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Wheat Pm4 resistance to powdery mildew is controlled by alternative splice variants encoding chimeric proteins

An Author Correction to this article was published on 03 March 2023

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Abstract

Crop breeding for resistance to pathogens largely relies on genes encoding receptors that confer race-specific immunity. Here, we report the identification of the wheat Pm4 race-specific resistance gene to powdery mildew. Pm4 encodes a putative chimeric protein of a serine/threonine kinase and multiple C2 domains and transmembrane regions, a unique domain architecture among known resistance proteins. Pm4 undergoes constitutive alternative splicing, generating two isoforms with different protein domain topologies that are both essential for resistance function. Both isoforms interact and localize to the endoplasmatic reticulum when co-expressed. Pm4 reveals additional diversity of immune receptor architecture to be explored for breeding and suggests an endoplasmatic reticulum-based molecular mechanism of Pm4-mediated race-specific resistance.

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Fig. 1: Molecular identification and characterization of a Pm4b candidate gene.
Fig. 2: Confirmation of the functional identity of the Pm4b gene by transgenic complementation and VIGS.
Fig. 3: The Pm4 protein variants differ in the S_TKc and transmembrane domains.
Fig. 4: Pm4_V1 and Pm4_V2 form an ER-associated complex.
Fig. 5: Evolutionary origin of Pm4b.
Fig. 6: A possible working model of Pm4-mediated resistance.

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Data availability

All data are available in the main text or the supplementary materials. Sequence data were deposited at the NCBI GenBank under the accession numbers MT783929 (Pm4b_V1 CDS) and MT783930 (Pm4b_V2 CDS) and at the NCBI short read archive database under the accession number PRJNA646941 (flow-sorted chromosome 2A of eight Fed-Pm4b mutants and the wild-type Fed-Pm4b). All B. graminis f. sp. tritici (Bgt) isolates listed in Supplementary Table 1 are kept alive in the Department of Plant and Microbial Biology of the University of Zurich and are available upon request. Any additional data that support the findings of this study are available from the corresponding author upon reasonable request. The databases that we used are all publicly available: see Methods and the Nature Research Reporting Summary linked to this article. Source data are provided with this paper.

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Acknowledgements

We thank J. Vrána, Z. Dubská, R. Šperková and J. Weiserová for the assistance with chromosome sorting and preparation of chromosomal DNA. This project was financially supported by the University of Zurich, Swiss National Science Foundation grant no. 310030B_182833 to B.K., the European Research Council under the grant no. 773153 (grant IMMUNO-PEPTALK) to C.Z. and the European Molecular Biology Organization (EMBO Long-Term Fellowships 438-2018) to J.G. M.C.K has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant no. 674964. M.K. and J.D. were supported by ERDF project “Plants as a tool for sustainable global development” (no. CZ.02.1.01/0.0/0.0/16_019/0000827). B.K. and J.S.M sincerely thank V. Mohler from the Bavarian State Research Center for Agriculture (LfL) for providing seeds from the hexaploid line Tm27d2. J.S.M. sincerely thanks N. Chumak from the Department of Plant and Microbial Biology (UZH) for providing the ER-marker (ER-ck, CD3-959).

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Authors and Affiliations

Authors

Contributions

J.S.M. and B.K. conceived the project. M.K. and J.D. performed chromosome flow sorting and preparation of chromosomal DNA. T.W., J.S.M., M.H., C.R.P., B.S. and M.C.K. performed bioinformatics analysis. H.Z. performed VIGS. G.H. carried out gene expression studies. J.G. and V.W. performed confocal microscopy. V.W. did validation by transgenic complementation. V.W., J.S.M., L.S. and J.I. performed biochemistry experiments. J.S.M. and L.S. carried out allele mining. C.Z. provided theoretical contributions to the project. J.S.M. and B.K. analysed the data and wrote the manuscript. All authors revised the manuscript.

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Correspondence to Javier Sánchez-Martín or Beat Keller.

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The authors declare no competing interests.

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Peer review information Nature Plants thanks Zuhua He and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Pm4a and Pm4b convey resistance to a wide range of Bgt isolates.

a, Disease reactions of Fed-Pm4a and Fed-Pm4b NILs to 108 genetically diverse contemporary Bgt isolates72,73,74. b, Selection of Bgt isolates for which Fed-Pm4a and Fed-Pm4b NILs showed a differential resistance/susceptibility pattern. The outer and inner circle represent the reaction pattern of Fed-Pm4a and Fed-Pm4b, respectively. Disease reaction was evaluated seven days post-inoculation. Five classes of host reactions were distinguished: R = resistance (0–10% of leaf area covered), IR (10–25% of leaf area covered), I (25–50% of leaf area covered), IS (50–75 % of leaf area covered) and S (>75% of leaf area covered). CHN: China, ISR: Israel; CHE; Switzerland; FRA: France; USA: United States; GRB: Great Britain; JPN; Japan.

Extended Data Fig. 2 Expression profiling of Pm4b mutants following infection with Bgt96224.

Transcripts levels of the Pm4_V1 and Pm4_V2 splice variants in mock-inoculated or Bgt-inoculated Fed-Pm4b plants. Statistical analysis was done using a two-tailed t-test at p < .05 (mock vs infected) based on n = 4 biological replicates. Error bars, mean ± s.e.m. Exact P values are shown above bars.

Extended Data Fig. 3 Agronomically-related traits of selected T2 transgenic families overexpressing Pm4b_V1CDS and Pm4b_V2CDS transgenes.

a, Plant growth of representative T2 transgenics from families T2#52-1.4 and T2#52-3.11 compared to Bobwhite S26 in the following order: Bobwhite S26, T2#52-1.4_1.10, T2#52-1.4_1.9, T2#52-3.11_1.2 and T2#52-3.11_1.3 b, Plant height of the T2 families overexpressing Pm4b_V1CDS and Pm4b_V2CDS transgenes presented in Fig. 3c and Supplementary Table 3. Names are indicated in the x axis. c, Thousand Grain Weight for the same T2 families. Selected representative of the same T2 family are displayed with the same colour: T2#3 in cyan, T2#25 lime green and T2#52 in magenta. In the boxplots, centre lines show the medians; box limits indicate the 25th and 75th percentiles as determined by the geom_boxplot function of the ggplot2 R package; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, individual data points are represented by dots. On top of each boxplot, p values based on two-tailed t-test at p < .05 (transformants versus Bobwhite S26). Above p values, n = the number of T2 progeny.

Extended Data Fig. 4 Gene expression in transgenic wheat plants overexpressing single splice variants of the Pm4b gene.

a, Expression levels of Pm4bV1_CDS transgenes in selected T1 progeny for three independent transgenic events (T1#9, T1#12, T1#12) overexpressing full-length cDNA of Pm4b_V1 compared to the endogenous Pm4b_V1 transcripts in the wild-type Fed-Pm4b (second bar). b, Expression levels of Pm4bV2_CDS transgenes in selected T1 progeny for three independent transgenic events (T1#6, T1#24, T1#29) overexpressing full-length cDNA of Pm4b_V2 compared to the endogenous Pm4b_V2 transcripts in the wild-type Fed-Pm4b (second bar). For a and b, data points are technical replicates (triple quantifications) on single T1 progeny. Error bars, mean ± s.e.m. of three technical replicates. On top of each bar, the number corresponds to the x-fold expression compared to Pm4b_V1 or Pm4b_V2 in the wild-type Fed-Pm4 genotype. Below each T1 progeny, representative images of disease reactions after infection with the Pm4a/b-avirulent Bgt96224 and Bgt94202 isolates are shown.

Extended Data Fig. 5 Predicted Pm4 kinase catalytic domain.

A multiple amino acid sequence alignment of 38 protein kinase catalytic domains involved in disease resistance was used to infer the Pm4b kinase domain architecture. In Pm4b (indicated with a red rectangle) all the 14 key conserved residues of protein kinases are present. In the alignment, red arrowheads mark invariant residues (G52, K72, E91, D166, N171, D184, G186, E208, R280), which are numbered with upper case numbers corresponding to their position in the α form of the cAMP-dependent protein kinase catalytic unit (cAPK). Likewise, black arrowheads indicate the mostly invariant residues (G50, V57, F185, D220, G225). Based on the presence of a L residue at position R165 of cAPK in subdomain VI, Pm4 Kinase was classified as a non-RD kinase. Moreover, conserved residues in subdomain VI (D166 -> N171, DLKPAN in Pm4b vs. DLPKPEN in cAPK) and VIII (GTMGYLAPE in Pm4b vs. GT/SXXY/FXAPE in cAPK) indicate that the Pm4 kinase domain is a serine/threonine protein kinase. Labels: red and black arrowheads, key invariant and nearly invariant residues in the protein kinase catalytic domains, respectively. Light blue diamond points to the RD or non-RD kinase determination site. Black asterisks, substrate binding site. Green arrowheads, ATP binding site. Core conserved, diagnostic regions of subdomains I, II, VI, and VIII are highlighted by grey bars labelled with Roman numerals. On top of the wrapped alignment, EMS mutagenized line designations affecting the Pm4 kin domain in Pm4a or Pm4b genes and corresponding amino acid changes are indicated. Violet squares indicate polymorphic amino acids within the kinase domain among the Pm4 allelic variants described in this study. Numbers above violet squares indicate the position on the alignment based on the cAPK sequence.

Extended Data Fig. 6 Sequence alignment of Pm4 C2 domains with homologous C2 domains of Arabidopsis MCTPs.

a, Sequence alignment of Pm4b-C2C with C2C domains from Arabidopsis MCTPs. b, likewise alignment of C2D domains. C2 domains were delimited based on Conserved Domain Database (CDD) from NCBI104. The location of the domain is indicated by the sequence range numbers. C2 domains in Pm4 (black background) are indicated with a red rectangle. c, Phylogenic tree of C2C and C2D domains of Arabidopsis MCTPs and Pm4b-C2C/C2D domains. The human DySF dysferlin C2C/D domains was used as outgroup.

Extended Data Fig. 7 Determination of aspartate residues predicted to be involved in Ca2+-binding in Pm4b C2 domains.

a, Sequence alignment of Pm4b-C2C and Pm4b-C2D domains with C2 domains previously described to bind Ca2+. UniProt entry names followed by the specific C2 domain displayed are located on the left. The region of the C2 domain displayed is indicated by the sequence range numbers. Conserved aspartate residues involved in Ca2+-binding are highlighted in pink. Pm4b_C2C (fourth row from the bottom) does not have conserved aspartate residues and exhibits diverse amino acid substitutions, including D -> E, A or I. However, Pm4b_CD2 (third row from the bottom) has three conserved aspartate residues (positions I, III and IV) and two conservative substitutions, asparagine (position II) and glutamine (position V), both polar and relatively small amino acids. Interestingly, Pm4_C2D contains an insertion of eight amino acids (green) just before the predicted Ca2+ binding region 3 that shifts the position of the conserved aspartate residues at position III and IV (highlighted in red) (see Extended Data Fig. 6). Rectangles denote calcium-binding regions (CBR) 1 and 3, respectively. b, Structured-based alignment of C2D Pm4b_V2 (turquoise) and the C2 domain from PKCα (pink) (Protein kinase C alpha type, PDB: 1DSY). The predicted structural model of the Pm4bC2 domain was done using the Phyre2 server on the basis of the crystal structure of rat otoferlin c2a (PDB: 3L9B, Fold library id: c3l9bA) with 14% of identity and 99.9% of confidence. c, On top, calcium-binding regions (CBR) CBR1 and 3 of PKCα. In the middle, CBR1 and 3 of Pm4b_C2D domain. On the bottom part, overall alignment of CBRs 1 and 3 of Pm4b_C2D domain (turquoise) and PKCα (dark blue). d, Three-dimensional structure of C2D domain of Pm4b using the Phyre2117 server based on the crystal structure of rat otoferlin c2a (PDB: 3L9B, Fold library id: c3I9bA) with 14% of identity and 99.9 % of confidence highlighting in blue CBR 1 and 3, with predicted residues involved in Ca2+-binding labelled. Calcium ions are shown as grey balls.

Extended Data Fig. 8 Negative controls for the Pm4b interaction.

a, Pm4b_V1 does not interact with the ER-marker ER_ck_CD3_95338. b, Pulldown with anti-HA beads is specific for the presence of HA-tagged Pm4b variants. Co-immunoprecipitation experiments were repeated two times with similar results.

Source data

Extended Data Fig. 9 Binding ability of Pm4b variants for homo- and heteromeric interactions.

a, Split-LUC combinations showing luciferase signal for Pm4b_V1 homomeric interaction in Fig. 4e were co-infiltrated with fluorescence-tagged Pm4b_V2 protein variants. b, Split-LUC combinations showing luciferase signal for Pm4b_V2 homomeric interaction in Fig. 4f were co-infiltrated with fluorescence-tagged Pm4b_V1 protein variants. The data are displayed following the same logic as presented in Fig. 4: in each of the 18 panels, the first boxplot corresponds to the positive control, AvrPm3b_N-LUC & AvrPm3b_C_LUC. The second boxplot (orange colour) corresponds to the tested combination, displayed at the top of each panel. For simplicity, V1 and V2 refer to Pm4b_V1 and Pm4b_V2, respectively. Finally, the last two boxplots in each panel correspond to the negative controls co-infiltrated. Significant differences were determined by Krustal–Wallis test followed by Dunn’s multiple comparisons test with two-sided 95.0% confidence interval with Bonferroni correction based on n = 24 (8 technical and 3 biological replicates). Exact P values are shown above bars. In the boxplots, centre lines show the medians; box limits indicate the 25th and 75th percentiles as determined by the geom_boxplot function of the ggplot2 R package; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, individual data points are represented by dots.

Extended Data Fig. 10 Bivariate flow karyotype GAA-FITC vs. DAPI obtained after the analysis of chromosomes isolated from mutant pm4b_m256.

The population representing chromosome 2A, which was flow-sorted, is highlighted in orange. Inset: Flow-sorted chromosomes were identified microscopically after FISH with probes for GAA microsatellites (green) and Afa repeat (red). The fluorescent labelling pattern allowed chromosome identification and estimation of the contamination of sorted fractions by other chromosomes. Chromosomes were counterstained by DAPI (blue).

Supplementary information

Supplementary Information

Supplementary Figs. 1–5.

Reporting Summary

Supplementary Tables

Supplementary Tables 1–8.

Source data

Source Data Fig. 4

Unprocessed western blots.

Source Data Extended Data Fig. 8

Unprocessed western blots.

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Sánchez-Martín, J., Widrig, V., Herren, G. et al. Wheat Pm4 resistance to powdery mildew is controlled by alternative splice variants encoding chimeric proteins. Nat. Plants 7, 327–341 (2021). https://doi.org/10.1038/s41477-021-00869-2

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