Periodic Reporting for period 4 - ImmunityByPairDesign (Design and redesign of a plant immune receptor complex)
Berichtszeitraum: 2020-04-01 bis 2021-09-30
We address the problem of plant disease which results in substantial crop losses, controlled both by spraying agrochemicals, and by genetic resistance. Variation for resistance usually maps to loci encoding nucleotide-binding, leucine-rich repeat (NLR) receptors that enable detection of and response to "effector" molecules made by pathogens. This process is poorly understood. Some NLRs function in pairs, one of which senses effectors via an integrated domain, the other of which implements defence activation, thus cooperating to convert recognition into defence activation. We aim to understand how a specific NLR pair, Arabidopsis RPS4 and RRS1, converts recognition of two different bacterial effectors (AvrRps4 and PopP2) into defence activation.
Reducing crop losses to disease is crucial for sustainable agriculture so we can produce enough food while minimizing use of land, water and mineral resources. A better understanding of plant immunity could reduce use of agrochemicals to control crop disease and enable engineering novel recognition capacities. Further, 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 (PD).
Question 1 (3 PDs) 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 PD) Can we use discoveries about RPS4/RRS1 mechanisms to design new recognition capacities into RPS4/RRS1 or closely related gene pairs?
Q1 Goal 1 (PD1) is to understand the interactions between the different domains in RPS4 and RRS1 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.
Q1 Goal 2 (PD 2) is to characterize the properties of the RPS4/RRS1 protein complex, both before and after activation.
Q1 Goal 3 (PD3) 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 (PD 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.
Reducing crop losses to disease is crucial for sustainable agriculture so we can produce enough food while minimizing use of land, water and mineral resources. A better understanding of plant immunity could reduce use of agrochemicals to control crop disease and enable engineering novel recognition capacities. Further, 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 (PD).
Question 1 (3 PDs) 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 PD) Can we use discoveries about RPS4/RRS1 mechanisms to design new recognition capacities into RPS4/RRS1 or closely related gene pairs?
Q1 Goal 1 (PD1) is to understand the interactions between the different domains in RPS4 and RRS1 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.
Q1 Goal 2 (PD 2) is to characterize the properties of the RPS4/RRS1 protein complex, both before and after activation.
Q1 Goal 3 (PD3) 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 (PD 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.
PD1 Interactions between RRS1 and RPS4 domains keep the complex inactive, and change upon effector detection (Ma et al, PNAS, PMID 30254172).
The RRS1 TIR domain and the C-terminus interact to promote activation of the complex (Guo et al, CellHostMicrobe, PMID 32234500).
Auto-active alleles of RPS4 need RRS1 for their auto-activity (Guo et al Plant Phys PMID 33793895). Overall, we revealed how domains of RPS4 and RRS1 interact for effector recognition to activate defence.
PD2 The RPS4/RRS1 immune complex (Huh et al PMID 28475615) exists as multiple forms from ~270 kDa to ~500 kDa. Unlike other TIR-NLRs that form tetramers upon ligand detection, the RPS4/RRS1 complex does not change in size. We work with Prof Jijie Chai’s lab in Tsinghua to express activated and non-activated forms of RPS4/RRS1 in insect cells, prior to cryo-EM to determine structure.
Overall, we learned much about RPS4/RRS1 during activation, but structure of the whole complex has not yet been determined.
PD3 Mechanistic insights into RPS4/RRS1 require structure. We defined the structure of the WRKY/AvrRps4 complex (Mukhi et al PNAS PMID 34880132). Structural characterization of the rest of the protein requires analysis of insect-cell produced full length proteins, working with Jijie Chai.
PD4 RPS4 TIR domain oligomerization is sufficient to activate defence (Duxbury et al PNAS PMID 32709746).
We tested 3 ways to engineer novel recognition capacity.
(i) Xanthomonas XopS effector interacts with pepper CaWRKY40. We replaced the RRS1 WRKY domain with CaWRKY40. In transient assays, the chimeric RRS1 activates RPS4-dependent defence in response to XopS. Disease assays are in progress.
(ii) Deleting the WRKY domain results in an autoactive RRS1 allele. We set up a protease trap detector system that activates RRS1 by cleaving off the WRKY domain with a potyviral protease from Potato Virus Y (PVY). This confers protease-dependent defence activation in transient assays but did not confer PVY resistance in vivo.
(iii) RRS1-R and RRS1-S suppress RPS4-dependent autoactivity of the RRS1-Rslh1 allele. Attaching an effector-dependent degron to RRS1-R enables degradation by phytoplasma effector SAP05. We used this to engineer a defence response to SAP05 (https://doi.org/10.1101/2021.09.06.459143).
All 3 approaches enabled novel recognition and response capacities in tobacco, but none enabled disease resistance. RPS4/RRS1 is more difficult than other paired NLRs for engineering novel resistances.
The RRS1 TIR domain and the C-terminus interact to promote activation of the complex (Guo et al, CellHostMicrobe, PMID 32234500).
Auto-active alleles of RPS4 need RRS1 for their auto-activity (Guo et al Plant Phys PMID 33793895). Overall, we revealed how domains of RPS4 and RRS1 interact for effector recognition to activate defence.
PD2 The RPS4/RRS1 immune complex (Huh et al PMID 28475615) exists as multiple forms from ~270 kDa to ~500 kDa. Unlike other TIR-NLRs that form tetramers upon ligand detection, the RPS4/RRS1 complex does not change in size. We work with Prof Jijie Chai’s lab in Tsinghua to express activated and non-activated forms of RPS4/RRS1 in insect cells, prior to cryo-EM to determine structure.
Overall, we learned much about RPS4/RRS1 during activation, but structure of the whole complex has not yet been determined.
PD3 Mechanistic insights into RPS4/RRS1 require structure. We defined the structure of the WRKY/AvrRps4 complex (Mukhi et al PNAS PMID 34880132). Structural characterization of the rest of the protein requires analysis of insect-cell produced full length proteins, working with Jijie Chai.
PD4 RPS4 TIR domain oligomerization is sufficient to activate defence (Duxbury et al PNAS PMID 32709746).
We tested 3 ways to engineer novel recognition capacity.
(i) Xanthomonas XopS effector interacts with pepper CaWRKY40. We replaced the RRS1 WRKY domain with CaWRKY40. In transient assays, the chimeric RRS1 activates RPS4-dependent defence in response to XopS. Disease assays are in progress.
(ii) Deleting the WRKY domain results in an autoactive RRS1 allele. We set up a protease trap detector system that activates RRS1 by cleaving off the WRKY domain with a potyviral protease from Potato Virus Y (PVY). This confers protease-dependent defence activation in transient assays but did not confer PVY resistance in vivo.
(iii) RRS1-R and RRS1-S suppress RPS4-dependent autoactivity of the RRS1-Rslh1 allele. Attaching an effector-dependent degron to RRS1-R enables degradation by phytoplasma effector SAP05. We used this to engineer a defence response to SAP05 (https://doi.org/10.1101/2021.09.06.459143).
All 3 approaches enabled novel recognition and response capacities in tobacco, but none enabled disease resistance. RPS4/RRS1 is more difficult than other paired NLRs for engineering novel resistances.
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. This has revealed that either the variant cADP Ribose made by bacterial TIRs is non-identical to the plant form, or additional and as yet uncharacterized signalling processes are required to initiate a full defence response
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. This has revealed that either the variant cADP Ribose made by bacterial TIRs is non-identical to the plant form, or additional and as yet uncharacterized signalling processes are required to initiate a full defence response