New insights into the evolution of subtilisin-like serine protease genes in Pezizomycotina
- Juan Li†1,
- Li Yu†1,
- Jinkui Yang1,
- Linqian Dong1, 2,
- Baoyu Tian3,
- Zefen Yu1,
- Lianming Liang1,
- Ying Zhang1,
- Xu Wang4 and
- Keqin Zhang1Email author
© Li et al; licensee BioMed Central Ltd. 2010
Received: 11 August 2009
Accepted: 9 March 2010
Published: 9 March 2010
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.
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.
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.
Subtilisin-like serine proteases play an important role in the pathogenicity of pathogenic fungi. By using subtilisin-like serine proteases, pathogenic fungi disrupt the physiological integrity of the hosts during penetration and colonization [1, 2]. Previous studies have suggested that pathogenic fungi with different life styles utilize subtilisin-like serine proteases as their virulence factor [2–5]. Investigations into the similarities and differences of pathogenic mechanisms among these pathogenic fungi will significantly enhance our understanding of the evolution of these genes.
Pezizomycotina, the largest subphylum of Ascomycota, includes all filamentous, sporocarp-producing species. These organisms have diverse ecological niches and life styles . A recent study based on six nuclear genes divided Pezizomycotina into nine major clades . Among them, nematode-trapping fungi, which are conidial anamorphs of Orbiliomycetes and belong to nematophagous fungi, are unique because they capture free-living soil nematodes using trapping devices (e.g. adhesive networks, constricting rings, adhesive columns, adhesive knobs and nonconstricting rings) [8, 9]. In contrast, nematode-parasitic fungi, another group of nematophagous fungi within Pezizomycotina, infect nematodes mainly using extracellular enzymes (subtilisin-like serine protease, chitinase and etc.) [2, 10]. Previous studies have suggested that subtilisin-like serine proteases are involved in the penetration and the digestion of nematode cuticles [11–23]. However, only limited studies have been done on the evolutionary pattern of subtilisin-like serine protease genes in nematode-trapping fungi so far [10, 11, 17]. In view of this, we here newly determined five subtilisin-like serine protease genes from the nematode-trapping fungi. In conjunction with the other Pezizomycotina sequences obtained from previous studies and extensive database searching of available genome assembly, we perform the most comprehensive investigation to date of subtilisin-like serine protease genes in Pezizomycotina.
New subtilisin-like serine protease genes from nematode-trapping fungi
Basic information of cloned cuticle-degrading proteases from nematode-trapping fungi.
Intron length (bp)
Our phylogenetic tree (Figure 2) clustered the cuticle-degrading protease genes from entomopathogenic and nematode-parasitic fungi into clade A with the exclusion of nematode-trapping fungi (BS = 81). The genes from nematode-trapping fungi all grouped into clade D (BS = 99). Clade B contained the subtilisin-like serine protease genes from fungi causing dermatosis in humans and animals. Clade E comprised sequences from four classes of Pezizomycotina (Sordariomycetes, Eurotiomycetes, Leotiomycetes and Dothideomycetes), as well as vacuolar protease genes [5, 25–28], indicating the possible endocellular origin of these proteases.
Although the inconsistent relationships among the five clades were produced in our phylogenetic analyses, the earliest divergence of Clade E was strongly supported in all tree topologies (Figure 2; Additional files 1, 2 and 3). The alternative tree topologies, in which Clade E was not the basal clade in the phylogeny, were all found to be significantly worse than our tree (P < 0.05).
Signatures of positive selection in the nematode-trapping fungi
In sum, the results showed for the first time that positive selection might have acted on the subtilisin-like serine protease genes in nematode-trapping fungi, at least in the early stage of their evolution.
The infection of insects and nematodes with entomopathogenic fungi and nematode-parasitic fungi
Fungal strains used in the bioassay.
YMF cultures (strain number)
Beauveria bassiana a
Lecanicillium psalliotae b
Metarhizium anisopliae a
Metarhizium flavoviride c
Paecilomyces farinosus a
Paecilomyces fumosoroseus a
Paecilomyces lilacinus b
Pochonia chlamydosporia b
Nigrospora oryzae b, d
In sum, our bioassay results indicated that entomopathogenic and nematode-parasitic fungi could infect both nematode and insect eggs, thus supporting the inference from the phylogenetic analyses.
Effects of the subtilisin-like serine protease PSP-3 on the nematode and insect eggs
Subtilisin-like serine proteases are known to play a key role during penetration of nematode or insect cuticles [2, 10]. In the study of Bonants et al., the subtilisin-like serine protease PSP-3 of nematode-parasitic fungus Paecilomyces lilacinus was found to be involved in the infection of P. lilacinus against the eggs of the root-knot nematode Meloidogyne hapla .
Discussion and Conclusions
Our study provides new insights into the evolution of subtilisin-like serine protease genes in Pezizomycotina. First, our phylogenetic results suggest that Pezizomycotina subtilisin-like serine proteases most likely evolved from endocellular to extracellular proteases. As seen from the tree, the subtilisin-like serine protease genes of Pezizomycotina previously identified or presumed to be endocellular proteases [5, 25–28] were clustered into Clade E and diverged the earliest in the phylogeny. In contrast, most of those previously identified or presumed as extracellular proteases clustered into the other clades and diverged later. In addition, the protease (GenBank no. XM_001385908) from the outgroup Pichia stipitis was assumed as an endocellular protease , supporting the endocellular to extracellular transition during the evolution of Pezizomycotina subtilisin-like serine proteases. The evolution from endocellular to extracellular enzymes might have facilitated pathogenic fungi to utilize these enzymes as virulence factor to colonize their hosts.
Second, our phylogenetic results and functional bioassays suggested that the entomopathogenic and nematode-parasitic fungi shared similar properties in parasitism. Previous studies indicated that the nematode-parasitic fungi could infect nematodes while the entomopathogenic fungi could parasitize other insects [8, 32]. The grouping of the cuticle-degrading protease genes of these two groups of fungi led us to believe that they might have overlapping pathogenic mechanisms against both insects and nematodes. Further experimental bioassay showing that the entomopathogenic and nematode-parasitic fungi could infect both nematode and insect eggs supported this hypothesis (Figure 4 and 5). Moreover, the subtilisin-like serine protease PSP-3 produced by nematode-parasitic fungus P. lilacinus was observed to have the ability to digest the eggs of nematodes and insects (Figure 6; Additional file 5). Our finding enriches the known fungal resources for the microbial control of both types of pests.
Third, our data provide further understanding about the duplications and subsequent functional divergence of subtilisin-like serine protease genes in Pezizomycotina. Previous studies have shown that gene duplications occurred in subtilisin-like serine protease genes of pathogenic fungi and that such duplications might have played an essential role in pathogenicity and contributed to their increased adaptability, host range, and/or survived in various ecological habitats outside the host of these fungi .
From our phylogenetic tree, we found that frequent gene duplication events have occurred in many pathogenic fungi. For example, there were 9 paralogous subtilisin-like serine protease genes in Metarhizium anisopliae. Four of them, including PR1A, PR1B, PR1I and PR1G, clustered into Clade A with the other cuticle-degrading protease genes known to serve as important virulence factors during fungal penetration of nematode or insect cuticles [13–17, 19]. These four genes most likely perform essential functions during the infection of hosts. In comparison, PR1J, PR1D, PR1E and PR1F of M. anisopliae fell into Clade C, indicating that they might have diverged to perform different as yet uncharacterized functions. Our results are different from previous inferences suggesting that PR1A was a key virulence factor during degradation of insect cuticles, while all the other PR1's were of only a minor contributor to cuticle degradation [25, 34].
In the dermatophytic fungus Arthroderma benhamiae, there are 7 subtilisin-like serine protease genes (SUB1-7) . Our phylogenetic tree clustered 6 of them in Clade B. The only exception was SUB2, which is grouped into Clade C. This result is consistent with the phylogeny of Jousson et al. . They hypothesized that SUB2 was the most ancestral, while the other SUBs were dermatophyte-specific and might have emerged more recently through successive gene duplication events. In addition, they speculated that SUB3 and SUB4 were key virulence proteases of dermatophytes and playing important role in invasion of human or animal keratinised tissues . From our analysis, the key virulence protease genes of dermatophytic fungi and nematophagous/entomopathogenic fungi were placed in different clades, suggesting that there were probably different pathogenic mechanisms between mammalian pathogens and nematode/insect pathogens.
Fourth, our evolutionary analysis revealed signatures of positive selection acting on the subtilisin-like serine protease genes of the nematode-trapping fungi, suggesting that the subtilisin-like serine protease genes might have played important roles during the evolution of pathogenicity of nematode-trapping fungi against nematodes. Although they both belonged to the cuticle-degrading proteases, the subtilisin-like serine protease genes from nematode-trapping fungi and nematode-parasitic fungi were not grouped together. This result supports earlier findings based on smaller data sets [10, 11, 17]. Since nematode-parasitic fungi do not produce trapping devices, they likely rely mainly on the extracellular enzymes (including subtilisin-like serine protease) as virulence factors to penetrate and digest nematode cuticles. In contrast, by virtue of synergistic interactions of extracellular enzymes and trapping devices, the nematode-trapping fungi can immobilize and penetrate nematodes within a few hours [2, 10]. Therefore, the subtilisin-like serine proteases of nematode-trapping fungi might experience special selective pressure resulted from the co-evolution of trapping structures and proteolytic enzymes. Interestingly, our evolutionary analysis demonstrated evidence of positive selection acting on the cuticle-degrading protease genes in nematode-trapping fungi, at least during the early stage of its evolution. We hypothesize that the subtilisin-like serine protease genes may have played important roles during the evolution of pathogenicity of nematode-trapping fungi against nematodes. In addition, the potentially adaptive amino acid replacements discovered by our analysis will provide valuable information for functional analysis in future studies.
The five nematode-trapping fungi (Arthrobotrys yunnanensis YMF1.00593, Arthrobotrys musiformis YMF1.01043, Monacrosporium psychrophilum YMF1.01412, Monacrosporium coelobrochum YMF1.01480 and Dactylella shizishanna YMF1.00022) were isolated from field soil samples in Yunnan Province of China and permanently stored in the Yunnan Microbiological Fermentation Culture Collection Center (YMF). They were maintained on cornmeal agar (CMA) at 28°C.
Genomic DNA extraction
The five nematode-trapping fungi were cultured in the PL-4 liquid medium on a rotary shaker (150 rpm) at 28°C for 1 week . Their mycelia were then filtered on a nylon mesh and genomic DNA was isolated using the E.Z.N.A.@ Fungal DNA Mini kits (Omega Bio-Tek, Inc. USA) following the manufacturer's protocol.
Primers design and cloning of subtilisin-like serine protease genes
List of primers used in this study.
The PCR reaction mixture was consisted of 0.5 μL Taq DNA polymerase, 5 μL of reaction mixture buffer, 3 μL of 25 mM MgCl2, 1 μL of 2.5 mM dNTPs, 1 μL of 100 μM degenerate primers, 3 μL of DNA template in a final volume of 50 μL supplied with double-distilled sterile water. Amplification started at 95°C for 5 min, followed by 35 cycles with 95°C for 40 s, 51°C for 40 s, and 72°C for 1.5 min. After the last cycle, the reaction mixture was maintained at 72°C for 10 min for a final extension step.
Sequencing and sequence analysis
The amplified products were electrophoresed on 1% agarose gels to check for fragment size and purity. All the PCR products were purified using the DNA fragment purification kit ver 2.0 (Takara, Japan) and sub-cloned into the vector pMD18-T (Takara, Japan). Escherichia coli strain DH5α was used as a host for transformation and cloning. It was grown in Luria-Bertani medium at 37°C. Ten positive colonies were selected for sequencing from each strain. The plasmids were sequenced in both directions on an ABI 3730 automated sequencer (Perkin-Elmer, USA). Sequence assembly was performed using the SeqMan software (DNA Star software package, DNASTAR, Inc. USA) and DNAman software package (Version 5.2.2, Lynnon Biosoft, Canada). Signal sequence was predicted using SignalP http://www.cbs.dtu.dk/services/SignalP/. Protein molecular masses were determined online with ProtParam tools http://us.expasy.org/tools/protparam.html, and N-linked glycosylation sites were predicted by NetNGlyc http://www.cbs.dtu.dk/services/NetNGlyc/.
Subtilisin-like serine protease genes from the other Pezizomycotina species
The amino acid sequences of each protease characterized as members of the subtilisin-like family were retrieved from National Center for Biotechnology Information (NCBI)  using BLASTX . MEROPS and UniProtKB (UniProt Knowledgebase) protein sequence database were also screened to iteratively search all known and predicted subtilisin-like serine protease genes, making sure that all available subtilisin-like serine protease genes of Pezizomycotina were included in the analyses. A total of nearly 500 subtilisin-like serine protease genes representing five of nine classes in Pezizomycotina, including Sordariomycetes, Eurotiomycetes, Leotiomycetes, Dothideomycetes and Orbiliomycetes, were obtained. In addition, five subtilisin-like serine protease genes from fungi in the Saccharomycotina were downloaded and used as outgroups. The partial sequences and those with unusual lengths (<200 aa or >700 aa) in Pezizomycotina were removed from the analysis, yielding 287 sequences.
The amino acids sequences of the 287 genes were aligned using Clustal X version 1.83  with default parameters. Several sequences with ambiguously aligned regions around the three active catalytic residues (Asp-His-Ser) and those with identical sequences but different accession numbers were eliminated in the analysis, yielding 189 subtilisin-like sequences in the final dataset. The Gblocks program http://molevol.cmima.csic.es/castresana/Gblocks.html[41, 42] was applied to extract conserved regions that contain more reliable phylogenetic signals. An alignment consisting of 229 amino acid positions was obtained.
Four tree-building methods were performed for phylogenetic reconstructions. The maximum parsimony (MP) tree with heuristic search was constructed using PAUP*4.0b8 , and maximum likelihood (ML) tree with the best-fit model (WAG+I+G) was constructed using PHYML version 2.4.4 . The best-fit model of protein evolution was selected by ProtTest http://darwin.uvigo.es. In addition, the program MEGA 4.1 [46, 47] was used to construct a neighbor joining (NJ) tree  and MrBayes 3.1.2  was used to perform Bayesian analysis. Bayesian analysis started with randomly generated trees and four Markov chains under default heating values were ran for 2 × 106 generations, with sampling at intervals of 100 generations. To ensure that these analyses were not trapped in local optima, the dataset was run three times independently. We determined the burn-in period by checking for likelihood stability.
To test for nodal reliabilities, bootstrap (BS) analysis  for MP (1,000 replicates), ML (100 replicates) and NJ (1,000 replicates) was applied. Bayesian posterior probabilities (PP) from the 50% majority-rule consensus tree were calculated to provide the estimates of nodal support in Bayesian phylogenies.
Testing the inconsistency of tree topologies
AU-test , SH-test  and KH-test  were performed using CONSEL version 0.1 h  with site-wise log-likelihoods calculated by Tree-Puzzle 5.1  to assess the inconsistency of tree topologies.
Detecting positive selection in nematode-trapping fungi
Positive selection has been considered a major force in forming new motifs or functions in proteins . The non-synonymous to synonymous rate ratio ω (dN/dS) provides an indication of the change in selective pressures. A dN/dS ratio = 1, <1, and >1 indicates neutral evolution, purifying selection, and positive selection on the protein involved, respectively. In this study, CODEML program of the PAML package [57, 58] and the Bn-Bs software http://www.bio.psu.edu/People/Faculty/Nei/Lab/software.htm were implemented to detect signatures of positive selection in nematode-trapping fungi. The multiply aligned data are available as supplementary material (see Additional file 6).
The Bn-Bs software implements a modified method from the original  by taking into account the transition bias for estimating synonymous and nonsynonymous substitutions along the branches of a given tree. Here, one-tailed Z test was performed.
Considering that positive selection may act in very short episodes during the evolution of a protein  and affect only a few sites along a few lineages in the phylogeny, the "branch-site" model, which allows ω ratios to vary both among lineages of interest and amino acid sites, was considered here in codon-based likelihood analysis using the PAML software [57, 58]. We used the branch-site Model A as a stringent test to identify the sites under positive selection along the lineages of interest  and each model was run twice. All the possible tree topologies were used here.
The infection of insects and nematodes with entomopathogenic fungi and nematode-parasitic fungi
For bioassays against the root-knot nematode, nine selected fungi (Table 2) were cultivated in 9 sterile Water Agar (WA) media (15 g agar in 1 L deionized water) at room temperature (10-25°C) separately, until each agar medium surface was colonized by the fungus. Then a suspension of nematode eggs (ca. 400 eggs) was added onto the fungal culture and the mixture was then kept at the room temperature. In order to keep the eggs moisturized, a 200 μL sterile M9 buffer (Na2HPO4 6 g, KH2PO4 3 g, NaCl 0.5 g, NH4Cl 1 g, deionized water to 1 L) was added daily. After 5 days, each culture was observed daily with the Olympus BX51 microscope (Japan) and counted the infected eggs 3 times each day. The criterion of infection was that the root-knot nematode eggs were failed to hatch and the fungal mycelia were seen growing from the eggs, while none could be seen in the negative control. All assays were repeated 3 times and each replicates lasted a week.
For bioassays against the potato tuber moth, a fungal culture with 6 mm in diameter was inoculated onto the center of a WA medium. Then exactly 30 insect eggs were put on the periphery of the fungal culture and incubated at room temperature. From 3 days onward, each culture was observed daily and counted the infected eggs 3 times each day. The criterion of infection was that the potato tuber moth eggs were failed to hatch and the fungal mycelia were seen growing from the eggs. All assays were repeated 3 times and each replicate lasted for 1 week.
Effects of the subtilisin-like serine protease PSP-3 on the nematode and insect eggs
The subtilisin-like serine protease PSP-3 produced by the nematophagous fungus P. lilacinus was tested for its ability to degrade the eggs of the root-knot nematode Meloidogyne sp. and potato tuber moth P. opercullella. The protease PSP-3 was purified following the procedures described by Bonants et al. .
The following experiment design was used: (treatment a). nematode (a1) or insect (a2) eggs, protease (1,000 μg/mL, 0.2 U/mL, dissolved in potassium phosphate buffer) 10 μL, toluene (as an antiseptic) 3 μL, potassium phosphate buffer (0.1 mol/L) 137 μL; (treatment b). nematode (b1) or insect (b2) eggs, toluene 3 μL, potassium phosphate buffer (0.1 mol/L) 147 μL; (treatment c). nematode (c1) or insect (c2) eggs, denatured protease (1,000 μg/mL, 0.2 U/mL, dissolved in potassium phosphate buffer and then denatured by boiling for 30 min) 10 μL, toluene 3 μL, potassium phosphate buffer (0.1 mol/L) 137 μL; and (d). toluene 3 μL, potassium phosphate buffer (0.1 mol/L) 147 μL. Protein concentration was measured using Coomassie Brilliant Blue G-250 according to Bradford , with bovine serum albumin (25-400 μg/mL) as the standard. One unit of protease was defined as the amount of enzyme that released 1 mg tyrosine/min at 25°C. For the nematode assay, ca. 1700 eggs were used in each treatment. For the insect assay, exactly 30 eggs were used. All the treatments were shaken at Speed 3 on the mixer (Cole-Parmer, USA) at 25°C. Every 24 h, each treatment was precipitated and a 20 μL- supernatant was collected to measure the amount of protein concentration using casein as substrate. The supernatant from treatment (d) was used as the negative control. Whole assay was repeated 3 times. After checking the data with the Kolmogorov-Smirnov test and Shapiro-Wilk test [64–66], we found the data from each treatment are approximated a normal distribution (P ≥ 0.05) (see Additional file 7). Statistics analyses were then carried out using one-way analysis of variance (ANOVA) in SPSS 14.0. Moreover, the LSD-test was also used for pairwise comparisons among the three treatments (see Additional file 5). A P value of less than 0.05 was considered as statistically significant.
We thank Dr. Jianping Xu (McMaster University, Canada), Jianjun Gao, Shuqun Liu and Mrs. Wei Zhou for their help and advice in our experiment. This work was funded by National Basic Research Program of China (approved no. 2007CB411600), projects from National Natural Science Foundation of China (approved nos. 30630003, 30660107 and 30960229), and the Department of Science and Technology of Yunnan province (approved nos. 2007C007Z, 2005NG05 and 2009CI052).
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