A 20-kb lineage-specific genomic region tames virulence in pathogenic amphidiploid Verticillium longisporum

Abstract

Amphidiploid fungal Verticillium longisporum strains Vl43 and Vl32 colonize the plant host Brassica napus but differ in their ability to cause disease symptoms. These strains represent two V. longisporum lineages derived from different hybridization events of haploid parental Verticillium strains. Vl32 and Vl43 carry same-sex mating-type genes derived from both parental lineages. Vl32 and Vl43 similarly colonize and penetrate plant roots, but asymptomatic Vl32 proliferation in planta is lower than virulent Vl43. The highly conserved Vl43 and Vl32 genomes include less than 1% unique genes, and the karyotypes of 15 or 16 chromosomes display changed genetic synteny due to substantial genomic reshuffling. A 20 kb Vl43 lineage-specific (LS) region apparently originating from the Verticillium dahliae-related ancestor is specific for symptomatic Vl43 and encodes seven genes, including two putative transcription factors. Either partial or complete deletion of this LS region in Vl43 did not reduce virulence but led to induction of even more severe disease symptoms in rapeseed. This suggests that the LS insertion in the genome of symptomatic V. longisporum Vl43 mediates virulence-reducing functions, limits damage on the host plant, and therefore tames Vl43 from being even more virulent.

Keywords: Verticillium longisporum; genome comparison; hybridization; lineage-specific region; pathogenicity.

FIGURE 1

Verticillium longisporum Vl43 and Vl32 colonize Arabidopsis thaliana roots, but only Vl43 induces disease symptoms in rapeseed. (a) Three allodiploid V. longisporum lineages emerged from hybridization events of haploid strains. Vl43/Vl145c and Vl32/Vl19 strains originate from hybridization between strains A1 and D1 or D3, respectively (Inderbitzin, Davis, et al., ; Tran et al., 2013). (b) Scanning electron micrographs of V. longisporum Vl32 or Vl43 colonizing A. thaliana roots 2 or 6 days after root dipping into spore solutions. Scale bars: left and middle, 20 µm; right, detailed view of the framed area, 4 µm; arrows: hyphopodia as possible entry sites; arrow heads: hyphae growing at interface of plant cells. (c) and (d) V. longisporum strain Vl43, but not Vl32, induces disease symptoms in Brassica napus. Plants were evaluated at 35 days postinoculation (dpi) by root dipping into respective spore solutions or water (Mock). (c) Representative plants (above) and overview pictures from a single experiment (below) are shown. (d) The diagram displays the number of plants with indicated symptoms relative to the total number of plants (n) assessed in two independent experiments. (e) Verticillium DNA in rapeseed hypocotyl was quantified at 35 dpi with respective spore solutions or water (Mock). Verticillium‐specific ribosomal DNA was detected by quantitative PCR. Values relative to Vl43 are shown as percentages. (f) Reisolation of Vl43 and Vl32 from surface‐sterilized stem slices of infected plants on potato dextrose agar with chloramphenicol after incubation at 25 °C for 7 days was possible. Less outgrowth was detected for Vl32 (red arrows)

FIGURE 2

Verticillium longisporum Vl32 and Vl43 harbour same‐sex mating‐type genes originating from both parental lineages. (a) Comparison of V. longisporum Vl32 and Vl43 MAT1‐1 and V. dahliae (Vd)/V. alfalfae (Va) mating‐type loci with flanking regions. Lines: intergenic regions; purple arrows:MAT1‐1‐specific regions; brown arrows:MAT1‐2‐specific regions; vertical line: border of the idiomorph sequence. Sequences from Va MAT1‐1 and Vd MAT1‐2 loci from Ensembl Fungi (Kersey et al., 2018) and Vd MAT1‐1 (AB505215.1) were used. (b) Phylogenetic tree of MAT1‐1‐1, MAT1‐1‐3, and a conserved 295‐bp region of VDAG_06529 homologs. Consensus trees based on nucleotide sequences were calculated with PHYLIP program NEIGHBOR. Percentages based on 1,000 replications of the neighbour‐joining procedure are shown. (c) Scanning electron micrographs of hyphal fusion sites (HS) from V. longisporum strains of lineage A1/D1 (Vl43, Vl145c) or A1/D3 (Vl32, Vl19) cultivated on malt extract agar for 3 days are shown (scale bar: 2 μm)

FIGURE 3

Verticillium longisporum strains are two‐parent hybrids. (a) In Vl43 and Vl32 genomes, two identity groups are bimodially resembled in the distribution of relative identity (%id) of long genomic contig (>1,000 kb) alignments. Contigs of Vl43, Vl32, VaMs.102, and VdJR2 were compared to the VdLs.17 genome using Exonerate software (Slater & Birney, 2005). (b) Strain‐specific genes in Verticilllium genomes. Numbers given in the top row represent the total amount of predicted open reading frames (ORFs). VdLs.17 and VaMs.102 were used as V. dahliae and V. alfalfae reference genomes, respectively (Ensembl Fungi; Kersey et al., 2018). Total numbers of predicted ORFs per genome according to Augustus prediction^a and Klosterman et al. (2011)^b are given

FIGURE 4

Genome rearrangements between Verticillium longisporum Vl43 and Vl32. Mapping is based on restriction patterns determined by optical mapping (OptGen). (a) Illustration of Vl43 and Vl32 chromosome numbers and sizes compared to V. dahliae VdLs.17 (de Jonge et al., 2013). (b) The Venn diagram illustrates the length (Mb) of overlapping and unique syntenic regions in Vl43, Vl32, and VdLs.17 genomes. Genome synteny is partially conserved between Vl32 and VdLs.17 and between Vl32 and Vl43. Synteny overlap between Vl43 and VdLs.17 is very low. (c) Chromosome synteny comparison in a Circos diagram reveals a high level of genomic rearrangements by illustration of collinear blocks between 15 Vl43 (red) and 16 Vl32 (green) chromosomes.

FIGURE 5

The Vl43LS20kb region of Verticillium longisporum Vl43 reduces severity of Brassica napus disease symptoms. (a) Arrangement of the 20 kb lineage‐specific (LS) region and adjacent regions in pathogenic (red) and asymptomatic (green) V. longisporum strains compared to V. dahliae VdLs.17 LS regions LS1 (dark grey), 3 (light grey), and 4 (white). Parts of VdLs.17 LS1 located on chromosome 3 as well as parts of LS3 and LS4 regions located on chromosome 4 are in synteny in the genomes of Vl43, Vl145c, and VdJR2, but disrupted in the Vl32 genome at indicated breakpoints. The 20 kb LS region is absent from the Vl32 genome. The Vl43LS20kb region was subdivided into LSI and LSII. Vl43 deletion strains lack LSI, LSII, or the total 20 kb LS region. (b) Absence of the Vl43LS20kb region in the Vl32 genome was verified. Seven genes (LSG1–LSG7) were amplified from DNA of Vl43, but not of Vl32. Histone H2A served as positive control (+). (c) Ex planta phenotypes of Vl43ΔLSI, Vl43ΔLSII, Vl43ΔLS strains on simulated xylem fluid medium (SXM; bottom view) and Czapek Dox medium (CDM) with sucrose or galactose (top view) for 14 days at 25 °C show no significant alterations compared to Vl43. (d) and (e) V. longisporum Vl43ΔLSI, Vl43ΔLSII, and Vl43ΔLS induce more severe disease symptoms in B. napus compared to wild‐type Vl43 and asymptomatic Vl32 35 days postinoculation by root dipping into respective spore solutions or water (Mock). (d) The diagram displays the number of plants with indicated symptoms relative to the total number of plants (n) from three experiments with two independent Vl43ΔLSI, Vl43ΔLSII transformants and one Vl43ΔLS transformant. (e) Representative overview pictures of plants from a single experiment