Genome sequencing and comparative transcriptomics of the model entomopathogenic fungi Metarhizium anisopliae and M. acridum

Abstract

Metarhizium spp. are being used as environmentally friendly alternatives to chemical insecticides, as model systems for studying insect-fungus interactions, and as a resource of genes for biotechnology. We present a comparative analysis of the genome sequences of the broad-spectrum insect pathogen Metarhizium anisopliae and the acridid-specific M. acridum. Whole-genome analyses indicate that the genome structures of these two species are highly syntenic and suggest that the genus Metarhizium evolved from plant endophytes or pathogens. Both M. anisopliae and M. acridum have a strikingly larger proportion of genes encoding secreted proteins than other fungi, while ~30% of these have no functionally characterized homologs, suggesting hitherto unsuspected interactions between fungal pathogens and insects. The analysis of transposase genes provided evidence of repeat-induced point mutations occurring in M. acridum but not in M. anisopliae. With the help of pathogen-host interaction gene database, ~16% of Metarhizium genes were identified that are similar to experimentally verified genes involved in pathogenicity in other fungi, particularly plant pathogens. However, relative to M. acridum, M. anisopliae has evolved with many expanded gene families of proteases, chitinases, cytochrome P450s, polyketide synthases, and nonribosomal peptide synthetases for cuticle-degradation, detoxification, and toxin biosynthesis that may facilitate its ability to adapt to heterogeneous environments. Transcriptional analysis of both fungi during early infection processes provided further insights into the genes and pathways involved in infectivity and specificity. Of particular note, M. acridum transcribed distinct G-protein coupled receptors on cuticles from locusts (the natural hosts) and cockroaches, whereas M. anisopliae transcribed the same receptor on both hosts. This study will facilitate the identification of virulence genes and the development of improved biocontrol strains with customized properties.

Figure 1. Major stages in the infection cycle of Metarhizium.

(A) A germinating conidium producing an appressorium. (B) Mycelia attacked by hemocytes after cuticular penetration. (C) Budding yeast-type cells (blastospores) produced by the fungus to facilitate dispersal in insect hemocoel. (D) Cadaver showing emerging hyphae producing conidia (E). CO, conidium; AP, appressorium. Bar, 5 µm.

Figure 2. Homology, syntenic, and phylogenomic relationships of M. anisopliae and M. acridum.

(A) Predicted proteins in M. anisopliae (MAA) and M. acridum (MAC) were compared with the genome encoding proteins of Aspergillus nidulans (AN), A. fumigatus (AF), Neurospora crassa (NC), Magnaporthe oryzae (MO), Fusarium graminearum (FG), Epichloë festucae (EF), Botrytis cinerea (BC) and Sclerotinia sclerotiorum (SS). The diagram was constructed with a cut off E-value <1×10⁻⁵. (B) Dot blot of M. anisopliae and M. acridum using ordered scaffold data. (C) Phylogenetic tree constructed using the Dayhoff amino acid substitution model showing the evolutionary relationships of 16 fungal species. MY =  million years.

Figure 3. Families of transposase genes and estimation of RIP.

Families of transposase genes (A) and estimation of RIP (B) in M. anisopliae and M. acridum.

Figure 4. Functional classification and comparison of M. anisopliae and M. acridum proteins.

Each circle represents the relative fraction of genes represented in each of the categories for each genome. The gene numbers are also shown.

Figure 5. Unrooted phylogenetic trees showing differences in gene expansion in M. anisopliae and M. acridum.

(A) Trypsins; (B) Subtilisins; (C) GH18 chitinases; (D) Cytochrome P450s. Black branches identify orthologous loci in M. anisopliae and M. acridum. Red and green branches identify genes that are only present in M. anisopliae or M. acridum, respectively. Lineage specific genes expressed by these species on cockroach cuticle are marked with a blue asterisk. Lineage specific genes expressed on locust cuticle are marked with a purple circle. Protease family classification refers to Table S7 for trypsins and Table S8 for subtilisins.

Figure 6. Differential gene expression by M. anisopliae (MAA) and M. acridum (MAC) on locust (LO) and cockroach (CO) hind wings.

Genes differentially expressed by M. anisopliae (A) and M. acridum (B) infecting cockroach versus locust cuticles. Genes differentially expressed by M. anisopliae versus M. acridum on cockroach (C) and locust (D) cuticles. The figures in parentheses are the number of genes significantly up- or down-regulated by each fungus.

Figure 7. Differentially regulated signaling pathways employed by M. anisopliae and M. acridum infecting cockroach and locust cuticles.

Both the MAP kinase and cAMP dependent protein kinase A (PKA) pathways were activated by M. anisopliae and M. acridum infecting cockroach and locust cuticles. PLC, phosphatidyl inositol-specific phospholipase C; PIP2, phosphatidylinositol 4,5-bisphosphate; IP3, inositol 1,4,5-triphosphate; DAG, diacylglycerol; PKC, protein kinase C; CaMK, calcium/calmodulin regulated kinase; ERK, extracellular signal-regulated protein kinase; CREB, a basic leucine zipper transcription factor that is a potential cAMP response element-binding protein; CO, conidium; AP, appressorium. Thicker arrows indicate pathways that are more highly expressed by M. acridum on either locust or cockroach cuticles.