Universität zu Köln

Sun, Huanhuan Sun (2023). Biochemical exploration of EDS1 family proteins and their interaction with helper-NLRs. PhD thesis, Universität zu Köln.

PDF Huanhuan Sun_PhD thesis_Publish.pdf Download (2MB)

Abstract

EDS1 family members are important immune hubs which influence many aspects of plant defense. In Arabidopsis, there are three EDS1 proteins: EDS1, SAG101, and PAD4. In unchallenged plants EDS1 is forming heterodimers with SAG101 and PAD4, respectively, and both heterodimers are important for TNL-mediated immune responses. NRG1s and ADR1s are two groups of NLRs, which are integrated with CCR domains in the N-terminus instead of TIR or CC domains in canonical NLRs. Unlike sensor NLRs, they function downstream of EDS1 family members in effector-triggered immunity and are therefore referred to as helper NLRs. Recently, research shows that two EDS1 family heterodimers are exclusively responsible for two parallel immune branches downstream of TIR-NLR receptors. Typically, upon effector recognition, E-S heterodimers activate downstream NRG1s to trigger the HR response. In contrast, E-P heterodimers activate ADR1s to induce resistance. However, how the signals are transmitted from upstream TNL activation to downstream EDS1 family members and further down to helper-NLRs and eventually elicit the ETI response was still a mystery. In this study, I applied multiple biochemical techniques to investigate the activation mechanism of EDS1 family members and their interaction with helper-NLRs. PAD4 was first purified and tested as a heterodimer together with EDS1 in vitro. A chimera chi2 formed of N-terminal PAD4 and C-terminal SAG101 was used to mimic the active form of SAG101. However, chi2 was not able to activate helper-NLRs under the tested conditions. Given the indispensable role of TNL NADase products in ETI response, we reconstituted the TNL resistosome together with EDS1 family members and helper-NLRs in insect cells, and surprisingly, we captured the interaction of the E-S complex with NRG1A, and the interaction of the E-P complex with ADR1_L1. Furthermore, these results were confirmed by pull-down assays, in which TIR/TNL NADase products were incubated with EDS1, SAG101, and NRG1A, respectively after incubation with EDS1, PAD4, and ADR1_L1. However, in the absence of TIR/TNL NADase products, the interactions could not be induced. This suggests, perhaps the most important finding of this study, that activation of helper-NLRs is controlled by the products of TNL NADases that are captured and delivered to helper-NLRs by the EDS1 family of heterodimers.

Item Type:	Thesis (PhD thesis)
Creators:	Creators Email ORCID ORCID Put Code Sun, Huanhuan Sun huanhuansun6@gmail.com UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-650908
Date:	2 March 2023
Language:	English
Faculty:	Faculty of Mathematics and Natural Sciences
Divisions:	Außeruniversitäre Forschungseinrichtungen > MPI for Plant Breeding Research
Subjects:	Natural sciences and mathematics Life sciences
Uncontrolled Keywords:	Keywords Language EDS1 English SAG101 English NRG1 English ADR1 English
Date of oral exam:	17 January 2023
Referee:	Name Academic Title Chai, Jijie Prof. Dr. Niefind, Karsten Prof. Dr.
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/65090