The barley stem rust-resistance gene Rpg1 is a novel disease-resistance gene with homology to receptor kinases

Abstract

Stem rust caused by Puccinia graminis f. sp. tritici was among the most devastating diseases of barley in the northern Great Plains of the U.S. and Canada before the deployment of the stem rust-resistance gene Rpg1 in 1942. Since then, Rpg1 has provided durable protection against stem rust losses in widely grown barley cultivars (cvs.). Extensive efforts to clone Rpg1 by synteny with rice provided excellent flanking markers but failed to yield the gene because it does not seem to exist in rice. Here we report the map-based cloning and characterization of Rpg1. A high-resolution genetic map constructed with 8,518 gametes and a 330-kb bacterial artificial chromosome contig physical map positioned the gene between two crossovers approximately 0.21 centimorgan and 110 kb apart. The region including Rpg1 was searched for potential candidate genes by sequencing low-copy probes. Two receptor kinase-like genes were identified. The candidate gene alleles were sequenced from resistant and susceptible cvs. Only one of the candidate genes showed a pattern of apparently functional gene structure in the resistant cvs. and defective gene structure in the susceptible cvs. identifying it as the Rpg1 gene. Rpg1 encodes a receptor kinase-like protein with two tandem protein kinase domains, a novel structure for a plant disease-resistance gene. Thus, it may represent a new class of plant resistance genes.

Figure 1

A genetic and physical map of barley stem rust-resistance gene Rpg1 region on chromosome 1(7H). (A) high-resolution genetic map based on 8,518 gametes showing critical markers and the number of total crossovers (c.o.) separating them. (B) Physical map encompassing Rpg1 based on BAC clone contig. The distance from Pic20A to RSB308 was estimated to be ≈270 kb.

Figure 2

Rpg1 gene, cDNA, and translated protein structure. (A) Genomic and cDNA structures with introns (gray) and exons (black) are shown to scale. The mRNA 5′ and 3′ untranslated regions are hatched. Exons are numbered above the drawing. Predicted TATA box, translation start site, and stop codon are shown below. The Rpg1 mRNA structure is shown below the gene. The mRNA was reconstructed with the sequence of 5′ truncated cDNA clone HVSMEf55g12 and sequences of the RT-PCR products from cv. Morex leaf total RNA. (B) Organization of the protein domains is shown to scale with predicted boundaries indicated above. pkinases 1 and 2 denote two tandem protein kinase domains. The asterisk indicates an asparagine residue within a putative N-glycosylation site NFSE.

Figure 3

A diagram representation of the derived amino acid sequence alignment of Rpg1 alleles from resistant (Rpg1) and susceptible (rpg1) cvs. The differences from the cv. Morex sequence are indicated. “Morex” includes additional resistant cultivars Q21861, Kindred, Chevron, Peatland, Leger, Bowman, and 80-TT-29. “OSU6” is a resistant H. vulgare subsp. spontaneum line PBI004-7-0-015 (accession no. 8321). Group 1, Sequence from susceptible cultivars Steptoe, Lion, Wisconsin38, and MoravianIII. Group 2, Sequences from susceptible cultivars SM89010, Dicktoo, and Gobernadora. “OSU15” is a susceptible H. vulgaresubsp. spontaneum line Wadi Qilt 23–38. AMS170 is a doubled haploid line with a recombination in the Rpg1 gene. *, A 3 bp insertion that introduces a serine to arginine conversion and inserts a phenylalanine at amino acid positions 320 and 321. §, Base pair substitutions in group 1 cultivars which converts amino acid codons to stop codons at amino acid positions 192, 524, and 788. ¶, A single base pair substitution results in lysine to glutamine conversion. **, Highly diverged with multiple amino acid substitutions, a small deletion, and a large deletion. §§, A single base pair deletion introduces a frame shift that results in a stop codon. ¶¶, Crossover region in the recombinant AMS 170 was narrowed to a 295-bp region between the lysine at amino acid position 436 of Morex and the stop codon at amino acid position 524 of Steptoe.