This project focusses on the evaluation of candidate receptors and their interacting partners from a physiology perspective using gene knock-out, mis/over-expression, and fluorescent protein tagging combined with confocal microscopy.

Aim of the project

In this project we aim to establish a robust high throughput cloning and plant transformation/selection system to generate genome editing and fluorescent protein reporter constructs. CRISPR/Cas9 genome editing technology provides a precise approach to knockout multiple genes of interest. This essential for validating the biological function of our candidate receptors and their interacting partner as the mutant of some of these genes are not available due to their chromosome architecture.

Through examining the single mutant phenotype of our candidate receptors, the relevance of the candidates for ROS signaling can be determined. On the other hand, through comparing the phenotype between the crk-single and crk-potential interacting partner double mutant, we can quickly determine if the selected receptors genetically interact with each other.

In addition to gene knockout, overexpression lines will be generated to see if they show phenotypes differential to the single or double mutants. To determine if our candidate receptors physically interact with their potential interacting partner, they will be tag by different fluorescent proteins followed by fluorescence imaging and Förster resonance energy transfer- fluorescence lifetime imaging microscopy (FRET-FLIM).