(A) Overview of the strategy for transposon-based C-terminal random truncation of NRC4 proteins. Hairpin Mu-STOP transposon and MuA proteins forming Mu transpososome were used for in vitro …
Sequences of NRC4 truncation library.
The Mu-STOP transposon insertion sites were confirmed by PCR amplicon sequencing with Mu-STOP seq Rv primer. The 65 truncate sequences of NRC4 are listed in this file.
The images were taken 7 days after agroinfiltration. ‘DV’, ‘WT’ and ‘TL’ describe autoactive NRC4DV mutant, wild-type NRC4 and the truncation library, respectively. Red characters indicate clones …
(A) Schematic representation of wild-type NRC4-YFP (NRC4WT-YFP) and the variants used for the in planta expression assays. The colour code is: red represents NRC4 1 to 29 amino acid region. (B) NRC41…
(A) Schematic representation of sgRNA positions targeting NRC4a and NRC4b. The PAM motifs are marked in blue, and the sequences of sgRNAs are marked in red. (B) Genotyping results of selected T2 nrc4…
(A) Schematic representation of NRC4DV-YFP, NRC41-29-YFP and the variants used for the in planta expression assays. Arrowheads show A206K mutation site in YFP. The red colour represents NRC4 1 to 29 …
(A) Schematic representation of the different NLR domains used in TRIBE-MCL and phylogenetic analyses. (B) Distribution of plant NLRs across N-terminal domain tribes. The colour codes are: orange …
Amino acid sequences of full-length NLRs in the CC-NLR database.
988 NLR sequences used for HMMER analysis are listed.
Amino acid sequences of N-terminal domains in the CC-NLR database.
N-terminal domain sequences of 988 proteins used for Tribe-MCL analysis are listed.
N-terminal domain tribes of CC-NLRs.
Results of the Tribe-MCL analysis are included in this file.
NLR proteins were predicted by NLR-parser from N. benthamiana (NbS-), tomato (Solyc-), Arabidopsis (AT-), sugar beet (Bv-), rice (Os-) and barley (HORVU-) proteomes, and were used for the MAFFT …
Amino acid sequences for CC/TIR-NLR phylogenetic tree.
NB-ARC domain sequences used for phylogenetic analysis are shown with the IDs, N. benthamiana (NbS-), tomato (Solyc-), Arabidopsis (AT-), sugar beet (Bv-), rice (Os-) and barley (HORVU-).
CC/TIR-NLR phylogenetic tree file.
The phylogenetic tree was saved in newick file format.
The phylogenetic tree was constructed with the NB-ARC domain sequences of CC-NLRs as described in Figure 3—figure supplement 2—source data 2. Each leaf is labelled with different colour ranges …
Amino acid sequences for CC-NLR phylogenetic tree.
NB-ARC domain sequences used for phylogenetic analysis are shown with the IDs, N. benthamiana (NbS-), tomato (Solyc-), Arabidopsis (AT-), sugar beet (Bv-), rice (Os-) and barley (HORVU-).
CC-NLR phylogenetic tree file.
The phylogenetic tree was saved in newick file format.
(A) Schematic representation of a classical CC-NLR protein highlighting the position of the MADA motif. Consensus sequence pattern of the MADA motif identified by MEME along with an alignment of …
Output of the HMMER search using the MADA motif HMM against tomato and Arabidopsis proteomes.
HMM scores are listed with the IDs, tomato (Solyc-) and Arabidopsis (AT-), and annotation information.
Amino acid sequences of the MADA motif.
The sequences were extracted from MEME output against N-terminal domain Tribe 2 and were used to build the MADA motif HMM.
Consensus sequence patterns in N-terminal domains were identified by MEME from 226 Tribe 1, 102 Tribe 2, 83 Tribe 3 and 59 Tribe 4 members. Motif logos describe the N-terminal consensus patterns …
(A) Consensus sequence of the MADA motif. The MADA motif logo was generated by MEME from 87 N-terminal domains of Tribe 2 members. (B) Graphical representation of the MADA HMM used to screen …
(A) Schematic representation of a classical CC-NLR protein highlighting the regions used for HMMER searches (MADA-HMM) and for TRIBE-MCL. (B) Occurrence of MADA/MADAL-CC-NLRs in representative …
Output of the HMMER search using the MADA motif HMM against the CC-NLR database.
HMM scores of the predicted MADA motifs are listed by IDs, N. benthamiana (NbS-), tomato (Solyc-), Arabidopsis (AT-), sugar beet (Bv-), rice (Os-) and barley (HORVU-), with Tribe-MCL result, the start (‘MADA_strat’) and end (‘MADA_end’) positions of the MADA motifs in the CC-NLRs.
List of the predicted Arabidopsis MADA-CC-NLRs.
The IDs are listed with the HMM score.
(A) HMM score bar graph for CC-NLR database (988 proteins). MADA/MADAL-CC-NLRs from HMMER analysis are shown in orange and blue, respectively. (B) HMM score bar graph with plant species information. …
(A) Distribution of NRCs (NRC-H) and NRC-dependent sensors (NRC-S) across N-terminal domain tribes of CC-NLRs. Individual NLR members of the NRC superclade were classified based on phylogenetic …
HMM scores of NRC-superclade proteins.
HMM scores are listed by IDs, N. benthamiana (NbS-), tomato (Solyc-) and sugar beet (Bv-) with Tribe-MCL result, the start (‘MADA_strat’) position of the MADA motifs and NRC clade information (‘NRC-H’ and ‘NRC-S’).
(A) Schematic representation of NRC4 and the mutated sites in the MADA motif. Mutated sites and substituted residues are shown as red characters in the NRC4 sequence alignment. (B) Cell death …
(A) Schematic representation of NRC41-29-YFP, ZAR11-144-YFP and the variants used for the in planta expression assays. Arrowheads show triple alanine mutation sites in NRC41-29 and ZAR11-144. The …
(A) Schematic representation of NRC4 and the glutamic acid (E) mutant scan of the MADA motif. Mutated sites are shown as red characters in the NRC4 sequence. (B) Cell death observed in N. benthamiana…
(A) Schematic representation of NRC4 and the alanine (A) mutant scan of the MADA motif. Mutated sites are shown as red characters in the NRC4 sequence. (B) Cell death observed in N. benthamiana …
(A) Cartoon representation of N-terminal α helices of NRC4 resistosome (zoom in grey box of Figure 4B). (B, C) N-terminal α helices are rotated 90 degrees and mutated amino acids are shown as stick …
(A) Subcellular localization of NRC41-29-YFP and the mutant proteins in N. benthamiana epidermal cells. N. benthamiana leaves expressing YFP, YFPA206K, NRC41-29-YFP, NRC41-29-YFPA206K and NRC41-29L9E…
(A) Alignment of the N-terminal region of the MADA/MADAL-CC-NLRs. Key residues for cell death activity identified in Figure 8 are marked as red characters with asterisks in the sequence alignment. …
(A) Box plots showing cell death intensity scored as an HR index based on three independent experiments. Statistical differences among the samples were analysed with Tukey’s HSD test (p<0.01). (B) …
(A) Schematic representation of NRC4 complementation assay for Rpi-blb2-mediated resistance. Wild-type and the variants of NRC4 were co-expressed with RFP-Rpiblb2 in wild-type or nrc4a/b_9.1.3 N. …
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Genetic reagent (Nicotiana benthamiana) | NRC4-KO N. benthamiana (nrc4a/b_9.1.3 and nrc4a/b_1.2.1) | This paper | Materials and methods: Generation of N. benthamiana nrc4a/b CRISPR/Cas9 mutants | |
Recombinant DNA reagent | pGEM::Mu-STOP | This paper | Materials and methods: Mu-STOP in vitro transposition | |
Commercial assay, kit | Mutation Generation System Kit | Thermo Fisher | Cat #: F-701 | Materials and methods: Mu-STOP in vitro transposition |
Gene (Solanum lycopersicum) | Tomato genome sequence (Tomato ITAG release 2.40) | Sol Genomics Network (https://solgenomics.net/) | Materials and methods: Bioinformatic and phylogenetic analyses | |
Gene (N. benthamiana) | N. benthamiana genome sequence (N. benthamiana Genome v0.4.4) | Sol Genomics Network (https://solgenomics.net/) | Materials and methods: Bioinformatic and phylogenetic analyses | |
Gene (Arabidopsis thaliana) | Arabidopsis genome sequence (Araport11) | https://www.araport.org/ | Materials and methods: Bioinformatic and phylogenetic analyses | |
Gene (Beta vulgaris) | Sugar beet genome sequence (RefBeet-1.2) | http://bvseq.molgen.mpg.de/index.shtml | Materials and methods: Bioinformatic and phylogenetic analyses | |
Gene (Oryza sativa) | Rice genome sequence (Rice Gene Models in Release 7) | http://rice.plantbiology.msu.edu/ | Materials and methods: Bioinformatic and phylogenetic analyses | |
Gene (Hordeum vulgare) | Barley genome sequence (IBSC_v2) | https://www.barleygenome.org.uk/ | Materials and methods: Bioinformatic and phylogenetic analyses | |
Other | 3D structure of ZAR1 | Protein Data Bank | 6J5T | Materials and methods: Structure homology modelling |
Primers used for generating NRC4 variants by Golden Gate cloning.
The MADA-HMM for HMMER analysis.
This MADA-HMM was used for searching MADA-CC-NLRs from CC-NLR database (Figure 3—source data 1).