Cyclops response elements in the evolution and function of root endosymbioses in Lotus japonicus

Gong, Xiaoyun (2021): Cyclops response elements in the evolution and function of root endosymbioses in Lotus japonicus. Dissertation, LMU München: Fakultät für Biologie

Abstract

Plant growth is dependent on sufficient supply of nitrogen, an essential component for important macromolecules such as proteins and nucleic acids. A small group of plants belonging to four orders - Fabales, Fagales, Cucurbitales and Rosales (the FaFaCuRo clade) - have evolved the ability to engage in a mutually beneficial interaction, namely the nitrogen-fixing root nodule symbiosis (RNS). Fixed nitrogen is supplied by the bacterial symbiont to the plant host, hence helping the host to overcome nitrogen limitation. RNS development is a complex procedure that involves massive transcriptional reprogramming carried out by a cohort of cis- and trans-acting regulators. The evolutionary steps leading to the emergence of RNS have been at the center of interest for decades. Understanding the main genetic differences between plants that can form RNS and those that cannot is assumed to provide the key for installing RNS in important crop plants that are currently unable to engage in RNS. Evolution of cis- and trans-acting elements has played key roles in the evolution of novel biological traits. This work focussed on two cis-regulatory elements that have played distinct roles in the evolution and maintenance of RNS. One cis-element, PACE (Predisposition Associated Cis- regulatory Element) was identified via a phylogenomic approach. PACE was discovered to be exclusively present in species within the FaFaCuRo clade in the promoter of the Nodule Inception (NIN) gene that encodes a master transcription factor (TF) positioned at the top of the transcriptional regulatory hierarchy specific for RNS. PACE confers responsiveness to bacterial signals and dictates gene expression in cortical cells forming infection threads (ITs), a tube-like plant-derived structure through which bacteria enter the root. PACE is essential for restoring IT formation in the Lotus japonicus nin-15 mutant even when engineered into the NIN promoter of tomato, a species outside of the FaFaCuRo clade. PACE contains the binding site of a TF Cyclops that is indispensable for transcriptional rewiring during RNS as well as for the evolutionarily older arbuscular mycorrhizal symbiosis. P ACE confers transactivation mediated by Cyclops in combination with the Calcium and Calmodulin-dependent protein kinase (CCaMK). These results suggested that PACE allows the induction of NIN via the symbiosis-induced signalling cascade common for RNS and AM. The phylogenetic restriction of PACE is congruent with that of RNS and consistent with an emergence in the last common ancestor of the FaFaCuRo clade. A related, yet functionally distinct cis-element was identified in the promoter of the Calcium Binding Protein 1 (CBP1) gene utilising the transgenic line T90 that originated from a promoter tagging program of L. japonicus. T90 carries a promoterless GUS gene that is specifically induced during RNS and AM. Dissection of the regulatory region of the T90 GUS gene led to the identification of one cis-regulatory element required for reporter expression in the epidermis and a second element, CYC-RECBP1 (Cyclops response element in the CBP1 promoter), necessary and sufficient for transactivation mediated by CCaMK/Cyclops and driving gene expression during both AM and RNS. The lack of GUS expression in three T90 white mutants that were identified from an ethyl methanesulfonate-mutagenised T90 population could be traced to DNA hypermethylation detected in and around CYC-RECBP1. Two additional regulatory regions also impact CBP1 expression. This work showcases Cyclops response elements as an essential building block for engineering RNS in crops, with a long-term goal of reducing agricultural fertiliser application.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: nitrogen-fixing root nodule symbiosis, arbuscular mycorrhiza symbiosis, Lotus japonicus, cis-regulatory elements, transcriptional regulation, epigenetic modification, DNA methylation, tissue specificity
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
• Fakultäten: Fakultät für Biologie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 10. Dezember 2021
• 1. Bericht­erstatter:in: Parniske, Martin