The transcription factor NIN mediates a dynamic balance between gene activation and repression in L. japonicus

Andrade Aguirre, Rosa Elena (2021): The transcription factor NIN mediates a dynamic balance between gene activation and repression in L. japonicus. Dissertation, LMU München: Fakultät für Biologie

Abstract

The Nodule inception (NIN) gene encodes a transcription factor positioned at the top level of the transcriptional regulatory cascade indispensable for the establishment of root nodule symbiosis (RNS), an intimate relationship between legumes and nitrogen-fixing bacteria. Gaining insights into regulation and activity of this transcription factor, is pivotal for the mechanistic understanding of RNS development. A previous model suggested that NIN regulates its own expression via a negative feedback loop by binding to the NIN promoter and through protein-protein interaction between NIN and Cyclops (Doctoral thesis, Lambert, 2017). In this study, I confirmed that NIN is involved in the regulation of its own expression by comparing the NIN expression between wild type and the nin-2 mutant. In a transient transactivation assay in Nicotiana leaves, NIN inhibited the transactivation of all known CCaMK/Cyclops targets, namely NIN, ERN1 and RAM1. To elucidate the mechanism by which NIN interferes with the CCaMK/Cyclops activity, I tested whether NIN recognizes a specific sequence in the promoter of NIN. Such sequence was not detected; moreover, NIN was able to inhibit Cyclops transactivation activity independently of the Cyclops DNA binding sequence. These results suggest that the inhibition of Cyclops by NIN is not mediated at the level of DNA binding (for example by competition). The interaction between NIN and Cyclops was confirmed and the interaction domain was delimited to their N-terminal regions. However, the delimited interaction domain of Cyclops was dispensable for the NIN-mediated inhibition. In addition, the presence of autoactive CCaMK versions disrupted the Cyclops/NIN interaction in vivo, although NIN was able to inhibit NIN expression mediated by this complex. My data suggest that the Cyclops/NIN interaction and the NIN-mediated inhibition of the CCaMK/Cyclops complex are two independent processes. I tested the impact of amino and carboxy-terminal domains of NIN on the local inhibition of nodulation and in the transcriptional repression of NIN, and observed that both processes require the carboxyterminal domain, specifically the PB1 protein-protein interaction domain. The following model is suggested: NIN is present at basal levels and interacts with Cyclops; thus, repressing it. When calcium spiking occurs, CCaMK phosphorylates Cyclops and the Cyclops/NIN interaction is disrupted. Cyclops binds to the NIN promoter and transactivates its expression (Singh et al., 2014). Once the NIN protein concentration increases, NIN inhibits the transactivation of Cyclops by possibly recruiting other PB1-containing protein. This could be a mechanism that allows a rapid response against further infection by rhizobia in an autoregulation of nodulation (AON)-independent manner.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: NIN, Cyclops, interaction, inhibition, negative feedback loop, root nodule symbiosis
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
• Fakultäten: Fakultät für Biologie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 19. März 2021
• 1. Bericht­erstatter:in: Parniske, Martin