Hph Gene Research Articles

Effect of Heat Shock Treatment on Hordeum Vulgare Protoplast Transformation Mediated by Polyethylene Glycol

The stable transformation of barley protoplasts has been observed in cv. Dissa by PEG-mediated pGL2 plasmid DNA uptake containing hygromycin phosphotransferase (hph) gene under the control of 35S transcript of CaMV. Hygromycin resistant colonies were obtained, when protoplasts were incubated with hygromycin B (25 μg cm−3) at 3 – 4 cell stage. Heat shock treatment, prior to polyethylene glycol (PEG) treatment, markedly increased the protoplast transformation frequency. The presence of active hph gene product, and integration of hph gene was confirmed by hygromycin assay and Southern blotting, respectively. Analysis indicated that rearrangement had possibly occurred during the integration of plasmid DNA into genomic DNA of barley callus. These transformed calli showed no morphogenic response onto the regeneration medium.

Agrobacterium-Mediated Transformation of Fusarium oxysporum: An Efficient Tool for Insertional Mutagenesis and Gene Transfer

ABSTRACT Agrobacterium tumefaciens-mediated transformation (ATMT) has long been used to transfer genes to a wide variety of plants and has also served as an efficient tool for insertional mutagenesis. In this paper, we report the construction of four novel binary vectors for fungal transformation and the optimization of an ATMT protocol for insertional mutagenesis, which permits an efficient genetic manipulation of Fusarium oxysporum and other phytopathogenic fungi to be achieved. Employing the binary vectors, carrying the bacterial hygromycin B phosphotrans-ferase gene (hph) under the control of the Aspergillus nidulans trpC promoter as a selectable marker, led to the production of 300 to 500 hygromycin B resistant transformants per 1 x 10(6) conidia of F. oxysporum, which is at least an order of magnitude higher than that previously accomplished. Transformation efficiency correlated strongly with the duration of cocultivation of fungal spores with Agrobacterium tumefaciens cells and significantly with the number of Agrobacteruium tumefaciens cells present during the cocultivation period (r = 0.996; n = 3; P < 0.01). All transformants tested remained mitotically stable, maintaining their hygromycin B resistance. Growing Agrobacterium tumefaciens cells in the presence of acetosyringone (AS) prior to cocultivation shortened the time required for the formation of transformants but decreased to 53% the percentage of transformants containing a single T-DNA insert per genome. This increased to over 80% when Agrobacterium tumefaciens cells grown in the absence of AS were used. There was no correlation between the average copy number of T-DNA per genome and the colony diameter of the transformants, the period of cocultivation or the quantity of Agrobacterium tumefaciens cells present during cocultivation. To isolate the host sequences flanking the inserted T-DNA, we employed a modified thermal asymmetric interlaced PCR (TAIL-PCR) technique. Utilizing just one arbitrary primer resulted in the successful amplification of desired products in 90% of those transformants analyzed. The insertion event appeared to be a random process with truncation of the inserted T-DNA, ranging from 1 to 14 bp in size, occurring on both the right and left border sequences. Considering the size and design of the vectors described here, coupled with the efficiency and flexibility of this ATMT protocol, it is suggested that ATMT should be regarded as a highly efficient alternative to other DNA transfer procedures in characterizing those genes important for the pathogenicity of F. oxysporum and potentially those of other fungal pathogens.

Efficient production of transgenic plants by Agrobacterium-mediated transformation of cassava (Manihot esculenta Crantz)

An efficient and reproducible method was developed for Agrobacterium-mediated transformation of embryogenic suspension cultures of cassava. LBA4404(pTOK233), containing the nptII, hph and gus marker genes, was used in the experiments. Chemical selection by means of kanamycin was used to establish 1037antibiotic resistant callus lines, of which 526 showed GUS expression. Of the 241 callus lines that were transferred to maturation medium 219formed somatic embryos. Thirty-seven of the 38 lines that were transferred to germination medium produced plants. GUS-positive plants could be obtained from 31 lines; in 14 of those lines 100% of the produced plants were GUS-positive, the remaining 17 lines yielded GUS-positive plants at an average of 72%. The transgenic nature of these plants was confirmed by Southern blot analysis.

A Transformation System for an Ectomycorrhizal Basidiomycete, Lyophyllum shimeji

A transformation vector, pLS-hph, was constructed with the promoter and terminator of the glyceraidehyde-3-phosphate dehydrogenase (GPD) gene derived from an ectomycorrhizal basidiomycete, Lyophyllum shimeji, and with the hygromycin B (HmB) phosphotransferase (hph) gene from Escherichia coli. This vector was introduced into protoplasts of L. shimeji and 3.4 transformants per microg plasmid DNA were obtained. In most of the transformants, multiple copies of the vector were integrated into the genomic DNA. The results indicate that pLS-hph is a useful vector for L. shimeji.

High Efficiency Transformation of U.S. Rice Lines from Mature Seed‐Derived Calli and Segregation of Glufosinate Resistance under Field Conditions

Gene transfer techniques have been developed previously for certain model rice (Oryza sativa L.) cultivars, but problems persist in U.S. lines for low transformation rates and in vitro callus culture. Moreover, few studies have evaluated traits such as herbicide resistance in transgenic U.S. rice lines under field conditions. A rapid and efficient method was developed for production and field evaluation of transgenic herbicide‐resistant elite U.S. rice lines and cultivars. Six elite U.S. rice lines were transformed for glufosinate herbicide resistance by particle bombardment of mature seed‐derived embryogenic calli; resistance was confered by either the pat or bar gene. By utilizing optimized media for embryogenic callus induction and bialaphos or hygromycin B as a selection agent, an average transformation efficiency of 5% (258 independent events/5201 calli) was obtained across six lines. Southern blot analysis of genomic DNA isolated from primary R0 and R1 progeny plants demonstrated that the pat and hygromycin phosphotransferase (hph) genes were stably integrated into the rice genome. Glufosinate resistance in R0, R1, and R2 progeny was confirmed in the greenhouse and under field conditions. All R1 and a majority (79%) of R2 progeny exhibited one to two gene segregation patterns for glufosinate resistance. The high efficiency and reproducibility of the improved transformation system should make it possible to routinely introduce genes of interest into any elite U.S. rice breeding line.

Efficient transformation of the edible basidiomycete Lentinus edodes with a vector using a glyceraldehyde-3-phosphate dehydrogenase promoter to hygromycin B resistance.

To construct a vector for high-level expression of heterologous genes in Lentinus edodes, the L. edodes GPD promoter, which is expressed constitutively and strongly, was fused to a hygromycin B phosphotransferase gene (hph) derived from Escherichia coli as a selective marker. Using the resulting pLG-hph construct, L. edodes was efficiently transformed to hygromycin resistance by restriction enzyme-mediated integration (REMI). The restriction enzyme concentrations yielding the maximal numbers of transformants by the REMI method were 10 U per transformation in the case of BglII and 25-50 U in the case of HindIII. Southern analysis of the transformants indicated that some 50% of plasmid integrations were REMI-mediated events. These results indicate that pLG is a useful vector for transformation of L. edodes. Deletion analysis of the GPD promoter region suggested that the segment between positions -442 bp and -270 bp relative to the transcription start point may be essential for function.

Agrobacterium-mediated engineering for sheath blight resistance of indica rice cultivars from different ecosystems

A concise T-DNA element was engineered containing the rice class-I chitinase gene expressed under the control of CaMV35S and the hygromycin phosphotransferase gene (hph) as a selectable marker. The binary plasmid vector pNO1 with the T-DNA element containing these genes of interest was mobilized to Agrobacterium tumefaciens strain LBA4404 to act as an efficient donor of T-DNA in the transformation of three different indica rice cultivars from different ecosystems. Many morphologically normal, fertile transgenic plants from these rice cultivars were generated after Agrobacterium-mediated transformation using 3-week-old scutella calli as initial explants. Stable integration, inheritance and expression of the chimeric chitinase gene were demonstrated by Southern blot and Western blot analysis of the transformants. Bioassay data showed that transgenic plants can restrict the growth of the sheath blight pathogen Rhizoctonia solani. Bioassay results were correlated with the molecular analysis. Although we obtained similar results upon DNA-mediated transformation, this report shows the potential of the cost-effective, simple Agrobacterium system for genetic manipulation of rice cultivars with a pathogenesis-related (PR) gene.

Rapid transformation of high cellulase-producing mutant strains of Trichoderma reesei by microprojectile bombardment.

Intact conidia of three industrially relevant strains of Trichoderma reesei were effectively transformed by particle bombardment. Transformations were carried out individually with plasmids carrying either the fungal amdS or bacterial hph gene as a selectable marker and by cotransformation with both plasmids. Transformant yields with single plasmids were up to 11 stable transformants per microg DNA at the bombardment distance of 6 cm. Mitotic stability of the transformants was 75-100% and the cotransformation efficiency averaged 92% when the first selection was performed on hygromycin B plates. The entire procedure could be completed in 1 week with the hph marker.

Using GUS expression in a nonpathogenic Fusarium oxysporum strain to measure fungal biomass

Nonpathogenic Fusarium oxysporum strain SA70, a biological control agent against tomato fusarium wilt, was genetically transformed with both hygromycin B resistance (Hph) and p-glucuronidase (GusA) genes. Transformant 70T01 was selected for further testing based on its stable expression of (3-glucuronidase (GUS) activity, resistance to hygromycin B (HmB), and retention of wild-type characteristics, including growth rate, root colonization ability, and disease control efficacy. Single copies of each of the GusA and Hph genes were stably integrated into the 70T01 genomic DNA. Expression of GUS, as measured by fluorometry, was highly correlated with mycelial dry weight. Assayable GUS activity was highest in young mycelia and was detectable in as little as 1 ng of mycelia (dry weight). GUS activity was extracted from protoplasts generated from germinating spores and the activity in the extracts related to the number of viable protoplasts (colony-forming protoplasts, CFPs). Fungal biomass in 70T01 -infected tomato plant roots, as determined by the GUS-CFP method, was compared with that determined by the conventional method of plating macerated, infected plant material. The results demonstrated that while similar changes in the relative levels of fungal biomass could be detected with both methods, GUS-CFP biomass estimates were typically 6- to 50-fold higher than those determined by the plating method. The GUS-CFP assay provides a rapid and sensitive technique for determining fungal biomass in natural ecological settings.

Elucidation of Septoria tritici × Wheat Interactions Using GUS-Expressing Isolates

ABSTRACT Isolate ISR398 of Septoria tritici (which produces none to few pycnidia on the wheat cv. Seri 82 and high coverage on cv. Shafir) and isolate ISR8036 (which is virulent on both cultivars) were genetically cotrans-formed using the selectable marker gene hph, which confers resistance to hygromycin B (hygB), and the reporter gene uidA, encoding beta-glucuronidase (GUS). Most of the genetically transformed isolates (98.8%) produced similar pycnidial coverage on seedlings of 'Seri 82' and 'Shafir' as the two wild-type isolates. Southern analysis of 25 randomly selected hygB(R)GUS(+) transformants probed with the uidA sequence revealed multiple insertion sites. GUS activity was determined fluorimetrically by measuring the conversion of 4-methylumbelliferyl beta-D-glucuronide (MUG) to 4-methylumbelliferone (MU). The high GUS-expressing transformants 398D97 and 8036E27 were used to elucidate fungal development within inoculated leaf tissue by using GUS activity to estimate the fungal proteins content in planta. Increase in fungal biomass was recorded in 'Shafir' inoculated with the GUS-expressing transformants 398D97 and 8036E27 following a 12-day latent period. A 15-day latent period was recorded in 'Seri 82' inoculated with 8036E27, whereas an 18-day latent period was recorded on 'Seri 82' inoculated with 398D97 and the two mixtures 398D97 + ISR8036 and ISR398 + 8036E27. The rate of fungal development and the estimated level of fungal proteins at the pycnidia maturation stage was high in leaves of 'Shafir' and moderate to low on 'Seri 82', even in cases in which no significant differences were recorded in pycnidial coverage. An endogenous capacity to hydrolyze beta-1,4-D-glucuronidase was recorded in leaves inoculated with wild-type isolates. The latent periods in MU production of the uidA-expressing transformants mimicked those recorded for the wild-type isolates. However, at all stages, the levels of MU produced in wheat inoculated with wild-type isolates were markedly lower than those produced by GUS-expressing transformants. The mode of interaction (compatible or incompatible) determined the onset of the induction, rate, and level of enzyme production.

Disruption of vma-1, the Gene Encoding the Catalytic Subunit of the Vacuolar H+-ATPase, Causes Severe Morphological Changes in Neurospora crassa

By using the process of Repeat-induced Point mutation (Selker, E. U., and Garrett, P. W. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 6870-6874), we inactivated vma-1, the gene encoding subunit A of the V-ATPase of Neurospora crassa. Two vma-1 mutant strains were characterized. One was mutated at multiple sites, did not make a protein product, and produced spores that only rarely germinated. The other had four point mutations, made a protein product, and produced viable spores. Neither strain had detectable V-ATPase activity. The vma-1 mutant strains did not grow in medium buffered to pH 7.0 or above or in medium supplemented with the cation Zn(2+). They were completely resistant to inhibition by concanamycin C, supporting our hypothesis that the V-ATPase is the in vivo target of this antibiotic. Inactivation of the vma-1 gene had a pronounced effect on morphology and development of the organism. In the mutants tip growth was inhibited, and multiple branching was induced. The vma-1 mutant strains could not differentiate conidia or perithecia. They could grow slowly as mycelia and could donate nuclei in a sexual cross. A mutation in the plasma membrane ATPase, which suppressed the sensitivity of wild type N. crassa to concanamycin, also proved effective in suppressing the sensitivity of a vma-1 null mutant to basic pH but did not correct the morphological defects.

Efficient production of transgenic cassava using negative and positive selection.

In order to improve the efficiency of cassava (Manihot esculenta Crantz) transformation, two different selection systems were assessed, a positive one based on the use of mannose as the selective agent, and a negative one based on hygromycin resistance encoded by an intron-containing hph gene. Transgenic plants selected on mannose or hygromycin were regenerated for the first time from embryogenic suspensions cocultivated with Agrobacterium. After the initial selection using mannose and hygromycin, 82.6% and 100% of the respective developing embryogenic callus lines were transgenic. A system allowing plant regeneration from only transgenic lines was designed by combining chemical selection with histochemical GUS assays. In total, 12 morphologically normal transgenic plant lines were produced, five using mannose and seven using hygromycin. The stable integration of the transgenes into the nuclear genome was verified using PCR and Southern analysis. RT-PCR and northern analyses confirmed the transgene expression in the regenerated plants. A rooting test on mannose containing medium was developed as an alternative to GUS assays in order to eliminate escapes from the positive selection system. Our results show that transgenic cassava plants can be obtained by using either antibiotic resistance genes that are not expressed in the micro-organisms or an antibiotic-free positive selection system.

Co-transformation of the sclerotial mycoparasite Coniothyrium minitans with hygromycin B resistance and β-glucuronidase markers

Coniothyrium minitans was successfully co-transformed with the uidA (b-glucuronidase) and the hygromycin-resistance (hph) genes. Both were under the control of the glyceraldehyde-3-phosphate promoter from Aspergillus nidulans. Hygromycin resistance was used as a selectable marker for transformation. In successive transformation experiments, transformation frequencies of up to 1000 transformants lg’ of plasmid DNA were obtained for isolate A69. Of the ten monospore hygromycin-resistant cultures tested, nine also expressed the uidA gene. Expression of hph and uidA was stable in all transformants after several months of successive subculturing on non-selective medium, and after passage through a sclerotium of Sclerotinia sclerotiorum. Southern hybridization analyses showed all transformants carried multiple copies of each marker gene. When grown on PDA, the culture morphology of three of the transformants of (T‹2, T‹3 and T‹4) was similar to the wild type. Four of the five transformants (T‹3, T‹4, T‹21 and T‹24) grew as well as the wild type on dierent media, and responded to changes in water potential in a similar manner to the wild type. All five transformants were equally parasitic on sclerotia of S. sclerotiorum compared with the wild type. Transformants T‹3 and T‹4 were the most similar to the wild type in biological characteristics and will be used in future studies. The results indicate that hph- and uidA-transformed strains of C. minitans will be useful for ecological studies on its survival and dissemination. Sclerotinia sclerotiorum (Lib.) de Bary is a soil-borne fungal pathogen which attacks a wide range of fruit and vegetable crops (Purdy, 1979). The sclerotia of S. sclerotiorum are crucial for the pathogen’s survival, serving as a primary inoculum source and remain viable in soils for many years (Merriman, 1976). Mature sclerotia germinate either myceliogenically to produce mycelium which infects host plants directly (Huang & Dueck, 1980), or carpogenically to produce apothecia which discharge wind-borne ascospores which subsequently infect the host (Huang & Kokko, 1992). Control of Sclerotinia spp. has been obtained by fungicide application, in particular the use of members of the dicarboximide group, but enhanced degradation of the fungicides in soils by other microorganisms has led to reduced ecacy of control in Europe (Martin et al., 1990). Enhanced degradation of dicarboximides has been reported in onion paddocks in New Zealand (Slade et al., 1992) and is likely to occur in other vegetable paddocks where these chemicals are being used to control Sclerotinia diseases. Soil sterilisation by stem or methyl bromide is eective but costly. Further, environmental concerns over the use of methyl bromide mean

Electrotransformation of the human pathogenic fungus Scedosporium prolificans mediated by repetitive rDNA sequences.

The regions encoding the 5.8S rRNA and the flanking internal transcribed spacers (ITSI and ITSII) from two isolates of the human pathogenic fungus Scedosporium prolificans and one isolate of the taxonomically related species Pseudallescheria boydii (S. apiospermum) were sequenced. The sequences of the two S. prolificans isolates were identical. However, there were minor differences between both species. Phylogenetic analysis of known fungal sequences confirmed a close relationship between S. prolificans and P. boydii. An attempt was made to transform S. prolificans by electroporation using a plasmid vector, pMLF2, bearing the Escherichia coli hygromycin B phosphotransferase gene (hph) under the control of Aspergillus nidulans promoter and terminator sequences. To increase transformation efficiency, the sequenced ribosomal cluster of S. prolificans was used to construct a new vector for homologous recombination.

The erg-3 (sterol delta14,15-reductase) gene of Neurospora crassa: generation of null mutants by repeat-induced point mutation and complementation by proteins chimeric for human lamin B receptor sequences.

Null mutations were generated in the erg-3 gene of Neurospora crassa by repeat-induced point mutation (RIP). The mutants were viable, lacked ergosterol, were resistant to the steroidal glycoside alpha-tomatine and were sensitive to the phytoalexins pisatin and biochanin A. RIP was frequently associated with silencing of the hph gene located adjacent to the duplicated erg-3 sequence. The silencing of hph was reversible in the two cases examined and appeared to be due to the spread of cytosine methylation associated with RIP. The erg-3 mutant could be complemented by transformation with recombinant genes that encode proteins chimeric for amino acid sequences from the transmembrane (TM) domain of human lamin B receptor (LBR). This indicates that the LBR TM domain possesses delta14,15-reductase activity.

Genetic engineering of an ectomycorrhizal fungus Laccaria bicolor for use as a biological control agent

We are developing genetically engineered ectomycorrhizal fungi for use as biological control agents. Using the gold particle bombardment technique, we transformed Laccaria bicolor, an ectomycorrhizal fungus capable of forming ectomycorrhizae on a wide range of plant hosts. Structural gene sequences of the hygromycin B phosphotransferase (hph) gene (hygromycin resistance for selection of transformants), β-glucuronidase (GUS) gene (reporter gene for tracking transformants) and Bacillus thuringiensis δ-endotoxin (BtCryIIIA) gene (Coleopteran specific insecticidal gene from Bacillus thuringiensis var tenebrionis) were introduced into L. bicolor simultaneously. Southern and western blot analyses showed that the introduced sequences were inserted into the fungal genome and transcribed and translated. One of the transformants produced the insecticidal toxin; however, the levels were not sufficient to perform toxicity assays. The technique developed can be employed to generate superior strains of L. bicolor suitable for use as biological control agents in various soil and plant conditions.

Isolation, characterization and disruption of the areA nitrogen regulatory gene of Gibberella fujikuroi.

The gene areA-GF, a homologue of the major nitrogen regulatory genes nit-2, areA, nre and NUT1 of Neurospora crassa, Aspergillus nidulans, Penicillium chrysogenum and Magnaporthe grisea, respectively, was cloned from the gibberellin (GA)-producing rice pathogen Gibberella fujikuroi. areA-GF encodes a protein of 972 amino acid residues which contains a single putative zinc finger DNA-binding domain that is at least 98% identical to the zinc finger domains of the homologous fungal proteins. The areA-GF gene has been shown to be functional in N. crassa by heterologous complementation of a RIP induced nit-2 mutant. The transformation rate was nearly as high as in a homologous complementation control. Transformants were able to utilize nitrate and expressed a normally regulated nitrate reductase activity. To generate areA-GF- mutants, gene replacement experiments were performed using a linearized replacement vector carrying the hygromycin B phosphotransferase (hph) gene. The replacement of the zinc finger by the hygromycin cassette resulted in transformants which were unable to utilize nitrogen sources other than ammonium and glutamine, and gave significantly reduced gibberellin production yields. Complementation of such a mutant with the wild-type gene led to the full recovery of gibberellin production.

GUS transformation of the maize fungal endophyte Fusarium moniliforme

Visual markers detectable by histochemical staining have been developed for analysing the time course and tissue specificity of maize infections by Fusarium moniliforme. Three F. moniliforme strains, RRC 374, MRC 826 and RRC PAT, were transformed with a plasmid, pHPG, containing the gusA reporter gene which codes for β-glucuronidase (GUS) and the hph gene for hygromycin resistance as the selectable marker. Introduction of plasmid DNA into germinating conidia yielded 1.2 × 10 −7 transformants per conidium; expression of both gusA and hph was however, transient. Stable transformants were obtained using protoplasts as the recipient, but transformation frequency was reduced. Southern blot and PCR analyses confirmed incorporation of pHPG into the genome of all three F. moniliforme strains with gusA properly inserted in MRC 826 and RRC PAT, but apparently disrupted in RRC 374. The growth pattern for transformed F. moniliforme isolates and the parental wild types followed a sigmoid curve on minimal and enriched media. Hygromycin totally inhibited growth for wild type isolates, but not of transformants. Transformed isolates maintained the ability to infect the maize plant. Thus, this study is the first report of F. moniliforme transformed with a visibly detectable reporter gene to use for analysing this endophyte-host interaction of world-wide importance to animal and human health.

Transformation of sapstaining fungi with hygromycin B resistance plasmids pAN 7-1 and pCB1004

Protoplasts were produced by treating the yeast-like cells of O. piceae and O. quercus with high concentrations of Novozym 234 (20 mg ml−1). Transformants with resistance to hygromycin B were obtained by transforming the protoplasts with the plasmids pAN7-1 and pCB1004 containing the hygromycin B phosphotransferase (hph) gene. For all isolates tested, transformation efficiencies ranged between 103 and 105 transformants μg−1 vector DNA. High polyethylene glycol concentration (66%) increased the transformation efficiency. Integration of the hph gene into the transformant genome was verified by a polymerase chain reaction and dot DNA hybridization analysis. There was no evidence of mitotic instability of hygromycin B resistance in the transformants.

Cloning and use of theWdURA5gene as ahisGcassette selection marker for potentially disrupting multiple genes inWangiella dermatitidis

A genomic clone encoding the Wangiella dermatitidis orotidine monophosphate pyrophosphorylase gene (WdURA5) was isolated by screening a subgenomic plasmid DNA library of this phaeohyphomycotic agent using a PCR amplification product of the gene as a probe. When plasmid DNA containing the cloned WdURA5 gene was introduced by electroporation into a wdura5 auxotrophic recipient strain derived previously by selection with 5-fluoroorotic acid (5-FOA), an apparent gene repair event occurred at high frequency without any evidence of integration of the plasmid DNA. Therefore, the hygromycin B resistance gene (the hph gene) was used as a dominant selective marker for the disruption of WdURA5 to generate a new, more stable, wdura5 auxotrophic strain. Transformation of this strain was then achieved with high efficiency and high frequency by site-specific integration using WdURA5 as a selective marker. To initiate attempts to use this marker repeatedly for multiple chitin synthase (WdCHS) gene disruptions in single strains of W. dermatitidis, a hisG_WdURA5_hisG cassette was constructed and used to disrupt WdCHS2. The WdURA5 gene in the disruptant was then successfully recycled under selection for resistance to 5-FOA.