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

Kangara, Ngonidzashe (2021) Genetic identification and characterisation of gain-of-virulence mutants of Puccinia graminis f. sp. tritici. Doctoral thesis, University of East Anglia.

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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.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Chris White
Date Deposited: 20 Oct 2022 08:11
Last Modified: 20 Oct 2022 08:11

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