Whole-genome sequencing reveals that mutations in myosin-5 confer resistance to the fungicide phenamacril in Fusarium graminearum

Abstract

To determine the mechanism of resistance to the fungicide phenamacril (JS399-19) in Fusarium graminearum, the causal agent of Fusarium head blight, we sequenced and annotated the genome of the resistant strain YP-1 (generated by treating the F. graminearum reference strain PH-1 with phenamacril). Of 1.4 million total reads from an Illumina-based paired-end sequencing assay, 92.80% were aligned to the F. graminearum reference genome. Compared with strain PH-1, strain YP-1 contained 1,989 single-nucleotide polymorphisms that led to amino acid mutations in 132 genes. We sequenced 22 functional annotated genes of another F. graminearum sensitive strain (strain 2021) and corresponding resistant strains. The only mutation common to all of the resistant mutants occurred in the gene encoding myosin-5 (point mutations at codon 216, 217, 418, 420, or 786). To confirm whether the mutations in myosin-5 confer resistance to phenamacril, we exchanged the myosin-5 locus between the sensitive strain 2021 and the resistant strain Y2021A by homologous double exchange. The transformed mutants with a copy of the resistant fragment exhibited resistance to phenamacril, and the transformed mutant with a copy of the sensitive fragment exhibited sensitivity to phenamacril. These results indicate that mutations in myosin-5 confers resistance to phenamacril in F. graminearum.

Figure 1. Phenamacril resistant mutants contain the point mutations at codon 216, 217, 418, 420 and 786 of myosin-5 in Fusarium graminearum.

(a): Alignment of partial deduced amino acid sequences of myosin-5 from the reference strain PH-1, induced resistant strain YP-1 from PH-1, wild-type strain 2021 and induced resistant strains Y2021A, B, C, D, F from 2021. The vertical boxes indicate the amino acid changes at the codon 216, 217, 418, 420 and 786 that are responsible for phenamacril resistance. (b): Schematic representation of Fusarium graminearum myosin-5. Sites of lysine, serine, glutamic acid and methionine mutantions are indicated with blue arrowheads. The conserved motor domain, myosin tail (TH1) and src homology domain 3 (SH3) are highlighted.

Figure 2. Generation and identification of Fusarium graminearum myosin-5 gene recombinant mutants by gene replacement.

(a) Schematic representation of gene replacement strategy. The upper part represents the genomic locus target of the replacement construct. The black dashed lines represent the surrounding genomic region. The gene replacement cassette contains the hygromycin resistance gene and the myosin-5 mutated zone. Primer binding sites are indicated by arrows (see Table S1 for the primer sequences). The asterisk represents the selected myosin-5 mutated zone. (b) and (c) Polymerase chain reaction (PCR) strategy to screen replacement transformants: (b) PCR performed with primer pair A5/A6; a 1.9-kb amplified fragment indicates replacement integration at the left junction. (c) PCR performed with primer pair A7/A8; a 3.7-kb amplified fragment indicates replacement integration at the right junction. (d) Southern blot hybridization analysis of 2021 and all the mutants using a 622-bp myosin-5 upstream fragment as probe, and genomic DNA digested with SacI.

Figure 3. Colony morphology of Fusarium graminearum strains.

(a): Effects of phenamacril (JS399-19) on mycelial linear growth of F. graminearum wild-type strain 2021, resistant strains Y2021A, B, C, D, F and myosin-5 replacement mutants on PDA. The mycelial plugs taken from the edge of a 3-day-old colony of strains or mutants were grown at 25°C for 2 days on the PDA plates, which were amended with JS399-19 at 0, 0.25, 1, 50, or 200 μg a.i./ml. (b): Colony morphology of 2021, PH-1 and all the mutants. Strains were grown on solid media (PDA) for 3 days at 25°C.

Figure 4. Expression level of myosin-5, myosin-2B and type II myosin in PH-1 and resistant mutants relative to that in 2021.

In (b), (c) and (d), -JS399-19 represents strains treated without JS399-19 and + JS399-19 represents strains treated with JS399-19 at 0.56 μg/ml for 6h. Values are the means ± standard error (SE) of three repeated experiments.