A pair of effectors in a conditionally dispensable chromosome of Fusarium oxysporum suppresses host-specific immunity

In plant pathogenic fungi, conditionally dispensable (CD) chromosomes are often associated with virulence, but not viability. Such virulence-associated CD chromosomes carry genes encoding effectors and/or host-specific toxin biosynthesis enzymes, potentially important for determining host specificity. Fusarium oxysporum causes devastating diseases of more than 100 plant species. In particular, F. oxysporum f. sp. conglutinans (Focn) can infect Brassicaceae plants including Arabidopsis and cabbage. Here we show that Focn has multiple CD chromosomes involving in not only virulence but also vegetative growth, which is an atypical feature of known CD chromosomes. Among them, we identified specific CD chromosomes that are required for virulence to either Arabidopsis, cabbage, or both. We revealed that a pair of effectors encoded in one of the CD chromosomes is required for suppression of the Arabidopsis-specific phytoalexin-based immunity. The effector pair is highly conserved in F. oxysporum isolates capable of infecting Arabidopsis. This study provides insights into how host specificity of F. oxysporum is determined by a pair of effector genes on a CD chromosome. Overall design: For pre-incubation, Fusarium oxysporum f. sp. conglutinans Cong:1-1 (FocnCong:1-1) was incubated on potato dextrose agar (PDA) at 28°C under dark. For bud cell production, FocnCong:1-1 was grown in NO3 medium (0.17% yeast nitrogen base without amino acids, 3% sucrose and 1% KNO3) at 120 strokes per minute (spm) for 4 days at 28°C under dark. For expression profiling, mycelia were harvested after 10 days of incubation on PDA at 28°C. Bud cells were collected from the above culture in NO3 medium by filtration with a nylon mesh and centrifugation. Hyphae trapped with the nylon mesh were dried and collected. Mycelia (PDA), bud cells and hyphae were stored in a tube at -80°C until RNA isolation. In addition, 20 or 21 day-old Arabidopsis Col-0 and 17 day-old cabbage cv. Shosyu roots were irrigated with 1 ml of the FocnCong:1-1 bud cell suspension (1 × 10^7 cells/ml). At 3 dpi and 10 dpi, infected roots were washed with water to remove soil and then dried. The roots were stored in a tube at -80°C until RNA isolation. RNA sequencing was performed by deep sequencing on Illumina NextSeq 500 using a custom high-throughput method (Front Plant Sci 2012, 3:202). Each biological replicate set of samples were sequenced as one batch (biorep1, biorep2, biorep3, biorep4 and biorep5 in filenames).