Department of Entomology, University of Maryland, 4112 Plant Sciences Building, College Park, MD 20742, USA
Correspondence
Raymond J. St Leger
rl106{at}umail.umd.edu
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ABSTRACT |
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The expression ratios for Metarhizium anisopliae ESTs in different cuticle-containing media are shown in Supplementary Table S1 with the online version of this paper at http://mic.sgmjournals.org.
Present address: Institute of Plant Sciences, ETH Zurich, Universitätsstr. 2, CH-8092 Zurich, Switzerland.
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INTRODUCTION |
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In this report, we use cDNA microarrays for high-throughput expression profiling of how M. anisopliae strain 2575 responds over a 24 h period to cuticle from tobacco hornworm caterpillars (Manduca sexta). As a control, we also define the response of M. anisopliae to nutrient deprivation. In addition we obtained snapshots of gene expression at 24 h to compare and contrast the responses of M. anisopliae to gypsy moth caterpillar cuticle (Lymantria dispar), and hard (sclerotized) cuticles from a beetle (Popilla japonica) and a cockroach (Blaberus giganteus). Each of these insects is a susceptible host for M. anisopliae.
These studies demonstrated that M. anisopliae can rapidly adjust its genomic expression patterns to adapt to insect cuticle, and identified specific responses to different cuticles. Genes specifically induced by cuticle included a plethora of cuticle-degrading enzymes, transporters for cuticle degradation products and a subset of transcription factors.
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METHODS |
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cDNA microarray experiments.
All unique ESTs with significant BLAST matches (Freimoser et al., 2003) were amplified using T3 and T7 primers and standard PCR protocols. It should be noted that, as with most other bioinformatic studies, gene identities are based on computer-predicted homologies, and in very few cases (e.g. serine proteases and hydrophobins) have the protein products of these genes been demonstrated experimentally. Genes found among the EST sequences of M. anisopliae sf. acridum (ARSEF 324), such as chitinases and chitosanase (Freimoser et al., 2003
), which were absent from the M. anisopliae sf. anisopliae (ARSEF 2575) EST collection, were amplified from M. anisopliae sf. anisopliae genomic DNA with specific primers and included on the array. This resulted in 837 clones, which were precipitated and resuspended in 3x SSC (1x SSC=0·15 M sodium chloride, 0·015 M sodium citrate, pH 7·0) to give a final DNA concentration between 100 and 300 ng ml1.
Printing, hybridization and scanning of slides were performed with an Affymetrix 417 Arrayer and 418 Scanner (see http://www.umbi.umd.edu/cab/macore/macorestart.htm for detailed protocols) at the University of Maryland Biotechnology Institute's Microarray Core Facility located at the Center for Biosystems Research. PCR products were spotted in triplicate on poly-lysine-coated glass slides, with a mean spot diameter of 100 µm and a spot spacing of 375 µm. Following printing and cross-linking, slides were washed with 1 % SDS to remove background, treated with blocking solution (0·2 M succinic anhydride, 0·05 M sodium borate, prepared in 1-methyl-2-pyrrolidinone) and washed with 95 °C water and 95 % ethanol. After drying, slides were kept in the dark at room temperature.
RNA was extracted as previously described for M. anisopliae (Joshi & St Leger, 1999). For experiments comparing different media, RNA from a culture transferred to SDB was used as the reference sample. For time-course experiments, mycelium was collected after 1, 2, 4, 8, 12, 18 and 24 h from MM or from medium containing MC. RNA from the 0 h time-point was used as a reference. Hybridizations were done with Cy3- and Cy5-labelled probes derived from 5080 µg of total RNA. All hybridizations were repeated at least three times with RNA from independent experiments and with switched labelling for the reference and test RNA samples.
Analysis of microarray data.
The images of the scanned slides were analysed with Scanalyse (available from Eisen Lab: http://rana.lbl.gov/) and the data obtained from each scanned slide were normalized using global normalization, as performed by J-Express (Dysvik & Jonassen, 2001). All data were log2-transformed, and for further analysis the mean (Em) and the standard deviation (SD) of the log-transformed expression ratios of the replicates were calculated for all genes. A gene was defined as differently regulated if the expression varied by at least a factor of two (1<Em<1). Expression ratios not fulfilling this requirement (1<Em<1) were defined as zero, and the same was done for cases where the interval Em±1·96xSD (95 % confidence interval around the mean value for the three replicate spots) included the value 0. Further analysis of the processed data was performed using J-Express (Dysvik & Jonassen, 2001
), EPCLUST (http://ep.ebi.ac.uk/EP/EPCLUST/) and Excel.
Validation of differentially expressed clones through real-time PCR.
A total of 16 clones predicted to be differentially expressed by microarray analysis were tested by quantitative reverse-transcription PCR (RT-PCR) by using an Applied Biosystems GeneAMP 5700 sequence detection system and an Applied Biosystems TaqMan RT kit. Transcript abundance was calculated by using the comparative Ct method relative to the amount of the tubulin alpha chain transcript AJ273998 or 18S rRNA in the sample, with primers and conditions as described by Parsley et al. (2002)
. Differential expression based on RT-PCR measurements was defined as a change in transcript abundance accumulation of twofold or more.
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RESULTS |
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An overview of the microarray results is presented in Fig. 1. They illustrate the rapid changes in expression of some genes in response to MC. Overall, these changes increased in magnitude with time. Thus at 4 and 18 h post-inoculation in MC medium, 88 and 154 genes were upregulated, respectively. Similarly, 66 genes were down-regulated at 8 h, and 143 genes were down-regulated at 18 h. During the first hour, there was no overlap between genes upregulated in response to MC and those upregulated in response to starvation conditions (MM). However, by 18 and 24 h, 30 % of the genes were concomitantly upregulated in MC and MM, indicating that catabolite repression is involved in regulating at least some cuticle-induced genes. A cluster of 41 genes was rapidly activated (<2 h) by MM, but down-regulated in response to cuticle (Fig. 1
). Only eight of these genes had homologues with known biological activity in databases, and these included the subtilisin Pr1G and ribosomal proteins. At least with respect to the regulation of these 41 genes, nutrient deprivation may be perceived as having an effect distinct from and even opposite to that of induction by cuticle.
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However, M. anisopliae appears to differ from T. reesei in the extent to which aerobic respiration prevails. As in yeast and T. reesei, a M. anisopliae pyruvate decarboxylase (AJ274332) is upregulated in the presence of sugar. However, in contrast to these fungi, M. anisopliae has an additional pyruvate decarboxylase (AJ274298) that is repressed in nutrient-rich medium but upregulated within 1 h on MC (Fig. 2). In S. cerevisiae, the acetaldehyde formed from pyruvate decarboxylase is reduced to ethanol by alcohol dehydrogenase, and is not converted to acetate, due to repression of aldehyde dehydrogenase by glucose. Two paralogous genes for aldehyde dehydrogenase have been identified in T. reesei, only one of which is repressed by nutrient-rich conditions. In contrast, both the aldehyde dehydrogenases (AJ272833 and AJ273869) in M. anisopliae are down-regulated in SDB compared to cuticle-containing media, suggesting that readily utilized nutrients repress acetate production. It is of interest that AJ272833 is upregulated at an earlier time-point on MC than in MM (Fig. 2
). We also identified two paralogues of acetyl coenzyme A synthetase: the AJ273955 transcript is upregulated early during growth on cuticle and late during growth in MM (Fig. 2
), while regulation of AJ274191 is not affected. If both enzymes have comparable specificity, production of acetyl coenzyme A in glucose-poor media such as cuticle will increase the entry of acetate, produced via the pyruvate bypass route, into the TCA cycle. Interestingly, M. anisopliae also has two paralogous genes for alcohol dehydrogenase. AJ273792 is regulated in a similar fashion to pyruvate decarboxylase AJ274332 (upregulated in SDB), whereas AJ273547, like pyruvate decarboxylase AJ274298, is repressed in SDB. Thus, M. anisopliae has multiple gene families of catabolic enzymes, some of which include isoforms that are differentially regulated by sugars. These alternative forms may give M. anisopliae the flexibility to shunt any available pyruvate into fermentation or the TCA cycle, irrespective of sugar levels.
Amino acid, carbohydrate and lipid metabolism.
Genes with homologues involved in amino acid catabolism and which were upregulated on cuticle included glutaminase A (AJ273512) and NADH-specific glutamate dehydrogenase (AJ274362). Glutamate is the preferred amino acid substrate for M. anisopliae (St Leger et al., 1986a). Otherwise, diverse genes involved in amino acid synthesis were commonly down-regulated in MM and on cuticle, consistent with the reduced availability of raw materials for biosynthesis. Insect cuticle also contains diverse lipids, and seven of 13 genes for lipid metabolism were upregulated on at least one cuticle. Only a cytochrome P450 monooxygenase (AJ274003) was also upregulated during growth in MM. Lipases are the last class of depolymerases to be secreted in insect cuticle (St Leger et al., 1986b
), consistent with which, lipase AJ274124 was upregulated in late cuticle-containing cultures (24 h) only. Enzyme assays have also detected a secreted DNase activity during growth on cuticle (St Leger et al., 1986b
), and in this study DNase (AJ273950) was upregulated in cuticle-containing media.
Protein aside, the major component of insect cuticle is chitin, and predictably therefore chitinases were upregulated on cuticle. Chitinase AJ274366 was expressed within 1 h on MC, but was not expressed in MM (Fig. 2). Chitosanase was only produced on GC (Fig. 4
). As this coincides with the GC-specific expression of genes involved in morphogenesis, it is possible that the chitosanase may be involved in modifying cell wall components. However, five additional enzymes involved in metabolizing carbohydrates not known to occur in cuticle were also upregulated in one or more of the cuticle media: formate dehydrogenase (AJ274347), usually involved in detoxification reactions; 1,2-
-D-mannosidase (aj273630);
-D-galactosidase (aj273808); L-sorbosone dehydrogenase (AJ273834); and
-glucosidase (AJ273623). These could be involved in digesting glycoproteins, but were also more weakly upregulated in starvation conditions, consistent with catabolite repression in SDB. Only one of the genes for carbohydrate metabolism (AJ272928) was upregulated in response to HL, while seven genes were down-regulated (Supplementary Table S1). Seven carbohydrate-metabolizing enzymes were down-regulated on cuticle-containing media, including a transketolase (AJ274194) and fructose-bisphosphate aldolase (AJ273952).
RNA synthesis.
Elements required for mRNA synthesis, such as RNA polymerase (AJ272996) and RNA polymerase transcription factor (AJ274125) were upregulated in cuticle-containing media, but not in MM or HL. This presumably adapts the fungus for the rapid synthesis of cuticle-degrading enzymes.
Transport proteins.
The ESTs included homologues of two distinct peptide transport systems, one for di-/tripeptides (PTR transporter AJ273551 and PTR-2 transporter AJ272830) and another for tetra-pentapeptides (OPT transporter AJ273568), as well as diverse amino acid transporters (e.g. the INDA1 homologue AJ272773). These all required induction by cuticle, and most were upregulated 8- to 12-fold within 1 h on MC (Figs 2 and 3). In contrast, the PTR transporter in T. reesei is upregulated by glucose exhaustion alone (Chambergo et al., 2002
), consistent with the M. anisopliae transporters having acquired more specialized functions in pathogenicity. Only the M. anisopliae oligopeptide transporter OPT2 (AJ273118) was not upregulated in cuticle-containing media. Regulation of peptide/amino acid transporters was not altered in HL compared to growth on SDB.
Proteolytic enzymes.
It had been shown previously that total subtilisin activity is produced in response to nutrient deprivation, but that production is enhanced by the addition of cuticle to media (Paterson et al., 1994). Consistent with this, subtilisins Pr1A and Pr1B were upregulated on MM, and to a greater extent on insect cuticles (Fig. 5
). Increased induction by cuticle compared with nutrient deprivation alone suggests that subtilisin production is controlled by multiple regulatory systems evoked under different environmental conditions. In contrast, Pr1C, Pr1D, Pr1E, Pr1F, Pr1I and Pr1J were down-regulated at most time-points in MM. Of these, Pr1C and Pr1D were rapidly upregulated (Pr1C within 1 h of transfer to MC; Fig. 2
), while upregulation of Pr1E and Pr1K in MC was delayed by 4 and 8 h, respectively. Pr1J was upregulated on all the cuticles, except BC. Pr1G was sharply down-regulated in CC. Pr1F and Pr1I were upregulated on MC and on GC. Expression of Pr1H was slightly upregulated by transfer to MC and MM.
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Transcription factors and signal transduction.
Of the 17 arrayed ESTs encoding homologues of proteins known to be involved in transcription in other organisms, ten (AJ272823, AJ272967, AJ273078, AJ273134, AJ273171, AJ273219, AJ273260, AJ273589, AJ273694 and AJ274235) were upregulated on at least one cuticle. The positive sulfur transcription regulator homologue (AJ273134) was down-regulated in MM and BC, suggestive of particularly low sulfur levels in these media (sulfite reductase, AJ273620, but not sulfite oxidase, AJ272866, was upregulated on cuticle, within 1 h in MC, but not in MM and HL). In contrast, the pH signalling transcription factor PacC (AJ273219) was upregulated on cuticle, but not in MM or HL. AJ272977 was unique in being upregulated in HL. In contrast, AJ273694 was very strongly down-regulated in HL and strongly upregulated on the lepidopteran cuticles GC and MC. Among gene products involved in signalling (category 4f), adenylate cyclase (AJ251971) (the enzyme that produces cAMP) and protein kinase A (AF116597) (PKA: the major effector of cAMP responses) were not upregulated on cuticle-containing media, while a downstream activity, MAP kinase kinase 2 (AJ273356) was upregulated in GC- and BC-containing media, and at two time-points in MC.
Cell wall proteins.
Of 30 genes encoding proteins involved in cell structure and function, 18 were upregulated in at least one cuticle-containing medium. The hydrophobins are differentially regulated. Thus, AJ273847 was upregulated in HL and MM, and down-regulated in cuticle-containing media, while AJ274156 was upregulated in MM and on sclerotized cuticles (CC and BC), unaltered on lepidopteran cuticles (GC and MC) and down-regulated in HL. The other cell wall proteins upregulated on cuticle were AJ273845, a homologue to an antigenic cell wall protein from the human pathogen Aspergillus fumigatus, and AJ274019, which is very similar to the antifungal glucan 1,3--glucosidase from Trichoderma atroviride (Donzelli et al., 2001
). Clearly, besides cell wall biosynthesis and structure, these proteins may have additional functions in pathogenicity or in protecting scarce resources from competitors.
Stress response.
Several arrayed M. anisopliae ESTs are similar to peptide synthases, reductases and other enzymes that take part in the synthesis of fungal toxins, such as destruxins, trichothecene and enniatin (Freimoser et al., 2003). This is in agreement with the observation that M. anisopliae strain 2575 rapidly kills its host after infection through the action of toxins, and subsequently colonizes the insect host by saprobic growth (Samuels et al., 1989
). Genes upregulated in at least one cuticle-containing medium included those encoding a peptide synthase (AJ272930, in BC and GC), a protein involved in sterigmatocystin biosynthesis (AJ273515, in GC), versicolorin B synthase (AJ272697, in CC, GC, MC and HL) and a bacteriolytic enzyme (AJ272917, in CC, BC, GC, early in MC and after 12 h in MM).
Validation of microarray results.
An external quality control check on the lists of differentially expressed clones generated through microarray profiling was provided by analysing a subset of 16 clones by quantitative RT-PCR. Each clone tested by RT-PCR was measured in triplicate for each of two independent RNA isolations. All 16 clones were confirmed by RT-PCR, indicating a very high success rate for predicting differentially expressed clones. However, expression ratios were consistently underestimated at least 10-fold by cDNA microarrays, compared to PCR-based methods.
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DISCUSSION |
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The demonstration of the differential regulation of genes encoding cuticle-degrading enzymes, cell wall proteins, toxins and toxin-producing enzymes on the different cuticles, and in HL and MM suggests that M. anisopliae may have the ability to target the production of these proteins to different hosts. Like other ascomycete pathogens, M. anisopliae secretes a great variety of proteases (Hu and St Leger, 2004), some of which have been associated with virulence, because they allow rapid physical ingress, nutrient solubilization and the disabling of antimicrobial peptides (St Leger et al., 1996
). The subtilisin cluster provides a good example of de novo protein synthesis required for adaptation to growth on cuticle (Figs 1 and 5
, Supplementary Table S1), particularly as the differences in regulation of subtilisins imply differences in their function. This supports homology-modelling studies based on sequences that predict differences between the Pr1s in their secondary specificities, adsorption properties to cuticle and alkaline stability (Bagga et al., 2004
). It is likely that these differences in regulation and structurefunction allow M. anisopliae to respond flexibly, producing proteases that are appropriate to the composition of the environment, consistent with its opportunistic lifestyle. Thus the proteases such as Pr1A produced as part of a general response to nutrient deprivation could also function outside of pathogenesis by scavenging for nutrients during saprophytic existence. During early infection processes, they could also function in concert with the exopeptidases to provide host degradation products. These may include specialized signals that allow the fungus to sample the cuticle and then respond with the secretion of a plethora of cuticle-induced proteins. This will include the proteases that require cuticle for induction, as they presumably have specialized roles in breaching host barriers. The very early induction of peptide/amino acid transport systems (Figs 2 and 3
) would enhance the ability of the fungus to rapidly and precisely monitor host degradation products.
Hydrophobins provide another example besides subtilisins where members of a family are differentially regulated, consistent with different functions. Thus, AJ273847 was upregulated in HL, while AJ274156 was down-regulated in HL. This suggests that adaptation to HL may include alterations in cell wall composition.
Of key importance to understanding the mechanisms behind adaptation to cuticles is the identification of components of signal transduction that will allow M. anisopliae to screen its surroundings to regulate protein synthesis and secretion. PacC-mediated pH signalling is crucial to the pathogenicity of the human pathogen Candida albicans and the plant pathogen Fusarium oxysporum (Caracuel et al., 2003; Davis et al., 2000
). Consistent with a crucial role for PacC in M. anisopliae, extracellular pH rises during cuticle degradation and acts as a key signal for the production of alkaline-active enzymes such as subtilisins (St Leger et al., 1998
). Significant for their absence of response were adenylate cyclase and protein kinase A, as transcriptional regulation in response to the cAMP signalling pathways seems central to infection-related development in M. anisopliae (St Leger, 1993
). Constitutive expression may be a feature of some primary initiators of physiological processes, so their importance will not be detected in microarray analyses. A downstream activity, MAP kinase kinase 2 (AJ273356) was upregulated in GC- and BC-containing media. This enzyme, and other transcription factors, may constitute downstream ground-level components which are immediately concerned with recognizing and responding to specific host features, and which do not control fungal metabolism as a whole. As such, they may be useful for strain improvement purposes.
Microarray technology has made it possible to decipher the transcriptional programmes of organisms by studying gene expression en masse while assessing individual gene function in a detailed manner (Brown & Botstein, 1999). Thus, knowing when and where a gene is expressed often provides a strong clue as to its function (DeRisi et al., 1997
). Almost 50 % of the arrayed ESTs upregulated in cuticle-containing media have undiscovered biological activities (Table 1 and Supplementary Table S1), and 25 % of these are not upregulated in MM or HL. These genes have never been recognized to have a role in pathogenicity, but are now implicated by co-regulation with known virulence factors. They thus provide an additional rich resource for future research.
Evolutionary theory has long held that the process of adaptation is driven by competition for limited resources. Among heterotrophic micro-organisms, the availability of carbon limits the ability of these organisms to multiply. As a result, the machinery of central metabolism is tuned to exploit reduced carbon resources in natural environments, where they vary greatly in both form and abundance (Ferea et al., 1999). Comparisons between M. anisopliae, T. reesei and S. cerevisiae suggest that the three fungi will respond differently to environmental changes, presumably reflecting their adaptation to predictable differences in the composition of these environments. The similarities between M. anisopliae and T. reesei may reflect their close relationship as clavicipitaceous pyrenomycetes. However, the alternatively regulated forms of catabolic enzymes in M. anisopliae and T. reesei suggest they will differ in how they coordinate the regulation of key parts of metabolism, such as fermentation at different levels of glucose. This could affect the extent to which aerobic respiration prevails in glucose-rich media.
Evidently, gene-duplication events and altered patterns of regulation could provide mechanisms for evolution to fine-tune ATP-producing pathways, allowing these organisms to adapt to their different environments and nutritional requirements. It is tempting to speculate that fermentation may play a more pivotal role in the life of M. anisopliae, compared to that of T. reesei, to enable it to exploit sugars in the anaerobic environment of the dead host. However, complicating interpretation of these results, ATP-producing pathways can be co-opted to other functions. Thus, some fungal acetyl coenzyme A synthetases are involved in the biosynthesis of secondary metabolites such as penicillin, as well as in primary metabolism (Martinez-Blanco et al., 1993). It is axiomatic that as more is learned about the function of each gene, comparative studies on transcriptomes will become an increasingly powerful tool allowing predictive insights into the behavioural plasticity of each saprophyte or pathogen.
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ACKNOWLEDGEMENTS |
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Received 16 August 2004;
revised 25 October 2004;
accepted 28 October 2004.
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