PEG-mediated Protoplast Transformation Research Articles

Exploring molecular tools for transformation and gene expression in the cultivated edible mushroom Agrocybe aegerita.

Agrocybe aegerita is a cultivated edible mushroom in numerous countries, which also serves as a model basidiomycete to study fruiting body formation. Aiming to create an easily expandable customised molecular toolset for transformation and constitutive gene of interest expression, we first created a homologous dominant marker for transformant selection. Progeny monokaryons of the genome-sequenced dikaryon A. aegerita AAE-3 used here were identified as sensitive to the systemic fungicide carboxin. We cloned the wild-type gene encoding the iron-sulphur protein subunit of succinate dehydrogenase AaeSdi1 including its up- and downstream regions, and introduced a single-point mutation (His237 to Leu) to make it confer carboxin resistance. PEG-mediated transformation of protoplasts derived from either oidia or vegetative monokaryotic mycelium with the resulting carboxin resistance marker (CbxR) plasmid pSDI1E3 yielded carboxin-resistant transformants in both cases. Plasmid DNA linearised within the selection marker resulted in transformants with ectopic multiple insertions of plasmid DNA in a head-to-tail repeat-like fashion. When circular plasmid was used, ectopic single integration into the fungal genome was favoured, but also gene conversion at the homologous locus was seen in 1 out of 11 analysed transformants. Employing CbxR as selection marker, two versions of a reporter gene construct were assembled via Golden Gate cloning which allows easy recombination of its modules. These consisted of an eGFP expression cassette controlled by the native promoter PAaeGPDII and the heterologous terminator Tnos, once with and once without an intron in front of the eGFP start codon. After protoplast transformation with either construct as circular plasmid DNA, GFP fluorescence was detected with either transformants, indicating that expression of eGFP is intron-independent in A. aegerita. This paves the way for functional genetics approaches to A. aegerita, e.g., via constitutive expression of fruiting-related genes.

Functional characterization of CgPBS2, a MAP kinase kinase in Colletotrichum gloeosporioides, using osmotic stress sensitivity as a selection marker

Colletotrichum leaf disease of Hever brasiliensis (rubber tree) caused by C. gloeosporioides is one of the major causes of declining rubber tree yields. Little is known about the fungal molecular characters that are important for pathogenicity on rubber tree and fungicide resistance. In this study, we cloned the CgPBS2 gene, the key component of the Hog1 pathway which controls various aspects of osmoregulation and fungicide resistance in various fungal pathogens, including the causal agent of Colletotrichum leaf disease of rubber tree. We characterized the function of the CgPBS2 gene by reverse genetics. Because the Hog1 pathway plays an important role in stress responses, we obtained a CgPBS2 gene deletion mutant by PEG-mediated transformation of protoplasts after reducing the concentration of sucrose in the screening medium from 1.0 M to 0.2 M. Then, the complemented transformants and GFP-labelled CgPBS2 gene transformants were selected directly under highly hyperosmotic medium (PDA + 1.5 M sorbitol) without using other selectable gene markers. Phenotypic observations showed that the CgPBS2 protein was mainly localized in the conidial cytoplasm of the CgPBS2-GFP transformants. In addition, disruption of CgPBS2 led to sensitivity to hyperosmosis and high salt concentration as well as resistance to the fungicide fludioxonil. No obvious difference in virulence was observed between the null mutant and the wild-type strain. These results provide insights into the role of the CgPBS2 gene in osmotic stress, salt stress and fludioxonil resistance and suggest that osmotic stress sensitivity can be used as a selection marker.

Optimization of Production Conditions for Protoplasts and Polyethylene Glycol-Mediated Transformation of Gaeumannomyces tritici.

Take-all, caused by Gaeumannomyces tritici, is one of the most important wheat root diseases worldwide, as it results in serious yield losses. In this study, G. tritici was transformed to express the hygromycin B phosphotransferase using a combined protoplast and polyethylene glycol (PEG)-mediated transformation technique. Based on a series of single-factor experimental results, three major factors—temperature, enzyme lysis time, and concentration of the lysing enzyme—were selected as the independent variables, which were optimized using the response surface methodology. A higher protoplast yield of 9.83 × 107 protoplasts/mL was observed, and the protoplast vitality was also high, reaching 96.27% after optimization. Protoplasts were isolated under the optimal conditions, with the highest transformation frequency (46–54 transformants/μg DNA). Polymerase chain reaction and Southern blotting detection indicated that the genes of hygromycin phosphotransferase were successfully inserted into the genome of G. tritici. An optimised PEG-mediated protoplast transformation system for G. tritici was established. The techniques and procedures described will lay the foundation for establishing a good mutation library of G. tritici and could be used to transform other fungi.

Targeted Gene Deletion in Cordyceps militaris Using the Split-Marker Approach.

The macrofungus Cordyceps militaris contains many kinds of bioactive ingredients that are regulated by functional genes, but the functions of many genes in C. militaris are still unknown. In this study, to improve the frequency of homologous integration, a genetic transformation system based on a split-marker approach was developed for the first time in C. militaris to knock out a gene encoding a terpenoid synthase (Tns). The linear and split-marker deletion cassettes were constructed and introduced into C. militaris protoplasts by PEG-mediated transformation. The transformation of split-marker fragments resulted in a higher efficiency of targeted gene disruption than the transformation of linear deletion cassettes did. The color phenotype of the Tns gene deletion mutants was different from that of wild-type C. militaris. Moreover, a PEG-mediated protoplast transformation system was established, and stable genetic transformants were obtained. This method of targeted gene deletion represents an important tool for investigating the role of C. militaris genes.

Generating Gene Silenced Mutants in Phytophthora sojae.

Phytophthora species are notorious pathogens of plants and cause enormous damage to agriculture. In order to understand Phytophthora pathogenesis, gene silencing and knockout methods are important for the investigation of gene functions. Although CRISPR/Cas9-based gene knockout procedures have been developed in Phytophthora sojae and Phytophthora capsici, it may not always be the best choice especially when knockout mutation leads to lethality. Therefore, gene silencing is a very useful tool for functional analysis of target genes in Phytophthora. This chapter introduces a gene silencing protocol for the soybean pathogen P. sojae. An expression cassette is incorporated into the P. sojae genome through PEG-mediated protoplast transformation, which leads to constitutive production of antisense RNA transcripts. These transcripts are able to target mRNAs through sequence complementarity and effectively reduce the expression of the target genes.

Careful with That Axe, Gene, Genome Perturbation after a PEG-Mediated Protoplast Transformation in Fusarium verticillioides.

Fusarium verticillioides causes ear rot disease in maize and its contamination with fumonisins, mycotoxins harmful for humans and livestock. Lipids, and their oxidized forms, may drive the fate of this disease. In a previous study, we have explored the role of oxylipins in this interaction by deleting by standard transformation procedures a linoleate diol synthase-coding gene, lds1, in F. verticillioides. A profound phenotypic diversity in the mutants generated has prompted us to investigate more deeply the whole genome of two lds1-deleted strains. Bioinformatics analyses pinpoint significant differences in the genome sequences emerged between the wild type and the lds1-mutants further than those trivially attributable to the deletion of the lds1 locus, such as single nucleotide polymorphisms, small deletion/insertion polymorphisms and structural variations. Results suggest that the effect of a (theoretically) punctual transformation event might have enhanced the natural mechanisms of genomic variability and that transformation practices, commonly used in the reverse genetics of fungi, may potentially be responsible for unexpected, stochastic and henceforth off-target rearrangements throughout the genome.

Establishment of polyethylene-glycol-mediated protoplast transformation for Lecanicillium lecanii and development of virulence-enhanced strains against Aphis gossypii.

Lecanicillium lecanii has been developed as a biopesticide and used in biological control of several agricultural insects. To improve fungal virulence, an optimised polyethylene glycol (PEG)-mediated protoplast transformation system was established for L. lecanii. Pr1A-like cuticle-degrading protease gene (Cdep1) from Beauveria bassiana was transferred into L. lecanii, and its resulting activity against Aphis gossypii was assessed. The optimised protoplast generation yielded 2.5 × 10(8) protoplasts g(-1) wet mycelium of fungi, and gave nearly 98% viability and 80% regeneration on plates. Protease activities were increased about fivefold in transformants expressing CDEP1. The median lethal concentration (LC50 ) for transformants expressing CDEP1 was twofold lower than that for the wild type (WT). The median survival time (LT50 ) for transformants expressing CDEP1 was also 14.2% shorter than that for WT, though no significant difference. There were no significant differences in conidial germination as colony growth and conidial yield on plates between transformants expressing CDEP1 and WT. The transformants expressing CDEP1 grew significantly quicker than WT in insects. The transformants expressing CDEP1 were lower in conidial yields on insect cadavers, but insignificantly different from WT. The PEG-mediated protoplast transformation system was effective for L. lecanii, and the expression of CDEP1 significantly enhanced fungal virulence against cotton aphids. © 2016 Society of Chemical Industry.

Transformation of the endophytic fungus Acremonium implicatum with GFP and evaluation of its biocontrol effect against Meloidogyne incognita.

Acremonium implicatum is an endophytic fungus with biocontrol potential against Meloidogyne incognita based on its opportunistic egg-parasitic, hatching inhibition, and toxic properties. To understand its mode of plant endophytism and opportunistic egg parasitism, GFP-tagged A. implicatum was constructed by PEG-mediated protoplast transformation. By laser scanning confocal microscopy (LSCM), we evaluated the endophytism and opportunistic egg parasitism of a stable gfp transformant (Acr-1). Acr-1 could colonize epidermal tissue, cortical tissue, and xylem of roots and form a mutualistic symbiosis with tomato host plants. LSCM of Acr-1 infecting M. incognita eggs revealed that hyphae penetrated the shell and grew inside eggs to form trophic hyphae. A large number of hyphae enveloped parasitized eggs. In addition, the egg shell integrity was destroyed by fungal penetration. The percentage of egg parasitism was 33.8 %. There were no marked differences between the wild type and mutant in nematode second-stage juvenile mortality and egg hatching and in fungal control efficiency in a pot experiment. In conclusion, gfp-transformation did not change the nematicidal activity of A. implicatum and is a tool to examine the mode of plant endophytism and opportunistic egg parasitism of A. implicatum.

Improvement in fruiting body yield by introduction of the Ampullaria crossean multi-functional cellulase gene into Volvariella volvacea

The multi-functional cellulase gene, mfc, from Ampullaria crossean was transformed into Volvariella volvacea by PEG-mediated protoplast transformation to improve the biological efficiency and fruiting body yield. PCR and Southern blotting indicated that mfc was integrated into the genomes of four transformants. In laboratory and large scale cultivation experiments, the average biological efficiency of the transformants was 18.39 ± 1.27% and 27.84 ± 3.21%, respectively, considerably higher than the corresponding values for untransformed controls of 12.69 ± 1.31% and 20.63 ± 2.59%. This is the first report of an improvement in biological efficiency and fruiting body yield of V. volvacea through transgenesis.

Development of Highly Efficient Transformation System of Yeast-Like Conidia of Tremella fuciformis

Tremella fuciformis is one of higher basidiomycetes. Its basidiospore can reproduce yeast-like conidia, which is also called the blastospore by budding. The yeast-like conidia of T. fuciformis is monokaryotic and easy to culture by submerged fermentation similar to yeast. Thus, it is a good recipient cell for exogenous gene expression. In this study, the expression plasmid pAN7-1 (containing promoter gpd-An derived from Aspergillus nidulans and selectable marker gene hph conferring resistance to hygromycin B) and plasmid pLg-hph (containing promoter gpd-Le derived from Lentinula edodes and selectable marker gene hph) were transformed into the yeast-like conidia of T. fuciformis by PEG-mediated protoplast transformation, respectively. The primary putative transformants were selected by the sandwich screening method with the selective medium containing 50 μg mL −1 hygromycin. The putative transformants were obtained from the primary putative transformants transferred on PDSA plates containing 100 μg mL −1 hygromycin for second round selection. Experimental results showed that the optimal concentration of PEG 4000 for mediating protoplast transformation was 25%. PCR and Southern blotting confirmed that the selectable marker gene hph was integrated effectively into the genome of the yeast-like conidia of T. fuciformis with plasmid pLg-hph transformation. Its transformation efficiency was 110 transformants per μg DNA, and the hph gene was integrated into the genome of some yeast-like conidia with plasmid pAN7-1 transformation. However, its transformation efficiency was only 9 transformants per μg DNA. The presence of hph gene in the genome of transformants after 5 generations of subculturing on PDSB medium was confirmed by PCR, suggesting that the foreign gene hph was stable during subculture.

Direct gene transfer in potato: A comparison of particle bombardment of leaf explants and PEG-mediated transformation of protoplasts

Direct gene transfer methods in potato would facilitate the transfer of multiple genes and the manipulation of metabolic pathways in this species. In this study, up to 1.8 transformation events per shot (=0.5 per bombarded leaf) and 67.2 events per million protoplasts treated were obtained with particle bombardment and PEG-mediated direct DNA uptake, respectively. Limited disassociation of both HPT and GUS genes appeared to occur during the process of integration in only 19% of transformants. A large number of transformed potato plants with transgene expression at levels comparable to Agrobacterium-mediated transformation was obtained. High levels of GUS expression were only obtained in lines derived from PEG treatment. No correlation between the number of gene insertions and gene expression levels was found, suggesting that multiple insertions may have little or no effect on transgene expression.

Split-Marker Recombination for Efficient Targeted Deletion of Fungal Genes

A commonly used method for fungal gene deletion is introduction of linear DNA consisting of a selectable marker gene flanked on both sides by short stretches of DNA that target a gene of interest (W irsel et al 1996 Curr. Genet 29:241-249). Gene deletion in Cochliobolus heterostrophus and Gibberella zeae occurs efficiently with this approach. To facilitate deletion construct synthesis, we have applied the split-marker” deletion strategy previously developed for Saccharomyces cerevisiae (Fairhead et al. 1996 Y east 12:1439-57; Fairhead et al. 1998 Gene 223:33-46). Here, we describe both fusion PCR-based and plasmid-based deletion methods using this strategy with PEG-mediated protoplast transformation (Turgeon et al, 1985 M ol. Gen. Genet. 201:450-453). These methods are predicted to work well with any transformable fungus that undergoes homologous recombination between chromosomal and introduced DNA sequences.

Superfluous Transgene Integration in Plants

Referee: Dr. Paul Hooykass, Institut of Molecular Plant Sciences, Leiden University, Clusius Laboratory, Wassenaarseweg 64, 2333, Al Leiden, NetherlandsRecent reports suggest the transfer of superfluous DNA sequences to plant genomes during transformation processes. This review investigates the evidence from the published literature for the prevalence of this phenomenon and highlights methods to limit or prevent DNA transfer and subsequent potentially detrimental evolutionary consequences.Evidence for superfluous foreign DNA transfer using both Agrobacterium-mediated transformation and direct DNA transfer methods such as microprojectile bombardment and PEG-mediated transformation of protoplasts is reported. In the case of Agrobacterium-mediated transformation, the lack of information on the integration of sequences from outside of the T-DNA borders has been due to the general belief by researchers that T-DNA processing is precise. This assumption was based on analysis of T-DNA in tumors and as a result the majority of T-DNA integration events have been identified exclusively using DNA probes, which are homologous only to DNA from within the T-DNA borders. Where direct gene transfer protocols are employed, any part of the transforming plasmid and indeed accompanying carrier DNA may become integrated into the plant genome. The main body of evidence proving that superfluous vector DNA sequences are present in plant genomes transformed using direct transfer methods is confined to the identification of plasmid concatamers integrated into plant genomes.The limited amount of recorded evidence pertaining to superfluous vector DNA integration in transgenic plants and transformed tissues makes it impossible to draw definitive conclusions as to the factors involved in promoting this phenomenon. However, there are methods available for removing superfluous sequences from transgenic plants. These have been developed for the removal of selectable marker genes, whose presence in transgenic plants has been a source of much controversy, but can equally be applied to other DNA sequences. Suggestions have been made in the review that might limit or prevent the integration of superfluous vector sequences during transformation procedures; however, these are not proven and further research is required.

Superfluous Transgene Integration in Plants

Referee: Dr. Paul Hooykass, Institut of Molecular Plant Sciences, Leiden University, Clusius Laboratory, Wassenaarseweg 64, 2333, Al Leiden, Netherlands Recent reports suggest the transfer of superfluous DNA sequences to plant genomes during transformation processes. This review investigates the evidence from the published literature for the prevalence of this phenomenon and highlights methods to limit or prevent DNA transfer and subsequent potentially detrimental evolutionary consequences. Evidence for superfluous foreign DNA transfer using both Agrobacterium-mediated transformation and direct DNA transfer methods such as microprojectile bombardment and PEG-mediated transformation of protoplasts is reported. In the case of Agrobacterium-mediated transformation, the lack of information on the integration of sequences from outside of the T-DNA borders has been due to the general belief by researchers that T-DNA processing is precise. This assumption was based on analysis of T-DNA in tumors and as a result ...

A mannose selection system for production of fertile transgenic maize plants from protoplasts.

Maize (Zea mays L.) callus cultures cannot use mannose as a sole carbohydrate source, but can utilize fructose for that purpose. Phosphomannose isomerase (PMI) can convert mannose to fructose. Transgenic maize plants were obtained by selecting polyethylene glycol (PEG)-mediated transformed protoplasts on mannose (20 g/l) containing medium. Transgenic calluses and plants carrying the PMI structural gene, manA, were able to convert mannose to fructose. The PEG-mediated protoplast transformation frequency was 0.06%. Stable transformation was confirmed by PCR, PMI activity, germination tests, and by histochemical staining with 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc). Stable integration of the transgenes into the maize genome was demonstrated in T1 and T2 plants. Results indicate that the mannose selection system can be used for maize PEG-mediated protoplast transformation.

Green fluorescent protein (GFP) as gene expression reporter and vital marker for studying development and microbe-plant interaction in the tobacco pathogen Phythphthora parasitica var. nicotianae.

PEG-mediated transformation of protoplasts in the presence of lipofectin was achieved in Phytophthora parasitica var. nicotianae, an oomycete pathogen of tobacco. Using oomycete promoter and terminator sequences, a plant-adapted green fluorescent protein (GFP) was introduced into the microorganism. The data show for the first time that this eukaryotic gene reporter can be used in an oomycete, both as a quantitative reporter of gene induction and as a vital marker allowing the study of development of Phytophthora in vitro and in the host plant.

Green fluorescent protein (GFP) as gene expression reporter and vital marker for studying development and microbe-plant interaction in the tobacco pathogen Phytophthora parasitica var. nicotianae

PEG-mediated transformation of protoplasts in the presence of lipofectin was achieved in Phytophthora parasitica var. nicotianae, an oomycete pathogen of tobacco. Using oomycete promoter and terminator sequences, a plant-adapted green fluorescent protein (GFP) was introduced into the microorganism. The data show for the first time that this eukaryotic gene reporter can be used in an oomycete, both as a quantitative reporter of gene induction and as a vital marker allowing the study of development of Phytophthora in vitro and in the host plant.

Stability of transgene expression during vegetative propagation of protoplast-derived tall fescue (Festuca arundinacea Schreb.) plants

The expression of a β-glucuronidase (GUS) transgene, under the control of the rice actin 1 promoter and first intron, introduced via PEG-mediated transformation of protoplasts, has been followed through a number of generations of vegetative propagation (tillering) in the forage grass Festuca arundinacea. Gene expression was found to be particularly unstable during early generations of tillering, but more stable in the fourth or fifth tiller generation. Loss of transgene expression was not due to deletion of the transgene nor was it due to gross rearrangements in the transgene as demonstrated by Southern blot analysis of expressing and non-expressing tillers. Loss of transgene expression due to hypermethylation of the transgene promoter has been shown previously, but in this case it was not possible to detect differences in the methylation patterns of the coding and promoter regions of expressing and non-expressing tillers. The implications for screening of transgenic plants intended to be vegetatively propagated are discussed.

PEG-mediated protoplast transformation with naked DNA.

Direct introduction of DNA into plant protoplasts facilitates a rapid analysis of transient gene expression, as well as the generation of stably transformed transgenic plants. Transient gene expression assays performed after DNA transformation permit a comparative analysis of cis–actmg regulatory sequences and then function m transcriptional control of plant genes by signaling pathways mediating cellular responses to different environmental and hormonal stimuli (). There are a number of methods for introducing DNA into plant protoplasts, but the most commonly used technique 1s the polyethylene glycol (PEG)–mediated DNA uptake. The PEG–mediated transformation is simple and efficient, allowing a simultaneous processing of many samples, and yields a transformed cell population with high survival and division rates (). The method utihzes inexpensive supplies and equipments, and helps to overcome a hurdle of host range limitations of Agrobactenum–mediated transformation The PEG–mediated DNA transfer can be readily adapted to a wide range of plant species and tissue sources.

An improved method for direct gene transfer and subsequent regeneration ofArabidopsis thaliana Landsbergerecta and two marker lines

A protocol for PEG-mediated transformation of protoplasts is described forA. thaliana ecotype Landsbergerecta and two marker lines derived from it, M4 and M10. The optimal transformation conditions were: 14 µg plasmid DNA and 28 µg carrier DNA per 6 x 105 protoplasts in 15% (w/v) PEG solution. Based on the hygromycin resistance conferred by the transgene, relative transformation frequencies of 2.5–3.2% and absolute transformation frequencies of 1–2 x 10−4, were obtained. Shoot regeneration frequencies of 40–60% were achieved, and fertile transgenic plants of the three tested lines were obtained. Southern blot hybridizations demonstrated multi-copy integration patterns in most cases. Hygromycin resistance segregation patterns of 3:1 and 15:1 were found, as well as unexpected segregation patterns, suggesting that modifications in gene expression took place and that these can progressively occur over successive generations.