description
- Fungal pathogens are adept at sharing the tools they use to infect their hosts across species boundaries. Until recently, we had little clue as to how fungal pathogens conducted these gene exchanges. Herein, we propose that a novel group of transposons encode all the genetic machinery required to move themselves between species, thereby defining, for the first time, the mechanism driving horizontal gene transfer in these pathogens. Horizontal gene transfer (HGT) enables the spread of dangerous genes and results in the emergence of novel diseases. In pathogenic fungi, an emerging global threat, the mechanisms that facilitate HGT remain completely unknown. Recent long-read DNA sequencing has uncovered a group of transposons called "Starships" that appear to be the drivers of large-scale (100+kb) HGT between fungi. However, Starship-mediated HGT has only been observed using genome sequence data, an indirect measure of the event after it has occurred. This proposal seeks to fill this evidence gap by capturing real-time transfer events, therefore demonstrating active horizontal Starship movement. Bipolaris sorokiniana, Parastagonospora nodorum and Pyrenophora tritici-repentis are among the top 10 most important fungal wheat pathogens globally. Whole genome data from these three pathogens show they carry a shared virulence gene called ToxA. ToxA was horizontally transferred as the "cargo" within two different Starship transposons. One Starship called "Sanctuary" is found in B. sorokiniana and a different Starship called "Horizon" is found in both P. tritici-repentis and P. nodorum. The presence of these giant, highly identical Starships in distantly related wheat pathogens is surprising and has only been observed using genome sequencing. Within the last year, other Starships have been associated with HGT of adaptive genes in many other fungal species. Starships join a growing family of giant DNA elements now described in all kingdoms of life that challenge our perception on the size limits associated with adaptive HGT. We propose that Starships are autonomous units that encode all the machinery required to excise themselves from a genome, facilitate their movement between donor and recipient fungal cells and finally re-integrate themselves into the new genome. There are currently neither direct evidence nor experimental approaches to explore this hypothesis, making this project ideal for Pioneer Award funding. Using Starships tagged with selectable markers, we will develop an experimental workflow that will facilitate the isolation of HGT events both in vitro and in planta. To do this, we will generate 'Donor' strains carrying the mobile Sanctuary or Horizon transposons tagged with the ble gene conferring phleomycin resistance. 'Recipient' strains resistant to hygromycin that do not contain a Starship will be used to screen for successful transfer events. The use of selectable markers in this screen allows for millions of fungal spores to be screened. An alternative approach, performed in parallel, will use a combination of fluorescent markers coupled with high-throughput flow cytometry to capture Starships moving between cells in real time. These cells can then be further imaged to understand if nuclei have moved between strains or alternatively if there is evidence of Starship-only transfer.