A proteomic approach to identifying proteins differentially expressed in conidia and mycelium of the entomopathogenic fungus Metarhizium acridum

f u n g a l b i o l o g y 1 1 4 ( 2 0 1 0 ) 5 7 2 e5 7 9

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A proteomic approach to identifying proteins differentially expressed in conidia and mycelium of the entomopathogenic fungus Metarhizium acridum Bruno H. R. BARROSa, Se´rgio H. da SILVAa, Everaldo dos Reis MARQUESa, Jose´ C. ROSAb, Ana Patrı´cia YATSUDAa, Donald W. ROBERTSc, Gilberto U. L. BRAGAa,* a

Departamento de Ana´lises Clı´nicas, Toxicolo´gicas e Bromatolo´gicas, Faculdade de Cieˆncias Farmaceˆuticas de Ribeira˜o Preto, Universidade de Sa˜o Paulo, Ribeira˜o Preto, SP 14040-903, Brazil b Centro de Quı´mica de Proteı´nas, Faculdade de Medicina de Ribeira˜o Preto, Universidade de Sa˜o Paulo, Ribeira˜o Preto, SP 14049-900, Brazil c Department of Biology, Utah State University, Logan, UT 84322-5305, USA

article info

abstract

Article history:

Metarhizium spp. is an important worldwide group of entomopathogenic fungi used as an

Received 3 June 2009

interesting alternative to chemical insecticides in programs of agricultural pest and disease

Received in revised form

vector control. Metarhizium conidia are important in fungal propagation and also are re-

7 April 2010

sponsible for host infection. Despite their importance, several aspects of conidial biology,

Accepted 20 April 2010

including their proteome, are still unknown. We have established conidial and mycelial

Available online 24 April 2010

proteome reference maps for Metarhizium acridum using two-dimensional gel electrophore-

Corresponding Editor: Judith K. Pell

sis (2-DE) and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF MS). In all, 1130
102 and 1200
97 protein spots were detected in ungermi-

Keywords:

nated conidia and fast-growing mycelia, respectively. Comparison of the two protein-ex-

Conidial proteomics

pression profiles reveled that only 35 % of the protein spots were common to both

Entomopathogenic fungus

developmental stages. Out of 94 2-DE protein spots (65 from conidia, 25 from mycelia

Fungal development

and two common to both) analyzed using mass spectrometry, seven proteins from conidia,

Fungal proteomics

15 from mycelia and one common to both stages were identified. The identified protein

Metarhizium acridum

spots exclusive to conidia contained sequences similar to known fungal stress-protector

Metarhizium anisopliae

proteins (such as heat shock proteins (HSP) and 6-phosphogluconate dehydrogenase) plus the fungal allergen Alt a 7, actin and the enzyme cobalamin-independent methionine synthase. The identified protein spots exclusive to mycelia included proteins involved in several cell housekeeping biological processes. Three proteins (HSP 90, 6-phosphogluconate dehydrogenase and allergen Alt a 7) were present in spots in conidial and mycelial gels, but they differed in their locations on the two gels. ª 2010 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Introduction Metarhizium anisopliae has been used almost worldwide for decades to control agricultural insect pests and disease vectors.

Licensed commercial products containing conidia and/or mycelium of the fungus are available for application in the field for control of pest insects and ticks, and for use in homes against insects such as cockroaches and termites (Roberts &

* Corresponding author. Tel.: þ55 16 36024425. E-mail address: [email protected] 1878-6146/$ e see front matter ª 2010 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.funbio.2010.04.007

Proteomic approach to identifying proteins differentially expressed in Metarhizium acridum

St. Leger 2004; Vega et al. 2009). The emergence of insects resistant to chemical insecticides and the growth of organic agriculture have greatly increased interest in the use of fungi as bioinsecticides. Metarhizium produces green rod-shaped conidia, both during its saprophytic existence and on the surface of the insect cadavers at the end of its pathogenic cycle. The conidium is the fungal stage normally used as the field inoculum in biological control programs. Conidia are specialized structures of filamentous fungi that are frequently responsible for dispersal and environmental persistence of these microorganisms. In pathogenic species, such as M. anisopliae and Metarhizium acridum, the conidia are also involved in host recognition and infection. In contrast to the vegetative mycelium, which has high metabolic activity, the conidia of M. anisopliae (and of most fungi) are dormant or quiescent structures with low metabolic activity (Braga et al. 1999). Processes such as transcription and protein synthesis normally do not occur in the conidia until germination, which greatly limits their physiological adaptation and response to environmental variations. The sublethal damage caused by conidial exposure to stressgenerating environmental agents such as desiccation, high temperatures and solar radiation is repaired only after germination begins (Braga et al. 2001; Rangel et al. 2005). The biochemical, physiological and morphological differences between conidia and mycelium should be, at least in part, due to differences in the sets of enzymes and structural proteins present in the two developmental stages. Proteomics allows qualitative and quantitative measurements of large numbers of proteins that directly influence cellular morphology and biochemistry, and thus provide accurate analysis of cellular protein status or changes during growth, differentiation and response to environmental factors (Wittmann-Liebold et al. 2006; Bo¨hmer et al. 2007; Kim et al. 2007a, b; Kim et al. 2008). Proteomics complements other functional genomics approaches such as transcriptomics, but it is a more reliable technique because proteins are directly related to the phenotype (Bhadauria et al. 2007). Additionally, several studies have shown that there is little correlation between mRNA levels and protein levels in cells (Barrett et al. 2005). Protein-level analysis allows location-specific analysis, as well as the study of post-translational modifications (e.g. phosphorylation, glycosylation, and proteolysis) (Kim et al. 2007a). Proteomic studies of filamentous fungi have identified proteins related to antibiotic and anti-fungal responses, osmoadaptation, carbon catabolite repression and pathogenehost interactions (Grinyer et al. 2005; Kniemeyer et al. 2006, 2009; Kim et al. 2007b, 2008; Gautam et al. 2008). Proteomic studies also contributed to a better understanding of Metarhizium interactions with their arthropod hosts. For example, M. anisopliae proteins synthesized and secreted in response to cowpea weevil (Collosobruchus maculates) exoskeleton have been identified (Murad et al. 2006, 2008). Also, major molecular factors involved in interactions between the greyback canegrubs (Dermolepida albohirtum) and M. anisopliae were investigated by comparing the proteomes of healthy canegrubs, canegrubs infected with Metarhizium, and the fungus alone (Manalil et al. 2009). Differential immunoproteomics enables identification of M. anisopliae proteins related to the cattle tick Rhipicephalus microplus infection (Santi et al. 2009).

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Few proteomic studies have been conducted to date that focused on ungerminated fungal conidia. Cooper et al. (2006) identified more than 400 proteins from asexual uredospores of the bean rust Uromyces appendiculatus and Noir et al. (2009) identified the proteins in 180 spots from the powdery mildew Blumeria gaminis conidiospores. In both cases most of the identified proteins were heat shock proteins (HSP) or had a predicted functions in carbohydrate, lipid or protein metabolism. Quin et al. (2009) identified 24 proteins from conidia of the entomopathogen Nomuraea rileyi isolated from infected silkworm larvae. The majority of the identified proteins were either housekeeping proteins or enzymes and membrane-associated proteins. Leng et al. (2008) determined the proteomic profile of dormant conidia of the dermatophyte Trichophyton rubrum. Identified proteins covered nearly all major biological processes. Although none of the previous studies have done a direct comparison between conidial and mycelia proteomes, the functional classification of the identified proteins in conidia of the different species has shown that ungerminated fungal spores exhibit an abundance of metabolic proteins similar to metabolically active fungal hyphae (Leng et al. 2008; Noir et al. 2009). The main objectives of the present study was to establish initial 2-DE reference maps of the proteomes of M. acridum conidia and mycelia and to identify proteins that are expressed in these two developmental stages. The identification of proteins expressed in conidia is a crucial step towards better understanding of the biology of this specialized and important structure of the life cycle of filamentous fungi; and, in the specific case of M. acridum this information may help explain the molecular bases of its entomopathogenicity.

Materials and methods Metarhizium acridum strain M. acridum wild-type strain ARSEF 324 was obtained from USDA-ARSEF Collection of Entomopathogenic Fungal Cultures (U.S.D.A. Plant, Soil and Nutrition Laboratory, Ithaca, NY). Strain ARSEF 324 was isolated from Austracris guttulosa [Orthoptera: Acrididae] in Australia. Note: A recent taxonomic revision (Bischoff et al. 2009) elevated Metarhizium anisopliae var. acridum to new-species status, viz., M. acridum; and specified that isolate ARSEF 324 is an M. acridum.

Conidia production The fungus was grown in 100-mm Petri dishes on 23 ml potato dextrose agar (Acumedia, Michigan, MI) supplemented with 1 g l1 yeast extract (Difco Laboratories, Detroit, MI) (PDAY). Incubation was in the dark at 28  C for 12 days, as previously described (Braga et al. 2006). After growth, mature conidia were carefully harvested, frozen in liquid nitrogen, and stored at 80  C until use.

Mycelia production Erlenmeyer flasks (250-ml) containing 100 ml of potato dextrose broth medium (Difco) supplemented with 1 g l1 yeast

574

extract (Difco) (PDBY), were inoculated with 100 ml of conidial suspension (107 conidia ml1). Flasks were incubated on a shaker at 200 rpm, in constant darkness, at 28  C for 24 h. At 24 h, the rapidly growing mycelia (Fig S1, see “Supplementary information”) were harvested by filtration onto cellulose ester membranes (0.22-mm pore size, Millipore, Sa˜o Paulo, SP, Brazil), washed three times with distilled water, frozen in liquid nitrogen, and stored at 80  C until use.

B. H. R. Barros et al.

(conidia and mycelia), three gels from three independent experiments were analyzed. After spot detection, the gels were matched to each other and merged onto a master gel that contains the spots found in the three replicate gels for each treatment. Fourteen landmarks (spots present both in conidia and mycelia) were used as references to compare the two expression profiles. Comparison of the two master gels allowed visualization of differentially expressed protein spots from conidia and mycelia.

Whole-cell-protein extraction from conidia and mycelia Frozen conidia and mycelia were ground in liquid nitrogen in a precooled mortar and pestle. The whole-cell homogenate was treated with the “Clean-UP Kit” (GE Healthcare, Sweden). Fifty mg of the dry homogenate was resuspended in 300 ml of the precipitant solution of the Kit, and the steps listed in Protocol A of the manufacturer’s manual were performed. In the last step, the air-dried pellet was resuspended in 300 ml rehydration solution [7 M urea, 2 M thiourea, 4 % (w/v) CHAPS, 0.5 % (v/v) carrier ampholytes (IPG buffer 3-11 NL, GE Healthcare), 40 mM dithiotreitol (DTT), 0.002 % (v/v) bromophenol blue, and 0.5 % (v/v) protease inhibitor (Sigma, Germany)]. The protein concentrations were determined by the Bradford (1976) method using the BioRad protein assay kit (BioRad Lab., Hartfordshire, USA).

2-D gel electrophoresis The protein extracts (250 mg per gel for Coomassie staining and 75 mg for silver staining) were loaded for 2-DE as follows. The 13 cm strips (Immobiline Drystrip) covering a non-linear pH range of pH 3e11 (GE Healthcare) were rehydrated in 250 ml of protein solution for 16 h at 25  C. Isoelectric focusing was carried out using the Ettan IPGphor (GE Healthcare) with a ceramic tray manifold at 20  C with a current of 50 mA/strip following the protocol: 1.0 h at 500 V (step), 1.0 h at 1000 V (gradient), 2.5 h at 8000 V (gradient), and 0.5 h at 8000 V (step) until a total of 15e17 kVh was reached. The strips were then equilibrated for 15 min in 15 ml equilibration buffer (TriseHCl 50 mM pH ¼ 8.8, urea 6 M, glycerol 30 %, SDS 2 %), with 1 % (w/v) DTT, and subsequently for 15 min in 15 ml equilibration buffer with 2.5 % (w/v) iodoacetamide. Proteins were separated in 12.5 % (w/v) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Ruby 600, GE Healthcare) with 25 mM Tris, 192 mM glycine, 0.1 % SDS buffer. Protein standards were loaded on each gel (BenchMark protein ladder, Invitrogen, USA).

Protein visualization and image analysis Proteins were visualized by Coomassie Brilliant Blue G (CBB) (Sigma) staining. Gels were fixed for 1 h [40 % (v/v) methanol, 10 % (v/v) acetic acid], stained for 1 h in the same solution containing 0.5 % (w/v) CBB and de-stained over night in a destaining solution [50 % (v/v) methanol, 10 % (v/v) acetic acid]. Alternatively, the spots were silver stained according to a previously described protocol (Yatsuda et al. 2003). Stained gels were scanned with Labscan software (GE Healthcare). Image Master 2D Platinum 6.0 software (GE Healthcare) was used for images and statistical analyses. For each treatment

Matrix-assisted laser desorption/ionization-time of flight-mass spectrometry Protein spots were excised manually from the gels and digested with trypsin as described previously (Shevchenko et al. 1996). After digestion, peptides were extracted from gel spots by two washes with 30 ml 50 % acetonitrile (v/v) plus 5 % trifluoroacetic acid (v/v) solution. The volume of the supernatant, containing the peptides, was reduced to 5e10 ml for MALDI-TOF MS/MS analyses. The samples for mass spectrometry were prepared by diluting (1:1) the peptides with the matrix solution [5 mg ml1 a-cyano-4-hydroxycinnaminic acid in 50 % acetonitrile/0.1 % trifluoracetic acid (v/v)]. One ml of the resulting solution was loaded onto a MALDI target and allowed to dry at room temperature. MALDI MS/MS spectra were acquired using either MALDI-TOF/TOF-MS (Axima Performace, Kratos-Shimadzu, Manchester, UK) with external calibration using a mixture of synthetic peptides (SigmaeAldrich, St. Louis, MO) or MALDIMS/MS (Waters, Manchester, UK) calibrated with a misture of polyethyleneglycols. The MS/MS data obtained from each protein digest was analyzed using MASCOT software version 2.2 (Matrix Science, London, UK) to search for proteins in the National Center for Biotechnology Information non-redundant (NCBInr) database. The spectra of all spots were also searched against publicly available Metarhizium expressed sequence tags (ESTs) deposited into NCBI database. For the latter mode of identification, the corresponding translated sequences were used for basic local alignment search tool (BLAST) searches against the NCBInr database (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Search parameters for MASCOT were as follows: other fungi taxonomic restriction; trypsin cleavage, allow up to one missed cleavage; fixed modification, carbamidomethyl (C); variable modification, oxidation (M); peptide tolerance, 1.2 Da; MS/MS fragment ion tolerance, 0.8 Da and average masses. Results were regarded as significant with an allowed likelihood for a random hit of P < 0.05, according to the MASCOT score.

Results Protein extraction and 2-D gel electrophoresis The robustness of the wall and the presence of pigments and other metabolites in the mycelium are frequently pointed out as factors that can impair protein extraction and separation by 2-DE (Shimizu & Wariishi 2005; Bhadauria et al. 2007; Kim et al. 2007a). The extraction protocol selected for the

Proteomic approach to identifying proteins differentially expressed in Metarhizium acridum

current study was that of Kniemeyer et al. (2006) who presented an optimized 2-D electrophoresis protocol for the humanepathogenic fungus Aspergillus fumigatus. Metarhizium anisopliae samples were homogenized directly in the precipitant solution of the Clean-UP kit, and this afforded highly reproducible expression profiles for both conidia and mycelia. About 1130
102 (correlation coefficient ¼ 0.94) protein spots were separated by 2-DE from conidia (Fig 1A) and 1200
97 (correlation coefficient ¼ 0.91) from M. anisopliae mycelia (Fig 1B). After spot detection, the gels were matched to each other (see Materials and methods) and the numbers of spots common to the three replicate gels were 901 and 917 for conidia and mycelia, respectively. Of these 1818 protein spots, 470 spots (approximately 35 % of the total) were common to both developmental stages (Fig 2).

575

Fig 2 e Numbers of protein spots on 2-DE gels of M. acridum that are exclusive to extracts of conidia, or mycelia, or common to both developmental stages.

In the present study, we focused only on qualitative differences between the expression profiles, i.e., on the absence or presence of spots in conidia and in mycelia, and not on the quantitative differences between the spots. The comparison between conidial and mycelial expression profiles revealed great differences between the sets of proteins and/or isoforms present in ungerminated mature conidia and in fast-growing mycelium.

Protein identifications

Fig 1 e Silver-stained 2-D gels of protein extracts of conidia (A) and mycelia (B) of M. acridum. Proteins (75 mg) were separated by IEF using 13-cm IPG strips (pH 3-11 NL) followed by 12.5 % SDS-PAGE. The circles denote protein spots analyzed by MALDI-MS/MS but not identified; black arrows denote spots identified with MASCOT scores > 56 (P < 0.05); and white arrows denote spots identified with MASCOT scores < 56 (P > 0.05).

Ninety-four protein spots [65 unique to conidia, 25 unique to mycelia, and two common to both (landmarks L-3 and L-8)] were subjected MALDI-TOF MS analysis (Fig 1, Table 1 and Table S1). The spectra of 41 proteins spots were assignable directly to at least one known protein (GI accession) by searching the NCBInr database. Twenty four (eight from conidia and 16 from mycelia) identified proteins had MASCOT scores higher than 56 (P < 0.05) (Table 1). MS data for 17 other protein spots had MASCOT scores below 56 (P > 0.05) (Table S1, see “Supplementary information”). Only two conidial and three mycelial spectra matched directly to Metarhizium proteins. All other protein spots were identified indirectly via homologues in other fungal species (Table 1). The paucity of Metarhizium-derived protein hits is not surprising; since, as of March 5, 2010, only 427 Metarhizium polypeptide entries were present in the NCBInr database. The spectra of all spots also were searched against publicly available Metarhizium ESTs (NCBI ESTs). These searches revealed that, out of 24 previously assigned spectra with P < 0.05, 12 matched to Metarhizium ESTs (Table 1) and, out of 17 assigned spectra with P > 0.05, three also matched to Ma ESTs (Table S1). The MS spectra of two conidial spots (C8 and C17) matched only to Ma ESTs (“EST-only” hits) (Table S1). Identified conidial protein spots (P < 0.05) included 30 and 90 kDa HSP, 6-phosphogluconate dehydrogenase, allergen Alt a 7, cobalamin-independent methionine synthase and actin (Table 1). Conidial protein spots with P > 0.05 are involved in several biological processes (Table S1). Identified mycelial protein spots (P < 0.05) included tubulin subunit beta, HSP 70

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B. H. R. Barros et al.

Table 1 e Identified 2-DE protein spots with MASCOT scores > 56 (P < 0.05) in the conidial and mycelial proteome maps of M. acridum. All C or M labeled protein spots indicate spots exclusive either to conidium or mycelium, respectively. L3C and L3 M are spots common to both stages and were used as landmarks. Spot number Conidium C6 C9 C13 C16 C34 C54 C55 L3C

Mycelium M1 M2 M8 M9 M10 M13 M14 M15 M16 M17 M18 M19 M21 M22 M23 L3M

Protein

Species

Accession numbera

Mw theor Mw obs pI Coverage MASCOT (kDa) (kDa) theor score

Similar to HSP 30 Similar to 6-phosphogluconate dehydrogenase Similar to allergen Alt a 7 Similar to actin HSP 90 HSP 90 Similar to cobalamin-independent methionine synthase Similar to ATP synthase F1, beta subunit

85111102c 21954534b

Neurospora crassa Aspergillus oryzae

25.2 54.6

35.2 60.0

5.56 5.82

4% 5%

72 66

154280184c 19073082c 88766397b 88766397c 34500101b

Ajellomyces capsulatus Cercospora beticola Metarhizium anisopliae Metarhizium anisopliae Epichloe festucae

21.9 36.8 80.2 80.2 77.6

26.8 58.7 43.9 129.8 127.3

5.82 5.76 4.98 4.98 6.31

13 % 14 % 1% 7% 7%

96 118 56 113 128

70997705b

Aspergillus fumigatus

55.6

60.0

5.30

10 %

121

Similar to 30 kDa HSP Similar to L-xylulose reductase Tubulin subunit beta Similar to aspartyl protease 1 Similar to ketol-acid reductoisomerase Similar to citrate synthase, mitochondrial precursor Similar to 6-phosphogluconate dehydrogenase Similar to transaldolase Similar to allergen Alt a 7 HSP 90 Similar to enolase/allergen Asp F 22 Similar to protein disulfide isomerase Pdi1 HSP 70 Similar to peptidyl-prolyl cis-trans isomerase D Similar to phosphoglycerate kinase Similar to ATP synthase beta chain, mitochondrial precursor

85111102c 115388155b 74655102b 73915318b 119191187c

Neurospora crassa Aspergillus terreus Metarhizium anisopliae Coccidioides posadasii Coccidioides immitis

25.3 28.7 50.4 43.9 45.0

26.0 25.8 59.2 51.4 46.5

5.56 5.24 4.76 4.68 9.08

4% 10 % 11 % 7% 10 %

78 190 179 156 102

46109080c

Gibberella zeae

52.0

53.4

8.18

10 %

111

29409963c

Aspergillus niger

54.5

70.9

5.93

6%

180

238505090c 154280184c 88766397c 121709952c 70989789b

Aspergillus flavus Ajellomyces capsulatus Metarhizium anisopliae Aspergillus clavatus Aspergillus fumigatus

35.6 21.9 80.0 47.3 57.9

39.0 19.3 80.2 76.1 100.0

5.57 5.82 4.98 4.92 4.64

5% 13 % 9% 18 % 4%

79 188 317 453 114

88766395b Metarhizium anisopliae 261351952b Verticillium albo-atrum

71.9 42.6

110.1 66.3

5.02 7.66

18 % 2%

674 75

115491365b Aspergillus terreus 67523719b Aspergillus terreus

45.9 53.8

35.4 60.0

5.43 5.44

19 % 16 %

375 372

a NCBInr protein database. b Also matched a Metarhizium spp. EST. c Also matched a fungal (other than Metarhizium spp.) EST.

and HSP 90, aspartyl protease 1, protein disulfide isomerase, peptidyl-prolyl cis-tras isomerase and enzymes involved in carbohydrate metabolism (enolase, 6-phosphogluconate dehydrogenase, phosphoglycerate kinase, citrate synthase and transaldolase) (Table 1). One of the landmarks (L-3, Fig 1) was identified as ATP synthase beta chain (mitochondrial precursor). Three proteins (HSP 90, 6-phosphogluconate dehydrogenase and allergen Alt a 7) were represented by more than one spot. For example, the HSP 90 was present with different Mws at two positions on the conidial gel (C34 and C54) and at one position on the mycelial gel (M17). The predicted molecular masses for the majority of the identified proteins were consistent with the experimental data. Exceptions include spots C54, C55, M14, M18, M19, M21 and M22, which exhibited a higher molecular mass than calculated. Products related to spots such as C34 and M23 exhibited a lower molecular mass compared with the calculated value. These deviations could arise from both atypical

migration in the 2-DE gels or from post-translational modifications (Noir et al. 2009). Given the indirect identification of most polypeptides, i.e., via data from fungal species other than Metarhizium spp., interspecies variation also could explain these discrepancies.

Discussions Despite the limitations associated with current methodologies (Kniemeyer et al. 2006; Wittmann-Liebold et al. 2006), 2DE proved to be a robust method for the study of changes in proteomes during fungal development. In the present study, without any enrichment strategies, approximately 1130 and 1200 protein spots were detected in Metarhizium acridum ungerminated conidia and fast-growing mycelia, respectively. Owing to the 2-DE separation procedure employed, these are expected to predominantly comprise soluble cytoplasmic

Proteomic approach to identifying proteins differentially expressed in Metarhizium acridum

proteins (Noir et al. 2009). The comparison between the conidial and mycelial proteomes of Metarhizium acridum highlighted hundreds of developmentally regulated proteins and/or isoforms. Only 35 % of the protein spots were common to the two developmental structures. The differences in expression profiles were expected in view of the marked differences in the morphology and physiology of these two structures. In addition to the presence of proteins specific to the different phases of development, results showed that alternative forms of the same annotated protein (as identified by their occurrence in different locations in mycelial versus conidial gels), contributed to the difference between conidial and mycelial 2-DE profiles. The use of subcellular studies for the identification of proteins present in specific structures such as the cell wall, plasma membrane and cytoplasmic organelles will permit a more extensive and precise determination of differences between conidial and mycelial proteomes. Several proteins found in M. acridum conidia had been observed previously in conidia of other species (Cooper et al. 2006; Leng et al. 2008; Noir et al. 2009; Quin et al. 2009). The identified conidial proteins are involved in cellular response against stress (i.e., HSP 30 and 90 and antioxidants enzymes 6-phosphogluconate dehydrogenase and mitochondrial peroxiredoxin) and in other biological processes (see Table 1 and Table S1). The abundance of HSP and other stress-related proteins may enhance conidial ability to survive environmental stresses (Cooper et al. 2006). The presence in conidia of several species of an enzyme arsenal covering nearly all biological processes indicates that the molecular machinery required for conidial germination is preformed, which allows rapid initiation of vegetative growth (Cooper et al. 2006; Leng et al. 2008; Noir et al. 2009; Quin et al. 2009). Conidia frequently are exposed to environmental stress factors such as desiccation, high temperatures and solar radiation. Little is known about the mode of action of the defense systems of dormant and/or quiescent conidia and how they can influence conidial persistence in the environment. An increase in the environmental persistence of M. acridum conidia is highly desirable because the conidial stage is the fungal developmental stage normally used as inoculum in biological control programs. Oxidative stress is a characteristic of the beginning of germination (Oh et al. 2010). Several protein and non-protein antioxidants are present exclusively or predominantly in conidia of different fungal species. Aspergillus nidulans has a conidia-specific catalase A encoded by the catA gene, whose expression is induced during conidiation and whose mutation renders conidia H2O2 sensitive (Navarro et al. 1996; Kawasaki et al. 1997). In a previous study, we detected conidia-specific Mn-superoxide dismutase and catalases in M. acridum ARSEF 324 (Miller et al. 2004). Fungal conidia also are exposed to environmental factors that can generate oxidative stress, such as ultraviolet A (UVA) radiation. Three proteins (HSP 90, 6-phosphogluconate dehydrogenase and allergen Alt a 7) were present in different spots in conidia and mycelia. Identical functional annotation of distinct spots are common in fungi and may result from either post-translational modifications of the same gene product (i.e., proteolysis, glycosylation, phosphorylation) or from the presence of sequence-related isoforms encoded by distinct paralogue genes (Noir et al. 2009; Vo¨disch et al. 2009). In the case of species for which no fully annotated genome sequence

577

is available, such as M. acridum, it is impossible to distinguish between these two possibilities on the basis of MS data. Some of the proteins identified in the present study in spots specific to conidia are identified also in mycelia of other species, such as Aspergillus fumigatus (Vo¨disch et al. 2009). Mycelial proteins identified in the present study are involved in several biological processes (see Table 1). Of 15 proteins identified in spots exclusive to Metarhizium mycelia, seven are among the most abundant (10 % highest) identified protein spots in mycelia of A. fumigatus (Vo¨disch et al. 2009). Several of the proteins identified in the present study in spots exclusive to M. acridum mycelia (i.e., tubulin, citrate synthase, HSP 70 and 90) also were identified in conidia of other species (Cooper et al. 2006; Leng et al. 2008; Noir et al. 2009; Quin et al. 2009). The spectra of all spots were searched also against publicly available fungal (including Metarhizium) ESTs. The MS data for several of the conidial and mycelial proteins also matched with Metarhizium ESTs, and most of the ESTs blasted to the same previously identified protein increasing the reliability of protein identification. Most of these ESTs, including those corresponding to our exclusively conidial spots, were originally obtained from mycelia of Metarhizium spp. (including the same M. acridum strain used in the present study) growing in various liquid media (Freimoser et al. 2003; Freimoser et al. 2005; Wang et al. 2005). All together, the results reinforce the idea that alternative forms of the same annotated protein are expressed in conidia and mycelia. The reasons for the large number of unidentified proteins spots [only 24 (with P < 0.05) identified out of 94 selected] are that the sequencing of the M. acridum genome is not yet complete, and that the number of ESTs of the species deposited in databases is small. Only 3449 ESTs from M. anisopliae and 1670 from M. acridum had been deposited in the NCBI by March 5, 2010. The genome sequencing of the M. acridum is underway and is expected to be concluded soon (R.J. St. Leger, personal communication). This information will greatly facilitate postgenomic studies and protein identification in these genera. The establishment of 2-DE reference maps for conidia and mycelia of M. acridum will permit correlating quantitative and qualitative variations in the proteomes of these two structures with other phenotypic characteristics, such as pathogenicity, virulence and tolerance to environmental stresses. Comparisons of two stages of ARSEF 324 with those other M. acridum isolates with contrasting phenotypes will assist in identifying new proteins involved in these processes. Additionally, variations in physical and chemical factors during growth (i.e., chemical composition of the culture medium, pH, temperature and light) can alter the virulence and stress tolerance of the conidia produced (Daoust & Roberts 1982; Schwerdtfeger & Linden 2001; Ibrahim et al. 2002; Rangel et al. 2004); and the comparison of conidial proteomes of the same strain grown under different conditions will help clarify the molecular basis of these epigenetic variations.

Acknowledgments We thank Ludmilla Tonani and Helen J. Laure for technical assistance. This work was supported by grants 03/07702-9 from

578

The State of Sa˜o Paulo Research Foundation (FAPESP). We sincerely thank the Brazilian Synchrotron Light Laboratory for the use of the MALDI-MS/MS, and FAPESP for a post-doctoral fellowship to E.R.M.

Supplementary material Supplementary material for this article is available in the online version at doi:10.1016/j.funbio.2010.04.007.

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