos was chosen for the transformation experiments. Three different transformation experiments were done with both wild type 74A and the m e t - 7 s t r a i n . The results are summarized in Table 3. In these experiments the same spheroplast suspension was used to select for bialaphos-resistant or benomyl-resistant transformants on both Fries' and Westergaard's media. No colonies were observed on Fries' or Westergaard's media supplemented with bialaphos or benomyl in the absence of added plasmid DNA when the viable

spheroplast concentration ranged from 1-3 X 105 per plate. However, transformants were obtained with bialaphos- or benomyl-supplemented media with the same spheroplast concentration of wild type 74A and the m e t - 7 strain when plasmid DNA was added. In these experiments, the m e t - 7 strain was used to determine if there was a strain difference between wild type and the m e t - 7 strain on bialaphos- or benomyl-supplemented media. As indicated in Table 3, the m e t - 7 s t r a i n was transformed at a much higher frequency than wild type under both selection conditions. The addition of methionine to the plating media did not alter the transformation frequencies of wild type. Furthermore, transformants were obtained at a higher frequency with benomyl than with bialaphos-supplemented media in both wild type 74A and the m e t - 7 strain. These results may reflect the fact that the bar plasmid construct pJA4 contains N. crassa DNA homologous only to the his-3 promoter region 5' to the bar gene coding region. There is no homology to N. crassa DNA 3' to the bar gene in pJA4. If integration alters or interferes with a functional his-3 promoter, then either no transformants would be obtained or bar gene expression would be reduced. Other plasmid constructs with additional regions ofN. crassa DNA might lead to a higher transformation frequency with the bar gene. Presumptive transformants were isolated from Fries' bialaphos-supplemented medium for genetic and Southern blot analyses. Nine heterokaryotic transformants were selected for Southern blot analyses. The results of these analyses indicated that all strains were transformants since the DNAs isolated from these strains hybridized to the bar gene probe. The bar gene probe does not hybridize to N crassa DNA (data not shown). A representative Southern blot of three heterokaryotic transformants is shown in Fig. 2 (lanes 2, 5, 8 and 10). The differences in the banding pattern among these transformants suggested that the bialaphos gene integrated into more than on site within the N. crassa genome. DNAs isolated from the two transformants 11 (lane 10) and 13 (lane 8) were grown in the presence and absence of bialaphos in the medium. The Southern blot analyses showed an identical hybridization pattern independent of the growth conditions (data not shown). These results suggest that the integrated bar gene was stable through mitosis. The nine transformants were crossed to the p a n - 2 allele B23 to obtain homokarykotic isolates. F1 homokaryotic transformants were recovered by plating on medium without bialaphos and subsequently testing the isolates for bialaphos-resistance. DNAs from the bialaphos-resistant strains were isolated for Southern blot analyses and these results indicated that all DNAs hybridized to the bar gene probe. The banding patterns

372 of DNAs isolated from four homokaryotic transform a n t s are s h o w n in Fig. 2 (lanes 1, 3, 4, 6, 7, a n d 9). T h e b a n d i n g p a t t e r n s as h e t e r o k a r y o t i c a n d h o m o k a r y k o tic isolates i n t r a n s f o r m a n t s 13 (lanes 6-8) a n d 11 (lanes 9 a n d 10) differ, while the b a n d i n g p a t t e r n s in t r a n s f o r m a n t s 40 (lanes 1 a n d 2) a n d 27 (lanes 3 a n d 5) are similar. T h e o b s e r v e d differences in b a n d i n g p a t t e r n s between h e t e r o k a r y o t i c a n d h o m o k a r y o t i c isolates m a y r e p r e s e n t i n d e p e n d e n t segregation, f o l l o w i n g meiosis, o f two or m o r e p l a s m i d s i n t e g r a t e d at different sites within the genome. These overall results indicate t h a t the bar gene can be used as a d o m i n a n t selectable m a r k e r for b i a l a p h o s resistance in t r a n s f o r m a t i o n o f N. crassa. In these e x p e r i m e n t s , the efficiency o f t r a n s f o r m a t i o n was lower for the bar gene t h a n for the Brnl gene. H o w e v e r , o t h e r constructs m i g h t lead to the p r o d u c t i o n of a p l a s m i d with a t r a n s f o r m a t i o n efficiency c o m p a r a b l e to that o b t a i n e d for the Brnl gene. Since the bar gene has no effect o n g r o w t h ofN. crassa, it can be used as an a d d i t i o n a l d o m i n a n t selectable m a r k e r for t r a n s f o r m a tion o f fungi.

Acknowledgements. J. A was a recipient of a long-term fellowship from the N.A.T.O. Scientific Program. This work was supported by Public Health Grant GM28777 from the National Institutes of Health (to M.E.C.).

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C o m m u n i c a t e d b y H. B e r t r a n d Received March 3/August 14, 1989