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

Authors: LI, YUXIANG - Washington State University; XIA, CHONGJING - Washington State University; WANG, MEINAN - Washington State University; YIN, CHUNTAO - Washington State University; Chen, Xianming

Submitted to: BMC Genomics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/20/2020
Publication Date: 3/20/2020
Citation: Li, Y., Xia, C., Wang, M., Yin, C., Chen, X. 2020. Whole-genome sequencing of Puccinia striiformis f. sp. tritici mutant isolates identifies avirulence gene candidates. BMC Genomics. 21. https://doi.org/10.1186/s12864-020-6677-y.
DOI: https://doi.org/10.1186/s12864-020-6677-y

Interpretive Summary: The stripe rust pathogen threats world wheat production. To determine the role of mutation in the pathogen virulence changes, we previously developed 30 mutant isolates a least virulent isolate using ethyl methanesulfonate (EMS) mutagenesis and phenotypically characterized them for virulence changes and established a high-quality reference genome from the progenitor isolate. In the present study, we sequenced the 30 mutant isolates compared their sequences with the reference genome to determine the genomic variation and identify candidates for avirulence genes. A total of 94 mutated genes were found significantly associated to 16 avirulence genes after selection through a series of criteria for putative effectors and degree of association, including 75 genes encoding secreted proteins (SPs) and 19 non-SP genes but with high levels of association to avirulence phenotypes. Fourteen of the SP genes were identified as avirulence-associated effectors with high-confidence as they met five or six of the six criteria used to determine effectors. 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 wheat and the stripe rust pathogen.

Technical Abstract: Background: The stripe rust pathogen, Puccinia striiformis f. sp. tritici (Pst), threats world wheat production. Resistance to stripe rust 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 on wheat lines each possessing a single Yr gene for stripe rust resistance. 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, 423,295 single nucleotide polymorphism (SNP) sites and 250,715 Indels (Insertion/deletion) were detected among the 30 mutant isolates. Genome wide association analysis identified 8,619 and 585 genes in each mutant isolate on average from the SNP and Indel data, respectively associated with avirulence variants. A total of 94 genes were found significantly associated to 16 avirulence genes after selection through a series of criteria for putative effectors and degree of association, including 75 genes encoding secreted proteins (SPs) and 19 non-SP genes but with high levels of association (P = 0.0001) to avirulence phenotypes. Fourteen of the SP genes were identified as avirulence-associated effectors with high-confidence as they met five or six of the 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.