Dissecting the causal polymorphism of the Lr67res multipathogen resistance gene.

Partial resistance to multiple biotrophic fungal pathogens in wheat (Triticum aestivum L.) is conferred by a variant of the Lr67 gene, which encodes a hexose-proton symporter. Two mutations (G144R, V387L) differentiate the resistant and susceptible protein variants (Lr67res and Lr67sus). Lr67res lacks sugar transport capability and was associated with anion transporter-like properties when expressed in Xenopus laevis oocytes. Here, we extended this functional characterization to include yeast and in planta studies. The Lr67res allele, but not Lr67sus, induced sensitivity to ions in yeast (including NaCl, LiCl, KI), which is consistent with our previous observations that Lr67res expression in oocytes induces novel ion fluxes. We demonstrate that another naturally occurring single amino acid variant in wheat, containing only the Lr67G144R mutation, confers rust resistance. Transgenic barley plants expressing the orthologous HvSTP13 gene carrying the G144R and V387L mutations were also more resistant to Puccinia hordei infection. NaCl treatment of pot-grown adult wheat plants with the Lr67res allele induced leaf tip necrosis and partial leaf rust resistance. An Lr67res-like function can be introduced into orthologous plant hexose transporters via single amino acid mutation, highlighting the strong possibility of generating disease resistance in other crops, especially with gene editing.

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.

A pathogen-induced putative NAC transcription factor mediates leaf rust resistance in barley.

Leaf rust, caused by Puccinia hordei, is one of the most widespread and damaging foliar diseases affecting barley. The barley leaf rust resistance locus Rph7 has been shown to have unusually high sequence and haplotype divergence. In this study, we isolate the Rph7 gene using a fine mapping and RNA-Seq approach that is confirmed by mutational analysis and transgenic complementation. Rph7 is a pathogen-induced, non-canonical resistance gene encoding a protein that is distinct from other known plant disease resistance proteins in the Triticeae. Structural analysis using an AlphaFold2 protein model suggests that Rph7 encodes a putative NAC transcription factor with a zinc-finger BED domain with structural similarity to the N-terminal DNA-binding domain of the NAC transcription factor (ANAC019) from Arabidopsis. A global gene expression analysis suggests Rph7 mediates the activation and strength of the basal defence response. The isolation of Rph7 highlights the diversification of resistance mechanisms available for engineering disease control in crops.

Expression of the wheat multipathogen resistance hexose transporter Lr67res is associated with anion fluxes.

Many disease resistance genes in wheat (Triticum aestivum L.) confer strong resistance to specific pathogen races or strains, and only a small number of genes confer multipathogen resistance. The Leaf rust resistance 67 (Lr67) gene fits into the latter category as it confers partial resistance to multiple biotrophic fungal pathogens in wheat and encodes a Sugar Transport Protein 13 (STP13) family hexose-proton symporter variant. Two mutations (G144R, V387L) in the resistant variant, Lr67res, differentiate it from the susceptible Lr67sus variant. The molecular function of the Lr67res protein is not understood, and this study aimed to broaden our knowledge on this topic. Biophysical analysis of the wheat Lr67sus and Lr67res protein variants was performed using Xenopus laevis oocytes as a heterologous expression system. Oocytes injected with Lr67sus displayed properties typically associated with proton-coupled sugar transport proteins-glucose-dependent inward currents, a Km of 110 ± 10 µM glucose, and a substrate selectivity permitting the transport of pentoses and hexoses. By contrast, Lr67res induced much larger sugar-independent inward currents in oocytes, implicating an alternative function. Since Lr67res is a mutated hexose-proton symporter, the possibility of protons underlying these currents was investigated but rejected. Instead, currents in Lr67res oocytes appeared to be dominated by anions. This conclusion was supported by electrophysiology and 36Cl- uptake studies and the similarities with oocytes expressing the known chloride channel from Torpedo marmorata, TmClC-0. This study provides insights into the function of an important disease resistance gene in wheat, which can be used to determine how this gene variant underpins disease resistance in planta.

Stripe rust and leaf rust resistance in CIMMYT wheat line "Mucuy" is conferred by combinations of race-specific and adult-plant resistance loci.

Developing wheat varieties with durable resistance is a core objective of the International Maize and Wheat Improvement Center (CIMMYT) and many other breeding programs worldwide. The CIMMYT advanced wheat line "Mucuy" displayed high levels of resistance to stripe rust (YR) and leaf rust (LR) in field evaluations in Mexico and several other countries. To determine the genetic basis of YR and LR resistance, 138 F5 recombinant inbred lines (RILs) derived from the cross of Apav#1× Mucuy were phenotyped for YR responses from 2015 to 2020 at field sites in India, Kenya, and Mexico, and LR in Mexico. Seedling phenotyping for YR and LR responses was conducted in the greenhouse in Mexico using the same predominant races as in field trials. Using 12,681 polymorphic molecular markers from the DArT, SNP, and SSR genotyping platforms, we constructed genetic linkage maps and QTL analyses that detected seven YR and four LR resistance loci. Among these, a co-located YR/LR resistance loci was identified as Yr29/Lr46, and a seedling stripe rust resistance gene YrMu was mapped on the 2AS/2NS translocation. This fragment also conferred moderate adult plant resistance (APR) under all Mexican field environments and in one season in Kenya. Field trial phenotyping with Lr37-virulent Puccinia triticina races indicated the presence of an APR QTL accounting for 18.3-25.5% of the LR severity variation, in addition to a novel YR resistance QTL, QYr.cim-3DS, derived from Mucuy. We developed breeder-friendly KASP and indel molecular markers respectively for Yr29/Lr46 and YrMu. The current study validated the presence of known genes and identified new resistance loci, a QTL combination effect, and flanking markers to facilitate accelerated breeding for genetically complex, durable rust resistance.

Haplotype variants of the stripe rust resistance gene Yr28 in Aegilops tauschii.

KEY MESSAGE: Stripe rust resistance gene YrAet672 from Aegilops tauschii accession CPI110672 encodes a nucleotide-binding and leucine-rich repeat domain containing protein similar to YrAS2388 and both these members were haplotypes of Yr28. New sources of host resistance are required to counter the continued emergence of new pathotypes of the wheat stripe rust pathogen Puccinia striiformis Westend. f. sp. tritici Erikss. (Pst). Here, we show that CPI110672, an Aegilops tauschii accession from Turkmenistan, carries a single Pst resistance gene, YrAet672, that is effective against multiple Pst pathotypes, including the four predominant Pst lineages present in Australia. The YRAet672 locus was fine mapped to the short arm of chromosome 4D, and a nucleotide-binding and leucine-rich repeat gene was identified at the locus. A transgene encoding the YrAet672 genomic sequence, but lacking a copy of a duplicated sequence present in the 3' UTR, was transformed into wheat cultivar Fielder and Avocet S. This transgene conferred a weak resistance response, suggesting that the duplicated 3' UTR region was essential for function. Subsequent analyses demonstrated that YrAet672 is the same as two other Pst resistance genes described in Ae. tauschii, namely YrAS2388 and Yr28. They were identified as haplotypes encoding identical protein sequences but are polymorphic in non-translated regions of the gene. Suppression of resistance conferred by YrAet672 and Yr28 in synthetic hexaploid wheat lines (AABBDD) involving Langdon (AABB) as the tetraploid parent was associated with a reduction in transcript accumulation.

Identification and Characterization of Resistance Loci to Wheat Leaf Rust and Stripe Rust in Afghan Landrace "KU3067".

Leaf rust and stripe rust are important wheat diseases worldwide causing significant losses where susceptible varieties are grown. Resistant cultivars offer long-term control and reduce the use of hazardous chemicals, which can be detrimental to both human health and the environment. Land races have been a valuable resource for mining new genes for various abiotic and biotic stresses including wheat rusts. Afghan wheat landrace "KU3067" displayed high seedling infection type (IT) for leaf rust and low IT for stripe rust; however, it displayed high levels of field resistance for both rusts when tested for multiple seasons against the Mexican rust isolates. This study focused on identifying loci-conferring seedling resistance to stripe rust, and also loci-conferring adult plant resistance (APR) against the Mexican races of leaf rust and stripe rust. A backcrossed inbred line (BIL) population advanced to the BC1F5 generation derived from the cross of KU3067 and Apav (triple rust susceptible line) was used for both, inheritance and QTL mapping studies. The population and parents were genotyped with Diversity Arrays Technology-genotyping-by-sequencing (DArT-Seq) and phenotyped for leaf rust and stripe rust response at both seedling and adult plant stages during multiple seasons in Mexico with relevant pathotypes. Mapping results identified an all-stage resistance gene for stripe rust, temporarily designated as YrKU, on chromosome 7BL. In total, six QTL-conferring APR to leaf rust on 1AS, 2AL, 4DL, 6BL, 7AL, and 7BL, and four QTL for stripe rust resistance on 1BS, 2AL, 4DL, and 7BL were detected in the analyses. Among these, pleiotropic gene Lr67/Yr46 on 4DL with a significantly large effect is the first report in an Afghan landrace-conferring resistance to both leaf and stripe rusts. QLr.cim-7BL/YrKU showed pleiotropic resistance to both rusts and explained 7.5-17.2 and 12.6-19.3% of the phenotypic variance for leaf and stripe rusts, respectively. QYr.cim-1BS and QYr.cim-2AL detected in all stripe environments with phenotypic variance explained (PVE) 12.9-20.5 and 5.4-12.5%, and QLr.cim-6BL are likely to be new. These QTL and their closely linked markers will be useful for fine mapping and marker-assisted selection (MAS) in breeding for durable resistance to multiple rust diseases.

Haplotype variants of Sr46 in Aegilops tauschii, the diploid D genome progenitor of wheat.

KEY MESSAGE: Stem rust resistance genes, SrRL5271 and Sr672.1 as well as SrCPI110651, from Aegilops tauschii, the diploid D genome progenitor of wheat, are sequence variants of Sr46 differing by 1-2 nucleotides leading to non-synonymous amino acid substitutions. The Aegilops tauschii (wheat D-genome progenitor) accessions RL 5271 and CPI110672 were identified as resistant to multiple races (including the Ug99) of the wheat stem rust pathogen Puccinia graminis f. sp. tritici (Pgt). This study was conducted to identify the stem rust resistance (Sr) gene(s) in both accessions. Genetic analysis of the resistance in RL 5271 identified a single dominant allele (SrRL5271) controlling resistance, whereas resistance segregated at two loci (SR672.1 and SR672.2) for a cross of CPI110672. Bulked segregant analysis placed SrRL5271 and Sr672.1 in a region on chromosome arm 2DS that encodes Sr46. Molecular marker screening, mapping and genomic sequence analysis demonstrated SrRL5271 and Sr672.1 are alleles of Sr46. The amino acid sequence of SrRL5271 and Sr672.1 is identical but differs from Sr46 (hereafter referred to as Sr46_h1 by following the gene nomenclature in wheat) by a single amino acid (N763K) and is thus designated Sr46_h2. Screening of a panel of Ae. tauschii accessions identified an additional allelic variant that differed from Sr46_h2 by a different amino acid (A648V) and was designated Sr46_h3. By contrast, the protein encoded by the susceptible allele of Ae. tauschii accession AL8/78 differed from these resistance proteins by 54 amino acid substitutions (94% nucleotide sequence gene identity). Cloning and complementation tests of the three resistance haplotypes confirmed their resistance to Pgt race 98-1,2,3,5,6 and partial resistance to Pgt race TTRTF in bread wheat. The three Sr46 haplotypes, with no virulent races detected yet, represent a valuable source for improving stem resistance in wheat.

Adult plant stem rust resistance in durum wheat Glossy Huguenot: mapping, marker development and validation.

KEY MESSAGE: Adult plant stem rust resistance locus, QSrGH.cs-2AL, was identified in durum wheat Glossy Huguenot and mendelised as Sr63. Markers closely linked with Sr63 were developed. An F3 population from a Glossy Huguenot (GH)/Bansi cross used in a previous Australian study was advanced to F6 for molecular mapping of adult plant stem rust resistance. Maturity differences among F6 lines confounded assessments of stem rust response. GH was crossed with a stem rust susceptible F6 recombinant inbred line (RIL), GHB14 (M14), with similar maturity and an F6:7 population was developed through single seed descent method. F7 and F8 RILs were tested along with the parents at different locations. The F6 individual plants and both parents were genotyped using the 90 K single nucleotide polymorphism (SNP) wheat array. Stem rust resistance QTL on the long arms of chromosomes 1B (QSrGH.cs-1BL) and 2A (QSrGH.cs-2AL) were detected. QSrGH.cs-1BL and QSrGH.cs-2AL were both contributed by GH and explained 22% and 18% adult plant stem rust response variation, respectively, among GH/M14 RIL population. RILs carrying combinations of these QTL reduced more than 14% stem rust severity compared to those that possessed QSrGH.cs-1BL and QSrGH.cs-2AL individually. QSrGH.cs1BL was demonstrated to be the same as Sr58/Lr46/Yr29/Pm39 through marker genotyping. Lines lacking QSrGH.cs-1BL were used to Mendelise QSrGH.cs-2AL. Based on genomic locations of previously catalogued stem rust resistance genes and the QSrGH.cs-2AL map, it appeared to represent a new APR locus and was permanently named Sr63. SNP markers associated with Sr63 were converted to kompetetive allele-specific PCR (KASP) assays and were validated on a set of durum cultivars.

Characterization of synthetic wheat line Largo for resistance to stem rust.

Resistance breeding is an effective approach against wheat stem rust caused by Puccinia graminis f. sp. tritici (Pgt). The synthetic hexaploid wheat line Largo (pedigree: durum wheat "Langdon" × Aegilops tauschii PI 268210) was found to have resistance to a broad spectrum of Pgt races including the Ug99 race group. To identify the stem rust resistance (Sr) genes, we genotyped a population of 188 recombinant inbred lines developed from a cross between the susceptible wheat line ND495 and Largo using the wheat Infinium 90 K SNP iSelect array and evaluated the population for seedling resistance to the Pgt races TTKSK, TRTTF, and TTTTF in the greenhouse conditions. Based on genetic linkage analysis using the marker and rust data, we identified six quantitative trait loci (QTL) with effectiveness against different races. Three QTL on chromosome arms 6AL, 2BL, and 2BS corresponded to Sr genes Sr13c, Sr9e, and a likely new gene from Langdon, respectively. Two other QTL from PI 268210 on 2DS and 1DS were associated with a potentially new allele of Sr46 and a likely new Sr gene, respectively. In addition, Sr7a was identified as the underlying gene for the 4AL QTL from ND495. Knowledge of the Sr genes in Largo will help to design breeding experiments aimed to develop new stem rust-resistant wheat varieties. Largo and its derived lines are particularly useful for introducing two Ug99-effective genes Sr13c and Sr46 into modern bread wheat varieties. The 90 K SNP-based high-density map will be useful for identifying the other important genes in Largo.

A recombined Sr26 and Sr61 disease resistance gene stack in wheat encodes unrelated NLR genes.

The re-emergence of stem rust on wheat in Europe and Africa is reinforcing the ongoing need for durable resistance gene deployment. Here, we isolate from wheat, Sr26 and Sr61, with both genes independently introduced as alien chromosome introgressions from tall wheat grass (Thinopyrum ponticum). Mutational genomics and targeted exome capture identify Sr26 and Sr61 as separate single genes that encode unrelated (34.8%) nucleotide binding site leucine rich repeat proteins. Sr26 and Sr61 are each validated by transgenic complementation using endogenous and/or heterologous promoter sequences. Sr61 orthologs are absent from current Thinopyrum elongatum and wheat pan genome sequences, contrasting with Sr26 where homologues are present. Using gene-specific markers, we validate the presence of both genes on a single recombinant alien segment developed in wheat. The co-location of these genes on a small non-recombinogenic segment simplifies their deployment as a gene stack and potentially enhances their resistance durability.

BED domain-containing NLR from wild barley confers resistance to leaf rust.

Leaf rust, caused by Puccinia hordei, is a devastating fungal disease affecting barley (Hordeum vulgare subsp. vulgare) production globally. Despite the effectiveness of genetic resistance, the deployment of single genes often compromises durability due to the emergence of virulent P. hordei races, prompting the search for new sources of resistance. Here we report on the cloning of Rph15, a resistance gene derived from barley's wild progenitor H. vulgare subsp. spontaneum. We demonstrate using introgression mapping, mutation and complementation that the Rph15 gene from the near-isogenic line (NIL) Bowman + Rph15 (referred to as BW719) encodes a coiled-coil nucleotide-binding leucine-rich repeat (NLR) protein with an integrated Zinc finger BED (ZF-BED) domain. A predicted KASP marker was developed and validated across a collection of Australian cultivars and a series of introgression lines in the Bowman background known to carry the Rph15 resistance. Rph16 from HS-680, another wild barley derived leaf rust resistance gene, was previously mapped to the same genomic region on chromosome 2H and was assumed to be allelic with Rph15 based on genetic studies. Both sequence analysis, race specificity and the identification of a knockout mutant in the HS-680 background suggest that Rph15- and Rph16-mediated resistances are in fact the same and not allelic as previously thought. The cloning of Rph15 now permits efficient gene deployment and the production of resistance gene cassettes for sustained leaf rust control.

A five-transgene cassette confers broad-spectrum resistance to a fungal rust pathogen in wheat.

Breeding wheat with durable resistance to the fungal pathogen Puccinia graminis f. sp. tritici (Pgt), a major threat to cereal production, is challenging due to the rapid evolution of pathogen virulence. Increased durability and broad-spectrum resistance can be achieved by introducing more than one resistance gene, but combining numerous unlinked genes by breeding is laborious. Here we generate polygenic Pgt resistance by introducing a transgene cassette of five resistance genes into bread wheat as a single locus and show that at least four of the five genes are functional. These wheat lines are resistant to aggressive and highly virulent Pgt isolates from around the world and show very high levels of resistance in the field. The simple monogenic inheritance of this multigene locus greatly simplifies its use in breeding. However, a new Pgt isolate with virulence to several genes at this locus suggests gene stacks will need strategic deployment to maintain their effectiveness.

A highly differentiated region of wheat chromosome 7AL encodes a Pm1a immune receptor that recognizes its corresponding AvrPm1a effector from Blumeria graminis.

Pm1a, the first powdery mildew resistance gene described in wheat, is part of a complex resistance (R) gene cluster located in a distal region of chromosome 7AL that has suppressed genetic recombination. A nucleotide-binding, leucine-rich repeat (NLR) immune receptor gene was isolated using mutagenesis and R gene enrichment sequencing (MutRenSeq). Stable transformation confirmed Pm1a identity which induced a strong resistance phenotype in transgenic plants upon challenge with avirulent Blumeria graminis (wheat powdery mildew) pathogens. A high-density genetic map of a B. graminis family segregating for Pm1a avirulence combined with pathogen genome resequencing and RNA sequencing (RNAseq) identified AvrPm1a effector gene candidates. In planta expression identified an effector, with an N terminal Y/FxC motif, that induced a strong hypersensitive response when co-expressed with Pm1a in Nicotiana benthamiana. Single chromosome enrichment sequencing (ChromSeq) and assembly of chromosome 7A suggested that suppressed recombination around the Pm1a region was due to a rearrangement involving chromosomes 7A, 7B and 7D. The cloning of Pm1a and its identification in a highly rearranged region of chromosome 7A provides insight into the role of chromosomal rearrangements in the evolution of this complex resistance cluster.

The wheat Sr22, Sr33, Sr35 and Sr45 genes confer resistance against stem rust in barley.

In the last 20 years, stem rust caused by the fungus Puccinia graminis f. sp. tritici (Pgt), has re-emerged as a major threat to wheat and barley production in Africa and Europe. In contrast to wheat with 60 designated stem rust (Sr) resistance genes, barley's genetic variation for stem rust resistance is very narrow with only ten resistance genes genetically identified. Of these, only one complex locus consisting of three genes is effective against TTKSK, a widely virulent Pgt race of the Ug99 tribe which emerged in Uganda in 1999 and has since spread to much of East Africa and parts of the Middle East. The objective of this study was to assess the functionality, in barley, of cloned wheat Sr genes effective against race TTKSK. Sr22, Sr33, Sr35 and Sr45 were transformed into barley cv. Golden Promise using Agrobacterium-mediated transformation. All four genes were found to confer effective stem rust resistance. The barley transgenics remained susceptible to the barley leaf rust pathogen Puccinia hordei, indicating that the resistance conferred by these wheat Sr genes was specific for Pgt. Furthermore, these transgenic plants did not display significant adverse agronomic effects in the absence of disease. Cloned Sr genes from wheat are therefore a potential source of resistance against wheat stem rust in barley.

Adult Plant Slow Rusting Genes Confer High Levels of Resistance to Rusts in Bread Wheat Cultivars From Mexico.

Rust diseases continuously threaten global wheat production: stem rust, leaf rust, and yellow rust caused by Puccinia graminis f. sp. tritici, Puccinia triticina, and Puccinia striiformis f. sp. tritici, respectively. Recent studies indicated that the average losses from all these three rusts reached up to 15.04 million tons per year, which is equivalent to an annual average loss of around US $2.9 billion per year. The major focus of Mexican and worldwide breeding programs is the release of rust resistant cultivars, as this is considered the best option for controlling rust diseases. In Mexico, the emphasis has been placed on genes that confer partial resistance in the adult plant stage and against a broad spectrum of rust races since the 1970s. In this study, a set of the first-generation tall varieties developed and released in the 1940s and 1950s, the first semi-dwarfs, and other releases in Mexico, all of which showed different levels of rust resistance have been phenotyped for the three rust diseases and genotyped. Results of the molecular marker detection indicated that Lr34, Lr46, Lr67, and Lr68 alone or in different gene combinations were present among the wheat cultivars. Flag leaf tip necrosis was present in all cultivars and most were positive for brown necrosis or Pseudo Black Chaff associated with the Sr2 stem rust resistance complex. The phenotypic responses to the different rust infections indicate the presence of additional slow rusting and race-specific resistance genes. The study reveals the association of the slow rusting genes with durable resistance to the three rusts including Ug99 in cultivars bred before the green revolution such as Frontera, Supremo 211, Chapingo 48, Yaqui 50, Kentana 52, Bajio 52, Bajio 53, Yaqui 53, Chapingo 53, Yaktana Tardio 54, and Mayo 54 and their descendants after intercrossing and recombination. These slow rusting genes are the backbone of the resistance in the current Mexican germplasm.

Extensive Genetic Variation at the Sr22 Wheat Stem Rust Resistance Gene Locus in the Grasses Revealed Through Evolutionary Genomics and Functional Analyses.

In the last 20 years, severe wheat stem rust outbreaks have been recorded in Africa, Europe, and Central Asia. This previously well controlled disease, caused by the fungus Puccinia graminis f. sp. tritici, has reemerged as a major threat to wheat cultivation. The stem rust (Sr) resistance gene Sr22 encodes a nucleotide-binding and leucine-rich repeat receptor which confers resistance to the highly virulent African stem rust isolate Ug99. Here, we show that the Sr22 gene is conserved among grasses in the Triticeae and Poeae lineages. Triticeae species contain syntenic loci with single-copy orthologs of Sr22 on chromosome 7, except Hordeum vulgare, which has experienced major expansions and rearrangements at the locus. We also describe 14 Sr22 sequence variants obtained from both Triticum boeoticum and the domesticated form of this species, T. monococcum, which have been postulated to encode both functional and nonfunctional Sr22 alleles. The nucleotide sequence analysis of these alleles identified historical sequence exchange resulting from recombination or gene conversion, including breakpoints within codons, which expanded the coding potential at these positions by introduction of nonsynonymous substitutions. Three Sr22 alleles were transformed into wheat cultivar Fielder and two postulated resistant alleles from Schomburgk (hexaploid wheat introgressed with T. boeoticum segment carrying Sr22) and T. monococcum accession PI190945, respectively, conferred resistance to P. graminis f. sp. tritici race TTKSK, thereby unequivocally confirming Sr22 effectiveness against Ug99. The third allele from accession PI573523, previously believed to confer susceptibility, was confirmed as nonfunctional against Australian P. graminis f. sp. tritici race 98-1,2,3,5,6.[Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.