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Comparative Study
. 2017 May 2;18(1):339.
doi: 10.1186/s12864-017-3699-1.

Comparative analysis of the predicted secretomes of Rosaceae scab pathogens Venturia inaequalis and V. pirina reveals expanded effector families and putative determinants of host range

Cecilia H Deng  1 , Kim M Plummer  2   3 , Darcy A B Jones  4   5 , Carl H Mesarich  1   6   7 , Jason Shiller  4   8 , Adam P Taranto  4   9 , Andrew J Robinson  4   10 , Patrick Kastner  4 , Nathan E Hall  4   10 , Matthew D Templeton  1   6 , Joanna K Bowen  11
Affiliations
Comparative Study

Comparative analysis of the predicted secretomes of Rosaceae scab pathogens Venturia inaequalis and V. pirina reveals expanded effector families and putative determinants of host range

Cecilia H Deng et al. BMC Genomics. .

Abstract

Background: Fungal plant pathogens belonging to the genus Venturia cause damaging scab diseases of members of the Rosaceae. In terms of economic impact, the most important of these are V. inaequalis, which infects apple, and V. pirina, which is a pathogen of European pear. Given that Venturia fungi colonise the sub-cuticular space without penetrating plant cells, it is assumed that effectors that contribute to virulence and determination of host range will be secreted into this plant-pathogen interface. Thus the predicted secretomes of a range of isolates of Venturia with distinct host-ranges were interrogated to reveal putative proteins involved in virulence and pathogenicity.

Results: Genomes of Venturia pirina (one European pear scab isolate) and Venturia inaequalis (three apple scab, and one loquat scab, isolates) were sequenced and the predicted secretomes of each isolate identified. RNA-Seq was conducted on the apple-specific V. inaequalis isolate Vi1 (in vitro and infected apple leaves) to highlight virulence and pathogenicity components of the secretome. Genes encoding over 600 small secreted proteins (candidate effectors) were identified, most of which are novel to Venturia, with expansion of putative effector families a feature of the genus. Numerous genes with similarity to Leptosphaeria maculans AvrLm6 and the Verticillium spp. Ave1 were identified. Candidates for avirulence effectors with cognate resistance genes involved in race-cultivar specificity were identified, as were putative proteins involved in host-species determination. Candidate effectors were found, on average, to be in regions of relatively low gene-density and in closer proximity to repeats (e.g. transposable elements), compared with core eukaryotic genes.

Conclusions: Comparative secretomics has revealed candidate effectors from Venturia fungal plant pathogens that attack pome fruit. Effectors that are putative determinants of host range were identified; both those that may be involved in race-cultivar and host-species specificity. Since many of the effector candidates are in close proximity to repetitive sequences this may point to a possible mechanism for the effector gene family expansion observed and a route to diversification via transposition and repeat-induced point mutation.

Keywords: Apple; Effector; European pear; Malus x domestica; Pyrus communis; Secretome; Venturia inaequalis; Venturia pirina.

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Figures

Fig. 1
Fig. 1
The secretomes of four isolates of V. inaequalis and one isolate of V. pirina. Annotations were based on gene ontology analysis including interrogation of NCBI RefSeq [168], InterPro [169], UniRef [170], ExPASy UniProtKB/Swiss-Prot [171] and ExPASy Prosite [172], CAZyme identification using the CAT server [27] and BLASTp searches against the NCBI non-redundant database [29]. Small secreted proteins (SSPs) include predicted proteins with similarity to known effectors and proteins with no known function, either with or without putative conserved motifs identified by PfamScan [–38]
Fig. 2
Fig. 2
Proteins in the secretomes of four isolates of Venturia inaequalis and one of V. pirina. Similar proteins in each of the secretomes were identified by OrthoMCL-2.0.3 [30] together with the Markov clustering algorithm mcl-09-149 [173]. Similarity levels were calculated based on reciprocal BLASTp similarity searches between the protein sequences with an e value threshold of 1e-10. Figures within the boxes represent the number of proteins in each cluster, whereas figures outside the wheel are the number of clusters in each sector. Those proteins that are singletons within each secretome are not represented
Fig. 3
Fig. 3
Microscopic evaluation of Venturia inaequalis Vi1 infection of susceptible apple leaves. a Two, and b seven days post inoculation, and c in vitro (in cellophane) showing stromatic growth habit
Fig. 4
Fig. 4
Predicted proteins present in the VIS set from the secretome of Venturia inaequalis isolate Vi1. Annotations were based on gene ontology analysis including interrogation of NCBI RefSeq [168], InterPro [169], UniRef [170], ExPASy UniProtKB/Swiss-Prot [171] and ExPASy Prosite [172], CAZyme identification using the CAT server [27] and BLASTp searches against the NCBI non-redundant database [29]. Small secreted proteins (SSPs) include predicted proteins with similarity to known effectors and proteins with no known function, either with or without putative conserved motifs identified by PfamScan [–38]. The order of the categories in the legend is the same as that in the chart
Fig. 5
Fig. 5
Small secreted proteins (SSPs) in the Venturia inaequalis Vi1 secretome encoded by single genes. Only SSPs ≤200 amino acids in length are included. The number of similar proteins in the Venturia and related Dothideomycete proteomes are indicated by numbers: black indicates a gene predicted by AUGUSTUS, white indicates a putative coding sequence identified by tBLASTn using the protein sequence as query, followed by manual curation. Percentage identity is represented by: red = 100%; orange = 90–99%; yellow = 70–89%; green = 50–69; blue = 30–49%
Fig. 6
Fig. 6
Small secreted protein (SSP) families in four Venturia inaequalis and one V. pirina secretomes. Only SSPs ≤200 amino acids in length are included. Red = proteins in the same family from Vi1; yellow = protein families from Vi1.10; green = families from Vi1.2.8.9; blue = families from ViL; purple = families from Vp. Families f1-f28, and f34-f38 are cysteine rich i.e. two or more cysteines per protein; f29-f33 are those with one or no cysteines. ★ = families with predicted similar proteins in related Dothideomycete genomes
Fig. 7
Fig. 7
Similar small secreted proteins (SSPs) in Dothideomycete proteomes and the Venturia inaequalis Vi1 secretome. Only SSPs ≤200 amino acids in length are included. Shades of blue, purple and pink = Pleosporales: Cochliobolus sativus, C. heterostrophus C4, C. heterostrophus C5, C. lunatus, C. miyabeanus, C. victoriae, Pyrenophora tritici-repentis, P. teres f. teres, Leptosphaeria maculans, Parastagonospora nodorum; shades of orange, yellow and red = Capnodiales: Septoria populicola (teleomorph Mycosphaerella populicola), S. musiva (teleomorph M. populorum), M. fijiensis, Cladosporium fulvum (syn: Passalora fulva), Dothistroma septosporum, Zymoseptoria tritici, Baudoinia compniacensis; green = Dothideales: Aureobasidium pullulans var. pullulans
Fig. 8
Fig. 8
Flanking distance (3′ and 5′) between predicted genes of Venturia inaequalis Vi1. Intergenic distances for all predicted genes are represented in the underlying heatmap, with the number of genes in each bin shown as a colour-coded heat map (on orthogonal projection) generated as in Saunders et al. [185]. Genes were sorted into two-dimensional bins on the basis of the lengths of flanking intergenic distances to neighbouring genes at their 5′ and 3′ ends; overlying this are scatterplots of a 423 Core Eukaryotic Genes (white dots) or b Venturia infection secretome (VIS) gene set, plus AvrLm6- and Ave1-like genes (coloured dots: dark pink = SSPs with two or more cysteines (≤500 amino acids); light pink = SSPs with one or no cysteines (≤500 amino acids); blue = peptidases; dark green = CAZymes; light green = putative cell wall-degrading enzymes (non-CAZyme); white = cell wall associated and miscellaneous proteins >500 amino acids). Note that the axes are not linear. Genes at the scaffold end were excluded from this analysis
Fig. 9
Fig. 9
Distance between predicted genes and transposable element (TE)-like features of Venturia inaequalis Vi1. Flanking distances (3′ and 5′) to TE-like features for all predicted genes are represented in the underlying heat map, with the number of genes in each bin shown as a colour-coded heat map on orthogonal projection (generated as in Saunders et al. [185]). Genes were sorted into two dimensional bins on the basis of the lengths of flanking distances. a 423 Core Eukaryotic Genes (white dots) are similar to all genes in that they are not closely associated with TE-like features; whereas b Venturia infection secretome (VIS) gene set, plus AvrLm6- and Ave1-like genes (coloured dots: dark pink = SSPs with two or more cysteines (≤500 amino acid); light pink = SSPs with one or no cysteines or less (≤500 amino acids); blue = enzymes (peptidases/redox/primary metabolism); dark green = CAZymes; light green = putative cell wall-degrading enzymes (non-CAZyme); white = cell wall associated and miscellaneous proteins >500 amino acids in length). Note that the axes are not linear. Genes at the scaffold end were excluded from this analysis
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