1 Department of Entomology, 4112 Plant Science Building, University of Maryland, College Park, MD 20742-4454, USA
2 School of Biological Sciences, University of Wales Swansea, Swansea SA2 8PP, UK
Correspondence
Raymond J. St Leger
stleger{at}umd.edu
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ABSTRACT |
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Microarray data demonstrating the transcriptional variations of Metarhizium anisopliae sectors grown in different media are available in Supplementary Table S1 with the online version of this paper.
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INTRODUCTION |
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Apart from the reports of secondary metabolite decline in degenerative fungal cultures (Wing et al., 1995; Guzman-de-Pena & Ruiz-Herrera, 1997
; Ryan et al., 2002
; Kale et al., 2003
), little information is available to explain fungal culture instability at the molecular level. However, genomic DNA methylation was reported in a sector of Fusarium oxysporum after successive subculturing (Kim, 1997
). In another case, fungal morphological instability was linked to dsRNA virus infection (Dawe & Nuss, 2001
).
In this study, the spontaneous sterile sectors from two genetically distinct M. anisopliae strains were subcultured and characterized in comparison with wild-type parents. Microarray analysis using slides printed with 1730 M. anisopliae genes (Wang et al., 2005) demonstrated that the degenerative sectors are under strong oxidative stress and show signs of ageing/senescence. Since conidiation in plant-pathogenic fungi correlates with cAMP levels (e.g. Adachi & Hamer, 1998
), we also measured intracellular cAMP in the M. anisopliae sectors and attempted to rescue wild-type phenotypes using exogenous cAMP.
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METHODS |
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cAMP assay.
Intracellular cAMP levels of sectors and parent cultures were determined as described by Fillinger et al. (2002). Briefly, mycelia (1 g) harvested from 36 h cultures grown in Sabouraud glucose broth (SDB) were ground thoroughly under liquid nitrogen and suspended in 0·5 ml extraction buffer (50 mM Tris/HCl, 4 mM EDTA, pH 7·5). A 0·1 ml aliquot of suspension was used for the protein assay. The rest was boiled for 5 min, then centrifuged at 13 000 r.p.m. for 5 min. The cAMP concentration in the supernatant was determined on a microplate reader (Multiskan Ascent, Thermo Labsystem, Finland) using the cAMP enzyme immunoassay kit (Sigma) according to the supplier's instructions. The concentrations were expressed as pmol (mg protein)1 (Fillinger et al., 2002
). The assays were conducted in triplicate from two batch cultures. In parallel experiments, exogenous cAMP (Sigma) was added to SDA plates to a final concentration of 10 mM to examine whether it could induce sporulation in sterile sector cultures.
cDNA microarray analysis.
The conidia of the wild-type strains and the mycelia of the sector cultures were grown in SDB for 36 h. The mycelia were harvested and washed with sterile distilled water. The same amount of mycelia (2 g, wet weight) was then transferred for 24 h to: (1) 1 % Manduca sexta cuticle medium buffered with 1 g KH2PO4 l1, 0·5 g MgSO4 l1 and 10 mg FeSO4 l1 (Wang & St Leger, 2005); (2) SDB or (3) SDB amended with 10 mM cAMP. Total RNA was extracted using an RNeasy Plant mini kit including a treatment with DNase I (Qiagen). The RNA from the corresponding wild-type culture was used as the reference sample. Hybridizations were conducted using the slides printed with 1730 cDNA clones from M. anisopliae var. anisopliae ARSEF 2575 and microarray data were analysed as described previously (Wang et al., 2005
; Wang & St Leger, 2005
). A t test (Pan, 2002
) was conducted to identify the genes whose expression varied significantly between the V275Sec and V245Sec groups. A P value was estimated based on the t distribution and the overall
was set at 0·05. Hybridizations were conducted with RNA from three independent experiments.
Real-time PCR (RT-PCR) validation.
The extracted RNA (1 µg) from SDB and insect cuticle media was converted into cDNA for RT-PCR analysis using the anchored oligo-dT primer following the manufacturer's protocol (ABgene, Surrey, UK). Gene-specific primers were designed with an anticipated product size of approximately 200 bp to guarantee high amplification efficiency. The examined genes included those for subtilisins PR1A (M73795) and PR1B (U59484), the chymotrypsin CHY1 (AJ242735), the trypsin TRY1 (AJ242736) and the esterase STE1 (AJ251924), as they are involved in fungal virulence (Freimoser et al., 2003). The primers designed from the small subunit ribosomal gene (AF218207) of M. anisopliae were used as an internal control. PCR was conducted using a Bio-Rad iQ SYBR Green Supermix kit in a volume of 20 µl, including 1 µl 10x diluted cDNA template and 0·25 pmol of each primer. The cycling parameters were programmed on a Bio-Rad iCycler iQ system. The relative expression ratio of each gene was calculated by calibration with the ribosomal gene, and the fold-change between the sector and wild-type was estimated. Each sample had three replicates and the whole experiment was repeated twice.
Confirmation of oxidative stress.
To check if the sectors were under oxidative stress, the production of reactive oxygen species (ROS) was compared with the wild-type strains using the nitro blue tetrazolium (NBT) reduction assay (Lara-Ortiz et al., 2003). The mycelia from 36 h SDB medium were vacuum-filtered for 20 min with a 0·3 mM NBT aqueous solution containing 0·3 mM NADPH to increase sensitivity. NBT, on reduction by ROS, formed a blue/purple formazan precipitate. Mycelial discs were mounted in 30 % glycerol (v/v) and examined in a light microscope.
Mitochondrial DNA (mtDNA) digestion analysis.
Mitochondria were extracted from sectors and wild-type cultures using step gradients, based on the method of Lambowitz (1979), and resuspended in 450 µl lysis buffer (0·1 M Tris/HCl, pH 8·0, 1·0 % SDS, 2 % Triton X-100, v/v, 10 mM EDTA, 0·1 M NaCl). The samples were incubated at 65 °C for 30 min and extracted once with an equal volume of phenol/chloroform/isoamyl alchohol (25 : 24 : 1). The aqueous layer was precipitated with one volume of cold 2-propanol for 30 min and centrifuged at 20 000 g for 10 min. Pellets of mtDNA were washed with 70 % ethanol, air-dried and resuspended in Tris/EDTA buffer. mtDNA was digested with HindIII and the methylation-sensitive enzyme HpaII to determine if any mtDNA rearrangement/methylation had occurred in the sterile cultures.
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RESULTS |
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Genes involved in cell-structure reorganization
Patterns of gene expression indicate extensive cell-structure reorganization during sectorization. Down-regulated genes included those for the hydrophobins (AJ274156 and CN809178, Fig. 2A), and we observed that the sector cultures were easily wettable (data not shown), consistent with loss of hydrophobins (Kamp & Bidochka, 2002
). Conversely, several structural genes were upregulated in sectors, including an orthologue of Neurospora lustrin A (CN808315), the spore-coat protein SP96 (AJ274277) and
-tubulin (CN809527) (Table 2
). Upregulation of spore-coat protein SP96 suggests that the gene-expression profile leading to spore production is not completely blocked in sterile cultures. However, as well as being a structural component, SP96 helps coordinate cell-wall synthesis (Srinivasan et al., 2000
; Metcalf et al., 2003
), and an altered pattern of expression could thus contribute to defects in sporulation.
Genes involved in signal transduction
Sterile sectors may employ regulatory modulators of growth, metabolism and/or development that differ from the wild-type strains. Differentially upregulated genes included a proline oxidase (AJ274200) and a CAP20-like protein (CN808339) (Table 2). Endocellular proline levels inversely control cell division (Maggio et al., 2002
), and CAP20 has been found to play an important role in the control of development in Colletotrichum gloeosporiodies (Hwang et al., 1995
). A mitogen-activated protein (MAP) kinase kinase (AJ272796) and a serine/threonine protein kinase (CN808780) were also upregulated in both sectors. The MAP kinase kinase of Candida albicans is essential to the oxidative stress response (Arana et al., 2005
). Upregulation of AJ272796 indicates that the sectors were, at least in part, adapting to circumstances of oxidative stress.
Genes differentially expressed on insect cuticle and SDB
Genes specifically upregulated on insect cuticle but not in SDB include the chymotrypsins AJ273081 and AJ273663 (>fivefold for V275Sec and V245Sec). Wild-type M. anisopliae strains sharply upregulate proteases such as chymotrypsins, trypsins, carboxypeptidases and subtilisins in cuticle media (Freimoser et al., 2003). In contrast, the sector cultures down-regulate subtilisins, a carboxypeptidase (AJ272919) and a trypsin-like protease (AJ274008) (Fig. 2C
). Differential regulation of proteases by sectors suggests that they are under the control of different pathways and may possess other functions. Genes down-regulated on cuticle but upregulated in SDB compared to the wild-type include a catalase (CN808348) and a flavohaemoglobin (CN808105) that function in oxidative stress responses (Rosenfeld & Beauvoit, 2003
). Upregulation of the carboxypeptidase (Fig. 2C
), a homologue of yeast carboxypeptidase Y (CpY), in sectors grown in SDB is consistent with its putative role against the effects of oxidative damage (Martinez et al., 2003
).
Transcriptional differences between the two sector cultures in response to cAMP
Although there were no evident phenotypic differences between V275Sec and V245Sec, expression patterns suggest that they were physiologically quite distinct. Compared to V245Sec, V275Sec had higher expression levels of many genes during growth on insect cuticle or SDB±cAMP (Figs 2A and 3AC), and the t test identified 181 (
10 % of total) that were differentially regulated. These were principally distributed in the cell metabolism (26·0 %), protein metabolism (23·8 %) and cell structure and function (13·3 %) categories (Fig. 3
D). Since the addition of cAMP could restore the conidiation ability of V245Sec but not V275Sec, the effects of cAMP on gene expression were compared between the sectors grown in different media (Fig. 4
). Only a sporulation protein, SPO72 (AJ272728), was upregulated upon the addition of cAMP in both sectors. Deletion of Spo72 blocks the initiation of sporulation in Saccharomyces cerevisiae (Briza et al., 2002
).
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Genes specifically upregulated in V245Sec include a homologue of yeast Ssn6 (AJ274057, 2·8-fold in V245Sec and 1·3-fold in V275Sec) that is essential for derepression of yeast sporulation-specific proteins (Schultz & Carlson, 1987; Friesen et al., 1997
). Thus, the up-regulation of AJ274057 in V245Sec may be a precondition for restoring its ability to produce conidia. NADPH oxidase has been shown to be a source of ROS in A. nidulans (Lara-Ortiz et al., 2003
). The upregulation of a NADPH oxidase (CN807950) in V245Sec thus presented an additional oxidative pathway in V245Sec that is absent in V275Sec.
RT-PCR validation
The fold-changes in microarray analysis had a significant correlation (r=0·94, P<0·01) with the results from RT-PCR examination (Table 3), confirming a high level of accuracy for microarray analysis. However, consistent with our previous analysis, the expression ratios were underestimated in microarray analysis compared with RT-PCR (Freimoser et al., 2005
).
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DISCUSSION |
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Overall, sectors were characterized by extensive down-regulation of gene expression. Fig. 7 summarizes the physiological alterations predicted by these changes in gene expression. The increase of ROS production in the sterile cultures is expected to trigger/mediate global physiological changes. ROS accumulation could damage proteins, DNA and membranes, and is a major determinant of lifespan (e.g. Sohal & Weindruch, 1996
; Balaban et al., 2005
). However, the degenerative M. anisopliae cultures do not die even after being transferred for more than 50 generations. This could result from the protection imparted by fungal deoxidation responses such as catalase, as well as from HSPs. This may be why sectorization increases the lifespan of basidiomycetous fungi (Gramss, 1991
).
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The involvement of HSPs has also been well documented in mammalian cell senescence (reviewed by Sreedhar & Csermely, 2004) but has not been reported before in senescent fungi. HSPs as stress proteins' are induced by a variety of stressful stimuli in fungi, including changes in membrane fluidity (Maresca & Kobayashi, 1993
). The changes predicted by gene-expression patterns in major cell-wall components such as hydrophobins could easily impact on the membrane structure. More directly, a C-4 methyl sterol oxidase is upregulated in sterile cultures. This gene is involved in the biosynthesis of ergosterol, an essential component contributing to the fluidity of fungal-cell plasma membranes (Bard et al., 1996
).
The sterile cultures upregulated three major classes of HSP: small HSPs (AJ273036, AJ273662 and AJ273210), HSP70 cognates (AJ273534, AJ274192 and CN80809024) and HSP90 cognates (AJ274186, CN809267 and CN809338) (Table 2). Protection by HSP chaperones includes prevention of protein aggregation, refolding of misfolded proteins, retention of mitochondrial integrity and blocking DNA damage (Sreedhar & Csermely, 2004
). In addition, some HSPs are also involved in signal transduction to regulate morphogenic variation (Rutherford, 2003
). For example, differential expression of an HSP70 gene has been observed during transition from the mycelial to the yeast form in the human pathogenic fungus Paracoccidioides brasiliensis (Da Silva et al., 1999
). Consequently, HSPs could be involved in the morphological change from dense-sporulating to fluffy-mycelial type. On the other hand, Metarhizium HSP90 cognates AJ274186 and CN809338 are the homologues of vegetative incompatibility suppressors in Podospora and Aspergillus, respectively. Together with the down-regulation of a homologue to Neurospora Het-C (CN808914), the data imply that the mycelia of sterile cultures lose tight control of non-self recognition (Sarkar et al., 2002
; Glass & Kaneko, 2003
). This could facilitate the transmission and proliferation of dysfunctional mitochondria throughout the mycelial network, as observed in other senescent fungi (Debets et al., 1994
; Bertrand, 2000
).
Heterokaryon-incompatibility-triggered cell death can induce autophagic pathways to degrade cytosolic proteins and organelles (Pinan-Lucarre et al., 2003). The upregulation of autophagy-associated proteins (CN808094 and CN808832, Table 2
) in the sterile cultures indicates that autophagic pathways were triggered in the sectorial Metarhizium cultures (Fig. 7
). The involvement of HSPs in chaperone-mediated autophagy (Salvador et al., 2000
; Agarraberes & Dice, 2001
) is consistent with HSPs being involved in multiple functions in the sterile cultures.
Despite similar visible phenotypes, microarray data revealed that 181 of 1730 genes were differentially expressed between the two sector cultures, and the addition of exogenous cAMP only rescued sporulation in V245Sec. The wild-type strains V275 and V245 have previously been revealed to have chromosome-length polymorphisms (Wang et al., 2003) and to differ in Dtx production (Wang et al., 2004
). This study shows that there are also significant differences in transcriptional responses between the two strains. Some genes, such as fluffy, the hydrophobin-encoding gene eas and the clock-controlled gene ccg-2, are essential for conidiation (Rerngsamran et al., 2005
). Mutations in any of these genes could result in sterile cultures. However, in view of their high frequency of occurrence and the fact that V245Sec conidiation can be rescued, it is unlikely that fungal sectorization is caused by spontaneous gene mutation.
Growth in host-related medium did not result in the recovery of wild-type characteristics by the sterile cultures, suggesting that re-juvenilization does not readily occur. These data may help to alleviate the long-standing dispute as to whether passage through insect hosts can (e.g. Hayden et al., 1992) or cannot (Fargues & Robert, 1983
) restore the virulence of attenuated strains of insect pathogenic fungi.
In conclusion, we demonstrated that extensive gene down-regulation occurred in sterile cultures of M. anisopliae in comparison with the wild-type strains, and that the sectors are under strong oxidative stress. Overall, fungal sectorization shows similar changes in transcriptional profiles to those of ageing. Besides the loss of conidiation ability, the degenerative cultures may reorganize their cell structure and employ additional signal-transduction pathways for physiological adaptation. The pathways involved in self-rescue include components responsible for deoxidation, HSP chaperones that could protect protein and DNA from oxidative damage, and mechanisms for disposing of damaged proteins/organelles (Fig. 7). Similarities between the mammalian ageing process and fungal degenerative sectorization, such as cAMP production, oxidative stress and the involvement of HSPs, suggest that fungi may provide a good model for studies of ageing. Indeed, further studies are required to identify the trigger(s) for spontaneous sectorization/degeneration, the level of mitochondrial dysfunction and the extent to which this involves alterations in mtDNA as well as genomic DNA.
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ACKNOWLEDGEMENTS |
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Received 24 April 2005;
revised 1 June 2005;
accepted 27 June 2005.
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