Document Type

Thesis - Open Access

Award Date


Degree Name

Master of Science (MS)

Department / School

Biology and Microbiology

First Advisor

Senthil Subramanian


Legumes and soil rhizobia share a symbiotic relationship, wherein rhizobia fix atmospheric dinitrogen in exchange for photosynthates from the host plant. This exchange of mutual benefits occurs in a specialized root structure called ‘nodule’. Soybean (Glycine max) nodules arise from root outer cortical cells. De novo cell differentiation of cortical cells during nodule development leads to the formation of two distinct nodule zones in the mature nodule: the central infection zone where nitrogen fixation occurs and the peripheral parenchyma zone that houses vascular bundles. Plant signaling mechanisms that dictate the development of these nodule zones are not known. To address this knowledge gap, nodule zone-specific gene expression patterns were evaluated using “Isolation of Nuclei Tagged in specific Cell Types (INTACT)” and “Translating Ribosomes Affinity Purification (TRAP)”. Results from that study identified genes and pathways enriched in the nodule parenchyma and nodule infection zone. Enrichment of known pathways in the relevant nodule zones indicated that the datasets were reliable. The enriched genes in the two zones could be classified into one of these three categories: Auxin Signaling components, Transcription Factors (TFs) and Nutrient Transporters. In the present study, three of these genes, PIN-LIKES7 (GmPILS7, GLYMA.01G157700), AUXIN RESPONSE FACTOR5 (GmARF5, GLYMA.17G256500) and PHOSPHATE2 (GmPHO2, GLYMA.13G178600), were selected for functional characterization using RNA interference and CRISPR/Cas9-mediated gene editing in composite hairy root soybean plants. These genes were selected based on their specific enrichment levels in different nodule zones and the lack of knowledge on their role in nodule development despite the importance of auxin signaling and phosphate nutrition to this process. The zone-specific gene expression studies observed GmPILS7 to be enriched in nodule parenchyma at 7 days post inoculation (dpi) and 10 dpi. Composite hairy root plants expressing the GmPILS7-RNAi construct (55% knockdown) showed fewer nodules, a reduction in infection zone area, and an increased nitrogenase activity in transgenic roots. Prior studies in the lab showed GmPHO2 to be enriched in nodule infection zone at 7 dpi and nodule parenchyma at 10 dpi. Composite hairy root plants expressing the GmPHO2-RNAi construct (42% knockdown) showed fewer nodules, reduction in nodule size, and an increased nitrogenase activity per root. The GmARF5 was reported to be enriched in nodule parenchyma at 7 dpi and nodule infection zone at 10 dpi. An efficient knockdown for this gene was not observed (22% reduction in gene expression). Moreover, the CRISPR-Cas9 knockouts were not observed for any of the genes. Therefore, GmARF5 results are reported as inconclusive in this study. Since PILS7 is reported to be an intracellular auxin transport protein directing nuclear auxin to ER, reduced GmPILS7 expression might alter auxin levels in the nodule parenchyma and hence affect nodule development in soybean. Similarly, GmPHO2 coding for a ubiquitin conjugating E2 enzyme, has been reported to direct proteolytic degradation of several phosphate transporters. Based on the results obtained here, it implied that GmPHO2 plays a significant role in maintaining phosphate levels in different nodule zones, due to differential gene expression in the two zones. Although, a stark phenotype was not observed in terms of nodule zone development in silencing of either of the two genes, GmPILS7 and GmPHO2, this study adds to our understanding of the soybean nodule development and is the first report to establish role of PIN-LIKES protein family in soybean nodule development. It must also be noted that in both cases, mild to moderate silencing was observed. CRISPR-Cas9 knockouts of the genes and/or overexpression studies along with cellular and sub-cellular localization assays would help provide a clearer picture of the roles of these genes in soybean nodule development and zone differentiation.

Library of Congress Subject Headings

Soybean -- Genetics.
Soybean -- Roots.


South Dakota State University

SaCas9 Nucleotide alignment.txt (15 kB)
SaCas9 Nucleotide alignment.txt



Rights Statement

In Copyright