Genomic and proteomic analyses of the fungus Arthrobotrys oligospora provide insights into nematode-trap formation

Abstract

Nematode-trapping fungi are "carnivorous" and attack their hosts using specialized trapping devices. The morphological development of these traps is the key indicator of their switch from saprophytic to predacious lifestyles. Here, the genome of the nematode-trapping fungus Arthrobotrys oligospora Fres. (ATCC24927) was reported. The genome contains 40.07 Mb assembled sequence with 11,479 predicted genes. Comparative analysis showed that A. oligospora shared many more genes with pathogenic fungi than with non-pathogenic fungi. Specifically, compared to several sequenced ascomycete fungi, the A. oligospora genome has a larger number of pathogenicity-related genes in the subtilisin, cellulase, cellobiohydrolase, and pectinesterase gene families. Searching against the pathogen-host interaction gene database identified 398 homologous genes involved in pathogenicity in other fungi. The analysis of repetitive sequences provided evidence for repeat-induced point mutations in A. oligospora. Proteomic and quantitative PCR (qPCR) analyses revealed that 90 genes were significantly up-regulated at the early stage of trap-formation by nematode extracts and most of these genes were involved in translation, amino acid metabolism, carbohydrate metabolism, cell wall and membrane biogenesis. Based on the combined genomic, proteomic and qPCR data, a model for the formation of nematode trapping device in this fungus was proposed. In this model, multiple fungal signal transduction pathways are activated by its nematode prey to further regulate downstream genes associated with diverse cellular processes such as energy metabolism, biosynthesis of the cell wall and adhesive proteins, cell division, glycerol accumulation and peroxisome biogenesis. This study will facilitate the identification of pathogenicity-related genes and provide a broad foundation for understanding the molecular and evolutionary mechanisms underlying fungi-nematodes interactions.

Figure 1. Saprophytic and parasitic stages of the nematode-trapping fungus A. oligospora.

The life cycle of A. oligospora includes three stages: saprophytic stage, transitional stage, and parasitic stage. The parasitic stage can be divided into the following six steps: I. Trap formation; II. Attraction; III. Adhesion; IV. Capturing; V, Penetration and immobilization; and VI. Digestion and assimilation. The saprophytic stage (VII) and sexual stage (VIII) are also shown.

Figure 2. Annotation of the A. oligospora genome and proteome by COG/KOG categories.

The proteins were assigned into different KOG/COG functionary categories as shown in the pie chart. A. A total of 5762 proteins from A. oligospora genome were annotated by KOG/COG. B. Classification of the A. oligospora intracellular proteins detected by 2-DE. A total of 861 proteins were identified from A. oligospora cells treated with and without NE for 10 h and 48 h respectively.

Figure 3. The distributions of A. oligospora genes and PHI putative genes in different categories.

The other 10 fungal genomes were divided into non-pathogen and pathogen genomes. Based on orthology analysis, the genes in A. oligospora were classified into four categories: ao-specific (only found in A. oligospora), ao/pathogen (also found in pathogenic fungi), ao/pathogen/non-pathogen (found in all genomes) and ao/non-pathogen (also found in non-pathogenic fungi). A. The distribution of all A. oligospora genes in different categories. B. The distribution of PHI putative genes in different categories.

Figure 4. The phylogenomic tree was constructed based on orthologous proteins from 11 fungal genomes using Maximum likelihood method.

Figure 5. Comparison of pathogenicity-related gene families between A. oligospora and other sequenced fungi.

Several pathogenicity-related enzyme families were compared between A. oligospora and other 10 sequenced fungi including A. fumigatus (afu), C. globosum (cgl), C. immitis (cim), A. nidulans (eni), F. graminearum (fgr), H. capsulatum (hca), M. grisea (mgr), N. crassa (ncr), S. cerevisiae (sce) and V. dahliae (vda).

Figure 6. Disruption of the gene p186 and nematocidal activity analysis.

WT, wild-type strain. MT1 and MT2, mutant strains 1 and 2. Each point represents the average of five independent assays, and the bars represent the standard deviation values. *P<0.05 versus wild strain.

Figure 7. A proposed model for trap formation in A. oligospora.

Protein information can be found in Table S7 . In this model, multiple fungal signal transduction pathways are activated by its nematode prey to further regulate downstream genes associated with diverse cellular processes such as energy metabolism, biosynthesis of the cell wall and adhesive proteins, cell division, glycerol accumulation and peroxisome biogenesis. Black letters in red background represented the up-regulated proteins. Oval represented the 2-D results and quadrate represented the qPCR results.