Periodic Reporting for period 1 - EffectorTargets (Development of functional genomic screens to identify conserved host cell processes targeted by fungal effector proteins)
Berichtszeitraum: 2015-09-09 bis 2017-09-08
To understand the ways fungi infect their host, makes it possible to find protective measures.
The fungus blumeria is a thread to global food security since it can destroy harvest of cereal crops like wheat and barley, which are staple foods for many people.
This fungus uses a very large set of secreted proteins to alter the host cellular programs to its own benefit. We want to find out how the proteins operate by expressing them one-for-one in alternative hosts, yeast and the alga Chlamy.
The fungus blumeria is a thread to global food security since it can destroy harvest of cereal crops like wheat and barley, which are staple foods for many people.
This fungus uses a very large set of secreted proteins to alter the host cellular programs to its own benefit. We want to find out how the proteins operate by expressing them one-for-one in alternative hosts, yeast and the alga Chlamy.
I used the CSEP library for yeast growth inhibition screens, which consists of 534 effector genes from the powdery mildew fungus Blumeria graminis f. sp. hordei. Additionally, I added 14 effector genes from another fungus: Fusarium oxysporum f. sp. lycopersici.
I adapted high-throughput LR-clonase reactions to obtain yeast transformation ready plasmids for each gene. I then proceed to adapt a yeast transformation protocol for high throughput yeast transformation of the 548 plasmids. I obtained 548 yeast strains each containing an N-terminally GFP-tagged CSEP effector gene with an inducible expression system.
All the yeast strains were simultaneously screened for growth performances under different conditions. This was done by plating several dilutions of each yeast strain on plates with a different carbon source. Glucose as carbon source inhibited expression of the gene, while galactose as carbon source stimulates expression.
Another part of the project consisted of engineering a plasmid that allowed transformation to and expression of the CSEP genes in the green alga Clamydomonas reinhardtii. For this part I finished some part of the cloning work, but not fully.
I adapted high-throughput LR-clonase reactions to obtain yeast transformation ready plasmids for each gene. I then proceed to adapt a yeast transformation protocol for high throughput yeast transformation of the 548 plasmids. I obtained 548 yeast strains each containing an N-terminally GFP-tagged CSEP effector gene with an inducible expression system.
All the yeast strains were simultaneously screened for growth performances under different conditions. This was done by plating several dilutions of each yeast strain on plates with a different carbon source. Glucose as carbon source inhibited expression of the gene, while galactose as carbon source stimulates expression.
Another part of the project consisted of engineering a plasmid that allowed transformation to and expression of the CSEP genes in the green alga Clamydomonas reinhardtii. For this part I finished some part of the cloning work, but not fully.
Due to the early termination, no progress beyond the state of art was made