Isolation, characterization and expression analysis of the ... · Isolation, characterization and expression analysis of the ornithine decarboxylase gene (ODC1)ofthe entomopathogenic

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ARTICLE IN PRESS+ModelICRES-25385; No. of Pages 14

icrobiological Research xxx (2011) xxx—xxx

Available online at www.sciencedirect.com

www.elsevier.de/micres

solation, characterization and expression analysisf the ornithine decarboxylase gene (ODC1) of thentomopathogenic fungus, Metarhiziumnisopliae

aime Madrigal Pulidoa, Israel Padilla Guerreroa,saura de J. Magana Martíneza, Briseida Cacho Valadeza,uan Carlos Torres Guzmana, Eduardo Salazar Solisb,. Felix Gutierrez Coronaa, Augusto Schrankc,rancisco Jiménez Bremontd, Angélica González Hernandeza,∗

Departamento de Biología, Universidad de Guanajuato, Guanajuato, Gto., MexicoDepartamento de Agronomía, Universidad de Guanajuato, Irapuato, Gto., MexicoCentro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, BrazilDivision de Biología Molecular, Instituto Potosino de Ciencia y Tecnología, San Luis Potosí, SLP, Mexico

eceived 15 September 2010 ; received in revised form 29 September 2010; accepted 2 October 2010

KEYWORDSOrnithinedecarboxylase;Metarhiziumanisopliae;Biocontrol

AbstractThe gene ODC1, which codes for the ornithine decarboxylase enzyme, was isolatedfrom the entomopathogenic fungus, Metarhizium anisopliae. The deduced aminoacid sequence predicted a protein of 447 amino acids with a molecular weight of49.3 kDa that contained the canonical motifs of ornithine decarboxylases. The ODC1cDNA sequence was expressed in Escherichia coli cells; radiometric enzyme assaysshowed that the purified recombinant protein had ornithine decarboxylase activity.The optimum pH of the purified Odc1 protein was 8.0—8.5, and the optimum reactiontemperature was 37 ◦C. The apparent Km for ornithine at a pyridoxal phosphate
concentration of 20 mM was 22 �M. The competitive inhibitor of ODC activity, 1,4-
Please cite this article in press as: Madrigal Pulido J, et al. Isolation, characterization and expression analysis ofthe ornithine decarboxylase gene (ODC1) of the entomopathogenic fungus, Metarhizium anisopliae. Microbiol Res(2011), doi:10.1016/j.micres.2010.10.002

diamino-2-butanone (DAB), at 0.25 mM inhibited 95% of ODC activity. The ODC1 mRNAshowed an increase at the beginning of appressorium formation in vitro. Duringthe M. anisopliae invasion process into Plutella xylostella larvae, the ODC1 mRNAshowed a discrete increase within the germinating spore and during appressorium

∗Corresponding author at: Departamento de Biología, Universidad de Guanajuato, Noria Alta s/n, Col. Noria Alta, CP 36050uanajuato, Gto., Mexico. Tel.: +52 4737320006x8160; fax: +52 4737320006x8153.

E-Mails: [email protected], [email protected] (A. González Hernandez).

944-5013/$ – see front matter © 2010 Elsevier GmbH. All rights reserved.oi:10.1016/j.micres.2010.10.002

dx.doi.org/10.1016/j.micres.2010.10.002


mailto:[email protected]

mailto:[email protected]


2 J. Madrigal Pulido et al.

formation. The second expression peak was higher and prolonged during the invasion. The ODC1 gene complements the polyamine auxotrophy ofnull mutant.All rights reserved.

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Todftm(tebtflasks at 28 C and were shaken at 160 rpm. Themycelia were then collected by filtration.

The following strains of Y. lipolytica were used:

and death of the insectYarrowia lipolytica odc© 2010 Elsevier GmbH.

Introduction

Polyamines are small polycation molecules essen-tial for growth and differentiation in prokaryoticand eukaryotic cells (Heby 1981; Tabor and Tabor1985; Ruiz-Herrera 1994; Herrero et al. 1999;Coffino 2000). These molecules have a key rolein a variety of processes, such as nucleic acidpackaging, DNA replication, transcription, transla-tion, membrane stabilization, the functioning ofcertain ion channels and resistance to oxidativestress (Pegg and McCann 1982; Tabor and Tabor1985; Williams 1997; Cohen 1998). Polyamines mayhave additional roles in the protection of DNAfrom enzymatic degradation, X-ray irradiation andmechanical shearing (Jimenez-Bremont and Ruiz-Herrera 2008; Shah and Swiatlo 2008). The first stepin the biosynthesis of polyamines is the decarboxy-lation of ornithine by l-ornithine decarboxylase(ODC) to form putrescine, one of the most highlyregulated enzymatic reactions in eukaryotic sys-tems (Tabor and Tabor 1984; Pegg 2006). In plantsand some microorganisms, a second mechanism forthe synthesis of polyamines exists, which involvesthe action of arginine decarboxylase (ADC) to pro-duce agmatine, which can also be converted intoputrescine (Tabor and Tabor 1984).

In fungi, polyamine metabolism plays anessential role during differentiation. There areseveral-fold increases in activity of ODC andpolyamine levels preceding each differentiationevent (Martinez-Pacheco et al. 1989; Ruiz-Herrera1994; Jimenez-Bremont et al. 2001). The impor-tance of ODC activity during fungal dimorphictransition was determined by the addition of 1,4-diamino-2-butanone (DAB), a competitive inhibitorof ODC activity, which blocks the yeast tohyphae transition in different fungi such as Mucorrouxii (Martinez-Pacheco et al. 1989), Paracoc-cidioides brasiliensis (Nino-Vega et al. 2004),Coccidioides immitis (Guevara-Olvera et al. 2000),Yarrowia lipolytica (Guevara-Olvera et al. 1993;Jimenez-Bremont et al. 2001), Ustilago maydis

Please cite this article in press as: Madrigal Pulido J, et al.the ornithine decarboxylase gene (ODC1) of the entomopat(2011), doi:10.1016/j.micres.2010.10.002

(Guevara-Olvera et al. 1997) and Candida albicans(Herrero et al. 1999).

Metarhizium anisopliae is an entomopathogenicfungus used for the biological control of a wide

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ange of insect pests. The different stages of theathogenic process include adhesion of conidiao the surface of the insect, germination, pen-tration, invasive growth and conidiation. Thesetages constitute the life cycle of the fungusn insecta (Clarkson and Charnley 1996; Pedrinit al. 2007). In each of these stages, the fun-us undergoes differentiation events in whichDC activity may be involved. In previous work,e isolated a conserved fragment of a puta-

ive ODC1 gene from M. anisopliae; it showedigh homology to genes encoding ornithine decar-oxylase enzymes (Jimenez-Bremont et al. 2006).n this study we describe the isolation of theene and its expression analysis during in vitroppressorium formation and during the infec-ion process of its host, Plutella xylostella. TheDC1 gene was expressed in Escherichia coli,nd biochemical characterization of the recombi-ant protein was performed. Moreover, the abilityf the ODC1 gene to complement the null odcutant of the dimorphic yeast, Y. lipolytica, was

ested.

aterials and methods

trains and growth conditions

he M. anisopliae Ma10 (CNRCB MaPL10), wasbtained from the Centro Nacional de Referenciae Control Biológico, and was originally isolatedrom Geraeus senilis (Coleoptera: Curculionidae) inhe state of Colima, Mexico. To obtain conidia orycelia, the fungus was grown in minimal media

MM) or in Saboraud dextrose media (SDM), respec-ively, as previously described (Morales Hernandezt al. 2010). The harvested conidia were washedy centrifugation and suspended in sterile 0.1% Tri-on X-100 solution. The cultures were incubated in

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Isolation, characterization and expression analysis ofhogenic fungus, Metarhizium anisopliae. Microbiol Res

JOD (Mat A, leu 2-270, ura3-302, �odc) (Jimenez-remont et al. 2001); FJCO (Mat A, leu 2-270,odc/pFJ4 (YlODC+)), which was derived fromJOD, complemented with the wild type YlODC


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solation, characterization and expression analysis

ene, and up-regulated by the metallothioneinMETI-II) promoter (Jimenez-Bremont et al. 2001);nd YMaODC1, which was derived from FJOD, com-lemented with the cDNA of the MaODC1 gene,nd up-regulated by the metallothionein promoterMETI-II). The strains were maintained on slants ofEPD medium (1% yeast extract, 2% peptone, 2%extrose, and 2% agar) and propagated in liquidEPD medium or in minimal medium (0.67% yeastitrogen base without amino acids (DIFCO), 1% dex-rose, supplemented with uracil and or leucine50 �g mL−1)). In some experiments, the minimaledium was supplemented with 1 mM or 5 mM ofutrescine as required.

E. coli: The DH5� strain (Invitrogen, Carlsbad,A, USA) was used for DNA manipulations and trans-ormations. It was maintained in Luria—Bertaniedium at 37 ◦C (Sambrook and Russell 2001).mpicillin (100 �g mL−1) was added as required.

E. coli BL21(DE3)pLysS (Invitrogen) was utilizedor heterologous expression of the ODC1 cDNA.

ucleic acid isolation and MaODC1 geneloning

ungal genomic DNA was isolated from myceliarown for 24 h in SDM. DNA was extracted by fric-ion following standard protocols (Sambrook andussell 2001). Total RNA was extracted from cellsf either M. anisopliae or Y. lipolytica grown in dif-erent culture media or from P. xylostella larvaereviously inoculated with conidia of M. anisopliae,sing TRIzol® Reagent (Invitrogen) according tohe manufacturer’s instructions. All samples wereNase-treated using RNase-Free DNase (Promegao., Madison, WI, USA) according to the man-facturer’s instructions. The concentration andurity of both total RNA and DNA were deter-ined by the ratio of absorbencies at 260 and

80 nm. A pair of specific primers, invODCMa1 (5′-ACCTGCTCAATCTCCCCTTT-3′) and invODCMa2 (5′-TTACTATGTCTCCACGGCGTTCA-3′), was designedccording to the sequence of a fragment encod-ng a partial ornithine decarboxylase gene from M.nisopliae (GenBank AY325884) (Jimenez-Bremontt al. 2006). This was used in iPCR to amplify a frag-ent containing the complete open reading frame

ORF) from M. anisopliae according to a protocolreviously described (Ochman et al. 1988). Theeaction mixture contained total DNA, each primer

Please cite this article in press as: Madrigal Pulido J, et althe ornithine decarboxylase gene (ODC1) of the entomopat(2011), doi:10.1016/j.micres.2010.10.002

nd PCR SuperMix High Fidelity (Invitrogen). Themplification product was cloned into the pCR®2.1-OPO® vector (Invitrogen) and sequenced. Bothtrands of the nucleotide sequence of the cloneere generated using universal primers and spe-

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ific primers based on the ODC1 genomic sequenceeposited into the EMBL database (accession num-er FN689467). The DNA sequence was analyzedsing the Lasergene 8 program (DNASTAR Inc.,adison, WI, USA), and its identity was confirmedy comparison to published ornithine decarboxy-ase sequences in the database (www.ebi.ac.k).

outhern blot and northern blot analysis

or hybridization analysis, restricted chromoso-al DNA or total RNA was prepared. Southernlot or northern blot hybridization was per-ormed following standard procedures (Sambrooknd Russell 2001). A radioactively labeled DNArobe from ODC1 gene was used as a homologousrobe.

xpression analysis of the ODC1 gene byT-PCR

he in vitro production of appressoria was per-ormed according to the protocol previouslyescribed (St Leger et al. 1989) with some modifi-ations. Conidia (1 × 107 mL−1) suspended in 10 mL.01% Triton X-100 were inoculated in 10 mL of YEedia (0.125% yeast extract) in 8.5 cm polystyreneishes (Falcon). The cultures were incubated at8 ◦C without shaking. At specific times (4, 8,2, 18, 22, 24 and 28 h), the fungal materialas collected by scraping, frozen immediately in

iquid nitrogen and stored at −70 ◦C until use. Theaterial was used for RT-PCR analysis of the ODC1

ene, and AJ274118 (loading control) was used toompare transcript abundance. RT-PCR was per-ormed using the SuperScriptTM III One-step RT-PCRystem with the Platinum® Taq DNA Polymeraseit (Invitrogen) according to the manufacturer’secommendations. The reaction employed specificrimers: oliODC1 (5′-AAAGGGGAGAATGAGCAGGTG-′) and oliODC2 (5′-TGAACGCCGTGGAGACATAGTA-C-3′) for the ODC1 gene and conest100-sen5′-GGGGGTTTGATTATGTGGTTGGTATTAGCA-3′)nd conest100-anti-(5′-TAACTTCAGTCGTGCGTGC-ATTTCTAC-3′) for the AJ274118 gene, the latterf which was used as a constitutive expressionontrol (Freimoser et al. 2005; Morales Hernandezt al. 2010). Both oligomer pairs were used simul-aneously in the same reaction tube. The reverse

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. Isolation, characterization and expression analysis ofhogenic fungus, Metarhizium anisopliae. Microbiol Res

ranscription reactions were carried out at 58 Cor 30 min, and DNA amplifications were then car-ied out in a Gene AmpPCR System 9700 (Appliediosystems). Aliquots of the RT-PCR productsere analyzed on 3% agarose gels (UltraPureTM


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Agarose-1000, Invitrogen). Expression results werequantified by densitometric analysis (Quantity Oneversion 4.1.1, Bio-Rad).

In vivo expression of the ODC1 gene frominfected larvae (P. xylostella) by RT-PCRanalysis

The in vivo expression analysis of the ODC1 genewas performed according to the protocol previ-ously described (Morales Hernandez et al. 2010).Third-instar P. xylostella larvae were sprayed witha conidial suspension from a Potter spray tower(Potter-Precision Laboratory Spray Tower, BurkardScientific, Uxbridge, Middlesex, UK). The larvaewere fed with a leaf disk of Brassica oleraceaand incubated at 25 ◦C. Every 48 h the leaf diskwas changed. Different stages of infection weremonitored over an 8-day period. The stages ofinfection were categorized as previously described(Morales Hernandez et al. 2010): (1) 0 (conidia)to 24 h post-infection; (2) invasion of the infectedinsect (24—48 h); (3) death (48—72 h); (4) growth ofmycelia on the surface of the cadaver (72—144 h);and (5) conidial growth on the surface of thecadaver (+144 h). At each time point in the observedinfection, 10—40 larvae were collected and storedat −70 ◦C until RNA extraction. Upon death, conidiafrom infected insect cadavers were collected withsterile 0.1% Triton X-100 solution. From a parallelcontrol experiment, larvae treated only with 0.1%Triton X-100 solution were collected. RT-PCR analy-sis of the ODC1 gene and AJ274118 (loading control)was performed to compare transcript abundance.RT-PCR was performed using the SuperScriptTM

III One-step RT-PCR System with the Platinum®

Taq DNA Polymerase kit (Invitrogen) accordingto the manufacturer’s recommendations and aspreviously described (Morales Hernandez et al.2010).

Expression of the ODC1 cDNA in E. coli cells

Expression of the M. anisopliae ODC1 cDNA inE. coli BL21(DE3)pLysS (Invitrogen) was accom-plished by sub-cloning the ODC1 cDNA into apRSETB expression vector (Invitrogen). The ODC1cDNA was obtained from M. anisopliae Ma10amplified with two primers, ODCBamHIfd (5′-GGATCCGATGGTTATGGCAACGGCTATC-3′) which


contains a BamHI restriction site (underlined) andODCEcoRIrev (5′-GAATTCCTACATGTTCAAAAGAAA-3′) containing an EcoRI restriction site (underlined),in a reaction mixture with PCR SuperMix HighFidelity (Invitrogen). The amplification product

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J. Madrigal Pulido et al.

as digested with both BamHI and EcoRI restrictionnzymes and ligated to the BamHI/EcoRI-digestedRSETB DNA plasmid, resulting in the recombinantlasmid pJCTG424. E. coli BL21(DE3)pLysS cellsere transformed with plasmid pJCTG424, andolony selection occurred in LB medium supple-ented with ampicillin and chloramphenicol. Fordc1p expression and purification, bacterial cellsere grown to an OD600 of 0.4 at 37 ◦C in LB

iquid medium supplemented with 100 �g mL−1

mpicillin, 35 �g mL−1 chloramphenicol and 1 mMPTG for 6 h at 37 ◦C. Induced cells were col-ected by centrifugation (8000 × g for 5 min at◦C) and suspended in binding buffer (20 mMhosphate, 500 mM NaCl, 20 mM imidazole with.2 mg mL−1 lysozyme, and 20 �g mL−1 DNAse).ollected cells were lysed in a Tyssue Lysser IIQiagen, Mexico) following the manufacturer’secommendations. Cell debris was separated byentrifugation (40,000 × g for 30 min at 4 ◦C), andhe supernatant was passed through a 1 mL HisTrapF column (GE Healthcare, Piscataway, NJ, USA)quilibrated with 50 mM phosphate buffer at pH 7.5nd 20 mM imidazole according to the directionsf the supplier. The proteins bound to the columnere eluted with a gradient of 20—500 mM imida-ole. Odc1p was detected at 300 mM imidazole.ractions were saved at −70 ◦C until their use inrnithine decarboxylase assays. ODC activity wasetermined by a radiometric assay of ODC based onhe measurement of 14CO2 released from carboxyl-adiolabeled ornithine (Calvo-Mendez et al.987).

ffect of pH and temperature on ODCctivity

he effect of pH on ODC activity of Odc1p waseasured using the following buffers: 100 mM phos-hate buffer at pH 6.5, 7.2 and 8.0; and 100 mMris—HCl at pH 8.5 and 9.0. The effect of tem-erature on ODC activity was measured at 24, 28,7, 42 and 48 ◦C using 100 mM Tris—HCl buffer atH 8.5.

ffect of inhibitors on ODC activity

he effect of 1,4-diamino-2-butanone (DAB) andutrescine was assayed. First, 0.50 �g of puri-ed recombinant Odc1p protein was incubated at

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7 C with 100 mM Tris—HCl pH 8.5 in the pres-nce of 5, 12.5, 25, or 75 �M DAB or putrescine.ext, ODC activity was assayed. To analyze the

nhibitory effect of DAB during appressorium for-ation, suspensions of 5 × 106 conidia in 150 �L


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CACGCAAGTTCC-3′) and oliYLZNC1rev (5′-AGGCG-GTGGTGGAGCTGGATAGCCATAG-3′) to amplifytheYlZNC1 gene (as a constitutive control in thepresence of copper) as described above. The RT-PCR reaction was developed using SuperScriptTM IIIOne-Step RT-PCR with Platinum® Taq (Invitrogen)as described above. Functionality of the ODC1gene was determined by a radiometric assay ofODC. In order to test the prototrophy of thetransformants to putrescine and their abilityto show yeast—hyphae differentiation, it wasnecessary to decrease the levels of endogenousintracellular polyamines as follows: 100 �L of cellswere transferred from complete medium (YEPD)to synthetic minimal medium without amino acids(SC) supplemented with either 0.002% leucine (forFJCO and YMaODC1 strains) or 0.002% leucine and0.002% uracil (for FJOD strain). The cultures wereincubated for 15 h at 28 ◦C, and the cells (OD6001.7) were then transferred to fresh medium forthree successive periods of growth. After eachperiod of growth, cells dilutions of 1:10, 1:100 and1:1000 were made and inoculated on the respectiveagar SC medium and incubated for 3 days at 28 ◦C.The morphology of the Y. lipolytica colonies wasfollowed by stereoscopic observation, and the cellswere followed by microscopic observation countingat least 200 cells.

Fig. 1. Southern blot analysis. Genomic DNA from M.


f 0.125% yeast extract (YE) were prepared, andAB was added 25, 50, 75 and 100 mM at eitherime 0 or 12 h. The cultures were placed in Petriishes and incubated at 37 ◦C. The cells werebserved under the microscope to follow appres-orium formation at 20 and 22 h. The percentagesf appressorium formation were quantified in ateast 200 cells and repeated in three independentssays.

usion of ODC1 ORF with theetallothionein promoter, METl-II, of Y.

ipolytica

1362-bp fragment of the MaODC1 cDNA wasmplified by PCR using the primers oliODCBamfd5′-CACTAGGGATCCATGGTTATGGCAACGGCTAT-3′),hich contains a BamHI restriction site (under-

ined), and ORFODC2 (5′-GGTCTAGACTACATGTTC-AA-3′). An 870-bp fragment, including the′-end proximal coding region (26 codons)nd terminator region of the ODC1 gene, wasmplified by PCR using two primers, oliODCfd25′-TGCTCGGCCACCCAGTTCAAT-3′) and oliODCrev35′-ACGGATCCGTTTGGGTTGGCATTTTTGAGC-3′)hich contains a BamHI restriction site (under-

ined). Both PCR products were fused by alignmentnd extension. This 2178-bp fusion product wassed as a template; it was amplified by PCR usinghe two primers, oliODCBamfd and oliODCrev3 andloned into pCR2.1TOPO (pJCTG407). Sequencingas used to corroborate the correct fusion. The178-bp BamHI fragment was recovered fromJCTG407 and ligated into the BamHI site of theSG572 plasmid (Jimenez-Bremont et al. 2001).he correct orientation of ODC1 was tested byhe restriction pattern of the pJCTG413 plasmid,hich contains up-regulated ODC1 cDNA controlledy the METI-II promoter.

ransformation of a Y. lipolytica nullutant with the MaODC1 gene

he strain FJOD (�odc) was transformed using theithium acetate procedure (Chen et al. 1997) andhe pJCTG413 plasmid; the transformants wereecovered and maintained in minimal mediumithout uracil (2% dextrose, 0.67% Yeast Nitrogenase without amino acids, 0.002% of leucine and% agar). The transformants obtained were grown


n the presence of increasing concentrations ofopper. The expression of the ODC1 gene was mon-tored by RT-PCR. We used the primers oliODC1 andliODC2 to amplify the ODC1 gene, and we usedhe primers oliYlZNC1fd (5′-ATGTCCTCCACCAAAC-

anisopliae was digested with EcoRI, lane 1, or HindIII,lane 2, separated by agarose gel electrophoresis (A);transferred to a Hybond-N membrane and probed witha labeled DNA fragment of the ODC1 gene generated byPCR (B).




Fig. 2. Nucleotide and deduced amino acid sequence of the MaODC1 gene. The putative TATA and CAAT boxes areindicated by a square. The putative STREs, cccct, are indicated in boldface. The beginning and the end of the intronare indicated in boldface and underlined. The transcription termination motifs are indicated by a discontinuous square.The characteristic ODC motifs are shadowed, including the pyridoxal phosphate-binding site and the decarboxylasesfamily 2 signature 2. Finally, the region corresponding to the conserved ornithine decarboxylase catalytic domain isalso shadowed.


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loning of the MaODC1 gene and sequencenalysis

n previous work, a fragment of the MaODC1ene has been described (Jimenez-Bremont et al.006). In this work, we isolated the completeene sequence by inverse PCR (Ochman et al.988). In order to select the restriction enzymeecessary to perform this particular method, weerformed a Southern blot analysis. This techniquehows that the ODC1 gene exists as a unique copyn the M. anisopliae genome (Fig. 1). We alsoelected the restriction enzyme, HindIII, for com-lete gene isolation. The genomic DNA was digestedith HindIII and circularized. This was used astemplate in the iPCR reaction with a pair of

pecific primers arranged in a back-to-back ori-ntation. A DNA fragment of 2100-bp, containingoth the 5′- and 3′-ends of the ODC1 gene, wasmplified. The assembled sequence of the iPCRroduct, together with the 800-bp fragment previ-usly reported, was corroborated by isolation andequencing of the complete gene from genomic DNAnd cDNA. The analysis of this sequence revealedhe presence of a 2769-bp ODC1 gene with an openeading frame (ORF) of 1408-bp, interrupted byne intron of 65-bp (Fig. 2). The existence andocation of the intron was corroborated by align-ent of the genomic and cDNA sequences from

he Ma10 strain. A putative TATA element in the′ untranslatable region of the ODC1 gene wasound 182 bp upstream from the proposed initi-tion codon. A possible CAAT box was located62 bp upstream of the start codon. The 5′ UTRontains three putative STREs (stress responsivelements CCCCT). Meanwhile, sequences matchedo the transcription termination motifs, TAG, TAGTnd TTT, were found 103, 116 and 137 bp down-tream from the termination codon, respectively.orthern blot analysis revealed a hybridizationignal approximately 1.8-kb long (Fig. 3). Theequence predicted a protein of 447 amino acidsith a molecular weight of 49.3 kDa and an iso-lectric point of 5.26. A PROSITE database searchdentified canonical motifs of ornithine decar-oxylases, the sequence PFYAVKCNPDPKVLRLLAELGas located between amino acid residues 90—110.his is thought to be the pyridoxal phosphateinding site (Fig. 2). The sequence, AAEFGFTM-TLDVGGGFC, which is located between amino


cids 248 and 265 and contains three glycineesidues, is thought to be part of the proposedubstrate-binding region (Fig. 2). A cysteine isocated within the WGPTCDGID consensus sequence

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lectrophoresis, transferred to a Hybond-N membranend probed with a labeled DNA fragment of the ODC1ene generated by PCR.

t position 385 in the catalytic site (Poulin et al.992) (Fig. 2). BLAST analysis of MaOdc1p usinghe EMBL database (www.ebi.ac.uk) shows the high-st homology of this gene with the ODC fromhe plant symbiont, Epichloe festucae (86%, acces-ion number A8B3P2), and lowest homology withrnithine decarboxylases from the following fil-mentous fungi: Spe1p from Neurospora crassa69%, accession number M68970), Odc1p from Tape-ia yallundae (65%, accession number Q96X19),dc1p from Y. lipolytica (54%, accession number8WZM1), and Spe1p from Saccharomyces cere-isiae (51%, accession number P08432). Genes ofow homology from other fungi also exist.

xpression of the MaODC1 cDNA in E. coli

o demonstrate that the ODC1 gene codes for anrnithine decarboxylase protein, ODC1 cDNA wasxpressed in E. coli cells using the pRSETB vector.. coli BL21 cells were transformed by insertinghe pJCTG424 plasmid with the ODC1 cDNA intohe pRSETB vector. After recombinant Odc1p purifi-ation on a Ni+-agarose column, the recombinantrotein (Fig. 4A and B) showed ornithine decar-oxylase activity by radiometric enzyme assays.eanwhile ODC specific activity in E. coli crudextract was 6.7 ± 2.9 pmol CO2 h−1 �g protein−1,he ODC specific activity in crude extracts from. coli cells transformed with plasmid pJCTG424as 351.2 ± 21.6 pmol CO2 h−1 �g protein−1; and in

he purified recombinant MaOdc1p the ODC specificctivity was 484.6 ± 5.1 pmol CO2 h−1 �g protein−1.his result confirms the predicted amino acidequence analysis and indicates that ODC1 codesor an ornithine decarboxylase. The purified pro-ein showed a molecular weight of approximately


0 kDa on SDS-PAGE, which is similar to theredicted MW of 49.3 kDa. The optimum pHas 8.0—8.5 (data not shown), and the opti-um temperature of the enzyme reaction was



8

Fig. 4. Expression and purification of the recombinantOdc1p. (A) SDS-PAGE of crude extracts from E. coli BL21expressing the recombinant Odc1p; M, molecular weightstandards; lane 1, cells without induction; lane 2, cellsinduced for 2 h with 1 mM IPTG; lane 3, cells inducedfor 4 h with 1 mM IPTG; lane 4, cells induced for 6 hwith 1 mM IPTG. (B) SDS-PAGE from different purifica-tion steps. M, molecular weight standards; lane 1, crudeextract from cells induced for 6 h with 1 mM IPTG; lane 2,protein elution with 20 mM imidazole; lane 3, protein elu-tion with 60 mM imidazole; lane 4, protein elution with100 mM imidazole; lane 5, protein elution with 300 mMimidazole; lane 6, protein elution with 500 mM imida-zole. SDS-PAGE gels were stained with Coomassie BrilliantBlue-250.

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EP

Tawfmdawudsimultaneously amplified ODC1 and AJ274118 (load-ing control) in the same reaction tube (Fig. 6A).An ODC1 amplification product of 520-bp and anAJ274118 amplification product of 100-bp were

37 ◦C (data not shown). The apparent Km forornithine at a pyridoxal phosphate concentrationof 20 mM is 22 �M, which is lower than otherODC expressed in E. coli. The Km for Odc1pfrom S. cerevisiae was 131 �m (Fonzi and Sypherd1985) and 280 �M for Odcp from Trypanosomabrucei (Phillips et al. 1988). The competitiveinhibitor of ODC activity, 1,4-diamino-2-butanone(DAB) at 0.25 mM, inhibited 95% of ODC activ-ity. These results indicate that the ODC1 geneeffectively codifies for an ornithine decarboxylase


enzyme. o


xpression of the MaODC1 gene duringppressorium formation

n M. anisopliae, the early invasion of its host isharacterized by discrete differentiation events,nd conidia germination is followed by appresso-ium formation (adhesion structure). To determinef ODC1 expression correlates with these differen-iation processes, we evaluated ODC1 mRNA levelsy RT-PCR analysis during appressorium formationn vitro. Appressorium formation was developedccording to a protocol previously described (Steger et al. 1989). The conidia were inoculated on.125% YE on Petri dishes and incubated withoutgitation for 24 h. Samples were taken at differ-nt time points, and RT-PCR analysis was performedccording to Morales Hernandez et al. (2010). ODC1RNA increased at the beginning of germination

nd during appressorium formation (Fig. 5). To eval-ate if this increase of ODC1 expression is importantn the differentiation process required to producedhesion structures, we performed an assay ofppressorium formation in vitro. First, we addedhe competitive inhibitor, DAB, at different con-entrations (25, 50, 75 and 100 mM) at the startf the assay or 12 h after start. We observed thatdding DAB initially inhibited appressorium forma-ion up to 60% without affecting the germinationrocess. The threshold for affect on germinations 150 mM. However, when DAB was added to theulture medium 12 h later, there was a much lowernhibition of only 10%. Further, addition of 5 mMutrescine restored appressorium formation abil-ty, indicating that activity of the ODC is not onlymportant for this differentiation process but alsoequired during specific time points.

xpression of ODC1 gene during invasion of. xylostella

he above experiment was performed in vitro. Tonalyze ODC1 expression in insecta, P. xylostellaas infected with M. anisopliae conidia, and dif-

erent stages of infection in the larvae wereonitored. Observations were made over an 8-ay period. The infected larvae were collectedt different times post-inoculation, and the RNAas extracted. It was used to synthesize cDNAsed as templates for RT-PCR assays, as previouslyescribed (Morales Hernandez et al. 2010). We


bserved through the entire course of insect infec-


Isolation, characterization and expression analysis of th

Fig. 5. Expression of the ODC1 gene during in vitroappresorium formation. For the production in vitro ofappressoria, conidia of M. anisopliae Ma10 were inoc-ulated in 10 mL of YE media (0.125% yeast extract) in8.5 cm polystyrene dishes. The cultures were incubatedat 28 ◦C without shaking; at specific times, the fungalmaterial was collected and employed for RT-PCR analysisof the ODC1 gene to compare transcript abundances. (A)Gel electrophoresis. Lane M, molecular markers; lane 1,4 h; lane 2, 8 h; lane 3, 12 h; lane 4, 18 h; lane 5, 22 h; lane6, 24 h, lane 7, 28 h. (B) Relative RT-PCR analysis of ODC1and AJ274118 (load control). (C) Appresorium formationat 24 h, arrows show the appressoria. The results are rep-rR

toiHsDiIfei(

sapfRcxvo

Co

ItpvmtactpttYsctiPtl(iooas

tfdTaa(OIcwere capable of growing and differentiating in this

esentative from three independent RNA extractions andT-PCR analysis.

ion with M. anisopliae. Meanwhile the expressionf AJ4118 showed that it is constant throughout thenvasion process, as previously reported (Moralesernandez et al. 2010); the ODC1 gene expressionhowed fluctuations during the infection process.ensitometric analysis (Fig. 6B) showed a discrete

ncrease in the mRNA level within the first 24 h.n this time period, the spore germinates and dif-


erentiates to form the appressorium. The secondxpression peak was higher and prolonged dur-ng invasion (24—48 h) and death of the insect48—72 h), followed by a slow decrease in expres-

mstt


ion level 72 h post-inoculation. The densitometricnalysis showed that the ODC1 expression occursredominantly during invasive growth, when theungus moves inside the insect (Fig. 6B). The 370-bpT-PCR product amplified from uninfected insectsorresponds to non-specific amplification of the P.ylostella ODC gene. Fig. 6C shows P. xylostella lar-ae at different times after inoculation with conidiaf M. anisopliae.

omplementation of the odc null mutationf Y. lipolytica by the ODC1 gene

n fungi, ornithine decarboxylase genes are impor-ant during normal growth and differentiationrocesses. ODC1 gene expression analysis showedariation during appresorium formation and duringycelial growth inside P. xylostella. To prove that

he ODC1 gene isolated from M. anisopliae encodesfunctional ornithine decarboxylase, heterologous

omplementation of a strain of Y. lipolytica (FJOD)hat lacked the ODC gene was carried out. The com-lementation of the mutant was carried out usinghe pJCTG413 plasmid. The plasmid up-regulateshe ODC1 gene by the metallothionein promoter of. lipolytica (METI-II), which induces gene expres-ion in response to increasing concentrations ofopper sulfate in the medium. The independentransformant, YMaODC1, was selected because ofts ability to grow in SC medium without uracil.CR was used to confirm that the transformant con-ained the MaODC1 gene (data not shown). Two Y.ipolytica strains, YMAODC1 and the null odc mutantFJOD) were grown in the presence of increas-ng concentrations of copper sulfate. The levelsf MaODC1 gene expression were evaluated. Webserved increased MaODC1 mRNA in response ton increasing copper concentration, in YMAODC1train (Fig. 7).

To investigate if the ODC1 gene expressed inhe yeast Y. lipolytica (strain YMAODC1) produces aunctional ornithine decarboxylase capable of pro-ucing putrescine, we performed another assay.he polyamine pools were depleted in the cells,nd we assessed the ability of these cells to grownd differentiate from yeast to mycelium. FJODnull mutant) and FJCO (FJOD complemented by theDC1 gene from Y. lipolytica) were used as controls.

n Fig. 8, we show that the YMaODC1 strain, whichontains the MaODC1 gene, and the strain FJCO


edium without polyamines. In contrast, the FJODtrain, which is devoid of the ODC gene, was unableo grow and carry out the yeast-mycelium transi-ion in a medium without polyamines. In the latter



Fig. 6. In vivo expression analysis of the ODC1 gene during P. xylostella infection. (A) The RT-PCR analysis of ODC1and AJ274118 (load control) gene expression during progressive infection of third instar P. xylostella larvae with M.anisopliae Ma10. Lane M corresponds to DNA marker; lane 1 corresponds to RT-PCR from conidia; lanes 2—12 correspondto RT-PCR amplification products from total RNA of third instar P. xylostella larvae infected with M. anisopliae conidiaat different time points post-infection (lane 2 (4 h), lane 3 (8 h), lane 4 (12 h), lane 5 (24 h), lane 6 (48 h), lane 7(72 h), lane 8 (96 h), lane 9 (120 h), lane 10 (144 h), lane 11 (168 h), lane 12 (192 h)), lane 13 and 14 correspond toRT-PCR amplification products from total RNA of uninfected third instar P. xylostella larvae at 24 and 48 h. (B) Relativeexpression of ODC1 during invasion measured by densitometric analysis. (C) Morphology of the progression of the

postom t

tspCQnaibnaagar

infection in third instar P. xylostella larvae at different3 (120 h), and lane 4 (192 h). Results are represented frindependent infection assays.

case, only putrescine supplementation restored thewild-type phenotype. All the experiments aboveconfirmed that the ODC1 gene codes for a func-tional ornhitine decarboxylase protein.

Discussion

In this work, we isolated a genomic copy and thecDNA of the ODC1 gene from M. anisopliae Ma10,using an internal fragment of the ODC1 gene ofthis fungus previously reported (Jimenez-Bremontet al. 2006). Based on sequence identity of the


predicted protein, this gene encodes an ornithinedecarboxylase (ODC) highly homologous (86%) tothe ODC from the endophytic fungus, E. festucae(accession number A8B3P2). It is also homologousto the ODC from phytopathogenic fungi, such as

ibp

-infection time points: lane 1 (12 h), lane 2 (48 h), lanewo separate RNA extractions and RT-PCR analysis from

he ODC from Nectria haematococca (Fusariumolani) (78%, accession number C7YUY8), Magna-orthe grisea (71%, accession number A4R2T2),haetomium globosum (75%, accession number2HB10) and Phaeosphaeria nodorum (Septoriaodorum) (64%, accession number Q9UVG2). It islso homologous to the ODC of saprophytic fungi,ncluding N. crassa (69% identity, accession num-er P27121), Y. lipolytica (54% identity, accessionumber Q8WZM1) and S. cerevisiae (51% identity,ccession number P08432). Notably, phylogeneticnalysis of the ODC of the entomopathogenic fun-us M. anisopliae was closely related to fungi thatct as pathogens to other organisms in their envi-onments.


The predicted protein sequence indicated thatt contains typical domains of ornithine decar-oxylase, including the binding site for pyridoxalhosphate, a cofactor of the enzyme; the con-


Isolation, characterization and expression analysis of th

Fig. 7. Induction of the ODC1 gene by copper. Cellsfrom Y. lipolytica strains, YMaODC1 ((�odc/pJCTG413(MaODC1)) and FJOD (�odc), were grown for 16 h at28 ◦C in liquid minimal medium with the required aminoacids. Different CuSO4 concentrations were added, 3 hlater the cells were recovered and total RNA was iso-lated and used for RT-PCR analysis of the MaODC1 andYlZNC1 genes to compare transcript abundances. M, DNAmarkers; lane 1 (0.0 mM CuSO4); lane 2 (0.2 mM CuSO4);lane 3 (0.4 mM CuSO4); lane 4 (0.8 mM CuSO4); lane 5 (1.2 mM CuSO4); and lane 6 (2 mM CuSO4). (A) Y. lipolyt-ica strain, YMaODC1. (B) Y. lipolytica strain FJOD. (C)Relative RT-PCR analysis of MaODC1 and YlZNC1 (loadcontrol). Results are represented for two separate RNAee

secmbf(OPtPnrm(

Mo

tb(ds(z2to

teltitoyqTncittYomcecwt

ci(figiaaTHemttgf

xtractions and RT-PCR analysis from two independentxperiments.

erved cysteine within the catalytic site (Poulint al. 1992) and possible PEST sequences that areharacteristic of high-turnover proteins in mam-als (Rogers et al. 1986). These PEST regions haveeen described in several fungal ODC, such as ODCrom N. crassa (Williams et al. 1992), S. cerevisiaeFonzi and Sypherd 1987) and U. maydis (Guevara-lvera et al. 1997). ODC from other fungi, such as

. brasiliensis (Nino-Vega et al. 2004) and C. immi-is (Guevara-Olvera et al. 2000), do not have theseEST sequences. It is important to note that it isot clear whether fungal PEST regions play a similarole in the regulation of fungal ODC, because mam-alian PEST sequences regulate mammalian ODC


Hayashi and Murakami 1995).The expression of the cDNA of the ODC1 gene of

. anisopliae in E. coli facilitated the purificationf this protein. Biochemical analysis confirmed that

eggt


his protein is a PLP-dependent ornithine decar-oxylase, it is inhibited in vitro by its end productputrescine) or by the putrescine analogue, 1,4-iamino-2-butanone (DAB), like most ODCs in fungiuch as S. cerevisiae, N. crassa and A. nidulansTabor and Tabor 1985) or in the parasite proto-oan Entamoeba histolytica (Arteaga-Nieto et al.002). This suggests that polyamine levels regulatehe activity of this enzyme in vitro, as described inther fungi.

The complementation assays demonstrated thathe protein encoded by the MaODC1 gene andxpressed in the null mutant of the ODC gene of Y.ipolytica is functional. It fully complemented pro-otrophy and the fungus’s ability to differentiatetself from yeast to mycelium. These results suggesthat ODC activity can provide sufficient amountsf putrescine to support these two events in theeast, and, therefore, this protein was both ade-uately expressed and/or structurally competent.his result is in contrast with the ODC from S.odorum, which only partially complemented theonidiation process of A. nidulans, possibly due tonsufficient expression and/or structural incompe-ence of the protein (Bailey et al. 2000). In our case,he ODC1 gene was up-regulated by the promoter,lMTLII, which responds to copper. Indeed, webserved that the promoter responded as expected,odulating ODC1 gene expression in relation to

opper concentrations in the culture media. Inter-stingly, based on the complementation results, wean say that even basal expression of the ODC1 geneas sufficient to promote growth and differentia-

ion in Y. lipolytica.The ODC1 gene is expressed throughout the life

ycle of the fungus during invasion and growthn its host P. xylostella. During the first 24 hearly invasion), the conidia germinate and dif-erentiate to form the appressorium. This processs accompanied by a discrete increase in ODC1ene expression. This increase is seen again duringn vitro appressorium formation. In these in vitrossays, the inhibition of ODC activity by DAB causedreversible inhibition of appressorium formation.

his finding is congruent with those of Ruiz-errera (1994), who suggested that polyamines aressential in processes such as differentiation, ger-ination, sporulation and dimorphism in fungi. In

his particular case, the polyamines are necessaryo form the appressorium. Interestingly, the ODC1ene was more strongly induced during invasiveungal growth and death of the host than during


arly invasion. These data suggest that this fun-us requires more reduced expression of the ODCene for germination and appressorium formationhan for invasive growth. This result contrasts with



medmin

pFJ4

IaDirr(gtitratdiOfTtpent

Fig. 8. Morphology of Y. lipolytica strains grown in soliddilutions of the different Y. lipolytica strains were grown instrain in medium with 5 mM putrescine; (C) FJCO (�odc/

findings reported for U. maydis (Guevara-Olveraet al. 1997) and Y. lipolytica (Jimenez-Bremontet al. 2001), in which higher concentrations ofpolyamines are required for dimorphic transition (adifferentiation process), than for ensuring vegeta-tive growth. However, it cannot be excluded thatM. anisopliae could require higher concentrationsof polyamines for other differentiation processes,including conidiation.

The induction of the ODC1 gene expressionobserved during early invasion, appressorium for-mation in vitro, and invasive growth suggests thatthe ODC1 gene is transcriptionally regulated. Themolecular mechanism of this regulation is unknown.However, three STREs (stress responsive elements,CCCCT) have been observed within the ODC1 pro-moter, and they may be involved in this mechanism.STREs have been shown to mediate transcriptionalactivation in response to various stressors in S. cere-visiae (Siderius et al. 1997). Potentially, the STREsperform a similar function in M. anisopliae.

As shown in M. anisopliae, the inhibition of ODCinhibited appressorium formation without affectinggermination, as reported in Uromyces viciae-fabae


(Reitz et al. 1995). This structure has been asso-ciated with the ability of some fungi to infecttheir hosts, including Magnaporthe oryzae (Izawaet al. 2009) and M. grisea (Talbot et al. 1993).

aPre

ia. After depletion of polyamines, different cell cultureimal media at 28 ◦C for 3 days. (A) FJOD (�odc); (B) FJOD(YlODC+)); (D) YMaODC1 (�odc/pJCTG413 (MaODC1)).

nterestingly, in M. grisea, putrescine inhibitedppresorium formation but not the ODC inhibitor,FMO (Choi et al. 1998). The importance of ODC

n the pathogenic process was showed in S. nodo-um, in which an ODC mutant showed significantlyeduced virulence in wheat, its host organismBailey et al. 2000). In the hemibiotrophic fun-us, Colletotrichum higginsianum, disruption ofhe ODC gene also led to decreased pathogenic-ty (Huser et al. 2009). Obviously, it is interestingo study the role of the ODC1 gene in appresso-ium formation in relation to the virulence of M.nisopliae. In this fungi, it was reported that muta-ions that slowed the formation of the appressoriumecreased virulence (Fang et al. 2009). It would bemportant to demonstrate the involvement of theDC1 gene in each of the differentiation events the

ungus must undergo during its life cycle in insects.hus, null mutants and their complementation withhe gene controlled by a regulated promoter mayrovide information about the role of the gene inach of these events. In order to generate the ODC1ull mutant we generated a disruption cassette con-aining the bar gene of Streptomyces hygroscopicus


nd transformed M. anisopliae. After isolation andCR analysis of 120 transformants, we did not find aeplacement for the ODC1 gene. There are multiplexplanations for this finding, but the construction


I of th

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f an ODC1 gene-null mutant will help to establishts role in the virulence of this fungus.

cknowledgments

his work was supported by the Consejo Nacionale Ciencia y Tecnología (CONACyT), Consejo Estatale Ciencia y Tecnología del Estado de Guanaju-to (CONCyTEG), Secretaria de Educacion PublicaSEP), and the University of Guanajuato. Madrigalulido, J., Magana Martínez I., and Padilla Guerrero., were recipients of a fellowship from CONACyT,exico. Cacho Valadez B., received a fellowship

rom CONCyTEG, Mexico.

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Isolation, characterization and expression analysis of the ... · Isolation, characterization and expression analysis of the ornithine decarboxylase gene (ODC1)ofthe entomopathogenic