Investigating the role of KINC in arbuscular mycorrhizal symbiosis

Arbuscular mycorrhizal (AM) symbiosis is an over 400-million-year-old relationship where the AM fungus obtains carbon from the plant and in turn provides the plant with inorganic phosphate. Genetically altering plants to better benefit from its fungal partners may mitigate the unsustainable mining of finite phosphate reserves used for fertilizer. The KINASE Cs (KINC1 and KINC2) are a pair of AM host-conserved genes that are essential for AM symbiotic development. Our project investigated mechanism by which the KINCs support fungal accommodation by identifying and evaluating potential KINC-interacting proteins. Bimolecular fluorescence complementation assays were used to evaluate the putative interaction between the KINCs and KIN3. Our data suggest a KINC2 and KIN3 interaction but not a KINC1 and KIN3 interaction. KIN3 was also cloned into our yeast-two-hybrid system for future assays to further access these interactions. Finally, three independent experiments of plants were transformed and planted for tissue in future TurboID proximity labeling proteomics experiments. We additionally aimed to evaluate the importance of KINC1’s kinase activity. This was done by complementation experiments using various single amino acid mutants of the KINC1’s kinase domain, where colonization level was assessed. Our data show that none of the constructs successfully complement the kinc1kinc2 mutant phenotype. However, colonization level was notably low across all root samples. While these experiments must be repeated for confidence conclusions, they represent a first look at mechanisms by which the KINCs function in Medicago.

My time in the Harrison lab was a fruitful experience. My mentor taught me many valuable skills for this field, including Gateway Cloning, Agrobacterium-mediated transformations, confocal microscopy, and more. I am glad to have had the opportunity to work in a molecular lab for the first time, and I look forward to applying the knowledge and skills gained from this experience in future work.