Wheat stem rust effectors: Genomics and functional assay
BGRI 2015 Poster Abstract Upadhyaya
CSIRO Agriculture, Canberra, Australia
Puccinia graminis f. sp. tritici (Pgt) is one of the most destructive pathogens of wheat. Fungal secreted proteins termed effectors play an important role in modulating the host cellular environment and suppressing the plant defense response to enable fungal growth. They also become targets of plant resistance (R) proteins. We have taken a genomics approach to initially identify candidate effectors. We have built a draft genome for a founder Australian Pgt isolate of pathotype (pt.) 21-0 (collected in 1954) by next generation DNA sequencing. A combination of reference-based assembly using the genome of the previously sequenced North American Pgt isolate CDL 75-36-700-3 (p7a) and de novo assembly resulted in a 92 Mbp reference genome for Pgt isolate 21-0. This draft genome was subsequently used to build a pan-genome based on five Australian Pgt isolates. Transcriptomes from germinated urediniospores and haustoria were separately assembled for pt. 21-0 and comparison of gene expression profiles showed differential expression in ~10% of the genes in germinated urediniospores as well as haustoria. A total of 1,924 secreted proteins were predicted from the 21-0 transcriptome, of which 586 were classified as haustorial secreted proteins (HSPs). We are currently exploring effector gene expression during infection of wheat to reduce this candidate list based on a common expression profile identified for Avr genes in the flax rust fungus. Comparison of 21-0 with two presumed clonal field derivatives (collected in 1982 and 1984) that had evolved virulence on four additional resistance genes (Sr5, Sr11, Sr27, SrSatu) identified mutations in 13 HSP effector candidates. These candidate effectors are being assessed for recognition in wheat accessions with the corresponding R genes using a bacterial type three secretion delivery system based on an engineered Pseudomonas fluorescence strain (Upadhyaya NM et al. Mol Plant Microbe Interact 27:255-264).
Targeting stem rust resistance genes Sr32 and Sr1644 for cloning by mutagenesis and sequence capture
BGRI 2015 Poster Abstract Wulff
John Innes Centre, UK
Resistance offers the best means of control of the cereal rusts, but must be strategically deployed so as to avoid exposure of single major genes, which have faltered so many times in the past. The pyramiding of multiple effective resistance genes is a strategy that has proven effective in a number of wheat production areas around the world. However, the process of incorporating multiple resistance genes into a single cultivar using standard breeding techniques is time consuming, laborious, and hampered by the problem of linkage drag. If a suite of effective resistance genes could be efficiently cloned and transferred into wheat as a cassette, it would accelerate the development of durably resistant varieties without the problem of linkage drag. Toward this end, we have developed a resistance gene cloning technology based on resistance gene enrichment sequencing (RenSeq) of EMS-derived mutant R gene alleles. As a proof of concept test, we successfully ‘re’-cloned the already characterized gene Sr33 and are now targeting the cloning of eight other effective resistance genes. For the identification of susceptible mutants for the cloning of Sr32 from Aegilops speltoides, we screened 1,109 M2 families with race TPMKC — as a surrogate for race TTKSK. Five susceptible M2 mutants were confirmed by progeny testing. These mutants were also susceptible to race TTKSK. For the population involving Sr1644 from Ae. sharonensis, 1,649 M2 families were screened, yielding 33 M2 families that appeared to segregate for susceptibility. Thirteen of 33 families were confirmed as bona fide susceptible mutants by progeny tests in the M3 generation. Identification of susceptible EMS mutants of Sr32 and Sr1644 suggests that the underlying resistance in these lines is conferred by single genes. We will report on progress to clone and characterize these genes using R gene exome capture and sequencing technology (RenSeq).
Introgression of genes for high grain protein content (Gpc-B1) and Lr24 into leading cultivars by marker assisted backcross breeding
BGRI 2015 Poster Abstract Mishra
Department of Genetics and Plant Breeding, Institute of Agricultural Sciences, Banaras Hindu University, India
A wheat genotype PBW343+Gpc-B1+LR24 containing the high grain protein content (GPC) gene Gpc-B1 linked to marker Xucw108 was used as the donor parent to transfer Gpc-B1 and Lr24 into Eastern Gangetic Plains (EGP) cv. HUW234 and HUW468 that were released in 1986 and 1999, respectively. The backcrossing program involved the following steps: (i) foreground selection, (ii) marker selection, and (iii) recovery of recipient parent genome. Grain protein contents were recorded for all selected plants from the BC2F2:3 generation. The dominant marker Xucw108 was used for foreground selection, and heterozygous plants were identified through progeny testing. For RPG recovery, both genotypic and phenotypic selection was used. Introgression of the high GPC gene into the recipient background without yield loss was completed in 5 years, starting from 2009-10 (F1) and completed in 2013-14 (BC2F5). A conventional selection program would take the same time to reach BC2F5 but ensuring the transfer of GPC would not not be possible. Ten selected single plants from the BC2F3:4 generation had comparable yields of the parents with 26% higher GPC than the recurrent parent HUW 234. Eight selected plants had comparable yields and 34% higher GPC than HUW 468. Multi-row progenies (BC2F4 and BC2F5) of each selected plant were evaluated in yield traits with the donor and recipient parents as controls during 2012-13 and 2013-14. Two lines based on each recurrent parent were identified with significantly higher GPC with no yield penalty. The study reinforced the belief that MAS in combination with phenotypic selection could be a useful strategy to develop high GPC genotypes without sacrificing grain yield. These lines will be submitted to national trial where MAS derived lines require only two years of testing compared to four years for conventionally bred lines.
Resistance to wheat stem rust in selected accessions of Iranian wheat landraces
BGRI 2015 Poster Abstract Mojerlou
Tarbiat Modares University of Tehran, Iran
View mojerlou.pdf(199.61 KB)
Stem rust is a potentially destructive fungal disease of wheat worldwide. In 1998 Pgt pathotype TTKSK virulent to Sr31 was detected in Uganda. The same pathotype was confirmed in Lorestan and Hamedan provinces of Iran in 2007. We used a derivative of race TTKSK to phenotype 62 Iranian wheat landraces (resistant to stripe rust in a previous study) at the seedling stage to this new pathotype (TTSSK). Twenty eight accessions were evaluated for the presence of resistance genes Sr2, Sr22, Sr24, Sr25, Sr26, Sr35, Sr36 and Srweb using SSR markers. None carried Sr2, Sr24 or Sr26, but the presence of Sr22, Sr25, Sr35 and Sr36 was indicated. Some susceptible landraces predicted to carry Sr2 by marker analysis require further investigation. To evaluate defense gene expression in compatible and incompatible stem rust interactions we sampled resistant and susceptible cultivars at 0, 12, 18, 24, 72 hours post-inoculation (hpi). ?-1,3 glucanase expression was studied using qGLU-S and qGLUU-AS primers and a real-time PCR step-one ABI machine, with ?-tubulin and EF1-? genes used as internal controls. In incompatible interactions defense gene expression was increased at 24 hpi, but in compatible interactions the highest level of expression occurred at 12 hpi and was significantly decreased at 18 hpi. The results revealed that expression of defense genes such as ?-1,3 glucanase was earlier in compatible than in incompatible interactions but the expression level was less in incompatible interactions. On the other hand, in susceptible genotypes the expression of defense genes increased immediately after inoculation and declined sharply after infection. In contrast defense gene expression in resistant genotypes began to increase after establishment of the pathogen.
Performance of CIMMYT germplasm in Ethiopia: Key materials for variety development
BGRI 2015 Poster Abstract Abeyo
CIMMYT wheat germplasm flow to Ethiopia started in the late 1960s. Over 90 bread wheat varieties were released over the decades. Of these, about 77% had CIMMYT origins or were derived from CIMMYT materials. Wheat is a traditional rainfed crop grown by 5 million small-scale farmers on 1.6 ha more or less. Yields have increased from 1.0 t/ha in the 1960s to 2.54 t/ha in 2014 mainly due to high yielding semi-dwarf bread wheat varieties and modern agronomic practices. Using such technologies, better farmers often get 5-6 t/ha. The rusts are the most important production constraints. For example, the 2010 yellow rust epidemic debilitated the mega varieties Kubsa and Galama in the highlands. In 2013/14, stem rust caused up to 100% yield losses in the widely adopted bread wheat variety Digalu in Arsi and Bale. This epidemic was caused by Pgt race TKTTF, which is virulent to the gene SrTmp that is present in Digalu, but is avirulent to Sr31, which is overcome by race Ug99 (TTKSK) and derivatives. To avert the increasing threat of rusts, CIMMYT developed a shuttle breeding program where germplasm moves back and forth between Mexico and Kenya and has increased nursery testing sites (Holetta, Kulumsa, Debre Zeit, Sinana, Adet, and Melkassa) in Ethiopia from two to six. The germplasm passes through rigorous tests against major diseases during both the main- and off-seasons. To obtain high yielding rust resistant germplasm, many hundreds of genotypes were introduced and tested over the last two years. In 2014/15, 266 (25%) lines with multiple disease resistances and high yield were promoted to national trials. CIMMYT continues to be an important source of germplasm. Fast tracked variety testing and release, accelerated seed multiplication, demonstration and popularization of new varieties with high yield, multiple disease resistance, and acceptable quality will continue.
Evaluation of wild wheat introgression lines for rust resistance and yield
BGRI 2015 Poster Abstract Abugaliyeva
Kazakh Research Institute of Agriculture and Plant Growing
Wild species are sources and donors of many valuable traits for wheat improvement. We studied winter wheat introgression lines for productivity traits, disease resistance, and protein, globulin, gliadin and glutenin contents as well as grain mineral concentrations. Laboratory and field studies allowed selection in populations segregating for resistance to yellow rust and leaf rust. Lines 1718, 1721-9, 1721-4, 1675 and 1727 had the highest yields (6.2 t/ha) and stable leaf rust and stem rust resistances, but were still variable in response to stripe rust (30-80 S). Lines 1718 (Bezostaya 1 x Ae. cylindrica, genomes CCDD) and 1721 (Bezostaya 1 x T. militinae2 - 6, ABG) were resistant to stripe rust in trials at yield levels of 3.7-7.6 t/ha and from 5.7 to 8.2 t/ha, respectively. Line 1675 (Zhetisu x T. kiharae, ABGD) was resistant to all three rusts. Line 1676 (Steklovidnaya 24 x T. timopheevi, ABG) was resistant to LR and SR at a yield level of 8.3 t/ha, and 1671 (Zhetisu x T. militinae, ABG) was resistant to YR and SR at a yield level of 7.5 t/ha. Protein contents of the lines ranged from 13.6 to 18.4%, and grain mineral contents were above average.
There is emerging evidence that the geographical footprint of stripe rust is expanding, opening up prospects for an increase in economic losses attributable to this disease worldwide. Drawing on newly compiled data, along with insights obtained from a survey initiated at the BGRI meeting in New Delhi in August 2013, this talk will report on efforts to model the global occurrence and persistence of stripe rust in a geo-spatially sensitive fashion. Using the available data in conjunction with these newly developed climate suitability maps, I will present probabilistic crop production losses associated with the disease and place an economic value on the prospective losses. Given changes in the geographical spread of this disease, and the associated uncertainties about its likely wheat yield and economic effects, various scenarios will be assessed to inform and thereby help shape the research investment decisions regarding crop breeding and other options for ameliorating these prospective losses over the longer term.
Advances in breeding for resistance to stem rust caused by Ug99 and Ethiopian Pgt races in durum wheat
BGRI 2014 Plenary Abstract Karim Ammar
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.
Understanding resistance gene mediated recognition of stem rust in wheat
BGRI 2014 Plenary Abstract Peter Dodds
CSIRO Plant Industry, Australia
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.
The Global Rust Reference Centre (GRRC, www.wheatrust.org) was established in 2008 upon the request of CIMMYT and (ICARDA) and extended in 2011 by the support of the Borlaug Global Rust Initiative. GRRC serve as a global hub for investigating wheat rust fungi and can receive alive samples from all countries year round. The activities of GRRC comprise pathotyping of wheat yellow rust and wheat stem rust, as well as training of students and scientists, data handling and storage (databases) and reporting. The current research activities have a focus on evolutionary population biology, as well as basic genetic and genomic studies in yellow rust. The “Wheat Rust Toolbox” and the team behind has become part of the GRRC and all data generated by GRRC will be stored in this system. Data management, research activities and dissemination will be coordinated and integrated with partner information platforms at CIMMYT, ICARDA, Cornell University and other global partners. The quarantine greenhouse space has in recent years been enlarged by more than 50% allowing GRRC to take in more rust samples and students. The GRRC activities expanded significantly in 2011 and 2013 via grants from the Danish Strategic Research Council and the Ministry of Food, Agriculture and Fisheries. One of these initiatives, RUSTFIGHT, has a focus on understanding “aggressiveness” and involves a number of Danish and international partners, including ICARDA and CIMMYT, INRA and the John Innes Centre (UK), and private Danish plant breeding Industry.