Regulation and functions of ERF ERN transcription factors during root nodule symbiosis between Medicago truncatula and Sinorhizobium meliloti
Marion Cerri
Abstract:
Legumes are able to associate in symbiosis with Rhizobia bacteria in the soil, which culminates in the formation of a new organ referred to as the root nodule, within which differentiated bacteria fix nitrogen to the benefit of the host plant. The establishment of this association relies on a molecular dialogue between the two partners, involving bacterial lipo-chitooligosaccharide signals called Nod factors (NF). In the model legume Medicago truncatula, the perception of these symbiotic signals in the root epidermis, initiates a signaling pathway that leads to calcium oscillation responses required for the activation of downstream genes such as the well-characterized ENOD11. Previously, ERN1 and the closely-related ERN2 transcription factors (TFs) were reported as direct activators of ENOD11 via binding to the NFbox regulatory unit. In addition, phenotypic analysis of the ern1 knockout mutant has confirmed the importance of ERN1 not only during NF signaling but also throughout subsequent infection and nodule development stages. Nevertheless, the ern1 mutant displays a less severe phenotype compared to plants mutated in other NF signaling genes, raising the question of a possible functional redundancy with the endogenous closely-related ERN2 factor. My PhD project was focused on the study of the functional relationship between ERN1 and ERN2 TFs. By using a variety of strategies we aimed at determining both ERN expression profiles and relative functions during nodulation. We first examined the spatio-temporal expression profiles of these genes during rhizobial symbiosis and correlated this with the dynamics of cellular localization of ERN fusion proteins. These analyses revealed that these factors possess both common and distinct expression profiles, correlated with cell-type specific and dynamic in vivo protein accumulation, tightly associated with rhizobial pre-infection and subsequent infection stages in M. truncatula. Further cross-complementation studies in the ern1 mutant background showed that, when ERN2 is expressed under the control of the ERN1 promoter, it can fully restore the ern1 phenotype regarding NF-elicited ENOD11 activation and nodule formation. This indicates that these factors have similar biological activities and suggests that the incapacity of endogenous ERN2 to complement the ern1 mutant is mainly due to differences in their promoter activities. Finally, we also initiated a phenotypic characterization of M. truncatula ern2 mutant lines, in order to get a better insight into ERN2 specific functions during nodulation. The phenotypic analysis of a Tilling line (ern2.1) carrying a point mutation in a conserved amino acid in the ERN2 DNA binding domain, revealed a role for ERN2 during infection thread progression in the root cortex. Further molecular studies demonstrated that this mutated amino acid in the Tilling line is conserved and required for optimal DNA binding domain topology and transcriptional activity of ERN2 on its target ENOD11 gene. In addition, the ern2.1 line and a second ern2.2 insertional mutant line are both capable of forming nodules, in contrast to the ern1 mutant. Nevertheless, these nodules are partly infection defective leading to premature senescence. These findings provide evidence that ERN2 possesses specialized functions during nodulation that cannot be fully complemented by ERN1. This suggests that ERN possess common and divergent functions, ERN1 having a predominant role in rhizobial infection initiation and progression while ERN2 having a secondary and more centered role during infection thread progression. The ern1ern2.1 double mutant line, recently generated during my PhD, opens new perspectives to further study the functional relationship between ERN TFs during root endosymbioses.
Journal:
Thesis of the University of Toulouse
Link: