Comparative genomics reveals the in planta-secreted Verticillium dahliae Av2 effector protein recognized in tomato plants that carry the V2 resistance locus

Abstract

Plant pathogens secrete effector molecules during host invasion to promote colonization. However, some of these effectors become recognized by host receptors to mount a defence response and establish immunity. Recently, a novel resistance was identified in wild tomato, mediated by the single dominant V2 locus, to control strains of the soil-borne vascular wilt fungus Verticillium dahliae that belong to race 2. With comparative genomics of race 2 strains and resistance-breaking race 3 strains, we identified the avirulence effector that activates V2 resistance, termed Av2. We identified 277 kb of race 2-specific sequence comprising only two genes encoding predicted secreted proteins that are expressed during tomato colonization. Subsequent functional analysis based on genetic complementation into race 3 isolates and targeted deletion from the race 1 isolate JR2 and race 2 isolate TO22 confirmed that one of the two candidates encodes the avirulence effector Av2 that is recognized in V2 tomato plants. Two Av2 allelic variants were identified that encode Av2 variants that differ by a single acid. Thus far, a role in virulence could not be demonstrated for either of the two variants.

Fig. 1

Pathogenicity phenotyping of a collection of Verticillium dahliae strains on tomato. A. Typical appearance of V dahliae infection by strain JR2, TO22 and HOMCF as representatives for race 1, 2 and 3, respectively, on Moneymaker (MM) plants that lack known V. dahliae resistance genes, Ve1‐transgenic Moneymaker plants that are resistant against race 1 and not against race 2 or 3 strains, and Aibou plants that carry Ve1 and V2 and are therefore resistant against race 1 as well as race 2 strains, but not against race 3 strains at 21 days post inoculation (dpi). B–D. Measurement of V. dahliae‐induced stunting on wild‐type Moneymaker plants (B), Ve1‐transgenic Moneymaker plants (35S:Ve1) (C) and Aibou plants (D) at 21 dpi. The graphs show collective data from four different experiments indicated with different symbols (circles, squares, triangles and plus symbols), and asterisks indicate significant differences between V. dahliae‐ and mock‐inoculated plants as determined with an ANOVA followed by a Fisher's LSD test (P < 0.01). [Color figure can be viewed at wileyonlinelibrary.com]

Fig. 2

Expression of V. dahliae candidate avirulence effector genes in vitro and during colonization of tomato plants. To assess in planta expression, 12‐day‐old tomato cv. Moneymaker seedlings were root‐inoculated with V. dahliae strain JR2 (A) or strain TO22 (B), and plants were harvested from 4 to 14 days post inoculation (dpi), while conidiospores were harvested from 5‐day‐old cultures of V. dahliae on potato dextrose agar (PDA) to monitor in vitro expression. Real‐time PCR was performed to determine the relative expression of XLOC_00170, Evm_344 and the race 1‐specific effector gene VdAve1 as a positive control (de Jonge et al., 2012) for strain JR2, using V. dahliae GAPDH as reference (A). Similarly, the relative expression of XLOC_00170, Evm_344 and six additional avirulence effector genes for strain TO22, using V. dahliae GAPDH as reference (B).

Fig. 3

Genetic complementation demonstrates that XLOC_00170 encodes the avirulence effector Av2 that is recognized in V2 plants. A. Top pictures of Moneymaker plants that lack known V. dahliae resistance genes (MM), Ve1‐transgenic Moneymaker plants that are resistant against race 1 and not against race 2 strains of V. dahliae (MM 35S:Ve1), and Aibou plants that carry Ve1 and V2 and are therefore resistant against race 1 as well as race 2 strains of the pathogen (Usami et al., 2017) inoculated with the race 3 WT strains GF‐CB5 and HOMCF, and two independent genetic complementation lines that express XLOC_00170 or Evm_344, and the race 2 strain TO22. B. Quantification of stunting caused by the various V. dahliae genotypes on the various tomato genotypes as detailed for panel (A). Each combination is represented by the measurement of five plants. C. Quantification of fungal biomass with real‐time PCR determined for the various V. dahliae genotypes on the various tomato genotypes as detailed for panel (A). Each combination is represented by the fungal biomass quantification in five plants. Asterisks indicate significant differences between V. dahliae‐ and mock‐inoculated plants as determined with an ANOVA followed by a Fisher's LSD test (P < 0.01). [Color figure can be viewed at wileyonlinelibrary.com]

Fig. 4

Targeted deletion confirms that XLOC_00170 encodes the avirulence effector Av2 that is recognized in V2 plants. A. Top pictures of Moneymaker plants that lack known V. dahliae resistance genes (MM), Ve1‐transgenic Moneymaker plants that are resistant against race 1 and not against race 2 strains of V. dahliae (35S:Ve1), and Aibou plants that carry Ve1 and V2 and are therefore resistant against race 1 as well as race 2 strains of the pathogen (Usami et al., 2017) inoculated with the race 3 strains GF‐CB5 and HOMCF, the race 1 WT strain JR2, the deletion line JR2ΔAve1, two independent knock‐out lines of XLOC_00170 in JR2ΔAve1, the race 2 WT strain TO22 and two independent knock‐out lines of XLOC_00170 in TO22. B. Quantification of stunting. C. Quantification of fungal biomass with real‐time PCR caused by the various V. dahliae genotypes on the various tomato genotypes as detailed for panel (A). Different symbols (empty circles, filled circles and triangles) refer to five plants from three different experiments. Asterisks indicate significant differences between V. dahliae‐ and mock‐inoculated plants as determined with an ANOVA followed by a Fisher's LSD test (P < 0.01). [Color figure can be viewed at wileyonlinelibrary.com]

Fig. 5

Amino acid alignment of Av2 homologues found in few fungal species. Based on BLAST analyses, homologues of V. dahliae Av2 could only be identified in V. longisporum, V. nonalfalfae, V. alfalfa and in Fusarium phyllophilum, F. mundagurra, three F. oxysporum lineages and in Fusarium sp. NRRL66182. Global alignments were performed using ClustalW and visualized with Espript3. Conserved amino acids are shown in white font on red background. [Color figure can be viewed at wileyonlinelibrary.com]

Fig. 6

Phylogenetic tree of sequenced V. dahliae strains with indication of presence‐absence variation for the Ave1 and Av2 effectors. Strains that were phenotyped and included in the comparative genomics (Table 2) are shown in bold. Presence of the avirulence genes VdAve1 and Av2, and the race designation based on the presence or absence of these genes are indicated. Phylogenetic relationships between sequenced V. dahliae strains were inferred using Realphy (Langmead and Salzberg, 2012), and branch length represents sequence divergence.

Fig. 7

Presence–absence variation in the region surrounding the two candidate Av2 genes (A) genomic region flanking the Av2 candidate genes in 17 isolates detailed in Fig. 3. The matrix shows the presence (black/grey) and absence (white) in 100 bp non‐overlapping windows for Av2 variant E₇₃ (black) and Av2 variant V₇₃ grey. On top, annotated genes are displayed in black and repetitive elements in green, while Av2 is displayed in red and Evm_344 in blue. (B) Read coverage for V. dahliae strain JR2 that encodes Av2 variant E₇₃ and strain DVD‐S26 that encodes Av2 variant V₇₃ depicting a transposable element deletion in isolates that produce the V₇₃ variant. [Color figure can be viewed at wileyonlinelibrary.com]

Fig. 8

Allelic variation of Av2 in Verticillium dahliae. A. Gene model for Av2. The asterisk indicates the approximate position of the single (A to T) nucleotide substitution in 7 of the 17 isolates that carry the gene, leading to a single amino acid substitution (E₇₃V). B. Genomic sequence of Av2. The arrow shows the position of the single nucleotide substitution found in particular strains. C. Alignment of exon 3 of Av2 in the 17 strains containing the avirulence effector gene. The arrow shows the single nucleotide substitution that occurs in seven of the strains when compared with strain JR2. D. Av2 amino acid sequence as encoded by V. dahliae strain JR2 with E₇₃ that is substituted by V in seven isolates indicated by an arrow. [Color figure can be viewed at wileyonlinelibrary.com]