Whole-genome sequencing of Puccinia striiformis f. sp. tritici mutant isolates identifies avirulence gene candidates

Abstract

Background: The stripe rust pathogen, Puccinia striiformis f. sp. tritici (Pst), threats world wheat production. Resistance to Pst is often overcome by pathogen virulence changes, but the mechanisms of variation are not clearly understood. To determine the role of mutation in Pst virulence changes, in previous studies 30 mutant isolates were developed from a least virulent isolate using ethyl methanesulfonate (EMS) mutagenesis and phenotyped for virulence changes. The progenitor isolate was sequenced, assembled and annotated for establishing a high-quality reference genome. In the present study, the 30 mutant isolates were sequenced and compared to the wide-type isolate to determine the genomic variation and identify candidates for avirulence (Avr) genes. Results: The sequence reads of the 30 mutant isolates were mapped to the wild-type reference genome to identify genomic changes. After selecting EMS preferred mutations, 264,630 and 118,913 single nucleotide polymorphism (SNP) sites and 89,078 and 72,513 Indels (Insertion/deletion) were detected among the 30 mutant isolates compared to the primary scaffolds and haplotigs of the wild-type isolate, respectively. Deleterious variants including SNPs and Indels occurred in 1866 genes. Genome wide association analysis identified 754 genes associated with avirulence phenotypes. A total of 62 genes were found significantly associated to 16 avirulence genes after selection through six criteria for putative effectors and degree of association, including 48 genes encoding secreted proteins (SPs) and 14 non-SP genes but with high levels of association (P ≤ 0.001) to avirulence phenotypes. Eight of the SP genes were identified as avirulence-associated effectors with high-confidence as they met five or six criteria used to determine effectors. Conclusions: Genome sequence comparison of the mutant isolates with the progenitor isolate unraveled a large number of mutation sites along the genome and identified high-confidence effector genes as candidates for avirulence genes in Pst. Since the avirulence gene candidates were identified from associated SNPs and Indels caused by artificial mutagenesis, these avirulence gene candidates are valuable resources for elucidating the mechanisms of the pathogen pathogenicity, and will be studied to determine their functions in the interactions between the wheat host and the Pst pathogen.

Role of Genetics, Genomics, and Breeding Approaches to Combat Stripe Rust of Wheat

“…However, race evolution and pathogenicity behavior are less explored. These unsolved issues provoked scientists and diverted their attention toward sequencing of the Pst genome to provide a deep insight into pathogenicity and host–pathogen interaction ( 10 ). To date, more than 15 Pst reference genomes are publicly available ( 11 ).…”

Integrated Analysis of Gene Expression, SNP, InDel, and CNV Identifies Candidate Avirulence Genes in Australian Isolates of the Wheat Leaf Rust Pathogen Puccinia triticina

“…Given the complexity of fungal genomes and the limited knowledge of the genetic mechanisms underlying the development of virulence in fungal pathogens, more and more integrated approaches have been developed for effector detection. One such integrated approach is to combine genome-wide association analysis with variant comparisons, such as our study on AvrLr20 mining [23], a previous study on AvrPm3 in powdery mildew [63], and a recent study using 30 Pst isolates derived from ethyl methanesulfonate mutagenesis [21]. Although these association analyses still focus on genomic variations at the level of SNPs and InDels, our study is the first to integrate the major type of structure variation, CNV, into comparative analysis at the genome-wide scale in a rust pathogen.…”

“…tritici (Pst) [18]. With the availability of reference genomes, advances have been made for all three wheat rust fungi in genome-wide studies for effector mining, including studies on Pst [19][20][21], Pgt [22], and Pt [23], which have detected a panel of candidate avirulence genes for functional validation. One earlier study identified 15 candidates for 14 Avr genes using gene expression and RNA SNP analysis [24].…”

“…In addition to the analysis of gene expression, single nucleotide polymorphisms (SNPs), and insertions/deletions (InDels) [ 21 , 23 , 27 , 28 ], copy number variations (CNVs) have been highlighted as a new and significant source of genetic polymorphism that contributes to phenotypic diversity such as virulence in diverse fungal species [ 29 ]. In fact, the contribution of CNVs to population genetic and phenotypic diversity has been exemplified by a range of fungal studies, including studies on yeast Saccharomyces cerevisiae (Ascomycota, Saccharomycetes) [ 30 , 31 ], the wheat pathogen Zymoseptoria tritici (Ascomycota, Dothideomycetes) [ 32 ], and the human fungal pathogen Cryptococcus deuterogattii (Basidiomycota, Tremellomycetes) [ 33 ].…”

Transcriptome Analysis Based on Lr19-Virulent Mutants Strain Provides Clues for the Pathogenicity-Related Genes and Effectors of Puccinia Triticina

“…For example, Salcedo et al obtained stripe rust mutants through EMS-induced mutation and performed genome sequencing to obtain AvrSr35 candidate genes, and then veri ed the candidate genes through co-expression of AvrSr35 and its corresponding resistance gene in tobacco and wheat to trigger cell death [8]. Li et al screened 30 mutant variants from the least virulent isolate generated by EMS mutagenesis and candidates Avr genes were determined by sequencing [9]. Transcriptomics has proven to be an instrumental molecular tool to help identify virulence effectors and Avr genes [10][11][12].…”