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Transformation of Filamentous Fungi by Microprojectile Bombardment

Roland W. Herzog, Molecular Genetics Program, Department of Botany and Microbiology, 101 Life Sciences Building, Auburn University, AL 36849-5407, USA


Introduction
The vast majority of transformation protocols for filamentous fungi are based on permeabilizing cell membranes with polyethylene glycol (PEG) or electroporation. Both methods typically require preparing protoplasts or osmotically sensitive cells prior to transformation (Herzog et al ., 1996; Goldman et al ., 1990; a detailed review of fungal transformation systems is given by Lemke and Peng, 1995). Over the last few years, microprojectile bombardment has become a powerful tool for transformation of intact cells, particularly for the transformation of fungal strains that do not yield sufficient numbers of regenerating protoplasts or species that cannot be grown in culture, such as obligate plant pathogens. Biolistic transformation of Saccharomyces cerevisiae cells was optimized using the PDS-1000/He system (Heiser, 1992). In addition, several filamentous fungi have been transformed by means of microprojectile bombardment (Table 1). In this study, the PDS-1000/He system was used for stable nuclear transformation of an Aspergillus nidulans strain deficient in pyrimidine synthesis by selection for prototrophic transformants. The effect of conidial density, helium pressure, and size of microprojectiles on transformation frequency were investigated. Transformation frequency, mitotic stability of transformants, and Southern blot hybridization patterns were compared between conidia transformed by particle bombardment and protoplasts transformed by chemical (PEG-mediated) transformation. In addition, optimized conditions for microprojectile bombardment were compared with literature data on Biolistic transformation of other fungal species.


Materials and Methods
Strain and plasmid
Aspergillus nidulans strain A773 (pyrG89, wA3, pyroA4) was obtained from the Fungal Genetics Stock Center, Dept. of Microbiology, University of Kansas Medical Center, Kansas City, Kansas, and maintained on solid YG medium [0.5% yeast extract, 2% glucose, 2% agar, pH 6.3] supplemented with 0.12% uracil and 0.12% uridine. Plates were incubated for 68 days at 37 C at which time conidia were harvested in 0.9% sterile saline. The resuspended conidia were sonicated in an ultra sound sonicator for 1 min prior to being used for protoplast formation or for microprojectile bombardment.

Plasmid pRG-1, a 5 kb plasmid containing the Neurospora crassa pyr4 gene cloned into pUC9 was used for transformation experiments. This plasmid has been shown to complement the pyrG89 mutation in A. nidulans (Ballance et al ., 1983; Waring et al ., 1989).

Biolistic transformation
Intact conidia were spread onto 10 ml of solid YG medium (in a 100 x 15 mm petri dish), briefly air-dried under sterile conditions, and used for microprojectile bombardment within 13 hours. Each plate was bombarded twice. Tungsten particles (M5, M10, or M17 particles, Bio-Rad Laboratories, Hercules, California) were prepared and coated with plasmid DNA as described by Daniell (1993). The following were added in order to a 1.5 ml microcentrifuge tube: 25 l tungsten particle suspension (1.5 mg in 50% glycerol), 5 l plasmid DNA (0.5 g/l), 25 l 2.5 M CaCl2, and 5 l 1 M spermidine free base. After each addition the suspension was vigorously vortexed and incubated on ice for 10 min. The DNA-coated tungsten particles were then pelleted in a microfuge for 10 seconds. After removing the supernatant, the tungsten particles were washed with ethanol, resuspended in 30 l ethanol, and 8 l loaded onto each of three macrocarrier discs for bombardment in the PDS-1000/He instrument. Target distance (6 cm), vacuum (28 in Hg) and other parameters were identical to conditions optimized for nuclear transformation of S. cerevisiae cells (Heiser, 1992). The bombardment pressure was varied from 400 to 1,600 psi. Bombarded plates were incubated at 37 C for 48 hours. Control plates were bombarded with tungsten particles which were prepared as described above but not coated with plasmid.

Formation and transformation of protoplasts
PEG-mediated transformation of protoplasts was performed as described by Herzog et al ., 1996.

DNA isolations and Southern blot analysis
DNA was isolated from transformed and non-transformed mycelia by the procedure of Raeder and Broda (1985). Samples of DNA were digested with PstI which cuts pRG-1 at a single site (Waring et al ., 1989). Southern blot hybridizations were carried out as outlined by Sambrook et al . (1989). Plasmid DNA probes were radioactively labeled using the Random Primed DNA Labeling Kit from United States Biochemical (Cleveland, Ohio).

Results and Discussion
In initial experiments using M10 tungsten particles, a helium pressure of 1,200 psi, and other bombardment parameters were used as optimized for nuclear transformation of S. cerevisiae (Heiser, 1992). The spore density was varied from 106108 conidia per plate. When at least 107 conidia were plated, colonies with a prototrophic phenotype were found, indicating that the pyrG89 mutation of the pyrimidine auxotrophic A. nidulans strain was successfully complemented by transformation with pRG-1 plasmid DNA (Figure 1). The first transformants were visible 2436 hours after bombardment. After 48 hours, transformants were counted and transferred onto fresh selective plates for further study.

Conidial density was found to be a crucial parameter in optimizating transformation frequency (Figure 2A). In subsequent experiments, 108 conidia were spread on each plate. Further increasing the spore density, overincubation of plates, or prolonged storage of conidial suspensions prior to the transformation experiment resulted in the occurrence of background colonies. Transformants were found under a wide range of helium pressures (8001,600 psi, Figure 2B) with a reduced transformation frequency at 1,600 psi, possibly due to increased cell death. No transformants were produced by bombardment at 400 psi. No significant difference in transformation frequency was observed when M5 (mean diameter 0.4 m), M10 (0.7m) and M17 (1.1 m) tungsten particles were compared (data not shown). Particles made of material other than tungsten were not tested. The addition of an osmotic stabilizer to the media (1 M sorbitol) did not increase the transformation efficiency. This result is consistent with the results of others who reported that fungal spores or mycelia were transformed in the absence of an osmoticum (see references in Table 1).

Microprojectile bombardment resulted in an overall transformation frequency (6 0.5 transformants/g DNA) that was somewhat lower than the frequency observed by chemical transformation of protoplasts (20 4 transformants/g DNA, Herzog et al ., 1996). However, only about 30% of the transformants obtained from regenerated protoplasts were stable transformants (for further details see Herzog et al ., 1996). On the other hand, among Biolistic transformants, 6070% were mitotically stable. Therefore, the frequency with which stable A. nidulans transformants were obtained was similar for both techniques (4 transformants/g DNA for Biolistic transformation, 6 transformants/g DNA for chemical transformation). The reason for this difference may be because fungal protoplasts are typically enucleate to multinucleate, while conidia of A. nidulans are uninucleate (Bennett & Klich, 1992). Consequently, in a protoplast, only one of several nuclei might be transformed, thus resulting in a lower proportion of stable transformants compared to Biolistic transformation of Aspergillus conidia. A more detailed analysis of this phenomenon is given by Lorito et al . (1993) who made similar observations during their study of T. harzianum and G. virens.

Analysis of genomic DNA by Southern blot hybridization showed that Biolistic transformants were indistinguishable from transformants isolated previously following PEG-mediated transformation (Ballance et al ., 1983). Furthermore, microprojectile bombardment often resulted in the delivery of several copies of the pRG-1 plasmid per conidium. Tandem repeat integration of the whole plasmid with varying copy number (Figure 3, lanes 79) as well as integrations at multiple sites had taken place (Figure 3, lanes 5 and 6). Such integration is thought to result from a single cross-over event (type I or type II as illustrated by Lemke and Peng, 1995) between the plasmid and chromosomal DNA.


Conclusions
Aspergillus nidulans conidia are an ideal target for transformation by microprojectile bombardment due to their uninucleate character and the ease with which conidial density can be controlled and transformants selected and quantified. Since A. nidulans conidial suspensions are a heterogeneous mixture of spores (with a minimum diameter of 3 m and various stages of maturity, cell wall thickness and composition), and tungsten particles are extremely heterogeneous in size, similar transformation frequencies over a relatively wide range of conditions might not be surprising. These findings are consistent with the study by Lorito et al . (1993) who demonstrated that the number of conidia per plate significantly affected transformation efficiency of T. harzianum and G. virens conidia, whereas changes in the helium pressure (800 vs. 1,200 psi) did not affect transformation efficiencies. Bombardment conditions optimized for T. harzianum and G. virens are compared in Table 2. Transformation frequencies for each of these species by particle bombardment or chemical transformation were comparable (Lorito et al ., 1993; Herzog et al ., 1996). Integration of several copies of the entire plasmid at multiple sites and/or as tandem repeats, as evident from the Southern blot, was found to be the predominant fate of introduced DNA in stable transformants for both conidia transformed by particle bombardment and protoplasts transformed by PEG-mediated transformation (Figure 3, lanes 39; Waring et al ., 1989).

These results show that microprojectile bombardment is a very efficient method for transformation of intact Aspergillus conidia. A major advantage is that pre-treatment of cells prior to transformation is unnecessary. The conditions in Table 2 can be used as a starting point for optimizing transformation of other filamentous fungi. Additionally, particle bombardment can be an especially effective approach for transformation of obligately parasitic fungi that require transformation of intact cells.


Acknowledgement
The author deeply appreciates the support of Paul Lemke in the course of this investigation.


References
1. Armaleo, D., Ye, G. N., Klein, T. M., Shark, K. B., Sanford, J. C. and Johnston, S. A., Biolistic nuclear transformation of Saccharomyces cerevisiae and other fungi, Curr. Genet., 17, 97-103 (1990).

2. Bailey, A. M., Mena, G. L. and Herrera-Estrella L., Transformation of four Phytophthora spp by microprojectile bombardment of intact mycelia, Curr. Genet., 23, 42-46 (1993).

3. Ballance, D. J., Buxton F. P. and Turner, G., Transformation of Aspergillus nidulans by the ornithine-5-phosphate decarboxylase gene of Neurospora crassa, Biochem. Biophys. Res. Comm., 112, 284-289 (1983).

4. Bennett, J. W. and Klich, M. A., Aspergillus: biology and industrial applications, Butterworth-Heinemann, Stoneham, M. A., pp. 21-23 (1992).

5. Bhairi, S. M. and Staples, R. C., Transient expression of the β-glucuronidase gene introduced into Uromyces appendiculatus uredospores by particle bombardment, Phytopathology, 82, 986-989 (1992).

6. Bills, S. N., Richter, D. L. and Podila, G. K., Genetic transformation of the ectomycorrhizal fungus Paxillus involutus by particle bombardment, Mycol. Res., 99, 557-561 (1995).

7. Daniell, H., Foreign gene expression in chloroplasts of higher plants mediated by tungsten particle bombardment, Methods Enzymol., 217, 536-556 (1993).

8. Fungaro, M. H. P., Rech, E., Muhlen, G. S., Vainstein, M. H., Pascon, R. C., deQueiroz, M. V., Pizzirani-Kleiner, A. A. and de Azevedo J. L., Transformation of Aspergillus nidulans by microprojectile bombardment on intact conidia, FEMS Microbiol. Lett., 125, 293-298 (1995).

9. Goldman, G. H., Geremia, R., vanMontagu, M., and Herrera-Estrella, A., Transformation of filamentous fungi by high voltage electroporation, Bulletin 1352, Bio-Rad Laboratories, Hercules CA (1990).

10. Heiser, W., Optimization of the Biolistic transformation using the helium-driven PDS-1000/He system, Bulletin 1688, Bio-Rad Laboratories, Hercules CA (1992).

11. Herzog, R. W., Daniell, H., Singh, N. K. and Lemke, P. A., A comparative study on transformation of Aspergillus nidulans by microprojectile bombardment of conidia and a more conventional procedure using protoplasts treated with polyethylene glycol (PEG), Appl. Microbiol. Biotechnol., 45, 333-337 (1996).

12. Hilber, U. W., Bodmer, M., Smith, F. D. and Koller, W., Biolistic transformation of conidia of Botryotinia fuckeliana, Curr. Genet., 25, 124-127 (1994).

13. Lemke, P. A. and Peng, M., Genetic manipulation of fungi by transformation. In: The Mycota II, Genetics and Biotechnology (Kuck U., ed.), Springer, Berlin, pp. 109-139 (1995).

14. Li, A., Altosaar, I., Heath, M. C. and Horgen, P., Transient expression of the beta-glucuronidase gene delivered into urediniospores of Uromyces appendiculatus by particle bombardment, Can. J. Plant Pathol., 15, 1-6 (1993).

15. Lorito, M., Hayes, C. K., Di Pietro, A. and Harman, G. E., Biolistic transformation of Trichoderma harzianum and Gliocladium virens using plasmid and genomic DNA, Curr. Genet., 24, 349-356 (1993).

16. Raeder, U. and Broda, P., Rapid preparation of DNA from filamentous fungi, Lett. Appl. Microbiol., 1, 17-20 (1985).

17. Sambrook, J., Fritsch, E. F. and Maniatis, J., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York (1989).

18. Smith, F. D., Gadoury, D. M, Harpending, P. R., Sanford J. C., Transformation of a powdery mildew, Uncinula necator, by microprojectile bombardment, Phytopathology, 82, 247 (1992).

19. Waring, R. B., May, G. S. and Morris, N. R., Characterization of an inducible expression system in Aspergillus nidulans using alcA and tubulin-coding genes, Gene, 79, 119-130 (1989).


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