Team:WashU StLouis/Avirulence
To determine the virulence of the detected race of Puccinia graminis f. sp. tritici (Pgt), we are engineering Saccharomyces cerevisiae EBY100 to display Sr35, a stem rust resistance gene from Triticum monococcum on its surface. While not yet incorporated into commercial wheat lines, it has the potential to provide wheat with immunity to many highly virulent races of Pgt. Sr35 has been shown to interact directly with its corresponding effector from Pgt, AvrSr35.
The goal of this stage of the project is to produce a fluorescent signal by bimolecular fluorescence complementation when AvrSr35 from Pgt is present. Therefore, when Pgt spores are detected in the detecting stage, the profiling stage will determine the virulence to Sr35. If a fluorescent signal is produced in both parts, although Pgt is present, any crops containing Sr35 will not be infected and do not need to be treated; they will have innate immunity to the fungus. However, if no fluorescence occurs, the spores are virulent to crops protected by Sr35 and the crops will need to be treated to protect them from further infection.
Sr35 will be tagged on the C-terminus with cYFP with a Gly8 linker. The fully assembled structure will produce fluorescence when Sr35 is activated. The placement of cYFP was decided based on documentation from past papers. This combined sequence will form part BBa_K2663002 in the iGEM Registry.
Due to safety concerns, we cannot use Pgt in the lab, so the avirulence factor will also be synthesized from S. cerevisiae EBY100. The N-terminus of the AvrSr35 sequence will be linked to nFYP using a Gly8 linker. This sequence will be Part BBa_K2663004.
All three of the gene sequences above will be preceded by the Gal1 inducible promoter and followed by the Tef1 terminator.
When Sr35 and AvrSr35 combine, nYFP and cYFP will also combine, creating a fluorescent signal. Ununited, cYFP contains the c terminus portion of the full yellow fluorescent protein and and nYFP is the n-terminus part.
