Genetic identification and characterisation of gain-of-virulence mutants of Puccinia graminis f. sp. tritici

The fungus Puccinia graminis f. sp. tritici (Pgt), is the causal agent of wheat stem rust, a disease that has regained attention after the emergence of virulent races which have caused epidemics in Africa, Europe, and Central Asia. Durable control of this disease requires the development of wheat varieties that contain several cloned stem rust resistance (Sr) genes that are stacked together at a single locus. However, some resistance genes interact negatively or fail to function when expressed in some genetic backgrounds. Thus, verification of the function of stacked Sr genes must be conducted after stacking. The use of the pathogen in these assays is difficult due to the simultaneous secretion of multiple avirulence (Avr) effectors. The heterologous expression of single Avr effectors can avoid this limitation. However, this strategy is hampered by the availability of only a very few (three) cloned Pgt Avrs to test Sr stacks. To accelerate Pgt Avr gene cloning, I outline a method to generate an ethyl methanesulphonate (EMS) mutant Pgt population followed by screening for gain-of-virulence mutant isolates. I treated urediniospores with EMS and created a library of > 12,000 mutant isolates. I selected random mutants for sequencing and established the average EMS transitions to be 1 single nucleotide variant (SNV) per 258 kb. I screened the mutant library on wheat seedlings carrying Sr43, Sr44, or Sr45. From this, I obtained 9, 4, and 14 Pgt mutants with virulence toward Sr43, Sr44, and Sr45, respectively. Upon isolation and reinoculation of the mutants onto the lines they were identified, only the mutant isolates on Sr43 and Sr45 showed stable virulence. I characterized 8 mutants virulent on Sr43 by checking their virulence profile on the stem rust international differential set containing 20 defined Sr genes. These mutants maintained the same virulence profile as the wildtype from which they were derived showing that they were not contaminants. I further characterised two mutants, E1 and E7-1, to quantify their growth on Sr43 via chitin fluorescence. There was no difference between the chitin fluorescence of these mutants on Sr43 and that of the wildtype on the recurrent parent Chinese Spring. Therefore, loss of AvrSr43 has no apparent effect on Pgt fitness. In conclusion, my method enables the selection for virulent mutants toward targeted resistance (R) genes. The mutant library can be created from as little as 320 mg spores which provides a resource that enables screening against several R genes without repeating the EMS mutagenesis

Mutagenesis of Puccinia graminis f. sp. tritici and selection of gain-of-virulence mutants

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

Resistance gene cloning from a wild crop relative by sequence capture and association genetics

Disease resistance (R) genes from wild relatives could be used to engineer broad-spectrum resistance in domesticated crops. We combined association genetics with R gene enrichment sequencing (AgRenSeq) to exploit pan-genome variation in wild diploid wheat and rapidly clone four stem rust resistance genes. AgRenSeq enables R gene cloning in any crop that has a diverse germplasm panel

Potential for re-emergence of wheat stem rust in the United Kingdom

Wheat stem rust, a devastating disease of wheat and barley caused by the fungal pathogen Puccinia graminis f. sp. tritici, was largely eradicated in Western Europe during the mid-to-late twentieth century. However, isolated outbreaks have occurred in recent years. Here we investigate whether a lack of resistance in modern European varieties, increased presence of its alternate host barberry and changes in climatic conditions could be facilitating its resurgence. We report the first wheat stem rust occurrence in the United Kingdom in nearly 60 years, with only 20% of UK wheat varieties resistant to this strain. Climate changes over the past 25 years also suggest increasingly conducive conditions for infection. Furthermore, we document the first occurrence in decades of P. graminis on barberry in the UK. Our data illustrate that wheat stem rust does occur in the UK and, when climatic conditions are conducive, could severely harm wheat and barley production.</p

Aegilops sharonensis genome-assisted identification of stem rust resistance gene Sr62

The wild relatives and progenitors of wheat have been widely used as sources of disease resistance (R) genes. Molecular identification and characterization of these R genes facilitates their manipulation and tracking in breeding programmes. Here, we develop a reference-quality genome assembly of the wild diploid wheat relative Aegilops sharonensis and use positional mapping, mutagenesis, RNA-Seq and transgenesis to identify the stem rust resistance gene Sr62, which has also been transferred to common wheat. This gene encodes a tandem kinase, homologues of which exist across multiple taxa in the plant kingdom. Stable Sr62 transgenic wheat lines show high levels of resistance against diverse isolates of the stem rust pathogen, highlighting the utility of Sr62 for deployment as part of a polygenic stack to maximize the durability of stem rust resistance

Population genomic analysis of Aegilops tauschii identifies targets for bread wheat improvement.

Aegilops tauschii, the diploid wild progenitor of the D subgenome of bread wheat, is a reservoir of genetic diversity for improving bread wheat performance and environmental resilience. Here we sequenced 242 Ae. tauschii accessions and compared them to the wheat D subgenome to characterize genomic diversity. We found that a rare lineage of Ae. tauschii geographically restricted to present-day Georgia contributed to the wheat D subgenome in the independent hybridizations that gave rise to modern bread wheat. Through k-mer-based association mapping, we identified discrete genomic regions with candidate genes for disease and pest resistance and demonstrated their functional transfer into wheat by transgenesis and wide crossing, including the generation of a library of hexaploids incorporating diverse Ae. tauschii genomes. Exploiting the genomic diversity of the Ae. tauschii ancestral diploid genome permits rapid trait discovery and functional genetic validation in a hexaploid background amenable to breeding

Population genomic analysis of Aegilops tauschii identifies targets for bread wheat improvement

Aegilops tauschii, the diploid wild progenitor of the D subgenome of bread wheat, is a reservoir of genetic diversity for improving bread wheat performance and environmental resilience. Here we sequenced 242 Ae. tauschii accessions and compared them to the wheat D subgenome to characterize genomic diversity. We found that a rare lineage of Ae. tauschii geographically restricted to present-day Georgia contributed to the wheat D subgenome in the independent hybridizations that gave rise to modern bread wheat. Through k-mer-based association mapping, we identified discrete genomic regions with candidate genes for disease and pest resistance and demonstrated their functional transfer into wheat by transgenesis and wide crossing, including the generation of a library of hexaploids incorporating diverse Ae. tauschii genomes. Exploiting the genomic diversity of the Ae. tauschii ancestral diploid genome permits rapid trait discovery and functional genetic validation in a hexaploid background amenable to breeding