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PreprintCited by 19Wheat and Barley Genetics and PathologyPlant Disease Resistance and GeneticsPlant-Microbe Interactions and Immunity

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A single amino acid change can alter the specificity of the multi-allelic wheat stem rust resistance locus SR9

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Zhang, Jianping, Nirmala, Jayaveeramuthu, Chen, Shisheng, Jost, Matthias, Steuernagel, Burkhard, Karafiátová, Miroslava, Hewitt, Timothy, Li, Hongna, Edae, Erena, Sharma, Keshav, Hoxha, Sami, Bhatt, Dhara, Antoniou-Kourounioti, Rea, Dodds, Peter, Wulff, Brande, Doležel, Jaroslav, Ayliffe, Michael, Hiebert, Colin, McIntosh, Robert, Dubcovsky, Jorge, Zhang, Peng, Rouse, Matthew, Lagudah, Evans·2022·Nature Communications
DOI 10.21203/rs.3.rs-2208386/v1PMC PMC10645757

Preprint — published as 10.1038/s41467-023-42747-9 · Paper page

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Abstract

Most resistance genes thus far isolated from wheat have a very limited number of functional alleles, with the exception of the powdery mildew PM3 resistance locus. Here we report the isolation of most of the alleles at wheat stem rust resistance gene locus SR9, representing the largest multi-allelic rust resistance locus in common wheat. The seven previously reported resistance alleles (Sr9a, Sr9b, Sr9d, Sr9e, Sr9f, Sr9g, and Sr9h) at the locus were characterised using a synergistic strategy. Loss-of-function mutants and/or transgenic complementation were used to confirm Sr9b, two haplotypes of Sr9e (Sr9e_h1, Sr9e_h2), Sr9g, and Sr9h. Each allele encodes a highly related nucleotide-binding site leucine-rich repeat (NB-LRR) type immune receptor, containing a previously unreported motif at their N termini and an unusual long LRR domain, that confers resistance to a unique spectrum of isolates of the wheat stem rust pathogen. The only SR9 protein effective against stem rust pathogen race TTKSK (Ug99), SR9H, differed from SR9B by a single amino acid. SR9B and SR9G resistance proteins were also distinguished by only a single amino acid. The SR9 allelic series found in the B subgenome are orthologs of wheat stem rust resistance gene Sr21 located in the A subgenome with around 85% identity in protein sequences.

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Figures

Fig. 1 Predicted protein composition of the SR9 locus. a The positions of mutations in SR9 locus encoding proteins for SR9B, SR9E_H1, SR9E_H2, SR9G, and SR9H from mutant lines are shown in red, with predicted amino acid substitutions caused by missense mutations or premature stop codons shown above. N terminal domain, NB domain, and LRR motifs are shaded with yellow, cyan, and purple, respectively. Protein sequence conservation among the five SR9 alleles is shown in the lower plot indicating the most polymorphic region is the C-terminal region of the LRR domain. b Structure of the LRR region of SR9H from AlphaFold2 prediction of full length protein highlighting the two polymorphic aa sites in the 26th LRR unit. Inset regions show close up of this region for SR9H, SR9B, and SR9G. The two polymorphic residues are shown in sticks and highlighted in cyan in the predicted surface-exposed horseshoe shaped beta strands.
Fig. 1 Predicted protein composition of the SR9 locus. a The positions of mutations in SR9 locus encoding proteins for SR9B, SR9E_H1, SR9E_H2, SR9G, and SR9H from mutant lines are shown in red, with predicted amino acid substitutions caused by missense mutations or premature stop codons shown above. N terminal domain, NB domain, and LRR motifs are shaded with yellow, cyan, and purple, respectively. Protein sequence conservation among the five SR9 alleles is shown in the lower plot indicating the most polymorphic region is the C-terminal region of the LRR domain. b Structure of the LRR region of SR9H from AlphaFold2 prediction of full length protein highlighting the two polymorphic aa sites in the 26th LRR unit. Inset regions show close up of this region for SR9H, SR9B, and SR9G. The two polymorphic residues are shown in sticks and highlighted in cyan in the predicted surface-exposed horseshoe shaped beta strands.CC BY · PubMed Central
Fig. 2 NLR haplotype analysis of the wheat pangenome at the SR9 locus on chromosome 2B. a Phylogenetic grouping using genomic sequences of different functional SR9 alleles (black arrow) and closest homologs in sequenced wheat genomes. Accessions carrying functional alleles have been highlighted. b Schematic of the predicted NLRs within the SR9 homologous genomic region of wheat lines. NLRs are represented by colored arrows according to the NLR Annotator output as complete NLRs (green), complete pseudogenes (yellow) or partial NLRs (red). The position of closest SR9 homolog (vertical red arrow connecting red dashed lines) is always the first NLR of the cluster. The numbering I to V highlights phylogenetic subgroups (a) which have a conserved structure in the NLR cluster (b).
Fig. 2 NLR haplotype analysis of the wheat pangenome at the SR9 locus on chromosome 2B. a Phylogenetic grouping using genomic sequences of different functional SR9 alleles (black arrow) and closest homologs in sequenced wheat genomes. Accessions carrying functional alleles have been highlighted. b Schematic of the predicted NLRs within the SR9 homologous genomic region of wheat lines. NLRs are represented by colored arrows according to the NLR Annotator output as complete NLRs (green), complete pseudogenes (yellow) or partial NLRs (red). The position of closest SR9 homolog (vertical red arrow connecting red dashed lines) is always the first NLR of the cluster. The numbering I to V highlights phylogenetic subgroups (a) which have a conserved structure in the NLR cluster (b).CC BY · PubMed Central
Fig. 3 Transgenic complementation of Sr9h and Sr9e_h2. a Sr9h candidate confers resistance when transferred into the susceptible hexaploid wheat line Fielder. Seedling infection types of Gabo 56 (Sr9h), Fielder and eight plants from T1 family No. 7 of Sr9h transformants in a Fielder background in response to Pgt race TTKSK (upper panel), along with the Sr9h-KASP marker assessed indicated by green labeling) and the average pustule size in mm2 (indicated by black circled gray dot in each box) in the box and whisker plot (lower panel). The black center line denotes the median value, while the box contains the 25th to 75th percentiles of dataset. The whiskers mark the 5th and 95th percentiles. b Reactions to Pgt race 34MKGQM (isolate 20IAL06) in seven independent T1 transgenic families. Each 5 plants in the figure representing approximately 20 plants from three transgenic families T1-KN410, T1-KN422, T1-KN470 were inoculated and grown in a growth chamber at 25 °C day/22 °C night, along with the marker assessed (“+” indicates presence and “−” indicates absence, “R” for resistance and “S” for susceptible). Source data are provided as a Source Data file.
Fig. 3 Transgenic complementation of Sr9h and Sr9e_h2. a Sr9h candidate confers resistance when transferred into the susceptible hexaploid wheat line Fielder. Seedling infection types of Gabo 56 (Sr9h), Fielder and eight plants from T1 family No. 7 of Sr9h transformants in a Fielder background in response to Pgt race TTKSK (upper panel), along with the Sr9h-KASP marker assessed indicated by green labeling) and the average pustule size in mm2 (indicated by black circled gray dot in each box) in the box and whisker plot (lower panel). The black center line denotes the median value, while the box contains the 25th to 75th percentiles of dataset. The whiskers mark the 5th and 95th percentiles. b Reactions to Pgt race 34MKGQM (isolate 20IAL06) in seven independent T1 transgenic families. Each 5 plants in the figure representing approximately 20 plants from three transgenic families T1-KN410, T1-KN422, T1-KN470 were inoculated and grown in a growth chamber at 25 °C day/22 °C night, along with the marker assessed (“+” indicates presence and “−” indicates absence, “R” for resistance and “S” for susceptible). Source data are provided as a Source Data file.CC BY · PubMed Central
Fig. 4 Resistance race specificities of Sr9b, Sr9g, and Sr9h. Images of selected leaves including genetic stocks without or with Sr9b, Sr9g, and Sr9h (CS/KF 2B, CS/Mq 2B, and RL6203, respectively) and Sr9h transgenic line Sr9h-1a in response to three races of Pgt (QFCSC, MJGTC, RKRQC) that show differential responses to Sr9b, Sr9g, and Sr9h.
Fig. 4 Resistance race specificities of Sr9b, Sr9g, and Sr9h. Images of selected leaves including genetic stocks without or with Sr9b, Sr9g, and Sr9h (CS/KF 2B, CS/Mq 2B, and RL6203, respectively) and Sr9h transgenic line Sr9h-1a in response to three races of Pgt (QFCSC, MJGTC, RKRQC) that show differential responses to Sr9b, Sr9g, and Sr9h.CC BY · PubMed Central

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Authors
Zhang, J., Nirmala, J., Chen, S., Jost, M., Steuernagel, B., Karafiátová, M., Hewitt, T., Li, H., Edae, E., Sharma, K., Hoxha, S., Bhatt, D., Antoniou-Kourounioti, R., Dodds, P., Wulff, B., Doležel, J., Ayliffe, M., Hiebert, C., McIntosh, R., Dubcovsky, J., Zhang, P., Rouse, M., & Lagudah, E.
Affiliations
  • Commonwealth Scientific and Industrial Research Organisation (AU)
  • University of Minnesota (US)
  • Peking University (CN)
  • John Innes Centre (GB)
  • Institute of Experimental Botany, Centre of the Region Haná for Biotechnological and Agricultural Research
  • CSIRO Agriculture and Food, Canberra, ACT
  • Agricultural Research Service (US)
  • The University of Sydney (AU)
  • University of Glasgow (GB)
  • King Abdullah University of Science and Technology (SA)
  • Czech Academy of Sciences, Institute of Experimental Botany (CZ)
  • Agriculture and Agri-Food Canada (CA)
  • Howard Hughes Medical Institute (US)
Corresponding author
  • Zhang, Peng
  • Rouse, Matthew
  • Lagudah, Evans
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2022-10-31
Volume
14
Issue
1
Pages
7354-7354
ISSN
2041-1723 (ISSN-L)
DOI
10.21203/rs.3.rs-2208386/v1
PMC
PMC10645757
OpenAlex
W4388655723
Cited by
19
References
1
Citations by year
2026: 2 · 2025: 11 · 2024: 4 · 2023: 2
FWCI
2.98
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • Research Square (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • ENLIGHTEN (Jurnal Bimbingan dan Konseling Islam) (submittedVersion, cc-by)
  • eScholarship (California Digital Library) (submittedVersion, cc-by)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • King Abdullah University of Science and Technology Repository (King Abdullah University of Science and Technology) (submittedVersion, cc-by)
Funders
  • Howard Hughes Medical Institute
  • U.S. Department of Agriculture (2022-68013-36439)
  • Bill and Melinda Gates Foundation
  • Commonwealth Scientific and Industrial Research Organisation
  • Grains Research and Development Corporation
  • Canada Excellence Research Chairs, Government of Canada
  • Natural Science Foundation of Shandong Province (ZR2021ZD30, ZR2021MC056)
  • Directorate for Biological Sciences
  • National Institute of Food and Agriculture (2022‐68013‐36439)
  • Agricultural Research Service (2022-68013-36439)
  • Biotechnology and Biological Sciences Research Council (BBS/E/J/000PR9780)
Topics
  • Life Sciences › Agricultural and Biological Sciences › Plant Science › Wheat and Barley Genetics and Pathology
  • Life Sciences › Agricultural and Biological Sciences › Plant Science › Plant Disease Resistance and Genetics
  • Life Sciences › Agricultural and Biological Sciences › Plant Science › Plant-Microbe Interactions and Immunity
Keywords
wheat stem rust, Puccinia striiformis, resistance alleles, leucine-rich repeats, Ug99, loss-of-function mutants, transgenic complementation, haplotypes, plant disease resistance
MeSH terms
Alleles, Amino Acid Sequence, Basidiomycota, Chromosome Mapping, Haplotypes, Plant Diseases, Disease Resistance

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