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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.
Stem rust (SR) resistance is required for CIMMYT durum germplasm to keep relevance in Ethiopia, where Ug99 and other Pgt races are a major yield-limiting constraint, and in countries along the possible dissemination paths of these races. Resistance to Ug99 is widespread in most durum germplasm groups when tested in Kenya, but resistance is lost when exposed to Ethiopian races; hence selection at the Debre Zeit site in Ethiopia is essential for durum wheat. Due to difficulties with shuttling segregating populations between Mexico and Ethiopia, we have adopted a strategy involving the identification of resistant/moderately resistant lines at Debre- Zeit, and inter-crossing in Mexico followed by selection for resistance to leaf rust and agronomic type and finally screening for SR reaction in the resulting F6 lines at Debre-Zeit at the same time as they are tested for yield and quality in preliminary yield trials in Mexico. This has generated a significant increase in the proportion of resistant and moderately resistant genotypes within outgoing CIMMYT germplasm, from less than 3% at the onset of the initiative in 2008 to 16% in 2011, and 38% in 2013. SR-resistant germplasm was characterized by similar frequency distributions to other traits in the overall germplasm such as yield potential, drought tolerance and industrial quality parameters. Advances have also been realized using marker-assisted selection (MAS) to introgress Sr22 from bread wheat and to combine it with Sr25, producing advanced lines with 2-gene stacks with confirmed outstanding resistance and superior quality attributes. Since the two genes are closely linked but from different sources bringing them together required a very rare recombination event finally detected via MAS among thousands of plants. They are now essentially inherited together with a very low likelihood of generating recombinant individuals with either gene. The yield potential and stability of these lines are under evaluation in Ethiopia and the best lines are being used in a second round of breeding.
The Lr34/Yr18 gene has been used in agriculture for more than 100 years. In contrast to many other resistance sources against leaf rust and stripe rust, it has remained effective and no virulence has been reported. This makes Lr34 a unique and highly valuable resource for rust resistance breeding. The pleiotropic nature of the gene conferring partial resistance to different pathogen species, the associated leaf tip necrosis and its durability suggest a molecular mechanism that is different from major gene resistance. This is supported by the molecular nature of Lr34 which was recently found to encode an ABC transporter. Interestingly, all tested wheat lines contain an allele of the Lr34 gene on chromosome 7DS. In its susceptible form, the gene does not confer resistance. The difference between the encoded resistant and susceptible LR34 isoforms consists of only two amino acid changes, whereas the rest of the proteins are identical. These two changes must change the biochemical properties of the resistant LR34 transporter in such a way that the plant becomes resistant. We speculate that there is a slight conformational change in the resistant form of the protein, resulting either in modified specificity or kinetics of the transported molecule, or that the binding properties to an unknown second protein interacting with LR34 are changed, resulting in altered function. While the molecular nature of the molecule(s) transported by the LR34 protein remains unclear, it is likely that a physiological change related to Lr34 activity is at the basis of resistance. We are currently establishing transgenic approaches in heterologous grass species to further investigate the molecular activity of Lr34 and to better understand a physiological mechanisms resulting in disease resistance.
Stem rust resistance gene Sr43, derived from tall wheatgrass (Thinopyrum ponticum), is effective against Ug99 lineage Pgt races. Previous studies indicated that Sr43 was located on large Th. ponticum 7el2 chromosome segments in 7D/7el2 translocation stocks KS10-2 and KS24-1. In the present work, we applied a recently-established chromosome engineering procedure to reduce the size of the alien chromosome carrying Sr43. KS10-2 was crossed and backcrossed to the Chinese Spring (CS) ph1b mutant. BC1F1 plants were screened for stem rust response and Ph1- associated molecular markers. Resistant BC1F1 plants homozygous ph1bph1b were further backcrossed to CS. The resulting population of 706 BC2F1 plants was screened for stem rust response and with six co-dominant SSR markers. Wheat lines RWG33 and RWG34 carry Sr43 on shortened alien segments that are about 15% of that in KS10-2. Two molecular markers closely linked to Sr43 were identified; one was an SSR marker and the other a STS marker based on sequences of deletion bin-mapped expressed sequenced tags in wheat. The two new wheat lines with Sr43 and closely-linked markers may provide new resources for combating the threat of race Ug99 and derivatives.
Stem rust, caused by Puccinia graminis f. sp. tritici, is a devastating disease on wheat and barley. A single barley gene, Rpg1, has provided durable resistance since its commercial introduction in the 1940s. The cloned Rpg1 gene encodes a protein with two tandem protein kinase domains, one an active kinase (pK2) and one a pseudokinase (pK1). Function of both domains is required for resistance. The gene is constitutively expressed in all tissues with elevated levels in the epidermis. It is mostly cytoplasmic with small, but significant amount associated with the cell membrane. We have been studying this gene and protein to try to understand how it works and why it has been so durable. Here we report our most recent results showing that RPG1 is phosphorylated within 5 min after urediniospores from avirulent, but not virulent, races land on the leaf surface. Two effector proteins were isolated from the ungerminated spores and shown to work cooperatively to induce RPG1 phosphorylation and eventual degradation. The proteins were identified as a hypothetical protein (PGTG10537.2) with a fibronectin type III and BRCA1 C-terminal domains and vacuolar protein sortingassociated protein 9 (PGTG_16791). The rapidity of the effector function and the nature of the two protein effectors indicate that a unique mechanism for effector entry and signaling in the host cell is involved. This hypothetical mechanism may be similar to what is observed in animal cells where fibronectin proteins with an RGD-binding domain act to mediate communications between the extracellular matrix and plasma membrane.
The common barberry and several other Berberis spp. serve as the alternate hosts to two important rust pathogens of small grains and grasses, Puccinia graminis and P. striiformis. Barberry eradication has been practiced for centuries as a means to control stem rust. Diverse virulence variations have been observed in populations of P. graminis f. sp. tritici that were associated with susceptible barberries in North America. Barberry likely has played a role in generating new races of P. striiformis f. sp. tritici in some regions in the world. Several North American stem rust races, namely races 56, 15B and QCC, initially originated from barberry, were subsequently responsible for generating large-scale epidemics. Thus, sexual cycles on Berberis spp. may generate virulence combinations that could have serious consequences to cereal crop production.
Limited but targeted stem rust race characterization was undertaken in Kenya in 2004 and 2005 which led to the detection of Ug99 present in Kenya and designation of Ug99 as race TTKS (based on North American stem rust race nomenclature system). Further surveillance in 2006 and 2007 detected variants of TTKS with virulence on Sr24 (TTKST) and Sr36 (TTTSK), respectively. Stem rust surveillance was undertaken at an extended level in 2008 and 2009 within predominant wheat growing regions of Kenya. Three hundred and sixty farms were surveyed from regional districts of Naivasha, Narok, Nakuru, Laikipia, Meru, Uasin-Gishu, Nandi, Elgeyo and Trans-Nzioa, during 2008 main season (May to September and December). The information from farmers indicated that more than 95% of these farms were sprayed with fungicides. Despite the use of fungicides, stem rust was detected in 67% of the surveyed farms. Stem rust ranged from trace amount -100% in severity with minimum infection in Naivasha district (40%) and maximum in Narok district (90%). Yellow rust was detected in 22% of the farms. Out of one hundred and twenty-six stem rust samples collected, 37 and 39 (a total of 76 ) samples were sent to Cereal Disease Laboratory (CDL) Minnesota, USA and Cereal Research Laboratory of Agriculture and Agri-Food Canada respectively, for race typing using the respective differentials used by these labs. From the 39 collections sent to Canada, 17 (43%) survived, of which majority were typed to TTKST (65%) followed by TTKSK (18%), PTKST (12%) and mixture of TTKST and TTKSK (5%). The CDL typed vast majority of pathotypes as TTKSK (84%) followed by TTKST and TTTSK (7% each). The combined results of two labs indicated that predominant frequency in Kenya in 2008 was TTKSK (51%) followed by TTKST (31%), PTKST (6%) and TTTSK (6%). The frequency of TTKST significantly increased in 2008 compared to 2007 which is not surprising, given that Sr24 carrying wheat cultivar KS Mwamba is cultivated on large acreage in Kenya. In 2009, 262 farms were surveyed from regional districts of Narok, Laikipia, Nyandarua, Meru, Uasin-Gishu, Nandi, Elgeyo and Trans-Nzioa. The 2009 season experienced heavy drought in many areas. Nevertheless, stem rust was detected in 79% of the farms with disease severity ranging from trace to 100%. Yellow rust was detected in 15% of the farms. Stem rust infection ranged from 0 to 100% with minimum infection in Nyandarua (18%), Laikipia (42%) and maximum in Uasin-Gishu and Elgeyo (100% each). Out of seventy-four stem rust samples collected, 55 samples were sent to Canada for race typing. Only 20% of the samples survived, of which majority were typed to TTKST (50%), PTKST (34%) and PTKSK (16%). Borlaug Global Rust Initiative 2010 Technical Workshop / Poster Abstracts 7 The 2009 results did not depict real situation of predominance of pathogenic variability because of small sample size, however it provided fair indication that race TTKST is still the most prevalent. This information generated on the distribution of stem rust races, and the incidence of stem rust is important for anticipatory breeding and release of cultivars with effective sources of resistance in Kenya, and at same time mitigating global threat of stem rust by reducing intensity of stem rust inoculum in East Africa.
High quality molecular markers that are closely linked, codominant, and high throughput are critical for developing varieties with durable rust resistance. We are using a combination of microsatellite, sequence tagged site, and Diversity Array Technology markers for haplotyping, pyramiding, and mapping stem rust resistance genes. The primary goal of our research team is to identify and optimize markers for previously characterized and novel stem rust resistance genes in wheat. The specific objectives are to: 1) optimize markers for previously characterized stem rust resistance genes to maximize efficiency of the breeding programs, 2) haplotype uncharacterized rust resistant genotypes to infer novelty and to plan new mapping experiments, 3) pyramid novel sources of rust resistance, and 4) map novel sources of rust resistance, including adult plant resistance. To date, we have evaluated 58 markers associated with 21 stem rust resistance genes and used 20 for haplotyping 318 wheat lines and varieties for 15 Ug99 effective resistance genes. This germplasm panel is also being DArT genotyped. For tetraploids, the pyramiding includes Sr2, Sr13 and Sr25 in the breeding line UC1113 which is a high yielding semi-dwarf durum variety with the high-grain protein content gene Gpc-B1 and the non-race specific stripe rust resistance gene Yr36. The Australian group is developing markers for the stem rust resistance genes Sr33 and Sr45 that come from Aegilops tauschii and are located on wheat chromosomes 1DS. Diagnostic, codominant markers for Sr25 and Sr26 have been developed and are being pyramided into CIMMYT breeding lines. Three new sources of race-specific resistance in CIMMYT-derived spring wheat have been mapped and are designated SrA, SrB, and SrC. SrA mapped on 3DL, SrB on 3BS and SrC on 5DL. These genes provided moderate levels of resistance to stem rust at the seedling stage and acceptable to moderate levels at the adult plant stage.
The East Africa program of the Borlaug Global Rust Initiative (BGRI) was launched to reduce the scale and scope of wheat stem rust epidemics in Kenya and Ethiopia, and to mitigate the global threat of virulent and dangerous rust races originating from this region. Since the launch in 2005, the screening facilities in Kenya and Ethiopia have helped to determine the extent of the world’s vulnerability to stem rust race Ug99 and its variants, identify diverse sources of resistance including adult plant resistance based on minor genes, and catalyze a comprehensive global response, leading to expanded awareness, expanded research and breeding activities, and resource mobilization. This paper reviews the role and achievements of the eastern African screening facilities along with the opportunities and challenges faced by the facilities during the ongoing global response to the emergence of Ug99 and its variants.
A number of stem rust resistance genes derived from wild relatives of wheat appeared to be more effective against race TTKSK (Ug99) of Puccinia graminis f. sp. tritici than Sr genes of wheat origin. In an attempt to identify sources of stem rust resistance genes effective against TTKSK, we evaluated several cultivated and wild relatives of wheat for resistance to TTKSK and other stem rust races with broad virulence in seedling tests. Preliminary results indicated that TTKSK resistance could readily be found, but frequencies of resistance varied among the species. Aegilops speltoides had the highest frequency of resistance (nearly 100%). Other species having high frequencies of TTKSK resistance included triticale (77.7% of 567 accessions), Triticum urartu (96.8% of 205 accessions), and T. monococcum (61% of 1020 accessions). Frequencies of TTKSK resistance in other species were: 14.7% in Ae. tauschii (456 accessions), 15% in T. timopheevii (298 accessions), and 17% in T. turgidum ssp. dicoccoides (157 accessions). Based on specific infection types to several races, we postulated that novel genes for resistance to TTKSK are present in some of these species. Accessions with putatively new resistance genes were selected to develop crosses for introgressing resistance into wheat and for developing mapping populations.