The N-terminal MADA motif in the CC-NLR immune receptors is functionally conserved across distantly related plant species ●Hiroaki Adachi1, Mauricio Contreras1, Adeline Harant1, Chih-hang Wu1, Lida Derevnina1, Toshiyuki Sakai1, Cian Duggan2, Eleonora Moratto2, Tolga O Bozkurt2, Abbas Maqbool1, Joe Win1, Sophien Kamoun1 1The Sainsbury Laboratory, University of East Anglia, Norwich Research Park, Norwich NR4 7UH, UK. 2Imperial College London, Department of Life Sciences, London, UK. 1. Plant CC-NLRs form resistosome “death switch” 9. ACKNOWLEDGMENTS Model of NLR network for effector recognition. NRC, NLR required for cell death. modified from Wu et al. (2017) PNAS Wang et al. (2019) Science 2. NLRs form a receptor network composed of functionally specialized “sensor” and “helper” NLRs In vitro transposon insertion Truncation library in Agrobacterium Functional validation in N. benthamiana Premature STOP codons NRC4DV MuA D478V V 0 100 200 300 400 500 600 700 800 900 HR - HR + 1 145 504 881 9 CC NB-ARC LRR Amino acid position 3. Transposon-based truncation mutagenesis reveals 29 amino-acid region sufficient for NRC4-mediated cell death NRC41-29-YFP NRC4DV-YFP NRC4WT-YFP YFP TRIBE-MCL LRR NB-ARC CC 0 50 100 150 200 250 Number of proteins 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 59 RPM1, MLA10, Sr33, Sr50, Pik-1, Pik-2, RGA4, RGA5 Hero, Mi1.2, Rpi-blb2, Sw-5b R1 RPS5 Bs2 Prf Gpa2, Rx ADR1 NRG1 N-terminal domain tribes NRC2, NRC3, NRC4, ZAR1, RPP8, RPP13, R2, Rpi-vnt1 R3a Dicot Monocot 4. The MADA motif is conserved at the N-terminus of NRC4 and ZAR1 Dataset: 988 CC/CCR-NLRs from Arabidopsis, sugar beet, tomato, N. benthamiana, rice and barley. NRC4 MADA motif 1 21 0 1 2 3 4 bits 1 E R A L VM 2 G Y T L VA 3 A N S T G ED 4 I G T V E SA 5 G T I A F L V 6 M T LIV 7 D K G A E T NS 8 N T Y P S K L VF 9 S F V G L A 10 A T LIV 11 D G N S KQE 12 L Q A R S I W NK 13 V Y T A IL 14 Q S A M K L W G 15 H T K G E S N Q D 16 Q T W Y I V FL 17 E M V I AL 18 A E R T L V S I 19 K N S Y H R D E Q 20 H K Q NE 21 W G Y C I S A V 1 MADAVVNFLVENLLQLLTDNV 21 1 MVDAVVTVFLEKTLNILEEKG 21 NRC4 ZAR1 LRR NB-ARC CC NRC41-29 NRC4DV A B A, Clustering N-terminal domain of CC-NLRs by Tribe-MCL analysis. B, The consensus sequence pattern of the MADA motif identified by MEME. NRC-S without NTD NRC-H family NRC-S with NTD 0.1 NRC superclade Mi-1.2 Rpi-blb2 Hero Sw5b R1 Prf Bs2 Gpa2 Rx NRC4 NRC2 NRC3 Tree scale: 0.2 ZAR1 RPP13 R2 RPP8 RPi-vnt1 Pik-2 Tree scale: 0.2 Other NLRs MADA-CC-NLRs Bootstrap value > 0.7 6. NRC sensors do not have the MADA motif NRC-H: NRC helpers, NRC-S: NRC dependent sensors, NTD: N-terminal extension domain 0 5 10 15 20 25 30 35 40 Rice Barley Arabidposis Sugar beet Tomato N. benthamiana 0 5 10 15 20 25 30 35 40 Rice Barley Arabidposis Sugar beet Tomato N. benthamiana Frequency (%) MADA-CC-NLR MADAL-CC-NLR 15/371 4/229 25/72 14/88 32/133 14/81 72/371 36/229 1/72 4/88 5/133 13/81 5. The MADA motif is present in ~20% of CC-NLRs MADA HMM LRR NB-ARC CC Dataset: 988 CC/CCR-NLRs from Arabidopsis, sugar beet, tomato, N. benthamiana, rice and barley. Occurrence of MADA-CC-NLRs in monocots and dicots. MADAL-CC-NLR: MADA-like CC-NLR Pathogen Effector(s) Singleton NLR NLR network NLR pair Sensor NLR Helper NLR Time Specialization Diversification ▶ Degenerated MADA motif ▶ N-terminal domain integration ▶ Conserved MADA motif ▶ Conserved MADA position Sensor NLR Helper NLR Convergent downstream Hypersensitive cell death and disease resistance AvrBs2 CP AvrPto/ AvrPtoB AVR1 NSm Avr8 Avrblb2 Unknown protein Unknown protein RBP-1 NRC2 NRC3 NRC4 Gpa2 Rx Bs2 Prf R1 Sw5b R8 Rpi-blb2 Mi A B C A, Overview of the strategy for transposon-based C-terminal random truncation of NRC4 proteins. B, NRC41-29::Mu-STOP triggers cell death in N. benthamiana leaves. C, NRC41-29-YFP induces cell death in N. benthamiana leaves. The phylogenetic trees of the 988 CC-NLRs (Left) and the NRC superclade (right). NRC4WT NRC4DV NRC21-17