Agrobacterium-Mediated Transformation ofAspergillus awamoriin the Absence of Full-Length VirD2, VirC2, or VirE2 Leads to Insertion of Aberrant T-DNA Structures

Abstract

Reductions to 2, 5, and 42% of the wild-type transformation efficiency were found when Agrobacterium mutants carrying transposon insertions in virD2, virC2, and virE2, respectively, were used to transform Aspergillus awamori. The structures of the T-DNAs integrated into the host genome by these mutants were analyzed by Southern and sequence analyses. The T-DNAs of transformants obtained with the virE2 mutant had left-border truncations, whereas those obtained with the virD2 mutant had truncated right ends. From this analysis, it was concluded that the virulence proteins VirD2 and VirE2 are required for full-length T-DNA integration and that these proteins play a role in protecting the right and left T-DNA borders, respectively. Multicopy and truncated T-DNA structures were detected in the majority of the transformants obtained with the virC2 mutant, indicating that VirC2 plays a role in correct T-DNA processing and is required for single-copy T-DNA integration.Agrobacterium tumefaciens is capable of transferring a part of its tumor-inducing (Ti) plasmid (the transferred DNA, or T-DNA) to eukaryotic cells, where it stably integrates into the host genome. A. tumefaciens is now widely used for the transformation of plants, and more recently also for transformation of yeasts and fungi (3, 10). Proteins encoded by the virulence region of the Ti plasmid mediate T-DNA processing and transfer (13, 45). The VirD2 protein, in the presence of VirD1, recognizes the ends of the T-DNA, which is delimited by two T-DNA border repeats, and nicks the bottom strand of these 24-bp border repeats between the second and third bases. These nicks are then used as initiation and termination sites for the displacement of a linear single-stranded copy of the bottom strand of the T-DNA region, the T strand. The VirD2 protein stays covalently attached to the T strand via its tyrosine residue (Tyr29) (40). The right border is usually the initiation point for T-strand production due to the presence of a socalled overdrive located at that border. VirC1, one of the products of the virC locus, can bind this 25-bp overdrive sequence and thereby enhance T-strand production (32). The other product of the virC locus, the VirC2 protein, is also thought to play a role in efficient T-DNA transfer (43); however, its precise role in T-DNA formation is unknown. The T-strand-VirD2 complex is transported to the host via a piluslike structure consisting of VirD4 and 11 different VirB proteins. Once in the host, the T strand is coated with the singlestranded DNA binding protein VirE2, which is transported to the host independently of the T strand (36). Binding of VirE2 to the T strand protects it against nucleolytic degradation and turns the T strand into a transportable conformation, which facilitates the nuclear import of the T strand. The T complex, composed of the T strand, VirD2, and VirE2, is imported into the nucleus, where the T strand integrates stably into the host genome. Besides nicking the T-DNA borders, the VirD2 protein also plays a role in tar...

Agrobacterium-Mediated Plant Transformation: Biology and Applications

“…Involvement of VirD2 in protecting the integrity of the right border was also demonstrated in a fungal transformation system (Michielse et al, 2004); this study further showed that VirE2 similarly protects the left end of the T-DNA, while VirC2 helps ensure correct processing. Importantly, VirC2 is also required for single-copy integration in this system (Michielse et al 2004).…”

“…However, it is not clear how exactly the w domain of VirD2 is involved in T-DNA integration because the pattern of integration of the T-DNA is not affected by deletion of the ω sequence (Shurvinton et al, 1992;Tinland et al, 1995;Bravo-Angel et al, 1998;Mysore et al, 1998). Involvement of VirD2 in protecting the integrity of the right border was also demonstrated in a fungal transformation system (Michielse et al, 2004); this study further showed that VirE2 similarly protects the left end of the T-DNA, while VirC2 helps ensure correct processing. Importantly, VirC2 is also required for single-copy integration in this system (Michielse et al 2004).…”

Establishment of Agrobacterium tumefaciens -Mediated Transformation of an Oleaginous Fungus, Mortierella alpina 1S-4, and Its Application for Eicosapentaenoic Acid Producer Breeding

“…Figure 4 shows T-DNA region integration into chromosomal DNA observed on Southern blot analysis, using genomic DNA from each stable transformant, hand, a different, 3.0-kb band was observed for transformant 23. This phenomenon has often been observed with the ATMT system (2,5,17). It was considered that integrated T-DNA suffered truncation at the corresponding restriction sites.…”

Agrobacterium in the Genomics Age

“…In addition, Agrobacterium can transform Streptomyces, yeast, and other fungal species (Bundock et al, 1995(Bundock et al, , 2002Piers et al, 1996;de Groot et al, 1998;Abuodeh et al, 2000;Kelly and Kado, 2002;Roberts et al, 2003;Schrammeijer et al, 2003;Michielse et al, 2004), HeLa cells (Kunik et al, 2001), and sea urchin embryos (Bulgakov et al, 2006). Thus, Agrobacterium is incredibly promiscuous in its ability to mediate horizontal gene flow among numerous species of different phylogenetic kingdoms.…”