The Effector SIX8 Contributes to Virulence of Fusarium oxysporum f. sp. lactucae Race 4 on Lettuce

Fusarium wilt disease of lettuce caused by the soilborne fungus Fusarium oxysporum f. sp. lactucae (Fola) causes substantial crop losses worldwide. Four races of Fola have been identified with race 1 (Fola1) and the more recently emerged race 4 (Fola4) predominant in Europe. Recent analysis of Fola1 and Fola4 genomes alongside PCR-based isolate profiling determined that while both Fola1 and Fola4 isolates harbour homologues of the putative effector genes Secreted in Xylem (SIX) 9 and SIX14, Fola4 has additionally gained SIX8, which is divergently transcribed with Pair with Six Eight 1 (PSE1) or Pair with Six Eight 1- Like (PSL1) genes. Transcriptomic analyses at a single timepoint also demonstrated high expression levels for SIX8, SIX9, SIX14 and PSL1 during Fola4 lettuce infection. In this study we demonstrate that SIX8, SIX9 and SIX14 expression levels increase up to 96 h following Fola4 lettuce infection, hence confirming their potential importance in wilt disease. The role of SIX8 in virulence on lettuce was then investigated through phenotyping of knockout mutants generated by CRISPR-Cas9 gene editing. ΔSIX8 mutants resulted in reduced disease in both in vitro lettuce seedling and pot-based lettuce plant glasshouse bioassays compared to wild-type Fola4. Complementation of SIX8 back into one of the knockout mutants using Agrobacterium-mediated transformation restored full virulence on lettuce. We conclude that SIX8 plays a role in Fola4 virulence and discuss the hypothesis that SIX8 may allow Fola4 to overcome some sources of Fola1 resistance and hence infect a different range of lettuce cultivars.