Iron Homeostasis in the Legume-Rhizobial Symbiosis

Walton, Jennifer (2017) Iron Homeostasis in the Legume-Rhizobial Symbiosis. Doctoral thesis, University of East Anglia.

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All plants require iron to survive, but those that establish symbioses with nitrogen-fixing bacteria have a particularly high demand. The bacterial nitrogenase enzyme is dependent on iron-sulphur cofactors, and the host plant expresses large amounts of leghaemoglobin in infected cells to protect nitrogenase from oxygen inactivation. Using reverse genetics, this study aims to identify proteins involved in Fe transport and haem cofactor biosynthesis in the nodules of Medicago truncatula and its partner symbiont Sinorhizobium meliloti.

I identified the major (FECH1B) and minor (FECH1A) nodule ferrochelatases, enzymes responsible for the last stage of haem biosynthesis. I obtained a Tnt1 insertion mutant in each of these and confirmed genotype by PCR. RT-PCR showed that transcript was absent in fech1b but not fech1a, most likely because the insertion in the latter is intronic. fech1b mutants produced fewer pink nodules than the wild type, with some morphological differences. FECH1B-eGFP localised to the plastids.

I also identified two highly upregulated nodule-specific vacuolar iron transporter-like (VTL) genes, VTL4 and SEN1. I obtained two Tnt1 insertion mutants in VTL4, and also a mutant, 13U, with a 30 kilobase deletion encompassing VTL4 and SEN1; the only two nodule-expressed VTLs. 13U mutants only produced white nodules, while vtl4 mutants produced more white nodules than the wild type. A novel bacterial iron reporter PmbfA:lux showed that bacteria in 13U nodules perceived less iron than in wild-type nodules, with vtl4 nodules showing an intermediate phenotype. VTL4-mCherry localised to the plasma membrane and infection thread membrane of cells in the infection zone, while SEN1mCherry localised to the symbiosome membrane of interzone cells. Together, these data suggest that VTL4 and SEN1 mediate Fe transport to bacteroids, but at different stages of the infection process.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Megan Ruddock
Date Deposited: 23 Mar 2018 14:58
Last Modified: 31 Mar 2020 00:38

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