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.

Identifying pathogenicity-related genes in the pathogen Colletotrichum magnum causing watermelon anthracnose disease via T-DNA insertion mutagenesis

“…Understanding the mechanisms of its pathogenesis will facilitate disease management. ATMT is an effective method for exploring pathogenicity associated genes in pathogenic fungi ( Fitzgerald et al, 2003 ; Leclerque et al, 2004 ; White and Chen, 2006 ; Zhang et al, 2013 ; Li et al, 2019 ; Villena et al, 2020 ; Binh et al, 2021 ; Casado-Del Castillo et al, 2021 ; Chauhan et al, 2021 ; Liu et al, 2021 ). Howerever, this method has not been applied to C. magnum .…”

Assembly of plant virus agroinfectious clones using biological material or DNA synthesis

“…Prepare Agrobacterium AGL1 cells suitable for freeze-thaw transformation as detailed in Method S2 . Note: AGL1 is a hypervirulent C58 derivative that carries a disarmed pTiBo542, which confers high transformation efficiencies on numerous plant (and fungal) species ( Lazo et al., 1991 ; Casado-Del Castillo et al., 2021 ). The strain is a nonfunctional mutant of recA , which reduces the occurrence of plasmid recombination without interfering with DNA transfer ( Lazo et al., 1991 ).…”