Ceratocystis cacaofunesta genome analysis reveals a large expansion of extracellular phosphatidylinositol-specific phospholipase-C genes (PI-PLC)

Abstract

Background: The Ceratocystis genus harbors a large number of phytopathogenic fungi that cause xylem parenchyma degradation and vascular destruction on a broad range of economically important plants. Ceratocystis cacaofunesta is a necrotrophic fungus responsible for lethal wilt disease in cacao. The aim of this work is to analyze the genome of C. cacaofunesta through a comparative approach with genomes of other Sordariomycetes in order to better understand the molecular basis of pathogenicity in the Ceratocystis genus.

Results: We present an analysis of the C. cacaofunesta genome focusing on secreted proteins that might constitute pathogenicity factors. Comparative genome analyses among five Ceratocystidaceae species and 23 other Sordariomycetes fungi showed a strong reduction in gene content of the Ceratocystis genus. However, some gene families displayed a remarkable expansion, in particular, the Phosphatidylinositol specific phospholipases-C (PI-PLC) family. Also, evolutionary rate calculations suggest that the evolution process of this family was guided by positive selection. Interestingly, among the 82 PI-PLCs genes identified in the C. cacaofunesta genome, 70 genes encoding extracellular PI-PLCs are grouped in eight small scaffolds surrounded by transposon fragments and scars that could be involved in the rapid evolution of the PI-PLC family. Experimental secretome using LC-MS/MS validated 24% (86 proteins) of the total predicted secretome (342 proteins), including four PI-PLCs and other important pathogenicity factors.

Conclusion: Analysis of the Ceratocystis cacaofunesta genome provides evidence that PI-PLCs may play a role in pathogenicity. Subsequent functional studies will be aimed at evaluating this hypothesis. The observed genetic arsenals, together with the analysis of the PI-PLC family shown in this work, reveal significant differences in the Ceratocystis genome compared to the classical vascular fungi, Verticillium and Fusarium. Altogether, our analyses provide new insights into the evolution and the molecular basis of plant pathogenicity.

Keywords: Ceratocystis wilt of cacao; Phosphoinositide-specific phospholipases C (PI PLC); Plant pathogen.

Fig. 1

Correlation of genomes sizes and number of genes of Ceratocystis species compared with other Sordariomycetes genomes. List of species: CG, Chaetomium globosum; CM, Cordyceps militaris; CT, Chaetomium thermophilum; FG, Fusarium graminearum; FO, Fusarium oxysporum; FS, Fusarium solani (Nectria haematococca); FV, Fusarium verticillioides; GG, Glomerella graminicola (Colletotrichum graminicola); MC, Metarhizium acridum; MG, Magnaporthe grisea; MN, Metarhizium anisopliae (Metarhizium robertsii); NC, Neurospora crassa; NT, Neurospora tetrasperma; OP, Ophiostoma piceae; PA, Podospora anserina; TA, Trichoderma atroviride; TR, Trichoderma reesei; TT, Thielavia terrestris; TV, Trichoderma virens; VA, Verticillium alfafae; VD, Verticillium dahliae (Additional file 15)

Fig. 2

Amounts of CAZymes in each category defined using the dbCAM database. Sordariomycetes proteomes are classified as glycoside hydrolases (GHs), glycosyl transferases (GTs), carbohydrate esterases (CEs), carbohydrate-binding modules (CBMs), auxiliary activities (AAs), and polysaccharide lyases (PLs). The small bars in the right side of the figure are the fractions of CAZymes in each category relative to the total number of gene content in each species. The consensus phylogeny is in the left with branch supports obtained by bayesian posterior probabilities above branches and bootstrap support from likelihood analysis below branches. Nodes marked with S and H represents the taxonomic division in Sordariomycetidae and Hypocreomycetidae. In the center we present a table describing species life style, sapro, Saprotrophic; Entom, Entomopathogenic; P. path, Plant pathogen; Mycop, Mycoparasitism

Fig. 3

Overview of total secreted proteins in C. cacaofunesta compared with C. fimbriata classification using GO terms and Blast functional annotation results

Fig. 4

Phylogenetic inference and expansion/retraction of gene families for Sordariomycetes species. The consensus phylogeny is in the left with branch supports obtained by bayesian posterior probabilities above branches and bootstrap support from likelihood analysis below branches. Nodes marked with S and H represents the taxonomic division in Sordariomycetidae and Hypocreomycetidae. In the center we present a table describing species life style, with colored blocks separated according to monophyletic groups, and the number of protein family clusters and exclusive clusters obtained for each species. The phylogeny is mirrored in the right with branch width relative to the proportion of estimated gene gain (in green) and lost (in red). The absolute values estimated for protein families’ gain and lost are also indicated for each branch. b Phylogenetic inference and Expansion/retraction of gene family in Ceratocystis genus

Fig. 5

Evolution of PI-PLCs genes in Ceratocystis species and the close relative H. moniliformis. Numbers in the red dots displays the quantity of genes related to PI-PLC estimated using birth-death models by BadiRATE for each node

Fig. 6

Schematic representation of major genomic clusters content containing PI-PLC genes in C. cacaofunesta. Numbers are in kilobases. These clusters involve 66 of the 83 PI-PLC genes (red arrows) and show a few other genes within (grey and black arrows)

Fig.7

Phylogeny of PI-PLC gene family in Ceratocystis species. Fifteen clusters were defined being one ancestral, and the other 14 (A-N) equally related in a star-like branch. This figure shows the ancestral cluster and cluster A, being all other in Additional File 11. Posterior probabilities of the Bayesian Inference are above branches. A table in the right compiles information of the proteins

Fig. 8

a and b Ceratocystis PI-PLC molecular model. a. (Left) Catalytic active region is shown in red interacted directly with the ligand inositol or phosphoinositol in Green. (Right) Detailed view of the active site of phospholipases C specific for phosphoinositol crystallographic (in cyan) and modeled (red). The detailed residues refer to the modeled protein. The mutations in Gln140 and Ser227 are conservative and capable of promoting interactions with the inositol molecule, necessary for the maintenance of phosphodiesterase activity. b Detailed view of the active site showed amino acid residues interact directly with the ligand. Amino acid residues of the template (Crystallographic resolve protein in PDB code 1AOD) are showed in cyan and PI-PLC Ceratocystis model in red. Mutations at Ser227 and Gln140 are conservative and able to promote interactions with inositol molecule, necessary for the maintenance of phosphodiesterase activity

Fig. 9

Hypothetic model for a possible role of secreted Ceratocystis PI-PLCs in the context of Ceratocystis Wilt of cacao. The fungal moving into parenchyma of vascular system grow and degrading life primary host plant walls, using a variety of CAZymes including celullases. PI-PLCs proteins have the putative ability to recognize inositol and catalyze the cleavage of phosphatidylinositol (PI) substrates present in the host membranes and could rapid destroy the stability of the cell, specifically the typical structured berried formed by the plant called tyloses. The genome arsenal together with several secreted PI-PLCs proteins produced by C. cacaofunesta, rapidly causing necrosis and degrading the vascular system in cacao plants. Also C. cacaofunesta PI-PLCs potentially cleave glycosylphosphatidylinositol (GPI) anchors proteins present in the surface of the both fungal and plant membranes. These GPI released proteins could for instance produce chitin residues which could elicit plant defense responses, GHs could help to hydrolyze the plant cell wall an arsenal of proteins involved in plant-fungal interaction. Ultimately, secreted PI-PLC of C. cacaofunesta could amplify both signals of the pathogenesis of the fungus and host defenses