Abstract
Protoplast transformation for the introduction of recombinant DNA into Aspergillus nidulans is technically demanding and dependant on the availability and batch variability of commercial enzyme preparations. Given the success of Agrobacterium tumefaciens-mediated transformation (ATMT) in diverse pathogenic fungi, we have adapted this method to facilitate transformation of A. nidulans. Using suitably engineered binary vectors, gene-targeted ATMT of A. nidulans non-homologous end-joining (NHEJ) mutant conidia has been carried out for the first time by complementation of a nutritional requirement (uridine/uracil auxotrophy). Site-specific integration in the ΔnkuA host genome occurred at high efficiency. Unlike other transformation techniques, however, cross-feeding of certain nutritional requirements from the bacterium to the fungus was found to occur, thus limiting the choice of auxotrophies available for ATMT. In complementation tests and also for comparative purposes, integration of recombinant cassettes at a specific locus could provide a means to reduce the influence of position effects (chromatin structure) on transgene expression. In this regard, targeted disruption of the wA locus permitted visual identification of transformants carrying site-specific integration events by conidial colour (white), even when auxotrophy selection was compromised due to cross-feeding. The protocol described offers an attractive alternative to the protoplast procedure for obtaining locus-targeted A. nidulans transformants.
Highlights
Genetic transformation is an essential tool for identifying as well as manipulating gene function
The ease or difficulty with which transformable protoplasts can be obtained is influenced by several factors including natural variations between A. nidulans strains, the type of biomass to be used and its growth conditions, variations between batches of protoplasting enzymes that affect both protoplast release and viability, as well as other considerations related to the osmotic fragility of the latter
Osmotic stabilization is often achieved with buffers having high sugar or sugar alcohol concentrations, and their influence on carbon catabolite repression (CCR) could lead to effects on gene expression that could impact the selection of transformants
Summary
Genetic transformation is an essential tool for identifying as well as manipulating gene function. The current study is directed to exploiting three properties/resources of A. nidulans to develop a reliable ATMT alternative to protoplast transformation that would facilitate the use of reverse genetics techniques in this fungus for the analysis of gene or promoter function, or the modulation of gene expression (gene deletion/disruption, allele replacement, promoter swapping, protein tagging, (over)expression of recombinant gene cassettes): whereas A. nidulans protoplasts tend to be multinucleate and initially result in the generation of heterokaryotic transformants, A. nidulans conidia are uninuclear and have the potential to yield homokaryotic transformants directly; since site-specific genomic targeting is an important attribute for performing reverse genetics, it would be of interest in this context to examine the use of NHEJ strains as ATMT hosts; several functional orthologous gene expression cassettes are available that complement corresponding auxotrophies in A. nidulans, making nutrient marker selection a possibility in ATMT of this fungus By incorporating these features in ATMT of A. nidulans we have (i) efficiently obtained specific gene deletion mutants and (ii) visually identified site-specific integration transformants independently of auxotrophic selection
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