FRUITFULL (FUL) is a MADS-domain transcription factor that plays a major role in the development of the Arabidopsis fruit, the silique. In ful mutants, the silique remains very small and does not open to disperse the seeds.
The evolution of plant species largely depends on the evolution of transcription factors. In this project, we are investigating the evolution of the transcription factor FRUITFULL, which is present as a single copy in the model species Arabidopsis, and regulates many important developmental processes such as leaf development, plant architecture, flowering time and fruit development. In tomato, there are four FRUITFULL genes that can each regulate a subset of these functions.
To understand the evolution of the four tomato FRUITFULL proteins, we are investigating their properties and try to find out to which DNA sequences they can bind, which protein-protein interactions they form, what their functions are in tomato, and to what extent they can rescue the Arabidopsis fruitfull mutant. This information will also help us to dissect the functions of FRUITFULL in Arabidopsis, and to predict what functions FRUITFULL genes from other species will have.
The following experiments are performed to elucidate the evolution of the tomato FRUITFULL genes:
- Yeast two-hybrid experiments to investigate the protein-protein interactions. Try to identify the amino acids that are responsible for changes in these interactions and perform the experiments with mutated proteins. (Cloning of constructs, PCR, yeast transformation, yeast two-hybrid).
- DIP-Seq experiments to investigate to which DNA-sequence motifs the different FRUITFULL homologs can bind. (Recombinant protein expression, DIP-seq, high-througput sequencing, bioinformatics)
- CRISPR mutagenesis to knock-out the genes in tomato and study the function of the different homologs. (Cloning constructs, tomato transformation, tissue culture, DNA-extraction, PCR, phenotyping)
- Transformation of Arabidopsis fruitfull mutants with the tomato genes to determine to what extent they can rescue the phenotype. (Cloning constructs, Floral dip transformation, DNA-extraction, PCR, phenotyping).
- Expression analysis of the tomato genes. (qPCR, cloning of reporter constructs).