WO1995002691A2 - Production et application de mycelium et de sporophores de champignons transgeniques - Google Patents

Production et application de mycelium et de sporophores de champignons transgeniques Download PDF

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WO1995002691A2
WO1995002691A2 PCT/NL1994/000164 NL9400164W WO9502691A2 WO 1995002691 A2 WO1995002691 A2 WO 1995002691A2 NL 9400164 W NL9400164 W NL 9400164W WO 9502691 A2 WO9502691 A2 WO 9502691A2
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transformed
dna
transformation
host
donor dna
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PCT/NL1994/000164
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English (en)
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WO1995002691A3 (fr
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Andreas Mooibroek
Miranda Debora Van De Rhee
Hindrik Jan Huizing
Franciscus Hendricus Rats
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Bromyc B.V.
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Priority to EP94925030A priority Critical patent/EP0708826A1/fr
Priority to AU75091/94A priority patent/AU682057B2/en
Publication of WO1995002691A2 publication Critical patent/WO1995002691A2/fr
Publication of WO1995002691A3 publication Critical patent/WO1995002691A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/02—Preparation of hybrid cells by fusion of two or more cells, e.g. protoplast fusion
    • C12N15/04—Fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the invention involves different methods to modify genetic charac ⁇ teristics of homobasidiomycetes in particular commercial homobasidio ⁇ mycetes such as the common or button mushroom Agaricus bispovus via treatment with donor DNA or fusions using protoplasts and via matings between strains.
  • the methods may be used for the improvement of com- worthal characteristics and for the commercial production of enzymes and metabolites.
  • A. bispovus The main problem for effective breeding strategies is caused by the rather abnormal life-cycle of A. bispovus , which involves the usual simul- taneous segregation of either parental nucleus into one basidiospore. After outgrowth of this basidiospore heterokaryotic mycelium is formed containing nuclei and genetic characteristics that do not differ from those present in the parental mycelium. In addition, only little recombi- national activity is observed during meiosis (Summerbell et al. 1989) • For this reason investigators all over the world have attempted for quite some years to develop a transformation system for commercial mush ⁇ rooms such as A. bispovus for the introduction of novel characteristics.
  • Schizophyllum commune is considered a model organism to study genetics and developmental biology (Raper 1988) .
  • S. commune is in fact one of the first representatives of this class for which a transformation system was developed. This system is based on complementation of a tvpl auxotroph with the homologous TRP1 gene (Munoz-Rivas et al. 1986) .
  • TRP1 homologous TRP1 gene
  • the subject invention is directed at a method for obtaining a selectable stable transformant of a homobasidiomycete capable of expres ⁇ sing integrated donor DNA comprising at least a dominant selectable marker at a detectable level, wherein said host is optionally non-auxo- trophic and can be transformed without cotransformation with said dominant selectable marker and said host is transformed with said donor DNA.
  • the method according to the invention can successfully be used on commercial strains of homobasidiomycetes, such as strains belonging to the genus Agavicus . It has thus become possible to produce transgenic mycelium and fruitbodies by the introduction of foreign and/or homologous DNA sequences and the expression thereof resulting in new genetic and/or phenotypic characteristics in said mushroom mycelium and/or fruitbodies.
  • the transgenic mushroom material may also be applied for the transfer of transformed nuclei to mushroom mycelium of the same strain, another strain of the same species, another species of the same family or another species of another family.
  • the transgenic organism or any part thereof may further be used for the production of foreign and/or homologous proteins, (poly)peptides and/or metabolites.
  • the proteins, (poly)peptides and/or metabolites may be recovered from the organism's tissues and/or from the medium.
  • the method may be used for the production of strains with improved quality aspects, such as reduced levels of browning (e.g. PPO-activity) , for the production of pathogen resistant strains and for the genetic marking of commercially interesting strains of mushrooms to establish proprietary rights. It has now become possible to integrate specific desired heterologous nucleic acid sequences in a homobasidiomycete and maintain said nucleic acid sequence in the transformed mycelium and the fruiting body resulting therefrom, without the need for sustained selective pressure.
  • a dominant selectable marker is meant to be a marker that is selectable in a wild type of the host to be trans ⁇ formed i.e. a host without auxotrophic deficiencies. It is therefore now possible to transform homobasidiomycetes strains that are not auxotrophic with a selectable marker.
  • the method of transformation according to the invention in the embodiments just described can in particular be very successfully carried out on a host obtained by subjecting homobasidiomycete material to a transformation procedure, wherein the host to be transformed exhibits delayed differentiation in comparison either to non-protocloned homobasidiomycete material and/or the wild type strain Ul as obtainable from ATCC, said delayed differentiation being macroscopically visible in the form of amended morphology due to a change in the number and/or height of aerial hyphae, preferably by the absence of aerial hyphae and/or by a diminished hyphae aggregate formation preferably by the absence of hyphae aggregates.
  • a method wherein the host belongs to the strain 'Abade' is an embodiment that provides good results.
  • a strain exhibiting amended morphology of the required type for use in a method of transformation according to the invention can be suitably obtained by subjecting homobasidiomycete material to protocloning followed by selection of resulting homobasidiomycete material exhibiting the desired delayed differentiation.
  • a suitable host can also be obtained by subjecting homobasidiomycete material to a rejuvenation procedure followed by selection of resulting homobasidiomycete material exhibiting the desired delayed differentiation.
  • the transformation procedure commencing with the protoplast formation is carried out as soon as possible after detection of the occurrence of delayed differentiation.
  • An outgrowth phase in this respect comprises selecting a colony, plating out the colony and growing the colony on a 9 cm agar plate until the plate is full and a person skilled in the art will recognise that equivalents to the number and length of outgrowth phases thus defined also fall within the scope of the invention. Extremely good results were achieved when less than five outgrowth phases took place between selection of the desired rejuvenated host homobasidiomycete material and protoplast formation as part of the transformation procedure. It is supposed that the transformability can be lost if the number of outgrowth phases is too high.
  • hydrophobins accumulate in the cell walls of the hyphae that excrete them and contribute to the formation of hydrophobic crosslinked structures within the cell wall.
  • the S. ses thn mutation is analogous to the Stveptomyces bid mutation.
  • disruption of the analogous vodA gene encoding a hydrophobin- like protein resulted in a decreased hydrophobicity of aerial mycelium (Stringer et al., 1991. for review see Chater, 1991).
  • hydrophobin genes are also involved in fruitbody formation in S. ses (Wessels et al., 1991b).
  • the donor DNA is linearized prior to transformation as this leads to a more positive effect on the transformation efficiency.
  • This positive effect of the use of DNA that is linearized prior to transformation on transformation efficiency is in line with observations in other organisms than homo- basidiomycetes such as the yeast Hansenula pol movpha (Faber et al. 1992) and some filamentous fungi (Banks et al. 1992, Liou et al. 1992, Tsai et al. 1992).
  • any commonly acceptable method for transformation of protoplasts can be used in the method according to the invention, such as electropora- tion, use of PEG or particle bombardment.
  • electropora- tion any commonly acceptable method for transformation of protoplasts
  • PEG or particle bombardment A person skilled in the art will be able to determine which method best suits the homobasidiomycete material to be transformed, whether it has been derived by protoplasting or not.
  • electroporation to mediate uptake of donor DNA is a good choice due to the cell type specific controllability of parameters.
  • electroporation eliminates the risk of aggregate formation of protoplasts by PEG and thus prevents potential segregational instability of the donor DNA. This effect may be even more realistic for multinucleate A. bispovus.
  • the efficiency of transformation can be significantly increased by taking measures to ensure a higher yield in the number of protoplasts per unit time prior to the actual transformation step.
  • Methods are known to a person skilled in the art for increasing protoplast yield and regeneration efficiency (Sonnenberg et al 1988) . It is -pointed out here that the known methods for increasing protoplast formation as such are insufficient to obtain transformability of the host homobasidiomycete material. This was illustrated by the non transformability of Ul. As however the chances of finding transformants are increased by such measures the inclusion of such measures in the method according to the invention is preferred.
  • the rejuvenation is an important aspect of the subject method.
  • Rejuvenation as such is a known procedure (Fritsche 1991) and can occur in the form of natural rejuvenation or artificial rejuvenation.
  • Natural rejuvenation occurs in the form of mating homokaryotic spore cultures leading to mating products which are considered to b rejuvenated offspring.
  • furthe homokaryotes can be derived from the rejuvenated offspring fo transformation.
  • the artificial method of rejuvenation comprises formatio of protoplasts, regeneration and selection of homokaryons therefrom.
  • the invention is thus directed at a method fo obtaining a dominant selectable stable transformant of homobasidiomycete capable of expressing stably integrated donor DN comprising at least a dominant selectable marker at a detectable level, said method comprising a) subjecting the mycelium of the host to be transformed to at least:
  • step 4 a selection of a protoclone resulting from step 4 on the basi of exhibiting a delayed differentiation in comparison either t non-protocloned homobasidiomycete material and/or the wild typ strain Ul as obtainable from ATCC, said delayed differentiatio being macroscopically visible in the form of amended morpholog due to a change in the number and/or height of aerial hyphae, preferably by the absence of aerial hyphae and/or by diminished hyphae aggregate formation preferably by the absenc of hyphae aggregates.
  • the host to b transformed can optionally be non-auxotrophic and can also b transformed with said dominant selectable marker withou cotransformation.
  • the material to be used in step b) for transformation has not been subjected to more than 5 successive outgrowth phases in step 6.
  • the use of recently protocloning material for the transformation apparently enhances the success rate of obtaining transformants from a commercial strain, which was previously not transformable.
  • the protoplast formation step can be further improved by growth of fungal mycelium in plant medium for cultivation and regeneration of plant cells e.g. MS medium (Murashige and Skoog 1962) immediately preceding step 7•
  • MS medium Murashige and Skoog 1962
  • This additional measure has been found to be particularly effective for a host belonging to the genus Agavicus. It seems that the application of MS-medium during cultivation in liquid medium causes a less densely packed type of mycelium and perhaps also prevents the deposition of extracellular or cell wall specific metabolites that might interfere with the activity of the lytic enzyme used for protoplasting.
  • MS-medium lacks some essential components (which are present in DT ⁇ O) needed for long term cultivation of Agavicus .
  • the shift from a rich medium to a poorer medium for the induction of competence for genetic transformation is also well known for other transformation systems e.g. Bacillus s bti lis and E. coli .
  • the transformation can be successfully carried out using donor DNA comprising at least a dominant selectable marker.
  • the selectable marker can for example encode resis ⁇ tance to an antibiotic and/or a fungicide.
  • the resistance encoding sequence can suitably encode for resistance against hygromycin B. Such resistance can be provided by the hpt gene.
  • the gene sequence is known for hpt from both E. coli and Stveptomyces .
  • the sequence from E. coli is used in the Examples and is eminently suitable for use in a method according to the invention.
  • the donor DNA can further comprise at least one nucleotide sequence homologous to a part of the DNA of the non-transformed host.
  • the presence of such a homologous sequence can imply the presence of a sequence at which integration is desirable in the host chromosome, i.e. in order for site specific homologous recombination to occur. It can also imply the presence of homologous control sequences in order to ensure optimal processing of the donor DNA by the host.
  • the donor DNA further comprises a promoter and optionally a terminator sequence homologous to the host to be transformed.
  • a promoter and optionally a terminator sequence homologous to the host to be transformed.
  • basidiomycetes beside the already mentioned presence of the dominant selectable marker are made on the vector to be used. It has however been found that a specific new vector can quite successfully be used for transformation purposes of the hpt gene as dominant selectable marker.
  • the vector is obtainable by at least the following essential steps:
  • a vector according to the invention comprises a promoter controlling the dominant selectable marker of the donor DNA, said promotor being derived from the host to be transformed.
  • a vector comprising an A. bispovus promoter is for example preferred.
  • a pAN7-l vector can be modified when e.g. an A. bispovus promoter replaces the A. nidulans promoter.
  • a strong promoter sequence will be used to control expression of the dominant selectable marker in a vector according to the invention.
  • the strength of the promoter can generally be derived from the level of expression of a gene so that any promoter of a gene encoding a product that is present in large amounts will be suitable.
  • An eminently suitable promoter for transformation vectors for A. bispovus is the GPD-2 promoter sequence.
  • a pAN7 ⁇ l modified vector comprising the A. bispovus GPD-2 promoter sequence instead of the A. nidulans promoter sequence controlling expression of the hpt-gene is comprised within the invention.
  • termination sequences that can be recognised both by A. nidulans and A. bispovus are preferably absent in a vector according to the invention.
  • the termination sequences such as present in the TRPC terminator sequence of A. nidulans are preferably removed.
  • a further improvement of a vector according to the invention, in particular a pAN7 ⁇ l modified vector comprises the presence of a mutated E. coli hpt gene as gene encoding the dominant selectable marker. The mutated E.
  • coli hpt gene comprises a CG duplet at position 799 from the 5'-iVcoI-site comprising the ATG start codon instead of the native GC duplet. Consequently the mutant hpt-gene encodes Ile-Val instead of Met- Leu.
  • the mutated sequence driven by proper expression signal sequences resulted in a higher resistance level of E. coli transformants.
  • a vector comprising any other mutation in a hpt gene resulting in a higher resistance level of host transformants is included in the scope of the invention.
  • a suitable sequence for A. bispovus comprises the AbGH3 sequence, a sequence that can be isolated through hybridisation with putative N cvassa tyrosinase sequences obtained by PCR with degenerate primers and published sequences (Lerch 1992) in a manner known to a person skilled in the art. Any number of alternatives will be apparent to a person with knowledge of homologous integration and with access to the known DNA sequences of the host to be transformed.
  • a preferred vector according to the invention for transforming A. bispovus will comprise the homologous strong promoter GPD-2 controlling the dominant selectable marker, the E. coli hpt-gene with the mutated duplet disclosed above and will also comprise a sequence of homologous nucleic acid for A. bispovus such as AbGH3 if homologous integration is desired.
  • the cotransforming DNA may code for any homologous or hetero ⁇ logous polypeptide or protein which may or may not be excreted, thus affecting metabolic and biochemical potential of the transformant when expressed in the proper tissue and at the proper growth stage.
  • Specific homologous or heterologous genes may also be over-expressed by using a strong (e.g. GPD-2) promoter or by insertion of higher copynumbers of the same gene.
  • GPD which is a constitutive enzyme, may constitute about 5% of total cellular protein.
  • the addition of rDNA sequences might also favour the integration of multiple copies.
  • a specific gene may be repressed by different techniques. In the yeast Sacchavomyces cevevisiae and some fungi, like A.
  • Phenomena like antisense RNA inhibition and co-suppression are common in plant genetic engineering, but may not be applicable in heterokaryotic fungal transformants containing only one (co)transformed nucleus, unless both nuclei have been transformed directly or combined by mating or protoplast fusion.
  • Co-transformation with sequences coding for specific antibodies in plants denoted 'plantibodies' Hiatt 1990, however, may find general applicability. It appeared that also parts of antibodies comprising the variant (Fv) chains and expressed in E. coli may have sufficient binding capacities (Pl ⁇ ckthun 1990) , possibly also for application in fungi (fungibodies) .
  • Desirable sequences for example suitable for cotransformation using the transformation procedure according to the invention are the putative A. bispovus tyrosinase genes (isolated at our institute) cloned sense or antisense, the A. bispovus mannitol-dehydrogenase gene or the glucose-6- phosphate dehydrogenase gene (Wood et al. 1991). the A. bispovus methallo- thionein genes (Nishiyama et al. 1990), e.g. the barley ⁇ -thionin gene (Gausing 1987) or resistance to dsRNA viruses through cross-protection using a gene coding for e.g. a coat protein (Harmsen et al.
  • sequences can be introduced into A. bispovus protoplasts by cotransformation according to the invention. It is in fact possible to insert multiple nucleic acid sequences from the cotransforming vectors at the same site in the chromosome. This is probably due to in vivo ligation of nucleic acid from the various vectors after linearization of the vectors has occurred such that compatible sticky ends or blunt ends are created prior to the integration event. Compatible sticky ends can be created simply e.g. by digestion of the vectors with the same restriction enzyme(s). It is also possible to insert sequences in tandem using one vector.
  • transgenic heterokaryons like UlmplO primary transformants
  • matings or protoplast fusions between two compatible strains wherein at least one of the mating strains is a transformant obtainable from such a transformation method according to the invention.
  • a suitable transformant to be used for such a method is an 'Abade* transformant comprising resistance to hygromycin B as transgenic selectable marker and comprising an adenine deficiency as does the non- transformed strain 'Abade' .
  • This transformant can advantageously be mated with another mating strain that is not deficient for adenine and is also sensitive to hygromycin B, resulting in selectability of the product of said mating on both lack of adenine deficiency and resistance to hygromycin B.
  • Such mating can take place by generally -known techniques such as naturally occurring anastomosis or artificial protoplast fusion. It is also possible to produce homokaryotic material from trans ⁇ formed heterokaryotic material obtainable through the transformation method according to the invention. These homokaryons can be used for matings.
  • a method of providing a genetic fingerprint specific for a transformed homobasidiomycete comprising DNA analysis of a transformant or a transgenic fruitbody obtainable through a transformation method according to the invention also falls within the scope of the invention.
  • a genetic fingerprint specific for heterokaryotic material resulting from a method of mating using a transformant obtainable through the transformation method according to the invention as at least one of the mating strains can be determined distinguishing said heterokaryotic material from the homokaryotic transformant used as mating strain by analysing for the presence of more or different genetic material in the heterokaryotic material than in the homokaryotic transformant.
  • the genetic fingerprint can be further completed with an " analysis of the RFLP, RAPD or isozyme band pattern of the resulting heterokaryotic material and comparison thereof to the starting material or any other known strains can be used to ascertain proprietary rights.
  • the invention is also directed at non-auxotrophic transgenic homo ⁇ basidiomycete material derived from a non-auxotrophic homobasidiomycete, said transgenic material comprising stably integrated donor DNA compris ⁇ ing a dominant selectable marker such as a resistance to antibiotic and said transgenic material further being capable of expressing said donor DNA in an amount sufficient to ensure selectability over the correspond ⁇ ing non transgenic material.
  • GENERAL METHODS 1- Source and growth of mvcelia Strains from A. bispovus were purchased from the American Type Culture Collection (ATCC 24663, denoted 'Abade', and ATCC 62462, commercial strain Ul), inoculated on MMP agar medium containing Malt Extract (1%, Oxoid) , Mycological Peptone (0.5%, Oxoid) and agar (1.5%) and propagated at 24°C for 1-2 weeks for 3 generations each. Thus, the availability of inoculation material of identical generations was guaranteed for all individual experiments. Except mycelia needed directly for further processing, stocks were kept at 8°C.
  • MMP plates containing a cellophane sheet were loaded with 5 ⁇ 10 inocula each and grown for ⁇ 10 days at 24°C. Colonies were subsequently scraped off the cellophane and macerated for 20 seconds in a Waring Blender, containing 50 mL of MSG20 (Murashige and Skoog 1962, containing 20 g.L "1 glucose) medium. For 'Abade' 20 ⁇ g.mL "1 adenine was added. The amount of macerated mycelium that corresponded with the material derived from two plates was inoculated in each Fernbach flask containing a final volume of 150 mL. Depending on the strain used growth was allowed while standing for 3 ⁇ 7 days at 24°C. Protoplasts were usually isolated from the mycelium grown in 3 _ 4 Fernbach flasks.
  • the Fernbach cultures were rinsed thoroughly over cheese-cloth with sterile milliQ water and finally with 0.6 M sucrose.
  • the mycelium was then transferred to an Erlenmeyer flask containing 0.6 M sucrose and 10 mg.mL "1 Novozym 234 (Sigma or Interspex Products Inc.) and incubated for 2-3 hours at 24°C.
  • the formation of protoplasts was monitored micro ⁇ scopically with regular intervals. Their number amounted usually 10 8 -10 9 protoplasts per experiment for 'Abade' and 10 6 -10 7 for strains Ul and UlmplO.
  • Protoplasts were purified by sequential filtration through cheese-cloth and 50 mL-syringes containing about 2 g of glass-wool, previously rinsed extensively with 0.6 M sucrose and pelleted for 30 min at 3000 rpm at 8°C using a Heraeus Christ centrifuge accommodating 4-6 100 mL tubes.
  • Pellets were further purified by 2 washes with 0.6 M sucrose and 1 wash with SEH-electroporation buffer containing 0.6 M sucrose, 1 mM EDTA, 1 mM HEPES, pH 7.0, each time by centrifugation in 35 mL Corex tubes for min at 3500 rpm at 4°C using a Beckman RC-5C centrifuge and the HB4 swinging bucket rotor. Protoplasts were finally resuspended in 100 ⁇ L ice-cold SEH-buffer per parallel (usually 3-4) experiment. Alternatively, protoplasts can also be produced by growing colonies on membranes (e.g. Gene Screen, Dupont de Nemours and Co. Inc.
  • membranes e.g. Gene Screen, Dupont de Nemours and Co. Inc.
  • Transforming plasmid DNA was isolated using CsCl density gradient centrifugation or according to the Qiagen maxiprep extraction protocol.
  • Transforming plasmids were pAN7 ⁇ l (Punt et al. 1987) and pHAG3 ⁇ l (a derivative of pAN7 ⁇ l containing a 3 kb random A. bispovus genomic H ⁇ ndlll-fragment (which weakly hybridised to a N. cvassa laccase specific oligonucleotide [Lerch 1982] cloned in the unique Hindlll restriction site of pAN7-l).
  • Plasmid pAN7 ⁇ l was linearized with H ⁇ ndlll, pHAG3-l with Kpnl, which is located in the A. bispovus insert sequence, thus yielding two A. bispovus DNA termini. Restriction enzymes were used according to the suppliers recommendations (Pharmacia) . The DNA was then dialyzed for 30 min on Millipore VM membranes floating in a solution containing 10% glycerol and ImM EDTA or the DNA was phenol/chloroform- purified.
  • a new set of multipurpose fungal transformation vectors has also been constructed, which allow the convenient exchange of EcoRI-ZVcoI promoter fragments, the exchange of NcoI-BamHI structural gene fragments and/or the exchange of B ⁇ mHI-H ⁇ dlll terminator sequences.
  • the construc ⁇ tion of these novel vectors is described in Example 2.
  • the hpt gene constructs described in example 2 have been used for promoter probing and promoter trapping (promoter fishing) experiments in a manner known per se to find promoters that are activated at specific growth stages or under specific conditions.
  • A. nidulans promoter sequences were removed by digestion followed by electrophoresis and isolation of the proper vector-containing band from the gel by the Qiaex extraction protocol.
  • promotor-probing experiments random genomic A. bispovus fragments were ligated into the promoterless vector for transformation of E. coli . From all E. coli transformants obtained the plasmid DNA (now containing a variety of genomic A. bispo-pus fragments) was re-extracted and used for A. bispovus transformation as linear molecules.
  • the constructs were introduced with the hpt gene as primary selectable marker, by co-transformation.
  • the promoter sequences may be recovered by techniques like marker or plasmid rescue, inverse PCR or PCR with one specific anchor-primer annealing to the donor DNA plus a random (RAPD) primer.
  • RAPD random primer
  • genomic DNA of transformants is digested with a restriction enzyme, which does not cut the donor DNA introduced. Several restriction enzymes may be applicable separately.
  • the total genomic DNA is then ligated to make the donor DNA plus the flanking genomic sequences circular, which can then be used to transform appropriate E. coli strains.
  • the E. coli strain may be propagated to isolate the new recombinant plasmid for further analysis, including retransformation of A. bispovus .
  • the PCR- mediated techniques may be more suitable.
  • Protoplasts were incubated for 3 days at 24°C in petridishes containing 10 mL of liquid CMPS medium to regenerate cell walls. Serial dilutions were then plated onto DT ⁇ O agar medium containing 10, 2 or 50 ⁇ g.mL "1 hygromycin B. Similar procedures have been described earlier for the production of homokaryotic breeding material from heterokaryotic parental strains.
  • heterokaryotic constitution of the mycelium is required, which should contain at least two compatible nuclei with opposite mating types.
  • the initial transformant is already heterokaryotic (e.g. from UlmplO)
  • heterokaryosis between homokaryotic strains or even between a homokaryotic and a heterokaryotic strain may be accomplished by naturally occurring anastomosis, or artificially by protoplast fusions (see below).
  • inocula of about 2-5 mm 2 were placed upside down onto non-selective MMP agar medium, about 1-5 mm apart. Inoculated plates were incubated at 24°C for 1-4 weeks. Within the zone where outgrowing colonies touch, compatible interactions may be observed resulting in pigmentation, in an aberrant mycelial morphology and the frequent excretion of intracellular material resulting in the formation of brown droplets in e.g. P. ostveatus (Kay and Vilgalys 1992). Similar phenomena were also observed for matings described here. Inocula from the interaction zones that also included some parental mycelium were transferred to new agar media for further analyses described below in example 5-
  • the parental homokaryotic transformed strain can be distinguished from the new heterokaryon by additional counting of the number of nuclei per cell, by cell sorting of protoplasts or by RFLP-, RAPD-techniques and/or by isozyme analyses.
  • An elegant example is given by using 'Abade' C25-1 as one of the mating partners.
  • the presence of two different AbGH3 ⁇ fragments can be demonstrated, due to the modified size of the AbGH3-ClaI fragment through homologous integration of pHAG3 ⁇ l.
  • Hygromycin B or phleomycin resistant transformants from A. bispovus can be used for the production of intra- or interspecies hybrids by protoplast fusions followed by dominant selection of fusion products.
  • Transformants preferably (made) homokaryotic, derived e.g. from UlmplO and expressing the hpt or ble gene, may be fused to one another and fusion products may be dominantly selected by the simultaneous application of hygromycin B and phleomycin contained in the growth medium. They may also be fused to other organisms, containing another endogenous or donated dominant selection marker.
  • 'Abade' transformants expressing the hpt or ble gene may be used for the dominant selection of fusion products with protoplasts expressing the complementing ADE gene, from any wild-type organism, but preferably from A. bispovus , A. bitovquis , A. avvensis or other Agavicus spp. Fusions of protoplasts have already been accomplished between A. bispovus and A. bitovquis (Patent number US4996390; 91-302364/41), between P. ostveatus and Lentinus edodes (Patent number JP4173034; 92-255378/31) and between a variety of different fungal species (Patent number J02245179.
  • Protoplasts from Agavicus spp were prepared as described above for 'Abade' and A. avvensis or according to Sonnenberg et al. (1988) and finally resuspended in 0.6 M sucrose. Then about equal amounts (100 ⁇ L, containing 10 7 -10 8 protoplasts) of either protoplast type were mixed in a 15 mL pointed centrifuge tube (Corex) . One volume of 40% PEG6000 was added by pipetting carefully onto the centrifuge tube wall. By this way the PEG solution formed the lower layer at the interphase, covered by the protoplast suspension.
  • Protoplasts were then centrifuged for 5 min at 800 rpm in a swinging bucket rotor using a table centrifuge (Beckman) onto the interphase, which allowed the formation of fusions. After standing for 5 min at room temperature 5 mL of 0..6 M sucrose were added and carefully mixed by inversion. Subsequently, the protoplast suspension was washed three times by centrifugation for 5 min at 2500 rpm in a table centrifuge and resuspended in CMPS for 3 days to regenerate cell walls and plated onto DT ⁇ O agar medium containing the proper anti ⁇ biotic. Serial dilutions were plated onto CMP agar medium to determine the efficiency of regeneration.
  • MMP agar plates covered with cellophane were inoculated with 5 inocula each of the desired transformed, mated or fused strain. Plates were incubated at 24°C for 10-15 days. Liquid cultures were prepared in Fernbach flasks containing 150 mL MSG20 (supplemented with 100 ⁇ g.mL "1 Cefotaxim) and the macerated material of 2 plates for each flask. The Fernbach flasks were incubated at 24°C for 3-5 days. Then for spawning 100 grams of wheat-grains were sterilized in the presence of 50 ml of demineralized water by autoclaving for 20 min.
  • the sterilized wheat grains were added to the liquid Fernbach cultures and incubated at 24°C for about 2-3 weeks, dependent on the strain used.
  • the total contents of overgrown wheat-grains plus remaining MSG20 medium were mixed with about 750 grams of pasteurized ready to use compost (CNC, Milsbeek, The Nether- lands) and incubated in a 5 ⁇ 10 L polystyrene box.
  • the box was incubated closed, at 24°C for 2-3. weeks.
  • a layer of casing soil of about 3 cm (CNC, Milsbeek, The Netherlands) was added on top of the compost now overgrown by the mycelium.
  • the box was incubated closed at 24°C for another -8 days and then transferred to 16°C and a relative humidity of 75-90%.
  • the lid was then placed on 4 wooden pins in the corners of the box leaving a ventilation gap of 2-3 cm.
  • the surface of the casing soil was moistened daily. After several days fruitbodies start to emerge.
  • Fruitbodies can also be produced in jars essentially as described but with adapted amounts of mycelial inocula, grain kernels, compost and casing soil. Details are described in the legends to figure 17.
  • Example 1 Protoplasting and regeneration of 'Abade' and A. arvensis
  • a novel convenient set of multipurpose transformation vectors has been constructed based on pUT720 purchased from Cayla, Toulouse, which contains pUC19 vector sequences, the Aspevgi llus nidulans GPDA promoter plus the Tvichodevma veesei cellobiohydrolase I (SSA ) excretion signal sequences, contained in a 2.2 kb EcoRI-iVcoI fragment, the Stveptoallo- teichus hindustanus phleomycin-resistance (ble) gene cloned as a 0.44 kb -VcoI-B ⁇ mHI fragment and the A.
  • SSA Tvichodevma veesei cellobiohydrolase I
  • nidulans O.76 kb B ⁇ HI-Hindlll TRPC-termi ⁇ nator fragment After removal of the A. nidulans GPDA EcoRI-NcoI promoter fragment this vector can be used for the insertion of EcoRI-NcoI promoter fragments from any organism, but preferably from A. bispovus . Before or after exchange of the promoter fragments, the /VcoI-B ⁇ mHI ble gene frag ⁇ ment may be replaced by any structural gene, but preferably by the modi ⁇ fied E.
  • Ll/C-contain-ing construct may be used for transient expression assays, preferably in fungal protoplasts.
  • A. nidulans TRPC terminator sequence may be exchanged by any, but preferably A. bispovus terminator sequence, con ⁇ tained in a B ⁇ HI-Hindlll fragment.
  • the vectors thus constructed may be used for the isolation and in vivo selection of random genomic sequences with promoter or terminator activity from any organism, but preferably from A. bispovus . This technique is called promoter or terminator fishing, respectively.
  • Primer Nco-HPT5 introduces an Ncol-s ⁇ te encompassing the translation initiation codon (ATG) of the hpt gene. Furthermore, introduction of this Ncol-s ⁇ te changes the second codon of the hpt gene in pAN7-l from proline to alanine.
  • AnTRPC3C primes at the 3' end of the TRPC terminator of pAN7- 1. The PCR reaction yielded a 1.7 kb hpt-TRPC terminator fragment, which was isolated and digested with B ⁇ mHI, thereby separating the hpt gene from the TRPC terminator.
  • Plasmid pMHN was digested with EcoRI, followed by partial digestion with Ncol at position 352, and treatment with Mungbean nuclease and CIP (calf-intestine alka ⁇ line phosphatase) to remove protruding ends and 5' phosphate groups.
  • This vector was used to ligate the 0.1 kb PCR fragment, resulting in plasmid pMHMut carrying the hpt gene as a Ncol/Bam ⁇ l fragment in pMTL23- Sequencing of the new hpt gene revealed a change in the nucleotides at position 799 from the ATG start codon (see below) . This change improved the resistance level of recombinant E. coli and the transformation efficiency of A. nigev.
  • the hpt gene from pMHMut was cloned as an ZVcoI/B ⁇ mHI fragment into vectors with different promoters and the A. nidulans TRPC terminator (see
  • Modified luciferase construct In order to introduce an iVcoI-site encompassing the ATG of the LUC gene from P. pyvalis , primer EN-LUC-1 was designed. This primer was used together with the (standard) T7 primer and plasmid pT3T7 ⁇ LUC (Promega) in a PCR reaction which yielded a 1.8 kb LUC fragment. After digestion with Ncol and B ⁇ mHI the LUC gene was ligated into vector pMTL23 digested with Ncol and B ⁇ mHI, which yielded plasmid pLUC-N. The LUC gene was cloned as an Ncol/BamRl fragment into vectors with different promoters and the A.
  • nidulans TRPC terminator see below.
  • the integrity of the mutated hpt- and LUC-genes was assessed by positive expression in E. coli after cloning in appropriate expression vectors.
  • the modified hpt gene also resulted in a higher transformation efficiency in A. nigev.
  • promoter constructs In A. bispovus two GPD genes have been detected which are separated by a 0.29 kb intergenic region. Only the downstream GPD ks2 gene is active in mycelium (Harmsen et al. 1991) * The promoter region of this gene is used in new transformation vectors because of its high level of expression and because it normalizes the generation of mRNA when fused to the hpt gene (see below) . Two different sized promoter fragments were isolated via PCR. A 0.29 kb fragment, representing the intergenic region of the two GPD genes, was isolated using primers AbGPD3 and AbGPD2c.
  • plasmids were digested with iVcoI and B ⁇ mHI to replace the ble gene by the LUC gene which was isolated after Ncol/Bam ⁇ l digestion of plasmid pLUC-N (see 'Modified luciferase construct').
  • the resulting con- structs were named pAbAGLl and pAbUGLl (containing 1.0 kb GPD promoter fragments from 'Abade' and Ul, respectively) and pAbAGL2 and pAbUGL2 (containing 0.29 kb GPD promoter fragments from 'Abade' and Ul, respec ⁇ tively) .
  • the LUC gene was ligated directly into Ncol/BamRl- digested pUT720, creating construct pAnGLl, and into pUT720, in which the EcoRl/Ncol A. nidulans GPD promoter fragment including the signal sequence from the SSA cellobiohydrolase I gene from T. veesei was replaced by the EcoRl/Ncol A. nidulans GPD promoter fragment from pAN ⁇ -1, yielding pAnGL2.
  • constructs were made with the modified hpt gene from plasmid pMHMut.
  • constructs are: pAlH (analogous to pAbAGLl), pUlH (analogous to pAbUGLl) , pA2H (analogous to pAbAGL2) , pU2H (analogous to pAbUGL2) , pAnHl-5 (analogous to pAnGLl) and pAnH2 ⁇ 5 (analogous to pAnGL2) .
  • the methods described here may be applied for the construction of transformation vectors with any, but preferably A. bispovus promoter sequence and/or with any, but preferably A. bispovus transit signal sequence and/or with any homologous or heterologous structural gene or fusions thereof and/or with any, but preferably A. bispovus terminator sequence.
  • Example 3 Transformation of 'Abade' protoplasts through electroporation with linear plasmid DNA.
  • Figure 1 shows hybridization signals from linearized plasmids pAN7-l (6.5kb) and pHAG3 ⁇ l (9>5kb), lanes 1 and 2, respectively, from genomic 'Abade' DNA, lane 3 and from the genomic DNA isolated from colonies all recovered from selective plates (DT ⁇ O plus hygromycin B and adenine) .
  • Strains AlO-1 and A25-1 had not been exposed to any donor DNA, thus representing the well known false positives which were only detected during early transformation attempts.
  • Strains BlO-1 and B25-2 had originally been exposed to native (non-digested) pAN7 ⁇ l. Strain B25-2, lane 7.
  • B10-1 is a true transformant characterized by the hpt-specific hybridizing signal at the position of the non-digested chromosomal DNA, indicating that the donor DNA had been integrated into the 'Abade' genome. Integration was also observed after electroporation with linear pAN7 ⁇ l (C25-I and C25-2, lanes 8 and 9, respectively) and with linear pHAG3-l (DlO-l, D10-2, D10-3 and D25-1, lanes 10, 11, 12 and 13, respectively). No free plasmid migrating at the approximate position of the linear plasmids has been observed at any occasion. These data also support the stable nature of the hygromycin B resistant transformants.
  • Figure 2 shows a Southern blot analysis of the genomic DNAs from four 'Abade' hygromycin B transformants after digestion with different restriction enzymes.
  • each transformant bands were detectable at the same position as the linearized pAN7 ⁇ l or pHAG3 ⁇ l plasmids, indicating that within the transformants tandem repeats had been formed during processing of the donor DNA.
  • bands migrating at other positions are detectable as well (except in lanes 5 and 6, from transfor ⁇ mant C25-2), indicating that double- or multiple integration events had occurred, which is quite common in fungal transformants.
  • bordering fragments from the recipient also contribute to the generation of new bands.
  • Figure 2 also shows that the individual transformants can be distinguished on the basis of their unique banding patterns, which may be considered a strain specific fingerprint.
  • Plasmid pUT720 also contains T. veesei cellobiohydro- lase I (SSA) excretion signal sequence (see example 2).
  • the donor DNA was linearized via Hindlll digestion. After electroporation and regeneration (as described for the hpt gene) , regenerates were plated onto DT ⁇ O plates containing or 7-5 ⁇ g.mL "1 phleomycin (Cayla, Toulouse).
  • Example 4 Transformation of UlmplO, a derivative of commercial strain Ul. Protoplasts from strain UlmplO, which was isolated as described were transformed by electroporation with Hindlll linearized pAN7-l. The total number of protoplasts used for each individual experiment amounted 2.7xl0 6 . A number of 2.3xl0 /
  • the primary transformants had either the Ul-phenotype (50%) or the UlmplO-phenotype (50%) , immediately after isolation from the selective plate and further propagation on MMP.
  • UlmplO and 'Abade' hygromycin B resistant transformants was the consistent occurrence of differentiating sectors in those derived from UlmplO upon transfer to non-selective MMP-medium.
  • inocula from individual sectors were transferred to MMP-medium plus cellophane. Different sectors had a different growth rate. Between 1-4 weeks after inoculation the material was freeze-dried and subjected to Southern blot analysis.
  • Figure 4B shows that with one exception in sector 3 from transformant UlmplO/BblOO-2 all sectors had retained the donor DNA. In the same transformant sector 1 and 2 had slightly different banding patterns.
  • Figure 4C shows that transformant specific fingerprints were generated which allows discrimination between one another but also between 'Abade' transformants (figure 2).
  • Example 5 Production and dominant selection of A.bisporus heterokaryons containing an ade/hy and a wild-type nucleus
  • FIG. 5 shows a representative result of experiments in which inocula from said interaction zones and both parental mycelia were grown on double selective SD-medium (Yeast Nitrogen Base without amino acids, prepared according to the suppliers instructions with glucose as a carbohydrate source, Difco Laboratories, USA) thus lacking adenin and supplemented with hygromycin B (50 ⁇ g.mL "1 ). Only inocula from said interaction zones exhibited growth contrary to either parental strain. When fresh matings were attempted between either parental strain directly onto said double selective agar medium, no growth was observed, indicating that the mating procedure described was essential to yield ADE/hyg-- colonies.
  • SD-medium Yeast Nitrogen Base without amino acids, prepared according to the suppliers instructions with glucose as a carbohydrate source, Difco Laboratories, USA
  • Example 6 Analysis of transformant DNA using PCR.
  • Total DNAs were extracted according to the protocol described earlier. Samples (100 ng) of template DNAs were subjected to PCR analysis with primers indicated in the legend to figure 6. PCR was performed using T ⁇ -polymerase in 30 cycles of 1 min 94°C, 1 min 55°C and 1 min 72°C. PCR- products were electrophoresed in 1.0% agarose in TAE (Tris-HCl, acetate, EDTA)-buffer stained with ethidium-bromide.
  • TAE Tris-HCl, acetate, EDTA
  • Figure 6 shows clear banding patterns of specific PCR-fragments comprising parts of the tandem GPD1 and GPD2 genes of A. bispovus (lanes 2, 3. 4 and 5). indicating that both Abade and Ul have the same, or at least compatible, template DNA sequence of G-PD-genes, which is support for the proper classification of Abade as an A. bispovus " species, despite its aberrant colony morphology. From two putative (pAN7 ⁇ l)-transformants in lanes 7 and 8, PCR-fragments with the same size were generated as in lane 9. which contained plasmid pAN7 ⁇ l as a template, contrary to the non-transformed Abade control in lane 6.
  • Example T Identification of transformed fruitbodies derived from UlmplO.
  • Example 8 Southern blot of transformant DNA derived from 'Abade'.
  • Example 9 Cotransformation with pAN7-l and pUT720.
  • Hindlll-linearized pAN7 ⁇ l and pUT720 plasmid molecules have co- integrated into the genome after ligation in vivo .
  • co-integration is possible by linearizing both plasmids with the same restriction enzyme.
  • Example 10 Production of homokaryotic protoclones from primary heterokaryotic transformants.
  • sucrose resuspended in 5 mL of 0.6 M sucrose plus 1% Low Melting Point agarose (37°C) and plated onto CMPS (compost extract, Mycological Peptone, sucrose)-agar medium. Petri dishes were incubated at 24°C and individual colonies isolated, aided by microscopy, before touching their neighbours. Nineteen colonies were isolated with equal viability. From these colonies new inocula were tested for growth on DT ⁇ O-medium (not shown) or DT ⁇ O-medium containing 50 ⁇ g.mL "1 hygromycin. Non-transformed UlmplO was included as a control (colony A4) .
  • Example 11 PCR-analysis of donor DNA selectable markers into protoplasts.
  • Protoplasts (3 x 10 7 ) were isolated and extensively washed in EB (electroporation buffer) as described in the general methods section. Then 30 ⁇ g of plasmid pAN7 ⁇ l was added, the mixture of protoplasts plus DNA was divided into three portions (10 7 each) and incubated on ice. To the first portion (1) of 10 7 protoplasts plus pAN7 ⁇ l (10 ⁇ g) , plasmid pT3T7-luc (10 ⁇ g) was added directly. Portions 2 and 3 were subjected to electroporation according to the parameters described in the general methods section, incubated at 30°C for 30 min to allow sealing of pores generated by the electroporation procedure, and then transferred back to ice.
  • EB electroroporation buffer
  • plasmid pT3T7 ⁇ luc (10 ⁇ g to each portion of protoplasts) was also added followed by extensive washing with 0.6 M sucrose containing MgCl 2 (20 ⁇ g.mL "1 ) . Then DNAse I was added to portion 2 only (final concentration of 20 ⁇ g.mL "1 ). Portions 2 and 3 were incubated for 30 min at 30°C, then put back on ice and lysed with phenol/chloroform simultaneously with portion 1. Total DNA was then extracted according to the method described and subjected to PCR for 15 or 20 cycles using digoxigenin-11-dUTP in the dNTP nucleotide mixture and the sets of primers indicated in the legend to figure 11.
  • PCR-products were blotted onto a nylon membrane, treated with antidigoxigenine-AB, Fab-fragment, and AMPPD solution, exposed to FUJI medical X-ray film and developed.
  • Portions 1, 2 and 3 correspond to fig. 11, lanes 1, 2 and 3. respectively.
  • Ulmpl0/Bb50-1 fruitbodies were produced in jars essentially as described in the general methods section with adapted amounts of mycelial inocula, grain kernels, compost and casing soil. Details are described in the legends to figure 17- Fruitbodies were harvested at different timepoints. RNA was isolated from freeze-dried material and Northern blotting was carried out by standard procedures. The blot was hybridized to a 32 P-labelled hpt-probe.
  • Figure 12 shows a Northern blot analysis of RNA extracted from transgenic fruitbodies. After isolation the RNA showed considerable degradation on an ethidium bromide-stained agarose gel. Therefore, the signals on the Northern blot are weak and vague. However, the hpt-gene clearly appears to be expressed in transgenic fruitbodies.
  • Example 14 Southern blot analysis of total DNA from pHAG3-l derived Abade transformants derived from pHAG3-l and mated with Ul derived protoclones
  • the luminograph of figure 14 shows the aberrant position of the AbGH3/Cl ⁇ I-fragment of transformant C25-1/4/12 (lane 6).
  • Lanes 12 - 15 comprise the DNA from different mating products with this transformed strain. Either nuclear type was clearly present in the combinations with Ulp6 and Ulp ⁇ , although the Ulp ⁇ nucleus seems to be under-represented in C25 ⁇ l/Ulp8. Over-exposure (not shown) also revealed weakly hybridizing material at the 3-5 kb position in lanes 14 and 15.
  • B131-protoclones allows the isolation of transgenic homokaryotic spores.
  • a variety of new morphologies was observed after forced matings between B131-protoclones and Abade transformants on double selective medium and further propagation on MMP agar medium, including the formation of hyphal aggregates, which is also observed during early fruitbody formation. Fruitbody initiation has been started with these mating products.
  • the forced mating procedure described here can yield mating products in which the presence of either nuclear type can be -demonstrated.
  • unequal distribution or segregation of either one of the parental nuclei can be followed in time.
  • Example 1 Southern blot analysis of Abade transformants of pAlH or pUlH. Plasmids pUlH and pAlH were constructed as described and linearized with EeoRI before electroporation of Abade protoplasts. Southern blot analysis was carried out as described in the general methods section.
  • Figure 15 shows a Southern blot analysis of DNA from Abade transformants, obtained after transformation with plasmids pAlH or pUlH containing A. bispovus GPD2-promoter sequences.
  • the new pAlH and pUlH transformation constructs allow the direct selection of hygromycin- resistant transformants from Abade with a somewhat increased efficiency.
  • the GPD2-sequence containing vector does not integrate at the homologous position as efficiently as the vector that comprises the AbGH3 ⁇ sequence (PHAG3-1).
  • Example 16 Southern blot analysis of an Abade cotransformant
  • Transformation of Abade was performed as described in the general methods section with a mixture of plasmids pAN7 ⁇ l and pUT720, both linearized with Hindlll.
  • Transformant D20-1.14/6 was selected by growth on hygromycin-containing medium.
  • DNA was isolated and Southern blot analysis was performed as described in the general methods section.
  • Figure 16 shows a Southern blot analysis of total DNA from Abade co- transformant D20-1.14/6. Digests with Ncol/BamEl and EcoRI/B ⁇ HI of the DNA from D20-1.14/6 (lanes 15 and 16) show the same patterns as the plasmid controls pAN7-l (lanes 2 and 3) and pUT720 (lanes 5 and 6) after hybridization with the hpt-probe (figure 16A) and the JbZe-probe (figure 16B) , respectively. This indicates the presence of both hpt- and ble- sequences in D20-1. This is further demonstrated by the combination of patterns visible in figure 16C (hpt- and e-probes) .
  • Fruitbodies were produced from commercial strain Ul according to the protocol described in the general methods section. This time the experiments were performed in 500 mL jars, now with 25 grams of grain kernels that were inoculated with the amount of mycelium from one cellophane-covered MMP agar plate and, after colonization, with 50 grams of compost or hemp core tissue. Other handlings were in principle the same as described before.
  • Figure 17 shows the effect of substrate composition on the efficiency of small scale fruitbody formation.
  • mushroom fruitbodies can be applied for the bio-degradation of agro-waste materials (such as hemp core tissue) .
  • This system may be improved by the production of A. bispovus transformants that comprise suitable added genes.
  • Mushroom fruitbodies can be produced on alternative substrates.
  • the system can be improved by the addition of compost-borne components that can be released mechanically (or perhaps enzymatically) .
  • Example 18 Northern blot analysis of Abade transformants of pHAG3-l, pAlH and pUlH Transformant C25-1.4/12 was obtained from Kpnl-digested plasmid pHAG3-l. Transformants ElO-1.28/3, E20-1&2.28/3 and F10-1&2.28/3, were obtained from plasmids pAlH and pUlH, respectively, both digested with EcoRI. Total RNA was isolated from freeze-dried mycelium and Northern blot analysis was performed using standard procedures. Total RNA was extracted and electrophoresed as described in the general methods section.
  • Banding patterns from lanes 1 and 2 are representative for all transformants obtained with pAN7 ⁇ l or its direct derivative (pHAG3 ⁇ l) . " On the contrary, Abade transformants containing vectors which comprise the A. bispovus GP2)2-promoter sequence (and the modified hpt-gene) yield the normal transcripts also observed in A. nigev pAN7-l transformants.
  • novel transformation vectors comprising the A. bispovus GPD2- promoter sequence (and the modified hpt-gene), the efficiency of A. bispovus transformation can be increased and the nature of hpt-specific transcripts normalized.
  • Fig.l Southern blot of undigested total DNA from eight 'Abade' transformants and controls. Lane 1, B ⁇ mHI-digested plasmid pAN7-l. Lane 2, Kpnl-digested plasmid pHAG3 ⁇ l. Lanes 3 _ 13. undigested total DNA extracted from untransformed 'Abade' (lane 3) and colonies AlO-1, A25-1, BlO-1, B25-2, C25-1, C25-2, D10- 1, D10-2, D10-3, D25-1 (lanes 4-13, respectively). The blot was hybridized to a DIG-labelled hpt-probe.
  • Fig.2 Southern blot of digested total DNA from four 'Abade' trans ⁇ formants. Lane 1, B ⁇ ZII-digested plasmid pHAG3 ⁇ l. Lane 2, B ⁇ mHI-digested plasmid pHAG3 ⁇ l. Lane 11, B ⁇ mHI-digested plasmid pAN7-l. Lane 12, EcoRI-digested plasmid pAN7-l. Lanes 3"10, digested total DNA from transformants C25-1 (lanes 3&4), C25-2
  • Fig.3 Schematic representation of the reconstruction of the wild-type E. coli hpt gene.
  • Fig.4A Southern blot analysis of A. bispovus UlmplO-derived primary hygromycin B resistant transformants using a DIG-labelled hpt probe. Lanes 3 _ H contain undigested total DNA and lanes 12-20 contain EcoRV-digested DNA from non-transformed control UlmplO (lanes 3, 12) and transformants Bd25-1 (lanes 4, 13), Bb50-1 (lanes 5. 14), Bb50-2 (lanes 6, 15), Bb50 ⁇ 3 (lanes 7. 16) , BblOO-1 (lanes 8, 17), Bbl00-2 (lanes 9, 18) , Bbl00-3 (lanes
  • Lanes 1, 22 pAN7-l digested with B ⁇ mHI
  • Lanes 2, 21 size marker
  • Fig. 4B Southern blot analysis of A. bispovus UlmplO-derived hygromycin B resistant sectoring transformants using a DIG-labelled hpt probe. Total DNAs were digested with EcoRI and B ⁇ mHI. Symbols, a,b,c: inocula taken from sectors that developed at the centre, halfway or at the edge of the colony, respectively; p: plasmid pAN7-l digested with EcoRI and B ⁇ mHI.
  • Fig. 4C Strain-specific fingerprints of B ⁇ mHI+EcoRI double-digested total DNA from UlmplO-derived hygromycin B resistant trans ⁇ formants, probed with a DIG labelled hpt probe.
  • Lane 1 pAN7-l cut with B ⁇ mHI+EcoRI
  • Lane 2 size-marker
  • phage Lambda DNA double-digested with EcoRI+HindIII lanes 3 ⁇ 10: B ⁇ mHI+EcoRI double-digested DNAs from control non-transformed UlmplO and transformants Bd25-1, Bb50-1, Bb50-2, Bb50-3, BblOO-1, Bbl00-2, respectively.
  • Fig.5A,B Dominant selection of a A. bispovus heterokaryon containing an ade/hyg-- (from C25-1) and a Ul-derived nucleus (from protoclone Ulp ⁇ ).
  • (II) adenine (II) adenine.
  • B SD50-medium (50 ⁇ g.mL "1 hygromycin B) , with (I) or without (II) adenine.
  • Strain Ulp ⁇ shows clear growth on SDO- medium, but unexpectedly thin, hardly visible growth in the presence of adenine.
  • BII double selective medium shows continued growth of mated colonies (duplicate inocula in the middle), exclusively, whereas parental strains do not.
  • Fig. 6 PCR-analysis of Abade hygromycin-resistant transformants.
  • Lanes 1, 10 phage Lambda digested with EcoRI and Hindlll; lanes 2 - 5 - template DNA isolated from non-transformed Abade control, transformants C25-1, DlO-1 and non-transformed Ul, exposed to A.bispovus GPD2 promoter-specific primers (AbGPDl, sequence id 7, AbGPD2c, sequence id 9) 5 lanes 6 - 9: template DNA from non- transformed Abade control, transformants C25-1, D10-1 and pAN7- 1, respectively, exposed to hpt-specific primers (PR-HPT1, encompassing ATG at position 1: '-ATG.AAA.AAG.CCT.GAA.CTC.ACC.GCG.ACG.TCT-3' , sequence id 10 and PR-HPT2c [complement], encompassing TAG around position 1050: 3'-GGG.TCG.T
  • Fig. 7 Southern blot analysis of fruitbodies derived from UlmplO primary transformants, hybridized to a DIG-labelled hpt-probe. All DNAs were digested with B ⁇ mHI plus EcoRI. Lane 1: pAN7-l; lane 2: Ul; lanes 3 - ⁇ : transformants Ulmpl0/Bb50-1 (vav. Miranda) , UlmplO/Bb50-3 (vav. Paula) , UlmplO/BblOO-1 (vav. Nicole) , unstable transformant UlmplO/BblOO-2 (vav. Nonna) , Ulmpl0/Bbl00-3 (vav. Nadine), Ulmpl0/Bbl00-4 (vav. Febeline) , respectively, kb, kilobase pairs.
  • Fig. 9A, B, C Southern blot analysis of total DNA from an Abade-derived hpt/bZe-cotransformant D20-1, hybridized to DIG-labelled probes: A, hpt-probe; B, bZe-probe and C, mixed hpt/fcZe-probe.
  • Lane 1 pAN7 ⁇ l digested with EcoRI plus B ⁇ mHI (2.3 kb band hardly visible); lanes 2 and 3 pUT720 digested with EcoRI and EcoRI plus B ⁇ mHI, respectively; lane 4: phage Lambda (digested with EcoRI plus Hindlll, not hybridizing); lanes 5. 6 and 7 total DNA from D20-1 digested with Ncol , B mHI or Hindlll, respectively. Non-transformed Abade control did not hybridize (not shown), kb: kilobase pairs.
  • Fig. 10 Production of homokaryotic protoclones from primary heterokaryotic transformants. Hygromycin-resistance was tested of Ulmpl0/Bbl00-1 (vav. Nicole)-derived protoclones grown on DT ⁇ O agar medium containing 50 ⁇ g.mL "1 hygromycin. A4: non- transformed UlmplO control, B3: negative protoclone which has lost the hygromycin-resistance (growth without hygromycin was same as others, not shown).
  • Fig. 11 PCR-analysis of donor DNA hpt- and LUC-markers after re- extraction from electroporated Ul protoplasts-.
  • Lane 1 donor DNA mixture containing plasmids pAN7-l (hpt) and pT3T7-luc (L ⁇ 7C) ; lane 2: re-extracted DNA after extensive washing plus DNAse I-treatment of protoplasts; lane 3 re-extracted DNA after extensive washing of protoplasts, only.
  • DNA samples serving as PCR-templates were exposed simultaneously to hpt- specific primers PR-HPT1 (encompassing ATG at position 1: sequence id 10) and PR-HPT2c (complement, encompassing TAG around position 1050: sequence id 11) and LtC-specific primers PR-LUC-1 (encompassing ATG at position 304: sequence id 12) and PR-LUC-2c (complement, encompassing position I896: sequence id 13).
  • Panel A, B 15, 20 PCR-cycles, respectively.
  • Fig. 12 Northern blot analysis of RNA extracted from fruitbodies produced from UlmplO-derived primary transformants.
  • Lane 1 RNA from mycelium of control Abade transformant D10-2;
  • lane 2 RNA from non-transformed Ul-fruitbodies;
  • lane 3 "5 lane 4: RNA from Ulmpl0/Bb50-1 fruitbodies and lane 5 same strain harvested 4 days later.
  • the arrow indicates the position of the major transcript detected in A. nigev (pAN7-l)-transformants, corresponding to about 1450 nucleotides.
  • Lane 1 mixture of pHAG3 ⁇ l digested with B ⁇ ZII or B ⁇ mHI
  • Lane 2 mixture of non-digested and Hindlll-digested phage Lambda DNA (not hybridizing);
  • lanes 3 - 6 Abade non-transformed control DNA digested with CZ ⁇ l, Kpnl, Bg-ZII or EcoRI, respectively
  • lanes 7 - 10 DNA from Abade transformant C10- 1.15/3
  • lanes 11 - 14 DNA from Abade transformant C25- 1.15/3. also digested with CZ ⁇ l, Kpnl, Bglll or EcoRI, respectively
  • kb kilobase pairs.
  • Fig. 14 Southern blot analysis of total DNA from pHAG3-l-derived Abade transformant C25-1.4/12 (showing homologous integration through the AbGH3 ⁇ sequence) , mated to Ul-derived protoclones and hybridized to a DIG-labelled AbGH3-probe. All A. bispovus genomic DNAs from controls and mating products were digested with CZ ⁇ l.
  • Lanes 1 and 2 pHAG3 ⁇ l digested with Bg-ZII or Hindlll, respectively; lane 3 phage Lambda DNA digested with Hindlll plus EcoRI (not hybridizing); lane " 4 and 5 non- transformed Abade and Ul control DNAs; lane 6: DNA from Abade transformant C25-1.4/12; lanes 7 - 11: DNA from Ul-protoclones Ulp6, Ulp ⁇ , Ulpl2, Ulpl5 and Ulpl6, respectively; lanes 12 - 15: mating products between C25-1.4/12 and Ulp6, Ulp ⁇ , Ulpl2 and Ulpl5, respectively, kb: kilobase pairs.
  • Fig. 15 Southern blot analysis of DNA from Abade transformants, obtained after transformation with plasmids pAlH or pUlH containing A. bispovus GPZ>2-promoter sequences from Abade or Ul, respectively and the modified hpt-gene. Genomic DNAs were digested with EcoRV (odd numbered lanes) or with EcoRI plus B ⁇ mHI (even numbered lanes) and hybridized to a DIG-labelled hpt-probe.
  • Lanes 1 and 2 non-transformed Abade control; lanes 3 and 4: transformant E10-1.26/3; lanes 5 and 6: transformant E20-1.26/3; lanes 7 and ⁇ : transformant E20-2.26/3; lanes 9 and
  • Fig. l ⁇ Northern blot analysis of total RNA from Abade hygromycin- resistant strains, transformed with pHAG3 ⁇ l (A. nidulans GPD- promoter and non-modified hpt-gene) , pAlH or pUlH (containing A. bispovus Abade or Ul GPZ ⁇ 2-promoter-sequences plus the modified hpt-gene described above) , hybridized to a 3 P-dCTP- labelled hpt-probe.
  • pHAG3 ⁇ l A. nidulans GPD- promoter and non-modified hpt-gene
  • pAlH or pUlH containing A. bispovus Abade or Ul GPZ ⁇ 2-promoter-sequences plus the modified hpt-gene described above
  • Lane 1 Abade; lanes 2 and 3: pHAG3 ⁇ l transformant C25-1.4/12, RNA from lane 2 and 3 isolated after 10 generations and after 4 generations, respectively; lane 4 - ⁇ : pAlH- transformants E10-1.26/3, E20-1.2 ⁇ /3, E20-2.26/3 and pUlH-transformants F10-1.26/3, F10-2.2 ⁇ /3, respectively. Arrows indicate the positions of major transcripts.
  • Approximate sizes a, b, c and d, 1700 , 1450, 900, 400 nucleotides, respectively, as deduced from a co-electrophoresed Gibco BRL 0.17 - 1.77 kb size marker.

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Abstract

L'invention concerne différents procédés de modification de caractéristiques génétiques d'homobasidiomycètes, en particulier, des homobasidiomycètes commercialisables, tel que le champignon commun ou le jeune champignon de couche de l'espèce Agaricus bisporus, au moyen d'un traitement par ADN donneur ou de fusions utilisant des protoplastes, ainsi qu'au moyen de conjugaisons entre des souches. On peut utiliser les procédés pour l'amélioration des caractéristiques commerciales et pour la production commerciale des enzymes et des métabolites. L'invention concerne notamment un procédé d'obtention d'un transformant sélectionnable stable d'un homobasidiomycète pouvant exprimer l'ADN donneur intégré comprenant au moins un marqueur sélectionnable dominant à un niveau détectable, dans lequel ledit hôte est éventuellement non auxotrophe et peut être transformé sans cotransformation par ledit marqueur sélectionnable dominant et est transformé par ledit ADN donneur. L'invention concerne également un procédé de production de sporophores transgéniques stables directement à partir d'hétérocaryons transformés ou indirectement par conjugaison ou fusion protoplastique de transformants obtenus par appariement de transformants obtenus. Elle concerne également un procédé de constitution d'une empreinte génétique de matière à la fois homocaryote et hétécaryote obtenue par transformation, ainsi qu'un procédé de production de matière homocaryote à partir de matière hétérocaryote transformée. Enfin, elle concerne un vecteur spécifique s'utilisant dans la transformation, ainsi qu'un procédé de production dudit vecteur.
PCT/NL1994/000164 1993-07-13 1994-07-13 Production et application de mycelium et de sporophores de champignons transgeniques WO1995002691A2 (fr)

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EP94925030A EP0708826A1 (fr) 1993-07-13 1994-07-13 Production et application de mycelium et de sporophores de champignons transgeniques
AU75091/94A AU682057B2 (en) 1993-07-13 1994-07-13 Production and application of transgenic mushroom mycelium and fruitbodies

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996041882A1 (fr) * 1995-06-12 1996-12-27 Proefstation Voor De Champignoncultuur Hydrophobines de champignons commestibles, genes, sequences nucleotidiques, fragments d'adn codant pour lesdites hydrophobines et leur expression
EP0900027A1 (fr) * 1995-02-09 1999-03-10 Amycel, Inc. Chapeaux de champignons avec ecaillage reduit
US6964866B2 (en) 2000-06-28 2005-11-15 The Penn State Research Foundation Methods and compositions for highly efficient transformation of filamentous fungi
WO2007111500A1 (fr) * 2006-03-27 2007-10-04 Plant Research International B.V. immunisation par champignons transgeniques
US7973215B2 (en) 2008-04-11 2011-07-05 Mycomagic Biotechnology Co., Ltd. Method for the introduction of a heterologous polynucleotide into a mushroom
WO2011130247A3 (fr) * 2010-04-14 2012-02-23 The Penn State Research Foundation Stratégies de manipulation transgénique de champignons filamenteux
US8907165B2 (en) 2009-04-22 2014-12-09 Medicine In Need Corporation Production of provitamin A carotenoids in mushrooms and uses thereof
CN110295161A (zh) * 2019-07-16 2019-10-01 华南农业大学 一种巨大口蘑和双孢蘑菇的原生质体融合方法
CN110714094A (zh) * 2019-11-22 2020-01-21 福建农林大学 一种特异性鉴别银耳菌株的scar分子标记及其鉴定方法和应用

Citations (1)

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Publication number Priority date Publication date Assignee Title
US4996390A (en) * 1989-01-19 1991-02-26 Campbell Soup Company Novel interspecific mushroom strains

Patent Citations (1)

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US4996390A (en) * 1989-01-19 1991-02-26 Campbell Soup Company Novel interspecific mushroom strains

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol.58, no.9, September 1992, AM.SOC.MICROBIOL.,WASHINGTON,DC,US; pages 2971 - 2977 R.S. KHUSH ET AL. 'DNA amplification polymorphisms of the cultivated mushroom Agaricus bisoprus' *
CURRENT GENETICS, vol.22, no.1, July 1992, SPRINGER INTERNATIONAL, NEW YORK, US; pages 53 - 59 M. PENG ET AL. 'Recovery of recombinant plasmids from Pleuterus ostreatus transformant' cited in the application *
CURRENT GENETICS, vol.22, no.6, December 1992, SPRINGER INTERNATIONAL, NEW YORK, US; pages 447 - 454 M.C. HARMSEN ET AL. 'Sequence analysis of the glyceraldehyde-3-phosphate dehydrogenase genes from the basidomycetes Schizophillum commune, Phanerochaete chrysosporium and Agaricus bisporus' cited in the application *
CURRENT MICROBIOLOGY, vol.17, 1988, SPRINGER-VERLAG, NEW YORK, US pages 285 - 291 A.S. SONNENBERG ET AL. 'An efficient protoplast/regeneration system for Agaricus bisporus and Agaricus bitorquis' cited in the application *
DISSERTATION ABSTRACT INTERNATIONAL B, vol.49, no.11, May 1989 page 4693-B M.G. MARK 'The application of recombinant DNA technology to the cultivated mushroom' *
GENETICS AND BREEDINGS OF AGARICUS, 1991, PUDOC, WAGENINGEN, NETHERLANDS; pages 129 - 134 L.J.L.D. VAN GRIENSVEN (EDITOR) *
GENETICS AND BREEDINGS OF AGARICUS, 1991, PUDOC, WAGENINGEN, NETHERLANDS; pages 135 - 139 L.J.L.D. VAN GRIENSVEN (EDITOR) cited in the application *
GENETICS AND BREEDINGS OF AGARICUS, 1991, PUDOC, WAGENINGEN, NETHERLANDS; pages 62 - 72 L.J.L.D. VAN GRIENSVEN (EDITOR) cited in the application *
GENETICS, vol.133, no.2, February 1993, GENETICS SOCIETY OF AMERICA, BALTIMORE, MD, US; pages 225 - 236 R.W. KERRIGAN ET AL. 'Meiotic behavior and linkage relationships in the secondarily homothallic fungus Agaricus bisporus' *
MOLECULAR & GENERAL GENETICS, vol.222, 1990, SPRINGER INTERNATIONAL, NEW YORK, US; pages 41 - 48 H. MOOIBROEK ET AL. 'Introduction of hygromycin B resistance into Schizzophyllum commune: Preferential methylation of donor DNA' cited in the application *
See also references of EP0708826A1 *

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0900027A1 (fr) * 1995-02-09 1999-03-10 Amycel, Inc. Chapeaux de champignons avec ecaillage reduit
EP0900027A4 (fr) * 1995-02-09 2000-03-08 Amycel Inc Chapeaux de champignons avec ecaillage reduit
WO1996041882A1 (fr) * 1995-06-12 1996-12-27 Proefstation Voor De Champignoncultuur Hydrophobines de champignons commestibles, genes, sequences nucleotidiques, fragments d'adn codant pour lesdites hydrophobines et leur expression
US6964866B2 (en) 2000-06-28 2005-11-15 The Penn State Research Foundation Methods and compositions for highly efficient transformation of filamentous fungi
US7700349B2 (en) 2000-06-28 2010-04-20 The Penn State Research Foundation Methods and compositions for highly efficient transformation of filamentous fungi
WO2007111500A1 (fr) * 2006-03-27 2007-10-04 Plant Research International B.V. immunisation par champignons transgeniques
US7973215B2 (en) 2008-04-11 2011-07-05 Mycomagic Biotechnology Co., Ltd. Method for the introduction of a heterologous polynucleotide into a mushroom
US8907165B2 (en) 2009-04-22 2014-12-09 Medicine In Need Corporation Production of provitamin A carotenoids in mushrooms and uses thereof
WO2011130247A3 (fr) * 2010-04-14 2012-02-23 The Penn State Research Foundation Stratégies de manipulation transgénique de champignons filamenteux
US8686218B2 (en) 2010-04-14 2014-04-01 The Penn State Research Foundation Strategies for the transgenic manipulation of filamentous fungi
CN110295161A (zh) * 2019-07-16 2019-10-01 华南农业大学 一种巨大口蘑和双孢蘑菇的原生质体融合方法
CN110295161B (zh) * 2019-07-16 2023-08-29 华南农业大学 一种巨大口蘑和双孢蘑菇的原生质体融合方法
CN110714094A (zh) * 2019-11-22 2020-01-21 福建农林大学 一种特异性鉴别银耳菌株的scar分子标记及其鉴定方法和应用
CN110714094B (zh) * 2019-11-22 2022-06-03 福建农林大学 一种特异性鉴别银耳菌株的scar分子标记及其鉴定方法和应用

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WO1995002691A3 (fr) 1995-03-09

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