Targeted gene deletion in Aspergillus fumigatus using microbial machinery and a recyclable marker

• Aspergillus fumigatus, a major fungal pathogen, is difficult to transform.
• A system was assembled to more easily transform A. fumigatus.
• The system utilizes yeast cloning, Agrobacterium transformation and a recyclable marker.
• This uses ‘microbial labor’ and allows for the generation of multiple gene deletions.

The emerging invasive fungal pathogen Aspergillus fumigatus causes very serious infections among immunocompromised patient populations. While the genome of this pathogen has been sequenced, a major barrier to better understanding the complex biology of this eukaryotic organism is a lack of tools for efficient genetic manipulation. To improve upon this, we have generated a new gene deletion system for A. fumigatus using yeast recombinational cloning and Agrobacterium tumefaciens mediated transformation (ATMT) employing a recyclable marker system. This system reduced the time for generating a gene deletion strain in our hands by two-thirds (12 weeks to 3 weeks) using minimal human labor, and we demonstrate that it can be used to efficiently generate multiple gene deletions within a single strain.