Cloning of the nitrate reductase gene (niaD) of Aspergillus nidulans and its use for transformation of Fusarium oxysporum

147

Gene, 78 (1989) 147-156 Elsevier GEN 02986

Cloning of the nitrate reductase gene (niaD) of Aspergillus nidulans and its use for transformation of Fusarium oxysporum (Chlorate resistance; gene library; heterologous gene expression; phytopathogenic fungus; recombinant DNA)

Laurence Malardier *, Marie J. Daboussi a) Jacqueline Julien”, Francine Roussel b, Claudio Scazzoccbio

b and

Yves Brygoo” a Cryptogamie, Universitt!Paris Sud, 91405 Orsay (France) and b Institut de Microbiologic, UniversitkParh Sud, 91405 Orsay (France) Tel. (1) 69 4163 56 Received

by J.K.C. Knowles:

Revised:

12 December

Accepted:

27 December

7 June 1988

1988 1988

SUMMARY

An heterologous transformation system for the phytopathogenic fungus Fusarium oxysporum has been developed based on the use of the Aspergillus nidulans nitrate reductase gene (niaD). F. oxysporum nia - mutants were easily selected by chlorate resistance. The A. nidulans niaD gene was isolated from a gene library by complementation of an A. nidulans niaD mutant. The cloned gene is capable of transforming F. oxysporum niamutants at a frequency of up to ten transformants per ,ug of DNA. Southern analysis of the DNA of the F. oxyspontm transformants showed that transformation resulted in integration of one or more copies of the vector DNA into the genome.

melonis

INTRODUCTION

Fusarium oxysporum

is a fungal pathogen for many plant species with each strain exhibiting a restricted host-range. This is the case for F. oxysponrm f.sp. Correspondenceto: Dr. Y. Brygoo, Laboratoire

de Cryptogamie,

Universite

Orsay

Paris-Sud,

Tel. (1)69.41.70.06; Abbreviations: CM,

Ap, ampicillin;

complete

medium

SSC,

medium

NaCI/O.OlS M

MM + 200 mM KClOs/5

0378-I 119/89/$03.50

(France)

(Daboussi,

Chl, chlorate;

A, deletion;

1980);

kb,

1980); MS, 10 mM

MSC, MS + 10 mM CaCl,;

glycol; R, resistant;

0.15 M

91405

bp, base pair(s);

(Daboussi,

pH 6.3/l M sorbitol;

polyethylene

400,

Fax (1)69417296.

1000 bp; MM, minimal MOPS

BLtiment

SDS, sodium

dodecyl

Na,.citrate

pH 7.6;

PEG, sulfate; UC,

mM urea; wt, wild type.

0 1989 Else&r

Science Publishers

B.V. (Biomedical

found in nature as a series of races that are defined by differential reactions on near-isogenic cultivars (Bouhot, 1981). The identification of genes involved in host-pathogen specificity requires the development of molecular methods to clone these genes. An essential step to achieve this goal is the development of a transformation system. Transformation of nutritional mutants with cloned wt genes has been the main approach to date. Although the number of available foreign genes as selectable markers increases, their utility is not assured; obtaining mutants of the corresponding gene in genetically poorly characterized organisms such as pathogenic fungi may be difficult. So, for these organisms, it was of the utmost interest to have a positive selection procedure for the isolation of Division)

148

mutants

lacking

enzymatic

activity.

The positive

screening of uracil auxotrophs through the resistance to 5-fluoro-orotate has been successfully used to develop

a transformation

mentous

fungi

1986; Diez

et al., 1987; Van

1987). Chlorate recovery fungus

system

(Razanamparany

species

and

et al.,

which allows the mutants

in several

such as A. niduhzns (Cove,

Neurosporu crussa (Tomsett

fila-

Begueret,

Hartingveldt

(Chl) resistance

of nitrate-non-utilizing

in different

and

Garrett,

strains

carrying

deletions

experiments;

overlapping

the niiA-niaD

wA3, white conidiospores; argB2, requirement for arginine; niaD 10 and niaD 14 are internal deletions of nitrate reductase-coding

gene; other deletions

lapping the niiA (nitrite reductase-coding

1980),

Cove, 1979).

AND METHODS

(a) Strains and plasmids The A. nidulans strains are specified by their genotype; biA 1 was the source of DNA for the genomic library; yA2 argB2 niaD10 and yA2 wA3 niaD14

over-

gene) and

the niuD gene were A625 and A516 (Tomsett

provides an interesting system for the positive selection of mutations mapping at several different genes. This procedure, which does not require the use of mutagens is of quite general applicability and particularly suitable for fungi without uninucleate spores, which includes many fungi of agricultural and industrial importance. In addition, the nitrate-non-utilizing mutants could be easily characterized by their ability to grow on various nitrogen sources. In A. niduhzns (Cove, 1979) N. crassa (Tomsett and Garrett, 1980), Septoria nodorum (Newton and Caten, 1988) and Fusarium monilifrme (Klittich and Leslie, 1988), the nitrate reductase enzyme is encoded by a single gene and mutants having a lesion in this gene were recovered with a high frequency. We, therefore, looked for such mutants in F. oxysporum strains and investigated their complementation by either an heterologous or an homologous wt gene. In the present publication we report the heterologous complementation of nitrate reductase mutants of F. oxysporum f.sp. melonis strain, by the niaD gene of A. nidulans. During the preparation of this manuscript, a F. oxysporum transformation system based on a selectable marker for resistance to hygromycin B has been reported (Kistler and Benny, 1988). A comparison between the two systems will be presented in the discussion.

other

cluster were used in Southern blots. biA 1 denotes requirement for biotin, yA2, yellow conidiospores;

1976),

Penicillium chrysogenum (Birkett and Rowlands, 1981) Fusarium oxysporum (Correll et al, 1987),

MATERIAL

were used in the transformation

and

The strain of F. oxysporum f.sp. melonis used is strain 150 derived from the isolate FOM 15 belonging to race 0 (Bouhot, 1981). Plasmids were propagated in Escherichia coli strain DH5 (F- , end4 1, hsdR 17 (r; , rn; ), supE44, thi-1, recA1, gyrA96, rel4 1). The vector pFB39 used for the library was a gift of M.F. Buxton. It consists in the insertion into the Sal1 site of pUC8 of the argB gene of A. nidulans which codes for ornithine carbamoyl transferase. (b) Media and growth conditions The standard media and growth conditions for A. nidulans were used as described by Cove (1966). For 1;. oxysporum, CM, nitrogen-less MM as well as growth conditions are those described for N. haematococca (Daboussi, 1980). The ChlR mutants were classified according to their phenotype on media containing one of the four nitrogen sources: 23 mM nitrate, 10 mM nitrite, 0.7 mM hypoxanthine or 5 mM ammonium. (c) Enzyme assays Mycelium for enzyme extraction was obtained from cultures inoculated with 5 x lo6 spores per ml, grown for 30 h at 26°C in MM with 5 mM urea as nitrogen source. Washed mycelium was then transferred to fresh MM containing either 20 mM nitrate or 20 mM L-glutamine as the sole nitrogen source and shaken for 6 h. Mycelia were harvested and frozen at -80°C until they were used. Cell-free extracts were made by grinding frozen mycelium and resuspending the powder (1 g) in 3 ml of buffer (200 mM Tris * HCl pH 8/0.5 mM phenylmethylsulfonylfluoride). After centrifugation (12 000 x g, 30 min, 4’ C), the supematant was used as a crude enzyme extract. The nitrate reductase

149

assay was carried out according to the procedure described by Cove (1966). Enzyme units are expressed as nmol of nitrite produced per min per mg/protein. (d) Preparation of protoplasts

Protoplasts of A. nidulans were prepared according to the method of Tilburn et al. (1983). For the production of protoplasts of F. oxysporum, the following procedure was used: approximatively lo6 conidia from a 4 day-old culture were inoculated onto CM media covered with a cellophane disc and incubated for 20 h at 26°C. Mycelia were collected on a nylon mesh, washed with the stabilized buffer 0.6 M KCl/O.l M sodium phosphate pH 5.8, resuspended (1 g wet weight/25 ml) in 0.6 M KC1 with 50 mg/ml of Glucanex (Novo Ferment, Basel, Switzerland) and incubated for 2 h at 26°C with gentle shaking. Protoplasts (108-109) were separated from conidia and mycelial debris by filtration on nylon mesh (porosity 50 vm), collected by centrifugation at 3000 x g for 10 min, they were suspended in 2 ml 0.6 M KC1 layered onto 10 ml of 30% sucrose and centrifiugated for 10 min at 3000 x g. Protoplasts located at the interface were removed and resuspended in 1 vol. of MS buffer, washed twice and resuspended in 200 ~1 MSC. (e) Transformation

procedures

The transformation procedure for A. nidulans was based on the method of Tilbum et al. (1983). The transformation procedure of F. oxysporum was as follows: approx. 10’ protoplasts in 100 ~1 MSC kept on ice for 20 min were mixed with pAN301 (see RESULTS AND DISCUSSION, section b) or pFB39 (10 pg in 60 ~1 10 mM Tris . HCl pH 7.5) and the mixture was incubated at room temperature for 20 min. Then 160 ~1 of 60% PEG 4000 (KochLight)/10 mM MOPS were added to each suspension and the mixture was incubated at room temperature for 15 min. After addition of 1 ml of MSC, protoplasts were pelleted at 12000 x g for 5 min, resuspended in 200 ~1 MSC and mixed with 3 ml of molten 0.4% agar containing 20% sucrose and 23 mM nitrate (KNO,) as sole nitrogen source. Oxoid agar was used to prevent residual growth of nontransformed protoplasts (Tilbum et al., 1983).

(f) Isolation and manipulation

of DNA

DNA ofA. nidulans was extracted as described for P. chrysogenum by Sanchez et al. (1987). For F. oxysporum the following method was used: 0.1 g

of lyophilized mycelium was ground with sand in a mortar, suspended in 0.6 ml of 10 mM HEPES pH 6.9/0.5 M sucrose/200 mM EDTA/l% SDS. The mixture was then incubated for 15 min at 65’ C. DNA was purified by extracting the lysate with phenol-chloroform-isoamyl alcohol (49 : 49 : 2) isopropanol-precipitated, RNase-treated and reprecipitated with ethanol. Digestion of DNA with restriction enzymes was carried out as recommended by the suppliers. For the construction of an A. nidulans gene library, high-M, DNA was partially digested with Sau3AI to give a mixture of fragments with an average size of 5-10 kb. The digestion mixture was layered on top of an NaCl gradient (0.8 M/4.1 M) and centrifuged for 19 h at 17 000 rev./min in an SW41 rotor. The gradient was fractionated and analysed by agarose gel electrophoresis. Those fractions containing fragments between 7-10 kb were pooled, dialysed against TE buffer (10 mM Tris/l mM EDTA) and concentrated by ethanol precipitation. A. nidulans DNA fragments and BamHI-digested phosphatasetreated pFB39 were mixed and ligated. The ligation mixture was used to transform E. coli DH5 for Ap resistance.

RESULTS ANDDISCUSSION

(a) Isolation of nitrate-reductase-deficient

strains in

Fusarium oxysporum

The selection of nit- mutants was performed according to the method of Cove (1976). Conidia ( lo3 per petri dish) were inoculated onto UC medium and incubated for four weeks at 26” C. Growth of the fungus was greatly inhibited and ChlR mutants arose after two weeks. Several hundred ChlR mutants were classified onto test media according to Cove (1976). They could be divided into four phenotypic groups from their growth responses on the various nitrogen sources. The phenotype symbols-used in A. nidulans have been adopted for comparable phenotypes in

150

TABLE

I

Comparison

of ChlR mutant types recovered

stable transformants Strain a

from the wt and two

of Fusuriumoxysporum

Number’

Mutant

types ’

CRUN

cnx

nir

nia

wt

187

30

21

4

132

TRl

226

16

16

0

194

TR2

12

2

1

2

7

a TRl,

TR2: stable transformants.

b Number

of ChlR mutants

’ The mutants

analysed.

were grouped

ability to grow on nitrogen

into four groups according

sources.

CRUN,

to their

ChlR strains able to

utilise nitrate; cnx, unable to utilise either nitrate or hypoxanthine and able to utilise nitrite; nir, unable to utilise nitrate nia, unable

to utilise nitrate

or nitrite;

and able to utilise nitrite.

F. oxysponrm (Table I). On UC medium we noted a high frequency of the nia- phenotype amongst the ChlR mutants. To confirm that nia- mutants are

defective for nitrate-reductase activity, specific activities were measured in crude extracts of mutants and wt strains. Only the wt strain, when induced, displayed nitrate-reductase activity (about 40 nmol/ min/mg protein). One strain, nia3, which reverts to prototrophy at a frequency of less than lo-* was chosen as the recipient strain for transformation experiments. (b) Isolation of the niaD gene of Aspergillus nidulam

The wt gene encoding the nitrate-reductase enzyme was cloned from A. nidulans for three reasons: (i) well-defined mutations affecting nitrate assimilation and an efficient system of genetic transformation are available in this organism; (ii) nitrate assimilation in F. oxysporum appears to be similar to that in A. nidulans in that they respond to Chl in the same way and produce mutants with similar physiological and genetic characteristics; (iii) heterologous expression of A. nidulans genes has been used to develop systems for the genetic transformation of pathogen fungi: argB in Magnaporthe grisea (Parsons et al., 1987) and amdS in Cochliobolus heterostrophus (Turgeon et al., 1985). A gene library of A. nidulans DNA constructed in the laboratory by Pefialva and Glatigny by the insertion of 7-lo-kb Sau3AI partials into the vector

pFB39 was used. This library transformed the A. nidulans strain yA2 argB2 niaD10 with high efficiency: about 1000 transformants per pg of DNA were recovered after prototrophy selection. Transformation with 100 pg of DNA from the library gave rise to four transformants on nitrate medium in the absence of At-g. DNA from these transformants was extracted. Analysis by Southern blotting of digested DNA showed a simple integration pattern for only one transformant, tAN1. Unrestricted DNA of this transformant was used to transform E. coli to Ap resistance as described by Johnstone et al. (1985): two bacterial clones were recovered after transformation with 10 pg of genomic DNA. Plasmid DNA was prepared from these clones and tested for their ability to transform the A. nidulans recipient strain carrying the niaD10 deletion. One of the plasmids, pAN301, which carries an 11-kb insert transforms the recipient strain at low efficiency: about ten transformants per pg of DNA, irrespective of whether the selection was for both growth on nitrate and complementation of the argB2 mutation or only for growth on the absence of Arg, using urea as a nitrogen source. This plasmid transforms with the same frequency a strain carrying a larger deletion of the niaD gene (yA2 wA3 niaD14) suggesting that pAN301 contains all the niaD gene. As can be seen from the restriction map presented in Fig. 1, pAN301 contains four EcoRI fragments of 9, 3.2, 2.7 and 1.3 kb. To check the organization of the insert of pAN301 Southern analysis of DNA isolated from the wt was carried out using the vector pFB39, the whole pAN301 and the three smallest EcoRI fragments of the plasmid as individual probes. pFB39 showed an 8-kb band which contains argB sequences (data not shown). The whole rescued plasmid showed five EcoRI bands of 1.4, 2.7, 3.8, 5.5 and 8 kb (Fig. 2, lane 1 and Fig. 3, lane 1). The 1.3- and 2.7-kb electroeluted pAN301 fragments revealed that the 2.7-kb band is really a doublet, one band hybridising with the 2.7-kb and the other with the 1.3-kb fragment of the plasmid (Fig. 3, lanes 2 and 4). The 3.2-kb electroeluted fragment revealed two bands of 3.8 kb and 1.4 kb, respectively (not shown). It should be noticed that the internal 3.2-kb fragment of pAN301 does not correspond in size to a genomic fragment. To examine the origin of this rearrangement, the niaD 10

151 -

Ikb HBg

H 1

I

X Ba

H: III

II

II

HP~ &es II

X HpaXbH

H I

I

I

I E

E pFB 39

.

.

I

H

Ill1

I

I E EE 3.2

Ba

I E

EE 2.7

__J

I pAN 301 EE 1.3

EE 2.7

EE 2.7 I E

E

I

EE 5.5

I E

Wild typa genomlc structur*

E

EE 1.4

E

E A516

1

-

‘“” MS

-

ganomlc

Fig. 1. Restriction

map of plasmid

HpaI; X, XhoI.

DISCUSSION,

-

DNA

limits of two deletions. Hpa,

-1

pFB30

Restriction

Seu3A Insort, ot pAN 301 and homologous S*nomic trapm*nts

pAN301,

wt genomic

. J

organization

in A. niduhs

sites for several enzymes are indicated

EE 1.3, EE 1.4... etc. correspond

Dal,tion Ilmits

A625 ___

to /&RI

of the sequences

by vertical lines: Ba, BumHI; bands

homologous

to the insert, and the

Bg, BglII; E, EcoRI; H, HindIII;

of 1.3, 1.4 kb, etc., respectively

(see RESULTS

AND

section b).

recipient strain and the tAN1 transformant, which gave rise to pAN301 by natural excision, were analyzed by Southern hybridization. The hybridization pattern of wt and niuD 10 strains were identical (results not shown). Fig. 2, lane 2, shows that tAN1 contains all the pAN301 and wt fragments. This can be explained by an integration of the original plasmid at the niuD locus; the rearrangement was not due to the excision process but was probably present in this original plasmid which gives rise to tAN1 transformant. The most probable explanation could be a ligation of two non-contiguous Sau3AI fragments during library construction as proposed in Fig. 1. This hypothesis was confirmed by the isolation, from a genomic library of A. niduluns in phage AEMBL4, of a recombinant phage which contained the 3.8-kb EcoRI fragment but no sequences homologous to the 1.4- and 5.5-kb fragments. This phage, 12301,was isolated by hybridization with the 1.3-kb fragment of pAN301. To localize the niuD gene in the inserted DNA present in pAN301, we compared the restriction pattern of mutants carrying deletions in the niaD gene (Tomsett and Cove, 1979) to that of the wt using as probes either the whole of pAN301 or electroeluted fragments as described above. DNA of the niaD14 strain, which contains a deletion genetically strictly located within the niaD gene, has been compared to that of the wt using the 1.3-, 2.7- and 3.2-kb EcoRI

pAN301 fragments as probes. The hybridization patterns of both strains were identical when the 1.3-kb (Fig. 3, lanes 2 and 3) or the 3.2-kb fragment (data not shown) were used. Using the 2.7-kb fragment, the expected 2.7-kb genomic fragment (lane 5) was not observed, whereas two new bands appeared in niuD14 strain (lane 4). This result indicates that the niuD 14 strain contains a modified genomic structure corresponding not to a simple deletion but more probably to an inversion and/or an insertion. Whatever the modification, this demonstrates that the 2.7-kb fragment contains niaD gene sequences. To determine the relative position of the genes within the cluster, we analysed the restriction pattern of strains carrying each a different deletion (A625 and 4516) which extends from niaD into the niL4 gene. The hybridization patterns show that in these strains, the two 2.7-kb fragments were absent while only one was modified in the strain carrying the niuD 14 deletion (Fig. 3). This indicates that the nii4 gene is located towards the righthand side of the pAN301 insert (Fig. 1). In fact, A625 is one of the shortest deletions in the genetical map of Tomsett and Cove (1979), and this probably implies that the intergenic region and probably the start of the niiA gene is contained within pAN30 1. Unpublished data of Johnstone (1985), who has also cloned the niaD niiA region, later confirmed this conclusion.

152

2.7

Fig, 3. Molecular amdysis of a wt and deletion strains from A. niduluns. In each lane 10 ag of DNA was digested with EcoRI,

Fig. 2. Molecular analysis ofA. nidufans wt and the tAN1 transformant. DNA samples from wt (lane l), transformant tAN1 (lane 2) and PAN 301 (lane 3) were cleaved by EcoRI, separated on a 0.7% agarose gel, transferred to nylon filter (Hybond-N, Amersham) and probed with 3ZP-labelled pAN301 (Amersham, nick-translation kit). Prehybridization and hybridization were performed according to the procedure recommended by the manufacturer. Post-hybridization washes were carried out under the following conditions: 0.2 x SSC, 0.1% SDS at 68°C for 2 h. Sizes are given in kb.

(c) Transformation of nitrate-reductase-deficient

a

Fusarium oxysporum strain with pAN301

Approximately 10’ protoplasts of the nia3 strain were mixed with 10 pg of pAN301, A301 or pFB39 in the presence of PEG4000 and CaCl, and then regenerated in soft agar overlays. No colonies appeared on MM when protoplasts were exposed to pFB39 while fast-growing colonies arose on nitrate medium five to ten days after plating protoplasts exposed to pAN301 or 2301. In addition, few smaller colonies incapable of further growth on subculture,

fractionated, and probed, as described in Fig. 2, with 32P-labelled pAN301 or an EcoRI fragment electroeluted from pAN301. Lanes: I, wt DNA probed with pAN30 1;2 and 3, n&zL)14 and wt DNA probed with a 1.3-kb EcoRI fragment; 4 and 5, n&D 14 and wt DNA probed with a 2.7-kbEcoRI fragment; 6 and 7, A625 and A516 DNA probed with pAN301.

were observed. These transformants termed abortive have been reported for Neurospora (Huiet and Case, 1985) and Aspergilh (Tilbum et al., 1983) and interpreted as resulting from transient expression without stable inte~ation of the transforming DNA. Counting only the fast growing colonies as real transformants the frequency varied between experiments from one to ten transformants per pg DNA. This frequency is low, but in the same range as that obtained for A. niduians with this plasmid. (d) Mitotic stability of transformants

The phenotypic stability of transformants during vegetative growth and conidiation can be judged by culturing the transformants on non-selective medium (CM medium) and thereafter testing the nid phenotype on selective medium (nitrate as nitrogen

153

source). Subcultured through uninucleate conidia, all ten transformants tested retained the nitrateutilizing ability of the original transformant cultures. Some of them showed phenotypical differences with the wt as judged by the pigmentation and the density of aerial mycelium. During the successive subcultures of the transformants, some conidial transfers grew very sparsely on nitrate medium as do nia- mutants, indicating instability. The degree of mitotic instability could be estimated using the Chl-resistance selection in the following way. From a single conidium inoculum, transformants were grown for a week on CM. Then a spore suspension was plated on urea-Chl medium ( lo3 conidia per petri dish). The frequency of those spores lacking the transforming marker can be estimated by the number of fast-growing areas. From this procedure two types of transformants have been characterized by comparing the delay of appearance and the number of ChlR colonies with those of the wt strain. Some transformants (TRl and TR2) yield the same proportion of ChlR mutants as a wt F. oxyspontm (Fig. 4a,b). Moreover this ChlR strains appear with the same delay as those derived from a

Fig. 4. Mitotic stability of transformants. (b) stable transformants

(approx. ten days), which implies that their phenotype results from new mutations in any of the loci involved in nitrate assimilation (including the niaD A. nidulans gene). The spectra of ChlR mutants recovered from these transformants and the wt are similar (Table I). The other transformants, the majority, gave rise to ChlR colonies as soon as 5 days after plating the conidia on urea-Chl medium. Their number varies between 10 and 100 colonies per petri dish (lo3 conidia plated) and are all of the Nia- phenotype (Fig. 4c,d). The delay with which ChlR mutants appear is comparable to that observed in reconstitution experiments (about ten nia - conidia mixed with lo3 wt conidia before plating). The behaviour of these transformants can be explained by the preexistence of 1 to 10% nia- conidia in the initial inoculum. wt

(e) Molecular analysis of the transformants Total cellular DNA of transformants was isolated and Southern transfers of undigested and EcoRIdigested DNA were hybridized with pAN301 as a probe. The wt Fusarium strain FOM150 contained

Three weeks after plating, plates of UC medium were inoculated

TRI and unstable

transformants

(c) TR34 and (d) TRl 1.

with lo3 conidia from (a) wt,

154

no sequences which hybridize to the vector at this stringency. In all undigested transformant DNAs, hybridization occurred only in the high-Mr genomic band (not shown), suggesting that pAN301 integrated into chromosomal DNA and did not replicate autonomously. Ten transformants were analyzed. The varied patterns of hybridization of transformant DNAs after EcoRI and Hind111 digestion are illustrated for live of them in Fig. 5. The TR34 transformant (lane 6) showed the presence of the four pAN301 EcoRI bands associated with additional ones. This can be explained by a tandem integration of several copies of the pAN301 plasmid. The more complex pattern observed for transformants TR7 and TRll (lanes 4 and 5, respectively) suggests internal rearrangements coinciding with the integration event and/or multiple integration sites. Of the ten transformants analyzed, eight are thought to

have more than one copy of pAN30 1. TRl and TR2 transformants (lanes 7,8,9 and 10) gave rise to typical patterns which can be interpreted as an integration of a single copy. In the EcoRI digest (lanes 7 and 8), the 9-kb band expected from a pAN301 structure is not visualized. In the Hind111 digest the 5-kb band also disappeared (lanes 9 and 10). This result is consistent with an integration event within the left part of the insert of pAN301. All the transformants that had integrated more than one copy are unstable on urea-Chl medium, whereas the transformants with only one copy are stable. The analysis of one niu - mutant recovered from a monocopy transformant (niu-TRl Fig. 6, lane 2) and one from a multicopy transformant (niu-TR34, lane 4) revealed that in the first case the transforming plasmid is conserved while in the second case it is completely lost.

1234

1234567891011

Fig. 5. Molecular

analysis

Each DNA was digested probed,

as described

of the F. oxysporum by EcoRI or HindHI,

in Fig. 2, with

Lanes 1 to 8, EcoRI digested strain

respectively.

32P-labelled

DNA from pAN301,

niu3, and transfonnants

TR7, TRll,

Lanes 9 to 11, HindIII-digested

TR2 and pAN301,

respectively.

transformants.

fractionated,

and

pAN301.

wt, recipient

TR34, TRl,

TR2,

DNA from TRl,

Fig. 6. Molecular F. oxysporum digested

with EcoRI,

Fig. 2, with mutant;

analysis

transformants.

of two mutants

fractionated

32P-labelled

isolated

from two

In each lane 10 pg of DNA pAN301.

3, TR34 and 4, nia-TR34

and probed, Lanes: mutant.

as described

1, TRl;

was in

2, niu-TRl

Sizes are given in kb.

155

an alternative

(f) Conclusions

biotics

to those based on resistance

to anti-

C for A. nidulans (Ward

such as oligomycin

in P. chrysogenum (Kolar

system

et al., 1986), phleomycin

F. oxy-

sporum by expression of the A. nidulans nitratereductase gene in this organism. Only one copy of the

et al., 1988) and hygromycin B in Cephalosporium acremonium (Queener et al., 1985), A. nidulans (Punt et al., 1987), Glomerella cingulata (Rodriguez and

A. nidulans gene is enough to complement

Yoder,

(1) We have developed for the imperfect

mutation

a transformation

phytopathogenic

fungus

the nia -

Ustilago maydis (Wang

of F. oxysporum. A relatively low transfor-

mation

frequency

was obtained

mants

per pg DNA).

However,

(up to ten transforthis frequency

Fulvia filva

1987),

is

et al., 1988) and

advantages

in this species and based on hygromycin

tems have to be developed

per pg DNA;

resistance

Kistler and Benny,

(2) For direct selection of cloned genes in this organism, higher transformation frequencies should be achieved. An improvement might he obtained through the development of an homologous system. As reported for A. niger homologous transformation allowed the recovery of transformants at a frequency which is at least one order of magnitude higher than that observed with an heterologous system (Kelly and Hynes, 1985; Goosen et al., 1987). For this reason, the cloning of the nia + gene of F. oxysporum is in progress. (3) The Chl resistance of Nia- strains offered a simple system with which to analyse the instability of the transformed strains. From two different classes of transformants, we obtained Nia - strains. Molecular analysis of two transformants TRl (stable) and TR34 (unstable) and one of the Nia strain from these transformants, revealed that Nia-TRl contains the entire sequence of the transforming gene and, conversely, the Nia8-TR34 strain corresponds to an exact excision of the transforming plasmid. The fact that no part of the transforming gene was detected, indicated that Fusarium sequences are probably involved in the excision event. (4) The transformation system we present here will be useful for the construction of gene transfer systems in genetically poorly characterized organisms. (i) Nitrate-reductase-deficient strains, easily obtained via Chl resistance with or without mutagen treatment, can be used as recipient strains. (ii) They are successfully complemented with an heterologous gene. This has been already applied to other strains of F. oxysporum and other fungi of industrial and agricultural importance (manuscript in preparation). This system appears to be ubiquitous and could offer

et al.,

1987), recently

F. oxysporum (Kistler and Benny, 1988). It should be noticed that this system has a number of economic

similar to that reported in the other system developed (one transformant 1988).

(Oliver

particularly

when

transformation

for different

sys-

organisms

since the selection of transformants and the analysis of the transformant marker can be carried out on minimal medium.

ACKNOWLEDGEMENTS

We thank M.A. PelIalva and A. Glatigny for the A. nidulans gene library in pFB39, F. Buxton for the gift of this vector, I. Johnstone for unpublished data on the positioning of the n&I and niaD genes, T. Langin for helpful comments on the manuscript and C. Gerlinger for excellent technical assistance.

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