Date of Award

12-2015

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Legacy Department

Biochemistry and Molecular Biology

Committee Member

Dr. Julia A. Frugoli, Committee Chair

Committee Member

Dr. Douglas G. Bielenberg

Committee Member

Dr. Cheryl Ingram-Smith

Committee Member

Dr. Hong Luo

Abstract

The control of nodule number in legumes is primarily accomplished through a complex systemic signaling pathway termed autoregulation of nodulation (AON). The protein kinase SUNN, a leucine-rich repeat receptor-like kinase, exhibits shoot control of AON. I show subcellular localization of SUNN to the plasma membrane and specifically to the plasmodesmata, a prime location for systemic signaling. To aid in better understanding the mechanisms of signaling in AON, I used different approaches to identify potential protein-protein interactions involving SUNN, including candidate genes, exploratory proteomics, and forward genetic screens. Candidate interactors, including CLAVATA2 (CLV2), CORYNE (CRN) and guanine exchange factors of the RopGEF family, were investigated using the bimolecular fluorescence complementation (BiFC) assay with SUNN and mutant analysis for their role in AON. I report positive BiFC interactions between SUNN and CLV2, CRN, RopGEF1, RopGEF2, and RopGEF5. Mutant analysis of these candidate proteins provide additional support for a role in AON. In legumes, mutations in either CLV2 or CRN, and RNAi of RopGEF1, RopGEF2, or RopGEF5 in roots render hypernodulation phenotypes. Taken together, my results suggest a signaling complex involving SUNN is formed in the shoots to transduce a signal from the roots into a signal back to the roots through the activation of GTPases by RopGEFs, ultimately regulating nodulation events. I also identified 22 novel putative interactors of SUNN stemming from an exploratory proteomics experiment involving transgenic M. truncatula carrying SUNN-YFP/HA that were subjected to co-immunoprecipitation to isolate protein complexes that contain the transgene. The precipitated proteins were identified by LC-MS/MS and are presented here as putative interacting partners. In addition, a forward genetics approach identified components of the AON signaling pathway. Utilizing a genetic suppressor screen of sunn-1, we identified four independent lines carrying mutations that suppress the supernodulation phenotype of sunn-1. The mapping of one of these suppressor lines pinpointed the location of the lesion to an approximately 150 kB region on Linkage Group 2 harboring about 26 annotated genes.

Share

COinS