scholarly journals Agrobacterium Tumefaciens-mediated T-DNA Insertional Mutagenesis of Fusarium Oxysporum

2020 ◽  
Author(s):  
Zeqing Feng ◽  
Dan He ◽  
Song Gao ◽  
Shuaishuai Han ◽  
Yunyun Wei ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Fusarium Oxysporum ◽  
Insertional Mutagenesis ◽  
Pathogenic Fungi ◽  
Small Scale ◽  
Efficient Procedure ◽  
Phenotypic Changes ◽  
Insertion Sites ◽  
Tail Pcr ◽  
Dna Insertional Mutagenesis

Abstract BackgroundFusarium species are important pathogenic organisms, which can cause many diseases in plants and humans. Characterizing the mechanism underlying their pathogenicity and drug resistance is critical. Agrobacterium tumefaciens-mediated genetic transformation has been widely used for the molecular analysis of many species. ResultsIn this study, we constructed the pXEN recombinant plasmid carrying the neomycin phosphatase II gene (neo) and established a simple and efficient procedure for the transformation of resistant Fusarium oxysporum mediated by A. tumefaciens. The transformation efficiency was as high as 250 mutants per 104 conidia. A total of 1,450 stably transformed mutants were generated, resulting in a small-scale library of F. oxysporum mutants containing T-DNA tags. Some of the mutants exhibited phenotypic changes in growth, metabolism, and development. Additionally, the sequences flanking the inserted T-DNA were obtained by touchdown-TAIL PCR, the insertion sites and genes associated with the phenotypic changes could be determined.ConclusionsThe developed method may enable to analyze gene functions and study biological characteristics, which will lay the foundation for future analyses of the mechanism underlying F. oxysporum pathogenicity and resistance. Furthermore, it may be applicable to investigations of other important pathogenic fungi.

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

2001 ◽  
Vol 91 (2) ◽  
pp. 173-180 ◽  
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.

scholarly journals Insertional Mutagenesis of Genes Required for Seed Development inArabidopsis thaliana

Genetics ◽  
2001 ◽  
Vol 159 (4) ◽  
pp. 1751-1763 ◽  
Author(s):  
John McElver ◽  
Iris Tzafrir ◽  
George Aux ◽  
Rebecca Rogers ◽  
Carl Ashby ◽  
...  
Keyword(s):  
Seed Development ◽  
Insertional Mutagenesis ◽  
Saturation Mutagenesis ◽  
Significant Advance ◽  
Large Numbers ◽  
Wide Range ◽  
Genome Duplications ◽  
Dna Insertion ◽  
Insertion Sites ◽  
Tail Pcr

AbstractThe purpose of this project was to identify large numbers of Arabidopsis genes with essential functions during seed development. More than 120,000 T-DNA insertion lines were generated following Agrobacterium-mediated transformation. Transgenic plants were screened for defective seeds and putative mutants were subjected to detailed analysis in subsequent generations. Plasmid rescue and TAIL-PCR were used to recover plant sequences flanking insertion sites in tagged mutants. More than 4200 mutants with a wide range of seed phenotypes were identified. Over 1700 of these mutants were analyzed in detail. The 350 tagged embryo-defective (emb) mutants identified to date represent a significant advance toward saturation mutagenesis of EMB genes in Arabidopsis. Plant sequences adjacent to T-DNA borders in mutants with confirmed insertion sites were used to map genome locations and establish tentative identities for 167 EMB genes with diverse biological functions. The frequency of duplicate mutant alleles recovered is consistent with a relatively small number of essential (EMB) genes with nonredundant functions during seed development. Other functions critical to seed development in Arabidopsis may be protected from deleterious mutations by extensive genome duplications.

scholarly journals Embryonic Lethals and T-DNA Insertional Mutagenesis in Arabidopsis

1991 ◽  
Vol 3 (2) ◽  
pp. 149 ◽  
Author(s):  
Deena Errampalli ◽  
David Patton ◽  
Linda Castle ◽  
Leigh Mickelson ◽  
Karl Hansen ◽  
...  
Keyword(s):  
Insertional Mutagenesis ◽  
Dna Insertional Mutagenesis

scholarly journals Chlamydomonas WDR92 in association with R2TP-like complex and multiple DNAAFs to regulate ciliary dynein preassembly

2018 ◽  
Vol 11 (9) ◽  
pp. 770-780 ◽  
Author(s):  
Guang Liu ◽  
Limei Wang ◽  
Junmin Pan
Keyword(s):  
Mass Spectrometry ◽  
Insertional Mutagenesis ◽  
The Other ◽  
Cytoplasmic Protein ◽  
Flagellar Motility ◽  
Heavy Chains ◽  
The Third ◽  
Axonemal Dynein ◽  
Dynein Arms ◽  
Dna Insertional Mutagenesis

Abstract The motility of cilia or eukaryotic flagella is powered by the axonemal dyneins, which are preassembled in the cytoplasm by proteins termed dynein arm assembly factors (DNAAFs) before being transported to and assembled on the ciliary axoneme. Here, we characterize the function of WDR92 in Chlamydomonas. Loss of WDR92, a cytoplasmic protein, in a mutant wdr92 generated by DNA insertional mutagenesis resulted in aflagellate cells or cells with stumpy or short flagella, disappearance of axonemal dynein arms, and diminishment of dynein arm heavy chains in the cytoplasm, suggesting that WDR92 is a DNAAF. Immunoprecipitation of WDR92 followed by mass spectrometry identified inner dynein arm heavy chains and multiple DNAAFs including RuvBL1, RPAP3, MOT48, ODA7, and DYX1C. The PIH1 domain-containing protein MOT48 formed a R2TP-like complex with RuvBL1/2 and RPAP3, while PF13, another PIH1 domain-containing protein with function in dynein preassembly, did not. Interestingly, the third PIH1 domain-containing protein TWI1 was not related to flagellar motility. WDR92 physically interacted with the R2TP-like complex and the other identified DNNAFs. Our data suggest that WDR92 functions in association with the HSP90 co-chaperone R2TP-like complex as well as linking other DNAAFs in dynein preassembly.

scholarly journals Insight into the molecular requirements for pathogenicity of Fusarium oxysporum f. sp. lycopersici through large-scale insertional mutagenesis

2009 ◽  
Vol 10 (1) ◽  
pp. R4 ◽  
Author(s):  
Caroline B Michielse ◽  
Ringo van Wijk ◽  
Linda Reijnen ◽  
Ben JC Cornelissen ◽  
Martijn Rep
Keyword(s):  
Fusarium Oxysporum ◽  
Large Scale ◽  
Insertional Mutagenesis ◽  
Insight Into

scholarly journals Bright Side of Fusarium oxysporum: Secondary Metabolites Bioactivities and Industrial Relevance in Biotechnology and Nanotechnology

2021 ◽  
Vol 7 (11) ◽  
pp. 943
Author(s):  
Sabrin R. M. Ibrahim ◽  
Alaa Sirwi ◽  
Basma G. Eid ◽  
Shaimaa G. A. Mohamed ◽  
Gamal A. Mohamed
Keyword(s):  
Fusarium Oxysporum ◽  
Cyclic Peptides ◽  
Pathogenic Fungi ◽  
New Drugs ◽  
Biotechnological Applications ◽  
The World ◽  
Different Types ◽  
Bright Side ◽  
Reported Data ◽  
Industrial Relevance

Fungi have been assured to be one of the wealthiest pools of bio-metabolites with remarkable potential for discovering new drugs. The pathogenic fungi, Fusarium oxysporum affects many valuable trees and crops all over the world, producing wilt. This fungus is a source of different enzymes that have variable industrial and biotechnological applications. Additionally, it is widely employed for the synthesis of different types of metal nanoparticles with various biotechnological, pharmaceutical, industrial, and medicinal applications. Moreover, it possesses a mysterious capacity to produce a wide array of metabolites with a broad spectrum of bioactivities such as alkaloids, jasmonates, anthranilates, cyclic peptides, cyclic depsipeptides, xanthones, quinones, and terpenoids. Therefore, this review will cover the previously reported data on F. oxysporum, especially its metabolites and their bioactivities, as well as industrial relevance in biotechnology and nanotechnology in the period from 1967 to 2021. In this work, 180 metabolites have been listed and 203 references have been cited.

Mutagenesis via Insertional or Restriction Enzyme-Mediated Integration (REMI) as a Tool to Tag Pathogenicity Related Genes in Plant Pathogenic Fungi

1999 ◽  
Vol 380 (7-8) ◽  
pp. 855-864 ◽  
Author(s):  
F. J. Maier ◽  
W. Schäfer
Keyword(s):  
Restriction Enzyme ◽  
Molecular Basis ◽  
Insertional Mutagenesis ◽  
Transformation Efficiency ◽  
Pathogenic Fungi ◽  
Transformation Protocol ◽  
Vector Integration ◽  
Future Developments ◽  
Genetically Determined ◽  
Remi Mutagenesis

Abstract Random insertional mutagenesis is a powerful tool to investigate the molecular basis of most genetically determined processes, for example in pathogenic fungi. An improved version of this method is the insertional mutagenesis via restriction enzyme mediated integration (REMI). Transformation efficiency and mode of vector integration are species dependent and further influenced by vector conformation, restriction enzyme activity, and transformation protocol. An overview is given, covering the mutants and already identified genes obtained after REMI mutagenesis. An outlook describes the future developments in the field.

T-DNA insertional mutagenesis in Arabidopsis

1992 ◽  
Vol 20 (5) ◽  
pp. 963-976 ◽  
Author(s):  
Csaba Koncz ◽  
Kinga N�meth ◽  
George P. R�dei ◽  
Jeff Schell
Keyword(s):  
Insertional Mutagenesis ◽  
Dna Insertional Mutagenesis

scholarly journals Recovery of Fusarium oxysporum Fo47 Mutants Affected in Their Biocontrol Activity After Transposition of the Fot1 Element

2002 ◽  
Vol 92 (9) ◽  
pp. 936-945 ◽  
Author(s):  
Sophie Trouvelot ◽  
Chantal Olivain ◽  
Ghislaine Recorbet ◽  
Quirico Migheli ◽  
Claude Alabouvette
Keyword(s):  
Fusarium Oxysporum ◽  
Insertional Mutagenesis ◽  
Wild Type Strain ◽  
Growth Habit ◽  
Antagonistic Activity ◽  
Phenotypic Characterization ◽  
Wild Type ◽  
Biocontrol Activity

To investigate the biocontrol mechanisms by which the antagonistic Fusarium oxysporum strain Fo47 is active against Fusarium wilt, a Fot1 transposon-mediated insertional mutagenesis approach was adopted to generate mutants affected in their antagonistic activity. Ninety strains in which an active Fot1 copy had transposed were identified with a phenotypic assay for excision and tested for their biocontrol activity against F. oxysporum f. sp. lini on flax in greenhouse experiments. Sixteen strains were affected in their capacity to protect flax plants, either positively (more antagonistic than Fo47) or negatively (less antagonistic). The molecular characterization of these mutants confirms the excision of Fot1 and its reinsertion in most of the cases. Moreover, we demonstrate that other transposable elements such as Fot2, impala, and Hop have no transposition activity in the mutant genomes. The phenotypic characterization of these mutants shows that they are affected neither in their in vitro growth habit nor in their competitiveness in soil compared with wild-type strain Fo47. These results show that mutants are not impaired in their saprophytic phase and suggest that the altered biocontrol phenotype should likely be expressed during the interaction with the host plant.

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