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Mutagenesis of Puccinia graminis f. sp. tritici and Selection of Gain-of-Virulence Mutants.

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.

SUBMITTER: Kangara N

PROVIDER: S-EPMC7533539 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Mutagenesis of Puccinia graminis f. sp. tritici and Selection of Gain-of-Virulence Mutants.

Kangara Ngonidzashe N Kurowski Tomasz J TJ Radhakrishnan Guru V GV Ghosh Sreya S Cook Nicola M NM Yu Guotai G Arora Sanu S Steffenson Brian J BJ Figueroa Melania M Mohareb Fady F Saunders Diane G O DGO Wulff Brande B H BBH

Frontiers in plant science 20200916

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 fu ...[more]

PMID: 33072145

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Project description:Puccinia striiformis f. sp. tritici (Pst) is an obligate biotrophic fungus that causes extensive damage in wheat. The pathogen is now known to be a heteroecious fungus with an intricate life cycle containing sexual and asexual stages. Orthologues of the STE12 transcription factor that regulate mating and filamentation in Saccharomyces cerevisiae, as well as virulence in other fungi, have been extensively described. Because reliable transformation and gene disruption methods are lacking for Pst, knowledge about the function of its STE12 orthologue is limited. In this study, we identified a putative orthologue of STE12 from Pst in haustoria-enriched transcripts and designated it as PstSTE12. The gene encodes a protein of 879 amino acids containing three helices in the homeodomain, conserved phenylalanine and tryptophan sites, and two C2 /H2 -Zn2+ finger domains. Real-time reverse transcription-polymerase chain reaction (RT-PCR) analyses revealed that the expression of PstSTE12 was highly induced during the early infection stages and peaked during haustorium formation and the pycniospore stage in the aecial host barberry. Subcellular localization assays indicated that PstSTE12 is localized in the nucleus and functions as a transcriptional activator. Yeast one-hybrid assays revealed that PstSTE12 exhibits transcriptional activity, and that its C-terminus is necessary for the activation of transcription. PstSTE12 complemented the mating defect in an α ste12 mutant of S. cerevisiae. In addition, it partially complemented the defects of the Magnaporthe oryzae mst12 mutant in plant infection. Knocking down PstSTE12 via host-induced gene silencing (HIGS) mediated by Barley stripe mosaic virus (BSMV) resulted in a substantial reduction in the growth and spread of hyphae in Pst and weakened the virulence of Pst on wheat. Our results suggest that PstSTE12 probably acts at an intersection participating in the invasion and mating processes of Pst, and provide new insights into the comprehension of the variation of virulence in cereal rust fungi.

Project description:BackgroundStem rust is an economically important disease of wheat and barley. However, studies to gain insight into the molecular basis of these host-pathogen interactions have primarily focused on wheat because of its importance in human sustenance. This is the first extensive study utilizing a transcriptome-wide association mapping approach to identify candidate Puccinia graminis f. sp. tritici (Pgt) effectors/suppressors that elicit or suppress barley stem rust resistance genes. Here we focus on identifying Pgt elicitors that interact with the rpg4-mediated resistance locus (RMRL), the only effective source of Pgt race TTKSK resistance in barley.ResultsThirty-seven Pgt isolates showing differential responses on RMRL were genotyped using Restriction Site Associated DNA-Genotyping by Sequencing (RAD-GBS), identifying 24 diverse isolates that were used for transcript analysis during the infection process. In planta RNAseq was conducted with the 24 diverse isolates on the susceptible barley variety Harrington, 5 days post inoculation. The transcripts were mapped to the Pgt race SCCL reference genome identifying 114 K variants in predicted genes that would result in nonsynonymous amino acid substitutions. Transcriptome wide association analysis identified 33 variants across 28 genes that were associated with dominant RMRL virulence, thus, representing candidate suppressors of resistance. Comparative transcriptomics between the 9 RMRL virulent -vs- the 15 RMRL avirulent Pgt isolates identified 44 differentially expressed genes encoding candidate secreted effector proteins (CSEPs), among which 38 were expressed at lower levels in virulent isolates suggesting that they may represent RMRL avirulence genes. Barley transcript analysis after colonization with 9 RMRL virulent and 15 RMRL avirulent isolates inoculated on the susceptible line Harrington showed significantly lower expression of host biotic stress responses specific to RMRL virulent isolates suggesting virulent isolates harbor effectors that suppress resistance responses.ConclusionsThis transcriptomic study provided novel findings that help fill knowledge gaps in the understanding of stem rust virulence/avirulence and host resistance in barley. The pathogen transcriptome analysis suggested RMRL virulence might depend on the lack of avirulence genes, but evidence from pathogen association mapping analysis and host transcriptional analysis also suggested the alternate hypothesis that RMRL virulence may be due to the presence of suppressors of defense responses.

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Mutagenesis of Puccinia graminis f. sp. tritici and Selection of Gain-of-Virulence Mutants.

Publications

Mutagenesis of <i>Puccinia graminis</i> f. sp. <i>tritici</i> and Selection of Gain-of-Virulence Mutants.

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