1/n. Nucleotide-binding and leucine-rich repeat receptors (NLRs) are key players in plant immune systems that recognise and respond to harmful pathogen effector proteins.
2/n. NLRs can play specialised roles in either detecting effectors (sensor NLRs) or triggering the immune response (helper NLRs) and might function together as pairs or within larger networks.
Dec 3, 2025 09:233/n. Although many NLRs are known to oligomerise upon the perception of effectors, much less is understood about their resting state prior to pathogen recognition. Here is what we know for some CC-type NLRs:
4/n.Here, we investigated the oligomeric state of the sensor Pik-1 and helper Pik-2 NLR pair in the resting state.We used both wild-type Pik-1 that recognises effectors from M. oryzae via its HMA domain and engineered Pik-1(Enhancer) that recognises GFP via a nanobody
www.science.org/doi/10.1126/...5/n. Previous work from the Banfield lab showed that Pik-1 and Pik-2 associate both prior to and upon effector binding, prompting us to ask whether Pik-1 and Pik-2 form dimers or oligomers in their resting state.
journals.plos.org/plosone/arti...6/n. We performed non-denaturing BN-PAGE to investigate the oligomerisation state of Pik-1 and Pik-2 in the resting state and show that both NLRs form a massive ~1 MDa complex when co-expressed. It took
@hsuanpai.bsky.social and I some time to identify to right extraction conditions this complex!
7/n. However, given that Pik-1 and Pik-2 are very similar in size, it was impossible to determine whether we were observing a Pik-1 oligomer and Pik-2 oligomer with similar stoichiometries, or a Pik-1/Pik-2 heterocomplex.
8/n. We generated a “heavy” variant of Pik-1 C-terminally tagged with mCherry and v5 (Pik-1 “heavy”) to help us detangle this! The idea was that if Pik-1 and Pik-2 formed a heterocomplex, we would see a size shift when co-expressing Pik-1 “Heavy” with Pik-2 and probing for Pik-2. And we did!
9/n. The Pik pair forms a hetero-complex in its resting state, similar to what was observed for TIR-NLR sensor pairs CSA1-CHS3 and RPS4-RRS1.
doi.org/10.1126/scie... ,
doi.org/10.1101/2025...
Paired plant immune CHS3-CSA1 receptor alleles form distinct hetero-oligomeric complexes
Pairs of intracellular immune receptors heterodimerize and oligomerize to activate immunity in plants.
10/n. Where is the Pik complex located? We show that in absence of their paired partner, Pik-1 mostly accumulates in the soluble fraction, while Pik-2 is present in both soluble and membrane fractions. Upon co-expression with Pik-2, Pik-1 shifts from the soluble to the membrane fraction.
11/n. We confirmed that the hetero-complex accumulates at the plasma membrane with confocal microscopy.
12/n. What about the ligand? You may ask. We introduced mutations in the MADA motif present in the N-terminal α1 helix of Pik-2 (Pik-2EE) to abolish cell-death induction in presence of Pik-1 and its ligand to facilitate biochemical assays and address this question.
13/n. We detected GFP at ~1 MDa only when GFP was co-expressed with Pikm-1(Enhancer) and Pikm-2(EE), suggesting that the Pik-1 ligand is integrated into the Pik hetero-complex
14/n. Caveat: Given that the ~1 MDa bands were identical regardless of the presence of GFP, we could not conclude that the Pik pair was activated by GFP or whether Pikm-2(EE) is a non-functional mutant of Pikm-2 that is blocked in a conformation similar to that of its resting state.
15/n. Our findings contribute to the growing evidence that resting state configurations vary across NLRs. This knowledge could be leveraged for disease resistance engineering strategies complementary to approaches focussing solely on effector binding (this is what I will explore in my ERC project!)
16/n. Thank you to my wonderful colleagues for making this happen!
