Investigating the downstream targets of autoregulation of nodulation in Medicago truncatula

Abstract

Since the first Green Revolution, N-fertilisers have been overused to increase crop yields and this has led to economic and environmental consequences in agriculture area. Legumes can form symbiotic relationship with Rhizobiumin root nodules for symbiotic N-fixation (SNF) which allows legumes to grow in N-poor soil. This provides a possible alternative for N-fertiliser. Sincenodulation is a high-energy demanding process, root nodule numbers must be tightly regulated by plants to prevent excessive draining of essential carbon reserves. In Medicago truncatula, the systemic CLE (CLAVATA/Embryo surrounding region related)-SUNN (SUPER NUMERIC NODULES) dependent signalling pathway acts as negative feedback system to suppress further nodule development. This regulation is known as autoregulation of nodulation (AON). However, little is known about the downstream targets of this mechanism. In this project, a set of transcription factors (TFs) that act downstream of CLEs are identified by RNA-seq profiling. Two of these candidates, MtWRKY70 and MtMYB102, were selected based on their preferred gene expression characteristics that dramatically upregulated in wildtype, but not in sunn4receptor mutant in response to CLE13 peptide treatment. Reverse genetics was done to validate their function in AON.As composite individuals which has wild type shoot and transgenic roots can be generated via hairy root transformation in M. truncatula. This technique was used to overexpress the two selected TFs in M. truncatula roots. Both the 35S:WRKY70 and 35S:MYB102 transgenic lines have significantly decreased nodule numbers (approximately 2-fold) when compared with the 35S:GUS control line. These indicate MtWRKY70 and MtMYB102 are acting as negative regulators of nodulation, downstream of CLE13-SUNN circuit mediated AON. Homologues of these MtWRKY70 and MtMYB102 in other plant systems are known to regulate plant defence and hormone signalling. It is likely that these TFs may have been recruited in legumes to regulate nodulation. The in-depth understanding of AON will not only provide insight into how plants coordinate growth and development in response to the environment, but also will identify key gene candidates for improving efficacy of SNF in legumes.