METHODS FOR TRANSFORMATION OF FUNGAL SPORES

Abstract

The present invention provides to a method for the introducing polynucleotide molecules into fungal spores comprising exposing a mixture of fungal spores and magnetic nanoparticles carrying the polynucleotide to a magnet and/or a magnetic field. The present invention also provides a method for transformation a fungus comprising the steps of the method steps disclosed herein and allowing the integration of said polynucleotide molecules into the genome of said spores, thereby transforming the spore. The present invention also provides a system for delivery of nucleic acids to fungal spores comprising magnetic nanoparticles loaded with nucleic acids, a kit for transformation of fungal spores with a polynucleotide comprising MNPs loaded with the polynucleotide and fungal spores as well as a composition comprising MNPs loaded with nucleic acid molecules and fungal spores.

Claims

1. A method for introducing polynucleotide molecules into fungal spores comprising exposing a mixture of fungal spores and magnetic nanoparticles carrying the polynucleotide to a magnetic force.

2. The method claim 1 comprising the steps of: a. loading the polynucleotide molecules to be transferred into the spore on magnetic nanoparticles (MNP), and b. adding said polynucleotide molecules loaded on said magnetic nanoparticles (DNA-MNP) to said spores, and c. exposing the mixture to a magnetic force, d. incubating the mixture in presence of said magnet to allow introduction of the polynucleotide molecules into said spores, thereby introducing the polynucleotide molecules into said spores.

3. The method of claim 2, wherein the number of spores per millilitre in the incubation step is 10.sup.5 or higher.

4. The method of claim 1, wherein the polynucleotide loaded to the MNPs is DNA or RNA or a nucleotide analogon.

5. The method of claim 1, wherein the spores are transformed as non-germinated or germinated spores.

6. The method of claim 1, wherein the spore germination time for the spore before transformation is between 0 and 2 h.

7. A method for production of a transformed fungus comprising the steps of the method of claim 1 and further comprising selecting for fungal spores and/or fungal cells that have said polynucleotide present.

8. The method of claim 7, wherein the polynucleotide or a fragment thereof is stably integrated in to the genome of the spore DNA.

9. The method of claim 1, further comprising growing a fungus from the fungal spore.

10. A system for delivery of nucleic acids to fungal spores comprising magnetic nanoparticles loaded with nucleic acids.

11. A kit for transformation of fungal spores with polynucleotides comprising MNPs loaded with polynucleotides and means for the magnetofection of spores.

12. A composition comprising MNPs loaded with nucleic acid molecules, fungal spores and a buffer.

13. The composition of claim 12, wherein the spore density in the composition is more than 10.sup.5/ml for 100 ng to 500 ng polynucleotide.

14. The composition of claim 12, wherein the number of spores per millilitre in the composition is 10.sup.5 to 10.sup.7 for the transfection of an amount of DNA between 200 ng and 300 ng

15. The method of claim 1 wherein the polynucleotide is double strand or single strand ribonucleotide.

16. The method of claim 1, wherein the polynucleotide loaded to the MNPs is plasmid DNA or linear DNA.

17. The method of claim 1, wherein magnetic force is a magnet and/or a magnetic field.

Description

FIGURES

[0106] FIG. 1: Vector pSJ+GFP(HPT)-MF

[0107] FIG. 2: Vector pSJ(basic)-MF

[0108] FIG. 3: Table 1: Overview results of magnetofection of fungal spores

EXAMPLES

Chemicals and Common Methods

[0109] Unless indicated otherwise, cloning procedures carried out for the purposes of the present invention including restriction digest, agarose gel electrophoresis, purification of nucleic acids, ligation of nucleic acids, transformation, selection and cultivation of bacterial cells are performed as described (Sambrook J, Fritsch E F and Maniatis T (1989)). Sequence analyses of recombinant DNA are performed with a laser fluorescence DNA sequencer (Applied Biosystems, Foster City, Calif., USA) using the Sanger technology (Sanger et al., 1977). Unless described otherwise, chemicals and reagents are obtained from Sigma Aldrich (Sigma Aldrich, St. Louis, USA), from Promega (Madison, Wis., USA), Duchefa (Haarlem, The Netherlands) or Invitrogen (Carlsbad, Calif., USA). Restriction endonucleases are from New England Biolabs (Ipswich, Mass., USA) or Roche Diagnostics GmbH (Penzberg, Germany). Oligonucleotides are synthesized by Eurofins MWG Operon (Ebersberg, Germany).

Example 1. Production of Nano Fe3O4/PEI Particles

[0110] Nano Fe3O4/PEI particles are made as described in patent application CN103233042.

Example 2. Nano Fe3O4/PEI Gene Vector-Mediated Gene Transfer to Phakopsora pachyrhizi Spores

[0111] A vector, containing a fungal Uf-PMA1 promoter and -terminator (Djulic et al. Fungal Biology 115, 633-642 (2011)) driving both the SucDH1 (H254Y) gene associated with fungicide resistance and the DsRed reporter gene can be linearized by restriction enzyme digestion and mixed with magnetic nanoparticles, coated with PEI. Fungal spores from Phakospora pachyrhizi can be collected by gently tapping infected leaves and collecting spores.

[0112] Magnetic nanoparticles coated with PEI, can be mixed with plasmid DNA in a 1:2 ratio, i.e. 1 mg magnetic nanoparticles and 2 mg plasmid DNA, and subsequently added to a in 1 mL aqueous solution containing approximately 10.sup.6 spores. Then, this mixture can be put into a 0.3 T magnetic field with regular mixing.

[0113] Applying selection of resistant fungi following the procedure outlined in Djulic et al, ibid. using in planta selection with 50 mg/mL Carboxin treatment shows successful transformation of fungal spores.

Example 3: Binding of DNA to MNPs

[0114] MNP and plasmid DNA were mixed to form complex (MNP/DNA complex) by the attraction between positive (MNP) and negative (DNA) charges. If MNPs are fully loaded by DNA, the MNP/DNA complex has no charge, and it will stay in the wells of a gel at an electrophoresis experiment. When there is more DNA than can be bound by the MNPs, it will run into the gel and DNA bands can be detected. The highest MNP/DNA ratio without DNA band detected determined the optimal ratio for MNP and plasmid DNA. The manufacturer of the Magnetofection technology offers two types of MNPs (PolyMAG and CombiMAG) and both were tested.

[0115] It was shown that the plasmid DNA did not bind to the CombiMAG beads and binds to the PolyMAG beads. Also, linear DNA derived from a PCR binds to PolyMAG beats.

Example 4: Transformation of Pyricularia oryzae and Zymoseptoria tritici

[0116] Two vectors with hygromycin as selection marker and carrying a GFP expression cassette were used. These vectors work well with the two fungi. The plasmids pSJ+GFP(HPT)-MF and pSJ(basic)-MF are shown in FIG. 1 and FIG. 2, respectively. Binding-time of magnetic beads to DNA 0.5 h, 1 h and 2 h at 4° C. were tested in a 96-well-plate: 200 μl/well (volume of DNA+MNPs) and in a 24-well-plate: 500 μl/well (volume of DNA+MNPs). The DNA amount/well was set up to 250 ng/well. Fungal spore densities of Magnaporthe and Zymoseptoria of 10.sup.4/ml, 10.sup.5/ml, 10.sup.6/ml, and 10.sup.7/ml were tested.

[0117] Transfection were successful with binding times for the loading of the DNA to the MNPs of 1 h or 2 h, spore densities of 10.sup.5/ml, 10.sup.6/ml, or 10.sup.7/ml and incubation times with spores of 30 min on the magnetic plate.

Example 5: Results of Delivery of DNA to Spores from Zymoseptoria tritici

[0118] Successful delivery of DNA to spores were achieved with plasmid DNA with fungal spores germinated for 0 h, 0.5 h, 1 h, 2 h and 24 h. Linear DNA can be delivered to spores freshly harvested if the DNA/MNP complex concentration is increased for incubation with the spores. Delivery of DNA to the spores is found for spore densities of 10.sup.5/ml, 10.sup.6/ml, and 10.sup.7/ml, with the transformation rates increasing with the increase of the density of spores. For example, the transformation rates were at 10.sup.5/ml lower than 10.sup.6/ml, and lower than 10.sup.7/ml.

Example 6: Germination Times and Transformation Efficiency

[0119] Delivery of DNA to the spores were observed if magnetofection occurred 0 h, 0.5 h, 1 h, 2 h, or 24 h after start of germination of the fungal spores for spore densities of 10.sup.5/ml, 10.sup.6/ml, and 10.sup.7/ml. The highest delivery of DNA to the spores were observed at higher densities and at 0 h germination time.

[0120] In southern blots the integration of the DNA into the genome could be confirmed, with a transformation rate that is lower than in Agrobacterium mediated transformation).

Example 7: Results of Delivery of DNA to Spores from Pyricularia oryzae

[0121] Successful delivery of DNA to spores were achieved with plasmid DNA with fungal spores germinated for 0 h, 0.5 h, 1 h, 2 h. Linear DNA can be delivered to spores germinated for 0 h, 0.5 h, 1 h, 2 h and 24 h if the DNA/MNP complex concentration is increased for incubation with the spores. Highest delivery rates for linear DNA as well as plasmid DNA were achieved after 2 h of germination, independent from DNA/MNP complex density. Delivery of DNA to the spores is found for spore densities of 10.sup.5/ml, 10.sup.6/ml, and 10.sup.7/ml, with the transformation rates increasing with the increase of the density of spores. For example, the transformation rates were at 10.sup.5/ml lower than 10.sup.6/ml, and lower than 10.sup.7/ml.