We identify the wheat stem rust resistance gene Sr50 (using physical mapping, mutation and complementation) as homologous to barley Mla, encoding a coiled-coil nucleotide-binding leucine-rich repeat (CC-NB-LRR) protein. We show that Sr50 confers a unique resistance specificity different from Sr31 and other genes on rye chromosome 1RS, and is effective against the broadly virulent Ug99 race lineage. Extensive haplotype diversity at the rye Sr50 locus holds promise for mining effective resistance genes.
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The stem rust resistance genes Sr31 and Sr50 in wheat were both derived from translocations of the short arm of chromosome 1 from rye and conferred resistance to all field isolates of Puccinia graminis f. sp tritici (Pgt) for many years, preventing their distinction as different resistance specificities. We now show that Sr50 confers resistance against the Ug99 strain that overcomes Sr31, whereas a mutant Pgt strain virulent towards Sr50 is avirulent towards Sr31. Because lack of recombination between wheat and rye chromosome arms precludes genetic mapping and so map-based cloning of Sr50, we used a combination deletion mutagenesis and large DNA fragment cloning in bacterial artificial chromosome (BAC) vectors to define this resistance locus. Sequence analysis of a BAC contig spanning the smallest deletion detected with DNA markers at the Sr50 locus identified six coiled coil nucleotide binding site leucine-rich repeat (CC-NB-LRR) genes and a chymotrypsin inhibitor gene closely related to genes at the orthologous barley powdery mildew resistance locus, Mla. Sequencing of these genes from two EMS-induced mutants that had lost no DNA markers revealed mutation in one of the CC-NB-LRR orthologs of Mla. Transgenic complementation tests in stem rust susceptible wheat proved this gene to be Sr50. A survey of a set of rye accessions identified several carrying the gene but occurring in different Mla gene haplotypes based on DNA gel blot patterns and copy number of Mla orthologs. Several different powdery mildew and rust resistance genes including TmMla1 from T. monococcum, 23 Mla alleles from barley and stem rust resistance genes Sr33 from Aegilops tauschii and Sr50 from rye are all members of the Mla clade of cereal R genes. The gene Sr50,was initially thought to be allelic to Sr31, however, appearance of Ug99 showed that this is a different gene and is rye ortholog of barley Mla powdery mildew resistance gene. The cloning of Sr50 gives us an opportunity to screen the rye germplasm for presence of Sr50 and allows us to now do functional analysis of the various domains and understand the mechanism of resistance. The cloning also helps to add very effective resistance to gene cassette. Sr50 is effective against all the stem rust isolates around the globe
Stem rust caused by Puccinia graminis tritici (Pgt) is one of the most serious diseases in wheat and is combated mainly through the use of resistant varieties. Because the fungus evolves virulence towards previously resistant varieties, continuous breeding and identification of new sources of resistance are necessary to combat the threat of rust epidemics. Our work on the flax rust model system has provided insights into how the plant immune system recognises and responds to rust pathogens. We have been extending this work to wheat stem rust by targeted cloning of resistance (R) genes in wheat and corresponding Avr genes in Pgt. Plant R genes encode immune receptors that recognise and respond to pathogen effector proteins delivered into host cells from haustoria. We recently isolated the Sr33 and Sr50 resistance genes from wheat and have begun functional analyses to determine how they trigger defense responses. We are also targeting effectors from Pgt that are recognised by wheat R genes. We used genome and transcriptome sequencing to predict ~400 candidate effector genes from Australian Pgt race 21- 0. To screen for recognition of these proteins by wheat R genes, we developed a bacterial Type III Secretion System delivery assay using Pseudomonas fluorescens to inject the effector candidates into wheat leaf cells. We are screening candidate effectors on a set of 18 wheat cultivars carrying 22 different R genes and have so far identified one effector that induces a cell death response specifically on a wheat genotype carrying Sr22. Understanding the nature of wheat R genes and the Avr proteins that they recognize will allow better prediction of R gene durability and enable the possibility of rational design of novel R genes. We are also developing techniques for stacking R genes in cassettes for deployment of multiple genes at a single locus in wheat.
Chromosome I R of rye is a useful source of genes for disease resistance and enhanced agronomic performance in wheat. One of the most prevalent genes transferred to wheat from rye is the stein rust resistance gene Sr31. The recent emergence and spread of a stein rust pathotype virulent to this gene has refocused efforts to find and utilize alternative sources of resistance. There has been considerable effort to transfer a stem rust resistance gene, SrR, from Imperial rye, believed to be allelic to Sr31, into commercial wheat cultivars. However, the simultaneous transfer of genes at the Sec-1 locus encoding secalin seed storage proteins and their association with quality defects preclude the deployment of SrR in some commercial wheat breeding programs. Previous attempts to induce homoeologous recombination between wheat and rye chromosomes to break the linkage between SrR and Sec-1 whilst retaining the tightly linked major loci for wheat seed storage proteins, Gli-D1 and Glu-D3, and recover good dough quality characteristics, have been unsuccessful. We produced novel tertiary wheat-rye recombinant lines carrying different lengths of rye chromosome arm 1RS by inducing homoeologous recombination between the wheat 1D chromosome and a previously described secondary wheat-rye recombinant, DRA-1. Tertiary recombinant T6-1 (SrR+ Sec-1(-)) carries the target gene for stem rust resistance from rye and retains Gli-D1 but lacks the secalin locus. The tertiary recombinant T49-7 (SrR- Sec-1(+)) contains the secalin locus but lacks the stem rust resistance gene. T6-1 is expected to contribute to wheat breeding programs in Australia, whereas T49-7 provides opportunities to investigate whether the presence of secalins is responsible for the previously documented dough quality defects.
The short arm of rye (Secale cereale) chromosome 1 has been widely used in breeding programs to incorporate new disease resistance genes into wheat. Using wheat-rye translocation and recombinant lines, molecular markers were isolated and mapped within chromosomal regions of 1RS carrying rust resistance genes Lr26, Sr31, Yr9 from 'Petkus' and SrR from 'Imperial' rye. RFLP markers previously mapped to chromosome 1HS of barley - flanking the complex Mla powdery mildew resistance gene locus - and chromosome 1DS of Aegilops tauschii - flanking the Sr33 stem rust resistance gene - were shown to map on either side of rust resistance genes on 1RS. Three non cross-hybridising Resistance Gene Analog markers, one of them being derived from the Mla gene family, were mapped within same region of 1RS. PCR-based markers were developed which were tightly linked to the rust resistance genes in 'Imperial' and 'Petkus' rye and which have potential for use in marker-assisted breeding.