Silencing of Hygromycin Phosphotransferase (<Emphasis Type="Italic">hph</Emphasis>) Gene During Sexual Cycle and Its Reversible Inactivation in Heterokaryon of <Emphasis Type="Italic">Neurospora crassa</Emphasis>

We transformed wild-type Neurospora crassa with hph gene encoding hygromycin phosphotransferase to obtain hygromycin-resistant (Hyg^R) transformants and studied their behavior in the vegetative and sexual phases of growth. During vegetative growth in the absence of hygromycin, the hph gene was stable for at least three successive transfers with conidia. On the other hand, the behavior of the transformants in the sexual phase was different. The segregation of hph gene in the meiotic progeny was in accordance with the Mendelian ratio as inferred from PCR analysis. However, in spite of inheriting the hph gene, a proportion of the meiotic progeny failed to grow in the presence of hygromycin. This suggested that the hph gene is silenced in some progeny. The silencing effect was not confined to hph gene expression, since one-half of the meiotic progeny also showed poor conidiation. Genomic Southern analysis indicated deletions/rearrangements of the transgene in the progeny. A heterokaryon between silenced and non-silenced strains was able to grow on hygromycin-containing medium, showing that silencing was recessive. Silencing was reversed in homokaryotic nuclei extracted from such heterokaryon.Received: 2 October 2002 / Accepted: 4 November 2002     

PEG介导的猴头菌遗传转化体系的建立

对猴头菌Hericium erinaceus原生质体制备的各种因素进行比较研究,结果表明,猴头菌原生质体制备的最佳体系为：液体培养5d的猴头菌丝,以0.6mol/L KCl作为稳渗剂,加入含1.0%纤维素酶+1.0%蜗牛酶+1.0%溶壁酶的复合酶,在30℃酶解猴头菌丝3h时,原生质体得率达到3.0×106个/mL。潮霉素敏感性测试表明,猴头菌在PDSA固体培养基上的潮霉素最低筛选浓度为60μg/mL。采用PEG介导的原生质体法,将质粒pBgGI-hph（含有灵芝gpd1-Gl启动子和潮霉素抗性基因hph）转化猴头菌原生质体,经潮霉素初步筛选以及PCR鉴定,表明有4株猴头菌拟转化子的基因组扩增出hph基因;转化子经过多次转接后进行Southern杂交验证,结果表明4个转化子的基因组中均稳定整合了hph抗性基因。     

Transformation of taxol-producing endophytic fungi by restriction enzyme-mediated integration (REMI)

The REMI method was used to introduce the plasmid pV2 harboring the hygromycin B phosphotransferase (hph) gene controlled by the Aspergillus nidulans trpC promoter and the trpC terminator into a taxol-producing endophytic fungus BT2. REMI transformation yielded stable transformants capable of continuing to grow on PDA medium containing 125 mug mL(-1) hygromycin B. The transformation efficiency was about 5-6 transformants mug(-1) plasmid DNA. The presence of hph gene in transformants was confirmed by PCR and Southern blot analyses. To the authors' knowledge, this is the first report on the transformation of taxol-producing endophytic fungi by the REMI technique. This study provides an effective approach for improving taxol production of endophytic fungi by the genetic engineering of taxol biosynthetic pathway genes in the future.     

Agrobacterium tumefaciens-mediated genetic transformation of the entomopathogenic fungus Beauveria bassiana

Agrobacterium tumefaciens-mediated transformation (agro-transformation) was successfully applied to the entomogenous fungus Beauveria bassiana. Conidia of B. bassiana were transformed to hygromycin B resistance using the hph gene of Escherichia coli as the selective trait, under the control of a heterologous fungal promoter and the Aspergillus nidulans trpC terminator. The efficiency of transformation was up to 28 and 96 transformants per 10(4) and 10(5) target conidia, respectively, using three distinct vectors. High mitotic stability of the transformants (80-100%) was demonstrated after five successive transfers on a nonselective medium. Abortive transformants were observed for all the hph(r) vectors used. Putative transformants were analysed for the presence of the hph gene by PCR and Southern analysis. The latter analysis revealed the integration of two or more copies of the hph gene in the genome. The agro-transformation method was found to be effective for the isolation of B. bassiana hygromycin resistant transformants and may represent a useful tool for insertional mutagenesis studies in this fungus.     

Fate of transformed Trichoderma harzianum in the phylloplane of tomato plants

A propiconazole-resistant Trichoderma harzianum strain with high phylloplane survival capability was transformed with the E. coli hygromycin B phosphotransferase gene (hph), coding for hygromycin B resistance. Four transformants were analysed for survival ability on the phylloplane of tomato plants grown under glasshouse conditions in comparison with their prototype and a yellow, hygromycin B-sensitive mutant. Over 2 weeks, the four transformants showed higher survival rates in comparison with the wild-type strain. The yellow mutant TF3/973 did not significantly differ in survival from the transformants. Both hygromycin B resistance and mitotic stability of transformants were evaluated during growth in vitro and after reisolation from tomato phylloplane. Hybridization patterns with the complete plasmid indicated that all four transformants were mitotically stable after several rounds of vegetative growth without selective pressure and during 2 weeks on tomato plants. None of the transformants had lost the ability to grow in the presence of both propiconazole and hygromycin B after growth under the same conditions. The results are discussed in relation to risk assessment of the release of transgenic fungi.     

Reduction in solanapyrone phytotoxin production by Ascochyta rabiei transformed with Agrobacterium tumefaciens

Agrobacterium tumefaciens was used to transform Ascochyta rabiei, the causal agent of chickpea blight. Employing a T-DNA containing a hygromycin resistance gene (hph), 908 transformants were obtained from germinated pycnidiospores on a selective medium containing hygromycin. Transformants were confirmed using PCR and Southern analyses and of four of these that were tested, two had integrated multicopies of the hph gene, one had two copies and one had a single insertion. Transformants were tested for the production of solanapyrone A toxin using a microtitre plate assay. Loss of toxin production by transformants was confirmed by reversed phase high-performance liquid chromatography. Sixteen transformants out of 668 tested produced significantly less solanapyrone A than the wild-type strain.     

Transformation of Aspergillus nidulans with the hygromycin-resistance gene, hph

Aspergillus nidulans strain G191 was transformed to hygromycin resistance using plasmid pDH25, which contains the bacterial hygromycin B phosphotransferase gene (hph) fused to promoter elements of the A. nidulans trpC gene. Southern hybridizations of transformants revealed multiple, integrated copies of the vector. A pleiotropic effect conferring increased hygromycin B sensitivity was found to be associated with the A. nidulans pyrG89 allele. Plasmid pDH25 features a ClaI site immediately preceding the hph start codon thus permitting convenient replacement of the trpC sequences with other eukaryotic promoters.     

Expression of the Escherichia coli beta-glucuronidase gene in Pseudocercosporella herpotrichoides.

The plant-pathogenic fungus Pseudocercosporella herpotrichoides has been successfully transformed by using two different positive selection systems in combination with the Escherichia coli gusA gene. The selectable markers used in this study were the hygromycin B phosphotransferase gene (hph) from E. coli and the gene (bml) for beta-tubulin from a benomyl-resistant mutant of Neurospora crassa. A lower transformation rate was obtained with the bml system than with the hph system. Conversely, cotransformation frequencies, as determined with medium plates containing the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid, were higher with bml than with hph as the selectable marker. The hygromycin-resistant transformants were mitotically stable, and both the selectable gene and gusA were maintained through conidiation. The vector DNA was integrated into the genome, and the number and sites of insertion varied among transformants. Enzyme assays of mycelial extracts showed that beta-glucuronidase activity was highest in transformants with a high gusA copy number. Expression of gusA during growth of the fungus on plants was easily detectable and did not affect pathogenicity. These results form the basis for construction of a versatile and sensitive reporter gene system for P. herpotrichoides.     

Expression of the Escherichia coli beta-glucuronidase gene in Pseudocercosporella herpotrichoides.

The plant-pathogenic fungus Pseudocercosporella herpotrichoides has been successfully transformed by using two different positive selection systems in combination with the Escherichia coli gusA gene. The selectable markers used in this study were the hygromycin B phosphotransferase gene (hph) from E. coli and the gene (bml) for beta-tubulin from a benomyl-resistant mutant of Neurospora crassa. A lower transformation rate was obtained with the bml system than with the hph system. Conversely, cotransformation frequencies, as determined with medium plates containing the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid, were higher with bml than with hph as the selectable marker. The hygromycin-resistant transformants were mitotically stable, and both the selectable gene and gusA were maintained through conidiation. The vector DNA was integrated into the genome, and the number and sites of insertion varied among transformants. Enzyme assays of mycelial extracts showed that beta-glucuronidase activity was highest in transformants with a high gusA copy number. Expression of gusA during growth of the fungus on plants was easily detectable and did not affect pathogenicity. These results form the basis for construction of a versatile and sensitive reporter gene system for P. herpotrichoides.     

丝状真菌瑞氏木霉外源基因表达系统的构建

采用PCR技术体外扩增获得了瑞氏木霉外切葡聚糖纤维二糖水解酶Ⅰ (CBHⅠ )启动子和终止子序列 .并以大肠杆菌质粒pUC1 9为骨架 ,在该启动子和终止子序列间加入多克隆位点 ,构建了瑞氏木霉强表达整合型载体pTRIL .以质粒pAN7 1为模板 ,体外扩增了带有潮霉素磷酸转移酶(hph)基因的DNA片段 ,将hph插入pTRIL的cbh1启动子和终止子序列之间 ,构建了Pcbh1 hph Tcbh1表达盒 .用此表达盒转化瑞氏木霉C30原生质体 ,在潮霉素平板上得到 1 5株抗性转化子 .对其中的H1转化子进行了PCR和Southern印迹分析 ,证实hph基因确实整合到转化子染色体DNA上 ,并在Pcbh1 启动子控制下进行高效表达 .转化子H1对潮霉素抗性达 1 5 0mg L ,比出发菌株提高 2倍 .瑞氏木霉强表达整合型载体pTRIL的构建成功为开展瑞氏木霉分子生物学研究以及进一步的工程菌株构建工作奠定了基础     

Improving the efficiency of gene replacements in Neurospora crassa: a first step towards a large-scale functional genomics project

Here, we report the use of the mating type heterokaryon incompatibility system as a counterselection to increase the probability of identifying gene replacements in Neurospora crassa. We compared the frequencies of gene replacements observed among transformants obtained by using plasmids with or without the mat a-1(+) gene (hereby called "Toxic Gene") placed adjacent to disruption cassettes. On an average, we were 20x more likely to identify a correct gene replacement by incorporating the toxic gene in our constructs. Using this strategy, we constructed strains containing a deletion of the inl (1L-myo-inositol-1-phosphate synthase) gene. Finally, we demonstrated that we were able to remove the transformation marker (the hygromycin B phosphotransferase- thymidine kinase gene fusion [hph(+)::tk(+)]) from the genome by using a strategy similar to the "URA-blaster" strategy used in yeast, which we call "tk-blaster."     

Stable transformation of Pleurotus ostreatus to hygromycin B resistance using Lentinus edodes GPD expression signals

It was reported that Pleurotus ostreatus was transformed unstably using recombinant plasmids containing a hygromycin B phosphotransferase gene (hph) under the control of Aspergillus nidulans expression signals, and that the plasmids were maintained extrachromosomally in the transformants. Here we report a stable and integrative transformation of the fungus to hygromycin B resistance, using a recombinant hph fused with Lentinus edodes glyceraldehyde-3-phosphate dehydrogenase expression signals. Restriction-enzyme-mediated integration (REMI) was also tried and increased the transformation efficiency about ten-fold.     

Transformation of Aspergillus based on the hygromycin B resistance marker from Escherichia coli

A new, heterologous, dominant marker for selection of Aspergillus transformants is described. This marker is based on the Escherichia coli hygromycin B (HmB) phosphotransferase gene (hph). Expression of the hph gene is controlled by A. nidulans gpd and trpC expression signals. An Aspergillus transformation vector was constructed which contains this marker and confers HmB resistance to Aspergillus species. With both A. niger and A. nidulans, transformation frequencies of 5-20 transformants per micrograms vector DNA were obtained. Cotransformation with other vectors was shown to be very efficient in both species, when selection for HmB resistance was applied.     

Agrobacterium tumefaciens-mediated transformation of the plant pathogenic fungus Rosellinia necatrix

Rosellinia necatrix is a soil-borne root pathogen affecting a wide range of commercially important plant species. The mycelium of R. necatrix was transformed to hygromycin B resistance by an Agrobacterium tumefaciens-mediated transformation system using a binary plasmid vector containing the hygromycin B phosphotransferase (hph) gene controlled by the heterologous fungal Aspergillus nidulans P-gpd (glyceraldehyde 3-phosphate dehydrogenase) promoter and the trpC terminator. Co-cultivation of R. necatrix strain W1015 and A. tumefaciens strain AGL-1 at 25 degrees C using the binary vector pAN26-CB1300, which contained the hygromycin B resistance cassette based on pAN26 and pCAMBIA1300, resulted in high frequencies of transformation. The presence of the hph gene in the transformants was detected by PCR, and single-copy integration of the marker gene was demonstrated by Southern blot analysis. This report of an Agrobacterium-mediated transformation method should allow the development of T-DNA tagging as a system f or insertional mutagenesis in R necatrix and provide a simple and reliable method for genetic manipulation.     

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.     

Thermoadaptation of a mesophilic hygromycin B phosphotransferase by directed evolution in hyperthermophilic Archaea: selection of a stable genetic marker for DNA transfer into Sulfolobus solfataricus

A mutated version of the hygromycin B phosphotransferase (hph(mut)) gene from Escherichia coli, isolated by directed evolution at 75 degrees C in transformants of a thermophilic strain of Sulfolobus solfataricus, was characterized with respect to its genetic stability in both the original mesophilic and the new thermophilic hosts. This gene was demonstrated to be able to express the hygromycin B resistance phenotype and to be steadily maintained and propagated also in other, more thermophilic strains of S. solfataricus, i.e., up to 82 degrees C. Furthermore, it may be transferred to S. solfataricus cells by cotransformation with pKMSD48, another extrachromosomal element derived from the virus SSV1 of Sulfolobus shibatae, without any loss of stability and without affecting the replication and infectivity of this viral DNA. The hph(mut) and the wild-type gene products were expressed at higher levels in E. coli and purified by specific affinity chromatography on immobilized hygromycin B. Comparative characterization revealed that the mutant enzyme had acquired significant thermoresistance and displayed higher thermal activity with augmented catalytic efficiency.     

Ectopic Integration of Transforming DNA Is Rare among Neurospora Transformants Selected for Gene Replacement

In a variety of organisms, DNA-mediated transformation experiments commonly produce transformants with multiple copies of the transforming DNA, including both selected and unselected molecules. Such ``cotransformants' are much more common than expected from the individual transformation frequencies, suggesting that subpopulations of cells, or nuclei, are particularly competent for transformation. We found that Neurospora crassa transformants selected for gene replacement at the am gene had not efficiently incorporated additional DNA, suggesting that nuclei that undergo transformation by homologous recombination are not highly competent at integration of DNA by illegitimate recombination. Spheroplasts were treated with DNA fragments homologous to am and with an Escherichia coli hph plasmid. Transformants were initially selected for hph (hygromycin(R)), allowed to conidiate to generate homokaryons and then selected for either Am(-) (gene replacements) or hph. Surprisingly, most am replacement strains were hygromycin(S) (124/140) and carried no extraneous DNA (116/140). Most transformants selected for hph also had ectopic copies of am DNA and/or multiple copies of hph sequences (32/35), generally at multiple sites, confirming that efficient cotransformation could occur. To test the implication that cotransformation involving gene replacement and ectopic integration is rare, we compared the yields of am replacement strains with or without prior selection for hph. The initial selection did not appreciably help (or hinder) recovery of strains with replacements.