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1 Agriculture and Agri-Food Canada, Saskatoon, SK S7N OX2, Canada
2 Institute of Microbiology, College of Life Science, Zhejiang University, Hangzhou 310029, PR China
Correspondence
Dwayne D. Hegedus
Hegedusd{at}agr.gc.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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e–03) to other fungal genes, of which 69.6 % encoded proteins with a reported function. Proteins involved in protein synthesis and metabolism were encoded by 38.3 % of the ESTs, while 26.3 % encoded proteins involved in cell-cycle regulation, DNA synthesis, protein fate, transport, cell defence, transcription and RNA synthesis, and 4.9 % encoded proteins associated with cellular transport, signal transduction, control of cellular organization and cell-wall degradation. Several proteinases, including aspartic proteinases, trypsins, trypsin-like serine proteases and metalloproteases, with the potential to degrade insect cuticle were expressed by the two isolates.
The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are available in Tables 2–4 and supplementary Table S1.
A table of annotations and functional categories of Zoophthora radicans ESTs from both original and new host-passage isolates is available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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) with narrow host ranges, Z. radicans is best known for its broad geographical and host range. While the fungus is pathogenic on species from more than 10 insect orders, including Coleoptera, Lepidoptera, Diptera, Homoptera, Hymenoptera, Heteroptera, Orthoptera, Plecopetera, Psocoptera and Thysanoptera (Humber, 1992; Papierok et al., 1984; Furlong & Pell, 2001; Wraight et al., 2003), individual isolates exhibit differing degrees of host specificity. Generally, isolates exhibit greater virulence against the species from which they were originally isolated than against species from other insect orders (Glare, 1988). Repeated passage through an insect host is known to enhance the virulence of entomopathogenic fungi toward a specific host, while continual propagation on artificial medium leads to reduced virulence (Brownbridge et al., 2001; Quesada-Moraga & Vey, 2003); however, the mechanisms underlying these phenomena remain unclear. To identify factors involved in host specificity, we studied a strain of Z. radicans from an isolate originally associated with the cabbage butterfly, Pieris brassicae, and an isogenic strain that was adapted to be highly virulent toward the diamond back moth, Plutella xylostella, through serial infection and reisolation (Li et al., 2004a). Previous investigations found that the most obvious difference between the Pieris brassicae isolate and the isolates adapted to Plutella xylostella was the profile of their respective extracellular proteases (Xu et al., 2006).
Penetration, dissemination and subsequent death of the insect host involve several biochemical and physical activities, and therefore many genes are associated with fungal pathogenesis. The infection by Z. radicans starts when the primary or capillary conidia that are forcibly discharged from conidiophores land on the cuticle of a suitable host. These germinate and differentiate to form appressoria by extension and swelling of the germ tube tip. Penetration is brought about by a combination of hydrolytic enzymes and mechanical forces exerted by the protruding penetration peg beneath the appressorium. Once the cuticle has been breached, host defence responses are induced or enabled. Some entomophthoralean species initially produce protoplasts lacking the cell-wall components recognized by insect haemocytes, and proliferate with impunity (Glare & Milner, 1991; Pell et al., 2001; Shah & Pell, 2003).
The limited information on genes responsible for pathogenicity and virulence restricts our ability to make targeted improvements to these fungi, and thus a better understanding of the pathogenicity mechanisms and of host adaptation is required. Expressed sequence tag (EST) studies with Metarhizium anisopliae, Conidiobolus coronatus and Beauveria bassiana generated around 1700, 2000 and 10 000 ESTs, respectively, and suggested they share many of the same pathogenicity genes (Freimoser et al., 2003a, b; Cho et al., 2006a, b). Genes encoding enzymes with the potential to degrade host cuticle or to disable antimicrobial peptides, including multiple chitinases, subtilisins, trypsins, metalloproteases, aspartyl proteases, phospholipases, lipases and esterases, were identified. These EST data, combined with those from phytopathogenic fungi such as Blumeria graminis (Thomas et al., 2001), Gibberella zeae (Trail et al., 2003), Sclerotinia sclerotiorum (Li et al., 2004b), Magnaporthe grisea (Ebbole et al., 2004), Mycosphaerella graminicola (Keon et al., 2005), Fusarium verticillioides (Brown et al., 2005), Phakopsora pachyrhizi (Posada-Buitrago & Frederick, 2005) and a saprophyte, Aspergillus nidulans (Jeong et al., 2000; Sims et al., 2004), enrich our understanding of pathogenic mechanisms and will help to delineate key factors for fungal adaptation to new insect hosts. In this study, we generated ESTs from a Z. radicans strain originally isolated from Pieris brassicae, and from an isogenic strain adapted to Plutella xylostella to identify candidate pathogenicity genes and to compare gene expression patterns in different insect hosts.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). All isolates were routinely grown on SEMA (80 % Sabouraud dextrose agar, 11.5 % fresh milk, 8.5 % egg yolk in 1 l distilled water) at 10 °C in a photoperiod of 12 h light : 12 h dark (L12 : D12) and subcultured every 3 months.
Bioassay and data analysis.
For bioassays, all strains were grown on SEMA at 15 °C (L12 : D12) for 7 days. The resultant fungal colony was macerated with a needle and inoculated into a 100 ml flask containing 30 ml Sabouraud dextrose broth (SDB; 4 % glucose, 1 % peptone, 1 % yeast extract and 12.5 µg tetracycline ml–1). The liquid culture was incubated at 28 °C for 24 h with agitation (150 r.p.m.) and then poured into a 200 ml flask containing 80 ml SDB and incubated for an additional 48 h under the same regimen. Subsequently, the liquid culture was distributed in 10 ml aliquots onto 90 mm Petri dishes containing only 2 % agar. Excess liquid was removed using sterile filter paper and mycelial mats were used for inoculation when the fungus had sporulated uniformly.
Plutella xylostella were maintained on Brassica napus plants in a constant environment room under standard temperature and photoperiodic conditions (23
25 °C; L12 : D12). Pupae were collected and stored at 4 °C until sufficient quantities were obtained and then transferred into an oviposition cage for eclosion. Adults were allowed to lay ova on a perforated plastic bag containing a fresh cabbage leaf, and the bag was subsequently used to encase a plant during incubation. Second to third instar Plutella xylostella larvae were inoculated using the conidial shower method (Xu & Feng, 2000). Petri plates containing larvae on a cabbage leaf were placed beneath an inverted sporulating culture to allow the conidia to discharge directly onto the larval epidermis. Leaves and their resident larvae were transferred to Petri dishes covered with a porous membrane and were incubated at 20 °C at high relative humidity (90 %). Live and dead larvae were recorded daily. Dead larvae were removed and examined individually for verification of infection after overnight incubation.
All data were corrected for control mortality and analysed by using time–concentration–mortality modelling (Nowierski et al., 1996; Xu & Feng, 2000). The model includes I concentrations and J times of observation. The cumulative mortality probability (pij) related to the concentration Ci (i=1, 2, ..., I) at time tj (j=1, 2, ..., J) can be expressed as:
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j is the parameter(s) for the time effect of Ci during the period from start to the jth observation, (t1, t2, ..., tj-1, tj, tj+1,..., tJ). To guarantee that the observed mortality probability is independent of time, the true mortality induced with Ci at the interval [tj–1, tj] must be considered. This true mortality, qij, is called the conditional mortality probability (Robertson & Preisler, 1992) and is given as follows:
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j describes the conditional time effect of Ci at the interval [tj–1, tj]. The independence between time intervals of observation allowed the fitting of data to equation 2 by approaching the binomial response variable to the maximum-likelihood equation (Nowierski et al., 1996; Xu & Feng, 2000), yielding j and β. The values of j were then calculated using the formula (Robertson & Preisler, 1992) below:
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).
cDNA library construction.
Fungal mycelia were collected after 3 days of growth on SEMA at room temperature and ground in liquid nitrogen. Total RNA was isolated by dispensing 200 mg (wet wt) ground mycelia into a 1.5 ml micro-centrifuge tube containing 600 µl extraction buffer [0.1 M NaCl, 2 % SDS, 50 mM Tris/HCl (pH 9.0), 10 mM EDTA (pH 8.0)] and 600 µl phenol/chloroform/isoamyl alcohol (25 : 24 : 1, by vol.). The solution was mixed using a vortex for 30 s, transferred to a new tube and extracted once with 250 µl chloroform/isoamyl alcohol, and the RNA was precipitated with 0.1 vols 3 M sodium acetate (pH 5.2) and 2.5 vols 95 % ethanol. The RNA pellet was washed with 70 % ethanol, dried for 5 min and resuspended in 50 µl RNase-free ddH2O. Poly(A)+ mRNA was purified on an oligo(dT)-cellulose column using an mRNA purification kit (Amersham Pharmacia Biotech). The cDNA library was constructed using the ZAP cDNA synthesis kit (Stratagene) and amplified in Escherichia coli XL1-Blue. In vivo mass excision of pBluescript clones was performed according to the manufacturer's instructions (Stratagene). Colonies were randomly picked and put into wells of a 384-well microtitre plate with 80 µl freezing broth [1 % tryptone, 0.5 % yeast extract, 1 % NaCl, 4.5 % glycerol, 36 mM K2HPO4 . 3H2O, 13.2 mM KH2PO4, 1.7 mM sodium citrate, 0.41 mM MgSO4 . 7H2O, 6.8 mM (NH4)2SO4] with 100 µg carbenicillin ml–1. The plate was incubated for 16 h at 37 °C and stored at –80 °C.
DNA sequencing and EST analysis.
Bacteria in the 384-well plate were transferred using a pin tool to a 4x96-well flat bottom block with each well containing 1.25 ml LB medium plus 100 µg carbenicillin ml–1. Plasmid DNA was isolated using the DirectPrep 96 Miniprep kit (Qiagen). cDNA inserts were sequenced from both the 5' and 3' termini using T3 (forward) and T7 (reverse) primers, respectively, on an ABI Prism 3700 DNA sequencer using Big Dye Terminator chemistry (Applied Biosystems). The resultant sequences were processed for sequence quality by selecting those having a Phred score (Ewing et al., 1998) of more than 20 and the vector sequences were removed. Overlapping forward and reverse sequences were aligned using Sequencher 4.1 (GeneCodes) and combined to form a single EST contig. The sequences were translated and compared to international databases (non-redundant GenBank CDS translations, PDB, Swiss-Prot, PIR, PRF) using the gapped BLASTX algorithm (Altschul et al., 1997).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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. The deaths attributed to infection by the original and adapted strains R1, R3 and R5 occurred mostly on days 3–5, days 2–4, days 2–4 and days 3–5, respectively. All cadavers displayed typical symptoms for Z. radicans infection upon microscopic examination after overnight incubation in a humidified chamber. The cumulative mortality at the end of the observation period (6 days) ranged from 6.3 to 15.2 % for the original strain, 7.5–33.3 % for R1, 5.1–28.9 % for R3 and 5.5–17.6 % for R5 as the spore concentration increased. The cumulative mortality of the control larvae was 2.0–3.9 % on day 6 (Table 1).
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). The t statistics for all parameters estimated were significant (P<0.01). The Hosmer–Lemeshow test for the heterogeneity of the goodness of the fit (Nowierski et al., 1996) was insignificant for all strains (ARSEF 1342, C=4.45, P=0.81; R1-1342, C=10.11, P=0.26; R3-1342, C=2.93, P=0.94; R5-1342, C=7.02, P=0.53), indicating that the data for the Z. radicans isolates against Plutella xylostella fit the model well. The time–concentration–mortality relationships of four strains against Plutella xylostella based on the fitting of the cumulative and conditional mortality probabilities from equations 1 and 2 are shown in Fig. 1.
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j estimated by modelling, the mortality peak is the third day for the original strain and the second day for all strains adapted to Plutella xylostella, indicating that the infection time was reduced for strains adapted to Plutella xylostella. Based on time–concentration–mortality modelling, the LC20 (the lethal concentration required to cause 20 % mortality) on days 1–6 after exposure was estimated as 14.7, 14.5, 9.0, 7.1, 6.0 and 5.5 spores mm–2, respectively, for the original strain; 9.6, 5.0, 4.2, 3.6, 3.1 and 2.9 spores mm–2 for R1; 4.6, 2.9, 2.8, 2.5, 2.4 and 2.2 spores mm–2 for R3; and 5.2, 3.7, 3.2, 2.8, 2.6 and 2.6 spores mm–2 for R5. Thus the adapted strains tended to exhibit higher infectivity to the new host than the original isolate as the number of host passages increased.
cDNA library analysis and characterization of the EST data
Two cDNA libraries were constructed for Z. radicans EST analysis. One library (ZRA) was constructed using mycelia from a strain isolated from the original host (Pieris brassicae) and a second library (ZRB) was constructed using mycelia of an isogenic strain that had been passaged and reisolated from an alternate host (Plutella xylostella). The libraries from the strains isolated from Pieris brassicae and Plutella xylostella had primary titres of 5.18x105 and 7.39x105 plaques, respectively, with mean insert sizes of 1.35 kbp and ranging from 0.65 to 3.5 kbp. A combined total of 1536 randomly selected cDNA clones from both Z. radicans cDNA libraries were sequenced, yielding 1839 forward and reverse ESTs of satisfactory quality. For the Pieris brassicae isolate, a total of 844 ESTs of satisfactory quality were generated, representing 455 unique sequences consisting of 220 singletons and 235 contigs with an abundance of 2–40 times in the dataset. For the strain isolated from Plutella xylostella, a total of 995 ESTs with satisfactory quality were generated, representing 533 unique sequences consisting of 249 singletons and 284 contigs with an abundance of 2–29 times. In total, 899 unique Z. radicans sequences consisting of 433 singletons and 466 contigs were obtained of which 837 (284 contigs and 553 singletons) were similar to other sequences (Ee–03 and 95.2 % with Ee–05) in the NCBI ‘nr’ and Saccharomyces Genome databases. For this group, 69.65 % of the sequences encoded proteins with a known function with the most similar orthologues being from fungi (54.6 %) and animal (31.9 %) and to a lesser extent from plants (8.1 %) and bacteria (5.4 %).
Functional classification of expressed Z. radicans genes
We compared our EST data to several databases using BLASTX with a threshold E-value of 10–3 and grouped the annotated genes into 14 categories based on the Saccharomyces cerevisiae Functional Catalogue developed by the Munich Information Center for Protein Sequences (MIPS) (http://mips.gsf.de/projects/funcat) and the Gene Ontology consortium (http://www.geneontology.org/GO.doc.shtml). These results and corresponding GenBank accession numbers for each unique sequence are available in supplementary Table S1 (available with the online version of this paper). As shown in Fig. 2, most of the proteins were involved in protein synthesis (22.44 %) and metabolism (15.88 %), with the others involved in cell cycle and DNA synthesis (5.27 %), protein fate (5.53 %), transport facilitation (4.89 %), cell defence (4.18 %), transcription and RNA synthesis (3.43 %), energy (2.96 %), cellular transport (2.64 %), signal transduction (1.41 %), control of cellular organization (0.71 %) and cell-wall degradation (0.13 %). Approximately 30.35 % of transcripts were similar to hypothetical proteins or proteins for which a function has yet to be determined, indicating that a great deal of functional analysis of genes from entomopathogenic fungi remains to be done.
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). Genes encoding ribosomal proteins and translational machinery, such as elongation factors, were abundantly expressed. The longer form of elongation factor 1
(EX148848) was encoded by 69 cDNAs and represented by 58 % of the ESTs in the original Pieris brassicae isolate and 42 % in the strain passaged through Plutella xylostella. Genes encoding other ribosomal proteins, including ribosomal protein L6 (EX14295), L7A (EX149294), L8 (EX148837), L9 (EX148813), L11 (EX149252), L13 (EX149271), L15 (EX149263), L19 (EX149259), L21 (EX149285), S4 (EX148831), S6 (EX148816) and S-phase-specific ribosomal protein (EX149289), were also abundant in both libraries. High levels of expression of genes associated with protein synthesis indicated that the Z. radicans isolates require new protein synthesis for rapid growth, which is similar to other entomophthoralean fungi. Conidiobolus coronatus has been reported to express a much higher proportion of genes encoding ribosomal proteins and enzymes involved in intermediate metabolism that facilitate rapid growth compared to Metarhizium anisopliae (Freimoser et al., 2003a).
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cDNAs encoding proteins involved in the stress response, such as those of the 70 kDa heat-shock protein (Hsp70) family, including the PrBiP precursor (EX148912), Hsp70 protein 2 (EX149384) and ribosome-associated Hsp70-like protein 3 (EX149538), were highly represented in both libraries. cDNA encoding Hsp70-like protein 3 was found in the library from the Plutella xylostella isolate. These proteins are presumably involved in protecting the fungus from various environmental stresses (Da Silva et al., 1999).
Other transcripts, such as those encoding histone H4 (EX148794), cyclophilin A (EX149321 and EX149323), polyubiquitin (EX148866) and trypsin-like serine protease (EX149338), were also found to be highly represented in both libraries. cDNAs encoding histone H4, which is associated with DNA synthesis and processing, were more prevalent in the library from the Pieris brassicae isolate than from the strain passaged through Plutella xylostella.
Proteinase genes expressed in Z. radicans isolates
Several aspects of the fungus–insect interaction, including spore attachment and germination on the cuticle, secretion of cuticle-degrading enzymes and penetration of the integument, recognition by the insect defence system and enablement of such, come to bear on the success or failure of the pathogen to infect the host. Many virulence-associated genes have been identified from human fungal pathogens, such as Candida albicans (Goldman et al., 2003; Rodier et al., 1999), and plant-pathogenic fungi, such as Magnaporthe grisea (Viaud et al., 2002) and Ustilago maydis (Lee & Kronstad, 2002). Only a few virulence genes have been identified from entomopathogenic fungi, such as Metarhizium anisopliae (Freimoser et al., 2003a), Conidiobolus coronatus (Freimoser et al., 2003b) and Beauveria bassiana (Cho et al., 2006a, b).
Among the most widely studied pathogenicity and virulence factors for entomopathogenic fungi are proteinases as they are involved in degradation of insect cuticle and impairment of host defence responses (Joshi & St Leger, 1999; Screen & St Leger, 2000; Freimoser et al., 2003a, b; Bagga et al., 2004; Cho et al., 2006a, b; Xu et al., 2006). Both isolates expressed genes encoding secreted proteinases (containing a signal peptide), including several aspartic, serine and metalloproteases (Table 3); however, neither strain expressed genes encoding cysteine proteinases. Aspartic proteinases function in acidic environments and have been found in plant-pathogenic fungi, such as Botrytis cinerea, which release oxalic acid (ten Have et al., 2004). We identified two types of aspartic proteinases (EX149341 and EX149324), both of which were found in the library from the isolate from Plutella xylostella. Serine and metalloproteinases are common among the entomopathogenic fungi and are considered to be important for cuticle penetration (Xu et al., 2006; St Leger et al., 1994; Freimoser et al., 2003a, b). The library from the Plutella xylostella isolate contained cDNAs encoding a trypsin (EX149336 and EX149337), a trypsin-like serine protease (EX149338), an extracellular trypsin protease (EX149340) and a serine-type protease (EX149333), whereas the library from the Pieris brassicae isolate contained cDNAs encoding a cuticle-degrading proteinase similar to CDEP-1 (EX148865), an Astryp1-like protease (EX148859) and a trypsin-like serine protease (EX149338). Astryp1 is an acid proteinase and may function in the same environment as aspartic proteinases. Both isolates expressed genes encoding trypsins, but not subtilisins and chymotrypsins, which are common in other entomopathogenic fungi such as Metarhizium anisopliae (Freimoser et al., 2003a; Bagga et al., 2004) and Conidiobolus coronatus (Freimoser et al., 2003b). In this study, we found that the Plutella xylostella isolate also expressed genes encoding metalloproteinases (EX149342, EX149343, EX149344, EX149346 and EX149322). Previously, the original Z. radicans isolate from Pieris brassicae was shown to produce a metalloproteinase when grown in submerged medium for 30 days (Xu et al., 2006). Many researchers have reported that proteinases, including trypsin-like serine, metallo- and aspartic proteinases, are involved in virulence (Xu et al., 1998). St Leger et al. (1994) found that the subtilisin-like proteinase (Pr1), the trypsin-like serine (Pr2) and a thermolysin-like metalloproteinase in Metarhizium anisopliae are vital for digestion of cockroach cuticle. In addition, aspartic and metalloproteinases seem to play an important role in invasion by Candida albicans and could be considered as potential pathogenicity factors (Rodier et al., 1999).
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). Isocitrate lyase has been associated with pathogenicity and virulence in several pathogenic fungi as it is a key enzyme in the glyoxylate bypass pathway, which circumvents two decarboxylation steps of the citric acid cycle, allowing for more timely assimilation of carbon substrates. The mammalian pathogen Candida albicans also requires the glyoxylate cycle for assimilation of lipids (Lorenz & Fink, 2001). When phagocytosed by macrophages and neutrophils in mice, Candida albicans upregulates principal enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase, and mutants lacking isocitrate lyase are markedly less virulent than the wild-type. The glyoxylate cycle is also required for virulence of plant-pathogenic fungi such as Magnaporthe grisea (Wang et al., 2003) and Leptosphaeria maculans (Idnurm & Howlett, 2002). Wang et al. (2003) found that Magnaporthe grisea employs isocitrate lyase to catabolize plant lipids for turgor generation and appressorium formation prior to infection. Although L. maculans enters the host through stomata without the aid of an appressorium, isocitrate lyase is also essential for successful colonization on Brassica napus (Idnurm & Howlett, 2002). However, isocitrate lyase is not essential for disease development in an isolate of Saccharomyces cerevisiae (Goldstein & McCusker, 2001) or in the opportunistic animal pathogen Cryptococcus neoformans (Rude et al., 2002). Whether isocitrate lyase is involved in disease development in entomopathogenic fungi remains to be resolved.
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).
After fungi breach the physical barrier provided by the cuticle, they encounter a battery of insect cellular and humoral defences. The current view is that components on the surface of the pathogen, such as 1,3-β-glucan from fungi or peptidoglycan from bacteria, are recognized by specific receptors on haemocytes which in turn trigger a variety of defence responses, including prophenoloxidase activation, phagocytosis, nodule formation and encapsulation. Thus subtle changes in the cell wall could modify adhesion and recognition by the defence system (Navarro-Garcia et al., 2001; Goldman et al., 2003). Both isolates expressed genes encoding proteins involved in the synthesis of 1,3-β-glucan (EX148729, EX149121, EX149132 and EX149372). The Plutella xylostella isolate expressed a gene encoding thiol peroxidase (EX149380), which is considered to be an antioxidant protein involved in fungal pathogenicity (Wu et al., 1997) and may protect the pathogen from host oxidative defences (Iwanaga & Kawabata, 1998). In addition, both strains expressed a gene encoding superoxide dismutase (SOD) (EX149194 and EX148763), an antioxidant enzyme which catalyses the dismutation of superoxide radical anions to di-oxygen and hydrogen peroxide. Cu/Zn SOD has been shown to play a role in protecting cells against oxygen toxicity by decomposing superoxide radical anions, and has been proposed to be a virulence determinant in some pathogenic organisms. For example, Candida albicans strains lacking Cu/Zn SOD were more susceptible to macrophage attack and showed attenuated virulence in mice (Hwang et al., 2002).
Both Z. radicans strains expressed genes encoding cyclophilin A (EX149321 and EX149323), a member of the cyclophilin family of enzymes that have cis–trans isomerase activity. They have been implicated in cellular processes, including stress response, signalling, cell-cycle control, RNA splicing and facilitating viral replication (Howard et al., 2003). Cyclophilin A is present in bacteria, fungi, plants and animals, and is required for virulence of both plant-pathogenic fungi, such as Magnaporthe grisea (Viaud et al., 2002), and human-pathogenic fungi, such as Cryptococcus neoformans (Wang et al., 2001).
The database from the Plutella xylostella isolate also contained ESTs representing GTP-binding proteins (EX149327, EX148886 and EX149365), which are required for pathogenicity, budding growth and mating in Ustilago maydis (Lee & Kronstad, 2002). The Pieris brassicae isolate expressed a chitin synthase (EX148735) that was similar to that associated with virulence of Wangiella dermatitidis against mice (Wang et al., 2001). While entomopathogenic fungi, such as Metarhizium anisopliae and Conidiobolus coronatus, secrete toxins (Freimoser et al., 2003a, b), we did not identify genes encoding enzymes involved in their synthesis within the limited Z. radicans EST dataset. Cuticle degradation and evasion of insect defences appear to be important pathogenicity and virulence factors for Z. radicans.
Conclusions
Although the EST datasets presented here represent a small portion of the expressed Z. radicans genes, the analysis has provided insight into the mechanisms required for host infection. Around 80 % of the unique ESTs were found only in libraries from one isolate and encoded proteins involved in all functional categories, indicating that much more information can still be gathered from this approach. The study found that the highly expressed genes encoded proteins involved in basic metabolism, such as ATP-generating enzymes, histones and some ribosomal proteins. These have also been found to be highly expressed in Beauveria bassiana (Cho et al., 2006a, b). We found that genes encoding potential pathogenicity and virulence factors, such as a trypsin-like serine protease and superoxide dismutase, were also highly expressed. In addition, genes encoding several ribosomal proteins such as L11, L13, L15, L19 and L21 and S-phase-specific ribosomal protein, genes involved in the stress response (ribosome-associated Hsp70-like protein) and metabolism (phosphoglycerate kinase and aconitate hydratase) were expressed in the two isolates and may be required for adaptation to different environmental stress, nutrient availability or the defence response associated with the different hosts. The knowledge gained through this study has contributed to the understanding of entomophthoralean fungal pathogenesis and will assist in the development of highly virulent and host-specific isolates.
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ACKNOWLEDGEMENTS |
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Edited by: S. D. Harris
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REFERENCES |
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Received 9 July 2008;
revised 19 September 2008;
accepted 22 September 2008.
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