Evolution of a subtilisin-like protease gene family in the grass endophytic fungus Epichloë festucae

Abstract

Background: Subtilisin-like proteases (SLPs) form a superfamily of enzymes that act to degrade protein substrates. In fungi, SLPs can play either a general nutritive role, or may play specific roles in cell metabolism, or as pathogenicity or virulence factors.

Results: Fifteen different genes encoding SLPs were identified in the genome of the grass endophytic fungus Epichloë festucae. Phylogenetic analysis indicated that these SLPs belong to four different subtilisin families: proteinase K, kexin, pyrolysin and subtilisin. The pattern of intron loss and gain is consistent with this phylogeny. E. festucae is exceptional in that it contains two kexin-like genes. Phylogenetic analysis in Hypocreales fungi revealed an extensive history of gene loss and duplication.

Conclusion: This study provides new insights into the evolution of the SLP superfamily in filamentous fungi.

Figure 1

Gene and protein structures of E. festucae Fl1 SLPs. A. Genetic maps showing gene arrangement around the E. festucae SLP loci. Black arrows indicate the SLP genes, with non-protease genes indicated by light grey arrows. Gene names, where specific names have been assigned (in E. festucae Fl1), are shown above the genes. Systematic numbers assigned during annotation of the E. festucae E2368 genome refer to the remaining genes. Scale bars indicate a length of 1 kb. B. Domain organization of the E. festucae SLP proteins. Conserved catalytic triad residues are shown in circles above domain structures. The polypeptide is indicated in grey and colored blocks as follows: the signal peptide in red, subtilisin N-terminal domains in yellow, peptidase S8 domains in green, PA domains in black, DUF1034 domains in blue, kexin propeptides in orange, P domains in purple, serine/threonine-rich domains in teal and transmembrane domains in white. Conserved kexin autocatalytic cleavage sites are indicated by black arrows. Conserved RGD motifs in the kexin family are indicated by an asterisk. Scale bars indicate a length of 50 amino acid residues.

Figure 2

Evolutionary relationships of E. festucae Fl1 SLPs. Neighbor joining analysis determined phylogenetic relationships. The phylogram is rooted using the Bacillus subtilis subtilisin Carlsberg protein (accession P00780) as an outgroup. The percentages of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. SLP families and subfamilies are indicated by black or grey square brackets respectively.

Figure 3

Exon-intron structures of E. festucae SLPs. Exon-intron structures of the E. festucae SLP like genes: proteinase K (A), fungal pyrolysins (B), and kexins (C). Each exon is indicated by a separate rectangle, with spaces between rectangles representing introns. Intron positions found in each family are indicated by numbers between exons. All scale bars indicate a length of 100 bp.

Figure 4

E. festucae SLP genes sharing conserved microsynteny with other fungal species. Conserved microsynteny at the prtD (A), prtG (B), prtK (C). and kexA (D) loci. SLP genes are shown in red. Non-orthologous genes are shown in grey or white. Orthologous genes are indicated by colours other than red, grey or white. All scale bars indicate a length of 2 kb.

Figure 5

Evolutionary relationships of Hypocreaceae SLPs based on neighbor joining analysis. The phylogram is rooted using the Bacillus subtilis subtilisin Carlsberg protein (accession P00780) as an outgroup. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. SLP families and subfamilies are indicated by black or grey square brackets respectively. E. festucae sequences are marked by black circles. Note: FOXG_14793 does not group with sf3 (the vacuolar SLPs) or sf4 (characterized by FGSG_09382) but falls into a group by itself. Additional analysis showed it groups with proteases from P. anserina and N. fischeri.