Voß, Martin
(2022).
Crucial steps on the way to a comprehensive structural understanding of the plant proteins EDS1 and PAD4 and their role in innate immunity.
PhD thesis, Universität zu Köln.
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Abstract
Infection of cultures of food-crops with pathogens can be a serious threat to the worldwide food supply. Therefore, the creation of resistant varieties, either via breeding or artificial genetic modification, is a pressing task for researchers and farmers worldwide. To rationally design resistant or more resilient species a deep understanding of the natural plant immune response is needed. Plants defend themselves against pathogens using an intricate two layered defence system. The first line of defence consists of membrane standing receptors that recognize conserved molecular pattern associated with pathogens like flagellin or chitin and trigger the immune response. Adapted pathogens have found ways to overcome the first line of defence by injecting effector proteins in the cell that block the initial immune response. In response, plants developed a series of soluble cytoplasmatic receptors that recognize the effectors, either directly or indirectly. The resulting immune response often leads to the controlled death of the infected cell to limit pathogen growth. The object of this thesis is the EDS1 protein family with its members enhanced disease susceptibility 1 (EDS1), phytoalexin deficient 4 (PAD4) and senescence associated gene 101 (SAG101). They are central points of the plant immune system and are involved in the signalling between receptors and immune response. A knockout of EDS1, PAD4 and to a lesser degree SAG101 leads to a high susceptibility of the plants against a broad variety of pathogens including bacteria, fungi and feeding insects. Despite the essential role of EDS1, PAD4 and SAG101 in the plant immunity, their function is not yet known. EDS1 forms mutually exclusive heterodimers with PAD4 or SAG101 which act downstream of the immune receptors of both layers of defence. All three protein consist of an N-terminal lipase-like domain with α/β-hydrolase fold and a unique C-terminal domain named after EDS1 and PAD4 as EP-domain. Except a structure of the EDS1-SAG101 dimer from A.thaliana (atEDS1/atSAG101), no structural information is known about the proteins. Previous attempts to gain a crystal structure of unbound EDS1 failed due to the low diffraction quality of the crystals gained. In this thesis, I used nanobodies raised from Llama glama against atEDS1 as crystallisation chaperones to gain a structure of unbound atEDS1. Crystallisation attempts were successful and yielded three structures of atEDS1 bound to different nanobodies. Combined with previously performed SAXS experiments and in planta IP data gathered by cooperation partners, I was able to confidently determine the quaternary structure of unbound EDS1 to be monomeric. Previous studies reporting an atEDS1 self-interactions can be explained by large non-biological crystal contacts and a tendency of the protein to forms aggregates. 13 As atPAD4 cannot be expressed in soluble form in E.coli, I used sequences from different organisms to gain soluble protein for characterisation in vitro. An PAD4 orthologue from Vitis vinifera (vvPAD4) could be expressed in high amounts in E.coli. This thesis is the first report of successfully producing a recombinant PAD4 variant. The attempt to produce a complex of EDS1/PAD4 from Vitis vinifera (vvPAD4/vvEDS1) revealed a surprising new splice variant of vvEDS1 that was successfully crystallised and structurally characterized. This finding raises several new questions about the regulation and alternative splice variants of EDS1
| Item Type: | Thesis (PhD thesis) | ||||||||
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| URN: | urn:nbn:de:hbz:38-614704 | ||||||||
| Date: | 16 May 2022 | ||||||||
| Language: | English | ||||||||
| Faculty: | Faculty of Mathematics and Natural Sciences | ||||||||
| Divisions: | Faculty of Mathematics and Natural Sciences > Department of Chemistry > Institute of Biochemistry | ||||||||
| Subjects: | Life sciences | ||||||||
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| Date of oral exam: | 14 April 2022 | ||||||||
| Referee: |
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| Refereed: | Yes | ||||||||
| URI: | http://kups.ub.uni-koeln.de/id/eprint/61470 |
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