Targeting fungi that cause diseases in agricultural plants

The PIS1 gene is an essential gene in the budding model yeast, Saccharomyces cerevisiae. Thus, deleting this gene results in death of the organism. Fusarium is a fungal pathogen that targets plants and triggers the wilting of grass. In this project, a haploid strain of Saccharomyces cerevisiae will be created that lacks the native PIS1 gene and contains the Fusarium PIS1 gene homolog (more specifically, Fusarium oxysporum f.sp. lycopersici isolate 4287) to see if the homolog is essential and therefore a good target for developing a fungicide.

The strain construction involved amplifying PIS1 from Fusarium by PCR, sequencing it to confirm absence of mutations, and inserting it into a pGEM-T plasmid. Subsequently, the Fusarium PIS1 gene was cut from the pGEM-T plasmid and ligated into a yeast expression vector, pYES2-CT. This plasmid allowed the expression of the PIS1 gene under the control of a galactose regulatable promoter.

From here, the plasmid will be transformed into a diploid Saccharomyces cerevisiae strain that is heterozygous for a pis1 deletion. The final step will involve sporulation to identify a haploid strain that has the Saccharomyces cerevisiae pis1 deletion and the Fusarium PIS1. Using this new vector, expression assays and various other tests can be done to see whether the new PIS1 gene complements the lethality caused by the pis1 deletion. If this is the case, the PIS1 gene can then be used as a target to make fungicides for pathogenic fungi.