The González-Guerrero Lab
Preprint
Publication Types:
Medicago truncatula Iron-chaperone 1 (ICHAP1) is required for symbiotic nitrogen fixation
Abstract
Hundreds of proteins in the cell require iron (Fe) or Fe-containing cofactors to function. However, how Fe2+ or Fe3+ are specifically allocated to each of these proteins in plant cells remains largely unknown. It has been proposed that Fe metalation could be driven by specific interactions with Fe-shuttling proteins known as Fe-chaperones. Here, we present the first family of plant Fe2+-chaperones (ICHAPs) with orthologues in dicots and monocots. The role of these proteins in Fe distribution to Fe-dependent metabolic processes has been illustrated using symbiotic nitrogen fixation in Medicago truncatula root nodules. ICHAP1 is a soluble Fe2+-binding protein that interacts with plasma membrane Fe2+ transporter NRAMP1, but not with symbiosome Fe2+-transporters. ICHAP1 mutants present altered Fe distribution in cells and they cannot fix nitrogen. A second family member, ICHAP2 is required to target Fe2+ to symbiosomes, as it accepts Fe2+ from ICHAP1 and interacts with symbiosome Fe2+-importer VTL8, but not with NRAMP1. These results indicate a path for Fe2+ allocation from the plasma membrane to the symbiosome through specific protein-protein interactions and Fe2+ exchange from NRAMP1 to ICHAP1, to ICHAP2, and to VTL8.
Funder Information Declared
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- Ministerio de Ciencia, Innovación y Universidades
- U.S. National Science Foundation, https://ror.org/021nxhr62, DBI-0703285, IOS-1127155, DBI-2233714
Enhances production of nitrogenase components in Nicotiana benthamiana through co-expression with Bacterioferritin A.
Abstract
Summary
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Engineering nitrogen fixing crops requires not only transferring the nitrogenase structural genes, but also the accessory genes to synthesize its iron-sulphur cofactors. Scaffold protein NifU is a critical element in this system as the starting point of nitrogenase cofactor assembly. NifU has been successfully produced in plants, however, its optimal production required high levels of iron in the medium. This is likely due to a faulty connection with the endogenous iron trafficking network
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To identify specific elements targeting iron to NifU, pull-down assays were performed to identify showing bacterioferritin A (BfrA) as a likely candidate. Co-immunopurification, mutant characterization, iron transfer assays, and co-expression in Nicotiana benthamiana assays were carried out.
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BfrA transfers iron to NifU through protein-protein interactions. When these two proteins were co-expressed in N. benthamiana leaves, there was an increase in NifU production. In turn, it led to doubling NifH synthesis, a nitrogenase structural protein that is also required for the synthesis of the more complex nitrogenase cofactors.
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Our results provide a new element towards engineering nitrogen-fixing crops. They also underscore the importance of transferring the metal delivery systems when expressing metalloproteins in heterologous systems.
Funder Information Declared
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- Agencia Estatal de Investigación, ID2021-12460OB-100, PCI2021-122052-2B, FJCI-2017-33222, PRE2022-101253
- Instituto de Salud Carlos III, PT17/0019
- Gates Foundation, INV-005889, INV-067006