A genome survey of Moniliophthora perniciosa gives new insights into Witches' Broom Disease of cacao

Abstract

Background: The basidiomycete fungus Moniliophthora perniciosa is the causal agent of Witches' Broom Disease (WBD) in cacao (Theobroma cacao). It is a hemibiotrophic pathogen that colonizes the apoplast of cacao's meristematic tissues as a biotrophic pathogen, switching to a saprotrophic lifestyle during later stages of infection. M. perniciosa, together with the related species M. roreri, are pathogens of aerial parts of the plant, an uncommon characteristic in the order Agaricales. A genome survey (1.9x coverage) of M. perniciosa was analyzed to evaluate the overall gene content of this phytopathogen.

Results: Genes encoding proteins involved in retrotransposition, reactive oxygen species (ROS) resistance, drug efflux transport and cell wall degradation were identified. The great number of genes encoding cytochrome P450 monooxygenases (1.15% of gene models) indicates that M. perniciosa has a great potential for detoxification, production of toxins and hormones; which may confer a high adaptive ability to the fungus. We have also discovered new genes encoding putative secreted polypeptides rich in cysteine, as well as genes related to methylotrophy and plant hormone biosynthesis (gibberellin and auxin). Analysis of gene families indicated that M. perniciosa have similar amounts of carboxylesterases and repertoires of plant cell wall degrading enzymes as other hemibiotrophic fungi. In addition, an approach for normalization of gene family data using incomplete genome data was developed and applied in M. perniciosa genome survey.

Conclusion: This genome survey gives an overview of the M. perniciosa genome, and reveals that a significant portion is involved in stress adaptation and plant necrosis, two necessary characteristics for a hemibiotrophic fungus to fulfill its infection cycle. Our analysis provides new evidence revealing potential adaptive traits that may play major roles in the mechanisms of pathogenicity in the M. perniciosa/cacao pathosystem.

Figure 1

Flow diagram of bioinformatics procedures applied in M. perniciosa genome survey.

Figure 2

Number of M. perniciosa gene models predicted by ab initio and/or extrinsic prediction methods. Left ellipse: gene models predicted by ab initio methods. Right ellipse: gene models predicted by extrinsic methods. The intersection contains gene models detected by both methods. Underlined: number of gene models with BLASTX-NR E-value similarity ≤ 1e-10. In italics: number of gene models with BLASTX-NR E-value similarity > 1e-10.

Figure 3

Correlation between the average number of M perniciosa gene models and the length of C. cinereus proteins.

Figure 4

Correlation of various organisms' genome size and number of genes (Gene Density). Error bar in M. perniciosa data point depicts the Standard Deviation (SD ± 0.05) of Gene/Kbp ratio using ab initio predicitions, extrinsic predictions and the sum of both predictions (see text).

Figure 5

Comparison of M. perniciosa protein families with other fungi. (A) comparison between cytochrome P450 monooxygenases, carboxylesterases, deuterolysins, thaumatins and aegerolysins; (B) comparison of plant cell wall degrading enzymes from fungi that interact with plants. Mp = Moniliophthora perniciosa, Lb = Laccaria bicolor, Cc = Coprinopsis cinerea, Pc = Phanerochaete chrysosporium, Um = Ustilago maydis, Mg = Magnaporthe grisea, Cn = Cryptococcus neoformans, Fg = Fusarium graminearum, Nc = Neurospora crassa, Sc = Saccharomyces cerevisiae. The legend at the right refers to the nomenclature of plant cell wall degrading enzymes according to CAZy .

Figure 6

Indole-3-acetate (IAA) biosynthesis pathways. M. perniciosa gene models are annotated in red.

Figure 7

An illustrated hypothetical model for WBD. Model correlating classical symptoms of green and dry broom in the field (A, C and E), M. perniciosa development inside cacao (B: biotrophic stage, D: transition from biotrophic to saprotrophic stage, F: saprotrophic stage) and molecular and cellular events displayed by the fungus in each developmental stages, based on genes annotated in genome survey (right side of the panel). Notice in B (right side) the presence of biotrophic mycelia in the apoplast surrounded by intact living cells. Also notice in C the presence of biotrophic mycelia (arrowhead) and saprotrophic mycelia (double arrowhead) inside a necrotic region. Micrographs scales: B left side: 15 μM; B right side: 25 μM; D: 50 μM; F: 50 μM.