UC Riverside

The strigolactone (SL) and karrikin (KAR) signaling pathways, two highly homologous pathways in plants, are not only essential in plant growth and development, symbiotic and parasitic interactions, and responses to environmental stresses, but also hold significant implications for the field of plant hormone biology. Understanding how these two complex pathways evolved to achieve specificity separate from one another while possibly maintaining a level of crosstalk has been a matter of great interest. The work in this dissertation, which aims to deepen knowledge of how receptors in each pathway interact with downstream protein partners.While there have been numerous technological advancements for analyzing strigolactone receptors, the processes, such as stable transformations, can be laborious and time-consuming. In the first chapter, I present an innovative and alternate approach to evaluate the functionality of the strigolactone receptor, DWARF14 (D14), in a transient assay in Nicotiana benthamiana. This approach, using CRISPR-cas9 to knock out the two endogenous N. benthamiana D14 genes, D14a and D14b, to analyze different strigolactone receptor functions in a transient system. The Nbd14a,b mutant displayed a typical d14 phenotype, short and highly branched. Using this mutant background and a ratiometric reporter system, I demonstrated how this system can evaluate D14 variants not previously investigated in a plant system. Most research on strigolactone receptors has concentrated on ligand binding and perception. Nevertheless, interactions with downstream protein partners, specifically SUPPRESSOR OF MAX2 LIKE (SMXLs) proteins, play a crucial role in strigolactone signal transduction. My second chapter focuses on identifying and mapping the D14 amino acids necessary for SMXL interaction. I determine highly conserved amino acids in D14 and analyze if these residues convey interaction towards the SMXL7 protein partner by mutational analysis in both the transient system described in Chapter One and stable Arabidopsis thaliana lines. The data indicates that altering specific D14 residues affects SMXL interaction, but there is no clear interaction interface. Additionally, variant D14D198A showed a defective interaction with SMXL. However, the data suggests that the D198 residue is crucial for the protein's structure and stability. Parasitic species in the Orobanchaceae family have evolved to sense host-derived SLs via the convergent evolution of karrikin receptors known as KAI2d proteins. In the last chapter, I used the transient N. benthamiana system to determine the functional roles of the seven KAI2d orthologs and the two SMAX1 orthologs found in the hemiparasite Phtheirospermum japonicum. The findings of this research, which discovered four PjKAI2d proteins are highly sensitive SL receptors, and two demonstrate inhibitory functions towards the sensitive receptors at low SL concentrations, hold great potential for further understanding and manipulation of plant-parasite interactions.