ZymoSoups: A high-throughput forward genetics method for rapid identification of virulence genes in Zymoseptoria tritici

These data were used to generate figures and summarise the number of mutations in 18 re-sequenced genomes of Zymoseptoria tritici. The isolates genotyped in this VCF file are all UV-mutegenized strains of the reference isolate IPO323.

Abstract

Septoria tritici blotch is caused by the fungus Zymoseptoria tritici and poses a major threat to wheat productivity. There are over twenty mapped loci in wheat that confer strong (gene-for-gene) resistance against this pathogen, however the corresponding genes in Z. tritici that confer virulence against distinct R genes remain largely unknown. In this study, we developed a rapid forward genetics methodology to identify genes that enable Z. tritici to gain virulence on previously resistant wheat varieties. We used the known gene-for-gene interaction between Stb6 and AvrStb6 as a proof of concept that this method could quickly recover single candidate virulence genes. We subjected the avirulent Z. tritici strain IPO323, which carries the recognized AvrStb6 allele, to UV mutagenesis and generated a library of over 66,000 surviving spores. We screened these survivors on leaves of the resistant wheat variety Cadenza, in mixtures (soups) ranging from 100-500 survivors per soup. We identified five soups with a gain-of-virulence (GoV) phenotype relative to the IPO323 parental strain and re-sequenced 18 individual isolates, including four control isolates and two isolates lacking virulence, when screened individually. Of the 12 confirmed GoV isolates, one had a single nucleotide polymorphism (SNP) in the AvrStb6 coding region. The other 11 GoV isolates exhibited large (~70Kb) deletions at the end of chromosome 5, including the AvrStb6 locus. Our findings demonstrate the efficiency of this forward genetic approach in elucidating the genetic basis of qualitative resistance to Z. tritici and the potential to rapidly identify other, currently unknown, Avr genes in this pathogen. 

Analysis pipeline

The pipeline used to generate this filtered vcf file and tab delimited file is publicly available at https://github.com/megancamilla/GATK4_Zymoseptoria

Isolate Data Accessibility

The individual fastq files for each isolate included in this study have been uploaded to NCBI's Short Read Archive (SRA), project number PRJNA1017776. This project contains 18 re-sequenced samples (SAMN37407989-SAMN37407972), which are UV mutagenized IPO323. Short Read Archive (SRA) accession numbers are SRR26072101-SRR26072118.