Antonie van Leeuwenhoek (2011) 99:671680 DOI 10.

1007/s10482-010-9543-0

ORIGINAL PAPER

Rho1 and other GTP-binding proteins are associated with vesicles carrying glucose oxidase activity from Fusarium oxysporum f. sp. lycopersici
Karla Macas-Sanchez Jesus Garca-Soto pez-Ramrez Adriana Lo Guadalupe Martnez-Cadena

Received: 28 July 2010 / Accepted: 9 December 2010 / Published online: 4 January 2011 Springer Science+Business Media B.V. 2010

Abstract In eucaryotic cells, the delivery of a secreted protein to the plasma membrane via vesicles must include transport, recognition, and fusion events. Proteins exposed on the cytoplasmic face of the secretory vesicles play a role in these events; these include the GTP-binding proteins, which are crucial components in this process. Fractions enriched with vesicles carrying glucose oxidase (GOX) activity from Fusarium oxysporum f. sp. lycopersici, a soilborne fungal pathogen causing vascular wilt on tomato plants, were obtained using two successive sucrose gradients, the rst a linear-log and the second an isopycnic gradient. In this study, we used the following Fusarium strains: a wild-type and a strain carrying a Drho1 loss-of-function mutation (presenting dramatically reduced virulence). By ADP-ribosylation with C3 exotoxin, and Western blot analysis with specic antibodies, we identied the small GTPases Rho1, Rho4, Cdc42 and Rab8, and a heterotrimeric Ga protein associated with vesicles carrying GOX activity. This was done for both strains, with the exception of Rho1, which was absent in the mutant strain; in addition, the levels of the Cdc42 protein were observed to be higher in the Drho1 strain. These data indicate
K. Macas-Sanchez J. Garca-Soto A. Lopez-Ramrez G. Martnez-Cadena (&) Division de Ciencias Naturales y Exactas, Departamento de Biologa, Campus Guanajuato, Universidad de Guanajuato, Apdo. postal 187, 36000 Guanajuato, Gto, Mexico e-mail: margua@quijote.ugto.mx

that three Rho proteins, Rho1, Rho4, and Cdc42, are present in secretory vesicles carrying GOX activity in F. oxysporum, and that Rho1 is not essential for the transport and secretion of, at least, cargo proteins carried in secretory vesicles, or Cdc42/Rho4 can fulll its role in these events. Keywords GTP-binding proteins Fusarium Secretory vesicles Rho GTPases GOX

Introduction G proteins are signal transduction proteins that act as molecular switches cycling between an active GTPbound state and an inactive GDP-bound state. These proteins are implicated in a wide variety of cellular processes such as polarization, membrane trafcking and exocytosis, among others (Wennerberg et al. 2005). Exocytosis is the mechanism by which newly synthesized proteins are delivered to the cell surface. In the budding yeast Saccharomyces cerevisiae, the tethering of secretory vesicles to the plasma membrane is coordinated by a multiprotein complex known as the exocyst, which is made up of eight proteins (Sec3p, Sec5p, Sec6p, Sec8p, Sec10p, Sec15p, Exo70p, and Exo84p) (Guo et al. 1999; TerBush et al. 1996). Various small G proteins also participate in the regulation of vesicular trafc,

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among them Rho1, Rho3, Cdc42 and Sec4 (Park and Bi 2007). Rho1 participates in actin organization, yeast cell wall remodeling, and as a master regulator of cell polarity (Cabib et al. 1998; Guo et al. 2001). Rho3p also acts as a key regulator of cell polarity by directing the rearrangement of the actin cytoskeleton through its interaction with type V Myo2, in addition to acting in the polarized delivery and fusion of vesicles to specic sites on the plasma membrane mediated by its interaction with the exocyst component Exo70 (Adamo et al. 1999). Two-hybrid analysis studies have shown that exocyst component Sec3 could interact with Rho1, Rho3 and Rho4 (Adamo et al. 2001). Cdc42 plays an important role in promoting the docking and fusion of secretory vesicles to the plasma membrane during polarized growth in S. cerevisiae (Adamo et al. 2001). Cdc42 also controls the polarized organization of the actin cytoskeleton, which provides the track for directional transport of secretory vesicles (Ayscough et al. 1997; Govindan et al. 1995). The interactions of both Cdc42 and Rho1 with formin family proteins such as Bni1, which make up part of the polarisome, may be important in controlling actin organization (Cabib et al. 1998; Erickson and Cerione 2001; Johnson 1999). Studies in our laboratory have demonstrated the presence of various G proteins on secretory vesicles of the strain UPO 1171 from Mucor circinelloides, an R7B transformed strain in which the promoter of the gene gpd1 encoding glyceraldehyde-3-phosphate dehydrogenase is fused to the open reading frame of the gox gene of Aspergillus niger. In these vesicles, Rho1 and Cdc42 proteins were clearly detected in vesicles carrying glucose oxidase (GOX) activity and chitosomes, respectively (Mo reno-Jimenez et al. 2008). Fusarium oxysporum f. sp. lycopersici is a phytopathogenic fungus causing vascular wilt disease on tomato, known as fusariosis (Beckman 1987; Di Pietro et al. 2003). Since our group found the association of Rho proteins, Rho1 and Cdc42, with transport and secretory vesicles, respectively (Mo reno-Jimenez et al. 2008), the results reported by Martnez-Rocha et al. (2008) on the F. oxysporum Drho1 strain may also indicate that the overall vesicle transport system of the fungus, including that which is involved in carrying the cell wall synthesis

enzymes, may be altered. This mutant is nonpathogenic on tomato plants and one of the explanations is that Rho1 could play a specic role in maintaining the fungal cell wall architecture in a way that makes it difcult to detect for the plant host. This might mean that the secretion process of the enzymes involved in the synthesis-degradation of the cell wall is alter, therefore it is interesting to study the role of Rho proteins in the secretion process of Fusarium. In this study, we set out to determine whether Rho1 and other GTP-binding proteins were present in secretory vesicles in F. oxysporum. To this end, GOX activity was chosen as a cargo protein of secretory vesicles, in view of the fact that Houterman et al. (2007) recently identied this protein in a xylem sap proteome analysis of tomato plants infected with F. oxysporum f. sp. lycopersici, even though its role in virulence has not yet been addressed. We puried vesicles carrying GOX activity from the wild-type strain 4287 and a strain carrying a rho1::hyg loss-offunction allele from F. oxysporum f. sp. lycopersici. Under the experimental conditions employed, GOX activity was detected in vesicles with a density of 1.10 g/ml in both strains used. The presence of Rho1, Rho4, and Cdc42 proteins in secretory vesicles of the wild-type 4287, and Rho4 and Cdc42 proteins in vesicles from the strain rho1::hyg was detected. Moreover, various proteins, such as Rab8 (Sec4 orthologue), and a Ga protein, were found associated with the vesicles from both strains. The absence of Rho1 in the secretory vesicles from the mutant strain strongly suggests that it is not essential for transport and docking of vesicles to the plasma membrane in F. oxysporum, or that Cdc42 and/or Rho4 can carry out these processes in the absence of this GTPase.

Materials and methods Fungal isolates and growth conditions Fusarium oxysporum f. sp. lycopersici strains wildtype 4287 (race 2), pathogenic strain, and rho1::hyg (loss-of-function allele), a strain with reduced virulence were kindly provided by Dr. M. Isabel. G. Roncero (Universidad de Cordoba, Spain) and stored at -70C with glycerol as a microconidial suspension. Conidias were germinated in potato dextrose broth for 4 days at 28C on a rotary shaker at 200 rpm. The

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resulting conidias were washed in sterile water, transferred to YPGlycerol medium (0.3% yeast extract, 1% gelatin peptone, and 2% glycerol) and incubated on a rotary shaker for 12 h at 200 rpm and 28C. Mycelium was obtained and stored at -20C before use. Preparation of cell-free extracts Mycelium was resuspended in lysis buffer (50 mM Tris/HCl, pH 7.8 containing 10% sucrose and protease inhibitors) mixed with a volume of glass beads (0.450.50 mm diameter) and broken in a Braun model MSK cell homogenizer for 2 min, while cooling with a stream of CO2. The homogenate was centrifuged at 3149g for 5 min to remove cell walls and unbroken cells. The cell wall-free material (crude extract) was centrifuged at 19609g for 45 min; the pellet, a mixed membrane fraction, was discarded, and the supernatant was saved for the purication of the vesicles carrying GOX activity. Purication of secretory vesicles The supernatant obtained in the preparation of cellfree extracts (3 ml) was layered on top of a linear-log (11.831.8%) sucrose gradient (Brakke and Van Pelt 1970) and centrifuged at 827059g for 2.5 h at 4C. The gradient was manually fractionated from the top in fractions of 1 ml. The four fractions with highest GOX activity were pooled; 50 mM Tris-HCl, pH 7 was added up to 18 ml and mixed with 18 ml 65% sucrose with a density gradient former. This second gradient was centrifuged at 827059g for 18 h at 4C (to form an isopycnic sucrose gradient) and fractionated from the top into 1 ml aliquots. In each fraction, the density and GOX activity were measured. GOX activity assay GOX activity was measured by a 2,20 -azino-bis(3ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay. A 100 ll sample was mixed with 1.4 ml reaction buffer (10 mg ABTS, 1 ml 20% glucose, 50 units of peroxidase, 16 ml of 50 mM sodium phosphate buffer, pH 6.3 and H2O up to 20 ml nal volume). The reactions were incubated for 1 or 2 h at room temperature and then stopped with HCl, and the absorbance was measured at 420 nm. As negative

controls, we assayed GOX activity in the absence of peroxidase. One mUnit is the amount of enzyme that oxidizes 1 nmol glucose/min. Electron microscopy assays For negative staining, droplets of samples (fractions with the highest GOX activity from the isopycnic gradients) were placed on carbon-coated formvar lms on 500-mesh copper grids. Subsequently, 2% uranyl acetate was added to each grid for 1.5 min and the excess liquid was withdrawn with lter paper. Samples were examined and photographed with a Jem-100 S model Jeol electron microscope. Morphogenic analyses were done by counting 100 vesicles per sample. ADP-ribosylation assay The ADP-ribosylation assay with the exotoxin C3 from Clostridium botulinum was carried out with the following reaction mix: 50 mM Tris-HCl, pH 7.4, 1 mM MgCl2, 0.5 mM DTT, 0.5 mM EDTA, 0.2% n-octyl-b-D-glucopyranoside, 0.25 ll of C3 toxin 0.02 lg/ll, 2 lM NAD, 2 lCi [32P]NAD, 100 lg of protein. The reaction was incubated for 1 h at 30C with agitation. Subsequently, the reaction was terminated by the addition of 49 solubilization buffer (0.25 M Tris-HCl, pH 6.8, 8% SDS, 40% glycerol, 20% 2-mercapto-ethanol, and bromphenol blue). Samples were boiled for 5 min and subjected to SDS-PAGE (Laemmli 1970). After electrophoresis, the gels were stained with Coomassie blue, and exposed on Kodak MXB lms. Immunoblot analysis Western blot analyses were performed on enriched fractions of vesicles carrying GOX activity. The fractions were subjected to SDS-PAGE or IEF-SDSPAGE and electrotransferred onto a nitrocellulose membrane. Blots were probed with one of the following antibodies: anti-Rho A at 1:500 dilution (Rho A [119] rabbit polyclonal IgG, Santa Cruz Biotechnology), anti-Rho B at 1:500 dilution (Rho B [119] rabbit polyclonal IgG, Santa Cruz Biotechnology), anti-Rho C at 1:500 dilution (Rho C [G-12] goat polyclonal IgG, Santa Cruz Biotechnology), anti-Rho D at 1:1000 dilution (Rho D [N-18] goat

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polyclonal IgG, Santa Cruz Biotechnology), antiCdc42 at 1:500 dilution (Cdc42 [P1] rabbit polyclonal IgG, Santa Cruz Biotechnology), anti-Rab8 at 1:500 dilution (Rab8 [FL-207] rabbit polyclonal IgG, Santa Cruz Biotechnology), anti-Ga i/o/t/z/gust at 1:1000 dilution (Ga i/o/t/z/gust [H-300] rabbit polyclonal IgG, Santa Cruz Biotechnology). The blocking peptides used were: Rho B [119] P control peptide, Santa Cruz Biotechnology and Cdc42 [P1] P blocking peptide, Santa Cruz Biotechnology. Proteins were detected with the Amersham ECLTM Western blotting analysis. The secondary antibodies used were the following: ECLTM anti-rabbit IgG, horseradish peroxidase linked F(ab)2 fragment, Amersham Biosciences UK Limited at 1:2000 dilution (RhoA, RhoB, Cdc42 and Rab8), secondary antibody HRP-rabbit anti-goat IgG (H?L), Zymed at 1:2000 dilution (RhoC and RhoD), and the secondary antibody HRP-goat anti-rabbit IgG (H?L), Zymed at 1:2000 dilution (Ga i/o/t/z/gust). Inmunoreactivity was monitored by the enhanced chemiluminescence method (Amersham). Cdc42 expression analysis Total RNA was extracted with TRIzol Reagent (Invitrogen, CA, USA) as indicated by the manufacturer. PCR reactions of cDNA were performed with 25-ll reaction volumes with 1 ll of cDNA, 15 pmol of each primer, 160 mM dNTPs, 2 mM MgCl2, 0.5 U of Platinum Taq polymerase (Invitrogen, CA, USA) on a Perkin-Elmer Gene Amp system 2400 with the primers cdc42F (50 -CAGCAAGATGCAACAATAG CTTCG-30 ), cdc42R (50 -GATACACAACCAACA AATTTCC-30 ), act1F (50 -AATGGTTCGGGTATG TGCAAGG-30 ), and act1R (50 -GTATCGTTCTGG ACTCTGGTGA-30 ).

and we analyzed the presence of various proteins associated with vesicles. GOX activity was chosen as a cargo protein of secretory vesicles. Under our experimental conditions, GOX activity was detected in complete cells after day 8 of growth in both strains, the wild-type 4287, and rho1::hyg (Fig. 1a). These results, together with the presence of a putative peptide secretion signal present in the ORF of GOX gene and those reported by Houterman et al. (2007), strongly suggest that the wild-type and rho1::hyg strains of F. oxysporum transport GOX to the plasma membrane through secretory vesicles. Purication of vesicles carrying GOX activity in F. oxysporum Secretory vesicles were isolated following the proce dure described previously (Moreno-Jimenez et al. 2008) by using two successive sucrose gradients, a linear and an isopycnic. The four fractions of the linear gradients obtained from the wild-type and rho1::hyg strains with the highest GOX activity (Fig. 1b, c, respectively) were pooled and mixed with 65% sucrose and subjected to an isopycnic sucrose gradient. In this gradient, the maximum enzymatic activity was obtained at a density of 1.10 g/ml (Fig. 1d, e). In both gradient types, of both strains of F. oxysporum (wildtype 4287 and rho1::hyg), we observed the same GOX activity behavior (Fig. 1). To corroborate whether these enriched fractions contained vesicles, the samples were negatively stained with uranyl acetate and observed under electron microscopy. These fractions revealed vesicles with a diameter of 27 1.8 and 24.5 4.28 nm (n = 100) in the wild-type 4287 and in the rho1::hyg strains, respectively (data not shown). In view of the fact that there was practically no change in density and diameter of the vesicles from both strains, these results suggest that Rho1 does not participate in the process of budding and vesicle formation from the trans Golgi network (Fig. 2). The protein Rho1 is present in the secretory vesicles of F. oxysporum Rho proteins are members of the Ras superfamily of small G proteins that have been reported to associate with vesicles and to participate in polarized secretion (Cabib et al. 1998; Matsui and Toh-e 1992; Wu et al. 2008). Members of the Rho subfamily that present the

Results Analysis of GOX activity in the F. oxysporum strains Our group is interested in investigating the role of small and heterotrimeric GTP-binding proteins in the secretory process. To this end, we used the wild-type 4287 strain and a strain carrying a rho1::hyg loss-offunction allele from F. oxysporum f. sp. lycopersici,

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a
GOX mUnits

450 400 350 300 250 200 150 100 50 0 0 5 10 15

Fractions (ml)

Fractions (ml) Sucrose density g/ml Fractions (ml)

28.2 kDa

c
GOX mUnits

Days

Fractions (ml)
Fig. 1 Glucose oxidase (GOX) extracellular activity from complete cells and sedimentation prole of vesicles carrying GOX activity from strains of F. oxysporum. Conidias (5 9 106/ ml) from F. oxysporum f. sp. lycopersici strains 4287 and rho1::hyg, respectively, were inoculated in YPGlycerol-2% Pectin medium and incubated at 28C for 10 days. Aliquots from the cultures were withdrawn, centrifuged, and GOX activity was assayed in complete cells as described in Materials and methods. Wild-type strain 4287 (lled circle), strain rho1::hyg (lled square). For the sedimentation prole,

cultures were processed as described in Materials and methods. After centrifugation in a linear-log gradient (b wild-type 4287 and c rho1::hyg), the four most active fractions of GOX activity were pooled and used to form a second, isopycnic gradient (d wild-type 4287 and e rho1::hyg) as described in Materials and methods. The maximum peak of GOX activity, which presents a density of 1.10 g/ml, is indicated with an arrow. One mUnit of GOX is expressed as the amount of enzyme that oxidizes 1 nmol glucose/min. Identical results were obtained in three different experiments

22 kDa

22 kDa 19 kDa
1 2 1 2

Fig. 2 Rho1 and Rho4 are associated with enriched vesicles carrying GOX activity. Enriched fractions of vesicles obtained from isopycnic sucrose gradient (d = 1.10) were subjected to (a) an ADP-ribosylation assay with the exotoxin C3 and [32P]NAD. Afterwards, proteins were separated by electrophoresis, stained with Coomassie blue and exposed on X-ray lm. Lanes 1, wild-type strain 4287 and 2, strain rho1::hyg. Proteins

were separated by SDS-PAGE, electro-transferred, and the membranes incubated with the primary antibody anti-RhoA; (b) or anti-Rho4; (c). Afterwards, the blots were incubated with a secondary antibody conjugated with horseradish peroxidase. Lanes 1, wild-type strain 4287 and 2, strain rho1::hyg, respectively. Identical results were obtained in three different experiments

conserved domain FENY are substrates for ADPribosyl transferase activity of the exotoxin C3 of C. botulinum (Sekine et al. 1989). Additionally to Rho 1

Type (Accession No. AY884607.1), reported by Martnez-Rocha et al. (2008), the F. oxysporum database reports the presence of four genes, which

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a
kDa

75 50 37 25 20 1 2 3 4 cdc42 5 6

24 kDa

act 1 2

Fig. 3 Rho1 regulates Cdc42 protein association levels with secretory vesicles and cdc42 gene expression in F. oxysporum. (a) Proteins of the crude extract of the wild-type strain and fractions (d = 1.10) with vesicles carrying GOX activity from the wild-type and rho1::hyg strains were separated by SDS electrophoresis, electro-transferred, and the membranes incubated with human polyclonal antibody anti-Cdc42; afterwards, the blots were incubated with a secondary antibody conjugated with horseradish peroxidase (Lanes 1, 2, 5, and 6); in Lanes 3 and 4, the blots were stained with Amido Black. Lanes 1 crude extract of the wild-type 4287; 2 crude extract of the wild-type 4287 with blocking peptide; 3, 5 proteins of vesicles from the wild-type 4287 and 4, 6 rho1::hyg proteins, respectively. (b) Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis for the cdc42 transcript (460 pb) from the wild-type (Lane 1) and rho1::hyg (Lane 2) strains, respectively. The actin gene fragment (440 pb) was used as positive control. PCR products were subjected to electrophoresis in 2% agarose gels. Similar results were obtained in three different experiments

encode for Rho2 (FOXG_11573), Rho3 (FOXG_ 01310), and Rho4 (FOXG_00764) GTPase proteins and a putative protein Rho which is not classied as a specic isoform (FOXG_15954.2). To detect the presence of Rho proteins, the enriched fractions of vesicles carrying GOX activity of the strains of F. oxysporum were incubated with C3 exotoxin and [32P]NAD. Fig. 3a (lanes 1 and 2) show that a Rho protein with a molecular mass of 22 kDa was detected in these fractions from both strains. Next, we subjected these fractions to Western blot analysis using a polyclonal antibody against a human RhoA (antiRhoA; Rho1p orthologue). As can be observed in Fig. 3b (lane 1), this antibody revealed two bands with molecular masses of 22 and 19 kDa, respectively, with

that of 22 kDa corresponding to a Rho1 protein, and that detected at 19 kDa possibly a degradation product of this protein. These vesicular proteins were only detected in the wild-type 4287 strain of F. oxysporum. The 22 kDa protein (predicted pI/Mw: 5.99/ 21947.23), identied as RhoA (Rho1p orthologue) presented a pI of 6.36 when separated by twodimensional gel electrophoresis and immunodetected with anti-RhoA antibodies (data not shown). As expected, on the vesicles from the strain rho1::hyg, we did not detect the bands of 22 and 19 kDa with the anti-RhoA (Fig. 3b, lane 2). This result indicates that Rho1 is associated with the vesicles carrying GOX activity in the wild-type 4287 strain of F. oxysporum. Since we detected an ADP-ribosylated Rho protein when C3 exotoxin was used in secretory vesicles from the Drho1 strain, we decided to study the presence of Rho2 (predicted molecular mass of 22 kDa), Rho3 (predicted molecular mass of 22.8 kDa), and Rho4 (predicted pI/Mw: 7.54/ 29.8 kDa), proteins in the crude extract of wild-type and in the enriched vesicles carrying GOX activity of both strains of F. oxysporum. We used polyclonal antibodies against human RhoB (Rho2 orthologue), against human RhoC (Rho3 orthologue) or against human RhoD (Rho4 orthologue) to determine the presence of these proteins. With the primary antibodies anti-Rho2 and anti-Rho3, proteins with molecular masses of 23 and 22 kDa, respectively, were detected in the crude extracts of the both strains (data not shown), but these bands were not detected in the enriched fractions with vesicles carrying GOX activity from the wild-type 4287 or rho1::hyg strains (data not shown). When we looked for the presence of the protein Rho4 using the polyclonal antibody against human RhoD (Rho4 orthologue), a protein of molecular mass of 28.2 kDa was detected in the fractions from the enriched vesicles carrying GOX activity from the wild-type and rho1::hyg strains (Fig. 3b, lanes 1 and 2, respectively). This protein presented the same pI of 7.13 when the proteins of the enriched vesicles of the two strains were separated by two-dimensional gel electrophoresis (data not shown). However, none of the antibodies used enabled us to identify the isoform of the Rho protein with a molecular mass of 22 kDa observed in the ADPribosylation assay with the exotoxin C3 in the vesicles from strain rho1::hyg. This result could

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indicate the presence of a Rho protein not annotated in the Fusarium genome database present in secretory vesicles which is a substrate of the C3 exotoxin. Association of Cdc42 with secretory vesicles It has been reported that the Cdc42 protein regulates polarized growth by regulation of the actin cytoskeleton, and also participates in the docking of the secretory vesicle to the plasma membrane (Zhang et al. 2001). This protein belongs to the Rho family, but does not show the FENY domain, site of ADPribosylation by the C3 toxin from C. botulinum; therefore, this protein is not ADP-ribosylated by this toxin. We used a polyclonal antibody against human Cdc42 (anti-Cdc42) to detect the presence of this protein in the secretory vesicles in the two strains of F. oxysporum (Fig. 3a). With this antibody, we detected a band of molecular mass of 24 kDa (FOXG_09453, theorical molecular weight of 22 kDa) in the crude extract of the wild-type 4287, which disappeared in presence of the blocking peptide (Fig. 3a, lanes 1 and 2, respectively). This antibody also detected a band of molecular mass of 24 kDa in the enriched vesicles from the wild-type and rho1::hyg strains (Fig. 3a, lanes 5 and 6, respectively). However, the signal detected in the wild-type 4287 strain (Fig. 3a, lane 5) was barely visible compared to that detected from the rho1::hyg strain (Fig. 3a, lane 6). Similar amounts of proteins can be observed in the AmidoBlack stain of the membranes (Fig. 3a, lanes 3 and 4, respectively). This result indicates that Cdc42 is associated with the vesicles carrying GOX activity; however, it seems that its association with the vesicles is different in the strains of F. oxysporum studied in this work. To evaluate whether the difference in the band intensities of Cdc42 was due to the lack of Rho1 in the mutant strain we determined the expression levels of the cdc42, using actin mRNA as a consecutive gene. As shown in Fig. 3b, cdc42 mRNA levels are higher in the rho1::hyg strain (Fig. 3b, lane 1) than in the wildtype strain (Fig. 3, lane 2). Rab8 is associated with secretory vesicles Rab8 (Sec4 orthologue) is a protein present on the vesicle membrane by way of its carboxyl terminal lipid anchor (Boyd et al. 2004). This protein is the

rst GTPase that interacts with the exocyst (Lipschutz and Mostov 2002) and participates in both vesicular transport and fusion (Guo et al. 2000; Novick and Guo 2002). We used polyclonal antibody raised against a human Rab8 (anti-Rab8) to detect whether this protein was associated with the vesicles carrying GOX activity of the wild-type and rho1::hyg strains. In the crude extract of wild-type and the separated proteins of the vesicles from both strains (wild-type 4287 and rho1::hyg), this antibody was able to detect a band of molecular mass of 25 kDa (FOXG_08501.2, predicted molecular mass of 22.7 kDa) (data not shown). Therefore, its presence on secretory vesicles may indicate a direct role in the transport or fusion of the vesicles of F. oxysporum to their target membranes. This protein was detected in transport vesicles (chitosomes) and secretory vesicles (carrying GOX activity) from M. circinelloides (Moreno-Jimenez et al. 2008). Association of a Ga protein with secretory vesicles It has been reported that heterotrimeric G proteins regulate the formation of secretory vesicles (Barr et al. 1991; Leyte et al. 1992), besides participating in the secretory process in various fungi (Gronover et al. 2001; Solomon et al. 2004). We used polyclonal antibody against human Ga i/o/t/z/gust (anti-Ga i/o/t/ z/gust) to detect whether this protein was associated with the vesicles carrying GOX activity from the wild-type and rho1::hyg strains. With this antibody, we detected a protein with a molecular mass of 46.7 kDa in the vesicles from the two strains of F. oxysporum (data not shown). This Ga protein, associated with the vesicles from the two strains, presented a pI of 5.5 when separated by twodimensional gel electrophoresis (data not shown). Therefore, its presence on secretory vesicles may indicate a role in the formation of vesicles or in the secretory process in F. oxysporum.

Discussion Exocytosis is the mechanism by which various recently synthesized proteins are released to the cell surface. Through storage in transport vesicles, various membrane proteins troop new components to the cell

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plasma membrane, while some soluble proteins are stored in secretory vesicles and secreted to extracellular space. GOX may be used as a marker of secretory vesicles in fungi because it is a secreted enzyme. This protein catalyzes the oxidation of b-D-glucose to D-glucono-1,5-lactone and hydrogen peroxide. The enzymes role has been reported in various microorganisms, such as the fungus Penicillium, in which it acts as an antimicrobial (Leiter et al. 2004); A. niger, in which it reduces environmental pH (Wong et al. 2008), and Phanerochaete chrysosporium, in which it contributes to lignin degradation Ramasamy et al. 1985). Recently, Houterman et al. (2007) reported the presence of an oxidoreductase in a xylem sap proteome analysis of F. oxysporum-infected tomato plants. This oxidoreductase (FOXG_14258.2) presents an identity of 29 and 46% homology with the GOX from A. niger. Moreno-Jimenez et al. (2008) reported a diameter of 2530 nm of vesicles enriched with GOX activity in a transformed strain of Mucor circinelloides. Similar values were found in the vesicles carrying GOX activity from the two strains of F. oxysporum, suggesting that these cargo proteins may be transported from the trans Golgi to the plasma membrane in small vesicles. The role of Rho protein in vesicular trafc in the yeast S. cerevisiae has been widely reported. This protein is associated with secretory vesicles and participates in delivering them to the bud growth site through the organization of the actin cytoskeleton (Adamo et al. 1999). Conditional rho1- mutants in S. cerevisiae show defects in cell wall integrity, resulting in lysis at the small-bud stage (Pruyne and Bretscher 2000). The F. oxysporum database contains annotations of ve genes that encode for ve Rho proteins (Rho1, Rho2, Rho3, Rho4 and Rho FOXG_15954.2). Of these, only Rho4 presents a predicted higher molecular weight of approximately 29 kDa. We detected Rho1 (Accession No. AY884607.1) and Rho4 (FOXG_00764) associated with vesicles of the wild-type strain, and only Rho4 associated with the vesicles of the rho1::hyg. Rho4 was only detected as a minor band in the ADP-ribosylation assay with C3 toxin in crude extracts from the two strains (data not shown), but we did not detect it in vesicles, possibly because it is not a good substrate for this toxin under the experimental conditions used in this work, although we could detect it by Western blot analysis. The presence of these Rho proteins in the vesicles

may suggest their involvement in the exocytosis process. However, in the case of protein Rho1, its participation is not essential, since the amount of periplasmic GOX activity found in the rho1::hyg strain was similar to that of the wild-type 4287 strain. Studies in our laboratory showed the presence of the Rho1 in secretory vesicles in M. circinelloides, suggesting its involvement in vesicular trafcking (Moreno-Jimenez et al. 2008). However, our results with rho1::hyg strain denitely discard an essential role of Rho1, at least for the transport and fusion of secretory vesicles to the plasma membrane. Immundecoration experiments of the vesicles with different anti-Rho antibodies could differentiate subpopulations of secretory or transport vesicles. Cdc42, a GTPase from the Rho subfamily, regulates polarized growth by regulating the actin cytoskeleton and coordinating vesicle docking machinery (Zhang et al. 2001; Wennerberg et al. 2005). In this study, we found this protein associated with secretory vesicles from the two strains of F. oxysporum, suggesting a role in their transport, even though the rho::hyg mutant presented higher levels of this protein and its mRNA, suggesting that the increase in Cdc42 levels can compensate for the lack of the Rho1-Sec3 secretory pathway in this mutant (reviewed by Wu et al. 2008). On the other hand, these results differ from those reported for M. circinelloides (Moreno-Jimenez et al. 2008), since Cdc42 was only detected in chitosomes of this fungus, which could suggest different pathways for the transport of proteins participating in cell wall synthesis in Zygomycetes. Work remains to be done to investigate which Rho GTPases are present in chitosomes from F. oxysporum. It has been reported that G heterotrimeric proteins regulate the formation of secretory vesicles (Barr et al. 1991; Leyte et al. 1992), besides participating in the secretion process, as it is known that the proteins Gna1 and Bcg1 (Ga class I) of the fungi Stagonospora nodorum and Botrytis cinerea, respectively, are involved in protease secretion (Gronover et al. 2001; Solomon et al. 2004). The F. oxysporum f. sp. lycopersici database contains annotations of Ga proteins Fga1 (Class I, molecular mass 41 kDa and pI of 5.12), which has been reported to participate in conidiation, heat resistance and virulence (Jain et al. 2002), and Fga2 (Class III, molecular mass of 40.8 kDa and pI of 5.91), which is involved in heat

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resistance and virulence (Jain et al. 2005). Also, a sequence for a third Ga protein (FOXG_06321) is partially annotated in the F. oxyspoum database. In this work, in vesicles from the F. oxysporum strains, Western blot analysis revealed the presence of a Ga protein with a molecular mass of 46.7 kDa and an isoelectric point of 5.55.6. Although the molecular weight and pI are different from those predicted, it is important to point out that G proteins present post-translational modications that usually change biochemical parameters from those predicted. The antibodies used can detect any of the three isoforms of the Ga protein. Western blot studies of vesicles from the Dfga1 and Dfga2 mutant strains of F. oxysporum will provide evidence of which Ga protein is present in secretory vesicles. In addition to the proteins identied in this study on secretory vesicles (Rho1, Rho4, Cdc42, Rab8, Ga), the F. oxysporum database contains annotations of proteins homologous to those proteins forming the exocyst in S. cerevisiae. We therefore suggest that these proteins are also involved in vesicle transport to the plasma membrane; it is yet unknown, however, whether the mechanism by which this process takes place in F. oxysporum is similar to that of S. cerevisiae.
Acknowledgments This study was supported by the Mexican National Council of Science and Technology (CONACYT) under Grant 62568, and the Universidad de Guanajuato. K.M.S. is a doctoral student with a scholarship from CONACYT and the Universidad de Guanajuato.

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