Tool For Insertional Mutagenesis Research Articles

Agrobacterium tumefaciens-Mediated Transformation Method for Fusarium oxysporum.

Agrobacterium tumefaciens-mediated transformation (ATMT) is becoming a popular effective system as an insertional mutagenesis tool in filamentous fungi. An efficient Agrobacterium tumefaciens-mediated transformation approach was developed for the plant pathogenic fungus, F. oxysporum, the causal agent of Apple replant disease (ARD) in China. Four parameters were selected to optimize efficiencies of transformation. A. tumefaciens concentration, conidial concentration of F. oxysporum, and co-culture temperature and time have a significant influence on all parameters. Transformants emit green fluorescence under fluorescence microscopy. The integration of a mitotically stable hygromycin resistance gene (hph) in the genome is confirmed by PCR. The transformation efficiency can reach up to 300 transformants per 106 conidia under optimal conditions. This ATMT method is an efficient tool for insertional mutagenesis of F. oxysporum.

Development of an Agrobacterium tumefaciens-mediated transformation system for Tilletia controversa Kühn

Dwarf bunt of wheat caused by Tilletia controversa Kühn has been identified an international quarantine disease, which replace the grain material into millions of teliospores. Agrobacterium tumefaciens-mediated transformation (ATMT) system is a powerful tool for fungi transformation with significant advantages of simple operation, high efficiency, and genetic stability of transformants. In this study, we constructed ATMT system for T. controversa. All the transformants were tested using Acetosyringone (AS) concentration at 150 μmol/l, hygromycin B at 25 μg/ml, 1 × 106 T. controversa hypha cells/ml, A. tumefaciens with OD600 of 0.5 co-cultivation at 16 °C for 48 h and culture was incubated at 16 °C for 20 days. Using the ATMT method, we cultivated 8 generations of transformants on complete medium (CM) containing hygromycin B antibiotic and validated by PCR, which indicate that T-DNA had been successfully inserted into each of T. controversa transformants. In addition, thermal asymmetric interlaced PCR (TAIL-PCR) evaluated the Ti element inserts were at random sites in the fungal genome. Thus, ATMT approach is an efficient tool for insertional mutagenesis of T. controversa.

PiggyBac transposon-mediated mutagenesis and application in yeast Komagataella phaffii.

Around one-fourth of the Komagataella phaffii genes encode hypothetical proteins with unknown functions. However, lack of powerful tools for genetic screening in K. phaffii significantly limits the functional analysis of these unknown genes. Transposon mutagenesis has been utilized as an insertional mutagenesis tool in many other organisms and would be extremely valuable if it could be applied in K. phaffii. In this study, we investigated in K. phaffii the transposition activity and efficiency of piggyBac (PB) transposon, a DNA transposon from the cabbage looper moth Trichoplusia ni through the integrated-plasmid system. We also designed a binary-plasmid system which could generate stable mutants. Finally we evaluated the quality of this mutagenesis system by a simple screening for functional genes involved in K. phaffii carbon catabolite repression. Our results demonstrate that PB-mediated mutagenesis could be a feasible and useful tool for functional gene screening in K. phaffii.

Agrobacterium tumefaciens-mediated transformation as an efficient tool for insertional mutagenesis of Kabatiella zeae

Agrobacterium tumefaciens-mediated transformation (ATMT) has been widely used in filamentous fungi. In this study, an efficient Agrobacterium tumefaciens-mediated transformation approach was developed for the plant pathogenic fungus, Kabatiella zeae, the causative pathogen of eyespot in maize. Five parameters were selected to optimize efficiencies of transformation. A. tumefaciens concentration, conidia concentration of K. zeae and mixing ratio of A. tumefaciens and K. zeae were found to exert a significant influence on all parameters. Transformants emitted green fluorescence under fluorescence microscopy. The presence of mitotically stable hygromycin resistance gene (hph) integration in the genome was confirmed by PCR. Up to 148 transformants per 107 conidia could be obtained under optimal conditions. In this way, ATMT approach is an efficient tool for insertional mutagenesis of K. zeae.

Integration of ALV into CTDSPL and CTDSPL2 genes in B-cell lymphomas promotes cell immortalization, migration and survival.

Avian leukosis virus induces tumors in chickens by integrating into the genome and altering expression of nearby genes. Thus, ALV can be used as an insertional mutagenesis tool to identify novel genes involved in tumorigenesis. Deep sequencing analysis of viral integration sites has identified CTDSPL and CTDSPL2 as common integration sites in ALV-induced B-cell lymphomas, suggesting a potential role in driving oncogenesis. We show that in tumors with integrations in these genes, the viral promoter is driving the expression of a truncated fusion transcript. Overexpression in cultured chick embryo fibroblasts reveals that CTDSPL and CTDSPL2 have oncogenic properties, including promoting cell migration. We also show that CTDSPL2 has a previously uncharacterized role in protecting cells from apoptosis induced by oxidative stress. Further, the truncated viral fusion transcripts of both CTDSPL and CTDSPL2 promote immortalization in primary cell culture.

Agrobacterium tumefaciens-mediated transformation as an efficient tool for insertional mutagenesis of Cercospora zeae-maydis

An efficient Agrobacterium tumefaciens-mediated transformation (ATMT) approach was developed for the plant pathogenic fungus, Cercospora zeae-maydis, which is the causative agent of gray leaf spot in maize. The transformation was evaluated with five parameters to test the efficiencies of transformation. Results showed that spore germination time, co-cultivation temperature and time were the significant influencing factors in all parameters. Randomly selected transformants were confirmed and the transformants were found to be mitotically stable, with single-copy T-DNA integration in the genome. T-DNA flanking sequences were cloned by thermal asymmetric interlaced PCR. Thus, the ATMT approach is an efficient tool for insertional mutagenesis of C. zeae-maydis.

Agrobacterium tumefaciens-mediated transformation: An efficient tool for insertional mutagenesis and targeted gene disruption in Harpophora oryzae

The endophytic filamentous fungus Harpophora oryzae is a beneficial endosymbiont isolated from the wild rice. H. oryzae could not only effectively improve growth rate and biomass yield of rice crops, but also induce systemic resistance against the rice blast fungus, Magnaporthe oryzae. In this study, Agrobacterium tumefaciens-mediated transformation (ATMT) was employed and optimized to modify the H. oryzae genes by either random DNA fragment integration or targeted gene replacement. Our results showed that co-cultivation of H. oryzae conidia with A. tumefaciens in the presence of acetosyringone for 48h at 22°C could lead to a relatively highest frequency of transformation, and 200μM acetosyringone (AS) pre-cultivation of A. tumefaciens is also suggested. ATMT-mediated knockout mutagenesis was accomplished with the gene-deletion cassettes using a yeast homologous recombination method with a yeast–Escherichia–Agrobacterium shuttle vector pKOHo. Using the ATMT-mediated knockout mutagenesis, we successfully deleted three genes of H. oryzae (HoATG5, HoATG7, and HoATG8), and then got the null mutants ΔHoatg5, ΔHoatg7, and ΔHoatg8. These results suggest that ATMT is an efficient tool for gene modification including randomly insertional mutagenesis and gene deletion mutagenesis in H. oryzae.

Gene transfer and genome-wide insertional mutagenesis by retroviral transduction in fish stem cells.

Retrovirus (RV) is efficient for gene transfer and integration in dividing cells of diverse organisms. RV provides a powerful tool for insertional mutagenesis (IM) to identify and functionally analyze genes essential for normal and pathological processes. Here we report RV-mediated gene transfer and genome-wide IM in fish stem cells from medaka and zebrafish. Three RVs were produced for fish cell transduction: rvLegfp and rvLcherry produce green fluorescent protein (GFP) and mCherry fluorescent protein respectively under control of human cytomegalovirus immediate early promoter upon any chromosomal integration, whereas rvGTgfp contains a splicing acceptor and expresses GFP only upon gene trapping (GT) via intronic in-frame integration and spliced to endogenous active genes. We show that rvLegfp and rvLcherry produce a transduction efficiency of 11~23% in medaka and zebrafish stem cell lines, which is as 30~67% efficient as the positive control in NIH/3T3. Upon co-infection with rvGTgfp and rvLcherry, GFP-positive cells were much fewer than Cherry-positive cells, consistent with rareness of productive gene trapping events versus random integration. Importantly, rvGTgfp infection in the medaka haploid embryonic stem (ES) cell line HX1 generated GTgfp insertion on all 24 chromosomes of the haploid genome. Similar to the mammalian haploid cells, these insertion events were presented predominantly in intergenic regions and introns but rarely in exons. RV-transduced HX1 retained the ES cell properties such as stable growth, embryoid body formation and pluripotency gene expression. Therefore, RV is proficient for gene transfer and IM in fish stem cells. Our results open new avenue for genome-wide IM in medaka haploid ES cells in culture.

Identification of pathogenicity-related genes in the rice pathogen Ustilaginoidea virens through random insertional mutagenesis

Agrobacterium tumefaciens-mediated transformation (ATMT) is becoming a popular effective system as an insertional mutagenesis tool in filamentous fungi. To gain more insight into the cellular and molecular mechanisms in the pathogenesis of Ustilaginoidea virens, the causal agent of rice false smut disease, a T-DNA insertion mutant library of U. virens was established using ATMT. We optimized a range of conditions to improve the transformation efficiency. Transformants were most effectively obtained when the optimal co-cultivation time is 72h, with 50μM AS in medium and 100μl A. tumefaciens for co-cultivation, leading to the production of 160–185 hygromycin B resistant transformants per 1×105 conidia. Southern blot analysis indicated that 58.14% of transformants had a single T-DNA copy. Among 5600 transformants tested for virulence, 37 mutants with reproducible pathogenic defects were obtained. The flanking sequences of three avirulent tranformants (B20, B1015 and B1465) and two pathogenicity-reduced transformants (B726 and B785) were amplified by high-efficiency thermal asymmetric interlaced PCR. Sequence analyses revealed that single T-DNA insertion in mutant B20 targeted the coding region of a gene encoding a protein highly similar to SUN family protein, and in mutant B726 targeted upstream of a gene with unknown function. The two T-DNA insertion sites in mutant B785 were found in the coding region of a gene encoding C6 transcription factor, but failed in amplified flanking sequence of another T-DNA. Chromosomal rearrangement occurred in the genome of mutant B1016 and B1465 with single T-DNA insertion. Among avirulent mutants, B20 showed altered colony growth and pigmentation. The T-DNA insert in B20 was detected in the coding region of a gene named UvSUN2. Morphophysiological characterization analysis suggested that UvSUN2 might be a virulence factor, and possibly required for proper fungal growth, cell wall construction, and stress responses in U. virens. This study optimize and validate the transformation procedure, maximizing the number of single copy transformants and developing an efficient procedure for rescuing adjacent host sequences along with the inserted T-DNA. This is the first report of identification several candidate virulence factors and validated one in U. virens. Together with identification of avirulent mutants and their associated genes, results suggested that ATMT can effectively be used to identify genes associated with pathogenicity in U. virens, and provided novel insights into molecular mechanisms underlying virulence of this pathogen.

Pseudotyped murine leukemia virus for schistosome transgenesis: approaches, methods and perspectives

Draft genome sequences for the human schistosomes, Schistosoma japonicum, S. mansoni and S. haematobium are now available. The schistosome genome contains ~11,000 protein encoding genes for which the functions of few are well understood. Nonetheless, the newly described gene products and novel non-coding RNAs represent potential intervention targets, and molecular tools are being developed to determine their importance. Over the past decade, noteworthy advances has been reported towards development of tools for gene manipulation of schistosomes, including gene expression perturbation by RNAi, and transient and stable transfection including transgenesis mediated by genome integration competent vectors. Retrovirus-mediated transgenesis is an established functional genomic approach for model species. It offers the means to establish gain- or loss-of-function phenotypes, supports vector-based RNA interference, and represents a powerful forward genetics tool for insertional mutagenesis. Murine leukemia virus (MLV) pseudotyped with vesicular stomatitis virus glycoprotein mediates somatic transgenesis in S. mansoni, and vertical transmission of integrated transgenes in S. mansoni has been demonstrated, leading the establishment of transgenic lines. In addition, MLV transgenes encoding antibiotic resistance allow the selection of MLV-transduced parasites with the appropriate antibiotics. Here we describe detailed methods to produce and quantify pseudotyped MLV particles for use in transducing developmental stages of schistosomes. Approaches to analyze MLV-transduced schistosomes, including qPCR and high throughput approaches to verify and map genome integration of transgenes are also presented. We anticipate these tools should find utility in genetic investigations in other laboratories and for other helminth pathogens of important neglected tropical diseases.

The use of T-DNA tagging to isolate mutants ofColletotrichum gloeosporioidesandColletotrichum acutatumwith reduced virulence againstHevea brasiliensis

Agrobacterium tumefaciens-mediated transformation (ATMT) is being increasingly recognized as an effective insertional mutagenesis tool in studies of filamentous fungi for gene discovery and functional analysis. We developed and optimized ATMT for 2 Colletotrichum species, Colletotrichum gloeosporioides and Colletotrichum acutatum, the causative agents of Colletotrichum leaf disease in rubber trees in Southern China. A. tumefaciens strain AGL-1 carrying an ILV1 gene and a green fluorescent protein gene were used to transform the conidia of these 2 Colletotrichum species. The transformation efficiency was correlated with the co-cultivation duration and bacterial cell concentrations, which reached 300–400 transformants per 1 × 106 conidia after optimization. Southern blot analysis indicated that about 60.0% of the C. gloeosporioides transformants and 46.2% of the C. acutatum transformants had a single copy of T-DNA in their genomes. Fungal genomic DNA segments flanking the T-DNA were identified in the transformants through thermal asymmetrical interlaced polymerase chain reaction followed by sequencing. The flanking sequences from 4 C. acutatum and 7 C. gloeosporioides transformants showed moderate or weak homology to the NCBI database entries. Some sequences matching those reported virulence-related genes. The results suggest that the T-DNA inserted mutants banks constructed are useful for the discovery of new or important genes and to elucidate their function in the 2 Colletotrichum species.

Sleeping Beauty transposon system harboring HRAS, c-Myc and shp53 induces sarcomatoid carcinomas in mouse skin

The Sleeping Beauty transposon system is used as a tool for insertional mutagenesis and oncogenesis. However, little is known about the exact histological phenotype of the tumors induced. Thus, we used immunohistochemical markers to enable histological identification of the type of tumor induced by subcutaneous injection of the HRAS, c-Myc and shp53 oncogenes in female C57BL/6 mice. The tumor was removed when it reached 100 mm3 in volume. Subsequently, we used 13 immunohistochemical markers to histologically identify the tumor type. The results suggested that the morphology of the tumor was similar to that of sarcomatoid carcinoma.

Agrobacterium tumefaciens-mediated transformation of the soybean pathogen Phomopsis longicolla

To facilitate functional genomics in the soybean pathogen Phomopsis longicolla, we developed a robust Agrobacterium tumefaciens-mediated transformation system that yielded 150–250 transformants per 1×106 conidia of P. longicolla. This first report of P. longicolla transformation provides a useful tool for insertional mutagenesis in an increasingly important pathogen of soybean.

Genetic transformation of Diaporthe phaseolorum, an endophytic fungus found in mangrove forests, mediated by Agrobacterium tumefaciens

We describe the genetic transformation of the mycelial tissue of Diaporthe phaseolorum, an endophytic fungus isolated from the mangrove species Laguncularia racemosa, using Agrobacterium tumefaciens-mediated transformation (ATMT). ATMT uses both the hygromycin B resistant (hph) gene and green fluorescent protein as the selection agents. The T-DNA integration into the fungal genome was assessed by both PCR and Southern blotting. All transformants examined were mitotically stable. An analysis of the T-DNA flanking sequences by thermal asymmetric interlaced PCR (TAIL-PCR) demonstrated that the disrupted genes in the transformants had similarities with conserved domains in proteins involved in antibiotic biosynthesis pathways. A library of 520 transformants was generated, and 31 of these transformants had no antibiotic activity against Staphylococcus aureus, an important human pathogen. The protocol described here, using ATMT in D. phaseolorum, will be useful for the identification and analysis of fungal genes controlling pathogenicity and antibiotic pathways. Moreover, this protocol may be used as a reference for other species in the Diaporthe genus. This is the first report to describe Agrobacterium-mediated transformation of D. phaseolorum as a tool for insertional mutagenesis.

Recent Progress in Development of Tnt1 Functional Genomics Platform for Medicago truncatula and Lotus japonicus in Bulgaria

Legumes, as protein-rich crops, are widely used for human food, animal feed and vegetable oil production. Over the past decade, two legume species, Medicago truncatula and Lotus japonicus, have been adopted as model legumes for genomics and physiological studies. The tobacco transposable element, Tnt1, is a powerful tool for insertional mutagenesis and gene inactivation in plants. A large collection of Tnt1-tagged lines of M. truncatula cv. Jemalong was generated during the course of the project ‘GLIP’: Grain Legumes Integrated Project, funded by the European Union (www.eugrainlegumes.org). In the project ‘IFCOSMO’: Integrated Functional and COmparative genomics Studies on the MOdel Legumes Medicago truncatula and Lotus japonicus, supported by a grant from the Ministry of Education, Youth and Science, Bulgaria, these lines are used for development of functional genomics platform of legumes in Bulgaria. This review presents recent advances in the evaluation of the M. truncatula Tnt1 mutant collection and outlines the steps that are taken in using the Tnt1-tagging for generation of a mutant collection of the second model legume L. japonicus. Both collections will provide a number of legume-specific mutants and serve as a resource for functional and comparative genomics research on legumes. Genomics technologies are expected to advance genetics and breeding of important legume crops (pea, faba bean, alfalfa and clover) in Bulgaria and worldwide.

Efficient insertional mutagenesis system for the dimorphic pathogenic fungus Sporothrix schenckii using Agrobacterium tumefaciens

Sporothrix schenckii is a dimorphic pathogenic fungus that causes human and animal sporotrichosis globally. Here we developed and optimized an Agrobacterium tumefaciens-mediated transformation (ATMT) system of S. schenckii for insertional mutagenesis. The transformation efficiency reached more than 600 transformants per 106 conidia. Using this protocol enabled us to obtain a large number of T-DNA insertional mutants within a short experimental period. Several mutants with altered phenotypes were obtained during the transformation experiments. The mutants displayed mitotic stability. Transferred DNA (T-DNA) flanking sequences were cloned by thermal asymmetric interlaced PCR (TAIL-PCR). Our results demonstrated that the ATMT system can be an effective tool for insertional mutagenesis in S. schenckii. This is the first report of a suitable mutagenesis system which may provide valuable mutants and information for both forward and reverse genetics research in the future for this medically important fungus.

A piggyBac transposon-based mutagenesis system for the fission yeast Schizosaccharomyces pombe

The TTAA-specific transposon piggyBac (PB), originally isolated from the cabbage looper moth, Trichoplusia ni, has been utilized as an insertional mutagenesis tool in various eukaryotic organisms. Here, we show that PB transposes in the fission yeast Schizosaccharomyces pombe and leaves almost no footprints. We developed a PB-based mutagenesis system for S. pombe by constructing a strain with a selectable transposon excision marker and an integrated transposase gene. PB transposition in this strain has low chromosomal distribution bias as shown by deep sequencing-based insertion site mapping. Using this system, we obtained loss-of-function alleles of klp5 and klp6, and a gain-of-function allele of dam1 from a screen for mutants resistant to the microtubule-destabilizing drug thiabendazole. From another screen for cdc25-22 suppressors, we obtained multiple alleles of wee1 as expected. The success of these two screens demonstrated the usefulness of this PB-mediated mutagenesis tool for fission yeast.

Agrobacterium tumefaciens-mediated transformation as a tool for insertional mutagenesis in medicinal fungus Cordyceps militaris

Cordyceps militaris is an insect-born fungus with various biological and pharmacological activities. The mutant library of C. militaris was constructed by improved Agrobacterium tumefaciens-mediated transformation (ATMT), for the ultimate identification of genes involved in isolate degeneration during fruiting body production. Successful transformation of C. militaris JM4 by A. tumefaciens AGL-1 carrying vector pATMT1 was performed, with efficiency in the range of 30–600 transformants per 1 × 10 5 conidia. Acetosyringone (AS) supplement in C. militaris ATMT was not necessary during either precultivation or cocultivation. The transformation procedure was optimised based on the ratios between donor A. tumefaciens and recipient conidia, and pH value of cocultivation media. The integration of the hyg gene into C. militaris genome was determined by PCR and Southern blot analysis, suggesting that 67–88 % resulting transformants in cultivation conditions with or without AS were inserted by T-DNA and 55–80 % were single-copy. Special mutants with altered phenotypes and growth potentials were characterised. The efficient TAIL-PCR approach was established for identifying T-DNA flanking sequences from C. militaris mutants. The successful construction of the mutant library indicated the usefulness of this approach for functional genetic analysis in this important fungus.

Sleeping beauty-mediated somatic mutagenesis implicates CSF1 in the formation of high-grade astrocytomas.

The Sleeping Beauty (SB) transposon system has been used as an insertional mutagenesis tool to identify novel cancer genes. To identify glioma-associated genes, we evaluated tumor formation in the brain tissue from 117 transgenic mice that had undergone constitutive SB-mediated transposition. Upon analysis, 21 samples (18%) contained neoplastic tissue with features of high-grade astrocytomas. These tumors expressed glial markers and were histologically similar to human glioma. Genomic DNA from SB-induced astrocytoma tissue was extracted and transposon insertion sites were identified. Insertions in the growth factor gene Csf1 were found in 13 of the 21 tumors (62%), clustered in introns 5 and 8. Using reverse transcription-PCR, we documented increased Csf1 RNAs in tumor versus adjacent normal tissue, with the identification of transposon-terminated Csf1 mRNAs in astrocytomas with SB insertions in intron 8. Analysis of human glioblastomas revealed increased levels of Csf1 RNA and protein. Together, these results indicate that SB-insertional mutagenesis can identify high-grade astrocytoma-associated genes and they imply an important role for CSF1 in the development of these tumors.

Expression-independent gene trap vectors for random and targeted mutagenesis in embryonic stem cells.

Promoterless gene trap vectors have been widely used for high-efficiency gene targeting and random mutagenesis in embryonic stem (ES) cells. Unfortunately, such vectors are only effective for genes expressed in ES cells and this has prompted the development of expression-independent vectors. These polyadenylation (poly A) trap vectors employ a splice donor to capture an endogenous gene's polyadenylation sequence and provide transcript stability. However, the spectrum of mutations generated by these vectors appears largely restricted to the last intron of target loci due to nonsense-mediated mRNA decay (NMD) making them unsuitable for gene targeting applications. Here, we present novel poly A trap vectors that overcome the effect of NMD and also employ RNA instability sequences to improve splicing efficiency. The set of random insertions generated with these vectors show a significantly reduced insertional bias and the vectors can be targeted directly to a 5′ intron. We also show that this relative positional independence is linked to the human β-actin promoter and is most likely a result of its transcriptional activity in ES cells. Taken together our data indicate that these vectors are an effective tool for insertional mutagenesis that can be used for either gene trapping or gene targeting.