Genetic and miRNA Transcriptomic Analysis of Autoregulation of Nodulation Signaling in Medicago truncatula
Date of Award
Doctor of Philosophy (PhD)
Genetics and Biochemistry
Julia A Frugoli
James C Morris
Legumes can source nitrogen from the air through biological nitrogen fixation inside root nodules formed in a symbiosis with rhizobia. A complex root-to-shoot-to-root signaling pathway called Autoregulation of Nodulation (AON) controls the number of nodules formed depending on the plant’s nitrogen requirements. In Medicago truncatula, the MtSUNN receptor complex in the shoot binds to the root-generated AON signals, peptides called MtCLEs, resulting in downregulation of miR2111 expression in the shoot, with the effect of decreased transport of miR2111 to the roots. Decreased miR2111 levels in the roots then cause an increase in transcript levels of the miR2111 targets MtTML1 and MtTML2 in the roots with the end result of inhibition of further nodulation. In this dissertation, I used forward genetics, reverse genetics, and small RNA transcriptomics to explore AON. I mapped a suppressor mutation to identify a change in the MEDIATOR OF RNA POLYMERASE SUBUNIT 16 (MtMED16/SOS16) gene (R805H) which suppresses the hypernodulation phenotype of sunn-1 plants in a root-dependent manner. In contrast to both wild type and sunn-1 mutants, the suppressed plants lack nodule primordia and induction of expression of the critical nodulation transcription factor MtNIN at 3 days post inoculation, delaying and reducing nodule development. Applying a reverse genetics approach to study the role of the two TML genes in AON, I created multiple mutant alleles in MtTML1 and MtTML2 as well as double mutants using CRISPR-Cas9 mutagenesis and obtained a mutant in MtTML2 from a Tnt1 insertion library for analysis. Single mutant alleles produce approximately 2-fold more nodules than wild type, displaying partial loss of AON, whereas the double mutants exhibit a synergistic effect, producing approximately 20-fold and more nodules compared to wild type. Using miRNA transcriptomics to explore early signaling events, I identified a total of 43 differentially expressed pre-miRNA genes in A17 and sunn-4 plant roots between 0, 4, 12, 24, and 48 hours post inoculation. I identified seven pre-miRNAs encoding miRNAs in five families as interesting candidates for future study. The combined discovery of a novel gene in legume-rhizobia symbiosis through genetic mapping of the suppressor; the demonstration that MtTML1 and MtTML2 genes have a synergistic effect in M. truncatula nodulation, and the identification of interesting miRNA candidates for future study will result in an improved understanding of AON.
Chauligain, Diptee, "Genetic and miRNA Transcriptomic Analysis of Autoregulation of Nodulation Signaling in Medicago truncatula" (2020). All Dissertations. 3252.