Figure 1.
Figure 1.

Barley stem rust resistance genes and their reaction to Puccinia graminis f. sp. tritici races MCCF, QCCJ, and TTKSK and P. graminis f. sp. secalis isolate 92-MN-90. Race MCCF is avirulent on resistance gene Rpg1 carried by barley cultivar Morex and line Q21861. Q21861 also carries the rpg4 and Rpg5 resistance genes, which, independent of Rpg1, also provide resistance to race MCCF (not shown). Races QCCJ and TTKSK are avirulent on the rpg4/Rpg5 complex but virulent on Rpg1. Isolate 92-MN-90 is avirulent on Rpg5 (rpg4 not required) but virulent on Rpg1. The ‘Steptoe’ control does not carry any known stem rust resistance genes. Mr, moderately resistant; R, resistant; S, susceptible.

 


Figure 2.
Figure 2.

Wild-type and mutant Rpg1-mediated reaction to infection with race MCCF and QCCJ. Columns from left to right show cartoon Rpg1 structure with mutated amino acids indicated; disease reaction phenotype; RPG1 protein response to infection timed in hours; and kinase autocatalytic activity measured in vitro. Lane 1 from top indicates kinase domain 2 mutant with K461 and K462 converted to N and Q, respectively. The resulting mutant transgenic in ‘Golden Promise’ genomic background is highly susceptible to race MCCF, the RPG1 protein is not degraded in 60 h, and it has no kinase activity. Compare with Lane 2, showing kinase domain 1 mutant with K152 and K153 mutated to N and Q, respectively. This transgenic mutant is also highly susceptible to race MCCF, the RPG1 protein is not degraded in 60 h, but it retains kinase activity, indicating that kinase domain 2 is sufficient for kinase activity, but kinase domain 1 is also required for disease resistance. Compare both with wild-type Rpg1 transgenic (GP/Rpg1T1) in Golden Promise (GP) genomic background (Lane 3) and GP control (Lane 6), which does not have a detectable Rpg1 gene or protein. RPG1 protein in GP/Rpg1T1 is degraded 20 to 28 h after infection, indicating that protein degradation is associated with disease resistance. Autocatalytic kinase activity is present. ‘Morex’ (Lanes 4 and 5) with wild-type Rpg1 shows resistance to race MCCF but susceptibility to race QCCJ. The RPG1 protein is degraded between 20 and 28 h in MCCF infection but not with QCCJ infection, indicating that RPG1 degradation is a specific response to infection with avirulent, but virulent races. Autocatalytic activity is retained in both cases.

 


Figure 3.
Figure 3.

Stem rust resistance gene organization and predicted protein structures. (A) Genomic DNA organization of Rpg1, Rpg5, and rpg4 genes (labeled to the right) with exons (black) introns (gray), 5 prime, and 3 prime untranslated regions (white). Exons are numbered above. ATG represents the start methionine codon and TGA represents the stop codon. The scale shown above is in kilobases. (B) Protein domain structures for RPG1, RPG5, and RPG4 are shown to scale with predicted boundaries above. pkinase denotes serine–threonine protein kinase domains, NBS denote nucleotide binding site, LRRs denote leucine rich repeats, and orange bars indicate position of conserved amino acids at actin binding sites. Asterisk indicates the conserved putative regulatory phosphorylation site. Proteins are labeled to the right.

 


Figure 4.
Figure 4.

Sequence annotations of the rpg4/Rpg5 genomic region and protein structures. Gray horizontal bars represent sequenced regions from barley cultivar Morex (susceptible) and line Q21861 (resistant) labeled on the left. White circles represents position of the flanking genetic markers, ARD5016 and ARD5112 labeled above. Scale is shown above in kilobases. The red arrows represent the rpg4 (Adf2) and Rpg5 (NBS-LRR-S/TPK) stem rust resistance genes. Black arrows indicate other annotated genes. Cartoon protein domain structures of RPG4 and RPG5 are shown below the Q21861 sequence annotation.