Cotransformation in the phytopathogenic fungus Septoria nodorum

145

Mycol. Res. 94 (2): 145-151 (1990) Printed in Great Britain

Cotransformation in the phytopathogenic fungus Septoria nodovum

R. N. C O O L E Y * , F. C. H. F R A N K L I N A N D C. E. C A T E N School of Biological Sciences, University of Birmingham, P.O. Box 363, Birmingham B15 2 1 7 , U.K.

Cotransformation in the phytopathogenic fungus Septoria nodorum. Mycological Research 94 ( 2 ) : 145-151 (1990). Septoria nodorum has been cotransformed with a number of DNA species. Transformants were selected for resistance to hygromycin B conferred by plasmid pAN7-I. When a second vector (pBT6, pAN5-41B or A) was included in the transformation experiments, approximately 50% of the hygromycin-resistant transformants had expressed and/or integrated this unselected DNA. Plasmid pBT6 cotransformed Septoria to MBC-resistance by expression of a benomyl-resistant allele of the Neurospora crassa P-tubulin gene (tub-2). Endogenous P-galactosidase in S. nodorum was found to be repressed by growth on sucrose and this allowed the detection of cotransformants expressing a translational fusion between the Aspergillus nidulans gpd gene and the Escherichia coli lazZ gene, carried by the plasmid pAN5-41B. Unlike the presence of the two unselected vectors mentioned above, the presence of wild-type A-DNA in cotransformation experiments lowered the frequency with which hygromycin-resistant transformants appeared. However, 50% of these were still found to have been cotransformed following Southern hybridization analysis. These high cotransformation frequencies are consistent with previous reports for Septoria and other fungal species and are suggestive of the presence of a small subpopulation of competent protoplasts.

Key words: Cotransformation, Septoria nodorum, MBC resistance, Transformation, P-galactosidase activity.

Much interest is now being focussed on the molecular basis of fungal pathogenicity towards plants (Yoder & Turgeon, 1985). A prerequisite to the molecular analysis of fungal plant pathogens is the establishment of a transformation system. Such a system makes possible the screening of a genornic library for pathogenicity genes by looking for complementation of induced non-pathogenic mutants back to pathogenicity. It also permits the introduction of constructs of reporter genes and expression sequences produced in vitro into the pathogen cells, so that regulation by the host can be examined. To these ends a transformation system has been developed in Septoria nodorum based on the plasmid pAN7-1 which confers resistance to the antibiotic hygromycin B (HmB) (Cooley et al., 1988). Septoria nodorum (teleomorph = Leptosphaeria nodorum) is an important pathogen of wheat and barley causing a leaf spot and glume blotch disease (King, Cook & Melville, 1983). It is often desirable to introduce into a recipient cell a gene located on a vector separate from that canying the selected marker used to detect transformants. Results with the few developed fungal transformation systems suggest that an unselected plasmid may frequently be transformed at the same time as the selected plasmid (Kelly & Hynes, 1985 ; Punt et al.,

To whom all correspondence should be sent.

1987; Wemars et al., 1987). Such cotransformation is potentially very useful since it avoids the need to incorporate the desired gene into the selected vector for transfer into a recipient cell. In a preliminary study it was demonstrated that ~lasmidp3SR2, which carries the Aspergillus nidulans amdS and amdI genes and allows the utilization of acetamide as a nitrogen source (Hynes, Corrick & King, 1983), is cotransformed at a high frequency with pAN7-1 when transformants are selected only for HmB resistance (Cooley et a l , 1988). Here we report further evidence of cotransformation in S. nodomm using two more plasmid vectors and also wildtype A-DNA. The first of these plasmids is pBT6 which cames a benomyl-resistant allele of the P-tubulin gene (tub-2) of Neurospora crassa (Orbach, Porro & Yanofsky, 1986). The second plasmid, pAN5-41B, contains a translational fusion between the expression signals of the A. nidulans gpd gene and the E. coli 'lacZ gene (van Gorcom et a l , 1986). This construct produces the enzyme encoded by lacZ (P-galactosidase) constitutively and so its presence can be detected by the development of blue colonies on media containing the indicator X-gal (Miller, 1972). Experiments with wild-type hDNA were camed out to investigate the possibility of using cotransformation to introduce into mutant recipients a genomic library of Septoria produced in the A-vector EMBL3, In all these cotransformation experiments pAN7-I and resistance to HmB was used for the initial selection of transformants.

Cotransformation in Septoria nodorum

146

MATERIALS A N D METHODS Strains, media and growth conditions

benomyl-resistant allele of the Neurospora crassa P-tubulin gene (tub-2) cloned on a Hind111 fragment into the plasmid pUC12 in which a BglII linker had been inserted at the SmaI site. Plasmid pAN5-41B was constructed by van Gorcom el al. (1986) and contains a fusion of the expression signals and the N-terminal part of the A. nidulans gpd gene with the E. colr 'lac2 gene. The A-DNA used was from a commercial preparation (BRL, U.S.A.). All DNA was dissolved in STC buffer (1.2 M sorbitol, 10 mM CaCl,, 10 m M Tris/HCl, pH 7.5) prior to transformation.

The wheat-adapted wild-type strain of S. nodorum %I71 (Osboum, Scott & Caten, 1986) was used throughout as the recipient. Standard methods for storage, growth and sporulation of strains and preparation of minimal medium (MM) and sporulation medium (CzV8CS) are described elsewhere (Newton & Caten, 1988). The media used for plating and regeneration of protoplasts are described by Cooley et al. (1988). The fungicide carbendazim (methyl benzimidazole-2~1 carbamate = MBC) was added to media from a 1 mg/ml Preparation of protoplasts and transformation protocol stock solution made in methanol. Lactose minimal medium (MMlac) consisted of M M in which the 3 % (w/v) sucrose Methods for the preparation and transformation of protoplasts was replaced by 2 % (w/v) lactose. This medium was distinct of S. nodorum to hygromycin-resistance are described from minimal medium plus lactose (MM lac) which contained elsewhere (Cooley ef al., 1988). Between 2.5 and 5.0 pg of both sugars. Media used to test the presence of P-galactosidase pAN7-I DNA were used to transform 100 p1 aliquots of activity contained 5-bromo-4-chloro-3-indoly~-~-~-ga~actoprotoplasts. pyranoside (X-gal) at a concentration of 40 ~ g / m l . All incubation was at 25 OC in the dark. DNA isolation and molecular analysis

+

Vectors

Plasmid pAN7-1 was constructed by Punt et al. (1987) and contains the E. coli hygromycin B resistance gene (hph) under the control of A. nidulans expression sequences. Plasmid pBT6 is a derivative of pBT3 (Orbach et a]., 1986). It consists of a

Septoria DNA was isolated as described by Cooley et al. (1988). DNA manipulations, including restriction digests and Southern blotting, were as described by Maniatis, Fritsch & Sambrook (1982). 32P-labelled DNA was produced by the hexa-nucleotide random primer labelling system according to Feinberg & Vogelstein (1983, 1984).

Fig. 1. Cotransformation with plasmid pBT6. Wild-type BS171 (top left) and three HmBR transformants on (a) minimal medium, (b) mimimal medium containing 150 pg HmB/ml and (c) 2.5 pg MBC/ml.

147

R. N. Cooley, F. C. H. Franklin and C. E. Caten

RESULTS Inhibition of growth of wild type by MBC

Mycelial blocks of the wild-type strain BS171 were transferred onto MM plates containing a range of concentrations of MBC. Growth was completely inhibited by 2.5 pg MBC/rnl (Fig. 1).

Cotransformation to carbendazim-resistance

Aliquots of BS171 protoplasts were treated with a mixture of plasmids pAN7-1 and pBT6 at a molar ratio of 1:I. Initial selection of transformants was solely for resistance to HmB. HmB-resistant ( H ~ B ~ transformants ) appeared as large mitotically stable colonies at a frequency of 2-10 per pg of pAN7-1 DNA. HmBR 'abortive' transformants appeared at higher frequency as was the case with transformations using pAN7-1 alone (Cooley et al., 1988). Stable transformants were transferred onto separate MM plates containing 150 vg

Fig. 2. Cotransformation with pBT6. The presence of pBT6 DNA was detected by Southern hybridization analysis using 32P-labelled tub-2 as a probe. Lane I, BS171 DNA; lanes 2-4, DNA from three putative pBT6 transformants obtained by selecting directly for MBC resistance (unpubl.); lanes 5-8, DNA from four putative cotransformants. Approximately 0.2 pg of undigested DNA was loaded in each case. Size standards in kb are indicated on the left-hand side. All four putative cotransformants show positive hybridization signals, whilst the wild type shows none under the conditions used.

HmB/ml and 2.5 pg MBC/ml (Fig. I). Strain BS171 was completely inhibited by this concentration of HmB, whilst the HmBR transformants grew strongly. BS171 was also unable to grow on the MBC plates. However, 27 out of 47 H ~ B transformants tested, were able to grow at this concentration of MBC, suggesting that they had also been transformed by pBT6. While the cotransformants were clearly distinguished from BS171 by their ability to grow on media containing 2.5 ug MBC/ml their level of resistance was not high, being completely inhibited at 3-0 pg MBC/ml. This compares with induced MBC-resistant (MBCR)mutants of S. nodorum, which , with will grow at concentrations of MBC up to 20 ~ g / m l and the original N. crassa benomyl-resistant mutant strain (Bml 511(r)a), which was still able to grow at benomyl concentrations almost 100 times those which inhibited growth of the wild type (Borck & Braymer, 1974). The chromosomal DNA of four of the putative pAN7I/pBT6 cotransformants was isolated and probed in Southern hybridizations (Fig. 2) with the 3.1 kb Hind111 fragment of pBT6, containing the N. crassa tub-2 gene. All four gave positive hybridization signals whilst DNA from BS171 gave none under the conditions used (50 % (v/v) formamide at 42O). This confirms the presence of pBT6 DNA in these transformants. Control of endogenous Septoria p-galactosidase

Strain BS171 was inoculated onto media containing 40 ~g Xgal/ml with sucrose, lactose and both sucrose and lactose as carbon sources. After 2-4 d growth on MMlac the mycelium of BS171 clearly developed a blue colour (Fig. 3), indicating Pgalactosidase activity. However, the colonies growing on MM and MM lac dates remained white. Thus. enzvme activity is induced by growth on lactose, whereas growth on sucrose (_+lactose)results in the absence of enzyme activity, suggesting that P-galactosidase is subject to sucrose, or more probably glucose, repression. This situation is comparable to that seen in A. nidulans (van Gorcom et al., 1985). It allows us to look for enzyme activity conferred by constitutive lac2 fusion vectors since, if grown in the presence of sucrose, endogenous P-galactosidase activity will be repressed.

+

Cotransformation with a gpd-lacZ fusion

Strain BS171 was transformed with a mixture of plasmids pAN7-1 and pAN5-41B in a molar ratio of 1:1with the initial selection solely for HmB-resistance. HmBR transformants appeared at a rate of approximately 2 per pg pAN7-1 DNA and 45 were picked off onto (a) M M containing 150 pg HmB/ml and (b) MM containing 40 erg X-gal/ml. After 3 d growth, 21 of the 45 transformants developed a blue colour on the X-gal medium indicating that these had been cotransformed with the gpd-lacZ fusion vector pAN5-41B (Fig. 4). Generally p-galactosidase was expressed uniformly across the whole transformant colony, although in a few cases blue and white sectors could be seen. 'Abortive' HmBR transformants (Cooley et al., 1988) also appeared in these cotransformation experiments and 112 of these were transferred onto MM ~ l a t e scontaining X-gal to see if any were

~

Cotransformation in Septoria nodorum

148

Fig. 3. Control of endogenous P-galactosidase expression. Wild-type 85171 growing on plates containing the indicator X-gal and the following carbon sources: sucrose (top left), sucrose and lactose (top right), lactose (bottom). Fig. 4. Expression of P-galactosidase from the E. coli lacZ gene in cotransfomants. BS171 (bottom) and 16 HmBR colonies from pAN7-l/pAN5-41B cotransfomation experiments tested o n minimal medium containing 150 vg HmB/ml (left) and X-gal with sucrose as a carbon source (right).

R. N. Cooley, F. C. H. Franklin and C. E. Caten Table 1. Summary of transformation and cotransformation frequencies of S. nodorum

Selected marker (vector) hph (pAN7-1)t hph (pAN7-l)t hph (pAN7-1) hph (pAN7-I) hph (pAN7-1)

Cotransformed marker (vector)

Molar ratio (selected: cohansformed)

-

-

amdS(p3SRZ) gpd-lacZ(pAN5-41B) tub-Z(pBT6)

1:5

-0)

Transformation frequency (per ug DNA)

Cotransformation frequency*

1:l 1:I 1:l

No. cotransformants/No. HmBR transformants tested. t from Cooley et al. (1988).

stably transformed for plasmid pAN5-41B. All 'abortives' grew up as white colonies and no blue colonies or sectors expressing p-galactosidase activity were seen. Cotransformation with wild-type A-DNA

BS171 protoplasts were treated with a mixture of pAN7-1 and A-DNA at a 1: 1molar ratio and HmBR transformants selected as before. These appeared at a rate of 0.5-1-0 per pg pAN71 DNA. The DNA from ten HmBR transformants was tested in Southern hybridizations with "P-labelled A-DNA as probe. Five of these gave positive hybridization signals indicating that they had been cotransformed wAh A-DNA (examples in Fig. 5).

by the Neurospora tub-2 allele carried by pBT6 is of interest since it involves both the expression of a heterologous gene and the functioning of a heterologous structural component. This MBCR tub-2 allele also functions in A. nidulans and A. niger (Orbach et al., 1986), Colletotrichum frifolii (Dickrnan,

Fig. 5. Cotransformation with A-DNA. The presence of A-DNA was detected by Southern hybridization analysis using 32P-labelledA as a probe. Lane 1,A DNA digested with HindIII; lane 2, pAN7-1 DNA; lane 3, BS171 DNA; lanes 4-7, DNA From Four independent HmBK transformants. 0.2 ug of Septoria DNA was loaded in each case. Size standards in kb are indicated on the left-hand side. Three of these four HmBR transformants (lanes 4 4 ) show positive hybridization signals with the A probe. 1

DISCUSSION We have demonstrated the cotransformation of Septoria nodorum with three different unselected DNA species: a plasmid carrying a gene determining a structural protein from another fungal species, a translational fusion between the E. coli 'lacZ gene and expression sequences from A. nidulans and intact linear A-DNA. Cotransformation with a Eourth DNA species, the acetamidase gene and associated regulatory sequence from A. nidulans, was reported in a previous paper (Cooley ef al., 1988). In all four cases, transformants were selected initially for their resistance to hygromycin B encoded by the plasmid pAN7-I, and then tested for expression and/or integration of the unselected DNA. Cotransformation frequency did not seem to be influenced by the nature of the unselected DNA, occurring at a frequency of approximately 50% with three different unselected vectors when equimolar ratios of the two cotransformed DNA species were used (Table I). The higher frequency of cotransformation observed with p3SR2 may be attributed to the higher ratio of unselected to selected plasmid (5:1) in these experiments (Cooley ef al., 1988). This interpretation is supported by the results of Wemars ef al. (1987) who showed that, up to a limit, the frequency of cotransformation increased with increasing concentration of the unselected plasmid. None of the plasmids used here carried homologous DNA sequences. It would be interesting to determine whether the introduction of a Septoria sequence onto the unselected plasmid would affect the frequency of cotransformation. The expression in Septoria of the MBC resistance determined

2

3

4

5

6

7

Cotransformation in Sepforia nodorum

150

1988) and Gaeumannomyces graminis (Henson et al., 1988), The ability to express an exogenous P-galactosidase gene and the A. niger tubulin gene has been used as a selectable whilst repressing the endogenous form will allow us to use transformation marker in Penicillium chrysogenum (Rambosek & IacZ as a reporter gene to analyse expression sequences Leach, 1987). Hence it appears that evolutionary conservation isolated from Septoria. A suitable promoter-probe vector, of p-tubulin allows the copolymerization of molecules from (pAN923) based on the E. coli 'lacZ gene has already been different species to form functional microtubules. However, constructed (van Gorcom ef a]., 1986). When wild-type )\-DNA was used together with pAN7-1 the Sepforia pBT6 cotransformants do not show as great a resistance to MBC as MBCK mutants isolated in the same in cotransformation experiments the frequency with which HmBR transformants appeared dropped approximately tenstrain. This is also the case in Neurospora where transformants are less resistant to benomyl than the mutant (Bml 511(r)a) fold. Occasionally in these experiments DNA was seen to from which the transforming P-tubulin allele was cloned (M. J. precipitate on addition of the polyethylene-glycol solution. It Orbach, pers. comm.). This intermediate level of resistance is likely that, on precipitating, the high molecular weight hpresumably results from the expression in transformants of DNA acted as a carrier and co-precipitated pAN7-1 DNA, thereby reducing the amount available for transformation and both the recipient MBC%allele and the transforming MBC" allele leading to a mixed population of tubulin molecules. In so depressing the transformation frequency. However, even genetical terms, the MBC" allele shows partial dominance, though few transformants were obtained, Southern hybridalthough this interpretation is complicated by the variable ization analysis showed that 50% of these had been cotransformed by A-DNA. Similar problems of DNA precopy number of the transformed allele. The gpd-lacZ fusion carried on pAN5-41B is particularly cipitation may be experienced in cotransforming the EMBL3 useful since its presence and expression can be observed library into Septoria and this would severely reduce the without imposing the selection associated with antibiotic efficiency and feasibility of this approach. The results reported here confirm the preliminary conclusion resistance or biosynthetic prototrophy. This revealed that some of the primary HmBR transformant colonies were (Cooley et al., 1988) that cotransformation of unselected mosaics of both transformed (blue sectors) and untransformed plasmids occurs at a high frequency in S. nodorum. High (white sectors) cells, with respect to the unselected plasmid. It cotransformation frequencies have also been observed in has been shown previously by pedigree analysis that primary Aspergillus species (Kelly & Hynes, 1985; Punt el al., 1987; HmBR transformants may arise as heterokaryons (Cooley ef Wemars et al., 1987; Rambosek & Leach, 1987) and in al., 1988) and the white sectors probably contain nuclei which Cochliobolus heterostrophus (0.C. Yoder, pers. comm.). Similar findings have been reported for the introduction of unlinked are not transformed with either vector. In addition to stable HmBR transformants, treatment with unselected genes into higher plants (Schocher ef al., 1986). The pAN7-1 leads to the production of 'abortive' transformants coincidence of low transformation frequencies with high which express a transient resistance to HmB (Cooley et al., cotransformation frequencies suggests that only a small 1988). Presumably these 'abortives' develop from cells which subpopulation of protoplasts are competent to take up and have taken up and express plasmid DNA but fail to integrate integrate DNA, but that these d o so very efficiently. and replicate it. In order to determine whether any cells which Competence may therefore be the limiting factor in fungal were transformed 'abortively' by pAN7-1 were, or could transformation and, in seeking to improve its efficiency, more become, stably transformed for the unselected plasmid, effort should be directed to the nature of the protoplast 'abortive' transformants from pAN5-41B cotransformation preparation. experiments were picked from the hygromycin selection The results of this study show that cotransformation can be plates onto plates containing X-gal. In this way full used to introduce a number of unselectable DNA types into transformation by the unselected plasmid would be detected S. nodorum and it is hoped that this will prove useful in the by the development of complete or sectored blue colonies. investigation of pathogenicity-specific genes. However, all 112 'abortives' tested in this way produced white colonies suggesting either that no pAN5-41B had been We are grateful to Dr Cees van den Hondel for providing taken up or that, like pAN7-I, it had failed to integrate. Given pAN7-1 and pAN5-41B and to Prof. John F. Peberdy for the very large number of copies of the plasmids present and providing pBT6. We thank Miss Claire Anderson for her the high cotransformation frequencies shown by the full assistance in this work, and also the microbial preparation HmBR transformants, failure of uptake of pAN5-418 in all 112 room staff for general support. This research was supported cases seems very unlikely, and hence this result points by grants from the Agricultural and Food Research Council towards failure of integration of the unselected, as well as the and the Gatsby Charitable Foundation. It was carried out selected plasmid. Such cofailure of integration suggests that under MAFF licence numbers PHF 78A/40(56), PHF the protoplasts producing 'abortives' are deficient in an 78A/68(17), PHF 78A/ 18(26) and PHF 78A/ 73(27). essential step in transformation and are not simply an intermediate stage with the final step, integration, occurring only at low frequency. In other experiments X-gal was included in the transformation selection plates and, whilst REFERENCES B ~ both blue and white BORCK, K. & BRAMER, H. D. (1974). The genetic analysis of amongst the stable H ~ transformants colonies were observed, all 'abortive ' H ~ B "colonies appeared resistance to benomyl in Neurospora crassa. ]oumal of General Microbiology 8 5 , 51-56. white.

R. N. Cooley, F. C. H. Franklin and C. E. Caten COOLEY, R. N., SHAW, R. K., FRANKLIN, F. C. H. & CATEN, C. E. (1988). Transformation of the phytopathogenic fungus Septoria nodorum to hygromycin B resistance. Current Genetics 13. 383-389. DICKMAN, M. B. (1988). Whole cell transformation of the Alfalfa fungal pathogen Colletotrichum trifolii. Current Genetics 14. 241-246. FEINBERG, A. P. & VOGELSTEIN, B. (1983). A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Analytical Biochemistry 132,6-13. FEINBERG, A. P. & VOGELSTEIN, B. (1984). Addendum. Analytical Biochemistry 137, 266-267. HENSON, 1. M., BLAKE, N. K. & PILGERAM, A. L. (1988). Transformation of Gaeumannomyces graminis to benomyl resistance. Current Genetics 14, 113-117. HYNES, M. J., CORRICK, C. M. & KING, J. A. (1983). Isolation of genomic clones containing the amdS gene of Aspergillus nidulans and their use in the analysis of structural and regulatory mutations. Molecular and Cellular Biology 3, 1430-1439. KELLY, J. M. & HYNES, M. J. (1985). Transformation of Aspergillw niger by the amdS gene of Aspergillus nidulans. The E M B O Journal 4,475-479. KING, J. E., COOK, R. J. & MELVILLE, S. C. (1983). A review of Septoria diseases of wheat and barley. Annals of Applied Biology 103, 345-373. MANIATIS, F., FRITSCH, E. F. & SAMBROOK, J. (1982). Molecular Cloning. Cold Spring Harbor Laboratory: Cold Spring Harbor, New York. MILLER, J. H. (1972). Experiments in Molecular Genetics. Cold Spring Harbor Laboratory: Cold Spring Harbor, New York. NEWTON, A. C. & CATEN, C. E. (1988). Auxotrophic mutants of Septoria nodorum isolated by direct screening and by selection for resistance to chlorate. Transactions of the British Mycological Society 90, 199-207. (Received for publication 16 February 1989 and in revised form 29 April 1989)

151 ORBACH, M. J., PORRO, E. B. & YANOFSKY, C. (1986). Cloning and characterization of the gene for P-tubulin from a benomylresistant mutant of Neurospora crassa and its use as a dominant selectable marker. Molecular and Cellular Biology 6 , 2452-2461. OSBOURN, A. E., SCOTT, P. R. & CATEN, C. E. (1986). The effects of host passaging on the adaptation of Septoria nodorum to wheat and barley. Plant Pathology 35, 135-145. PUNT, P. J., OLIVER, R. P., DINGEMANSE, M. A., POUWELS, P. H. &VAN DEN HONDEL, C. A. M. J. J. (1987).Transformation of Aspergillus based on the hygromycin B resistance marker from Escherichia coli. Gene 5 6 , 117-124. RAMBOSEK, J. & LEACH, J. (1987). Recombinant DNA in filamentous fungi: Progress and prospects. C R C Critical Reviews in Biotechnology 6 , 357-393. SCHOCHER, R. J., SHILLITO, R. D., SAUL, M. W., PASZKOWSKI, J. & POTRYKUS, I. (1986). Co-transformation of unlinked foreign genes into plants by direct gene transfer. Biotechnology 4, 1093-1096. VAN GORCOM, R. F. M., POUWELS, P. H., GOOSEN, T., VISSER, J., VAN DEN BROEK, H. W. J., HAMER, J. E., TIMBERLAKE, W. E. & VAN DEN HONDEL, C. A. M. J. J. (1985). Expression of an Escherichia coli P-galactosidase fusion gene in Aspergillus nidulans. Gene 40, 99-106. VAN GORCOM, R. F. M., PUNT, P. J., POUWELS, P. H. & VAN DEN HONDEL, C. A. M. J. J (1986). A system for the analysis of expression signals in Aspergillus. Gene 48, 211-217. WERNARS, K., GOOSEN, T., WENNEKES, B. M. J., SWART, K., VAN DEN HONDEL, C. A. M. J. J. & VAN DEN BROEK, H. W. J. (1987). Co-transformation of Aspergillw nidulans: a tool for replacing fungal genes. Molecular and General Genetics 209, 71-77. YODER, 0.C. & TURGEON, B. G. (1985). Molecular bases of fungal pathogenicity to plants. In Gene Manipulafions in Fungi (ed. J .W. Bennett & L. L. Lasure), pp. 417-448. London, U.K.: Academic Press.