Abstract
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.
Highlights
Wheat stem rust is a destructive disease caused by the fungus Puccinia graminis f. sp. tritici (Pgt) that is resurging due to the evolution of virulent isolates that have overcome several stem rust resistance (Sr) genes (Pretorius et al, 2000; Olivera Firpo et al, 2017)
To select Pgt mutants with induced mutations in a defined Avr gene requires a wheat line in which the corresponding Sr gene has been genetically isolated in a background which is susceptible to the Pgt isolate chosen for mutagenesis. To identify such Sr gene stocks for the Pgt isolate UK-01 which was designated race type TKTTF according to the North American nomenclature (Lewis et al, 2018), we inoculated: (i) ten wheat lines with chromosome segments carrying defined Sr genes from wild wheats, (ii) the wheat recurrent parents used for Sr introgression, (iii) two Ae. tauschii accessions predicted to carry only Sr46 or SrTA1662 (Arora et al, 2019), one Ae. sharonensis accession carrying Sr2020, and (iv) the respective susceptible and resistant control wheat cultivars Vuka and Kavkaz/Federation4 (Sr31) (Table 1, Supplementary Table 3)
The rapid advance of next-generation sequencing and computational technologies has enabled the cloning of several major dominant Sr genes and many more are in the process of being cloned (Keller et al, 2018; Periyannan, 2018)
Summary
Wheat stem rust is a destructive disease caused by the fungus Puccinia graminis f. sp. tritici (Pgt) that is resurging due to the evolution of virulent isolates that have overcome several stem rust resistance (Sr) genes (Pretorius et al, 2000; Olivera Firpo et al, 2017). 80% of the world’s wheat cultivars are vulnerable to infection (Singh et al, 2008; Lewis et al, 2018) and worldwide yearly grain losses attributed to the disease are estimated at 6.2 million tonnes, equivalent to ~1% of the annual wheat yield (valued at USD 1.12 billion; (Beddow et al, 2013). This masks crop losses at local or regional levels, which can reach 40% or more (Schumann and Leonard, 2000; Saunders et al, 2019). Such effectors are termed avirulence (Avr) effectors and are often associated with a macroscopic hypersensitive cell death response
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