Summary of the genome assembly and annotation statistics.

<p>Summary of the genome assembly and annotation statistics.</p

Hierarchical clustering of <i>Pseudocercospora musae</i>, <i>Pseudocercospora eumusae</i>, and <i>Pseudocercospora fijiensis</i> based on copy number changes in different groups of KOG gene families.

<p>Hierarchical clustering of the species based on (A) the KOG distribution profile (i.e. the number of genes assigned to each category of KOG) of their entire proteomes, (B) the KOG distribution profiles of the 575 core gene families with copy number variation (CNV), and (C) a subset of 190 core gene families with CNV that are predicted to be involved in metabolism based on KOG assignments. The reliability of the clustering patterns was assessed by bootstrap tests (1000 replicates) and obtained bootstrap values are indicated next to their corresponding branching nodes. While clustering of the species based on the KOG distribution profiles of the entire proteomes follows a pattern that is respective of the their phylogenetic relations (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005904#pgen.1005904.g002" target="_blank">Fig 2</a>), clustering of the species based on the KOG profiles of core gene families with CNV or their subset of gene families involved in metabolism, indicates a swapped topology in which <i>P</i>. <i>eumusae</i> is clustered together with <i>P</i>. <i>fijiensis</i> suggesting that these two species share a more similar pattern of gene family expansions and contractions.</p

Hierarchical clustering of <i>Pseudocercospora musae</i>, <i>Pseudocercospora eumusae</i>, <i>Pseudocercospora fijiensis</i>, and 16 other representative Dothideomycete fungi with different nutritional lifestyles, based on copy number changes in carbohydrate-active enzyme (CAZyme) families or the subset of plant cell wall degrading enzymes (PCWDEs).

<p>The selected 16 representative Dothideomycete species that are included in the analysis fall into three major orders: Capnodiales (red), Hysteriales (blue), and Pleosporales (green). The nutritional lifestyle of each species is indicated by a colored dot next to each species name: biotrophs (blue), hemi-biotrophs (green), necrotrophs (yellow), saprophytes (red). (A) Hierarchical clustering of the species based on their total CAzyme distribution profile (i.e. the number of genes assigned to each CAZyme family) (B) Hierarchical clustering of the species based on their distribution profile for CAZyme families related to plant cell wall degradation. In both cases, clustering supported a swapped topology in which <i>P</i>. <i>eumusae</i> is clustered together with <i>P</i>. <i>fijiensis</i>, suggesting that these two species share a more similar pattern of gene family expansions and contractions in CAZymes and PCWDEs in particular.</p

Molecular phylogeny of the three species that constitute the Sigatoka disease complex and 16 other representative Dothideomycetous fungi.

<p>The maximum likelihood (ML) tree was constructed based on a concatenated sequence alignment of 46 orthologous single-copy genes. Bootstrap values (%) are indicated next to corresponding branching nodes. <i>Aspergillus nidulans</i> (class of Eurotiomycetes) was used as an outgroup species for rooting the tree. The selected 16 representative Dothideomycete species that are included in the phylogeny fall into three major orders, i.e. Capnodiales (red), Hysteriales (blue), and Pleosporales (green). In the inferred topology <i>P</i>. <i>musae</i>, <i>P</i>. <i>eumusae</i>, and <i>P</i>. <i>fijiensis</i> are strongly clustered (bootstrap value of 100%) as a monophyletic clade within the Capnodiales, whereas <i>P</i>. <i>eumusae</i> is sister to <i>P</i>. <i>musae</i> (bootstrap value of 100%), suggesting an earlier split of <i>P</i>. <i>fijiensis</i> from the common ancestor of these two species.</p

Shared and species-specific gene families and genes in <i>Pseudocercospora musae</i>, <i>Pseudocercospora eumusae</i>, and <i>Pseudocercospora fijiensis</i>.

<p>(A) Venn diagram showing the total number of species-specific genes and shared gene families among the three species, as determined by reciprocal BlastP best hit (e-value: 1e-5) analysis implemented in OrthoMCL. A larger number of species-specific genes are found in <i>P</i>. <i>fijiensis</i>, whereas more gene families are shared between <i>P</i>. <i>eumusae</i> and <i>P</i>. <i>fijiensis</i> as compared to <i>P</i>. <i>eumusae</i> and <i>P</i>. <i>musae</i>, or <i>P</i>. <i>musae</i> and <i>P</i>. <i>fijiensis</i>. (B) The Venn diagram is expanded to include a broader comparison of the three species gene content against the NCBI nr database and the JGI fungal genome database (BlastP e-value: 1e-5, alignment coverage > 50%). In both Venn diagrams, the number of genes from each species included within the pool of shared gene families is indicated at every intersection.</p

Genome size and composition in <i>Pseudocercospora musae</i>, <i>Pseudocercospora eumusae</i>, and <i>Pseudocercospora fijiensis</i>.

<p>(A) Overall genome composition and repeat content in <i>P</i>. <i>musae</i>, <i>P</i>. <i>eumusae</i>, and <i>P</i>. <i>fijiensis</i>. The size (Mb) and proportion (%) of the main components of the species’ genomes are indicated. Given their close evolutionary relationships, the species show considerable differences in genome size mainly due to differences in repeat content. (B) The distribution and composition of repeat elements in <i>P</i>. <i>musae</i>, <i>P</i>. <i>eumusae</i>, and <i>P</i>. <i>fijiensis</i>. The proportion (%) and size (Mb) of each individual class of repeat elements are indicated. The three species differ in their composition and proportion of the different classes or repeat elements.</p

Disease symptoms caused on banana by the three species that constitute the Sigatoka disease complex.

<p>(A–C) Leaf symptoms and conidia of <i>Pseudocercospora eumusae</i>. (D–F) Leaf symptoms and conidia of <i>Pseudocercospora fijiensis</i>. (G–I) Leaf symptoms and conidia of <i>Pseudocercospora musae</i>. Scale bars = 10 μm. (leaf photo credits Profs. A. Viljoen and G. Kema).</p

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<p>Pseudocercospora fijiensis, causal agent of the black Sigatoka disease (BSD) of Musa spp., has spread globally since its discovery in Fiji 1963 to all the banana and plantain growing areas across the globe. It is becoming the most damaging and economically important disease of this crop. The identification and characterization of genes that regulate infection processes and pathogenicity in P. fijiensis will provide important knowledge for the development of disease-resistant cultivars. In many fungal plant pathogens, the Fus3 and Slt2 are reported to be essential for pathogenicity. Fus3 regulates filamentous-invasion pathways including the formation of infection structures, sporulation, virulence, and invasive and filamentous growth, whereas Slt2 is involved in the cell-wall integrity pathway, virulence, invasive growth, and colonization in host tissues. Here, we used RNAi-mediated gene silencing to investigate the role of the Slt2 and Fus3 homologs in P. fijiensis in pathogen invasiveness, growth and pathogenicity. The PfSlt2 and PfFus3 silenced P. fijiensis transformants showed significantly lower gene expression and reduced virulence, invasive growth, and lower biomass in infected leaf tissues of East African Highland Banana (EAHB). This study suggests that Slt2 and Fus3 MAPK signaling pathways play important roles in plant infection and pathogenic growth of fungal pathogens. The silencing of these vital fungal genes through host-induced gene silencing (HIG) could be an alternative strategy for developing transgenic banana and plantain resistant to BSD.</p

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<p>Pseudocercospora fijiensis, causal agent of the black Sigatoka disease (BSD) of Musa spp., has spread globally since its discovery in Fiji 1963 to all the banana and plantain growing areas across the globe. It is becoming the most damaging and economically important disease of this crop. The identification and characterization of genes that regulate infection processes and pathogenicity in P. fijiensis will provide important knowledge for the development of disease-resistant cultivars. In many fungal plant pathogens, the Fus3 and Slt2 are reported to be essential for pathogenicity. Fus3 regulates filamentous-invasion pathways including the formation of infection structures, sporulation, virulence, and invasive and filamentous growth, whereas Slt2 is involved in the cell-wall integrity pathway, virulence, invasive growth, and colonization in host tissues. Here, we used RNAi-mediated gene silencing to investigate the role of the Slt2 and Fus3 homologs in P. fijiensis in pathogen invasiveness, growth and pathogenicity. The PfSlt2 and PfFus3 silenced P. fijiensis transformants showed significantly lower gene expression and reduced virulence, invasive growth, and lower biomass in infected leaf tissues of East African Highland Banana (EAHB). This study suggests that Slt2 and Fus3 MAPK signaling pathways play important roles in plant infection and pathogenic growth of fungal pathogens. The silencing of these vital fungal genes through host-induced gene silencing (HIG) could be an alternative strategy for developing transgenic banana and plantain resistant to BSD.</p

<i>Cf</i>-mediated hypersensitive responses (HR) triggered by wild-type and mutant Avr proteins.

<p><i>Avr</i>-wild-type and mutant genes were cloned in the PVX vector and the recombinant PVX virus was assayed for HR-inducing activity on <i>Cf-2</i>, <i>Cf-4</i> or <i>Cf-5</i> tomato plants. (<b>A</b>) PVX-mediated expression of wild-type Avr2 protein causes strong HR-inducing activity and eventually kills <i>Cf-2</i> tomato plants, but mutant Avr2 protein (p.Cys63Phe substitution) present in isolate CF212 lost HR-inducing activity. (<b>B</b>) PVX-mediated expression of wild-type Avr4 protein causes strong HR-inducing activity and eventually kills <i>Cf-4</i> tomato plants. Five different mutant Avr4 proteins (substituted amino acid residues are indicated) all lost HR-inducing activity on <i>Cf-4</i> tomato plants. (<b>C</b>) PVX-mediated expression of wild-type Avr5 protein elicits a strong HR on <i>Cf-5</i> tomato plants, whereas mutant Avr5 protein (p.Gly90Arg substitution) lost HR-inducing activity. Plants were photographed at 3 weeks post inoculation.</p