A plant pathogen effector blocks stepwise assembly of a helper NLR resistosome
The Phytophthora infestans effector AVRcap1b jams the tomato helper NLR NRC3 partway through assembly, stalling it at three protomers, and in doing so shows that NRC resistosomes build up one subunit at a time.
Resistosome structures so far have been endpoints: a resting state or a finished oligomer, nothing in between. AVRcap1b changes that. The P. infestans effector binds activated tomato NRC3 on the face that would normally be buried inside a completed hexamer, wrapping across three protomers and stopping there. Cryo-EM of the complex resolves a stalled half-resistosome whose three protomers superimpose on one half of the hexamer with an RMSD of 0.79 A. Truncating AVRcap1b's N-terminal LWY domains lets a fourth protomer in, so the block is steric and the assembly is stepwise. A four-residue NB-domain motif, YEFF, carries the interaction: mutate it and NRC3 still builds a resistosome but no longer binds the effector. No Rx sensor was captured in any complex, which fits activation-and-release.
Key findings (10)
AVRcap1b consistently and fully suppresses cell death mediated by all four NRC3 orthologs tested (N. benthamiana, Capsicum annuum, Solanum tuberosum, Solanum lycopersicum), while suppression of NRC1 and NRC2 orthologs is variable. This breadth is why the work pivoted from NRC2 to NRC3.
Evidence hypersensitive cell death assays in N. benthamiana nrc2/3/4 knockout lines, using the Rx / PVX-CP activation system
Source Figure 1B; figures S1A and S1B
In BN-PAGE, AVRcap1b reduces the high-molecular-weight signal of activated SlNRC3 and produces an additional band intermediate in size between resting SlNRC3 and the full resistosome, indicating a stabilised assembly intermediate. The P. ipomoeae AVRcap1b ortholog, which does not suppress NRCs, has no such effect, and AVRcap1b does not affect NbNRC4 oligomerisation.
Evidence BN-PAGE with MADA-motif mutants (SlNRC3EEE, NbNRC4L9E) that oligomerise without triggering cell death; repeated three times
Source Figure 1C; figure S1C
Cryo-EM resolves activated SlNRC3 as a hexameric resistosome resembling NbNRC2 and NbNRC4, and resolves the AVRcap1b-bound state as three activated SlNRC3 protomers wrapped by the L-shaped effector. Superimposing the intermediate on the hexamer gives RMSD 0.793 A, so the stalled species is structurally one half of the complete resistosome.
Evidence single-particle cryo-EM; consensus reconstruction at 3.01 A and focused map at 3.21 A merged into a composite; SlNRC3 hexamer consensus map at 2.85 A; FSC 0.143 cutoff
Source Figures 2A-2E; figures S3-S10; table S1; PDB 9RI9 / EMDB EMD-53990 (hexamer); PDB 9RIA / EMDB EMD-53991, EMD-53988, EMD-53989 (complex)
AVRcap1b binds the interface that would be masked by the second half of a completed hexamer, wrapping around the underside of three protomers and contacting their CC, NB and WHD domains. LWY6 and LWY7 account for 61% of the buried surface (1274 of 2088 A2). The N-terminal WY1 domain, which binds NbTOL9a, is unresolved and therefore probably flexible.
Evidence cryo-EM atomic model; interface analysis
Source Figures 3A-3E; table S2
Removing N-terminal LWY domains lets assembly proceed further. AVRcap1bLWY3-7 stalls SlNRC3 at four protomers rather than three, while AVRcap1bLWY4-7 and AVRcap1bLWY5-7 lose NRC binding entirely and do not co-purify. Assembly therefore proceeds by progressive addition of activated protomers to a hexamer.
Evidence N-terminal truncation series; co-purification; negative-stain EM 2D class averages; intermediate modelled with AlphaFold 3
Source Figure 4; figures S11A-S11C
A conserved four-residue motif at the periphery of the NB domain, Tyr204-Glu205-Phe206-Phe207 (YEFF), is simultaneously engaged by LWY6 and LWY7. Of ten NbNRC3 mutations tested, five became insensitive to AVRcap1b suppression; four of those were 'trigger-happy', firing with Rx alone. Only NbNRC3Y206A/E207A was neither autoactive nor trigger-happy.
Evidence site-directed mutagenesis of NbNRC3; cell death assays
Source Figure 5A; figures S12, S13
The Y204A/E205A substitution separates binding from assembly. SlNRC3Y204A/E205A still forms high-molecular-weight resistosome complexes on activation, but no longer forms the intermediate band with AVRcap1b. The equivalent mutation made StNRC3 insensitive to suppression, while CaNRC3 remained partially suppressed.
Evidence BN-PAGE; cross-ortholog mutagenesis
Source Figure 5B; figures S16, S17
Mutating LWY7 residues of AVRcap1b abolishes suppression (four single and two double mutants), and swapping the P. infestans LWY7 domain into the non-suppressing P. ipomoeae ortholog confers NRC3 suppression. Mutations contacting LWY6 (NbNRC3R211A, K258A and the double) barely affect suppression, so LWY7 carries the critical contacts.
Evidence effector-side mutagenesis and domain swap; gain-of-function reciprocal test
Source Figures S13B, S14, S15
No Rx sensor was captured in any complex, by BN-PAGE or in planta purification. The authors conclude Rx-SlNRC3 interaction is transient and that helper NLRs dissociate and assemble the resistosome independently, with no sensor-mediated nucleation. This supports the activation-and-release model.
Evidence absence of sensor in purified complexes across two independent approaches
Source Discussion; Figure 5C
AVRcap1bP92E, compromised in NbTOL9a binding, retains moderate SlNRC3 suppression, so the NRC-suppressing activity is at least partly independent of NbTOL9a binding. The authors propose AVRcap1b is not a bona fide NLR inhibitor like the nematode effector SS15 but a bridge linking activated NLRs to the NbTOL9a trafficking pathway.
Evidence copper-inducible transcriptional activation time-course assay; cell death time course
Source Figures S2A and S2B; Discussion; figure S18
NLRs
| Name | Class | Role in this paper |
|---|---|---|
| NRC3 SlNRC3, NbNRC3, StNRC3, CaNRC3, SlNRC3EEE (MADA mutant), NbNRC3D480V (autoactive) | helper subject | central subject; resolved both as a complete hexamer and as an AVRcap1b-stalled three-protomer intermediate |
| Rx | sensor subject | the activating sensor throughout; notably absent from every purified complex, which is the paper's evidence for transient sensor-helper engagement |
| NRC2 NbNRC2 | helper comparator | the original AVRcap1b target from earlier work; NbNRC2-AVRcap1b complex could not be purified, prompting the switch to NRC3 |
| NRC4 NbNRC4, NbNRC4L9E | helper comparator | negative control; its oligomerisation is unaffected by AVRcap1b |
| NRC1 | helper cited-only | included in the ortholog suppression panel; variably suppressed |
| NRG1 | helper cited-only | contrast case: NRG1 needs partner proteins to oligomerise, NRC helpers do not |
| NLRC4 | paired cited-only | animal comparator for progressive oligomerisation |
Effectors
| Name | Pathogen | Role |
|---|---|---|
| AVRcap1b | Phytophthora infestans | central subject; an L-shaped RXLR-LWY effector that binds across three activated SlNRC3 protomers and stalls resistosome assembly. Used deliberately as a molecular probe of the assembly pathway. |
| AVRcap1b (P. ipomoeae ortholog) | Phytophthora ipomoeae | negative control; gains NRC3 suppression when its LWY7 domain is swapped for the P. infestans one |
| PVX coat protein | Potato virus X | the activating trigger in the Rx/NRC system used throughout |
| SS15 | potato cyst nematode (Globodera spp.) | contrasting case discussed, not assayed: SS15 blocks NRCs before activation, AVRcap1b acts after it. Convergent evolution on the same network node from a metazoan and an oomycete. |
Structures
| Entry | State | Description |
|---|---|---|
| PDB 9RI9 (EMDB EMD-53990) | hexamer | Activated SlNRC3 resistosome; consensus map at 2.85 A; resembles reported NbNRC2 and NbNRC4 resistosomes |
| PDB 9RIA (EMDB EMD-53991 composite, EMD-53988 consensus, EMD-53989 focused) | three-protomer intermediate with one effector (3:1) | SlNRC3-AVRcap1b complex: three activated SlNRC3 protomers bound by one L-shaped AVRcap1b; consensus 3.01 A, focused 3.21 A, merged composite; AVRcap1b WY1 domain unresolved |
| negative-stain EM, not deposited | tetramer and intermediate series | SlNRC3 stalled at four protomers in the presence of AVRcap1bLWY3-7; 2D class averages suggesting a progression of oligomeric intermediates through to the complete hexamer without effector |
| AlphaFold3 predicted | four-protomer intermediate | Model of the four-protomer SlNRC3 intermediate stalled by AVRcap1bLWY3-7 |
Open questions
- Whether other L-shaped RXLR-LWY effectors in Phytophthora target NLRs in the same way is untested; sequence conservation analysis shows NRC-binding regions are the least conserved surface residues, implying divergent targets.
- The NbNRC2-AVRcap1b complex could not be purified, so the mechanism on NRC2, the effector's originally described target, remains structurally unresolved.
- Why several YEFF mutants become 'trigger-happy' - firing with Rx alone - is not explained; the motif may have a role in autoinhibition beyond effector binding.
- Whether AVRcap1b bridges activated NRCs to the NbTOL9a ESCRT pathway, as proposed, is a model built from separate binding data rather than a resolved ternary complex.
- Why CaNRC3Y204A/E205A remains partially suppressed while the tomato and potato equivalents do not is unresolved.
- The physiological stoichiometry of effector to NLR during infection is not addressed, though the authors argue it matters more for SS15 than for AVRcap1b.
Connections in this corpus
| Paper | Why |
|---|---|
madhuprakash-2026-avrcap1b-tol9a | Companion papers on the same effector, and they should be read as a pair. Madhuprakash solves the AVRcap1b-NbTOL9a crystal structure and shows the effector associates with activated NbNRC2; Seager solves the AVRcap1b-NRC3 cryo-EM structure and shows what that association does. Seager's AVRcap1bP92E result, that TOL9a-binding-deficient effector retains partial NRC suppression, is the hinge between them. Shared authors: Madhuprakash, Contreras, Kamoun. |
guo-2026-ccg10-resistosome | Complementary views of resistosome assembly. Guo captures three stable end states of one receptor by cryo-EM; Seager captures stalled intermediates of another. Both purify NLRs from N. benthamiana and both use MADA-motif or funnel mutants to decouple oligomerisation from cell death. |
toghani-2026-sensor-helper-interface | Direct agreement on activation-and-release from opposite evidence. Seager infers transient sensor-helper engagement from the absence of Rx in every purified complex; Toghani predicts and validates the Rx-NRC2 interface that must form transiently for that to work. The same Rx/NRC pair in both. |
toghani-2026-sni-undecamer | Both interrogate NRC oligomeric state beyond the canonical hexamer, Seager by trapping sub-hexameric intermediates, Toghani by finding a supra-hexameric undecamer. Together they argue the hexamer is one point on a range, not a fixed property. |
ibrahim-2026-nrg1-chloroplast | Seager notes NRG1 requires EDS1-SAG101 partners to oligomerise whereas NRC helpers do not; Ibrahim works on that same NRG1 heterocomplex and shows where it acts. Both bear on whether compartment or partner requirement is the main axis separating helper classes. |
pai-2026-lettuce-nrc | Seager shows a single conserved NB-domain motif determines effector susceptibility across NRC3 orthologs, with Capsicum behaving differently from tomato and potato. Pai maps how NRC helpers and sensors diverge across lineages. Whether effector-binding surfaces track the evolutionary trajectories Pai describes is a natural question across the two. |
gomezdelacruz-2026-mla3-mimicry | Both dissect an interface by mutagenesis and confirm it with a gain-of-function swap between orthologs: this paper transplants LWY7 from P. infestans AVRcap1b into the non-suppressing P. ipomoeae ortholog, Gomez De La Cruz transplants the Pwl2 binding interface from MLA3 into SR50. Reciprocal gain-of-function is the shared standard of proof. |
gao-2026-vnt1-splicing | Two ways to stop a resistosome forming, one endogenous and one imposed. Gao has an autoinhibitory N-terminal extension blocking assembly from the inside; this paper has an effector jamming assembly from the outside. Both use BN-PAGE to distinguish partial from absent oligomerisation, and both recover intermediate states rather than a binary switch. |
Provenance and caveats
Cryo-EM resolutions (2.85 A hexamer consensus; 3.01 A complex consensus; 3.21 A focused map) are taken from the Materials and Methods text, not from table S1, which was not parsed. FSC 0.143 cutoff stated.
The YEFF residue numbering differs between orthologs: Tyr204-Glu205-Phe206-Phe207 in SlNRC3 and Tyr206-Glu207-Phe208-Phe209 in NbNRC3. Mutant names in the paper follow whichever ortholog was used, so NbNRC3Y206A/E207A and SlNRC3Y204A/E205A are the equivalent mutation. Any wiki page must not present these as different mutations.
Reference numbers in in_corpus_or_lab_lineage marked [UNVERIFIED] were inferred from in-text citation context, not read from the reference list. Resolve at Stage 3 before any of them is used as a citation.
Sources
- Full citation: Seager BA, Harant A, Contreras MP, Hou L-Y, Wu CH, Kamoun S, Madhuprakash J (2026) A plant pathogen effector blocks stepwise assembly of a helper NLR resistosome. Sci Adv 12:eaeb1931.
- DOI: 10.1126/sciadv.aeb1931
- PDF on file:
papers/sciadv.aeb1931.pdf - Extraction:
data/papers/seager-2026-avrcap1b-nrc3.json(schema v1.0) - References: 46 works, retrieved from OpenAlex
- Licence: CC BY-NC