New insights into the evolution of subtilisin-like serine protease genes in Pezizomycotina

<p>Abstract</p> <p>Background</p> <p>Subtilisin-like serine proteases play an important role in pathogenic fungi during the penetration and colonization of their hosts. In this study, we perform an evolutionary analysis of the subtilisin-like serine protease genes of subphylum Pezizomycotina to find if there are similar pathogenic mechanisms among the pathogenic fungi with different life styles, which utilize subtilisin-like serine proteases as virulence factors. Within Pezizomycotina, nematode-trapping fungi are unique because they capture soil nematodes using specialized trapping devices. Increasing evidence suggests subtilisin-like serine proteases from nematode-trapping fungi are involved in the penetration and digestion of nematode cuticles. Here we also conduct positive selection analysis on the subtilisin-like serine protease genes from nematode-trapping fungi.</p> <p>Results</p> <p>Phylogenetic analysis of 189 subtilisin-like serine protease genes from Pezizomycotina suggests five strongly-supported monophyletic clades. The subtilisin-like serine protease genes previously identified or presumed as endocellular proteases were clustered into one clade and diverged the earliest in the phylogeny. In addition, the cuticle-degrading protease genes from entomopathogenic and nematode-parasitic fungi were clustered together, indicating that they might have overlapping pathogenic mechanisms against insects and nematodes. Our experimental bioassays supported this conclusion. Interestingly, although they both function as cuticle-degrading proteases, the subtilisin-like serine protease genes from nematode-trapping fungi and nematode-parasitic fungi were not grouped together in the phylogenetic tree. Our evolutionary analysis revealed evidence for positive selection on the subtilisin-like serine protease genes of the nematode-trapping fungi.</p> <p>Conclusions</p> <p>Our study provides new insights into the evolution of subtilisin-like serine protease genes in Pezizomycotina. Pezizomycotina subtilisins most likely evolved from endocellular to extracellular proteases. The entomopathogenic and nematode-parasitic fungi likely share similar properties in parasitism. In addition, our data provided better understanding about the duplications and subsequent functional divergence of subtilisin-like serine protease genes in Pezizomycotina. The evidence of positive selection detected in the subtilisin-like serine protease genes of nematode-trapping fungi in the present study suggests that the subtilisin-like serine proteases may have played important roles during the evolution of pathogenicity of nematode-trapping fungi against nematodes.</p

Genomic and Proteomic Analyses of the Fungus Arthrobotrys oligospora Provide Insights into Nematode-Trap Formation

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

Fourteen New Species of Foliar Colletotrichum Associated with the Invasive Plant Ageratinaadenophora and Surrounding Crops

Ageratina adenophora is one of the most invasive weeds in China. Following an outbreak in Yunnan in the 1960s, A. adenophora has been spreading in Southwest China at tremendous speed. Previous research indicated A. adenophora contained many Colletotrichum species as endophytes. In this study, we investigated the diversity of Colletotrichum in healthy and diseased leaves of the invasive plant A. adenophora and several surrounding crops in Yunnan, Guangxi, and Guizhou provinces in China, and obtained over 1000 Colletotrichum strains. After preliminary delimitation using the internal transcribed spacer region (ITS) sequences, 44 representative strains were selected for further study. Their phylogenetic positions were determined by phylogenetic analyses using combined sequences of ITS, actin (ACT), chitin synthase (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta-tubulin (TUB2). Combined with morphological characteristics, 14 new Colletotrichum species were named as C. adenophorae, C. analogum, C. cangyuanense, C. dimorphum, C. gracile, C. nanhuaense, C. nullisetosum, C. oblongisporum, C. parvisporum, C. robustum, C. simulanticitri, C. speciosum, C. subhenanense, and C. yunajiangense

The complete mitochondrial genomes of Dactylellina Leptospora (Orbiliales, Orbiliaceae)

In this study, the complete mitochondrial DNA sequence was obtained from Dactylellina leptospora using next-generation sequencing technology. The mitogenome is a circular molecule of 201,677 bp in length. The overall nucleotide composition was 38.54% A, 36.34% T, 11.21% C, and 13.9% G, with a higher A + T content (74.88%) than other species. In the D. leptospora mitogenome, we identified 36 genes including 15 protein-coding genes, 2 ribosomal RNA (rRNA), 19 transfer RNA (tRNA). Phylogenetic analysis showed D. leptospora was genetically divergent from species Pyronema omphalodes within Pezizomycetes

Neodactylariales, Neodactylariaceae (Dothideomycetes, Ascomycota): new order and family, with a new species from China

During a mycological survey of aquatic hyphomycetes on submerged decaying leaves in southwest China, a distinct new fungus was collected. The collection was cultured and sequenced and a BLAST search of its ITS and LSU sequence against data in GenBank revealed a dothideomycetous affiliation, with the closest related taxa in the genus Neodactylaria. Phylogenetic analyses of a multigene matrix containing sequences from four genes (LSU, SSU, rpb2, and tef1), representing broad groups of Dothideomycetes, revealed its placement within Dothideomycetes, but without a supported familial or ordinal affiliation. Based on further phylogenetic analyses and morphological investigations, the new fungus is described here as a new species of Neodactylaria, N. simaoensis sp. nov., and placed in a new family Neodactylariaceae fam. nov. and a new order Neodactylariales ord. nov

Fourteen New Species of Foliar <i>Colletotrichum</i> Associated with the Invasive Plant <i>Ageratina</i><i>adenophora</i> and Surrounding Crops

Ageratina adenophora is one of the most invasive weeds in China. Following an outbreak in Yunnan in the 1960s, A. adenophora has been spreading in Southwest China at tremendous speed. Previous research indicated A. adenophora contained many Colletotrichum species as endophytes. In this study, we investigated the diversity of Colletotrichum in healthy and diseased leaves of the invasive plant A. adenophora and several surrounding crops in Yunnan, Guangxi, and Guizhou provinces in China, and obtained over 1000 Colletotrichum strains. After preliminary delimitation using the internal transcribed spacer region (ITS) sequences, 44 representative strains were selected for further study. Their phylogenetic positions were determined by phylogenetic analyses using combined sequences of ITS, actin (ACT), chitin synthase (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta-tubulin (TUB2). Combined with morphological characteristics, 14 new Colletotrichum species were named as C. adenophorae, C. analogum, C. cangyuanense, C. dimorphum, C. gracile, C. nanhuaense, C. nullisetosum, C. oblongisporum, C. parvisporum, C. robustum, C. simulanticitri, C. speciosum, C. subhenanense, and C. yunajiangense

Neodactylariales, Neodactylariaceae (Dothideomycetes, Ascomycota): new order and family, with a new species from China

During a mycological survey of aquatic hyphomycetes on submerged decaying leaves in southwest China, a distinct new fungus was collected. The collection was cultured and sequenced and a BLAST search of its ITS and LSU sequence against data in GenBank revealed a dothideomycetous affiliation, with the closest related taxa in the genus Neodactylaria. Phylogenetic analyses of a multigene matrix containing sequences from four genes (LSU, SSU, rpb2, and tef1), representing broad groups of Dothideomycetes, revealed its placement within Dothideomycetes, but without a supported familial or ordinal affiliation. Based on further phylogenetic analyses and morphological investigations, the new fungus is described here as a new species of Neodactylaria, N. simaoensis sp. nov., and placed in a new family Neodactylariaceae fam. nov. and a new order Neodactylariales ord. nov

Two new Trichoderma species (Hypocreales, Hypocreaceae) isolated from decaying tubers of Gastrodia elate

Species of Trichoderma are widely distributed around the world. In this study, two new species in Trichoderma, named as T. albidum and T. variegatum, were introduced and illustrated. These species were isolated from diseased tubers of Gastrodia elata in China and identified based on morphological characteristics and multi-gene sequence analyses of three loci that is the internal transcribed spacer regions of the ribosomal DNA (ITS), the translation elongation factor 1-α encoding gene (tef1-α) and the gene encoding the second largest nuclear RNA polymerase subunit (rpb2). Distinctions between the new species and their close relatives were discussed. According to results of the phylogenetic analyses, T. albidum belonged to the Harzianum clade and T. variegatum are grouped with species of the Spirale clade. The expansion of two clades provided research foundations for the prevention and control of tuber diseases in G. elata

Two new species of Verruconis from Hainan, China

Two new species of the genus Verruconis, V. hainanensis and V. pseudotricladiata, were described using combined morphological and DNA sequence data. The DNA sequences of respective strains including nuclear ribosomal DNA genes (nuSSU, ITS, nuLSU) and fragments of three protein-coding genes (ACT1, BT2, TEF1) were sequenced and compared with those from closely-related species to genera Ochroconis and Verruconis (Family Sympoventuriaceae, Order Venturiales). Morphologically, both species showed typical ampulliform conidiophores and conidiogenous cells, features not seen in other species of Verruconis. The conidia of V. hainanensis are fusiform and those of V. pseudotricladiata are Y or T shaped, similar to old members of a closely-related genus Scolecobasidium. The addition of these two new species provides a new perspective on the heterogeneity of Scolecobasidium