Agrobacterium-mediated transformation as a tool for functional genomics in fungi

Plant sciencesMicrobial Biotechnolog

Pathogen profile update: Fusarium oxysporum

Taxonomy: Kingdom Fungi; Phylum Ascomycota; Class Sordariomycetes; Order Hypocreales; Family Nectriaceae; genus Fusarium. 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 leaves, progressive wilting, defoliation and, finally, death of the plant. On fungal colonization, the vascular tissue turns brown, which is clearly visible in cross-sections of the stem. Some formae speciales are not primarily vascular pathogens, but cause foot and root rot or bulb rot. Economic importance: Can cause severe losses in many vegetables and flowers, field crops, such as cotton, and plantation crops, such as banana, date palm and oil palm. Control: Use of resistant varieties is the only practical measure for controlling the disease in the field. In glasshouses, soil sterilization can be performed. Useful websites: http://www.broad.mit.edu/annotation/genome/fusarium_group/MultiHome.html; http://www.fgsc.net/Fusarium/fushome.htm; http://www.phi-base.org/query.ph

The Aspergillus nidulans amdS gene as a marker for the identification of multicopy T-DNA integration events in Agrobacterium-mediated transformation of Aspergillus awamori

The Aspergillus nidulans amdS selection marker was used for the identification of multicopy T-DNA insertions in Agrobacterium-mediated transformation of Asp. awamori. The selection of transformants on agar plates containing acetamide as sole nitrogen source and hygromycin resulted in a six-fold decrease in the transformation frequency, compared with the transformation frequency obtained after hygromycin selection alone. However, it was found that 47% of the transformants obtained after hygromycin and acetamide double selection contained multiple T-DNA integrations. Furthermore, it was found that the multicopy transformants could easily be identified based on their growth rate on agar plates containing acetamide medium. Based on these data, it can be concluded that the amdS marker can also be used as a selection marker in Agrobacterium-mediated transformation of Asp. awamori and that it is a very useful marker to identify those transformants containing multiple T-DNA integrations. © Springer-Verlag 2004

Role of bacterial virulence proteins in Agrobacterium-mediated transformation of Aspergillus awamori

The Agrobacterium-mediated transformation of Aspergillus awamori was optimized using defined co-cultivation conditions, which resulted in a reproducible and efficient transformation system. Optimal co-cultivation conditions were used to study the role of Agrobacterium tumefaciens virulence proteins in T-DNA transfer. This study revealed that inactivation of either of the regulatory proteins (VirA, VirG), any of the transport pore proteins (VirB), proteins involved in generation of the T-strand (VirD, VirC) or T-strand protection and targeting (VirE2) abolishes or severely reduces the formation of transformants. The results indicate that the Agrobacterium-mediated transformation of A. awamori requires an intact T-DNA machinery for efficient transformation; however, the plant host range factors, like VirE3, VirH, and VirF, are not important. Index descriptors: Agrobacterium tumefaciens; T-DNA; Virulence proteins; DNA-transformation; Aspergillus awamori; Filamentous fungi © 2004 Elsevier Inc. All rights reserved

Agrobacterium-mediated transformation leads to improved gene replacement efficiency in Aspergillus awamori.

Item does not contain fulltextIn this study, the efficiency of gene replacement in Aspergillus awamori between Agrobacterium-mediated transformation and CaCl(2)/PEG-mediated transformation was compared. For the genes, pyrG and gfaA, it was found that the homologous recombination frequencies obtained by Agrobacterium-mediated transformation were 3- to 6-fold higher than the frequencies obtained with CaCl(2)/PEG protoplast transformation. For the pyrG gene, it was found that Agrobacterium-mediated transformation allowed an efficient homologous recombination with shorter DNA flanks than CaCl(2)/PEG protoplast transformation. Finally, the addition of the dominant amdS marker as a second selection marker to the gene replacement cassette led to a further 2-fold enrichment in transformants with gene replacement events, resulting in a gene replacement frequency of 55%. Based on the data it can be concluded that Agrobacterium-mediated transformation is an efficient tool for gene replacement and that the amdS gene can be successfully used as a second selection marker to select transformants with putative gene replacement

A one-step method to convert vectors into binary vectors suited for Agrobacterium-mediated transformation.

Bacterial artificial chromosomes (BACs) are widely used for the construction of physical maps, positional-cloning and whole-genome sequencing strategies. Unfortunately, their use for functional genomics is limited, as currently there is no efficient method to use BACs directly for complementation. We describe a novel strategy for one-step conversion of any BAC into a binary BAC (BIBAC). Using Agrobacterium tumefaciens, these BIBACs can be efficiently transformed to virtually all organisms, including plants, fungi, yeasts and human cells. As the strategy is based on in vivo recombineering and does not depend on restriction sites, it is applicable to any vector. To show the feasibility of the method five BACs, containing 0-75 kb of fungal DNA, were converted into BIBACs. These were subsequently transformed to the plant pathogenic fungus Fusarium oxysporum f.sp. lycopersici and to Aspergillus awamori, a filamentous fungus often used for large-scale protein production. Molecular characterisation of the transformants showed that the BIBACs were efficiently transferred to the fungi and stably integrated into their genomes

The FRP1 F-box gene has different functions in sexuality, pathogenicity and metabolism in three fungal pathogens

Plant-pathogenic fungi employ a variety of infection strategies; as a result, fungi probably rely on different sets of proteins for successful infection. The F-box protein Frp1, only present in filamentous fungi belonging to the Sordariomycetes, Leotiomycetes and Dothideomycetes, is required for nonsugar carbon catabolism and pathogenicity in the root-infecting fungus Fusarium oxysporum. To assess the role of Frp1 in other plant-pathogenic fungi, FRP1 deletion mutants were generated in Fusarium graminearum and Botrytis cinerea, and their phenotypes were analysed. Deletion of FgFRP1 in F. graminearum led to impaired infection of barley roots, but not of aerial plant parts. Deletion of BcFRP1 in B. cinerea did not show any effect on pathogenicity. Sexual reproduction, however, was impaired in both F. graminearum and B. cinerea FRP1 deletion mutants. The mutants of all three fungi displayed different phenotypes when grown on an array of carbon sources. The F. oxysporum and B. cinerea deletion mutants showed opposite growth phenotypes on sugar and nonsugar carbon sources. Replacement of FoFRP1 in F. oxysporum with the B. cinerea BcFRP1 resulted in the restoration of pathogenicity, but also in a switch from impaired growth on nonsugar carbon sources to impaired growth on sugar carbon sources. This effect could be ascribed in part to the B. cinerea BcFRP1 promoter sequence. In conclusion, the function of the F-box protein Frp1, despite its high sequence conservation, is not conserved between different fungi, leading to differential requirements for pathogenicity and carbon source utilization