Agrobacterium tumefaciens-Mediated Transformation of NHEJ Mutant Aspergillus nidulans Conidia: An Efficient Tool for Targeted Gene Recombination Using Selectable Nutritional Markers

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.

Keywords: ATMT; Aspergillus nidulans; NHEJ; conidia; fungal transformation; mutagenesis; nutritional (pyrG/pyr4) and colour (wA) selectable markers; pCAMBIA-derived vectors; targeted gene deletion/disruption; ΔnkuA mutants.

Figure 1

Plasmids used in this work. (A) Construction of binary vectors for implementation of ATMT in A. nidulans. A. tumefaciens binary vector pCAMBIA1300 carrying the hygromycin and kanamycin resistance genes. Black and red arrows show the restriction sites for XmnI and AseI: digestion with XmnI followed by recircularisation yielded pCAMBIA-mini^XmnI; digestion with AseI followed by recircularisation yielded pCAMBIA-mini^AseI. Restriction sites in the MCS are shown in blue. (B) Modified binary vector for the deletion of AN8423 by ATMT. pCmXdel8423 comprises the A. tumefaciens binary vector pCAMBIA-mini^XmnI into which has been cloned the AN8423 deletion fragment containing the Tr_pyr4 expression cassette that complements the pyrG89 mutation in A. nidulans. Restriction sites of the binary vector MCS are shown in blue. (C) Modified binary vector for integration of Af_riboB in wA by ATMT. pCmAdelwA comprises the A. tumefaciens binary vector pCAMBIA-mini^AseI into which has been cloned the wA (AN8209) deletion fragment containing the Af_riboB expression cassette that complements the riboB2 mutation in A. nidulans. The EcoRI site of the binary vector MCS is shown in blue.

Figure 2

Schematic overview of ATMT of A. nidulans conidia. Transformants were obtained after 3 days’ growth at 37 °C on Aspergillus selective MM plates supplemented with cefotaxime. At-IM: induction medium supplemented with kanamycin and acetosyringone.

Figure 3

Deletion of AN8423. (A) Growth of A. nidulans ATM transformants in the presence or absence of cefatoxime. A. nidulans conidia at a titre of 10⁸ per mL were used for co-culture. (B) Scheme showing replacement of the AN8423 CDS by the Tr_pyr4 expression cassette. (C) Diagnostic PCR with primer pairs 641/642 and 643/644 yields fragments of the sizes predicted for replacement of locus AN8423 by Tr_pyr4. SL: Smart Ladder (Promega, Madison, WI, USA).

Figure 4

Integration of Af_riboB in the wA locus. (A) Growth of AR198 on MM plates supplemented with pyridoxine but lacking riboflavin. (B) Late appearance of white colonies on MM plates lacking riboflavin. (C) Approximately 20× magnification of the red-circled area in (B). (D) Scheme showing insertion of the Af_riboB expression cassette in wA. (E) Diagnostic PCR with primer pairs 741/111 and 113/742 yielded fragments of the sizes predicted for insertion of Af_riboB in the wA locus. Numbers refer to the four independent transformants analysed.

Figure 5

Nutrient cross-feeding. (A) Suppression of riboflavin and pyridoxine auxotrophies by A. tumefaciens. Relevant genotypes: AR198 (riboB2, pyroA4) and V112 (pyrG89, argB2). (B) Arginine auxotrophy in V112 is not suppressed by A. tumefaciens.