• Journal of Microbiology
• /
• v.43 no.5
• /
• pp.417-423
• /
• 2005

In this study, the nematode-trapping fungus, Monacrosporium sphaeroides, was transformed with a plasmid harboring the hygromycin B phosphotransferase gene, via restriction enzyme-mediated integration (REMI). Frequencies of up to 94 transformants ${\mu}g^{-1}$ per linearized plasmid DNA were obtained by optimizing the PEG concentration, as well as the category and quantity of the added restriction enzyme. $90\%$ of the transformants were determined to be stable for drug resistance when 20 randomly selected transformants were tested. Southern analyses revealed that the transforming DNA was integrated into the M. sphaeroides genome either with or without rearrangement. Five mitotic stable mutant strains were obtained using this approach, all of which had been altered with regard to sporulation capacity and pathogenicity toward nematodes. Southern blot analyses of the five mutants revealed that foreign plasmid DNA had integrated into the genome. Three of the mutants, Tms2316, Tms3583 and Tms1536, exhibited integration at a single location, whereas the remaining two, Tms32 and Tms1913, manifested integration at double or multiple locations. Our results suggest that the transformation of M. sphaeroides via REMI will facilitate insertional mutagenesis, the functional analysis of a variety of genes, and the tagging or cloning of genes of interest.

• Proceedings of the Microbiological Society of Korea Conference
• /
• 2005.05a
• /
• pp.217.2-217
• /
• 2005

• Mycobiology
• /
• v.31 no.1
• /
• pp.32-35
• /
• 2003

Restriction enzyme-mediated integration(REMI) was used to transform uracil auxotrophs of Pleurotus ostreatus to prototrophy. When protoplasts of Pleurotus ostreatus were treated by the reaction mixture containing 10 units of BamHI, the frequency of REMI was about 64 transformants per 1 ${\mu}g$ of DNA. This efficiency was increased by 14.2 times compared with that of the conventional PEG transformation. The optimal condition for REMI of P. ostreatus was achieved when 1 ${\mu}g$ of linearized pTRura3-2 DNA was added into $1{\times}10^7$ protoplasts along with 10 units BamHI. Southern blot analysis revealed that about 50% of transformants examined were caused by REMI event and 30% carried single copy insertion at the genome. This suggested that the REMI method might be a useful tool for efficient transformation and tagging mutagenesis of P. ostreatus.

• Mycobiology
• /
• v.43 no.1
• /
• pp.1-8
• /
• 2015

Mushroom transformation requires a series of experimental steps, including generation of host strains with a desirable selective marker, design of vector DNA, removal of host cell wall, introduction of foreign DNA across the cell membrane, and integration into host genomic DNA or maintenance of an autonomous vector DNA inside the host cell. This review introduces limitations and obstacles related to transformation technologies along with possible solutions. Current methods for cell wall removal and cell membrane permeabilization are summarized together with details of two popular technologies, Agrobacterium tumefaciens-mediated transformation and restriction enzyme-mediated integration.

• Journal of Microbiology and Biotechnology
• /
• v.27 no.2
• /
• pp.306-315
• /
• 2017

Metabolic engineering with a high-yielding mutant, A. terreus AN37, was performed to enhance the production of itaconic acid (IA). Reportedly, the gene cluster for IA biosynthesis is composed of four genes: reg (regulator), mtt (mitochondrial transporter), cad (cis-aconitate decarboxylase), and mfs (membrane transporter). By overexpressing each gene of the IA gene cluster in A. terreus AN37 transformed by the restriction enzyme-mediated integration method, several transformants showing high productivity of IA were successfully obtained. One of the AN37/cad transformants could produce a very high amount of IA (75 g/l) in shake-flask cultivations, showing an average of 5% higher IA titer compared with the high-yielding control strain. Notably, in the case of the mfs transformants, a maximal increase of 18.3% in IA production was observed relative to the control strain under the identical fermentation conditions. Meanwhile, the overexpression of reg and mtt genes showed no significant improvements in IA production. In summary, the overexpressed cis-aconitate decarboxylase (CAD) and putative membrane transporter (MFS) appeared to have positive influences on the enhanced IA productivity of the respective transformant. The maximal increases of 13.6~18.3% in IA productivity of the transformed strains should be noted, since the parallel mother strain used in this study is indeed a very high-performance mutant that has been obtained through intensive rational screening programs in our laboratory.

• The Plant Pathology Journal
• /
• v.25 no.3
• /
• pp.205-212
• /
• 2009

Colletotrichum acutatum was the main cause of the recent outbreaks of anthracnose on pepper fruit in Korea. To facilitate molecular analysis of C. acutatum, we generated an arginine auxotrophic mutant of the C acutatum strain JC24 using a targeted gene replacement strategy. A 3.3-kb genomic region carrying an ortholog (designated CaARG2) of the fungal gene encoding N-acetylglutamate synthase, the first enzyme of arginine biosynthesis in fungi, was deleted from the fungal genome. The mutant exhibited normal growth only when arginine was exogenously supplied into the culture medium. Transformation of the arginine auxotrophic mutant with a plasmid DNA carrying an intact copy of CaARG2, which was smaller than the deleted region in the mutant, not only caused random vector insertions in the fungal genome, but also recovered both hyphal growth and pathogenicity of the mutant to the wild-type level. Using this new selection system, we have successfully developed a restriction enzyme-mediated integration procedure, which would provide an economically efficient random mutagenesis method in C. acutatum.

• Research in Plant Disease
• /
• v.19 no.4
• /
• pp.254-258
• /
• 2013

Fusaric acid (FA) is a mycotoxin produced by Fusarium species. Its toxicity is relatively low but often associated with other mycotoxins, thus enhancing total toxicity. To date, biosynthetic genes or enzymes for FA have not been identified in F. oxysporum. In order to explore the genetic element(s) for FA biosynthesis, restriction enzyme mediated integration (REMI) procedure as an insertional mutagenesis was employed using FA producing-F. oxysporum strains. Genetic transformation of two F. oxysporum strains by REMI yielded more than 7,100 transformants with efficiency of average 3.2 transformants/${\mu}g$ DNA. To develop a screening system using phytotoxicity of FA, eleven various grains and vegetable seeds were tested for germination in cultures containing FA: Kimchi cabbage seed was selected as the most sensitive host. Screening for FA non-producer of F. oxysporum was done by growing each fungal REMI transformant in Czapek-Dox broth for 3 weeks at $25^{\circ}C$ then observing if the Kimchi cabbage seeds germinated in the culture filtrate. Of more than 5,000 REMI transformants screened, fifty-three made the seeds germinated, indicating that they produced little or fewer FA. Among them, twenty-six were analyzed for FA production by HPLC and two turned out to produce less than 1% of FA produced by a wild type strain. Sequencing of genomic DNA regions (252 bp) flanking the vector insertion site revealed an uncharacterized genomic region homologous (93%) to the F. fujikuroi genome. Further study is necessary to determine if the vector insertion sites in FA-deficient mutants are associated with FA production.

• Proceedings of the Korean Society of Plant Pathology Conference
• /
• 2003.10a
• /
• pp.85.2-86
• /
• 2003

Fusarium graminearum is an important pathogen of cereal crops in many areas of the world causing head blight and ear rot of small grains. In addition to serious economic losses, this fungus produces mycotoxins, such as trichothecenes and zearalenone on diseased crops and has been a potential threat to human and animal health. To massively identify pathogenesis-related genes from F. graminearum, two representative strains (SCKO4 from rice and Z03643 from wheat) were mutagenized using restriction enzyme-mediated integration (REMI). In total, 20,DOD REMI transformants have been collected from the two strains. So far, 63 mutants for several traits involved in disease development such as virulence, mycotoxin production, and sporulation have been selected from 3,000 REMI transformants. Now, selected mutants of interest have being genetically analyzed using a newly developed outcross method (See Jungkwan Lee et al poster). In addition, cloning and characterization of genomic DNA regions flanking the insertional site in the genome of the mutants are in progress.

• The Plant Pathology Journal
• /
• v.22 no.3
• /
• pp.215-221
• /
• 2006

Gibberella zeae (anamorph: Fusarium graminearum) is an important pathogen of cereal crops. This fungus produces a broad range of secondary metabolites, including polyketides such as aurofusarin (a red pigment) and zearalenone (an estrogenic mycotoxin), which are important mycological characteristics of this species. A screen of G. zeae insertional mutants, generated using a restriction enzyme-mediated integration (REMI) procedure, led to the isolation of a mutant (Z43R606) that produced neither aurofusarin nor zearalenone yet showed normal female fertility and virulence on host plants. Outcrossing analysis confirmed that both the albino and zearalenone-deficient mutations are linked to the insertional vector in Z43R606. Molecular characterization of Z43R606 revealed a deletion of at least 220 kb of the genome at the vector insertion site, including the gene clusters required for the biosynthesis of aurofusarin and zearalenone, respectively. A re-creation of the insertional event of Z43R606 in the wild-type strain demonstrated that the 220-kb deletion is responsible for the phenotypic changes in Z43R606 and that a large region of genomic DNA can be efficiently deleted in G. zeae by double homologous recombination. The results showed that 52 putative genes located in the deleted genomic region are not essential for phenotypes other than the production of both aurofusarin and zearalenone. This is the first report of the molecular characterization of a large genomic deletion in G. zeae mediated by the REMI procedure.

• The Plant Pathology Journal
• /
• v.23 no.2
• /
• pp.51-56
• /
• 2007

A plant pathogenic fungus, Gibberella zeae (anamorph: Fusarium graminearum), not only generates economic losses by causing disease on cereal grains, but also leads to severe toxicosis in human and animals through the production of mycotoxins in infected plants. Here, we characterized a histidine auxotrophic mutant of G. zeae, designated Z43R1092, which was generated using a restriction enzyme-mediated integration (REMI) procedure. The mutant exhibited pleiotropic phenotypic changes, including a reduction in mycelial growth and virulence and loss of sexual reproduction. Outcrossing analysis confirmed that the histidine auxotrophy is linked to the insertional vector in Z43R1092. Molecular analysis showed that the histidine requirement of Z43R1092 is caused by a disruption of an open reading frame, designated GzHIS7. The deduced product of GzHIS7 encodes a putative enzyme with an N-terminal glutamine amidotransferase and a C-terminal cyclase domain, similar to the Saccharomyces cerevisiae HIS7 required for histidine biosynthesis. The subsequent gene deletion and complementation analyses confirmed the functions of GzHIS7 in G. zeae. This is the first report of the molecular characterization of histidine auxotrophy in G. zeae, and our results demonstrate that correct histidine biosynthesis is essential for virulence, as well as sexual development, in G. zeae. In addition, our results could provide a G. zeae histidine auxotroph as a recipient strain for genetic transformation using this new selectable marker.