These are slides from a Journal club about recent discoveries in the area of TIR domain signaling in plants and how this informs our understanding of general stress biology. The journal club was hosted by the Plant-microbe interactions group from Utrecht University on Tue July 12, 2022. Main content: - History of TIR identification, Toll as a byproduct of the Heidelberg screen - Signalling of TIRs via protein-protein interactions - The surprise from Wallerian degradation: oops, TIRs have enzymatic activity - The era of TIR enzymatic activities in plants - Attempt to make a coherent model of TIR signaling - can we use the 2',3'-cNMP synthetase activity as a biotech target? Supplement (good juice here in my opinion)

1. 13-7-2022
Toll!
The TIR revolution in
plant stress biology
Dmitry Lapin
Postdoctoral researcher
PLANT-MICROBE INTERACTIONS, TRANSLATIONAL PLANT BIOLOGY
Lemaitre et al (1996) https://doi.org/10.1016/S0092-8674(00)80172-5

2. Plan
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• What are the TIR protein domains and why care?
• The breakthrough: generation and perception of
immunogenic small nucleotide molecules in plants
• What’s the link with general stress response?
• Potential of TIR enzymatic activities as a biotech targets

3. Disclaimers
- I am former MPIPZ colleague having a collaboration with Parker lab
- Not a promo of research from a couple of laboratories but rather an
overview of the state-of-the-art (no names mentioned in the presentation)
- Only a small selection of papers was cited on slides due to space
constraints; priority to sources of graphics and key points
- This meeting is recorded; the recording could be shared without your
explicit permission
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4. Principles of development, 3rd Ed. (2007)

5. The Toll byproduct
- The Heidelberg screen
70s-80s
- Toll (adj.) = “mad”,
“amazing”, “great”
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Wieschaus and Nüsslein-Volhard (2016) https://doi.org/10.1146/annurev-cellbio-113015-023138
Nüsslein-Volhard (2022) https://doi.org/10.1016/j.tig.2021.09.006
Toll dominant
allele with
circulating
ventral belts

6. Part of Toll is shared
with components of
immune systems in
different organisms
IL-1R = Interleukin 1R
receptor
N = tobacco resistance
gene
TIR = Toll, IL-1R, Resistance
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Gay and Keith (1991) https://doi.org/10.1038/351355b0
Whitham et al. (1994) https://doi.org/10.1016/0092-8674(94)90283-6
Similarity of Toll, IL-1R and plant Resistance protein N

7. Who has TIRs?
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Lapin et al. (2022) https://doi.org/10.1093/plcell/koac035

8. What are TIRs good/bad for (in plants)?
- Warding off infection
- Prevention of dysbiosis
- Neuron cell death
- Regulation of development
- Manipulation of host by
effectors
- Reproduction barrier
- Indirect effect on abiotic
stress tolerance
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Schultink et al (2017) https://doi.org/10.1111/tpj.13715
Vaishnava et al (2011) https://doi.org/10.1126/science.1209791
Yang et al (2010) https://doi.org/10.1073/pnas.1011957107

9. How TIRs exert their
activity?
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Bernoux et al. (2011) https://doi.org/10.1016/j.chom.2011.02.009
Williams et al. (2014) https://doi.org/10.1126/science.1247357
Lapin et al. (2022) https://doi.org/10.1093/plcell/koac035

10. Signaling by
cooperative assembly
formation (SCAF)
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Clabbers et al. (2021) https://doi.org/10.1038/s41467-021-22590-6

11. Wallerian degeneration
and the awesome
power of genetics
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Coleman and Freeman (2010) https://doi.org/10.1146/annurev-neuro-060909-153248
Osterloh et al. (2012) https://doi.org/10.1126/science.1223899
Kudos to Drosophila genetics!

12. Taking the TIR field beyond
interactions: TIR of SARM1 is
NAD+ consuming enzyme
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Essuman et al. (2017) https://doi.org/10.1016/j.neuron.2017.02.022; Sporny et al. (2020) https://doi.org/10.7554/eLife.62021;
Figley et al. (2021) https://doi.org/10.1016/j.neuron.2021.02.009; Osterloh et al. (2012) https://doi.org/10.1126/science.1223899
Initial hypothesis was wrong, but …

13. Plant TIRs can consume
NAD+ as well
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Horsefield et al (2019) https://doi.org/10.1126/science.aax1911
Wan et al (2019) https://doi.org/10.1126/science.aax1771

14. Plant TIR immune
receptors are pathogen
effector-induced
enzymes
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Ma et al (2020) https://doi.org/10.1126/science.abe3069
Martin et al (2020) https://doi.org/10.1126/science.abd9993
RPP1 + effector ATR1 Roq1 + effector XopQ
Effector
Interacting TIRs
TIR NBD LRR
Basic TNL receptor organization

15. ‘TIRs are talking but
what do they say?’
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TIR
TIR
Substrate(s) Product(s)
Pathogen growth arrest
Cell death
? ?
How can we fish out the “right product”?
Receptor for TIR products

16. EDS1 family - putative TIR product receptor
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Cui et al (2018) https://doi.org/10.1016/j.molp.2018.05.007
Lapin et al (2020) https://doi.org/10.1146/annurev-phyto-010820-012840
Gartner et al (2019) https://doi.org/10.1105/tpc.19.00099
(PAD4)

17. Conserved NLR proteins
help in TIR signaling
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Shao et al (2016) https://doi.org/10.1104/pp.15.01487
Bonardi et al (2011) https://doi.org/10.1073/pnas.1113726108
Qi et al (2018) https://doi.org/10.1073/pnas.1814856115
TIR receptors Coil-coiled receptors
Conserved helpers:
ADR1 and NRG1
TIR-dependent
cell death
TIR/CC NBD LRR
Basic NLR receptor organization

18. Establishing a minimal EDS1-helper
NLR TIR signaling module
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Lapin et al (2019) https://doi.org/10.1105/tpc.19.00118
Sun et al (2021) https://doi.org/10.1038/s41467-021-23614-x
N.b. epss (knocked out EDS1 family)
TNL Roq1
N.b. WT
TNL Roq1
+XopQ
EDS1
SAG101
NRG1.1
+XopQ
EDS1
PAD4
NRG1.1
+XopQ
EDS1
SAG101
NRG1.1
YFP

19. Fishing for TIR products with a
functional EDS1-helper module
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Huang et al (2022) https://doi.org/10.1126/science.abq3297
Jia et al (2022) https://doi.org/10.1126/science.abq8180
TIR
TIR
Substrate(s) Product(s)
-EDS1-SAG101-NRG1
-EDS1-PAD4-ADR1
RPP1 + effector ATR1

20. Inference of small
molecules from
structures of activated
EDS1 family complexes
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Huang et al (2022) https://doi.org/10.1126/science.abq3297
Jia et al (2022) https://doi.org/10.1126/science.abq8180
RPP1+ATR1 RPP1+ATR1

21. Genetic evidence for
physiological relevance of
the inferred small
molecules
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Huang et al (2022) https://doi.org/10.1126/science.abq3297; Jia et al (2022) https://doi.org/10.1126/science.abq8180;
Lapin et al (2019) https://doi.org/10.1105/tpc.19.00118; Sun et al (2021) https://doi.org/10.1038/s41467-021-23614-x;
Dongus et al (2022) https://doi.org/10.1111/tpj.15747

22. Model of the stepwise
small molecule
production by plant TIRs
22
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Huang et al (2022) https://doi.org/10.1126/science.abq3297
Jia et al (2022) https://doi.org/10.1126/science.abq8180
Essuman et al (2022) https://doi.org/10.1126/science.abo0001

23. ‘Plant TIRs are talking but
one needs a translator’
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Huang et al (2022) https://doi.org/10.1126/science.abq3297; Jia et al (2022) https://doi.org/10.1126/science.abq8180;
Essuman et al (2022) https://doi.org/10.1126/science.abo0001; Lapin et al. (2022) https://doi.org/10.1093/plcell/koac035;
Sun et al (2021) https://doi.org/10.1038/s41467-021-23614-x
TIR
TIR
Substrate(s) Product(s)
NAD+
di-ADPR
ADPr-ATP
pRib-AMP
pRib-ADP
Translator
EDS1 family
Helper NLRs

24. Signaling by
cooperative assembly
formation (SCAF)
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Clabbers et al. (2021) https://doi.org/10.1038/s41467-021-22590-6
Evidence in plants?

25. Plant TIRs can form
filaments
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Yu et al (2022) https://doi.org/10.1016/j.cell.2022.04.032; Bernoux et al. (2011) https://doi.org/10.1016/j.chom.2011.02.009;
Swiderski et al (2009) https://doi.org/10.1094/MPMI-22-2-0157; Lapin et al. (2022) https://doi.org/10.1093/plcell/koac035
TIR NBD LRR
Basic TNL receptor organization
First implication of the plant-
specific aD extension in TIR
enzymatic activities

26. Isolated plant TIRs can
produce 2’,3’-cAMP and
2’,3’-cGMP
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Yu et al (2022) https://doi.org/10.1016/j.cell.2022.04.032;
Nishimura et al (2017) https://doi.org/10.1073/pnas.1620973114
Bartsch et al (2006) https://doi.org/10.1105/tpc.105.039982
TIR NBD LRR
RBA1 is a TIR-only protein
NUDT7 is phosphodiesterase
genetically linked to EDS1 signaling

27. Where does this all fit?
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Huang et al (2022) https://doi.org/10.1126/science.abq3297; Jia et al (2022) https://doi.org/10.1126/science.abq8180;
Essuman et al (2022) https://doi.org/10.1126/science.abo0001; Yu et al (2022) https://doi.org/10.1016/j.cell.2022.04.032
TIR
TIR
Substrate(s) Product(s)
NAD+ di-ADPR
ADPr-ATP
pRib-AMP
pRib-ADP
Translator
EDS1 family
Helper NLRs
TNLs
TIR-only
TIR
TIR
DNA/RNA 2’,3’-cAMP
2’,3’-cGMP
?
TIR-only

28. 28
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Kosmacz et al (2018) https://doi.org/10.1104/pp.18.00285
Chodasiewicz et al (2022) https://doi.org/10.1093/plphys/kiac013; Created with BioRender.com
Step 1:
Initial activation of
immunity by TNLs and
surface receptors leads to
accumulation of TIR-only
Step 2:
Accumulation of 2’,3’-
cNMPs
TIR
TIR
TIR
TIR
2’,3’-cAMP
2’,3’-cGMP
di-ADPR,
ADPr-ATP,
pRib-AMP,
pRib-ADP
EDS1-independent in principle, activation of EDS1
signaling is a byproduct (later phase, 2’,3’-cNMPs are
stable)
EDS1-dependent
(early phase, TIR products short-
lived)
di-ADPR, ADPr-ATP,
pRib-AMP, pRib-ADP
Step 3:
Perception of 2’,3’-cNMPs
and initiation of general
stress response in plants
2’,3’-cAMP
2’,3’-cGMP
An attempt to make a coherent picture of TIR signaling in dicot plants

29. Potential of TIR
enzymatic activities as
a biotech targets
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Created with BioRender.com
2’,3’-cAMP
2’,3’-cGMP

31. Supplement related to the follow-up
discussion
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32. What is still puzzling
about the interaction
of EDS1 family with TIR
products?
• The AtEDS1 R493 position is critical for the
interaction with di-ADPR, ADPr-ATP, pRib-
AMP and pRib-ADP and the recruitment of
helpers
• Tomato EDS1 mutant equivalent to AtEDS1
R493 and AtEDS1_R493A are still functional
in Roq1-dependent cell death
• AtEDS1_F419E and equivalent tomato EDS1
mutation are strong loss-of-function but di-
ADPR, ADPr-ATP, pRib-AMP and pRib-ADP
do not go that deep into the EDS1 family
pocket
• Similarly, still unclear what the role of EDS1
F61 (highly conserved) in the lipase-like
domain is
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13-7-2022
Huang et al (2022) https://doi.org/10.1126/science.abq3297; Jia et al (2022) https://doi.org/10.1126/science.abq8180;
Gantner et al (2019) https://doi.org/10.1105/tpc.19.00099; Lapin et al (2019) https://doi.org/10.1105/tpc.19.00118
Interface for:
pRib-AMP
pRib-ADP
Interface for:
di-ADPR
ADPr-ATP

33. 2’,3’-cAMP/cGMP synthetase
activity is not essential for
activating EDS1-helper signaling
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Huang et al (2022) https://doi.org/10.1126/science.abq3297; Jia et al (2022) https://doi.org/10.1126/science.abq8180;
Yu et al (2022) https://doi.org/10.1016/j.cell.2022.04.032
-> 2’,3’-cAMP/cGMP signaling
EDS1-independent?

34. TIR enzymology and
EDS1-helper
phylogenetic signature
-> TIR-only proteins are
predicted in early land
plants but the EDS1 family
and helpers ADR1/NRG1
are absent
-> is 2’,3’-cNMP synthetase
activity ancestral?
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Johanndrees et al (2021) https://doi.org/10.1101/2021.11.29.470438

35. What is the source of
TIR-only proteins in
cells?
- TIR-only is the most common TIR
architecture in plants
- TIR-containing genes are induced
in response to PAMPs and upon
effector recognition
- There is evidence that alternative
splicing of TNLs (RPS4, N) is
essential for their function
- Nonsense-mediated decay can
also lead to TIR-only forms
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Dinesh-Kumar et al (2000) https://doi.org/10.1073/pnas.020367497; Zhang et al (2007) https://doi.org/10.1104/pp.107.108720;
Nandety et al (2013) https://doi.org/10.1104/pp.113.219162; Parker et al (2021) https://doi.org/10.7554/eLife.65537; Johanndrees
et al (2021) https://doi.org/10.1101/2021.11.29.470438; Lapin et al (2022) https://doi.org/10.1093/plcell/koac035;

36. SA and TIR signaling
Stresses including SA
treatment can lead to
formation of ‘stress
granules’ filled with RNA
Is this a source of RNA for
2’,3’-cNMP synthetase
activity?
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Kosmacz et al (2018) https://doi.org/10.1104/pp.18.00285
Zavaliev et al (2020) https://doi.org/10.1016/j.cell.2020.07.016