Agrobacterium-mediated transformation of Aspergillus awamori in the absence of full-length VirD2, VirC2, or VirE2 leads to insertion of aberrant T-DNA structures.
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.
Michielse
Caroline B
CB
Clusius Laboratory, Institute of Biology, Leiden University, 2333 AL, Leiden, The Netherlands. Michielse@rulbim.leidenuniv.nl
Ram
Arthur F J
AF
Hooykaas
Paul J J
PJ
van den Hondel
Cees A M J J
CA
D001230:Aspergillus / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D004271:DNA, Fungal / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D007473:Ion Channels / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D011995:Recombination, Genetic; D012231:Rhizobium / Q000235:genetics*; D014170:Transformation, Genetic; D037521:Virulence Factors / Q000235:genetics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D007473:Ion Channels; C036483:T-DNA; D037521:Virulence Factors; C051583:virE2 protein, Agrobacterium