ZymoSoups: A High-Throughput Forward Genetics Method for Rapid Identification of Virulence Genes in Zymoseptoria tritici.

Septoria tritici blotch is caused by the fungus Zymoseptoria tritici and poses a major threat to wheat productivity. There are over 20 mapped loci in wheat that confer strong (gene-for-gene) resistance against this pathogen, but 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 ultraviolet (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 to 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 (approximately 70 kb) 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. [Formula: see text] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.