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INVESTIGATING RHIZOBIA AND MOLECULAR DETERMINANTS OF SUCCESSFUL SYMBIOTIC NITROGEN FIXATION IN LEGUMES

Abstract
Legumes can thrive in nitrogen-limited soil by forming a symbiotic relationship with nitrogen-fixing bacteria called rhizobia. This allows them to grow sustainably beyond their native regions. Examples include sunn hemp (Crotalaria juncea L.) and barrel clover (Medicago truncatula) that have successfully expanded from India and the Mediterranean. Symbiotic nitrogen fixation occurs on specialized root structures called nodules and involves complex interactions between the symbiotic partners with initial molecular communication leading to nodule formation and subsequent nitrogen fixation in nodules. However, not all legume - rhizobia interactions result in effective nitrogen fixation due to factors including compatibility between legume and rhizobia, and transport of host molecules to the rhizobia. Therefore, to address these issues, this dissertation investigates the compatibility between tropical sunn hemp and US-native rhizobia for nodule formation and fixation. Additionally, this study develops a CRISPR/Cas9 gene-editing tool as a resource to examine the functional importance of nodule-specific protein molecules, nodulin-22 (NOD22) and nodulin-25 (NOD25) in producing nitrogen-fixing nodules in Medicago truncatula. I show that Massachusetts-native rhizobia fail to effectively fix nitrogen for sunn hemp, resulting in suboptimal growth benefits. This contrasts with non-native rhizobia found in commercial inoculants, which proved more effective at fixing nitrogen and promoting sunn hemp growth. These non-native rhizobia in commercial inoculant persisted in the soil as nitrogen fixers for sunn hemp for up to three years. Additionally, it was discovered that the Massachusetts-native rhizobia that did not benefit sunn hemp, were instead compatible with hairy vetch (Vicia villosa), another legume cover crop previously established in the region. Moreover, compatibility studies conducted in different parts of the US revealed varying results, including nodulation and nitrogen fixation incompatibilities.Next, I developed a gene-knockout tool using a vector-based CRISPR/Cas9 system to facilitate the assessment of the functional significance of two legume nodule - specific genes namely nodulin-22 (NOD22) and nodulin-25 (NOD25), in producing nitrogen fixing nodules in Medicago truncatula. Previously, these protein molecules have been studied individually, but their roles in nitrogen fixation remain unknown. Analyses via PCR-genotyping and Sanger sequencing demonstrated the successful deletion of a substantial 12 kb DNA fragment by my CRISPR/Cas9 vector, potentially inactivating NOD22 and NOD25 genes. Medicago plants exhibiting this deletion displayed stunted shoot growth and contained small, round and white nonfixing nodules. When compared to wild type pink nodules that fixed nitrogen for Medicago plants, the non-fixing nodules had very few rhizobia. The availability of the CRISPR/Cas9-NOD22/25 vector is expected to furnish mutants, thereby advancing functional studies regarding nodulin proteins. Collectively, understanding how root nodules function in nitrogen fixation in legumes is crucial for farmers to identify rhizobia that facilitates nitrogen fixation, supporting the growth of non-native legumes in the US. Additionally, researchers can gain insights into the biological significance of legume protein molecules during the process of symbiotic nitrogen fixation.
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openaccess
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dissertation
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http://creativecommons.org/licenses/by-sa/4.0/
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2024-09-01T00:00:00-07:00
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