Wheat stem rust caused by the fungus Puccinia graminis f. sp. tritici (Pgt), is regaining prominence due to the recent emergence of virulent isolates and epidemics in Africa, Europe and Central Asia. The development and deployment of wheat cultivars with multiple stem rust resistance (Sr) genes stacked together will provide durable resistance. However, certain disease resistance genes can suppress each other or fail in particular genetic backgrounds. Therefore, the function of each Sr gene must be confirmed after incorporation into an Sr-gene stack. This is difficult when using pathogen disease assays due to epistasis from recognition of multiple avirulence (Avr) effectors. Heterologous delivery of single Avr effectors can circumvent this limitation, but this strategy is currently limited by the paucity of cloned Pgt Avrs. To accelerate Avr gene cloning, we outline a procedure to develop a mutant population of Pgt spores and select for gain-of-virulence mutants. We used ethyl methanesulphonate (EMS) to mutagenize urediniospores and create a library of > 10,000 independent mutant isolates that were combined into 16 bulks of ~658 pustules each. We sequenced random mutants and determined the average mutation density to be 1 single nucleotide variant (SNV) per 258 kb. From this, we calculated that a minimum of three independently derived gain-of-virulence mutants is required to identify a given Avr gene. We inoculated the mutant library onto plants containing Sr43, Sr44, or Sr45 and obtained 9, 4, and 14 mutants with virulence toward Sr43, Sr44, or Sr45, respectively. However, only mutants identified on Sr43 and Sr45 maintained their virulence when reinolculated onto the lines from which they were identified. We further characterized 8 mutants with virulence toward Sr43. These also maintained their virulence profile on the stem rust international differential set containing 20 Sr genes, indicating that they were most likely not accidental contaminants. In conclusion, our method allows selecting for virulent mutants toward targeted resistance (R) genes. The development of a mutant library from as little as 320 mg spores creates a resource that enables screening against several R genes without the need for multiple rounds of spore multiplication and mutagenesis.
Bibliographical noteFunding Information:
We gratefully acknowledge funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 674964, the John Innes Centre Science For Africa Initiative (JIC-SFA) and the Biotechnology and Biological Sciences Research Council (BBSRC) cross-institute strategic programmes Designing Future Wheat (BB/P016855/1) and Plant Health (BB/P012574/ 1) through a Discovery Fellowship to GR (BB/S011005/1). SG was supported by a Monsanto’s Beachell-Borlaug International Scholars’ Program fellowship and 2Blades Foundation.
We would like to thank: Jens Maintz for Pgt UK-01 purification and spore Pgt mutant harvesting, Yajuan Yue and Macarena Forner-Mart?nez for bulking up of seed for lines used in the experiments, Rebecca Doherty for harvesting of Pgt mutant spores as well as watering of inoculated plants in the CERs, JIC Horticultural Services for sowing the seedlings used in experiments and maintenance of plants during seed bulking in the glasshouses, the JIC Germplasm Resource Unit for the maintenance and disbursing of seed, the NBI Computing Infrastructure for Science (CiS) group for HPC maintenance, Cristobal Uauy and Chris Darby for help with funding acquisition, Tobin Florio (www.flozbox-science.com ) for the artwork in Figure 1, Catherine Gardener for proof reading the manuscript, Simon Berry and Paul Fenwick from Limagrain for providing Pgt UK-01, Tom Fetch for supplying the Sr gene Core Differential Set, Evans Lagudah for supplying the Sr22, Sr33 and Sr45 lines, Ian Dundas for supplying the Sr40 and Sr44 lines, Steven Xu for supplying the Sr43 line, Michael Pumphrey for supplying the Sr51 and Sr53 lines, Eitan Millet for supplying the Sr1644-1Sh line, the Wheat Genetic and Genomic Resource Center for the Ae. tauschii Sr1662 line and Brian Steffenson for supplying the Ae. sharonensis accession 2020.
- Puccinia graminis f. sp. tritici
- ethyl methanesulphonate mutagenesis