Differential expression of the putative Kex2 processed and secreted aspartic proteinase gene family of Cryphonectria parasitica

f u n g a l b i o l o g y 1 1 6 ( 2 0 1 2 ) 3 6 3 e3 7 8

journal homepage: www.elsevier.com/locate/funbio

Differential expression of the putative Kex2 processed and secreted aspartic proteinase gene family of Cryphonectria parasitica Debora JACOB-WILKa,1, Marino MORETTIb,2, Massimo TURINAb,2, Pam KAZMIERCZAKa,1, Neal K. VAN ALFENa,* a

Department of Plant Pathology, College of Agricultural and Environmental Sciences, One Shields Avenue, University of California, Davis CA, 95616, USA b Istituto di Virologia Vegetale, CNR, Strada delle Cacce 73, 10135 Torino, Italy

article info

abstract

Article history:

Kex2-silenced strains of Cryphonectria parasitica, the ascomycete causal agent of chestnut

Received 13 April 2011

blight, show a significant reduction in virulence, reduced sexual and asexual sporulation

Received in revised form

and reductions in mating and fertility. Due to this and the known involvement of Kex2

12 December 2011

in the processing of important proproteins in other systems, we searched the whole C. par-

Accepted 13 December 2011

asitica genome for putative Kex2 substrates. Out of 1299 open reading frames (ORFs) pre-

Available online 6 January 2012

dicted to be secreted, 222 ORFs were identified as potential Kex2 substrates by this

Corresponding Editor:

screen. Within the putative substrates we identified cell wall modifying proteins, putative

Pieter van West

proteinases, lipases, esterases, and oxidoreductases. This in silico screen also uncovered a family of nine secreted aspartic proteinases (SAPs) of C. parasitica. Northern blot analyses

Keywords:

of this gene family showed differential expression when exposed to chestnut wood and

Cryphonectria parasitica

Cryphonectria hypovirus 1 (CHV1). Due to the reduction in fungal virulence known to be

Cryphonectria hypovirus 1

caused upon hypoviral infection of C. parasitica, the differential gene expression observed,

Secreted aspartic proteinases

and the known involvement of SAPs in virulence in other systems, we conducted deletion

Secretome

analyses of four of these proteinases, representing different expression patterns. Deletion of each of the four SAPs did not affect growth rates, sporulation or virulence, suggesting that none of the considered SAPs is essential for the full development or virulence of C. parasitica under the conditions tested. ª 2011 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Introduction The fungal pathogen Cryphonectria parasitica decimated the American chestnut, Castanea dentata, in its natural range. Interestingly, this ascomycete becomes hypovirulent after infection

by a virus. Since the discovery and the use of hypovirulence for the biocontrol of the European chestnut, Castanea sativa, this fungal-virus interaction has been used as a model system for the study of fungal pathogenicity [reviewed in (Dawe and Nuss, 2001; Milgroom and Cortesi, 2004; Nuss, 2005)].

* Corresponding author. Tel.: þ1 530 752 1605; fax: þ1 530 752 9049. E-mail addresses: [email protected], [email protected], [email protected], [email protected], [email protected] 1 Tel.: þ1 530 754 5500; fax: þ1 530 752 5674. 2 Tel.: þ39 011 3977923; fax: þ39 011 343809. 1878-6146/$ e see front matter ª 2011 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.funbio.2011.12.006

364

Cryphonectria hypovirus 1 (CHV1) replicates within fungal vesicles that were previously shown to accumulate during viral infection, and to cofractionate with the fungal trans-Golgi network (TGN) marker Kex2 (Hansen et al., 1985; Newhouse et al., 1990; Fahima et al., 1993; Jacob-Wilk et al., 2006). Several hypoviral elements cofractionate with fungal vesicles containing the Kex2 endoprotease, and previous work has demonstrated that viral infection of the fungus causes the down-regulation of several genes containing putative Kex2 processing signals, so we investigated the possible involvement of Kex2 in the virulence of C. parasitica. Kex2-silenced strains of C. parasitica showed a significant reduction in virulence, sexual and asexual sporulation, mating, and fertility (Jacob-Wilk et al., 2009). These results suggested the importance of secreted Kex2 processed proteins in fungal development and virulence. Members of the kexin-like family have been found in animals, plants and fungi, and play crucial roles in several important regulatory processes. Kexin-like proteins activate secreted proteins that are initially synthesized as an inactive precursor or pre-pro-form (Julius et al., 1984; Taylor et al., 2003). In fungi, kexin-like proteins process proproteins involved in cell wall maintenance and degradation, secreted proteinases, pheromones, and lipases (Basco et al., 1996;
de et al., Iguchi et al., 1997; Newport and Agabian, 1997; Pigne 2000; Bader et al., 2001; Bader et al., 2008). Secreted aspartic proteinases (SAPs) are a well characterized family of Kex2 substrates, shown to be involved in adherence, colonization, nutrition, and dissemination of Candida albicans (Naglik et al., 2003; Naglik et al., 2004). They encode preproenzymes controlled primarily at the mRNA level, and their expression is affected by different factors such as temperature, pH, nutrient source, and morphological transitions (White and Agabian, 1995; Hruskova-Heidingsfeldova, 2008). It has also been suggested that different SAPs play distinct roles at different stages of infection (Schaller et al., 2005). Recently, the genome of C. parasitica was sequenced and made available to the public by the Department of Energy (DOE), Joint Genome Institute (JGI). The 43.9 MB genome was assembled into 39 main genome scaffolds. The current draft release includes a total of 11 184 predicted gene models, and was functionally annotated using the JGI annotation pipeline. Due to the significant effects of the silencing of Kex2 on the virulence and development of C. parasitica, we decided to explore the newly sequenced C. parasitica genome and analyze its predicted secretome for potential Kex2 substrates. Using an in silico screen we identified 222 open reading frames (ORFs) whose products are potential Kex2 substrates. We classified these putative proteins into groups based on sequence and domain homology, according to their predicted cellular functions. The list of candidate substrates includes predicted secreted proteins that localize at the interface between the host and the pathogen C. parasitica. Several of the identified putative Kex2 substrates have already been shown to act at the fungus-plant host interface; for others the functions remain to be assessed. Our in silico screen uncovered a family of nine SAPs. In an effort to understand whether SAPs might be involved in the development or virulence of C. parasitica, we used the C. parasiticaeCHV1 system to study the expression patterns of

D. Jacob-Wilk et al.

the C. parasitica SAP gene family. This was done under normal laboratory infection conditions and in response to chestnut wood and to hypoviral infection. We produced deletions of four SAPs and characterized their phenotype. Results showed that the C. parasitica SAP gene family is differentially expressed after chestnut wood exposure and hypoviral infection. The deletion mutant phenotypes of three of the selected SAPs, differentially expressed, were indistinguishable from wild-type strain, while one of the four showed reduced proteinase activity on culture filtrate and clumping when grown on liquid cultures.

Materials and methods In silico analysis of the Cryphonectria parasitica genome The genome of C. parasitica was sequenced, assembled and analyzed by the DOE, JGI and is available at http:// genome.jgi-psf.org/Crypa1/Crypa1.home.html. This draft release version of the genome contains 11 184 gene models. A list of 1299 ORFs predicted to contain putative signal peptides, as determined by both SignalP-HMM and SignalP-NN prediction tools for eukaryotic models (Nielsen et al., 1997; Nielsen and Krogh, 1998), was extracted from the filtered models and kindly provided by Andrea Aerts from JGI. These 1299 ORFs predicted to contain a signal peptide were subsequently analyzed using the ProP 1.0 server, a propeptide cleavage site prediction tool (http://www.cbs.dtu.dk/services/ProP/) from the ExPASy Proteomics tools for post-translational modification prediction at the ExPASy Proteomics server, the Swiss Institute for Bioinformatics (SIB). The ProP 1.0 server predicts arginine and lysine propeptide cleavage sites in eukaryotic protein sequences using an ensemble of neural networks and a general proprotein convertase (PC) prediction (Duckert et al., 2004). This predicting tool is integrated with the SignalP 3.0 server predicting the presence and location of signal peptide cleavage sites (Bendtsen et al., 2004) together with the propeptide cleavage site, and helped cross-validate the signal peptide predictions previously obtained. These analyses generated a list of proteins that contained a putative signal peptidase cleavage site and a putative PC cleavage site. These results were scanned for Kex2 specific cleavage recognition sites as defined by the motif P4-P3[K/R/P]-RY, where P3 and P4 can be any amino acid, in predicted ORFs greater than 100 amino acids that contained a propeptide less than 150 amino acids long as previously described for C. albicans (Newport et al., 2003). This criterion was derived from known Kex2 substrates from Saccharomyces cerevisiae and from experimental studies (Singh et al., 1983; Bostian et al., 1984; Mizuno et al., 1988; Rockwell et al., 2002; Bader et al., 2008) and adapted to what is known for C. parasitica. We removed the criterion for P4 specificity, which states that P4 cannot be CDFGPS or W, because this consensus is weak, it was established based on S. cerevisiae substrate specificity, and it does not apply to the only protein experimentally shown to be processed by Kex2 in C. parasitica, cryparin (JacobWilk et al., 2009). Also, the C. parasitica mating factor Mf1-1, homologous to S. cerevisiae a pheromone e a known Kex2 substrate e shows several putative Kex2 cleavage sites that do not conform to this consensus (Zhang et al., 1998). Furthermore,

Differential expression of the putative Kex-2 processed and secreted aspartic proteinase family

no definitive information was obtained from the Kex2 crystal structure analysis about the nature of P4 substrate recognition specificity (Rockwell and Thorner, 2004). Each of the candidate ORFs was subjected to National Center for Biotechnology Information (NCBI) blastp against non-redundant protein sequences database and scanned against ExPASy-prosite (SIB) database for protein domains, families and functional sites (http://www.expasy.ch/prosite/). Since the conclusion of this work a more recent version of the C. parasitica genome was released and it can be accessed at http://genome.jgi-psf.org/Crypa2/Crypa2.home.html. All SAP genes of interest were found in Crypa2 and all but one preserved their ID numbers; 62389 in version 1 (v1) is 346541 in v2, its protein sequence and annotation remain the same.

Phylogenetic analyses We identified SAPs from a number of genomes of fungal species relatively close to C. parasitica, and representatives of different ecological behaviours: Neurospora crassa (a non-pathogen), Magnaporthe oryzae and Fusarium graminearum (herbaceous plant pathogens), Aspergillus oryzae (a fungal species used for fermentation) and C. albicans (a phylogenetically distant ascomycete included in the analysis since its SAPs are the best characterized among fungal species). Aspartic proteinases (APs) were searched in each genome through a number of blastp searches (Altschul et al., 1997) in an iterative process with a number of APs from C. parasitica. We then performed a further in silico analysis for a signal peptide and Kex2 processing sites as previously described. In the final phylogenetic analysis only putative SAPs were included (excluding the APs without a signal peptide). Multiple alignment analysis was carried out using the CLUSTAL W software version 1.82 (Thompson et al., 1997). Phylogenetic trees were constructed using the MEGA4 software package (Tamura et al., 2007) for the Neighbor-Joining (Saitou and Nei, 1987) and the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) methodology (Sneath and Sokal, 1973). Phylogenetic analysis was also performed using maximum likelihood methods implemented in PhyML-3.0 (Guindon and Gascuel, 2003). The LG amino acid substitution model was used with the proportion of invariable sites set to 0, and 4 categories of substitution rates (Le and Gascuel, 2008). The starting tree to be refined by the maximumlikelihood algorithm was a distance-based BIONJ tree estimated by the program (Guindon and Gascuel, 2003). Statistical support for phylogenetic grouping was assessed by approximate likelihood-ratio tests (aLRT) based on a Shimodairae Hasegawa-like procedure (SH-aLRT) (Anisimova and Gascuel, 2006). A list of the GenBank accession and protein identification numbers of all the various SAP genes considered in the phylogenetic analysis is presented in Table S1, together with the identification codes used in the tree graphic.

Growth of cultures Fernbach flasks containing 1 L Endothia parasitica (EP) complete liquid medium (Puhalla and Anagnostakis, 1971) were inoculated using cultures that were previously grown for 7 d on PDAmb (potato dextrose agar with 1 mg/L methionine and biotin) Petri plates. To inoculate liquid cultures, the agar cultures

365

were homogenized for 1 min in EP complete at full speed in a Waring Blender (New Hartford, CT) before being added to the liquid medium. Cultures were grown on an orbital shaker at room temperature for 3 d under ambient light with and without chestnut wood. Chestnut wood was cut into pieces of approximately 6 cm, wrapped in heavy duty aluminium foil, struck with a hammer until broken to small pieces, and added to the liquid media prior to sterilization. Two independent experiments were carried out with 50 and 40 g wood, respectively. Mycelia were harvested by filtration and immediately frozen, lyophilized and stored at 80  C. Strains used were wild type C. parasitica EP155 (ATCC#38751) and CHV1-713 containing isogenic strain UEP1 (Powell and Van Alfen, 1987). Deletion mutants produced by a gene replacement approach were obtained from C. parasitica Dcpku80 strain derived from EP155 (Lan et al., 2008).

Cloning of SAPs from Cryphonectria parasitica PCR products for the genes corresponding to all SAPs were amplified from genomic C. parasitica DNA using the primers described in Table S2 and cloned into PCR vector pGEM-T Easy (Promega Corp., Madison, WI, U.S.A). For clarity each SAP was addressed by its protein identification number as assigned by the genome sequencing project. Sizes of PCR fragments cloned were as follows; SAP-62389 (1376 bp), SAP-38462 (1497 bp), SAP-87438 (1642 bp), SAP-90510 (1379 bp), SAP-71039 (1928 bp), SAP-34127 (1414 bp), SAP-43429 (1550 bp), SAP-37154 (1412 bp), SAP-107093 (1674 bp). PCR fragments of SAP-71039 and SAP-90510 were cloned into the pSC-B vector (Agilent Technologies, La Jolla, CA) using primers described in Table S2.

RNA extraction and Northern blot analysis For RNA extraction 1 ml of RNA Trizol reagent (Invitrogen, Carlsbad, CA) was added to 0.1 g ground lyophilized mycelia in 2 ml tubes. Manufacturer’s instructions were followed with minor modifications: the addition of 5 M NaCl was omitted, and 0.5 ml of chloroform was added instead of 0.3 ml. After adding isopropyl alcohol, samples were incubated at 20  C for 1 h. RNA was centrifuged at 4  C for 30 min. Pellets were resuspended in 50 ml diethyl pyrocarbonate (DEPC) treated water. RNA was denatured, separated, and transferred onto membranes using the glyoxal-phosphate electrophoresis method as previously described (Sambrook et al., 1989). Northern blot analysis was done as previously described (Sambrook et al., 1989). RNA was quantified on a NanoDrop ND-1000 spectrophotometer from NanoDrop technologies, Inc. (Wilmington, DE). Probes for Northern blots were generated as follows: DNA from each pGEM-TeSAP clone was used as template for PCR using primers described in Table S2. PCR reactions were separated on agarose gels and each specific band isolated using QIAEX II gel extraction kit (QIAGEN, Valencia, CA) and labelled by random prime labelling with [32P]dCTP.

Deletion vector construction For deletion vector construction, vector pGEM-T-87438 was linearized with StuI, predicted to digest inside the 87438

366

genomic sequence at base 669. The linearized vector was dephosphorylated, and ligated to a previously blunted 1400 bp Hygromycin B phosphotransferase (Hyg) fragment. Vector pGEM-T-62389 was digested using AccIII, predicted to digest inside the 62389 genomic sequence at base 556. The linearized vector was filled in, dephosphorylated and ligated to the previously described Hyg fragment. Vectors were linearized using SacI and transformed into spheroplasts of strain Dcpku80, as previously described (Churchill et al., 1990). All transformants were checked for stability and single spores were analyzed by Southern blot. For strains D71039 and D90510 a similar approach was used. The 1400 bp Hyg fragment was inserted between two StuI sites (at position 2769 and 3633) on the genomic sequence for the 71039 SAP gene, whereas for the 90510 gene the same Hyg fragment was inserted into the NruI site at position 1108. Vectors were digested with EcoRI for linearization before transformation. The initial screening for double recombination events was done using primers flanking the Hyg insertion sites such as primers 90510-F (50 ccacaccctctacaacccga-30 ) and 90510-R (5-gtcggtgccaacgttgc-30 ), primers 71039-F (50 -tcgtacgacaacctccccaa-30 ), and 71039-R (50 taccgatcagggcaccgaac-3’). Transformants originating from a single spore lacking the PCR band originating from the original gene were then used for Southern blot analysis.

DNA extraction and Southern blot analysis Fungal genomic DNA was extracted as described by Turina et al. (2003). Southern blot analysis was performed as described (Sambrook et al., 1989). Genomic DNA was digested with appropriate restriction enzymes and transferred to Hybond Nþ (Amersham Biosciences, Piscataway, NJ) after separation on a 1 X Tris-acetate-EDTA 0.7 % agarose gel. Probes for D87438 and D62389 Southern blots were generated by random prime labelling with [32P]dCTP of restriction fragments obtained from each specific gene. For 87438, a 356 bp fragment was obtained by digesting pGEM-T-87438 using PstI. For 62389 a 907 bp fragment was obtained by digesting pGEM-T-62389 using SacII and StuI. Fragments were separated on agarose gels and DNA fragments were isolated using QIAEX II gel extraction kit (QIAGEN, Valencia, CA) and labelled. For deletion strains D71039 and D90510, Southern blots were analyzed using riboprobes that were synthesized as described by Rastgou et al. (2009) using the T7 RNA polymerase.

Fungal fitness assessment Pycnidial numbers, asexual spore counts, and growth assessment were carried out as described by Jacob-Wilk et al. (2009). For fungal growth measurements strains were grown on PDAmb, Malt extract agar (MEA), and minimal media (MM) plates as described by Zhang et al. (1993) and diameters of colonies were measured at 5 d post inoculation (DPI).

D. Jacob-Wilk et al.

Assay for AP activity AP activity was assayed using haemoglobin as the substrate by the method of Anson (1938) with modifications. One hundred microlitres of crude enzyme sample (culture filtrate from tested strains grown as previously described) were added to 150 ml citrate buffer pH 3.3 and 150 ml of a 1 % haemoglobin solution prepared in the same buffer. After incubation at 26  C for 60 min the reactions were stopped by addition of 200 ml 10 % (w/v) trichloroacetic acid (TCA). Reactions were centrifuged at 10 000 g for 10 min and the absorbance of the supernatant was measured. Blanks were stopped before addition of enzyme sample and proteinase activity was defined as the increased absorbance measured at 280 nm (Ben Khaled et al., 2011). Three independent experiments were used for the statistical analysis of the results.

Results Analysis of the Cryphonectria parasitica genome for putative Kex2 substrates The total predicted gene models for the C. parasitica genome is 11 184 of which 1299 have putative signal peptides; therefore, the C. parasitica secretome comprises roughly 11.6 % of the genome. Of the predicted gene models, 5.3 % were found to contain putative propeptide cleavage sites and 2 % were found to contain Kex2 specific cleavage signals as defined by the motif described in materials and methods. Based on the chosen criteria our screen extracted a total of 222 ORFs. Each of the 222 candidate ORFs was subjected to NCBI blastp against non-redundant protein sequences database and scanned against ExPASy-prosite (SIB) database for protein domains, families, and functional sites. A total of 108 ORFs showed no homologies to any known sequences or domains, whereas 114 showed similarity and domain conservation with various proteins from different organisms. Within the 114 ORFs with homologies to known proteins or domains, 29 were classified as cell wall related and included putative glycosil hydrolases, glycosil transferases, carbohydrate binding protein, one cutinase, one pectinase, and one hydrophobin (the well characterized cryparin protein) (Table 1). We classified 26 as proteinase-like, nine putative SAPs, three aspergillopepsin II like and six kexin-subtilisin-like; seven carboxypeptidases, and one cysteine protease (Table 2). Of the putative secreted Kex2 substrates, 22 were classified as oxidoreductases. Among these were seven putative cytochrome P450, a peroxidase, a sulfoxide reductase, laccase 3, a thioredoxin like and several mono, and dioxygenases (Table 3). Three proteins were classified as kinases/phosphatases, five as lipases/ esterases, eight as transporter like, four are GTP/ATPases, one pheromone, ten DNA/RNA modifying enzymes, and several classified as ‘others’. A full list of these candidate substrates can be found in supplemental materials (Table S3).

Virulence tests Lesion formation tests on apple fruits and canker formation tests on dormant chestnut wood were performed as previously described (Elliston, 1985; Rigling et al., 1989; Jacob-Wilk et al., 2009).

The Cryphonectria parasitica Kex2 processed SAP gene family Our analysis revealed nine SAPs, all having a signal peptide between amino acids 18 and 24 and a pro-region containing

Differential expression of the putative Kex-2 processed and secreted aspartic proteinase family

367

Table 1 e Cell wall related putative Kex2 substrates in the C. parasitica secretome. Protein ID# 89717 66378 53933 67185 67267 88086 67638 50755 74826 64234 48608 19334 108665 45201 72086 60245 89178 68782 71215 43708 104818 72924 62278 90742 77170 69404 51943 104198 103357

Homologies and similarities

Domain homologies

Hypothetical protein Glycoside hydrolase, putative Endo-arabinase, putative Probable alpha-1, 2-mannosyltransferase Beta glucosidase homologue Related to glucosidase II, alpha subunit Glycosyl hydrolase, family 92 protein Arabinosidase putative Endo-1,6-beta-D-glucanase Hypothetical protein similar to alpha monosidase Putative arabinanase Endo-1,4-beta-xylanase B precursor Hypothetical protein Probable arabinogalactan endo-1,4-beta-galactosidase Hypothetical protein Beta-galactosidase Chain A, glycoside hydrolase family 15 glucoamylase Endo 1,5-alpha-arabinanase Cellulose hydrolase, putative Endo 1,5-alpha-arabinanase Beta-1,3-glucanosyltransferase Arabinogalactan endo-1,4-beta-galactosidase GPI transamidase component PIG-U, putative N-acetylglucosaminylephosphatidylinositol deacetylase GPI mannosyltransferase 4 Hypothetical protein Pectin methylesterase, putative Cryparin 1,3-beta-glucanosyltransferase

Glycosil hydrolase Glycosil hydrolase Glycosil hydrolase family 43 Glycosil transferase 15 superfamily Glycosil hydrolase family3 Glycosil hydrolase family 31 Glycosil hydrolase family 92 Glycosil hydrolase Glycoside hydrolase family 30 Glycoside-hydrolase family 47 Glycosil hydrolase Glycoside hydrolase family 11 Glycosyl transferase family 25 Glycoside hydrolase family 53 Carbohydrate binding WSC Glycoside hydrolase, family 35 Glycoside hydrolase family 15 Glycoside hydrolase family 43 Secreted glycoside hydrolase family 5 Glycoside hydrolase, family 43 Glycoside hydrolase family 72 Glycoside hydrolase family 53 PIG-U PIG-L Mannosyltransferase, glycosiltransferas superfamily 22 Cutinase superfamily domain Pectinesterase, pectine methyl esterase Hydrophobin-2 GPI-anchor, secreted glycoside hydrolase family 72

Protein identification numbers (ID) are as provided at the genome portal.

a putative Kex2 cleavage site between amino acids 29 and 115 (Table 4). The mature enzymes contain sequence motifs typical of APs, including two conserved aspartic acid residues of the active site spread over a two-domain structure, possibly arising from ancestral duplication and a conserved cysteine possibly responsible for secondary structure maintenance (Table 4). This family of APs is classified by MEROPS (an online database for peptidases) as the peptidase family A1. Since C. albicans SAPs 9 and 10 both possess signals for Glycosylphosphatidylinositol (GPI)-anchoring (Naglik et al., 2003), a search for such a pattern in the SAP family of C. parasitica was performed using big-PI predictor tool from ExPASy proteomics server tools (SIB) (Eisenhaber et al., 1999). None of the nine identified SAPs yielded putative GPI-anchoring signals. A search for trans-membrane domains using the TMHMM program from San Diego Super Computer (SDSC) Biology Workbench (http://workbench.sdsc.edu) revealed putative domains in 71039 and 34127, the rest showed no putative membrane attachment signals.

Phylogenetic analysis of the Cryphonectria parasitica SAP family and comparison to others fungal SAP families While our survey identified nine putative SAPs displaying Kex2 processing signals, C. albicans genome was shown to possess ten SAPs. In light of this we carried out an advanced search of the C. parasitica genome, hoping to identify SAPs that were not picked up by our procedure. This analysis

identified 18 more ORFs with AP domains. Only eight of them contained signal peptides and none of them included putative Kex2 cleavage sites (Table 4). Phylogenetic analysis of the C. parasitica SAP family (Fig 1), showed that three major groups are clustered within the family, A, B, and C. Clusters A and B are supported by higher statistical values than cluster C (in all the methods used for calculation). In particular, cluster B contains six members with high similarity among them. Similarly, the C. albicans SAP family was shown to cluster in three major groups (Stehr et al., 2000). SAPs with putative Kex2 processing sites are distributed equally among the three clades, suggesting that Kex2 processing is not a phylogenetically conserved property (in boxes in Fig 1). Comparisons between phylogenetically related species with different ecological habits are thought to suggest possible specific genes involved in the adaptation process to a specific ecological niche. Given the specificity of C. parasitica as a tree pathogen, we wanted to compare different genomes of related ascomycetes that are either plant pathogens (but not of woody trees) (M. Oryzae and F. graminearum), or nonpathogens (N. crassa and A. oryzae). A human pathogen was also included, given that it is the best characterized family of SAPs (C. albicans). Mining the C. parasitica genome for genes homologous to SAPs retrieved 17 ORFs, a number that is higher than the SAPs present in other plant pathogens, such as M. oryzae (14) (Dean et al., 2005) and F. graminearum (14) (Cuomo et al., 2007). Also N. crassa, A. oryzae, and C. albicans

368

D. Jacob-Wilk et al.

Table 2 e Proteinase-like putative Kex2 substrates in the C. parasitica secretome. Protein ID#

Homologies and similarities

Domain homologies

Aspartic proteinases 90510 Pepsin-like 34127 Pepsin-like 87438 Pepsin-like 37154 Pepsin-like 71039 Pepsin-like 43429 Aspartic type endopeptidase 107093 Aspartic protease 62389 Endothiapepsin 38462 Endothiapepsin Acid proteinases, peptidase A4 family 85286 Proteinase aspergillopepsin II 103361 Proteinase aspergillopepsin II 70434 Proteinase aspergillopepsin II Subtilases, kexin-like, proprotein convertases 91219 Aorsin precursor, alkaline protease 62358 Kex2, 46243 Extracellular alkaline protease 102797 Putative vacuolar serine protease 84359 Subtilisin-like serine protease 99456 Tripeptidyl peptidase I, putative, lyzosome Carboxypeptidases 66919 Serine carboxypeptidase 101284 Serine carboxypeptidase putative 73241 Serine carboxypeptidase putative 93741 Carboxypeptidase C (cathepsin A) 94492 Serine type peptidase 108972 carboxypeptidase S1, putative 84160 Serine-peptidase putative Cystein protease 61062 GPI-anchor transamidase precursor

have 12, 11, and ten SAPs, respectively (Naglik et al., 2003; Borkovich et al., 2004; Naglik et al., 2004; Machida et al., 2005). Kex2 signals were predicted to be present in 9 out of 17 SAPs in C. parasitica, 9 out of 10 in C. albicans, 9 out of 14 in

Peptidase A1 apartic active site Peptidase A1 apartic active site Peptidase A1 apartic active site Peptidase A1 apartic active site Peptidase A1 apartic active site Peptidase A1, ABC transporter Peptidase A1 aspartic protease Aspartic protease precursor Aspartic peptidase active site Acidpeptidase Acidpeptidase Acidpeptidase Kexin, subtilisin, sedolisin Kexin, subtilisin, sedolisin Kexin, subtilisin, sedolisin Kexin, subtilisin, sedolisin Kexin, subtilisin, sedolisin Alkaline serine protease, sedolisin Serine carboxypeptidase Serine carboxypeptidase Serine carboxypeptidase Serine carboxypeptidase Carboxypeptidase S1, putative Serine carboxypeptidase Esterase/lipase/thioesterase Cysteine-type endopeptidase

M. oryzae, 4 out of 14 in F. graminearum, 8 out of 12 in N. crassa, and 5 out of 11 in A. oryzae. Phylogenetic analysis of all the SAPs present in the five fungal genomes included in the analysis showed the presence

Table 3 e Oxidoreductase-like putative Kex2 substrates in the C. parasitica secretome. Protein ID# 13756 34153 89675 51525 50651 65971 51772 71032 108309 38511 98577 95172 68157 13424 46347 73466 53312 45181 105659 102235 38584 81442

Homologies and similarities

Domain homologies

Putative monooxigenase Hypothetical protein Putative D-amino acid oxidase Extracellular dioxygenase, putative Flavin containing polyamine oxidase Thioredoxin reductase, putative Monooxygenase, putative FAD-dependent oxidoreductase, putative Extracellular dioxygenase, putative GPI anchored dioxygenase Disulfide isomerase, putative Laccase 3 Laccase precursor Cytochrome P450 oxidoreductase Cytochrome P450 monooxygenase Hypothetical protein Cytochrome P450 oxidoreductase Cytochrome P450 monooxygenase Hypothetical protein Cytochrome P450 family protein Catalase-peroxidase 2 Peptide methionine sulphoxide, putative

Flavoprotein monooxigenase Intradiol dioxigenase superfamily FAD-dependent oxidoreductase Intradiol ring-cleavage dioxygenase Amino oxidase FAD-dependent oxidoreductase FAD-dependent pyridine nucleotide-disulphide oxidoreductase No domain homology Intradiol ring-cleavage dioxygenase Intradiol ring-cleavage deoxygenase Thioredoxin domain ER retention signal Multicoper-binding site domain and copper-binding Multicopper oxidase, type 1 Cytochrome P450 E-class P450, group I Cytochrome P450 Cytochrome P450 Cytochrome P450 Cytochrome P450 Cytochrome P450 Plant, fungal, bacterial peroxidase Peptide methionine sulfoxide reductase

Differential expression of the putative Kex-2 processed and secreted aspartic proteinase family

369

Table 4 e C. parasitica SAP family. Protein ID#

Signal-P site

Propeptide

Propeptide site (aa)

Domain

Source

Identifier

22e23 18e19 23e24 21e22 20e21 19e20 18e19 19e20 18e19 20e21 19e20 20e21 20e21 17e18 20e21 20e21 17e18

Yes Yes Yes Yes Yes Yes Yes Yes Yes No No No No No No No No

47 KR 86 KR 106 KR 96 KR 29 KR 62 KR 43 KR 42 PR 115 KR N/A N/A N/A N/A N/A N/A N/A N/A

AspProtease/Pepsin AspProtease/Pepsin AspProtease/Pepsin AspProtease/Pepsin AspProtease/Pepsin AspProtease/Pepsin AspProtease/Pepsin AspProtease/Pepsin AspProtease/Pepsin AspProtease/Pepsin AspProtease/Pepsin AspProtease/Pepsin AspProtease/Pepsin AspProtease/Pepsin AspProtease/Pepsin AspProtease/Pepsin AspProtease/Pepsin

Pfam Prosite/Pfam Prosite/Pfam Prosite/Pfam Prosite/Pfam Prosite/Pfam Pfam Pfam Pfam Prosite/Pfam Prosite/Pfam Pfam Prosite/Pfam Pfam Pfam Prosite/Pfam Prosite/Pfam

PF00026 PS00141/PF00026 PS00141/PF00026 PS00141/PF00026 PS00141/PF00026 PS00141/PF00026 PF00026 PF00026 PF00026 PS00141/PF00026 PS00141/PF00026 PF00026 PS00141/PF00026 PF00026 PF00026 PS00141/PF00026 PS00141/PF00026

87438 38462 62389 107093 90510 34127 43429 71039 37154 109050 93423 80821 100383 68161 37875 88976 83961

of a number of clusters of orthologous groups (COGs) as previously defined by (Tatusov et al., 2001) (Fig 2). We arbitrarily named them A, B, and C, according to the subdivision in the three major clusters observed in C. parasitica, with roman numerals to univocally identify each COG.

CP83961

75

CP93423

61

C

CP109050 CP37154

21

CP37875

82 76

CP34127 CP43429 CP87438

99

CP80821

95 80

98

8

CP38462

*

CP100383 CP107093

29

A

CP88976 CP71039

89

*

CP62389

B

*

CP68161

100

CP90510

*

0.2

Fig 1 e Phylogenetic relationships of the C. parasitica SAP gene family. Phylogenetic analyses were performed using maximum likelihood methods as implemented in PhyMLaLRT, based on an alignment of the conserved domains. Branch support values expressed as approximate likelihood-ratio probabilities are displayed. The bar marker indicates the number of amino acid substitutions. Protein identifiers correspond to the protein ID from the sequencing project. The putative Kex2 processed SAPs are boxed. Three major clusters are indicated with the letters A, B, and C. Asterisks were placed to show SAPs chosen for deletion analysis.

None of the C. albicans SAPs is included in any of the COGs that includes the other five species, suggesting that the expansion of the candida SAP family is an event that occurred after the separation from the other ascomycetes considered in this analysis. As for the other COGs, most of them include at least one SAP for each filamentous fungal species. C. parasitica has four SAPs in the B COG, whereas the other species considered have a maximum of two SAPs. A-II also includes three C. parasitica SAPs, the same number as M. oryzae, whereas the other species are represented in this COG only by one protein. SAP37875 of C. parasitica (CP) did not associate to any specific COGs. Phylogenetic trees derived with UPGMA and Neighbor-Joining methods all have a higher confidence number for each of the nodes supporting the clusters of SAPs we have outlined in our analyses (not shown).

Expression analysis of the Cryphonectria parasitica Kex2 processed SAP gene family during in vitro growth on chestnut wood and after hypovirus infection To identify SAPs that potentially contribute to the colonization of chestnut trees by wild type C. parasitica, we studied the expression patterns of the SAP gene family during in vitro growth on chestnut wood and after hypovirus infection. Wild-type strain EP155 was grown on liquid media without wood and levels of expression compared to the same strain and isogenic CHV1-infected strain grown on liquid media containing chestnut wood. We assumed that increased expression in response to chestnut wood and down-regulation in response to the hypovirus might indicate an involvement of this SAP in the infection of chestnut wood by wild type C. parasitica. It is important to mention that there are no differences in the growth rates of wild type and hypoviral containing strains when grown in liquid media without wood as measured by dry weight accumulation (Carpenter et al., 1992). Changes in mRNA levels for SAPs were monitored at 3 DPI (Fig 3). High levels of transcript accumulation for SAPs 71039 and 43429 were detected in all treatments, but no

370

D. Jacob-Wilk et al.

Differential expression of the putative Kex-2 processed and secreted aspartic proteinase family

371

type grown without wood. SAPs 38462, 34127, and 107093 showed very low amounts of transcript levels in the control strain grown in the absence of wood, but were up-regulated upon wood exposure. While no difference was observed between wood and hypoviral treatments in 38462 and 34127, a slightly increased expression was observed for 107093 upon hypoviral infection. Two SAPs, 90510 and 37154, were highly expressed in the control strain, EP155 without wood, and down-regulated upon wood exposure. No differences between virus-infected and control strains growing on wood were observed. Two SAPs, 62389 and 87438, were shown to be up-regulated upon exposure to chestnut wood but downregulated by virus infection, suggesting their possible involvement in infection of chestnut wood (Fig 3). Overall, six out of nine SAPs were shown to be up-regulated by exposure to chestnut wood.

In silico analysis of the SAP family promoter regions

Fig 3 e Expression patterns of the nine putative Kex2 processed SAPs form C. parasitica. Expression patterns were studied with or without chestnut wood and as affected by CHV1 infection. EP complete liquid cultures were inoculated with wild type C. parasitica (EP155) and expression patterns compared to EP155 or hypoviral containing isogenic strain (UEP1) grown for 3 d on liquid cultures with wood. Loaded amounts of rRNA for each treatment are shown on the bottom panel as a control for equal quality and quantity of RNA samples. Two independent experiments were carried out; representative results are shown. Asterisks were placed to show SAPs chosen for deletion analysis.

differences were observed between EP155 and EP155 wood treatments in 71039, while a slight increase was observed upon hypoviral infection. SAP 43429 showed a modest increase in both wood treatments as compared to the wild

In order to better understand the regulation of the C. parasitica SAP gene family, we analyzed the promoter regions for putative transcription factor binding sites. Prediction of putative transcription factor binding sites was performed with TRANSFAC suite and ExPlain 3.0 from BIOBASE International (https:// portal.biobase-international.com) (Kel et al., 2003; Chekmenev et al., 2005). This analysis revealed several putative sites common to most SAP promoter regions. Between them we identified numerous HSF and/or STRE sites, associated with the regulation of stress responsive genes; NIT2 sites, associated with genes induced by nitrogen; sites known to regulate genes involved in carbon utilization and glycolytic gene expression such as ADR1, GCR1, GCR2, MIG1, GAL4, CAT8, and phosphate metabolism in response to phosphate availability such as PHO4. Other sites found numerous times in most SAP promoter regions were associated with the regulation of amino acid biosynthetic genes such as GNC4, LEU3, and MET28, and MATA1 and MATALPHA2 associated with the regulation of mating type specific genes. Other elements also found were TEA1 and TEC1 associated with the regulation of hyphalassociated genes in yeast and PacC associated with pH responsive regulation. In general, even though similar sites were identified in most SAP promoter regions, the relative positions and numbers of putative regulatory sites varied significantly between the members.

Characterization of deletion strains Several SAPs were chosen for further analysis based on their expression patterns. SAPs 87438 and 62389 were chosen

Fig 2 e Phylogenetic relationships of SAPs belonging to six fungal species. Protein identifiers correspond to a shortened unique version of the GenBank accession number or locus/protein ID from the sequencing projects (specified in Table S1). C. parasitica SAPs are identified as in Fig 1. Ao [ Aspergillus oryzae, Ca [ Candida albicans, Fg [ Fusarium graminearum, Mo [ Magnaporthe oryzae, Nc [ Neurospora crassa. Phylogenetic analyses were performed using maximum likelihood methods as implemented in PhyML-aLRT, based on an alignment of the conserved domains. Branch support values expressed as approximate likelihood-ratio probabilities are displayed. The bar marker indicates the number of amino acid substitutions. The three major clusters (A, B, and C) grouped by brackets correspond to the same groups shown in Fig 1. Candida albicans SAPs, are also grouped in a major cluster. COGs are specified by straight vertical lines. Asterisks were placed to show SAPs chosen for deletion analysis.

372

because they were down-regulated by hypoviral infection, suggesting a possible involvement in virulence. SAP 71039 was chosen due to its slight up-regulation upon virus infection, and SAP 90510 was chosen due to its complete downregulation upon wood exposure. To allow a functional analysis of the role of SAPs 87438, 62389, 71039, and 90510 a gene disruption approach was used to create D87438, D62389, D71039, and D90510 strains. The linear replacement constructs containing the Hyg gene flanked by 50 and 30 parts of the genes (Fig 4) were used to transform the C. parasitica Dcpku80 strain. Hygromycin

D. Jacob-Wilk et al.

resistant transformants lacking an active copy of each gene were identified by PCR and Southern blot analysis. Mutants were observed for stability and single spores were subjected to further characterization as compared to control strain C. parasitica Dcpku80. Southern blot analyses for all deleted strains are shown in figure six. Genomic DNA of wild-type Dcpku80 and D87438 was digested with restriction enzymes EcoRV and SalI and probed using a PstI fragment. A single band of approximately 386 bp was detected on wild-type Dcpku80 as predicted from the restriction of EcoRV at position 1016 and SalI at position 630 on

Fig 4 e Southern blot analyses of deletion strains. Genomic DNA of wild-type strain, Dcpku80, and deletion strains was digested with restriction enzymes, blotted and hybridized as specified in each blot. A. Genomic DNA of wild-type Dcpku80 and deletion strains D87438 was digested with EcoRV and SalI and hybridized to a probe produced by digesting the vector containing clone 87438 with PstI. B. Genomic DNA of wild-type Dcpku80 and deletion strains D62389 was digested with SacII and StuI and hybridized to a probe produced by digesting the 62389 containing vector with SacII and StuI. C, D. Genomic DNA of wild-type Dcpku80 and deletion strains D71039 and D90510 was digested with PstI and HindIII, respectively, and hybridized to a riboprobe produced by linearizing each clone with EcoICRI and NruI, respectively. Schematic representations of genomic sequences and probes are provided under each blot.

Differential expression of the putative Kex-2 processed and secreted aspartic proteinase family

the genomic sequence. In mutant strains, this band is replaced by a band of approximately 1886 bp, the product of the Hyg insertion (1400 þ 386) (Fig 4A). Restriction digestion reactions for wild-type Dcpku80 and D62389 strains with SacII and StuI were probed with a fragment isolated using the same restriction enzymes on the cloned genomic DNA. This restriction resulted in a band of approximately 832 bp in the wild-type strain, which is replaced by a band of 2232 bp in the deletion strains (Fig 4B). Southern blot analyses for D71039 and D90510 were carried out by restriction digestion of genomic DNA with PstI and HindIII, respectively. Blots were probed with riboprobes produced by linearizing the vectors containing genomic clones for 71039 and 90510 with EcoICRI and NruI, respectively, as described in materials and methods (Fig 4C,D). Two bands were observed in wild-type Dcpku80 when hybridized to a 71039 probe, a band of 653 (predicted from the restriction by PstI at 2886 and 2233) and a 1934 bp band (predicted from the restriction by PstI at 2233 and 299). In D71039 disrupted strains the 653 bp band was replaced by a 2053 bp band (653 þ 1400 bp Hyg) (Fig 4C). Similarly, restriction analysis of control and deletion strains D90510 resulted in a 2090 bp band in the control strain (predicted from the digestion by HindIII at positions 2310 and 220) that was replaced by a band of w3490 in deleted strains. Characterization of the mutant strains showed that none of the deletion strains were significantly affected in growth rate on PDAmb, MM, and MEA plates (Table 5). Mutants were phenotypically indistinguishable from the control (Dcpku80 strain) when grown on solid media, and fungal sporulation was unchanged as statistically determined (not shown). Deletion strains were able to colonize autoclaved chestnut wood and were able to produce abundant stromal pustules (not shown). The analysis of lesion formation on apples and pathogenicity tests on dormant European chestnut stems revealed no reduction in virulence (Table 6). AP proteolytic activity was measured in the culture filtrates of all tested strains grown on wood, using bovine haemoglobin as a substrate at pH 3.3 and 26  C (Fig 5). Significant differences were observed only between control EP155 and D71039. D71039 showed significantly less proteolytic activity than EP155. Even though growth rates were not affected (not shown), D71039 grown in liquid cultures with wood tended to clump. All other strains tested were not significantly different from wild type EP155 under the conditions tested. In summary, deletion of SAPs D87437, D62389, D71039, and D90510 did not significantly affect growth rate, conidiogenesis,

373

or virulence of C. parasitica under the conditions tested. D71039 strains showed reduced proteolytic activity on culture filtrates and tended to clump when grown in liquid cultures.

Discussion The pleiotropic effects observed in Kex2-silenced strains of C. parasitica (Jacob-Wilk et al., 2009) suggested the importance of Kex2 substrates for the development and the expression of the full range of virulence traits in C. parasitica. This prompted us to search for putative Kex2 substrates in the whole C. parasitica genome. This bioinformatic approach helped identify 114 ORFs whose products are potential Kex2 substrates and among which are secreted and cell-wall associated putative candidates displaying a range of activities important at the hostepathogen interface. Consideration should be given to the fact that some of the putative substrates identified by this screen are sent to the secretory pathway for posttranslational modifications but remain intracellular. We were able to assess the relevance of the outcome of this in silico screen in several ways. Most importantly, the only experimentally verified substrate of a kexin-like protein in C. parasitica e cryparin e was positively identified by this screen. Also, several of the identified candidates are known from the literature to be Kex2 targets, i.e., Kex2 itself and the SAP gene family; which were all detected by this screen (Wilcox and Fuller, 1991; Togni et al., 1996). In addition, several of the identified candidates are known from the literature to be important at the host-pathogen interface such as cryparin, laccase, pectin methyl esterase, cellulase, and SAPs among others (Kazmierczak et al., 2001; Naglik et al., 2003; ValetteCollet et al., 2003; Chung et al., 2008). Our results are consistent with a similar study performed on C. albicans, whose bioinformatic approach also revealed putative Kex2 substrates involved in cell wall maintenance, degradation and cell wall components such as adhesins. They also uncovered a SAP family, hydrolases, and oxidoreductases among others (Newport et al., 2003). Consistent with the growth defects observed in response to the deletion of the Kex2 gene in various fungi, and indicative of the involvement of Kex2 substrates in cell wall integrity (Komano and Fuller, 1995; Newport and Agabian, 1997; Jalving et al., 2000; Bader et al., 2001; Mizutani et al., 2004), the largest group of putative substrates identified by this screen shows homology to domains and sequences related

Table 5 e Growth rate of SAP deletion strains on PDAmb, MM and MEA plates. Fungal strain Dcpku80 D87438 D62389 Dcpku80 D71039 D90510

Diameter  SD (cm) PDAmb 4.60  0.10 4.60  0.06 4.76  0.20 5.96  0.08 5.88  0.07 5.93  0.05

Diameter  SD (cm) MM 3.55 3.52 3.54 2.65 2.58 3.01

 0.14  0.07  0.11  0.05  0.07  0.07

Diameter  SD (cm) MEA 4.58 4.56 4.53 3.95 4.00 3.95

 0.07  0.06  0.09  0.08  0.11  0.08

The evaluation of the diameter of the colony was made at 5 DPI and the statistical analysis was performed using the Student’s t-test, P > 0.05. Data were not significantly different.

374

D. Jacob-Wilk et al.

Table 6 e Mean canker areas induced on European chestnut (C. sativa) stems by wild-type C. parasitica strain, Dcpku80, and deletion strains, D87438, D62389, D71039 and D90510. Isolate a a a b b b a a a a a a a a

Dcpku80 D87438-1 D87438-2 Dcpku80 D62389-1 D62389-2 Dcpku80 D71039-1 Dcpku80 D71039-2 Dcpku80 D90510-1 Dcpku80 D90510-2

Mean canker area (mm2)  SD 1033 1108 1060 1298 1713 1589 1319 1248 977 858 1272 1195 1056 916

             

226 201 263 225 348 462 232 280 194 273 274 254 294 348

Canker area was calculated using the formula for an ellipse. The area of the original wound was not subtracted from final measurements. SD ¼ standard deviation. Statistical analysis was performed using TukeyeKramer HSD test. Results were not significantly different P > 0.05. a Mean canker areas were determined 15 DPI on six replicates. b Mean canker areas were determined 13 DPI on five replicates.

to cell wall functions. Some of these could be involved in pathogenicity or morphogenesis. For example, we identified a putative pectin methyl esterase that could help in host penetration. A pectin methyl esterase was required for the full virulence of Botritis cinerea (Valette-Collet et al., 2003). We also identified a putative cutinase, cellulase, xylanase, and several

Fig 5 e SAP enzymatic assays. Proteolytic activity of wild-type EP155, hypoviral UEP1 and deletion strains was measured in culture filtrates using bovine haemoglobin as substrate. Activity was determined at 26  C and pH 3.3 and was measured as the change in absorbance observed at 280 nm. Results shown are from three independent experiments. Mean comparisons were analyzed by Dunnett’s method using EP155 as control group and an a value of 0.05. The p value is shown for strains that are significantly different from control EP155.

other putative glycosil hydrolases. Some of these proteins might be involved in keeping the cell wall plastic during cell wall expansion and branching, and are therefore involved in morphogenesis; others might be involved in modifying the host cell walls and therefore aiding in penetration. We identified proteins known to associate with the cell wall such as the hydrophobin cryparin. Cryparin has been shown to be required for stromal pustule eruption through the bark of the host and is therefore necessary for dissemination of propagules (Kazmierczak et al., 2005). Early histological observations of C. parasitica penetration through the bark pointed out the importance of mycelial fan formation for successful penetration (Anderson, 1914). It is likely that the development of such a specialized structure would require one or more cell wall modifying enzymes. Microorganisms possess a variety of hydrolytic enzymes, within them secreted extracellular proteinases, some of which are associated with virulence. They have been attributed functions in processes such as adhesion, evasion of host defences, enabling invasion, hyphal formation, and proteolysis of macromolecules for nutrition (Newport and Agabian, 1997; Naglik et al., 2003; Newport et al., 2003; Naglik et al., 2004). Attempts to establish a direct involvement of proteinases in the virulence of phytopathogens have given conflicting results. Lately however, there is mounting evidence to suggest an involvement of proteinases at the fungal-host interface. Proteinases of various phytopathogenic fungi have been detected in infected plants (Sreedhar et al., 1999; Carlile et al., 2000). In Sclerotinia sclerotiorum and B. cinerea the proteolytic activity is correlated with symptom development (Urbanek and Kaczmarek, 1985; Poussereau et al., 2001). The production of protease inhibitors by many plants, and the demonstration that they can affect pathogens (Lorito et al., 1994; Koiwa et al., 1997; Joshi et al., 1998), point to proteinase involvement at the host-pathogen interface. Proteinases are also regarded as antagonists of antifungal proteins secreted as part of the defence responses by the host (Poussereau et al., 2001). In Trichoderma asperellum two APs were induced during cucumber root colonization (Viterbo et al., 2004). Recently, the genomic analyses of several phytophatogenic fungi, have been shown to encode larger numbers of secreted proteinases in comparison to non-pathogenic fungi (Dean et al., 2005; Cuomo et al., 2007), and the number of proteinase inhibitor genes required to protect the pathogen from plant proteinases is expanded in the oomycetes Phytophthora spp. genomes (Tyler et al., 2006). In this study we found that secreted proteinases are the second largest group of secreted proteins containing putative Kex2 cleavage sites, suggesting an important role for these enzymes at the fungal-host interface. APs constitute the best characterized family of the C. albicans secreted and Kex2 processed proteins and their role have been reviewed (Naglik et al., 2003; Naglik et al., 2004). These isoenzymes were shown to be involved in adherence, colonization, nutrition and dissemination of C. albican. Since SAPs encode preproenzymes, their regulation can be controlled both at the mRNA or protein levels. Several studies suggested that proteinase synthesis and secretion are tightly coupled, strongly implying that their regulation occurs primarily at the mRNA level (Homma et al., 1993; White and

Differential expression of the putative Kex-2 processed and secreted aspartic proteinase family

Agabian, 1995). Studies on C. albicans SAP2 expression also demonstrated a positive-feedback regulatory mechanism, where proteolytic products of SAP2 induced this gene (Hube et al., 1994). Their differential expression in response to the environment proves vital for the success of C. albicans as a pathogen, allowing the fungus to survive and cause infections under a variety of conditions (Schaller et al., 2005). Although Kex2 may be a key regulator of SAPs (Newport and Agabian, 1997), alternative processing pathways are thought to exist and auto-activation was shown to occur extracellularly for several SAPs at certain pH values (Koelsch et al., 2000). In this study we identified the C. parasitica, putative Kex2 processed SAP gene family and studied its expression patterns in response to host exposure and hypoviral infection. We showed that SAPs in C. parasitica are regulated differentially in vitro at the mRNA level in response to the presence of the host, and in response to hypoviral infection. This suggested that different SAPs may play different roles at different stages of infection and colonization also in C. parasitica. Analysis of the promoter regions of this gene family revealed many putative transcriptional control factors. Many of which are indicative of the regulation of this gene family in response to different and complex environmental conditions, such as different stresses and nutritional sources. These results support the expression analysis showing differential regulation of this gene family in response to diverse growth conditions, and are consistent with previous reports on the promoter regions and expression patterns of the C. albicans and Candida dubliniensis SAP gene family (Naglik et al., 2003, 2004; Loaiza-Loeza et al., 2009; Parra-Ortega et al., 2009). However, the fact that none of the proteinase deletion strains produced a visibly altered phenotype on growth rates or virulence indicates that while different SAPs might be required at different stages of development and infection, they are not essential for the full development or virulence of C. parasitica under the conditions tested. Alternatively, the lack of phenotype observed could be attributed to functional redundancy known to occur in large gene families upon deletion of one member. In vitro proteolytic enzymatic assays suggest that SAP 71039 is the main extracellular proteolytic enzyme active in culture filtrates of C. parasitica at 3 DPI under the conditions tested. This could explain why deletion strains of 71039 show significant reduction in proteolytic activity while no reduction was observed on the other deletion mutants. Nonetheless, it is possible that the expression or the activity of SAPs 62389 and 87438 is dependent on SAP 71039 activity, and that the disruption of SAP 71039 causes in turn reduced expression or activity of other SAPs. We did not expect a strong reduction in the proteolytic activity for SAP 90510 deletion mutants due to the low levels of transcript accumulation observed for this strain at 3 DPI when grown in liquid cultures with wood. Sequence analysis of SAP 71039 by InterProScan revealed putative transmembrane and Yapsin domains. Yapsins have been shown to be required for cell wall integrity in S. cerevisiae (Krysan et al., 2005), therefore, the clumping observed when D71039 strains were grown in liquid cultures could be attributed to a possible defect on the cell wall of these deletion mutants. Our phylogenetic analysis suggests an expansion of the C. parasitica SAP family of proteins, particularly in some newly identified COG still not present in the COG database (Tatusov

375

et al., 2001; Tatusov et al., 2003). Such expansion could indicate specific adaptation to wood pathogenicity, which was not represented in the other fungal species included in the analysis. The suggested expansion will need to be confirmed by comparison to other wood pathogens comparable to C. parasitica, when their genomes become available. The third largest group of putative substrates identified in this study was oxidoreductases. The defence reaction in a living host can be protection against host oxidative response, detoxification of phenolic compounds, and pigmentation (Hoegger et al., 2004). We identified a putative catalaseperoxidase that could be involved in oxidative breakdown of lignin, melanin, or other aromatic polymers (Martinez et al., 2005). Two laccases were identified; laccase 3, previously characterized and found to be required for full fungal virulence, specifically up-regulated by tannic acid and down-regulated by the hypovirus (Chung et al., 2008), and a laccase precursor type 1 not previously characterized. Laccase A was not identified by this screen (Rigling and Van Alfen, 1991; Choi et al., 1992). Subsequent individual evaluation showed that laccase A possessed a signal peptided but the PR putative Kex2 processing site score was under the ProP server threshold and was therefore, eliminated at that stage. Fungal laccases have been implicated in three major pathways: lignin metabolism, fungal morphogenesis, and defence reactions (Chung et al., 2008). We also identified one thioredoxin reductase and several ORFs showing homology to cytochrome P450. Cytochrome P450 was one of the gene families found to be expanded in the Magnaporthe grisea genome when compared to non-pathogenic fungi Neurospora and Aspergillus (Dean et al., 2005). Furthermore, genes encoding for cytochrome P450 and monooxigenases, which typically modify polyketide backbones to form secondary metabolites, were found neighbouring polyketide synthases (PKSs) gene clusters (Dean et al., 2005). Other putative Kex2 substrates have been identified by this screen, putative components of cell signalling, kinases/phosphatases, GTP/ATPases, lipases, esterases and transporters. To our knowledge ours is the first computational analysis of the whole C. parasitica secretome for putative Kex2 processed sequences. This study identified three major groups of candidate Kex2 substrates: cell-wall-related, proteinases and oxidoreductases, some of which are known to contribute to fungal pathogenicity, some known to be important at the hostepathogen interface; yet a large group whose functions remain to be assessed. The current study characterized the C. parasitca SAP gene family and provided a large inventory of candidate proteins that may act at the fungalehost interface whose functions may be assessed by reverse genetics. Results showing that proteases are the second biggest group of secreted-putative Kex2 substrates, and the fact that SAPs are differentially regulated upon exposure to chestnut wood are suggestive of the importance of proteinases at the fungale host interface, yet their exact function remains elusive.

Acknowledgements We thank collaborators Donald L. Nuss, Center for Biosystems Research, University of Maryland Biotechnology Institute,

376

Alice C. L. Churchill, Department of Plant Pathology and PlantMicrobe Biology, Cornell University and Michael G. Milgroom, Department of Plant Pathology and Plant-Microbe Biology, Cornell University for writing and obtaining the grant that made the sequence of the C. parasitica genome possible. The authors would like to thank all members of JGI C. parasitica genome sequencing and annotation group. We specially thank Andrea Aerts from JGI for her kind help at all times and for kindly providing the initial list of proteins containing putative signal peptides. We also thank Donald L. Nuss and Baoshan Chen for providing the Δcpku80 strain of C. parasitica. We thank Tzion Fahima for critical reading of the manuscript and valuable advice. The C. parasitica genome sequence was performed under the auspices of the US Department of Energy’s Office of Science, Biological and Environmental Research Program, and by the University of California, Lawrence Berkeley National Laboratory under contract No. DE-AC02-05CH11231, Lawrence Livermore National Laboratory under Contract No. DE-AC5207NA27344, and Los Alamos National Laboratory under contract No. DE-AC02-06NA25396.

Supplementary material Supplementary data related to this article can be found online at doi:10.1016/j.funbio.2011.12.006.

references

Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ, 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 25: 3389e3402. Anderson PJ, 1914. The Morphology and Life History of the Chestnut Blight Fungus. The commission for the investigation and control of the chestnut tree blight disease in Pennsylvania. December, 1913. Anisimova M, Gascuel O, 2006. Approximate likelihood-ratio test for branches: a fast, accurate, and powerful alternative. Systematic Biology 55: 539e552. Anson ML, 1938. The estimation of pepsin, trypsin, papain, and cathepsin with hemoglobin. Journal of General Physiology 22: 79e89. Bader O, Krauke Y, Hube B, 2008. Processing of predicted substrates of fungal Kex2 proteinases from Candida albicans, C. glabrata, Saccharomyces cerevisiae and Pichia pastoris. BMC Microbiology 8: 116. € hlschlegel F, Bader O, Schaller M, Klein S, Kukula J, Haack K, Mu € fer W, Hube B, 2001. The Kex2 gene of Candida Korting HC, Scha glabrata is required for cell surface integrity. Molecular Microbiology 41: 1431e1444. Basco RD, Cueva R, Andaluz E, Larriba G, 1996. In vivo processing of the precursor of the major exoglucanase by KEX2 endoprotease in the Saccharomyces cerevisiae secretory pathway. Biochimica et Biophysica Acta 1310: 110e118. Ben Khaled H, Ghorbel-Bellaaj O, Hmidet N, Jellouli K, El-Hadj Ali N, Ghorbel S, Nasri M, 2011. A novel aspartic protease from the viscera of Sardinelle (Sardinella aurita): Purification and characterisation. Food Chemistry 128: 847e853.

D. Jacob-Wilk et al.

Bendtsen JD, Nielsen H, von Heijne G, Brunak S, 2004. Improved prediction of signal peptides: SignalP 3.0. Journal of Molecular Biology 340: 783e795. Borkovich KA, Alex LA, Yarden O, Freitag M, Turner GE, Read ND, et al. 2004. Lessons from the genome sequence of Neurospora crassa: tracing the path from genomic blueprint to multicellular organism. Microbiology and Molecular Biology Reviews 68: 1e108. Bostian KA, Elliott Q, Bussey H, Burn V, Smith A, Tipper DJ, 1984. Sequence of the preprotoxin dsRNA gene of type-I killer yeast - multiple processing events produce a two-component toxin. Cell 36: 741e751. Carlile AJ, Bindschedler L, Jones BL, Niku-Paavola ML, 2000. Characterization of SNP1, cell wall degrading trypsin, produced during infection by Stagnospora nodorum. Molecular PlantMicrobe Interactions 13: 538e550. Carpenter CE, Mueller RJ, Kazmierczak P, Zhang L, Villalon DK, Van Alfen NK, 1992. Effect of a virus on accumulation on a tissuespecific cell-surface protein of the fungus Cryphonectria (Endothia) parasitica. Molecular Plant-Microbe Interactions 4: 55e61. Chekmenev DS, Haid C, Kel AE, 2005. P-MATCH: Transcription factor binding site search by combining patterns and weight matrices. Nucleic Acids Research 33: W432eW437. Choi GH, Larson TG, Nuss DL, 1992. Molecular analysis of the laccase gene from the chestnut blight fungus and selective suppression of its expression in an isogenic hypovirulent strain. Molecular Plant-Microbe Interactions 5: 119e128. Chung HJ, Kwon BR, Kim JM, Park SM, Park JK, Cha BJ, Yang MS, Kim DH, 2008. A tannic acid-inducible and hypoviral-regulated Laccase 3 contributes to the virulence of the chestnut blight fungus Cryphonectria parasitica. Molecular Plant-Microbe Interactions 21: 1582e1590. Churchill ACL, Ciuffetti LM, Hansen DR, Van Etten HD, Van Alfen NK, 1990. Transformation of the fungal pathogen Cryphonectria parasitica with a variety of heterologous plasmids. Current Genetics 17: 25e31. Cuomo CA, Guldener U, Xu JR, Trail F, Turgeon BG, Di Pietro A, et al. 2007. The Fusarium graminearum genome reveals a link between localized polymorphism and pathogen specialization. Science 317: 1400e1402. Dawe AL, Nuss DL, 2001. Hypoviruses and chestnut blight: exploiting viruses to understand and modulate fungal pathogenesis. Annual Reviews of Genetics 35: 1e29. Dean RA, Talbot NJ, Ebbole DJ, Farman ML, Mitchell TK, Orbach MJ, et al. 2005. The genome sequence of the rice blast fungus Magnaporthe grisea. Nature 434: 980e986. Duckert P, Brunak S, Blom N, 2004. Prediction of proprotein convertase cleavage sites. Protein Engineering Design and Selection 17: 107e112. Eisenhaber B, Bork P, Eisenhaber F, 1999. Prediction of potential GPI-modification sites in proprotein sequences. Journal of Molecular Biology 292: 741e758. Elliston JE, 1985. Characteristics of dsRNA-free and dsRNAcontaining strains of Endothia parasitica in relation to hypovirulence. Phytopathology 75: 151e158. Fahima T, Kazmierczak P, Hansen DR, Pfeiffer P, Van Alfen NK, 1993. Membrane-associated replication of an unencapsidated double-strand RNA of the fungus, Cryphonectria parasitica. Virology 195: 81e89. Guindon S, Gascuel O, 2003. A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Systematic Biology 52: 696e704. Hansen DR, Van Alfen NK, Gillies K, Powell WA, 1985. Naked double-stranded RNA associated with hypovirulence of Endothia parasitica is packaged in fungal vesicles. Journal of General Virology 66: 2605e2614. Hoegger PJ, Navarro-Gonzalez M, Kilaru S, Hoffmann M, Westbrook ED, Kues U, 2004. The laccase gene family in Coprinopsis cinerea (Coprinus cinereus). Current Genetics 45: 9e18.

Differential expression of the putative Kex-2 processed and secreted aspartic proteinase family

Homma M, Chibana H, Tanaka K, 1993. Induction of extracellular proteinase in Candida albicans. Journal of General Microbiology 139: 1187e1193. Hruskova-Heidingsfeldova O, 2008. Secreted proteins of Candida albicans. Frontiers in Bioscience 13: 7227e7242. Hube B, Monod M, Schofield DA, Brown AJ, Gow NA, 1994. Expression of seven members of the gene family encoding secretory aspartyl proteinases in Candida albicans. Molecular Microbiology 14: 87e99. Iguchi K, Hirano H, Kishida M, Kawasaki H, Sakai T, 1997. Cloning of a protopectinase gene of Trichosporon penicillatum and its expression in Saccharomyces cerevisiae. Microbiology 143: 1657e1664. Jacob-Wilk D, Turina M, Kazmierczak P, Van Alfen NK, 2009. Silencing of Kex2 significantly diminishes the virulence of Cryphonectria parasitica. Molecular Plant-Microbe Interactions 22: 211e221. Jacob-Wilk D, Turina M, Van Alfen NK, 2006. Mycovirus Cryphonectria hypovirus 1 elements cofranctionate with trans-golgi network membranes of the fungal host, Cryphonectria parasitica. Journal of Virology 80: 6588e6596. Jalving R, van de Vondervoort PJI, Visser J, Schaap PJ, 2000. Characterization of the kexin-like maturase of Aspergillus niger. Applied and Environmental Microbiology 66: 363e368. Joshi BN, Sainani MN, Bastawade KB, Gupta VS, Ranjekar PK, 1998. Cysteine protease inhibitor from pearl millet: A new class of antifungal protein. Biochemical and Biophysical Research Communications 246: 382e387. Julius D, Brake A, Blair L, Kunisawa R, Thorner J, 1984. Isolation of the putative structural gene for the lysine-arginine-cleaving endopeptidase required for processing of yeast preproa-factor. Cell 37: 1075e1089. Kazmierczak P, Kim DH, Turina M, Van Alfen NK, 2005. A hydrophobin of the chestnut blight fungus, Cryphonectria parasitica, is required for stromal pustule eruption. Eukaryotic Cell 4: 931e936. Kazmierczak PK, Kim DH, McCabe P, Van Alfen NK, 2001. Role of the hydrophobin cryparin in the biology of the ascomycete, Cryphonectria parasitica. Phytopathology 91: S47. € ssling E, Reuter I, Cheremushkin E, Kel-Margoulis OV, Kel AE, Gro Wingender E, 2003. MATCH: A tool for searching transcription factor binding sites in DNA sequences. Nucleic Acids Research 31: 3576e3579. Koiwa H, Bressan RA, Hasegawa PM, 1997. Regulation of protease inhibitors and plant defence. Trends in Plant Science 2: 379e384. Koelsch G, Tang J, Loy JA, Monod M, Jackson K, Foundling SI, Lin X, 2000. Enzymic characteristics of secreted aspartic proteases of Candida albicans. Biochimica et Biophysica Acta 1480: 117e131. Komano H, Fuller RS, 1995. Shared functions in vivo of a glycosylphosphatidylinositol-linked aspartyl protease, Mkc7, and the proprotein processing protease Kex2 in yeast. Proceedings of the National Academy of Science U.S.A. 92: 10752e10756. Krysan DJ, Ting EL, Abeijon C, Kroos L, Fuller RS, 2005. Yapsins are a family of aspartyl proteases required for cell wall integrity in Saccharomyces cerevisiae. Eukaryotic Cell 4: 1364e1374. Lan X, Yao Z, Zhou Y, Shang J, Lin H, Nuss DL, Chen B, 2008. Deletion of the cpku80 gene in the chestnut blight fungus, Cryphonectria parasitica, enhances gene disruption efficiency. Current Genetics 53: 59e66. Le SQ, Gascuel O, 2008. An improved general amino acid replacement matrix. Molecular Biology and Evolution 25: 1307e1320. Loaiza-Loeza S, Parra-Ortega B, Cancino-Dıaz JC, Illades-Aguiar B,  ndez-Rodrıguez CH, Villa-Tanaca L, 2009. Differential Herna expression of Candida dubliniensis-secreted aspartyl proteinase genes (CdSAP1-4) under different physiological conditions and

377

during infection of a keratinocyte culture. FEMS Immunology and Medical Microbiology 56: 212e222. Lorito M, Broadway RM, Hayes CK, Woo SL, Noviello C, Williams DL, Harman GE, 1994. Proteinase inhibitors from plants as a novel class of fungicides. Molecular Plant-Microbe Interactions 7: 525e527. Machida M, Asai K, Sano M, Tanaka T, Kumagai T, Terai G, et al. 2005. Genome sequencing and analysis of Aspergillus oryzae. Nature 438: 1157e1161. Martinez AT, Speranza M, Ruiz-Duenas FJ, Ferreira P, Camarero S, Guillen F, Martinez MJ, Gutierrez A, del Rio JC, 2005. Biodegradation of liognocellulosics: microbial, chemical, and enzymatic aspects of the fungal attack of lignin. International Microbiology 8: 195e204. Milgroom MG, Cortesi P, 2004. Biological control of chestnut blight with hypovirulence: a critical analysis. Annual Reviews of Phytopathology 42: 311e338. Mizuno K, Nakamura T, Ohshima T, Tanaka S, Matsuo H, 1988. Yeast Kex2 gene encodes an endopeptidase homologous to subtilisin-like serine proteases. Biochemical and Biophysical Research Communications 156: 246e254. Mizutani O, Nojima A, Yamamoto M, Furukawa K, Fujioka T, Yamagata Y, Abe K, Nakajima T, 2004. Disordered cell integrity signaling caused by disruption of the kex2B gene in Aspergillus oryzae. Eukaryotic Cell 3: 1036e1048. Naglik JR, Albrecht A, Bader O, Hube B, 2004. Candida albicans proteinases and host/pathogen interactions. Cellular Microbiology 6: 915e926. Naglik JR, Challacombe SJ, Hube B, 2003. Candida albicans secreted aspartyl proteinases in virulence and pathogenesis. Microbiology and Molecular Biology Reviews 67: 400e428. Newhouse JR, MacDonald WL, Hoch HC, 1990. Virus-like particles in hyphae and conidia of european hypovirulent dsRNAcontaining strains of Cryphonectria parasitica. Canadian Journal of Botany 68: 90e101. Newport G, Agabian N, 1997. Kex2 influences Candida albicans proteinase secretion and hyphal formation. The Journal of Biological Chemistry 272: 28954e28961. Newport G, Kuo A, Flattery A, Gill C, Blake JJ, Kurtz MB, Abruzzo GK, Agabian N, 2003. Inactivation of Kex2p diminishes the virulence of Candida albicans. The Journal of Biological Chemistry 278: 1713e1720. Nielsen H, Engelbrecht J, Brunak S, Von Heijne G, 1997. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Engineering Design and Selection 10: 1e6. Nielsen H, Krogh A, 1998. Prediction of signal peptides and signal anchors by a hidden Markov model. Proceedings International Conference of Intelligent Systems for Molecular Biology 6: 122e130. Nuss DL, 2005. Hypovirulence: Mycoviruses at the fungal-plant interface. Nature Reviews Microbiology 3: 632e642.  ndezParra-Ortega B, Cruz-Torres H, Villa-Tanaca L, Herna Rodrıguez C, 2009. Phylogeny and evolution of the aspartyl protease family from clinically relevant Candida species. Mem Inst Oswaldo Cruz 104: 505e512.
de G, Wang H, Fudalej F, Gaillardin C, Seman M, Nicaud JM, Pigne 2000. Characterization of an extracellular lipase encoded by LIP2 in Yarrowia lipolytica. Journal of Bacteriology 182: 2802e2810. Poussereau N, Gente S, Rascle C, Billon-Grand G, Fevre M, 2001. aspS encoding an unusual aspartyl protease from Sclerotinia sclerotiorum is expressed during phytopathogenesis. FEMS microbiology Letters 194: 27e32. Powell WA, Van Alfen NK, 1987. Two nonhomologus viruses of Cryphonectria (Endothia) parasitica reduce accumulation of specific virulence associated polypeptides. Journal of Bacteriology 169: 5324e5326.

378

Puhalla JE, Anagnostakis SL, 1971. Genetic and nutritional requirements of Endothia parasitica. Phytopathology 61: 169e173. Rastgou M, Habibi MK, Izadpanah K, Masenga V, Milne RG, Wolf YI, Koonin EV, Turina M, 2009. Molecular characterization of the plant virus genus Ourmiavirus and evidence of inter-kingdom reassortment of viral genome segments as its possible route of origin. Journal of General Virology 90: 2525e2535. Rigling D, Heiniger U, Hohl HR, 1989. Reduction of laccase activity in dsRNA-containing hypovirulent strains of Cryphonectria (Endothia) parasitica. Phytopathology 79: 219e223. Rigling D, Van Alfen NK, 1991. Regulation of laccase biosynthesis in the plant-pathogenic fungus Cryphonectria parasitica by double-stranded RNA. Journal of Bacteriology 173: 8000e8003. Rockwell NC, Krysan DJ, Komiyama T, Fuller RS, 2002. Precursor processing by kex2/furin proteases. Chemical Reviews 102: 4525e4548. Rockwell NC, Thorner JW, 2004. The kindest cuts off all: crystal structures of Kex2 and furin reveal secrets of precursor processing. Trends in Biochemical Sciences 29: 80e87. Saitou N, Nei M, 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 4: 406e425. Sambrook J, Fritsch EF, Maniatis T, 1989. “Molecular cloning”: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. Schaller M, Borelli C, Korting HC, Hube B, 2005. Hydrolytic enzymes as virulence factors of Candida albicans. Mycoses 48: 365e377. Singh A, Chen EY, Lugovoy JM, Chang CN, Hitzeman RA, Seeburg PH, 1983. Saccharomyces cerevisiae contains two discrete genes coding for the alpha-factor pheromone. Nucleic Acids Research 11: 4049e4063. Sneath PHA, Sokal R, 1973. Numeric Taxonomy: The Principles and Practice of Numerical Classification. W.H. Freeman. Sreedhar L, Kogayashi DY, Bunting TE, Hillman BI, Belanger FC, 1999. Fungal proteinase expression in the interaction of the plant pathogen Magnaporthe poae with its host. Gene 235: 121e129. Stehr F, Felk A, Kretschmar M, Schaller M, Schafer W, Hube B, 2000. Extracellular hydrolitic enzymes and their relevance during Candida albicans infections. Mycoses 43 (Suppl. 2): 17e21. Tamura K, Dudley J, Nei M, Kumar S, 2007. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Molecular Biology and Evolution 24: 1596e1599. Tatusov RL, Fedorova ND, Jackson JD, Jacobs AR, Kiryutin B, Koonin EV, et al. 2003. The COG database: an updated version includes eukaryotes. BMC Bioinformatics 4: 41.

D. Jacob-Wilk et al.

Tatusov RL, Natale DA, Garkavtsev IV, Tatusova TA, Shankavaram UT, Rao BS, Kiryutin B, Galperin MY, Fedorova ND, Koonin EV, 2001. The COG database: new developments in phylogenetic classification of proteins from complete genomes. Nucleic Acids Research 29: 22e28. Taylor NA, Van De Ven WJM, Creemers JWM, 2003. Curbing activation: proprotein convertases in homeostasis and pathology. The FASEB Journal 17: 1215e1227. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG, 1997. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 25: 4876e4882. Togni G, Sanglard D, Quadroni M, Foundling SI, Monod M, 1996. Acid proteinase secreted by Candida tropicalis: functional analysis of preproregion cleavages in C. tropicalis and Saccharomyces cerevisiae. Microbiology 142: 493e503. Turina M, Prodi A, Van Alfen NK, 2003. Role of the Mf1-1 pheromone precursor gene of the filamentous ascomycete Cryphonectria parasitica. Fungal Genetics and Biology 40: 242e251. Tyler BM, Tripathy S, Zhang X, Dehal P, Jiang RHY, Aerts A, et al. 2006. Phytophthora genome sequences uncover evolutionary origins and mechanisms of pathogenicity. Science 313: 1261e1266. Urbanek H, Kaczmarek A, 1985. Extracellular proteinases of the isolate Botrytis cinerea virulent to apple tissue. Acta Biochimica Polonica 32: 101e109. Valette-Collet O, Cimerman A, Reignault P, Levis C, Boccara M, 2003. Disruption of Botrytis cinerea pectin methylesterase gene Bcpme1 reduces virulence on several host plants. Molecular Plant-Microbe Interactions 16: 360e367. Viterbo A, Harel M, Chet I, 2004. Isolation of two aspartyl proteases from Trichoderma asperellum expressed during colonization of cucumber roots. FEMS microbiology Letters 238: 151e158. White TC, Agabian N, 1995. Candida albicans secreted aspartyl proteinases: isoenzyme pattern is determined by cell type, and levels are determined by environmental factors. Journal of Bacteriology 177: 5215e5221. Wilcox CA, Fuller RS, 1991. Posttranslational processing of the prohormone-cleaving Kex2 protease in the Saccharomyces Cerevisiae secretory pathway. The Journal of Cell Biology 115: 297e307. Zhang L, Baasiri RA, Van Alfen NK, 1998. Viral repression of fungal pheromone precursor gene expression. Molecular and Cellular Biology 18: 953e959. Zhang L, Churchill ACL, Kazmierczak P, Kim D-H, Van Alfen NK, 1993. Hypovirulence-associated traits induced by a mycovirus of Cryphonectria parasitica are mimicked by targeted inactivation of a host gene. Molecular and Cellular Biology 13: 7782e7792.