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Review

Physical methods for genetic transformation of fungi and yeast

Ana Leonor Rivera a, Denis Magaña-Ortíz b, Miguel Gómez-Lim b, Francisco Fernández a,Achim M. Loske a,∗

a Centro de Física Aplicada y Tecnología Avanzada, Universidad Nacional Autónoma de México, A.P. 1-1010, C.P. 76000, Querétaro, QRO,Mexico

b Unidad de Biotecnología e Ingeniería Genética de Plantas, Centro de Investigación y de Estudios Avanzados del IPN, A.P. 629, C.P. 36500,Irapuato, GTO, Mexico

Received 15 January 2014; accepted 21 January 2014

Communicated by M. Frank-Kamenetskii

Abstract

The production of transgenic fungi is a routine process. Currently, it is possible to insert genes from other fungi, viruses, bacteriaand even animals, albeit with low efficiency, into the genomes of a number of fungal species. Genetic transformation requires thepenetration of the transgene through the fungal cell wall, a process that can be facilitated by biological or physical methods. Novelmethodologies for the efficient introduction of specific genes and stronger promoters are needed to increase production levels.A possible solution to this problem is the recently discovered shock-wave-mediated transformation. The objective of this articleis to review the state of the art of the physical methods used for genetic fungi transformation and to describe some of the basicphysics and molecular biology behind them.© 2014 Elsevier B.V. All rights reserved.

Keywords: Genetic transformation; Fungi; Electroporation; Biolistics; Agitation with glass beads; Shock waves

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22. Electroporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33. Biolistics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74. Agitation with glass beads . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95. Vacuum infiltration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96. Shock-wave-mediated transformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

* Corresponding author.E-mail addresses: analeonor@fata.unam.mx (A.L. Rivera), dmagana@ira.cinvestav.mx (D. Magaña-Ortíz), mgomez@ira.cinvestav.mx

(M. Gómez-Lim), francisco@fata.unam.mx (F. Fernández), loske@fata.unam.mx (A.M. Loske).

1571-0645/$ – see front matter © 2014 Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.plrev.2014.01.007

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1. Introduction

Fungi are useful organisms in our daily lives. For instance, they break down dead organic material, provide antibi-otics [1–3], and can be used to produce various compounds such as insulin, hepatitis B vaccines, the anticoagulanthirudin, and glucagon [4–7]. Fungi are also important model organisms used in experimental and theoretical biologyfor gene regulation [8,9], the characterization of new genes [10], and to study the molecular and biochemical bases ofdiseases [11–13]. Moreover, fungal genes have been employed in the production of transgenic plants [14–20]. Fungiprovide attractive expression platforms [3], and different processes can be humanized to ensure that the productsmimic the human counterparts [21]. They are also employed to produce aminoacids [22,23], and recombinant pro-teins [5,24–32]. They are an important source of a wide range of endogenous compounds and enzymes employed in thetextile [33,34], food [35,36], paper [37], chemical, and other industries [38,39]. Furthermore they are able to producechemicals used for fermentation [40–44], such as food acidulants [44–46], food preservatives [47], flavor enhancers[48,49], renewable plastics [50], solvents [51,52], biological fungicides [53], pesticides [54], biofuels [43,55,56], andanimal feed [57,58]. Commercially relevant enzymes for biodelignification [59], industrial oxidative processes [60],and environmental bioremediation [61,62] are also produced by fungi. On the other hand, fungi also cause plant, ani-mal and human diseases [63–69]. The development of genome sequencing has enabled a better comprehension of themetabolism of fungi [38,70–76].

Completion of the Saccharomyces cerevisiae genome project was accomplished in 1996 making it the first genomedeciphered from a eukaryotic organism [77]. Currently, 22 fungal genomes have been completely sequenced (seeNCBI home page http://www.ncbi.nlm.nih.gov/) and at least a thousand fungal genome-sequencing projects are un-derway (http://www.genomesonline.org).

The fungal genomes available are from

• Saccharomyces cerevisiae [77–79],• Kluyveromyces waltii [80],• Kluyveromyces lactis [81],• Yarrowia lipolytica [81],• Candida albicans [82,83],• Candida dubliniensis [83],• Candida glabrata [81],• Debaryomyces hansenii [81],• Saccharomyces pastorianus Weihenstephan [84],• Magnaporthe grisea [85,86],• Schizosaccharomyces pombe [87],• Neurospora crassa [88],• Phanerochaete chrysosporium [54],• Aspergillus fumigatus [89,90],• Aspergillus niger [91],• Pichia stipitis [92],• Vanderwaltozyma polyspora [93],• Gibberella zeae [94],• Podospora anserina [95],• Trichoderma reesei [96],• Nectria haematococca [97], and• Tuber melanosporum [98].

The first recombinant DNA molecules were produced in the 1970’s with the use of biochemical scissors called re-striction enzymes [99,100]. In this sense, many approaches have been successfully applied, such as varying the growthconditions in a systematic way, over-expressing activator genes, removing epigenetic silencing, introducing heterolo-gous genes, generating strains with novel properties, and improving bioinformatic programs of random mutagenesis.Saccharomyces cerevisiae, a common yeast, was the first eukaryote employed for heterologous gene expression [6,25].

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Transferring DNA into cells is an essential method of molecular cloning and environmental microbiology [14,15,101–103]. Genetic transformation, discovered by F. Griffith in the late 1920’s [104,105] has revolutionized molecularbiology and opened widespread applications to fungal biotechnology. It involves in vitro culture for multiplicationof clones, to select suitable promoters for a specific gene, to identify sequences that confer resistance to antibiotics(selective marker), to produce enzymes that generate a color not observed in the wild type strain (reporter gene),to characterize genes involved in a metabolic route, to produce recombinant DNA fragments [106–109], as wellas DNA transfer into the cell by membrane permeabilization, the integration of the DNA into a chromosome andits maintenance and replication [110]. This is not trivial for numerous important fungi that could produce usefulmetabolites and recombinant proteins. For example, the production of valuable recombinant proteins in fungi has beenpossible due to the isolation and description of promoters belonging to a gene with high levels of expression [111,112]. Additionally, the levels of expression can be controlled by the use of inducible promoters in specific species[113,114]. Finally, the design of novel strains by metabolic engineering usually requires the use of several rounds oftransformation [115]. Even if electroporation and biolistics enhanced the development of transformation systems inmany fungi, efficient transformation techniques are still needed to increase the number of transformants. The recentdiscovery of shock-wave-mediated transformation of fungi could be a solution to this problem [116].

The first trial for fungal genetic transformation was reported in Saccharomyces cerevisiae through the removalof the cell wall by an enzymatic treatment [117,118]; however, other research groups were unable to repeat theseresults [119]. Subsequently, Saccharomyces cerevisiae was transformed using auxotrophic markers and Escherichiacoli shuttle vectors [120,121], but it was until 1981 that the first report on intact yeast cell transformation was pub-lished [122]. Because of its composition, the manipulation of the cell wall for genetic transformation in yeast is easierin comparison with filamentous fungi. The wall of filamentous fungi has complex structures of protein and polysac-charides, such as chitin and chitosan, that hamper its permeabilization [123]. In contrast, the cell wall structures ofyeast is such that high osmotic pressure (induced by cations) and temperature induce the formation of transient pores[124]. For filamentous fungi, more laborious methods for genetic transformation were developed and the first trans-formation was achieved in the filamentous fungi Neurospora crassa [125], and Aspergillus nidulans [126–128]. Sincethen, transformation methods have been developed for fungi that are pathogenic to humans, including several speciesof filamentous fungi, and for many filamentous fungi that are pathogenic to plants. Currently, all major groups offungi, including different zygomycetes, ascomycetes and basidiomycetes, as well as several fungi imperfecti, can betransformed with varying efficiencies. These methods for fungal genetic transformation have been the subject of somereviews [1,3,10,25,74,75,110,124,129–144].

Genetic transformation methods for fungi are divided into two types: biological and physical. Biological methodsare based on Agrobacterium tumefaciens-mediated transformation [14,143–148] and protoplast transformation usingvarious cell-wall degrading enzymes [136,137,149,150]. The production of protoplasts remains the most commonmethod for preparation of cells for transformation. For some species of fungi, high concentrations of lithium [110,151,152] and calcium ions [153–155] are needed. Even if biological transformation methods are popular, protoplastproduction is a challenge for some fungi. Technologies based on physical genetic transformation methods, such aselectroporation, biolistics, agitation with glass beads, vacuum infiltration and shock waves contributed significantlytowards improving the capacities and have enabled the design of genetically manipulated strains of different fungi.Table 1 shows a comparison of the fungal transformation methods currently in use. Most available protocols to trans-form fungi are inefficient, laborious and have low reproducibility. This has motivated the search for more efficient,simpler and safer techniques [116]. The number of fungal species genetically transformed has been increasing in re-cent years; however, there remain many more that potentially could be transformed. The objective of this article is toreview the state of the art of the physical methods used for genetic transformation of fungi and to explain some of thefundamental physics involved in this process.

2. Electroporation

Electroporation is the most common physical technique for fungal transformation. It is based on the applicationof strong electrical fields to cells or tissues [110]. It is popular because it is simple, quick and efficient, even thoughit requires laborious protocols for regeneration after genetic transformation [14,110,130,134,135,138,142,156–162].Additionally, appropriate adjustment of the physical parameters and special fungal cell treatments are required toestablish a specific protocol for each species [110].

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Table 1Comparison of the most popular physical methods for fungal genetic transformation.

Method Procedure Advantages Disadvantages

Electroporation Electric pulses induce membranepermeabilization providing a localdriving force for ionic andmolecular transport through thepores.

Can be applied to any fungi in vivoor frozen. Efficient protocols:simple, fast, and easily optimized.

Depends on theelectrophysiological characteristicsof the fungus. Low transformationefficiency. Medium cost.

Biolistics Small particles covered with genesare accelerated to penetrate the cellwall.

Simple. No pretreatment of the cellwall required. Independent of thephysiological properties of thefungi. Transformation withmultiple transgenes is possible.

DNA can be damaged. Producesmultiple copies of introducedgenes. Complex protocols due toprojectile preparation. Lowtransformation efficiency.Expensive.

Agitation withglass beads

Agitation of the fungal cells withglass beads in the presence ofcarrier and plasmid DNA allowsDNA absorption.

Simple, fast and safe protocol.Inexpensive.

May cause cell disruption. Cellsrequire osmotic support. Lowtransformation efficiency.

Vacuuminfiltration

Vacuum generates a negativeatmospheric pressure that causesthe air spaces between the cells todecrease allowing the infiltration ofbacteria such as Agrobacterium.

Simple and fast. In vitro fungiregeneration. Medium efficiency.

Requires the use of bacteria whichmay have unwanted consequences.

Shock waves Acoustic cavitation changes thepermeability of the membranefacilitating the absorption of DNA.

Simple, good reproducibility, safe.High efficiency. Low operatingcosts.

Relatively high cost of the shockwave source. Expertise in shockwave physics required.

This technique enhances the formation of pores via a polarity alteration on the plasma membrane, caused by analternate or pulsed electrical field (voltage between approximately 0.5 and 2 V) allowing macromolecules to enter andbecome trapped inside the cell [161–170]. Membrane permeabilization is supposed to occur due to the transitory forceof electrodeformation produced by the electrostatic interaction of the dipoles generated on the cells due to the appliedelectrical field [14,157,165,166]. Electroporation causes different phenomena in the biological membranes such aschanges in the distribution of electrostatic charges, the formation of transitory pores and in certain cases rupture ofmembranes [162]. The commonly accepted theory is that exogenous DNA is captured through these transient pores[171]. The length and the strength of the pulsed electric field modify the transmembrane potential created, the extentof membrane permeation, the duration of the permeated state, and the mode and duration of the molecular flow [167].Moreover, physical factors such as global and local (surface) concentrations of DNA, tolerance of cells to membranepermeation and the heterogeneity of the cell population may also affect the electro-transfection efficiency [160,161,172,173].

Other applications of electroporation include the insertion of enzymes, metabolites, lipids and pharmaceutical com-pounds [174]. When using protoplasts of plants, yeast or fungi, the uptake of DNA can be achieved by electrofusion,i.e., two membranes located very close to each other can be fusioned by application of an electric field and the DNApresent in the cell suspension is trapped in the cytoplasm of the joined cells [175].

Under normal conditions, biological membranes have a low dielectric constant due to the structure of the lipid bi-layer and the proteins, composed of molecules with a poor capacity to transfer electricity. Osmotic balance is obtainedbecause the membranes exclude ions and other charged molecules. The barrier against the free diffusion of macro-molecules with charge is called the Born energy barrier. To introduce a charged molecule such as DNA, appreciablequantities of energy are required to transport the charge from a high dielectric medium as the water that surrounds thecells into a low dielectric medium as the cell membrane [162].

The application of high voltages induces the breakthrough of the Born energy barrier allowing the capture of ex-ogenous molecules that normally cannot enter the cell. In electroporation protocols the initial transmembrane voltageis dramatically changed by short electric pulses (microseconds to milliseconds) causing a specific phenomenon knownas reversible electrical breakdown (REB). REB induces the formation of transient pores of different sizes in the mem-

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branes with a non-homogenous distribution. If successive increments of voltage are applied, the cells suffer drasticchanges in membrane permeabilization, finally losing the capacity to maintain their metabolism [162]. A physicalmodel of the field generated in the membrane due to the voltage difference and the electro-deformation force as afunction of time, in response to the electrical pulse generated by a direct electric field was presented in a previouspaper in this journal [14].

Several experiments have demonstrated that exogenous DNA is attached to membrane cells during electroporation;however, the details of the process are not clear and there are several hypotheses about the mechanisms of DNAintroduction through transient pores and the integration of this molecule into the genome of the recipients [176].

The three types of factors that influence the successful introduction of exogenous molecules by electroporationare: cellular, physicochemical and electric factors [158]. Cellular factors include the stage of growth of the cell,the thickness and composition of the membranes, the structure and composition of the cell wall, the density of thecell culture, the cell diameter, and the susceptibility of the cells to transfer charges, among other factors. The morerelevant physicochemical factors in electroporation protocols are the temperature of the cell suspension, the quantitiesof recombinant DNA, the ions added, and the pH of the buffer used. Finally, electrical parameters are critical fora diverse range of cells. For example, an electric field strength (expressed in kV/cm) that leads to the successfultransformation of certain cells is harmful to other types of cells. Additionally, the pulse length and the number ofpulses applied may be critical for the insertion of recombinant DNA. However, the application of several pulses canbe deleterious for the DNA and the cells [177].

Other relevant factors are the geometry of the sample cuvette and the configuration of the electrodes. Currentlymost devices have cuvettes with parallel plates that produce a uniform electric field. To achieve a uniform field, theelectrodes are arranged in parallel. Further optimization of electroporation by adjusting the electric field strength andthe decay time constant of the pulse has been proposed [162]. The variation of the electric field strength has beensuccessful only in a limited range of values. Several studies have shown that successive increments of this parameterare not correlated with successful DNA transfer into cells or with an increment of the frequency of transformation[148]. One hypothesis is that an electric field strength that exceeds the value required for REB causes the irreversibleformation of pores. For this reason, the decay time is usually changed to optimize electroporation protocols. In suchcases, the electric field is applied for longer times or in multiple pulses, however, there are many types of cells thatare recalcitrant to this optimization process [162].

Usually, electroporation in prokaryotes requires an electric field strength from 12.5 to 18.7 kV/cm [178]. Gram-negative bacteria are more susceptible to electroporation than gram-positive bacteria. This may be due to the composi-tion of their membranes. Electroporation in Escherichia coli is highly efficient and widely used in laboratories aroundthe world. Using dominant select markers it is possible to obtain up to 1 × 1010 transformants of Escherichia coli permicrogram of recombinant DNA [178].

Electroporation of intact yeast has been performed by several groups but the frequency of transformation obtained(up to 1 × 105 transformants per microgram of DNA) was low in comparison with the frequencies obtained forbacteria. For this reason, the cell wall of Saccharomyces cerevisiae has been manipulated using chemical treatmentsor employing enzymatic cocktails to produce protoplasts before electroporation [124]. The cell wall of yeasts is morecomplex than the cell wall of bacteria. The former contains chitin, chitosan and other complex glucans [123]. Thiscomposition could offer more resistance to REB and probably generates a higher Born energy barrier against thetransference of exogenous material in comparison with bacteria.

Electroporation in filamentous fungi has been performed employing intact cells such as conidia [130,179]. Incontrast to the frequency of transformation obtained in bacteria and yeasts, electroporation protocols in filamentousfungi using intact cells produce an extremely low number of transformants, up to approximately 100 transformants permicrogram of DNA [148,179,180]. In most of these protocols the electric field tested was between 2 and 9 kV/cm. Theresults varied from one species to another; however, in all cases, successive increments in the electric field strengthcaused a dramatic decrease in the frequency of transformation.

To produce more transformants in fungi, several protocols have been established to modify the fungal cell walland make it more susceptible to electric fields [163,164,181]. Enzymes and chemical treatments reduce the highcontent of complex molecules such as chitin and chitosan present in the cell walls of filamentous fungi [182]. Chitin,chitosan and other complex molecules may cause high resistance to REB and increase the Born energy barrier. In thiscase, biological parameters such as the thickness and structure of the cell wall determine the successful insertion of

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exogenous DNA. In certain species electroporation can only be performed employing protoplasts [163]. Additionally,high rates of mortality have been reported in electroporation tests to transform filamentous fungi [183].

In 1990, Goldman et al. reported the first protocol for electroporation in filamentous fungi [164]. They used intactconidia of Trichoderma harzanium but no transformants were recovered. For this reason they employed an enzymaticcocktail to promote gene transfer into cells. Different electric field strengths, between 1.5 and 3.7 kV/cm, and dif-ferent times of incubation in enzymatic cocktails were tested. An electric field strength of 2.8 kV/cm and 30 minof incubation with enzymatic cocktails were determined as the optimum protocol to enhance the transformation fre-quency. However, Goldman and colleagues established that these conditions may change from batch to batch of theenzymatic cocktail employed. Finally, a transformation frequency of up to 400 transformants per microgram of DNAwas obtained by adding polyethylene glycol to the electroporation buffer [164].

Ozeki and colleagues [179] published another electroporation methodology for genetic transformation of As-pergillus niger. They exposed germinated conidia and protoplasts to electric field strengths from 4 to 8 kV/cm.The optimum electric field strength was 6 kV/cm; successive increments reduced the transformation frequency. Thetransformation frequency was between 1.2 and 100 transformants per microgram of DNA using 1 × 107 germinatedconidia. The use of protoplasts was also tested, and the frequency of transformation was improved two-fold. However,to transform most species of the Aspergillus genus by electroporation, the production of protoplasts is required [148,163,180].

Electroporation has several advantages compared to other methods of genetic transformation. It is a simple, fastand cheap procedure. Sometimes different tissues such as conidia, germinated conidia and mycelium, can be usedand in certain species of fungi like yeasts, intact cells can be used. However, a low frequency of transformation iscommonly obtained in these fungi.

A successful genetic transformation by electroporation in filamentous fungi commonly requires the treatment ofcell walls with enzymatic cocktails. This procedure is a laborious and time consuming task, after which a dramaticdecrease of cell viability is observed. The application of an electric field in combination with enzymatic treatmentsenhances the rate of cellular death. The transformation of intact cells has only been reported in a limited numberof species such as Aspergillus niger and Aspergillus nidulans. Thus, an increase in the frequency of transformationis severely hampered by the composition of the cell wall and its thickness. Furthermore, manipulation of physicalparameters such as the electric field strength is not correlated with high frequencies of transformation. These aspectsmake electroporation less useful for transforming filamentous fungi.

Electroporation protocols have been established for various filamentous fungi including:

• Neurospora crassa [180,184,185],• Neurospora spheroplasts [186],• Trichoderma harzianum [164],• Penicillium urticae [180],• Leptosphaeria maculans [180],• Aspergillus oryzae [180],• Aspergillus awamori [163],• Aspergillus niger [163,179,187],• Aspergillus nidulans [149,188],• Aspergillus fumigatus [138,189–193],• Aspergillus giganteus [148],• Metarhizium anisopliae [194],• Mycosphaerella graminicola [195],• Scedosporium prolificans [196],• Wangiella dermatitidis [190],

yeast species [197,198] including

• Hansenula polymorpha [199,200],• Saccharomyces cerevisiae [187,201–206],• Schizosaccharomyces pombe [207–211],

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• Schwanniomyces occidentalis [212],• Yarrowia lipolytica [213],• Yamadazyma ohmeri [214],• Pichia methanolica [200],• Pichia pastoris [215–219],• Chlamydomonas reinhardtii [220],

several Candida species including

• Candida albicans [142,159,221–223],• Candida maltosa [224],• Candida tropicalis [225],

endophytic fungus such as

• Piriformospora indica [226],

spores of

• Colletotrichum gloeosporioides [181,227],

and basal fungus such as

• Mucor circinelloides [228].

The device used to perform genetic transformation by electroporation is called an electroporator, a container witha slot containing two parallel plane electrodes of aluminum in contact with an aqueous electrolyte, containing intactcells in suspension and the DNA desired to be incorporated into the cell. Details on electroporators [229] can be foundin a previous publication in this journal [14]. Standard protocols for fungal transformation used electrical pulseslasting from microseconds to milliseconds with a voltage between 1 and 2 kV [172]. In some cases [163,164,179],increasing the field strength has been reported to enhance transformation efficiency. Because of this, some commercialdevices for fungi transformation operate at higher voltages [230] than standard electroporators. The transformationefficiencies of fungi range from 103 to 106 transformants/µg of DNA.

3. Biolistics

Biolistics, also known as “particle bombardment” or the “gene gun technique”, is a popular methodology forgenetic transformation because it can be used for many species, subcellular organelles, bacteria, fungi and even animalcells [14,231–234]. It requires a short processing time, involves acceptable costs for the production of transgenic cellsand it is simple to introduce multiple genes or chimeric DNA (DNA from two different species) [14]. It consists of theacceleration of high density carrier particles that are smaller than a fungal cell and are covered with DNA which passthrough the cells, leaving the DNA inside [14,233].

Biolistic protocols were developed at Cornell University in 1987 for genetic transformation of cereals [231,235,236], and algae (Chlamydomonas) [237] and have been applied to yeast since 1988 [238]. The transformation ef-ficiency of biolistics depends on several parameters, including temperature, the amount of cells, their ability toregenerate, the number of DNA-coated particles, as well as the amount of DNA that covers each particle and theparticles’ kinetic energy [239,240]. Transformation efficiencies for fungi range from 104 to 105 transformants/µg ofDNA.

Biolistics is a useful method for the genetic transformation of fungi because it avoids the enzymatic treatment ofthe cell wall and a reduction of the viability of the cells is usually not observed. For these reasons, the transformantscan be easily propagated after DNA insertion. Different types of tissue, such as conidia or mycelium, can also betransformed by biolistics. However, the need for sophisticated devices and the high number of copies of transgenes

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in recombinant strains are the main limitations of genetic transformation using biolistic methods. Additionally, lowfrequencies of transformation are commonly obtained.

The gene gun consists of a high-pressure and a low-pressure chamber with a diaphragm in the middle that acceler-ates microparticles of gold, tungsten or platinum, that are covered with DNA [240–242]. As the microparticles hit thecells, the DNA is released and may be incorporated into the chromosomal DNA by an adsorption mechanism. Moredetails can be found in a previous publication in this journal [14]. To transform fungi, an inert gas such as Heliumwith a pressure in the range of 500 to 2000 psi is used, and the micro-projectiles are accelerated to speeds of 400 m/sor higher in a partial vacuum of about approximately 30 mm Hg.

The biolistic methodology does not require a vector of a specific sequence, it does not depend on the electrophysi-ological properties of the cell, and the transformation parameters can be optimized to each biological target employed[240]. The factor limiting the use of the gene gun is the high probability of introducing multiple copies of genes,which can lead to various undesirable side effects such as gene silencing, low stability or altered genetic expression.Furthermore, the efficiency is extremely low [234].

Biolistics has been used to transform filamentous fungi including the following:

• Neurospora crassa [133],• Trichoderma harzianum [133],• Trichoderma reesei [243],• Aspergillus giganteus [148],• Aspergillus nidulans [244,245],• Aspergillus niger [246],• Metarhizium anisopliae [194],

yeast species including

• Saccharomyces cerevisiae [238],• Cryptococcus neoformans [247–250],

Candida species as

• Candida glabrata [251],

ascomycete fungi like

• Magnaporthe grisea [133],• Erysiphe graminis [252],• Paracoccidioides brasiliensis [253],• Pochonia chlamydosporia [254],

Basidiomycota fungi like

• Pisolithus tinctorius [255],• Uromyces fabae [256],

and spores of

• Cercospora caricis [257],• Mucor circinelloides [258].

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4. Agitation with glass beads

Fungal cells can be genetically transformed by rapid agitation with glass beads in the presence of carrier andplasmid DNA. A mixture of the desired DNA and 0.3 g sterile glass beads (diameter 0.45 to 0.52 mm) is vortexmixed at a top speed of 100 m/s for 15 to 45 s [259]. Agitation with glass beads is an inexpensive and fast methodfor genetically transformation of fungi. It rivals electroporation for being the least time consuming methodology,but it is also one of the least efficient (hundreds of transformants/µg of DNA). It can be performed easily withoutsophisticated devices and does not require chemical treatments or enzymatic cocktails. Notwithstanding, this methodhas a low frequency of transformation, because high quantities of DNA are damaged in the process and the viabilityof the cells is drastically reduced. Nevertheless, this procedure is particularly efficient at disrupting yeasts, which haverigid cell walls [259]. Its use in filamentous fungi is hampered by the composition and thickness of the cell wall ofthese organisms.

The first application of this methodology was in the yeast Saccharomyces cerevisiae [260]. Since then, it has beenused to transform filamentous fungi such as the following:

• Saccharomyces cerevisiae [260–269],• Saccharomyces kudriavzevii [269],

yeast such as

• Pichia pastoris [270,271],

and Candida species [272] such as

• Candida albicans [273–276], and• Candida guilliermondii [259].

5. Vacuum infiltration

Vacuum infiltration is a popular method of mediating the incorporation of Agrobacterium for fungi transformation[14]. Air spaces between the fungi cells increase due to the negative atmospheric pressure generated by the vacuumallowing the penetration of Agrobacterium into the inter-cell spaces. Agrobacterium promotes the genetic modificationof fungal DNA by a mechanism that is only partially understood. The time that a cell is exposed to the vacuum iscritical as prolonged exposure causes hyperhydricity, and eventually cell death.

Vacuum infiltration is useful in certain type of fungi with low sporulation rate or in species in which sporulationhas not been reported. Additionally, this method can be performed easily. If a high number of bacterial and fungalcells are used, the probability of successful transformation is enhanced; however, other variables may influence thefrequency of transformation.

The main limitations of this method are due to biological variables. Some strains of Agrobacterium are unableto infect certain species of fungi. The temperature, pH and times of induction of virulence genes have a dramaticeffect on the frequency of transformation [143]. Moreover, there are undesirable effects of the use of Agrobacteriumfor genetic transformation, for example, random integration of sequences that belong to the bacterial chromosome orvirulence plasmids have been reported in organisms such as Saccharomyces cerevisiae and Arabidopsis sp. [277,278].

The use of Agrobacterium-mediated transformation assisted by vacuum infiltration was first reported in 1993 fortransforming Arabidopsis sp. [279]. In fungi, it has been applied to the following species:

• Agaricus bisporus [280,281],• Fusarium oxysporum [282],• Phytophthora infestans [283],• Phanerochaete chrysosporium [284], and• Flammulina velutipes [285].

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Fig. 1. Photograph of the experimental piezoelectric underwater shock wave generator, used to transform filamentous fungi. Conidia were placedat the focus of the shock wave source (Richard Wolf GmbH) inside a small polyethylene bag.

6. Shock-wave-mediated transformation

Underwater shock waves, as used in orthopedics, urology and other medical applications [286] are pressure pulseswith a peak positive pressure of up to 150 MPa, followed by a pressure trough of up to approximately 20 MPa.The rise time of the positive pressure peak lasts only a few nanoseconds and the pulse duration (full width halfmaximum) equals 0.5 to 3 µs and 2 to 20 µs for the positive and negative pulses, respectively. In general, shockwaves are produced by electrohydraulic, electromagnetic or piezoelectric shock wave generators [287]. Some of thesedevices use metallic reflectors or acoustic lenses to focus the energy onto a relatively small volume and others areself-focusing. In the vicinity of the focal volume, the positive pressure pulse compresses all microbubbles containedin the fluid. A few microseconds later the enormous difference between the pressure inside and outside the bubblesproduces rapid bubble growth, which ends in violent collapses [288] and the formation of microjets of fluid thatpenetrate the bubbles, exiting them at several hundred meters per second [289–292]. Secondary shock waves arealso produced as a consequence of bubble collapse [293]. Shock-wave-mediated insertion of DNA has been reportedin Escherichia coli, Pseudomonas aeruginosa and Salmonella typhimurium [294–296]. It is known that microjetformation is responsible for the introduction of DNA into eukaryotic cells as well as DNA transfer into bacteria. Thevolume of fluid that a single microjet is capable of injecting into a cell has been estimated approximately 0.1R3

0 , whereR0 is the bubble radius before the arrival of the shock front [297].

Several authors have published articles on shock-wave-mediated DNA delivery in eukaryotic cells and prokaryotes[298–304]. As far as we know, our group was the first to report the transformation of fungi with shock waves [116].A Piezolith 2300-based experimental shock wave generator (Richard Wolf GmbH, Knittlingen, Germany) was usedfor this study (see Fig. 1). The system has approximately 3000 piezoceramic crystals, arranged on a bowl-shapedaluminum backing and insulated from water by a flexible membrane. A high voltage discharge across the array ofpiezoelectric crystals produces an abrupt expansion of each crystal. This sudden movement generates pressure wavestravelling towards the center (F) of the device. Superposition of the pressure pulses results in a shock wave shortlybefore arriving at F. The voltage applied to the crystal array and the pulse rate can be varied between 5 and 10 kV and0.5 to 2.5 Hz, respectively. A water tank with an XYZ positioner was placed on top of the shock wave source to couplethe shock waves into a 15 mm × 10 mm polyethylene bag containing a conidia suspension (see Fig. 2). Aspergillusniger, Trichoderma reesei, Phanerochaete chrysosporium, and Fusarium oxysporum were exposed to 50, 100, 200, 300or 400 shock waves at a rate of 0.5 Hz, using a discharge voltage of 7.6 kV. The first three fungi were selected becausethey are important in the industry, whereas the last one is a well known plant pathogen [54,115,305,306]. Each conidiasuspension was mixed with a vector or expression cassette. After shock wave treatment, fungi suspensions were placed

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Fig. 2. Photograph of the holder designed to fasten the sample horizontally inside the water tank of the shock wave generator.

on agar plates and incubated in the presence of hygromycin. Only hygromycin-resistant colonies were recoveredfrom the plates. Fungi were observed under a fluorescence microscope. Shock-wave-transformed spores led to thegrowth of hygromycin-resistant colonies, whereas non-shock wave-treated cells did not produce resistant colonies.Agrobacterium-mediated transformation [143] was compared with transformation by shock waves using the samefour species. Even if the viability of conidia decreased after shock wave treatment, the number of colonies obtainedwas two to four orders of magnitude higher than those obtained by standard methods [145,307]. Genes conferringresistance to hygromycin were detectable by polymerase chain reaction (PCR) in all transformed species. Theseinitial experiments revealed that shock wave transformation is efficient, fast and could replace standard techniques inthe near future, including species that have never been transformed [305]. An interesting improvement is that intactconidia can be directly exposed to shock waves, without previously removing the cell wall.

Certainly, the application of shock waves to transform fungi has several advantages. Expensive enzymatic cocktailsare not required, the transformation frequency is higher in comparison with other available methods, and the method isfast, easy to perform and reproducible. Additionally, the same frequency, energy, voltage and number of shock wavescan be used to transform diverse species of fungi [116]. At present, the main drawback for the use of shock waves isthe need for relatively expensive equipment.

7. Conclusions

Growing interest in biotechnological research demands the development of novel strategies to manipulate andincorporate specific sequences into fungi to improve their characteristics in an easy, safe, reliable and reproducibleform.

Some techniques have been successfully established for a few types of fungi, but a great deal of research remainsto be performed to effectively exploit these technologies in a wide variety of species and to increase the efficiencyand reproducibility of genetic transformations. The genetic transformation of fungi, whether performed by physicalor other methods, currently presents major challenges. A better understanding of the phenomena involved in genetictransformation should help make protocols more rigorous and may open up new strategies.

The increasing availability of genomic information should help to identify new promoters and regulatory sequencesto enhance heterologous gene expression. Together, this information should result in the development of new ormodified methods for the introduction of heterologous or homologous genes with high efficiency.

Ultrasound mediated-transformation is not described in this article, because in fungi ultrasound has been appliedonly to separate cells in order to facilitate DNA transfer mediated by Agrobacterium. The mechanisms involved in themodification of the cell wall of fungi by ultrasound have not been described and, to the best of our knowledge, there

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is no reliable evidence of the use of ultrasound to transfer DNA into fungi. A section on laser microbeams was notincluded because all reports are focused on cellular biology, i.e., chromosome separation during mitosis, and DNAtransfer is not reported.

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