Molecular Characterisation of Early Nodulation Events in Soybean

Legumes include some of the most economically important crops and are characterised by their ability to form a symbiotic relationship with nitrogen fixing rhizobia bacteria. Effective legume-rhizobia symbioses result in the formation of a wart-like structure on the root called the nodule. In this nodule, the rhizobia provide nitrogen to the host plant in exchange for energy and shelter. Nodulation is induced by the recognition of a rhizobia derived, strain specific signal, called Nod factor. Recognition of Nod factor from compatible rhizobia leads to complex signalling within and between the epidermis and the cortical cells of the root. Concomitantly, the rhizobia infect the young root hair cells, which are strictly located on the part of the root where the root hairs emerge, a region called the Zone Of Nodulation (ZON). Only when the plant’s Nod factor signalling and the rhizobia infection event in the ZON are in complete synchronicity, do nodules form. Nodulation research in the last few decades has identified a large number of genes associated with, or required for, the formation and development of nodules. These genes today form the foundation of our understanding of the early nodulation signalling pathway. However, there are a number of novel nodulation signalling components still to be discovered, which may hold the key to the missing links in our current knowledge on this signalling pathway. The work reported in this thesis aimed to identify novel components of the early nodulation signalling pathway using modern RNA-seq technology. Approximately 3,000 genes were identified as differentially regulated between roots inoculated with compatible, or a Nod factor-deficient mutant, rhizobia (nodC-). Targeting only the ZON to isolate the transcriptome allowed for the identification of genes having little and/or transient expression in the ZON, which would otherwise have been masked by other dominating transcripts. Gene ontology analysis identified both individual genes and molecular pathways that are likely regulated during early nodulation. Detailed expression analysis of selected candidate genes revealed that some of these genes, including two gibberellin (GA) biosynthesis genes and a TIR-NBS-LRR encoding a putative resistance gene, are specifically expressed in tissue responding to compatible rhizobia inoculation (ZON and nodules). Previous studies utilising mutant and pharmacological analyses have indicated that GA plays an important role in nodule development. However, its precise action, including the location and timing in which GA acts during nodulation, is yet to be determined. To better understand the role of GA in nodulation, follow up studies were conducted for two candidate GA biosynthesis genes identified here, GmGA20ox a and GmGA3ox 1a. Histochemical analysis revealed cell-type specific activation of the GmGA20ox a promoter during nodulation, which changed in localisation at different stages of nodule organogenesis. However, silencing of this gene had no effect on nodule number or appearance. Similarly, follow-up studies were conducted for the TIR-NBS-LRR coding gene, GmTIR-NBS-LRR. Unlike many TIR-NBS-LRR genes which are associated with pathogen-related effector molecule perception, this gene was up-regulated by the symbiotic bacteria, specifically in response to rhizobia-derived Nod factor. Silencing and over-expression of GmTIR-NBS-LRR both resulted in reduced, although not significantly, nodule numbers. However, the complex regulation of the TIR-NBS-LRR gene family makes the interpretation of these results difficult. Based on recent findings, including those reported in this thesis, updated models and predictions relating to the early nodulation pathway are presented and discussed. This includes outlining potential role(s) for the candidate genes identified here in the ZON transcriptome. The mechanistic action of GA and the TIR-NBS-LRR candidate as they relate to nodule formation are also discussed.