The role of plant membrane proteins in legume-rhizobia symbiosis [electronic resource]

Symbiotic nitrogen fixation occurs when rhizobia bacteria infect the roots of legume plants, resulting in the formation of a specialized organ called a nodule. In this mutualistic symbiosis, bacteria provide the plant with nitrogen, and the plant provides the bacteria with carbon sources. This metabolic exchange occurs inside the root nodules where bacteria reduce (or "fix") molecular dinitrogen to ammonia, a form of nitrogen that plants can use to synthesize amino acids and proteins. Legume-rhizobia symbiosis initiates with plant recognition of the bacterial signaling molecule Nod Factor (NF). After plant perception of NF, bacteria infect plant roots through host-derived infection threads and are eventually endocytosed into host cells. Inside the host cell, the bacteria remain surrounded by a host membrane and differentiate into their nitrogen-fixing form. A great deal is known about NF perception and signaling, but the mechanisms by which infection threads form and by which bacteria are endocytosed into host cells remain elusive. To identify genes required for infection or bacterial endocytosis, I used a candidate gene approach in the model legume Medicago truncatula. I searched the M. truncatula genome for regions with homology to endocytosis and membrane shaping genes in plants and other organisms. I identified two M. truncatula flotillin-like genes, FLOT2 and FLOT4, which are up-regulated in response to the M. truncatula symbiont, Sinorhizobium meliloti. Flotillins in animals have been implicated in actin polymerization, maintenance of cell-cell contacts, membrane trafficking and pathogenesis. I silenced FLOT2 and FLOT4 using RNAi and amiRNAs and found a non-redundant requirement for both genes in symbiosis. When FLOT4 was silenced, infection threads typically aborted in root hairs. FLOT2-silenced plants formed fewer nodules and infection threads. This work implicates plant flotillins in legume-rhizobia symbiosis and suggests that flotillins in plants and animals may have a common function. NF recognition in M. truncatula requires the receptors NFP and LYK3. Each receptor is required for separate downstream responses: NFP is necessary for all known responses to bacteria, while LYK3 is required for infection. A leucine-rich repeat receptor-like kinase DMI2 acts downstream of NFP. Using GFP-tagged proteins, I localized the symbiotic receptor kinases DMI2 and LYK3. Both proteins had punctate distributions associated with root hair plasma membranes. After bacterial inoculation, both LYK3:GFP and DMI2:GFP were present on intracellular vesicles. LYK3:GFP persisted in infected cells and localized to infection thread membranes. The DMI2:GFP signal was nearly absent by one day post inoculation. These data are consistent with a role for LYK3 in infection and a role for DMI2 in signaling. I found that like LYK3 and DMI2, GFP-tagged FLOT4 is associated with the plasma membrane and has a patchy distribution. Upon inoculation with S. meliloti, FLOT4:GFP puncta become more diffuse and redistribute to form a cap at the tips of elongating root hairs. I investigated the dependence of FLOT4 distribution on NF perception and signaling via symbiotic receptors NFP, LYK3, and DMI2. I found that FLOT4:GFP has a decrease in puncta density specific to the putative dead kinase allele of LYK3, hcl-1. I co-expressed LYK3:GFP and FLOT4:mCherry and found that in buffer-treated root hairs, there is little co-distribution of the two proteins, and tagged LYK3 puncta are dynamic while FLOT4 is relatively stable. After inoculation, I found an increase in LYK3:GFP and FLOT4:mCherry co-localization and that LYK3:GFP shares the stable distribution shown by FLOT4:mCherry. The similarities in mutant phenotypes, protein localization, and protein dynamics suggest that FLOT4 and LYK3 may be components of a shared complex. This work indicates that protein arrangement within plant membranes is complex and is altered by perception of a symbiotic bacterium. This work suggests that redistribution of plant membrane proteins upon signal perception may serve to compartmentalize cellular processes.

Keywords: Biology.
Advisor: Long Sharon R. primary advisor. Thesis advisor
Bergmann Dominique advisor. Thesis advisor
Ehrhardt David (David Walter) advisor. Thesis advisor
Pfeffer Suzanne advisor. Thesis advisor