Agrobacterium-mediated transformation of Fusarium oxysporum: An efficient tool for insertional mutagenesis and gene transfer
TL;DR: 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.
Abstract: 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...
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TL;DR: Although genetic resistance has been described in several plant species, only one resistance locus against Verticillium has been cloned to date and the molecular processes underlying this physiology remain largely unknown.
Abstract: Introduction: Verticillium spp. are soil-borne plant pathogens responsible for Verticillium wilt diseases in temperate and subtropical regions; collectively they affect over 200 hosts, including many economically important crops. There are currently no fungicides available to cure plants once they are infected. Taxonomy: Kingdom: Fungi, phylum: Ascomycota, subphylum, Pezizomycotina, class: Sordariomycetes, order: Phyllachorales, genus: Verticillium. Host range and disease symptoms: Over 200 mainly dicotyledonous species including herbaceous annuals, perennials and woody species are host to Verticillium diseases. As Verticillium symptoms can vary between hosts, there are no unique symptoms that belong to all plants infected by this fungus. Disease symptoms may comprise wilting, chlorosis, stunting, necrosis and vein clearing. Brown vascular discoloration may be observed in stem tissue cross-sections. Pathogenicity: Verticillium spp. have been reported to produce cell-wall-degrading enzymes and phytotoxins that all have been implicated in symptom development. Nevertheless, evidence for a crucial role of toxins in pathogenicity is inconsistent and therefore not generally accepted. Microsclerotia and melanized mycelium play an important role in the disease cycle as they are a major inoculum source and are the primary long-term survival structures. Resistance: Different defence responses in the prevascular and the vascular stage of Verticillium wilt diseases determine resistance. Although resistance physiology is well established, the molecular processes underlying this physiology remain largely unknown. Resistance against Verticillium largely depends on the isolation of the fungus in contained parts of the xylem tissues followed by subsequent elimination of the fungus. Although genetic resistance has been described in several plant species, only one resistance locus against Verticillium has been cloned to date.
720 citations
TL;DR: The potential of the Agrobacterium DNA transfer system to be used as a tool for targeted and random mutagenesis in fungi is discussed.
Abstract: In the era of functional genomics, the need for tools to perform large-scale targeted and random mutagenesis is increasing. A potential tool is Agrobacterium-mediated fungal transformation. A. tumefaciens is able to transfer a part of its DNA (transferred DNA; T-DNA) to a wide variety of fungi and the number of fungi that can be transformed by Agrobacterium-mediated transformation (AMT) is still increasing. AMT has especially opened the field of molecular genetics for fungi that were difficult to transform with traditional methods or for which the traditional protocols failed to yield stable DNA integration. Because of the simplicity and efficiency of transformation via A. tumefaciens, it is relatively easy to generate a large number of stable transformants. In combination with the finding that the T-DNA integrates randomly and predominantly as a single copy, AMT is well suited to perform insertional mutagenesis in fungi. In addition, in various gene-targeting experiments, high homologous recombination frequencies were obtained, indicating that the T-DNA is also a useful substrate for targeted mutagenesis. In this review, we discuss the potential of the Agrobacterium DNA transfer system to be used as a tool for targeted and random mutagenesis in fungi.
450 citations
Cites background or methods from "Agrobacterium-mediated transformati..."
...The addition of AS to the Agrobacterium pre-culture has been reported to result in either a decrease or an increase in single-copy T-DNA integration, for reasons unknown (Combier et al. 2003; Mullins et al. 2001; Rho et al. 2001)....
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...However, the length of the co-cultivation period had no influence on T-DNA copy number in F. oxysporum (Mullins et al. 2001)....
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...Integration also appears to occur at random (Abuodeh et al. 2000; Bundock et al. 2002; Combier et al. 2003; de Groot et al. 1998; Degefu and Hanif 2003; Leclerque et al. 2003; Michielse et al. 2004a; Mullins et al. 2001; Rho et al. 2001)....
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...…Covert et al. 2001; de Groot et al. 1998; Degefu and Hanif 2003; Fitzgerald et al. 2003; Hanif et al. 2002; Leclerque et al. 2003; Malonek and Meinhardt 2001; Michielse et al. 2004a; Mullins et al. 2001; Rho et al. 2001; Sullivan et al. 2002; Tanguay and Breuil 2003; Zwiers and De Waard 2001)....
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...…studies have shown that each fungus has an optimal combination of co-cultivation period and temperature to obtain a maximum number of transformants (Combier et al. 2003; Gardiner and Howlett 2004; Meyer et al. 2003; Michielse et al. 2004b; Mullins et al. 2001; Rho et al. 2001; Rolland et al. 2003)....
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TL;DR: Vascular wilt fungus causes severe losses on most vegetables and flowers, several field cropssuch as cotton and tobacco, plantation crops such as banana, plantain, coffee and sugarcane, and a few shade trees.
Abstract: SUMMARY
Taxonomy: Vascular wilt fungus; Ascomycete although sexual stage is yet to be found. The most closely related teleomorphic group, Gibberella, is classified within the Pyrenomycetes.
Host range: Very broad at the species level. More than 120 different formae speciales have been identified based on specificity to host species belonging to a wide range of plant families.
Disease symptoms: Initial symptoms of vascular wilt include vein clearing and leaf epinasty, followed by stunting, yellowing of the lower leafs, progressive wilting of leaves and stem, defoliation and finally death of the plant. In cross-sections of the stem, a brown ring is evident in the area of the vascular bundles. Some formae speciales are not primarily vascular pathogens but cause foot- and rootrot or bulbrot.
Economic importance: Causes severe losses on most vegetables and flowers, several field crops such as cotton and tobacco, plantation crops such as banana, plantain, coffee and sugarcane, and a few shade trees.
Control: Use of resistant varieties is the only practical measure for controlling the disease in the field. Under greenhouse conditions, soil sterilization can be performed. Alternative control methods with potential for the future include soil solarization and biological control with antagonistic bacteria or fungi.
Useful websites: http://www.fgsc.net/fus.htm, http://www-genome.wi.mit.edu/annotation/fungi/fusarium/, http://www.cbs.knaw.nl/fusarium/database.html
406 citations
Cites background from "Agrobacterium-mediated transformati..."
...A. tumefaciens-mediated transformation was shown to work efficiently in F. oxysporum (Mullins et al., 2001), while the transposon impala, originally isolated from F. oxysporum, was used to tag a pathogenicity gene in the rice pathogen M. grisea (Villalba et al., 2001)....
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TL;DR: In this paper, the authors identified a highly localized structure, the biotrophic interfacial complex (BIC), which accumulates fluorescently labeled effectors secreted by IH.
Abstract: Knowledge remains limited about how fungal pathogens that colonize living plant cells translocate effector proteins inside host cells to regulate cellular processes and neutralize defense responses. To cause the globally important rice blast disease, specialized invasive hyphae (IH) invade successive living rice (Oryza sativa) cells while enclosed in host-derived extrainvasive hyphal membrane. Using live-cell imaging, we identified a highly localized structure, the biotrophic interfacial complex (BIC), which accumulates fluorescently labeled effectors secreted by IH. In each newly entered rice cell, effectors were first secreted into BICs at the tips of the initially filamentous hyphae in the cell. These tip BICs were left behind beside the first-differentiated bulbous IH cells as the fungus continued to colonize the host cell. Fluorescence recovery after photobleaching experiments showed that the effector protein PWL2 (for prevents pathogenicity toward weeping lovegrass [Eragrostis curvula]) continued to accumulate in BICs after IH were growing elsewhere. PWL2 and BAS1 (for biotrophy-associated secreted protein 1), BIC-localized secreted proteins, were translocated into the rice cytoplasm. By contrast, BAS4, which uniformly outlines the IH, was not translocated into the host cytoplasm. Fluorescent PWL2 and BAS1 proteins that reached the rice cytoplasm moved into uninvaded neighbors, presumably preparing host cells before invasion. We report robust assays for elucidating the molecular mechanisms that underpin effector secretion into BICs, translocation to the rice cytoplasm, and cell-to-cell movement in rice.
394 citations
Cites methods from "Agrobacterium-mediated transformati..."
...All the fusion constructs were cloned in binary vectors pBHt2 (Mullins et al., 2001) or pBGt (S. Kang, unpublished data), and their transcriptional and translational fusions were verified by DNA sequencing....
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...All the fusion constructs were cloned in binary vectors pBHt2 (Mullins et al., 2001) or pBGt (S....
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TL;DR: The results suggest that I‐3‐mediated resistance is based on recognition of Six1 secreted in xylem vessels, which corresponds to the 12 kDa protein found inxylem sap of infected plants.
Abstract: A 12 kDa cysteine-rich protein is secreted by Fusarium oxysporum f. sp. lycopersici during colonization of tomato xylem vessels. Peptide sequences obtained with mass spectrometry allowed identification of the coding sequence. The gene encodes a 32 kDa protein, designated Six1 for secreted in xylem 1. The central part of Six1 corresponds to the 12 kDa protein found in xylem sap of infected plants. A mutant that had gained virulence on a tomato line with the I-3 resistance gene was found to have lost the SIX1 gene along with neighbouring sequences. Transformation of this mutant with SIX1 restored avirulence on the I-3 line. Conversely, deletion of the SIX1 gene in a wild-type strain results in breaking of I-3-mediated resistance. These results suggest that I-3-mediated resistance is based on recognition of Six1 secreted in xylem vessels.
353 citations
Cites methods from "Agrobacterium-mediated transformati..."
...pRD803 was derived from pBHt2 (Mullins et al., 2001) through insertion of a 3 kb EcoRI–XbaI fragment with the phleomycin resistance gene (ble) from pAN8....
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...Transformation of Fol and targeted deletion of SIX1 Fol was transformed with Agrobacterium-mediated transformation, with a protocol adapted from Mullins et al. (2001)....
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...This plasmid was called pRD803-SIX1. pRD803 was derived from pBHt2 (Mullins et al., 2001) through insertion of a 3 kb EcoRI–XbaI fragment with the phleomycin resistance gene (ble) from pAN8....
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...Fol was transformed with Agrobacterium-mediated transformation, with a protocol adapted from Mullins et al. (2001). Briefly, 10 fungal spores were mixed with the same volume of an Agrobacterium tumefaciens suspension (OD660 = 0....
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References
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TL;DR: Thermal asymmetric interlaced PCR is an efficient technique for amplifying insert ends from yeast artificial chromosome and P1 clones and the adaptation of this method for recovery and mapping of genomic sequences flanking T-DNA insertions in Arabidopsis thaliana is described.
Abstract: Thermal asymmetric interlaced (TAIL-) PCR is an efficient technique for amplifying insert ends from yeast artificial chromosome (YAC) and P1 clones. Highly specific amplification is achieved without resort to complex manipulations before or after PCR. The adaptation of this method for recovery and mapping of genomic sequences flanking T-DNA insertions in Arabidopsis thaliana is described. Insertion-specific products were amplified from 183 of 190 tested T-DNA insertion lines. Reconstruction experiments indicate that the technique can recover single-copy sequences from genomes as complex as common wheat (1.5 x 10(10) bp). RFLPs were screened using 122 unique flanking sequence probes, and the insertion sites of 26 T-DNA transgenic lines were determined on an RFLP map. These lines, whose mapped T-DNA insertions confer hygromycin resistance, can be used for fine-scale mapping of linked phenotypic loci.
1,459 citations
TL;DR: An efficient PCR strategy that overcomes the shortcomings of existing methods and can be automated is developed and protocols that are amenable to automation of amplification and sequencing of insert end sequences directly from cells of P1 and YAC clones are presented.
1,165 citations
TL;DR: It is reported that A. tumefaciens can also transfer its T-DNA efficiently to the filamentous fungus Aspergillus awamori, demonstrating DNA transfer between a prokaryote and a filamentous fungi.
Abstract: Agrobacterium tumefaciens transfers part of its Ti plasmid, the T-DNA, to plant cells during tumorigenesis. It is routinely used for the genetic modification of a wide range of plant species. We report that A. tumefaciens can also transfer its T-DNA efficiently to the filamentous fungus Aspergillus awamori, demonstrating DNA transfer between a prokaryote and a filamentous fungus. We transformed both protoplasts and conidia with frequencies that were improved up to 600-fold as compared with conventional techniques for transformation of A. awamori protoplasts. The majority of the A. awamori transformants contained a single T-DNA copy randomly integrated at a chromosomal locus. The T-DNA integrated into the A. awamori genome in a manner similar to that described for plants. We also transformed a variety of other filamentous fungi, including Aspergillus niger, Fusarium venenatum, Trichoderma reesei, Colletotrichum gloeosporioides, Neurospora crassa, and the mushroom Agaricus bisporus, demonstrating that transformation using A. tumefaciens is generally applicable to filamentous fungi.
893 citations
TL;DR: Forward genetics begins with a mutant phenotype and asks the question “What is the genotype?” that is, what is the sequence of the mutant gene causing the altered phenotype?
Abstract: Forward genetics begins with a mutant phenotype and asks the question “What is the genotype?” that is, what is the sequence of the mutant gene causing the altered phenotype? Reverse genetics begins with a mutant gene sequence and asks the question “What is the resulting change in phenotype
792 citations
TL;DR: This chapter analyzes the effects of one, or two, overlapping, reciprocal translocations on meiotic crossing-over and nondisjunction and to identify the processes of mitotic recombination in diploids with and without translocations, in triploids, and also in disomics from single and double translocation crosses.
Abstract: Publisher Summary This chapter analyzes the effects of one, or two, overlapping, reciprocal translocations on meiotic crossing-over and nondisjunction and to identify the processes of mitotic recombination in diploids with and without translocations, in triploids, and also in disomics from single and double translocation crosses, as well as the effects of inducing agents on these. It also assesses the various methods of genetic mapping, the uses of translocations for mapping, and the various problems arising from chromosomal aberrations for mapping by the parasexual cycle. The effects of chromosomal aberrations on meiotic recombination are of two general types. Based on an extensive analysis of effects by recombination-reducing aberrations on the disjunction of other chromosomes, two phases of pairing have been postulated for the meiosis of Drosophila : one early one, leading to and reinforced by chiasma formation, and a second one, “distributive pairing,” which involves all noncrossover chromosomes and may result in pairing of heterologous types. Several types of mitotic segregation which lead to spotting or variegation have been demonstrated in various organisms. In Aspergillus, two main types occur spontaneously: (1) Mitotic crossing-over, (2) Mitotic nondisjunction. A large variety of methods combining genetic, cytological, and biochemical techniques have been used in various organisms for the mapping of genes to specific chromosome segments. The chapter emphasizes the methods based on mitotic recombination that are new and especially useful in Aspergillus nidulans. In addition, techniques making use of translocations for genetic mapping in conjunction with meiotic and mitotic recombination are considered in detail.
706 citations