Investigating the development of Medicago truncatula symbiosome membrane in nitrogen-fixing root nodules using MtENOD16
Date
2018
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University of Delaware
Abstract
The endosymbiotic relationship between legumes and rhizobia results in the development of a root nodule, a specialized plant organ where symbiotic nitrogen fixation occurs. During nodule formation, bacteria enter the host’s cell within the invasion zone of the nodule using an endocytosis-like process. Within the cell, the symbiosome membrane encases the microbial symbiont and provides a physical barrier that separates the microbe from the plant’s cytoplasm. The work described in this thesis aims at investigating the development of the symbiosome membrane using early nodulin 16 protein (ENOD16) and a series of novel organelle markers in the model legume plant Medicago truncatula. Proteomic studies identified ENOD16 as a component of the symbiosome membrane in M. truncatula. I hypothesize that ENOD16 is essential for the development of a functional root nodule. In this work, ENOD16 localization was first determined by tagging its gene to the fluorescent marker citrine and HA epitope tag and comparing its localization with known organelle markers. Confocal laser scanning microscopy determined that ENOD16 localization in transiently expressed tobacco leaves and transformed M. truncatula root nodules was consistent with plasma membrane labeling. Secondly, symbiosome formation was evaluated in a set of M. truncatula mutants that each encoded an intervening retrotransposon Tnt1 within the ENOD16 gene or within the closely related ENOD20 gene. Nodules formed on ENOD16 and ENOD20 Tnt1 mutants displayed relatively smaller nodules in size with an overall lower density of infected cells compared to wild type as observed by transmission electron microscopy. In addition, the infection thread matrices within the mutant nodules were abnormal, as were the bacteroids. The infection thread matrices displayed a granulated texture compared to the smooth texture typically seen in wild type, and the released bacteroids appeared to be distressed and unhealthy, some of which were observed in vacuoles. Moreover, the infected cells of the mutants showed signs of membrane degradation and early senescence compared to wild type. ☐ To sum up, the results of this thesis will help advance what is currently known about ENOD16 and its role in the development of a functional root nodule in M. truncatula.
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Biological sciences