Transformation of Gibberella fujikuroi: effect of the Aspergillus nidulans AMA1 sequence on frequency and integration

Abstract

A stable and reproducible transformation selection system for Gibberella fujikuroi protoplasts based on the Aspergillus nidulans arg B gene, encoding ornithine transcarbamylase, has been developed. Inclusion into the vector of the A. nidulans DNA fragment (AMA1), which permits plasmid autonomous replication in A. nidulans, A. niger and A. oryzae, appeared to permit autonomous replication of G. fujikuroi although the transformation frequency was increased by only two-fold. Transformation was also achieved using the bacterial hygromycin B resistance gene under the control of G. pulicaris and A. nidulans promoters.

Transient and multivariate system for transformation of a fungal plant pathogen, Rosellinia necatrix, using autonomously replicating vectors

“…The AMA1 vectors may also be useful in the transformation of the fungi in which low yield and low regeneration of the protoplasts interferes with the development of a transformation system. The increase in the transformation efficiency using the pAMA-H vector, more than 5-fold higher than that with pCPXHY1, is similar to previously reported increases (Aspergillus species, 8-250-fold; Penicillium species, 7-270-fold; Monascus purpreus, fivefold; Torichoderma reesei, 61 fold; Gibberella fujikuroi, two-folds) (Bruckner et al 1992;Fierro et al 1996Fierro et al , 2004Gems et al 1991;Kubodera et al 2002;Shimizu et al 2006;Storms et al 2005;Verdoes et al 1994). Previous attempts at co-transformation were unsuccessful when two genomeintegrating vectors were used (Pliego et al 2011); in contrast, we were able to introduce multiple genes into R. necatrix by co-transformation using three autonomously replicating pAMA-H vectors.…”

“…AMA1 vectors have been used to develop several effective fungal transformation systems; however, the instability of AMA1 vectors under non-selective conditions has been reported for several of these systems. (Bruckner et al 1992;Fierro et al 2004;Storms et al 2005;). Moreover, the growth rate of transformants bearing the pAMA-H vector was lower than that of transformants bearing the genome-integrating vector.…”

“…Vectors containing AMA1 sequences are also used for transformation in other classes of fungi, Gibberella fujikuroi (Sordariomycetes) (Bruckner et al 1992), Trichoderma species (Sordariomycetes) (Kubodera et al 2002), and Botrytis cinerea (Leotiomycetes) (Rebordinos et al 2000). These results demonstrate that AMA1 sequence may mediate autonomous replication in fungi that do not belong to the class Eurotiomycetes.…”

The geranylgeranyl diphosphate synthase gene of Gibberella fujikuroi: isolation and expression

“…Although the biosynthetic pathway for gibberellins was established for plants and for the fungus a long time ago, molecular genetic studies of this pathway have started only recently. As a prelude to the molecular analysis of the fungal system, heterologous (Sanchez-Fernandez et al 1991;BruÈ ckner et al 1992) and homologous (Tudzynski et al 1996) transformation systems have been developed for G. fujikuroi. In addition, some mutants of the gibberellin pathway have been isolated and analyzed (Bearder et al 1974;Candau et al 1991;B.…”

Gibberellin biosynthesis inGibberella

“…Despite the availability of gib mutants and transformation protocols (SanchezFemandez eta/., 1991;Leslie & Dickman, 1991;Bruckner et a/., 1992) in Gibberella and the information on plant genes, no Gibberella gene specific for the gibberellin pathway has been cloned yet. The only genes available code for early enzymes, common to the biosynthesis of other terpenoids: hydroxymethylglutaryl coenzyme A reductase (Woitek et a/., 1997), famesyl pyrophosphate synthase (Homann et a/., 1996), and geranylgeranyl pyrophosphate synthase (Mende et a/., 1997).…”