Dataset Information

Subcellular localization and functions of the barley stem rust resistance receptor-like serine/threonine-specific protein kinase Rpg1.

ABSTRACT: The Rpg1 gene confers resistance to many pathotypes of the stem rust fungus Puccinia graminis f. sp. tritici and has protected barley from serious disease losses for over 60 years. Rpg1 encodes a constitutively expressed protein with two tandem kinase domains. Fractionation by differential centrifugation and aqueous two-phase separation of the microsome proteins located Rpg1 mainly in the cytosol but also in the plasma membrane and intracellular membranes. Recombinant Rpg1 autophosphorylates in vitro intramolecularly only serine and threonine amino acids with a preference for Mn(2+) cations and a K(m) of 0.15 and a V(max) of 0.47 nmol.min(-1).mg(-1) protein. The inability of wild-type Rpg1 to transphosphorylate a recombinant Rpg1 inactivated by site-directed mutation confirmed that Rpg1 autophosphorylation proceeds exclusively via an intramolecular mechanism. Site-directed mutagenesis of the two adjacent lysine residues in the ATP anchor of the two-kinase domains established that the first of the two tandem kinase domains is nonfunctional and that lysine 461 of the second domain is the catalytically active residue. Transgenic barley, expressing Rpg1 mutated in either the kinase 1 or 2 domains, were fully susceptible to P. graminis f. sp. tritici revealing requirement of both kinase domains for resistance. In planta-expressed Rpg1 mutant protein confirmed that mutation in domain 2, but not 1, rendered the protein incapable of autophosphorylation.

SUBMITTER: Nirmala J

PROVIDER: S-EPMC1464370 | biostudies-literature | 2006 May

REPOSITORIES: biostudies-literature

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Subcellular localization and functions of the barley stem rust resistance receptor-like serine/threonine-specific protein kinase Rpg1.

Nirmala Jayaveeramuthu J Brueggeman Robert R Maier Christina C Clay Christine C Rostoks Nils N Kannangara C Gamini CG von Wettstein Diter D Steffenson Brian J BJ Kleinhofs Andris A

Proceedings of the National Academy of Sciences of the United States of America 20060428 19

The Rpg1 gene confers resistance to many pathotypes of the stem rust fungus Puccinia graminis f. sp. tritici and has protected barley from serious disease losses for over 60 years. Rpg1 encodes a constitutively expressed protein with two tandem kinase domains. Fractionation by differential centrifugation and aqueous two-phase separation of the microsome proteins located Rpg1 mainly in the cytosol but also in the plasma membrane and intracellular membranes. Recombinant Rpg1 autophosphorylates in ...[more]

PMID: 16648249

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Project description:BackgroundPuccinia graminis f. sp. tritici (Pgt) race TTKSK and its lineage pose a threat to barley production world-wide justifying the extensive efforts to identify, clone, and characterize the rpg4-mediated resistance locus (RMRL), the only effective resistance to virulent Pgt races in the TTKSK lineage. The RMRL contains two nucleotide-binding domain and leucine-rich repeat (NLR) resistance genes, Rpg5 and HvRga1, which are required for resistance. The two NLRs have head-to-head genome architecture with one NLR, Rpg5, containing an integrated C-terminal protein kinase domain, characteristic of an "integrated sensory domain" resistance mechanism. Fast neutron mutagenesis of line Q21861 was utilized in a forward genetics approach to identify genetic components that function in the RMRL or Rpg1 resistance mechanisms, as Q21861 contains both genes. A mutant was identified that compromises both RMRL and Rpg1-mediated resistances and had stunted seedling roots, designated required for P. graminis resistance 9 (rpr9).ResultsThe rpr9 mutant generated in the Q21861 background was crossed with the Swiss landrace Hv584, which carries RMRL but contains polymorphism across the genome compared to Q21861. To map Rpr9, a Hv584 x rpr9 F6:7 recombinant inbred line (RIL) population was developed. The RIL population was phenotyped with Pgt race QCCJB. The Hv584 x rpr9 RIL population was genotyped with the 9 k Illumina Infinium iSelect marker panel, producing 2701 polymorphic markers. A robust genetic map consisting of 563 noncosegregating markers was generated and used to map Rpr9 to an ~ 3.4 cM region on barley chromosome 3H. The NimbleGen barley exome capture array was utilized to capture rpr9 and wild type Q21861 exons, followed by Illumina sequencing. Comparative analysis, resulting in the identification of a 1.05 Mbp deletion at the chromosome 3H rpr9 locus. The identified deletion contains ten high confidence annotated genes with the best rpr9 candidates encoding a SKP1-like 9 protein and a F-box family protein.ConclusionGenetic mapping and exome capture rapidly identified candidate gene/s that function in RMRL and Rpg1 mediated resistance pathway/s. One or more of the identified candidate rpr9 genes are essential in the only two known effective stem rust resistance mechanisms, present in domesticated barley.

Project description:Stem rust, caused by Puccinia graminis f. sp. tritici (Pgt) is an economically important disease of wheat and barley. Rpg1 is the only resistance gene deployed in Midwestern US barley varieties and provides remarkable resistance to most North American races, except Pgt race QCCJB. Rpg1 is also ineffective against Pgt race TTKSK and its lineage that originated in Africa. The barley rpg4-mediated resistance locus (RMRL) conferring resistance to Pgt races QCCJB and TTKSK was isolated from line Q21861, which is resistant to all known Pgt races due to Rpg1 and RMRL. To develop elite barley varieties RMRL was pyramided into the varieties, Pinnacle and Conlon (both contain Rpg1), producing the near isogenic lines (NILs), Pinnacle RMRL-NIL (PRN) and Conlon RMRL-NIL (CRN). The CRN was resistant to Pgt races QCCJB (RMRL specific) and HKHJC (Rpg1 specific) at the seedling stage and Pgt race TTKSK (RMRL specific) at the adult stage. In contrast, PRN was susceptible to QCCJB and HKHJC at the seedling stage and TTKSK at the adult stage. Interestingly, PRN's susceptibility to QCCJB and HKHJC showed that RMRL was non-functional in the Pinnacle background but its presence also suppressed Rpg1-mediated resistance. Thus, in the absence of a gene/s found in the Q21861 background, Rpg1 becomes non-functional if RMRL is present, suggesting that another polymorphic gene, that we designated Rrr1 (required for rpg4-mediated resistance 1), is required for RMRL resistance and Rpg1-mediated resistance in the presence of RMRL. Utilizing a PRN/Q21861 derived recombinant inbred line (RIL) population, Rrr1 was delimited to a ∼0.5 MB physical region, slightly proximal (∼1.8 MB) of RMRL on barley chromosome 5H. A second gene, designated required for Rpg1-mediated resistance 2 (Rrr2), with duplicate gene action to Rrr1 in Rpg1-mediated resistance function, was genetically delimited to a physical region of ∼0.7 MB, slightly distal (∼3.1 MB) to Rpg1 on the short arm of barley chromosome 7H. Thus, Rrr1 is required for RMRL resistance and Rrr1 or Rrr2 is required for functional Rpg1-mediated resistance in the presence of the RMRL introgression. Candidate Rrr1 and Rrr2 genes were identified that need to be considered when pyramiding Rpg1 and RMRL in barley.

Project description:Wheat stem rust, caused by Puccinia graminis f. sp. tritici (Pgt), is an important disease of barley and wheat. A diverse sexual Pgt population from the Pacific Northwest (PNW) region of the US contains a high proportion of individuals with virulence on the barley stem rust resistance (R) gene, Rpg1. However, the evolutionary mechanisms of this virulence on Rpg1 are mysterious considering that Rpg1 had not been deployed in the region and the gene had remained remarkably durable in the Midwestern US and prairie provinces of Canada. To identify AvrRpg1 effectors, genome wide association studies (GWAS) were performed using 113 Pgt isolates collected from the PNW (n = 89 isolates) and Midwest (n = 24 isolates) regions of the US. Disease phenotype data were generated on two barley lines Morex and the Golden Promise transgenic (H228.2c) that carry the Rpg1 gene. Genotype data was generated by whole genome sequencing (WGS) of 96 isolates (PNW = 89 isolates and Midwest = 7 isolates) and RNA sequencing (RNAseq) data from 17 Midwestern isolates. Utilizing ~1.2 million SNPs generated from WGS and phenotype data (n = 96 isolates) on the transgenic line H228.2c, 53 marker trait associations (MTAs) were identified. Utilizing ~140 K common SNPs generated from combined analysis of WGS and RNAseq data, two significant MTAs were identified using the cv Morex phenotyping data. The 55 MTAs defined two distinct avirulence loci, on supercontig 2.30 and supercontig 2.11 of the Pgt reference genome of Pgt isolate CRL 75-36-700-3. The major avirulence locus designated AvrRpg1A was identified with the GWAS using both barley lines and was delimited to a 35 kb interval on supercontig 2.30 containing four candidate genes (PGTG_10878, PGTG_10884, PGTG_10885, and PGTG_10886). The minor avirulence locus designated AvrRpg1B identified with cv Morex contained a single candidate gene (PGTG_05433). AvrRpg1A haplotype analysis provided strong evidence that a dominant avirulence gene underlies the locus. The association analysis identified strong candidate AvrRpg1 genes. Further analysis to validate the AvrRpg1 genes will fill knowledge gaps in our understanding of rust effector biology and the evolution and mechanism/s of Pgt virulence on Rpg1.

Dataset Information

Subcellular localization and functions of the barley stem rust resistance receptor-like serine/threonine-specific protein kinase Rpg1.

Publications

Subcellular localization and functions of the barley stem rust resistance receptor-like serine/threonine-specific protein kinase Rpg1.

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