Single amino acid change alters specificity of the multi-allelic wheat stem rust resistance locus SR9.

Most rust resistance genes thus far isolated from wheat have a very limited number of functional alleles. Here, we report the isolation of most of the alleles at wheat stem rust resistance gene locus SR9. The seven previously reported resistance alleles (Sr9a, Sr9b, Sr9d, Sr9e, Sr9f, Sr9g, and Sr9h) are characterised using a synergistic strategy. Loss-of-function mutants and/or transgenic complementation are used to confirm Sr9b, two haplotypes of Sr9e (Sr9e_h1 and Sr9e_h2), Sr9g, and Sr9h. Each allele encodes a highly related nucleotide-binding site leucine-rich repeat (NB-LRR) type immune receptor, containing 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, differs from SR9B by a single amino acid. SR9B and SR9G resistance proteins are 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. Together, our results show that functional diversification of allelic variants at the SR9 locus involves single and multiple amino acid changes that recognize isolates of wheat stem rust.

Tracing Viral Transmission and Evolution of Bovine Leukemia Virus through Long Read Oxford Nanopore Sequencing of the Proviral Genome.

Bovine leukemia virus (BLV) causes Enzootic Bovine Leukosis (EBL), a persistent life-long disease resulting in immune dysfunction and shortened lifespan in infected cattle, severely impacting the profitability of the US dairy industry. Our group has found that 94% of dairy farms in the United States are infected with BLV with an average in-herd prevalence of 46%. This is partly due to the lack of clinical presentation during the early stages of primary infection and the elusive nature of BLV transmission. This study sought to validate a near-complete genomic sequencing approach for reliability and accuracy before determining its efficacy in characterizing the sequence identity of BLV proviral genomes collected from a pilot study made up of 14 animals from one commercial dairy herd. These BLV-infected animals were comprised of seven adult dam/daughter pairs that tested positive by ELISA and qPCR. The results demonstrate sequence identity or divergence of the BLV genome from the same samples tested in two independent laboratories, suggesting both vertical and horizontal transmission in this dairy herd. This study supports the use of Oxford Nanopore sequencing for the identification of viral SNPs that can be used for retrospective genetic contact tracing of BLV transmission.

Shedding and transmission of a live attenuated influenza A virus vaccine in pre-weaned pigs under field conditions.

Influenza A virus (IAV) is one of the most important respiratory viruses affecting pig health and vaccination is the most common strategy to control influenza infections. In this field study we assessed the onset and duration of shedding of a live attenuated influenza virus (LAIV) vaccine, its ability to transmit to non-vaccinated pigs and whether the LAIV could be aerosolized and detected in the environment. Thirty-three litters (n = 33) of a farm using the LAIV vaccine were selected for the study, a subset of them (n = 12) were left unvaccinated and a subset of piglets (n = 3) in vaccinated litters were also left unvaccinated to serve as sentinels. Selected piglets from the litters were sampled multiple days post vaccination (DPV) by collecting nasal swabs and blood, and were tested using a LAIV vaccine specific RT-PCR assay and hemagglutination inhibition assay against the LAIV strains respectively. Environmental specimens consisting of air and surface wipes were also collected. One hundred percent (21/21) of the vaccinated litters tested LAIV positive 1 DPV and until 6 DPV. In contrast, only five (5/33) of the thirty-three non-vaccinated pigs tested positive during the course of the study. Viable LAIV was confirmed in vaccinated pigs by cell culture and whole genome sequencing. In addition, low levels of LAIV RNA (RT-PCR Ct values ranging between 33 and 38) were detected in all air specimens collected on the day of vaccination and until 6 DPV (3/10). Pigs had maternally derived antibodies reactive against the LAIV strains which may have influenced the degree of shedding observed. Under the conditions of this study, shedding of the LAIV from vaccinated pigs was limited in time, resulted in minimal transmission to non-vaccinated pigs and was detected in low levels in aerosols collected in the vaccinated rooms likely influenced by the presence of maternally derived antibodies against the LAIV strains.

Genetic variability of influenza A virus in pigs at weaning in Midwestern United States swine farms.

Suckling piglets play an important role at maintaining influenza A virus (IAV) infections in breeding herds and disseminating them to other farms at weaning. However, the role they play at weaning to support and promote genetic variability of IAV is not fully understood. The objective here was to evaluate the genetic diversity of IAV in pigs at weaning in farms located in the Midwestern USA. Nasal swabs (n = 9,090) collected from piglets in breed-to-wean farms (n = 52) over a six-month period across seasons were evaluated for the presence of IAV. Nasal swabs (n = 391) from 23 IAV-positive farms were whole-genome sequenced. Multiple lineages of HA (n = 7) and NA (n = 3) were identified in 96% (22/23) and 61% (237/391) of the investigated farms and individual piglets, respectively. Co-circulation of multiple types of functional HA and NA was identified in most (83%) farms. Whole IAV genomes were completed for 126 individual piglet samples and 25 distinct and 23 mixed genotypes were identified, highlighting significant genetic variability of IAV in piglets. Co-circulation of IAV in the farms and co-infection of individual piglets at weaning was observed at multiple time points over the investigation period and appears to be common in the investigated farms. Statistically significant genetic variability was estimated within and between farms by AMOVA, and varying levels of diversity between farms were detected using the Shannon-Weiner Index. Results reported here demonstrate previously unreported levels of molecular complexity and genetic variability among IAV at the farm and piglet levels at weaning. Movement of such piglets infected at weaning may result in emergence of new strains and maintenance of endemic IAV infection in the US swine herds. Results presented here highlight the need for developing and implementing novel, effective strategies to prevent or control the introduction and transmission of IAV within and between farms in the country.

Genetic diversity of influenza A viruses circulating in pigs between winter and summer in a Minnesota live animal market.

There has been little surveillance of influenza A viruses (IAVs) circulating in swine at live animal markets, particularly in the United States. To address this gap, we conducted active surveillance of IAVs in pigs, the air, and the environment during a summer and winter season in a live animal market in St. Paul, Minnesota, that had been epidemiologically associated with swine-origin influenza cases in humans previously. High rates of IAV were detected by PCR in swine lungs and oral fluids during both summer and winter seasons. Rates of IAV detection by PCR in the air were similar during summer and winter, although rates of successful virus isolation in the air were lower during summer than in winter (26% and 67%, respectively). H3N2 was the most prevalent subtype in both seasons, followed by H1N2. Genetically diverse viruses with multiple gene constellations were isolated from both winter and summer, with a total of 19 distinct genotypes identified. Comparative phylogenetic analysis of all eight segments of 40 virus isolates from summer and 122 isolates from winter revealed that the summer and winter isolates were genetically distinct, indicating IAVs are not maintained in the market, but rather are re-introduced, likely from commercial swine. These findings highlight the extent of IAV genetic diversity circulating in swine in live animal markets, even during summer months, and the ongoing risk to humans.

Comparison of individual, group and environmental sampling strategies to conduct influenza surveillance in pigs.

BACKGROUND: Influenza A virus (IAV) is an important pathogen in pigs that affects productivity and has important public health implications because of its zoonotic nature. Surveillance is central to the control of influenza, however, detection of IAV infections can be challenging in endemically infected herds with low prevalence of infection. METHODS: In groups of suckling (18-21 days of age) and growing (35-45 days of age) pigs, we compared various sampling approaches to detect, isolate and sequence IAV using individual (nasal swabs, nasal wipes and oropharyngeal swabs), group (oral fluids, surface wipes and sow udder skin wipes) and environmental (airborne particles deposited on surfaces and air samples) sampling approaches. All samples were tested by IAV rRT-PCR and a subset was used for virus isolation and direct sequencing. RESULTS: In general, environmental and group samples resulted in higher odd ratios (range = 3.87-16.5, p-value < 0.05) of detecting a positive sample by rRT-PCR compared to individual pooled samples, except for oropharyngeal swabs (OR = 8.07, p-value < 0.05). In contrast, individual samples were most likely to yield a viral isolate by cell culture. Oropharyngeal swabs in suckling pigs (78.4%), and nasal swabs (47.6%) or nasal wipes (45%) in growing pigs, and udder wipes in lactating sows (75%) were the preferred samples to obtain an isolate. CONCLUSIONS: Our findings indicate that group and environmental sampling strategies should be considered in influenza surveillance programs in particular if the goal is just to detect infection. This study provides new information on sampling approaches to conduct effective influenza surveillance in pigs and identifies udder wipes from lactating sows as a novel sample type that offers a convenient, cheap and sensitive manner to monitor IAV in litters prior to weaning.

Identification and Validation of a Common Stem Rust Resistance Locus in Two Bi-parental Populations.

Races belonging to Ug99 lineage of stem rust fungus Puccinia graminis f. sp. tritici (Pgt) continue to pose a threat to wheat (Triticum aestivum L.) production in various African countries. Growing resistant varieties is the most economical and environmentally friendly control measure. Recombinant inbred line (RIL) populations from the crosses of susceptible parent 'Cacuke' with the resistant parents 'Huhwa' and 'Yaye' were phenotyped for resistance at the seedling stage to Pgt race TTKSK (Ug99) and in adult plants in field trials at Njoro, Kenya for two seasons in 2016. Using the Affymetrix Axiom breeders SNP array, two stem rust resistance genes, temporarily designated as SrH and SrY, were identified and mapped on chromosome arm 2BL through selective genotyping and bulked segregant analysis (BSA), respectively. Kompetitive allele specific polymorphism (KASP) markers and simple sequence repeat (SSR) markers were used to saturate chromosome arm 2BL in both RIL populations. SrH mapped between markers cim109 and cim114 at a distance of 0.9 cM proximal, and cim117 at 2.9 cM distal. SrY was flanked by markers cim109 and cim116 at 0.8 cM proximal, and IWB45932 at 1.9 cM distal. Two Ug99-effective stem rust resistance genes derived from bread wheat, Sr9h and Sr28, have been reported on chromosome arm 2BL. Infection types and map position in Huhwa and Yaye indicated that Sr28 was absent in both the parents. However, susceptible reactions produced by resistant lines from both populations against Sr9h-virulent race TTKSF+ confirmed the presence of a common resistance locus Sr9h in both lines. Test of allelism is required to establish genetic relationships between genes identified in present study and Sr9h. Marker cim117 linked to SrH was genotyped on set of wheat lines with Huhwa in the pedigree and is advised to be used for marker assisted selection for this gene, however, a combination of phenotypic and genotypic assays is desirable for both genes especially for selection of Sr9h in breeding programs.

Identification and characterization of Sr13, a tetraploid wheat gene that confers resistance to the Ug99 stem rust race group.

The Puccinia graminis f. sp. tritici (Pgt) Ug99 race group is virulent to most stem rust resistance genes currently deployed in wheat and poses a threat to global wheat production. The durum wheat (Triticum turgidum ssp. durum) gene Sr13 confers resistance to Ug99 and other virulent races, and is more effective at high temperatures. Using map-based cloning, we delimited a candidate region including two linked genes encoding coiled-coil nucleotide-binding leucine-rich repeat proteins designated CNL3 and CNL13. Three independent truncation mutations identified in each of these genes demonstrated that only CNL13 was required for Ug99 resistance. Transformation of an 8-kb genomic sequence including CNL13 into the susceptible wheat variety Fielder was sufficient to confer resistance to Ug99, confirming that CNL13 is Sr13CNL13 transcripts were slightly down-regulated 2-6 days after Pgt inoculation and were not affected by temperature. By contrast, six pathogenesis-related (PR) genes were up-regulated at high temperatures only when both Sr13 and Pgt were present, suggesting that they may contribute to the high temperature resistance mechanism. We identified three Sr13-resistant haplotypes, which were present in one-third of cultivated emmer and durum wheats but absent in most tested common wheats (Triticum aestivum). These results suggest that Sr13 can be used to improve Ug99 resistance in a large proportion of modern wheat cultivars. To accelerate its deployment, we developed a diagnostic marker for Sr13 The identification of Sr13 expands the number of Pgt-resistance genes that can be incorporated into multigene transgenic cassettes to control this devastating disease.

Discovery of a Novel Stem Rust Resistance Allele in Durum Wheat that Exhibits Differential Reactions to Ug99 Isolates.

Wheat stem rust, caused by Puccinia graminis f. sp. tritici Eriks. & E. Henn, can incur yield losses in susceptible cultivars of durum wheat, Triticum turgidum ssp. durum (Desf.) Husnot. Although several durum cultivars possess the stem rust resistance gene Sr13, additional genes in durum wheat effective against emerging virulent races have not been described. Durum line 8155-B1 confers resistance against the P. graminis f. sp. tritici race TTKST, the variant race of the Ug99 race group with additional virulence to wheat stem rust resistance gene Sr24 However, 8155-B1 does not confer resistance to the first-described race in the Ug99 race group: TTKSK. We mapped a single gene conferring resistance in 8155-B1 against race TTKST, Sr8155B1, to chromosome arm 6AS by utilizing Rusty/8155-B1 and Rusty*2/8155-B1 populations and the 90K Infinium iSelect Custom bead chip supplemented by KASP assays. One marker, KASP_6AS_IWB10558, cosegregated with Sr8155B1 in both populations and correctly predicted Sr8155B1 presence or absence in 11 durum cultivars tested. We confirmed the presence of Sr8155B1 in cultivar Mountrail by mapping in the population Choteau/Mountrail. The marker developed in this study could be used to predict the presence of resistance to race TTKST in uncharacterized durum breeding lines, and also to combine Sr8155B1 with resistance genes effective to Ug99 such as Sr13 The map location of Sr8155B1 cannot rule out the possibility that this gene is an allele at the Sr8 locus. However, race specificity indicates that Sr8155B1 is different from the known alleles Sr8a and Sr8b.

Genetic Mapping of Stem Rust Resistance to Puccinia graminis f. sp. tritici Race TRTTF in the Canadian Wheat Cultivar Harvest.

Stem rust, caused by Puccinia graminis f. sp. tritici, is a destructive disease of wheat that can be controlled by deploying effective stem rust resistance (Sr) genes. Highly virulent races of P. graminis f. sp. tritici in Africa have been detected and characterized. These include race TRTTF and the Ug99 group of races such as TTKSK. Several Canadian and U.S. spring wheat cultivars, including the widely grown Canadian cultivar 'Harvest', are resistant to TRTTF. However, the genetic basis of resistance to TRTTF in Canadian and U.S. spring wheat cultivars is unknown. The objectives of this study were to determine the number of Sr genes involved in TRTTF resistance in Harvest, genetically map the resistance with DNA markers, and use markers to assess the distribution of that resistance in a panel of Canadian cultivars. A doubled haploid (DH) population was produced from the cross LMPG-6S/Harvest. The DH population was tested with race TRTTF at the seedling stage. Of 92 DH progeny evaluated, 46 were resistant and 46 were susceptible which perfectly fit a 1:1 ratio indicating a single Sr gene was responsible for conferring resistance to TRTTF in Harvest. Mapping with single nucleotide polymorphism (SNP) and simple sequence repeat (SSR) markers placed the resistance gene distally on the chromosome 6AS genetic map, which corresponded to the location reported for Sr8. SSR marker gwm459 and 30 cosegregating SNP markers showed the closest linkage, mapping 2.2 cM proximal to the Sr gene. Gene Sr8a confers resistance to TRTTF and may account for the resistance in Harvest. Testing a panel of Canadian wheat cultivars with four SNP markers closely linked to resistance to TRTTF suggested that the resistance present in Harvest is present in many Canadian cultivars. Two of these SNP markers were also predictive of TRTTF resistance in a panel of 241 spring wheat lines from the United States, Canada, and Mexico.

Markers Linked to Wheat Stem Rust Resistance Gene Sr11 Effective to Puccinia graminis f. sp. tritici Race TKTTF.

Wheat stem rust, caused by Puccinia graminis f. sp. tritici, can cause severe yield losses on susceptible wheat varieties and cultivars. Although stem rust can be controlled by the use of genetic resistance, population dynamics of P. graminis f. sp. tritici can frequently lead to defeat of wheat stem rust resistance genes. P. graminis f. sp. tritici race TKTTF caused a severe epidemic in Ethiopia on Ug99-resistant 'Digalu' in 2013 and 2014. The gene Sr11 confers resistance to race TKTTF and is present in 'Gabo 56'. We identified seven single-nucleotide polymorphism (SNP) markers linked to Sr11 from a cross between Gabo 56 and 'Chinese Spring' exploiting a 90K Infinium iSelect Custom beadchip. Five SNP markers were validated on a 'Berkut'/'Scalavatis' population that segregated for Sr11, using KBioscience competitive allele-specific polymerase chain reaction (KASP) assays. Two of the SNP markers, KASP_6BL_IWB10724 and KASP_6BL_IWB72471, were predictive of Sr11 among wheat genetic stocks, cultivars, and breeding lines from North America, Ethiopia, and Pakistan. These markers can be utilized to select for Sr11 in wheat breeding and to detect the presence of Sr11 in uncharacterized germplasm.

Molecular and genetic characterization of barley mutants and genetic mapping of mutant rpr2 required for Rpg1-mediated resistance against stem rust.

KEY MESSAGE: This study describes the generation, screening, genetic and molecular characterization, and high-resolution mapping of barley mutants susceptible to stem rust ( Puccinia graminis f. sp. tritici ) races MCCF and HKHJ. A single gene, Rpg1, has protected barley cultivars against many races of stem rust pathogen (Puccinia graminis f. sp. tritici) for the last 70 years in the United States and Canada. To identify signaling components of protein product RPG1, we employed a mutagenesis approach. Using this approach, six mutants exhibiting susceptibility to Puccinia graminis f. sp. tritici races MCCF and HKHJ were identified in the gamma irradiated M2 population of resistant cultivar Morex, which carries Rpg1 on chromosome 7H. The mutants retained a functional Rpg1 gene and an apparently functional protein, suggesting that the mutated genes were required for downstream or upstream signaling. Selected mutants were non-allelic, hence each mutant represents a unique gene. Low and high-resolution genetic mapping of the rpr2 mutant identified chromosome 6H (bin 6) as the location of the mutated gene. The target region was reduced to 0.6 cM and gene content analyzed. Based on the published barley genomic sequence, the target region contains approximately 157 genes, including a set that encodes putative leucine-rich receptor-like protein kinases, which may be strong candidates for the gene of interest. Overall, this study presents a strong platform for future map-based cloning of genes identified in this mutant screen.

A new 2DS·2RL Robertsonian translocation transfers stem rust resistance gene Sr59 into wheat.

KEY MESSAGE: A new stem rust resistance gene Sr59 from Secale cereale was introgressed into wheat as a 2DS·2RL Robertsonian translocation. Emerging new races of the wheat stem rust pathogen (Puccinia graminis f. sp. tritici), from Africa threaten global wheat (Triticum aestivum L.) production. To broaden the resistance spectrum of wheat to these widely virulent African races, additional resistance genes must be identified from all possible gene pools. From the screening of a collection of wheat-rye (Secale cereale L.) chromosome substitution lines developed at the Swedish University of Agricultural Sciences, we described the line 'SLU238' 2R (2D) as possessing resistance to many races of P. graminis f. sp. tritici, including the widely virulent race TTKSK (isolate synonym Ug99) from Africa. The breakage-fusion mechanism of univalent chromosomes was used to produce a new Robertsonian translocation: T2DS·2RL. Molecular marker analysis and stem rust seedling assays at multiple generations confirmed that the stem rust resistance from 'SLU238' is present on the rye chromosome arm 2RL. Line TA5094 (#101) was derived from 'SLU238' and was found to be homozygous for the T2DS·2RL translocation. The stem rust resistance gene on chromosome 2RL arm was designated as Sr59. Although introgressions of rye chromosome arms into wheat have most often been facilitated by irradiation, this study highlights the utility of the breakage-fusion mechanism for rye chromatin introgression. Sr59 provides an additional asset for wheat improvement to mitigate yield losses caused by stem rust.

Characterization of Sr9h, a wheat stem rust resistance allele effective to Ug99.

KEY MESSAGE: Wheat stem rust resistance gene SrWeb is an allele at the Sr9 locus that confers resistance to Ug99. Race TTKSK (Ug99) of Puccinia graminis f. sp. tritici, the causal fungus of stem rust, threatens global wheat production because of its broad virulence to current wheat cultivars. A recently identified Ug99 resistance gene from cultivar Webster, temporarily designated as SrWeb, mapped near the stem rust resistance gene locus Sr9. We determined that SrWeb is also present in Ug99 resistant cultivar Gabo 56 by comparative mapping and an allelism test. Analysis of resistance in a population segregating for both Sr9e and SrWeb demonstrated that SrWeb is an allele at the Sr9 locus, which subsequently was designated as Sr9h. Webster and Gabo 56 were susceptible to the Ug99-related race TTKSF+ from South Africa. Race TTKSF+ possesses unique virulence to uncharacterized Ug99 resistance in cultivar Matlabas. This result validated that resistance to Ug99 in Webster and Gabo 56 is conferred by the same gene: Sr9h. The emergence of pathogen virulence to several resistance genes that are effective to the original Ug99 race TTKSK, including Sr9h, suggests that resistance genes should be used in combinations in order to increase resistance durability.

Concerted action of two avirulent spore effectors activates Reaction to Puccinia graminis 1 (Rpg1)-mediated cereal stem rust resistance.

The barley stem rust resistance gene Reaction to Puccinia graminis 1 (Rpg1), encoding a receptor-like kinase, confers durable resistance to the stem rust pathogen Puccinia graminis f. sp. tritici. The fungal urediniospores form adhesion structures with the leaf epidermal cells within 1 h of inoculation, followed by hyphae and haustorium formation. The RPG1 protein is constitutively expressed and not phosphorylated. On inoculation with avirulent urediniospores, it is phosphorylated in vivo within 5 min and subsequently degraded. Application of arginine-glycine-aspartic acid peptide loops prevented the formation of adhesion structures for spore attachment, the phosphorylation of RPG1, and germination of the viable spores. Arginine-glycine-aspartic acid affinity chromatography of proteins from the ungerminated avirulent rust spores led to the purification and identification of a protein with fibronectin type III and breast cancer type 1 susceptibility protein domains and a vacuolar protein sorting-associated protein 9 with a coupling of ubiquitin to endoplasmic reticulum degradation domain. Both proteins are required to induce in vivo phosphorylation and degradation of RPG1. Combined application of both proteins caused hypersensitive reaction on the stem rust-resistant cultivar Morex but not on the susceptible cultivar Steptoe. Expression studies indicated that mRNA of both genes are present in ungerminated urediniospores and are constitutively transcribed in sporelings, infected leaves, and haustoria in the investigated avirulent races. Evidence is presented that RPG1, in yeast, interacts with the two protein effectors from the urediniospores that activate cooperatively the stem rust resistance protein RPG1 long before haustoria formation.

Stem rust spores elicit rapid RPG1 phosphorylation.

Stem rust threatens cereal production worldwide. Understanding the mechanism by which durable resistance genes, such as Rpg1, function is critical. We show that the RPG1 protein is phosphorylated within 5 min by exposure to spores from avirulent but not virulent races of stem rust. Transgenic mutants encoding an RPG1 protein with an in vitro inactive kinase domain fail to phosphorylate RPG1 in vivo and are susceptible to stem rust, demonstrating that phosphorylation is a prerequisite for disease resistance. Protein kinase inhibitors prevent RPG1 phosphorylation and result in susceptibility to stem rust, providing further evidence for the importance of phosphorylation in disease resistance. We conclude that phosphorylation of the RPG1 protein by the kinase activity of the pK2 domain induced by the interaction with an unknown pathogen spore product is required for resistance to the avirulent stem rust races. The pseudokinase pK1 domain is required for disease resistance but not phosphorylation. The very rapid phosphorylation of RPG1 suggests that an effector is already present in or on the stem rust urediniospores when they are placed on the leaf surface. However, spores must be alive, as determined by their ability to germinate, in order to elicit RPG1 phosphorylation.

Proteolysis of the barley receptor-like protein kinase RPG1 by a proteasome pathway is correlated with Rpg1-mediated stem rust resistance.

In plants, disease resistance mediated by the gene-for-gene mechanism involves the recognition of specific effector molecules produced by the pathogen either directly or indirectly by the resistance-gene products. This recognition triggers a series of signals, thereby serving as a molecular switch in regulating defense mechanisms by the plants. To understand the mechanism of action of the barley stem rust resistance gene Rpg1, we investigated the fate of the RPG1 protein in response to infection with the stem rust fungus, Puccinia graminis f. sp. tritici. The investigations revealed that RPG1 disappears to undetectable limits only in the infected tissues in response to avirulent, but not virulent pathotypes. The RPG1 protein disappearance is rapid and appears to be due to specific protein degradation via the proteasome-mediated pathway as indicated by inhibition with the proteasomal inhibitor MG132, but not by other protease inhibitors.

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

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.