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Design and redesign of a plant immune receptor complex

Periodic Reporting for period 3 - ImmunityByPairDesign (Design and redesign of a plant immune receptor complex)

Reporting period: 2018-10-01 to 2020-03-31

"We are addressing the problem of plant disease and specifically, an important mechanism in plant disease resistance. Plant disease caused by pathogens results in substantial crop losses, which are controlled both by spraying agrochemicals, and by genetic resistance introduced into crop varieties by breeders. Genetic variation for disease resistance usually maps to loci that encode intracellular nucleotide-binding, leucine-rich repeat (NLR) receptors. These proteins enable plants to detect ""effector"" molecules elaborated by pathogens, and to then activate defence. The mechanisms that underpin this process are poorly understood. Some NLRs function as pairs of NLRs, one of which senses pathogen molecules via an integrated effector target domain, the other of which implements defence activation. These two NLRs cooperate to convert recognition into defence activation. Our major goal is to understand the mechanism by which a specific NLR pair, Arabidopsis RPS4 and RRS1, convert recognition of two different bacterial effectors (AvrRps4 and PopP2) into defence activation. Better understanding of this mechanism would shed light on how other NLRs and NLR pairs function, both in plants and mammals.

Disease resistance in crops is important for society. Reducing crop losses to disease is crucial for sustainable agriculture, and enabling humans to produce enough food while minimizing the impact of agriculture on land, water and mineral resources. A better understanding of genetic resistance could reduce the requirement for agrochemicals in crop disease control, and might facilitate engineering novel recognition capacities. Further, NLR proteins play an important role in mammalian immunity; insights from plant NLRs may inform understanding of mammalian NLR mechanisms, and vice versa.

The project was established to address two questions, with four main goals, each to be implemented by one postdoc.
Question 1 (3 postdocs) What are the design principles of the RPS4/RRS1 complex, and which domain/domain interactions are important for effector recognition, and for defence activation?
Question 2 (1 postdoc) Can we use discoveries about RPS4/RRS1 mechanisms to design new recognition capacities into RPS4/RRS1 or closely related gene pairs?

Q1 Goal 1 (Postdoc 1) is to understand the interactions between the different domains in RPS4 and RRS1, both within proteins and between proteins, and how these change upon conversion of the complex from an inactive state prior to interaction with recognized effector, to the activated state post-recognition of the effector. RPS4 carries 4 domains (TIR, NB-ARC, LRR and CTD). RRS1 carries 6 domains (TIR, NB-ARC, LRR, Domain 4, WRKY and Domain 6).

Q1 Goal 2 (Postdoc 2) is to characterize the properties of the RPS4/RRS1 protein complex, both before and after activation.

Q1 Goal 3 (Postdoc 3) is to determine structural information (via X-ray crystallography and cryo-EM) that would shed light on the architecture and mechanism of the complex, and on how it recognizes effectors and converts recognition into defence activation

Q2 Goal 1 (Postdoc 4) is to take advantage of a refined understanding of the mechanisms of the RPS4/RRS1 complex in order to repurpose it to respond to novel ligands, thus creating novel sources of disease resistance.
"Goal 1 We discovered that the inactive conformation of RRS1 involves domains 4, 5 (WRKY) and 6 (D456), and is held together by WRKY/Domain 4 (DOM4) interactions that maintain inactivity prior to ligand detection. AvrRps4 binds the WRKY domain, and AvrRps4 perception via the WRKY domain disrupts WRKY/DOM4 association, leading to an ‘open’ D456 conformation, while PopP2 activates the complex differently. A ‘closed’ conformation of RRS1-R, reversibly engineered in a new method with bacterial colicin E9 and Im9 high-affinity domains (""molecular Velcro""), loses AvrRps4 and PopP2 responsiveness. Following RRS1 derepression, DOM4 interactions with the RPS4 C-terminal domain likely contribute to activation. This work is about to be submitted for publication (PNAS, in submission). We have also shown that the RRS1 TIR domain contributes to derepression. AvrRps4 promotes interaction of RRS1-TIR with Domains 5+6 of RRS1-R and RRS1-S, whereas PopP2 only promotes interaction with Domains 5+6 of RRS1-R, which may explain why RRS1-R responds to PopP2 and AvrRps4, whereas RRS1-S responds only to AvrRps4. In very recent data, we show a requirement for phosphorylation of the RRS1-R C-terminal region for PopP2 recognition, but not for AvrRps4 recognition.

Goal 2 We verified that Arabidopsis RPS4 and RRS1 NLR proteins are both required to make an authentic immune complex. Over-expression of RPS4 in tobacco or in Arabidopsis results in constitutive defence activation; this phenotype is suppressed in the presence of RRS1. RRS1 protein co-immunoprecipitates (co-IPs) with itself in the presence or absence of RPS4, but in contrast, RPS4 does not associate with itself in the absence of RRS1. In the presence of RRS1, RPS4 associates with defence signaling regulator EDS1 solely in the nucleus, in contrast to the extra-nuclear location found in the absence of RRS1. In contrast to earlier reports, the AvrRps4 effector does not disrupt RPS4-EDS1 association in the presence of RRS1. In the absence of RRS1, AvrRps4 interacts with EDS1, forming nucleocytoplasmic aggregates, the formation of which is disturbed by the co-expression of PAD4 but not by SAG101. These data indicate that the study of an immune receptor protein complex in the absence of all components can result in misleading inferences, and reveals an NLR complex that interacts with immune regulators EDS1/PAD4 or EDS1/SAG101, and with effectors, during defence activation upon effector recognition (Huh et al We are refining methods to minimise non-specific protein aggregation, in order to use blue and clear native gels to monitor changes in the RPS4/RRS1 complex over a time course of estradiol-inducible AvrRps4 provision. By putting split YFP components on RPS4 and RRS1 N-termini, we also could affinity purify the RPS4/RRS1 complex using anti-GFP antibodies.

Goal 3 To obtain mechanistic insights into the function of RPS4/RRS1, we are working to obtain structural biology knowledge. To date, full length RPS4 and RRS1 proteins can be produced in planta but not in E. coli or insect cells. Constructs to express each domain of these proteins have been tested in E coli and insect cells, with some fragments of certain domains accumulating with certain constructs, but except for the RRS1 WRKY domain, not at sufficient levels to justify more focused protein chemistry work. We have prepared RRS1 WRKY domain protein and its ligand AvrRps4 in E. coli, and shown they associate in vitro. Crystallization trials have revealed conditions to obtain minicrystals of AvrRps4/WRKY complex, but these are not yet good enough for diffraction studies; preparing diffraction quality crystals is currently a top priority. The affinity between AvrRps4 (and mutants) and the WRKY domain (and mutants) is being investigated using surface plasmon resonance. Moderate level expression of several protein domains has also been achieved in planta using geminivirus expression constructs; we are prioritizing understanding WRKY/DOM4 and DOM4-CTD interactions.

Goal 4 Both plant and animal NLR proteins detect pathogen-derived molecules and activate defence. Mammalian NAIP/NLRC4 NLR pairs respond to ligands by oligomerization to create an inflammasome which imposes induced proximity on the N-terminal Caspase-recruitment domain (CARD), but no such process have been defined for plant NLRs. As part of our high risk/high gain ""synthetic biology"" program (though not part of the original proposal), we recently fused the TIR domain of the Arabidopsis RPS4 NLR to the N-terminus of NLRC4, and showed in planta that inflammasome-dependent induced proximity of the TIR domain is sufficient to initiate defence signalling that is dependent on both EDS1 and also the NLR, NRG1. However, despite the genetic requirement for a presumed catalytic glutamate residue, the induced NADase activity reported for mammalian SARM1 could not be detected upon ligand-dependent defence activation.
We continue to address the high risk/high reward goal of engineering novel recognition capacities into the RPS4/RRS1 chassis, but so far without success. Attempts based on destabilizing an interfering form of RRS1 in trans to a constitutively active form have resulted in either constitutively active alleles or alleles that lose the capacity to interfere. We have set up a protease trap detector system that activates RRS1 by cleaving away the WRKY domain in the presence of a viral protease; this confers protease-dependent hypersensitive response (HR) in transient assays, but did not confer elevated disease resistance in vivo. Replacement of the RRS1 WRKY domain with other WRKY domains has so far only resulted in constitutive RPS4-dependent defence activation."
"In the above section, we report multiple advances that take our understanding of RPS4/RRS1 mechanisms beyond the state-of-the-art.

In particular, we highlight the innovative and novel deployment of conditional ""molecular Velcro"" using E. coli colicin Im9 and E9 proteins which bind to each other with very high affinity, to restrict the capacity to activate RRS1, in a manner that is reversible by TEV protease because of a protease cleavage site engineered between RRS1 and the E9 tag. We anticipate this method will prove to be of widespread utility in protein engineering and the investigation of protein complexes.

We also highlight our innovative deployment of the mammalian NLRC4/NAIP chassis to impose ligand-dependent induced proximity on the RPS4 TIR domain, and show this is sufficient to activate defence. This could be applied to any system in which the consequences of ligand-dependent induced proximity are under investigation

By the end of the project we expect to:
1- Define the role of WRKY/Domain 4 interactions and their disassociation by AvrRps4 in derepression of the complex
2- Define the involvement of the RRS1-TIR domain and its interactions with the WRKY domain in derepression of the complex
3- Define the role of RRS1-R domains 5+6 (D56) phosphorylation specifically in PopP2 responsiveness, and understand the requirement for the longer C-terminus of RRS1-R in PopP2 but not AvrRps4 responsiveness
4- Determine structures for WRKY domain and Domain 4 of RRS1, together and in association, and understand how their associations are disrupted by effector action
5- Determine structure for the RPS4 C-terminal domain, and understand the structural basis of its interaction with Domain 4 of RRS1, and how such interactions activate RPS4
6- Understand what changes occur in converting the inactive complex to its active form. The NAIP/NLRC4 chassis provides a positive control for inflammasome formation; in blue native gels we can see ligand dependent conversion of TIR-NLRC4 into a slow migrating form of ~ 1Md. We anticipate something similar must occur with RPS4/RRS1 but have not seen this yet. This will require monitoring changes in the complex over a time course of ligand induction, and refining buffers to minimise non-specific aggregation, thus avoiding false indications of interactions.
7- Tested all possible approaches to engineering novel recognition capacities into the RPS4/RRS1 chassis.