The stem rust resistance gene Rpg5 encodes a protein with nucleotide-binding-site, leucine-rich, and protein kinase domains

Abstract

We isolated the barley stem rust resistance genes Rpg5 and rpg4 by map-based cloning. These genes are colocalized on a 70-kb genomic region that was delimited by recombination. The Rpg5 gene consists of an unusual structure encoding three typical plant disease resistance protein domains: nucleotide-binding site, leucine-rich repeat, and serine threonine protein kinase. The predicted RPG5 protein has two putative transmembrane sites possibly involved in membrane binding. The gene is expressed at low but detectable levels. Posttranscriptional gene silencing using VIGS resulted in a compatible reaction with a normally incompatible stem rust pathogen. Allele sequencing also validated the candidate Rpg5 gene. Allele and recombinant sequencing suggested that the probable rpg4 gene encoded an actin depolymerizing factor-like protein. Involvement of actin depolymerizing factor genes in nonhost resistance has been documented, but discovery of their role in gene-for-gene interaction would be novel and needs to be further substantiated.

Fig. 1.

Genetic, physical, sequence, and recombinant characterization of the Rpg5/rpg4 locus. (A) High-resolution genetic map of the Rpg5 region based on 5,232 recombinant gametes. The Rpg5 flanking markers (ARD5016 and ARD5112) are designated above the white vertical bars. The candidate genes cosegregating with isolate 92-MN-90 (Rpg5) resistance and pathotype QCC (rpg4) resistance are inside the boxes labeled Rpg5 and rpg4, respectively. Numbers below the bar indicate the number of cross-overs. C and T represent centromere and telomere. (B) BAC physical map spanning the Rpg5/rpg4 locus. The two BAC clones (543P19 and 116G10) spanning the region are shown as horizontal gray bars. The white circles indicate the flanking genetic markers. Black circles indicate the position of Rpg5 and rpg4 candidate genes and the marker (ARD5005) used for chromosome walking. (C) Sequence annotation of the Rpg5 region. White horizontal bars represent sequenced regions from Morex and Q21861. White circles show the position of flanking markers. The black arrow represents the Rpg5 NBS-LRR-S/TPK gene. Gray arrows indicate annotated genes. The darker gray background between HvRGA2 and HvAdf3 indicates the region of colinearity breakdown. The scale is shown above in kilobases. (D) Recombinant sequence analysis. Horizontal bars represent sequence and/or genotyping from 10 lines with recombination defining the Rpg5/rpg4 region. Black indicates susceptible genotype, and white indicates the resistant Q21861 genotype. The “X” indicates the approximate point of recombination. The recombinant designations are labeled to the right with resistant (R) or susceptible (S), indicating the response to pathotype QCC (rpg4) and isolate 92-MN-90 (Rpg5), respectively. Gray arrows indicate candidate genes and the white circles represent flanking markers. Recombinant lines indicated with an asterisk have complete sequence analysis at the region of recombination. The scale is shown below in kilobases.

Fig. 2.

Rpg5 predicted gene structure from resistant and susceptible cultivars. (A) cDNA and genomic DNA structures with introns (gray), exons (black), and UTRs white shown to scale. Rpg5 predicted mRNA structure is shown above the genomic sequence with exons numbered above. ATG represents the start methionine codon and TGA represents the stop codon. AAA is the mRNA poly(A) tail, TSS is the transcription start site, and PAS is the polyadenylation signal. The scale below is shown in kilobases. (B) Organization of the predicted protein structure is shown to scale with predicted boundaries indicated by boxes. NBS denotes the nucleotide-binding site, LRR denotes 12 imperfect leucine-rich repeats, S/TPK denotes the serine threonine protein kinase domain, and the black bars represent predicted transmembrane domains. Numbers above indicate the last amino acid preceding the stop codon. Stop codons are represented by an asterisk. Cultivars or lines were placed into four groups. Group 1 (resistant) consists of Q21861 with a predicted intact and functional Rpg5 gene. Group 2 (susceptible) consists of the cultivars Morex and Steptoe. They have a cytosine-to-adenine conversion that introduces a stop codon resulting in a predicted truncated protein. In addition, they are missing the kinase domain coding region. Group 3 (susceptible) contains Golden Promise and MD2. They have a single cytosine insertion causing a frame-shift mutation that results in a stop codon and a predicted truncated protein. Group 4 (susceptible) consists of Harrington. Harrington is missing the kinase domain coding region and the second transmembrane domain.

Fig. 3.

Northern analysis of Rpg5 and virus-induced gene silencing phenotypes. (A) Northern blot analysis of Rpg5 transcript. Lane 1 is Q21861 poly(A) mRNA with the Rpg5 specific probe hybridizing to a single band at ≈4.8 kb, indicated by the arrow to the right. Lane 2 is a methylene blue stain of the RNA marker after transfer to membrane. Numbers to the right indicate molecular mass of Millennium Marker (Ambion) bands. (B) Barley seedlings inoculated with antisense Rpg5 or MCS VIGS constructs and challenged with Pgs isolate 92-MN-90. Approximately 30% of the inoculated seedlings showed a typical susceptible reaction to the Pgs 92-MN-90 fungal infection. These are marked asRpg5(S), whereas seedlings that failed to show a reaction to the fungal infection are marked asRpg5(R). A representative seedling is shown for each group. The MCS antisense (BSMV-MCS) treated plant shows typical resistant reaction to Pgs 92-MN-90. The seedling leaves marked Steptoe and Q21861 are the rust-susceptible and resistant virus uninoculated controls, respectively.

Fig. 4.

Expression of the Rpg5 gene after BSMV-VIGS induced gene silencing and infection with isolate 92-MN-90 given as percentage of GAPDH gene expression. Bars represent BSMV-VIGS data from plants sampled at either time point 0 or 14 d postfungal infection (DPI). Sample 1 is inoculated with BSMV-asRpg5, 2 is inoculated with BSMV-MCS, and 3 is the uninoculated control all sampled at TP0. Sample 4 is the untreated control, 5 is the virus uninoculated control, 6 is inoculated with BSMV-asRpg5 sample taken from seedlings showing susceptible fungal reaction, 7 is inoculated with BSMV-asRpg5 sample taken from seedlings failing to show susceptible fungal reactions, and 8 is inoculated with BSMV-MCS, all sampled at TP14. The plants were examined for disease reaction at 14 DPI. The tissue analyzed by qRT-PCR was taken at TP0 or TP14 d after inoculation with isolate 92-MN-90.