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

ABSTRACT Agrobacterium tumefaciens was used to transform Aspergillus fumigatus by either random or site-directed integration of transforming DNA (T-DNA). Random mutagenesis via Agrobacterium tumefaciens-mediated transformation (ATMT) was accomplished with T-DNA containing a hygromycin resistance cassette. Cocultivation of A. fumigatus conidia and Agrobacterium (1:10 ratio) for 48 h at 24°C resulted in high frequencies of transformation (>100 transformants/107 conidia). The majority of transformants harbored a randomly integrated single copy of T-DNA and were mitotically stable. We chose alb1, a polyketide synthase gene, as the target gene for homologous integration because of the clear phenotype difference between the white colonies of Δalb1 mutant strains and the bluish-green colonies of wild-type strains. ATMT with a T-DNA-containing alb1 disruption construct resulted in 66% albino transformants. Southern analysis revealed that 19 of the 20 randomly chosen albino transformants (95%) were disrupted by homologous recombination. These results suggest that ATMT is an efficient tool for transformation, random insertional mutagenesis, and gene disruption in A. fumigatus.

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Reverse Genetics for Functional Genomics of Phytopathogenic Fungi and Oomycetes

Comparative and Functional Genomics ◽

10.1155/2009/380719 ◽

2009 ◽

Vol 2009 ◽

pp. 1-11 ◽

Cited By ~ 16

Author(s):

Vijai Bhadauria ◽

Sabine Banniza ◽

Yangdou Wei ◽

You-Liang Peng

Keyword(s):

Functional Genomics ◽

Gene Disruption ◽

Insertional Mutagenesis ◽

Reverse Genetics ◽

Phytopathogenic Fungi ◽

Sequence Information ◽

Biological Functions ◽

Targeted Gene Disruption ◽

Local Lesions ◽

Genome Sequence Information

Sequencing of over 40 fungal and oomycete genomes has been completed. The next major challenge in modern fungal/oomycete biology is now to translate this plethora of genome sequence information into biological functions. Reverse genetics has emerged as a seminal tool for functional genomics investigations. Techniques utilized for reverse genetics like targeted gene disruption/replacement, gene silencing, insertional mutagenesis, and targeting induced local lesions in genomes will contribute greatly to the understanding of gene function of fungal and oomycete pathogens. This paper provides an overview on high-throughput reverse genetics approaches to decode fungal/oomycete genomes.

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

Journal of Microbiological Methods ◽

10.1016/j.mimet.2016.12.010 ◽

2017 ◽

Vol 133 ◽

pp. 8-13 ◽

Cited By ~ 7

Author(s):

Yuanyuan Lu ◽

Shuqin Xiao ◽

Fen Wang ◽

Jiaying Sun ◽

Likun Zhao ◽

...

Keyword(s):

Agrobacterium Tumefaciens ◽

Insertional Mutagenesis ◽

Efficient Tool ◽

Cercospora Zeae Maydis

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An efficient tool for random insertional mutagenesis: Agrobacterium tumefaciens-mediated transformation of the filamentous fungus Aspergillus terreus

Journal of Microbiological Methods ◽

10.1016/j.mimet.2014.01.007 ◽

2014 ◽

Vol 98 ◽

pp. 114-118 ◽

Cited By ~ 24

Author(s):

Dongyang Wang ◽

Dan He ◽

Guangquan Li ◽

Song Gao ◽

Huiying Lv ◽

...

Keyword(s):

Agrobacterium Tumefaciens ◽

Filamentous Fungus ◽

Insertional Mutagenesis ◽

Aspergillus Terreus ◽

Efficient Tool

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

Journal of Microbiological Methods ◽

10.1016/j.mimet.2018.05.004 ◽

2018 ◽

Vol 149 ◽

pp. 96-100 ◽

Cited By ~ 3

Author(s):

Jiaying Sun ◽

Ruidi Xu ◽

Shuqin Xiao ◽

Yuanyuan Lu ◽

Qifeng Zhang ◽

...

Keyword(s):

Agrobacterium Tumefaciens ◽

Insertional Mutagenesis ◽

Efficient Tool

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

Phytopathology ◽

10.1094/phyto.2001.91.2.173 ◽

2001 ◽

Vol 91 (2) ◽

pp. 173-180 ◽

Cited By ~ 425

Author(s):

E. D. Mullins ◽

X. Chen ◽

P. Romaine ◽

R. Raina ◽

D. M. Geiser ◽

...

Keyword(s):

Agrobacterium Tumefaciens ◽

Fusarium Oxysporum ◽

Fungal Pathogens ◽

Insertional Mutagenesis ◽

Genetic Manipulation ◽

Fungal Spores ◽

Hygromycin B ◽

Fungal Transformation ◽

Efficient Tool ◽

Binary Vectors

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 × 106 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.

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Development of a targeted gene disruption system in the PET-degrading bacterium Ideonella sakaiensis and its applications to PETase and MHETase genes

Applied and Environmental Microbiology ◽

10.1128/aem.00020-21 ◽

2021 ◽

Author(s):

Shin-ichi Hachisuka ◽

Tarou Nishii ◽

Shosuke Yoshida

Keyword(s):

Terephthalic Acid ◽

Parent Strain ◽

Gene Disruption ◽

Ethylene Terephthalate ◽

Targeted Gene Disruption ◽

Degrading Bacterium ◽

Poly Ethylene Terephthalate ◽

Poly Ethylene ◽

Pet Hydrolase

Poly(ethylene terephthalate) (PET) is a commonly used synthetic plastic; however its non-biodegradability results in a large amount of waste accumulation that has a negative impact on the environment. Recently, a PET-degrading bacterium Ideonella sakaiensis 201-F6 strain was isolated and the enzymes involved in PET-digestion, PET hydrolase (PETase) and mono(2-hydroxyethyl) terephthalic acid (MHET) hydrolase (MHETase), were identified. Despite the great potentials of I. sakaiensis in bioremediation and biorecycling, approaches to studying this bacterium remain limited. In this study, to enable the functional analysis of PETase and MHETase genes in vivo , we have developed a gene disruption system in I. sakaiensis . The pT18 mobsacB -based disruption vector harboring directly connected 5'- and 3'-flanking regions of the target gene for homologous recombination was introduced into I. sakaiensis cells via conjugation. First, we deleted the orotidine 5'-phosphate decarboxylase gene ( pyrF ) from the genome of the wild-type strain, producing the Δ pyrF strain with 5-fluoroorotic acid (5-FOA) resistance. Next, using the Δ pyrF strain as a parent strain, and pyrF as a counterselection marker, we disrupted the genes for PETase and MHETase. The growth of both Δ petase and Δ mhetase strains on terephthalic acid (TPA, one of the PET hydrolytic products) was comparable to that of the parent strain. However, these mutant strains dramatically decreased the growth level on PET to that on no carbon source. Moreover, the Δ petase strain completely abolished PET degradation capacity. These results demonstrate that PETase and MHETase are essential for I. sakaiensis metabolism of PET. IMPORTANCE The poly(ethylene terephthalate) (PET)-degrading bacterium Ideonella sakaiensis possesses two unique enzymes able to serve in PET hydrolysis. PET hydrolase (PETase) hydrolyzes PET into mono(2-hydroxyethyl) terephthalic acid (MHET) and MHET hydrolase (MHETase) hydrolyzes MHET into terephthalic acid (TPA) and ethylene glycol (EG). These enzymes have attracted global attention as they have potential to be used for bioconversion of PET. Compared to many in vitro studies including the biochemical and crystal structure analyses, few in vivo studies have been reported. Here, we developed a targeted gene disruption system in I. sakaiensis , which was then applied for constructing Δ petase and Δ mhetase strains. Growth of these disruptants revealed that PETase is a sole enzyme responsible for PET degradation in I. sakaiensis , while PETase and MHETase play essential roles in its PET assimilation.

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