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Hydrogen peroxide formation in cacao tissues infected by the hemibiotrophic fungus Moniliophthora perniciosa

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Hydrogen peroxide formation in cacao tissues infected by the hemibiotrophic fungus Moniliophthora perniciosa
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  Research article Hydrogen peroxide formation in cacao tissues infected by the hemibiotrophicfungus  Moniliophthora perniciosa Cristiano Villela Dias a , b , Juliano Sales Mendes a , Anderson Carvalho dos Santos a ,Carlos Priminho Pirovani a , Abelmon da Silva Gesteira a , c , Fabienne Micheli a , d , * ,Karina Peres Gramacho e , John Hammerstone b , Paulo Mazzafera f  , Júlio Cézar de Mattos Cascardo a , 1 a UESC, DCB, Centro de Biotecnologia e Genética, Rodovia Ilhéus-Itabuna Km 16, 45662-900 Ilhéus-BA, Brazil b Mars Center for Cocoa Science, Barro Preto, Bahia, Brazil c EMBRAPA Mandioca e Fruticultura Tropical, Rua Embrapa, s/n  , 44380-000 Cruz das Almas-BA, Brazil d CIRAD, UMR DAP, Avenue Agropolis TA96/03, 34398 Montpellier Cedex 5, France e Cocoa Research Center, CEPLAC/CEPEC, 45600-970 Itabuna-BA, Brazil f  IB/UNICAMP, Campinas, São Paulo, Brazil a r t i c l e i n f o  Article history: Received 28 January 2011Accepted 10 May 2011Available online 18 May 2011 Keywords: Ascorbic acidHydrogen peroxideOxalic acidResistance Theobroma cacao Witches ’  broom disease a b s t r a c t In plant e pathogen interaction, the hydrogen peroxide (H 2 O 2 ) may play a dual role: its accumulationinhibits the growth of biotrophic pathogens, while it could help the infection/colonization process of plant by necrotrophic pathogens. One of the possible pathways of H 2 O 2  production involves oxalic acid(Oxa) degradation by apoplastic oxalate oxidase. Here, we analyzed the production of H 2 O 2 , the presenceof calcium oxalate (CaOx) crystals and the content of Oxa and ascorbic acid (Asa) e the main precursor of Oxa in plants  e  in susceptible and resistant cacao ( Theobroma cacao  L.) infected by the hemibiotrophicfungus  Moniliophthora perniciosa . We also quanti fi ed the transcript level of ascorbate peroxidase (Apx),germin-like oxalate oxidase (Glp) and dehydroascorbate reductase (Dhar) by RT-qPCR. We report that theCaOx crystal amount and the H 2 O 2  levels in the two varieties present distinct temporal and genotype-dependent patterns. Susceptible variety accumulated more CaOx crystals than the resistant one, andthe dissolution of these crystals occurred in the early infection steps and in the  fi nal stage of the diseasein the resistant and the susceptible variety, respectively. High expression of the Glp and accumulation of Oxa were observed in the resistant variety. The content of Asa increased in the inoculated susceptiblevariety, but remained constant in the resistant one. The susceptible variety presented reduced Dharexpression. The role of H 2 O 2  and its formation from Oxa via Apx and Glp in resistant and susceptiblevariety infected by  M. perniciosa  were discussed.   2011 Elsevier Masson SAS. All rights reserved. 1. Introduction Reactive oxygen species (ROS) are continuously produced inplants as a result of the aerobic metabolism. ROS are srcinatedfromtheexcitementof themolecularO 2 ,formingsimplemolecularoxygen ( 1 O 2 ), or from the transfer of one, two or three electrons tothe O 2 , leading to the formation of superoxide radical (O 2  ),hydrogen peroxide (H 2 O 2 ) or hydroxyl radical (OH  ), respectively.The fast increase of ROS levels in cells is called oxidative burst andconstitutes one of the fastest responses to pathogen attack. Thesemoleculescanbeharmfultothecell,butontheotherhandtheycanactivate defense pathways or metabolic responses to biotic andabiotic stresses [1]. Hydrogenperoxide, the chemically more stable ROS, is involved in a series of cell processes related toplant defenseto pathogens via the biosynthesis of other important signalingmolecules such as salicylic acid (SA), abcisic acid (ABA), jasmonicacid (JA), ethylene and Ca 2 þ [1]. However, depending on theplant e pathogeninteraction type, the H 2 O 2  mayplaya dual role; itsaccumulation inhibited the growth of biotrophic pathogens, while  Abbreviations:  ABA, abcisic acid; Apx, ascorbate peroxidase; Asa, ascorbic acid;CaOx, calcium oxalate; DAB, 3-3 0 diaminobenzidene; dai, day after inoculation;Dhar, dehydroascorbate reductase; Glp, germin-like oxalate oxidase protein fromcacao; hai, hour after inoculation; JA, jasmonic acid; Oxa, oxalic acid; OXO, oxalateoxidase from wheat; ROS, reactive oxygen species; SA, salicylic acid; Tub, tubuline. *  Corresponding author. UESC, DCB, Centro de Biotecnologia e Genética, RodoviaIlhéus-Itabuna Km 16, 45662-900 Ilhéus-BA, Brazil. Tel.: þ 557336805196; fax: þ 5573 36805226. E-mail address:  fabienne.micheli@cirad.fr (F. Micheli). 1 In memoriam. Contents lists available at ScienceDirect Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy 0981-9428/$  e  see front matter    2011 Elsevier Masson SAS. All rights reserved.doi:10.1016/j.plaphy.2011.05.004 Plant Physiology and Biochemistry 49 (2011) 917 e 922  it has been ignored by necrotrophic pathogens or reported asa helper during the infection/colonizationprocess of plant by theseones [2 e 5]. In the case of the hemibiotrophic fungus  Septoria tritici ,the accumulation of H 2 O 2  inhibited the infection during the initialstage (biotrophic phase) of the disease in wheat [6]. Hydrogen peroxide is produced in plants via two possible pathways: i)cell wall oxidases catalyze the oxidation of the NADH to NAD þ ,whichinturnenablesreductionofO 2 toO 2  .Then,thedismutationof O 2  produces O 2  and H 2 O 2  [7]; ii) apoplastic oxalate oxidase andamine oxidase have been proposed to generate H 2 O 2  [8,9].In 2007, Ceita et al. [10] analyzed the interaction betweena susceptible variety of cacao ( Theobroma cacao  L.; Catongovariety)and the hemibiotrophic fungus  Moniliophthora perniciosa . Duringthe time course disease, two distinct phases were observed, i)a biotrophic phase, inwhich the mycelia growsonly in the apoplastandpresentsmonokaryotichyphae;andii)asaprotrophicphase,inwhich the mycelia invades the cell, presenting dikaryotic hyphaeand formation of clamp connections. Atthe end of the saprotrophicphase, plant cell death occurs, followed by basidiocarp productionand releasing of spores. The authors also reported an increase of calcium oxalate (CaOx) crystals concomitant with the funguscolonization (biotrophic phase). During the necrotrophic phase, anincrease in H 2 O 2  level was observed, associated with the dissolu-tion of the CaOx crystals and the degradation of the generated freeoxalicacid(Oxa)bythegermin-likeoxalateoxidase(Glp).Althougha considerable knowledge on the whole fungus cycle and in -omicsof   T. cacao  has been accumulated in the last 10 years [11], little isknown about the cellular mechanisms of the resistantcacao e M. perniciosa  interaction.In this study, we analyzed the production of H 2 O 2  and thecontentoffreeOxaand ascorbic acid (Asa) e mainprecursorof Oxain plants [12,13]. We also quanti fi ed the expression of ascorbateperoxidase (Apx; which converts Asa into dehydroascorbate),dehydroascorbate reductase (Dhar; which uses dehydroascorbateand glutathione to form Asa and glutathione disul fi de) and Glpgenes in two cacao varieties (susceptible  vs  resistant) infected andnon infected with  M. perniciosa . We showed that the CaOx crystalamount and the H 2 O 2  levels in the two varieties present distincttemporalandgenotype-dependentpatterns.HighexpressionoftheGlpandaccumulationofOxawereobservedintheresistantvariety.The content of Asa increased in the inoculated susceptiblevariety, but remained constant in the resistant one. The susceptiblevarietypresentedreducedDharexpression.TheroleofH 2 O 2 anditsformation from Oxa via Apx and Glp in resistant and susceptiblevariety infected by  M. perniciosa  were discussed. 2. Results  2.1. Detection of H   2 O  2  and CaOx crystals in resistant and susceptible cacao varieties An increase of H 2 O 2  production was observed at early stages(24 hai, Fig. 1D; 48 and 72 hai, data not shown) in the resistantcacao variety inoculated with  M. perniciosa , particularly in thetissuesadjacent tothe vascular system(Fig.1D and H). Nopresenceof H 2 O 2  in the susceptible variety was observed (Fig.1C); at 24 haithe susceptible variety did notdiffer fromthe control inoculated by M. perniciosa  and in fi ltrated with water (Fig.1A and B).CaOx crystal were visible on sections of both varieties as brightwhite spots (arrows; Fig. 1E e G). However, at 72 hai, much moreCaOx crystals were observed in the susceptible variety (Fig. 1E) incomparison to the resistant one (Fig. 1G). The resistant varietypresented less CaOx crystals at 72 hai (Fig. 1G) than before theinoculation (0 hai, Fig. 1F), suggesting a dissolution of the CaOxcrystals after the plant infection. This CaOx crystal dissolution wasassociated with the production of H 2 O 2  in the resistant plants(Fig. 1H). At 24 hai, some remaining CaOx crystals were stillobserved, surrounded by adjacent cells containing H 2 O 2  (DABstaining; Fig. 1G). In the susceptible variety, no reduction of CaOxcrystal number was observed.  2.2. Quanti  fi cation of Oxa and Asa in resistant and susceptiblecacao varieties inoculated by M. perniciosa ThebasallevelofOxawasabout2.5timeshigherintheresistantvariety than in the susceptible one (Fig. 2A and B, 0 hai). BothinoculatedvarietiesshowedanincreaseofOxaat3dai.However,intheinoculated susceptible variety, the Oxa level decreasedat 15 dai(returned to basal level), then increased at 30 dai and  fi nallydecreased at 45 and 60 dai (with no signi fi cant difference with theplant control; Fig. 2A). In the inoculated resistant variety, the Oxa Fig. 1.  DAB staining for H 2 O 2  detection in susceptible and resistant cacao varieties.Apical meristems of infected (24 hai) susceptible (A) and resistant (B) varieties in fi l-trated with distilled water. DAB staining of susceptible (C) and resistant (D) cacaovarieties at 24 hai (100  ). Dark  fi eld micrography of susceptible (E) and resistant (G)varieties at 72 hai (20  ). Dark  fi eld micrography of resistant variety at 0 hai (F; 100  ).DAB staining of the resistant variety at 48 hai (H; 400  ). White arrows show CaOxcrystal surrounded by H 2 O 2 . C.V. Dias et al. / Plant Physiology and Biochemistry 49 (2011) 917  e 922 918  levelremainedhighat15dai,thendecreasedat30daiandincreasedat 45 and 60 dai (with no signi fi cant difference with the plantcontrol at 45 dai; Fig. 2B).The basal levelof Asawas about 1.5 times higher in the resistantvariety than in the susceptible one (Fig. 2C and D, 0 hai). In theinoculated susceptible variety, the Asa level increased from 3 to 30dai, decreased at 45 dai (without signi fi cant difference with theplant control) and then increased at 60 dai (same level than at 30dai; Fig. 2C). In the inoculated resistant plant, the Asa level at 3 and15 dai remained constant and equal to the basal level (withoutsigni fi cant difference with the plant control), then slightlydecreased at 30 dai and  fi nally slightly increased at 45 and 60 dai(without signi fi cant difference with the plant control for 45 and 60dai; Fig. 2D).  2.3. Transcript level of Apx, Glp and Dhar in susceptibleand resistant cacao varieties inoculated with M. perniciosa The expression patterns of Apx, Glp and Dhar were obtained inthe early stages of the infection (up to 72 hai). No signi fi cant vari-ation of the expression of the three genes studied was observed inthe inoculated susceptible variety (Fig. 3B). In the inoculatedresistant variety, a signi fi cant increase of Apx, Glp and Dhar tran-script levels was observed at 48 hai. At 72 hai, Apx transcripts levelwas still high (and signi fi cantly different from the one observedbefore inoculation; Fig. 3A, 0 hai) while Glp and Dhar transcriptlevels were as low as the ones observed at 0 hai (Fig. 3A). Theexpression of the Dharand Glp in the resistant variety followed thesame patterns as the Apx throughout the time course, witha correlation coef  fi cientof 0.96 and 0.95for Dhar/Apx and Glp/Apx,respectively (data not shown). 3. Discussion Hydrogen peroxide plays an important role in plants understress conditions as a signaling molecule which intermediatesa series of important cellular responses [1,14]. In the resistant cacao variety infected by  M. perniciosa , we observed a signi fi cant accu-mulation of H 2 O 2  throughout the stem apex vascular system in the fi rst 72 hai (Fig. 1D) while no H 2 O 2  accumulation was observed atthe early infection stages in the susceptible variety (Fig. 1C). Ina previous work, we showed a signi fi cant accumulation of H 2 O 2  inthe advanced stages of infection (45 dai) in the susceptible cacaoinfected by  M. perniciosa  facilitating the transition phase of thefungus from biotrophic to necrotrophic [10] and corroboratingother works involving necrotrophic or hemibiotrophic pathogens[2,5,6]. Forexample, the infection of   Arabidopsis thaliana  by Botrytiscinerea  led to the accumulation of H 2 O 2 , which killed the host cellsfacilitating the pathogen invasion [2].The successive sequence of production of Oxa (higher after 72hai), transcript level of Glp (higher at 48 hai), dissolution of CaOxcrystalsandH 2 O 2 accumulation(visibleupto72hai)intheresistantinfected variety suggested that the Glp participates in Oxa degra-dationleadingtotheformationofCO 2 andH 2 O 2 .Theseobservationscorroborate with previous results obtained for infected susceptiblecacao plants inwhich the dissolution of CaOxcrystals was followedby a massive accumulation of H 2 O 2  in infected tissues at the lateinfectiontimes[10].TogetherwiththeincreaseoftheGlptranscript level in the resistant inoculated cacao variety, we observed theincrease of the Apx and Dhar transcript levels (Fig. 3A). Apx usesH 2 O 2  to convert Asa into dehydroascorbate, and Dhar uses dehy-droascorbate to react with glutathione and form Asa and gluta-thione disul fi de. Therefore, at the same time, Asa was used assubstrateofApx,andwasregeneratedbyDharreaction,whichmayexplain the similar Asa contents found in the inoculated and non-inoculated resistant plantlets (Fig. 2D). Despite the existence of several reports describing the function of the CaOx in the freecalcium amount regulation process in plants [15,16], a novel and more dynamic role for this molecule was raised by the discovery of thewheatgermins[17],openingtootherpossibilitiesregardingthe functionalityon the formation and/ordissolution of CaOx inplants.The over-expression of an oxalate oxidase (OXO) in transgenic Fig. 2.  Quanti fi cation of free Asa and Oxa in resistant and susceptible cacao varieties. Oxa level on susceptible (A) and resistant (B) varieties. Asa level on susceptible (C) andresistant (D) varieties. Results are the average of three biological replicates    standard error. Different letters indicate signi fi cant statistical difference between samples by theDuncan test. Dark gray bars: plant control; light gray bars: plant inoculated com  M. perniciosa . C.V. Dias et al. / Plant Physiology and Biochemistry 49 (2011) 917  e 922  919  plants triggered the production of H 2 O 2  and defense responses bythe plant to the fungal infection [18 e 20]. It was also demonstratedtheinvolvementofOXOinthereductionof theprogressionofsomefungal diseases, conferring, in some cases, partial resistance [21].It was also reported the existence of a crystal matrix protein asso-ciated with CaOx precipitation [22]; our group recently identi fi edcacaoproteinsassociatedwithcrystalformationwhichareinvolvedin the formation/dissolution of CaOxcrystals (data not shown). Theparticipation of Asa in the generation of Oxa has been previouslyproposed [23], and since then, has stimulated the interest of the researchers [13]. By using Asa, Oxa, eritroascorbic acid, galactoseand glycolate labeled with  14 C, it has been demonstrated that Asaacts as precursor of Oxa and CaOx in idioblasts of   Pistia stratiotes ,and that glycolate is a very poor substrate for Oxa synthesis andCaOx generation. This work con fi rmed the results of Wheeler et al.[24],whichproposedthatthe L  -galactosemaybeakeyintermediatein the conversion of the  D -glucose to Asa in plants.In the susceptible cacao variety, we observed that the CaOxcrystals were not dissolved after the infection by  M. perniciosa (Fig. 1E). In addition an increase of Asa in the susceptible plants(Fig. 2C) was observed but without variation of Glp, Apx and Dhartranscripts levels (Fig. 3B) suggesting a relation between Asa andCaOxcontents.Ceitaetal.[10]alreadysuggestedtheparticipationof Asa in the interaction of cacao e M. perniciosa  as a pro-oxidantmolecule [25]. In  Medicago truncatula  mutants, a relationshipbetween Asa levels and the presence of CaOx crystals was demon-strated, stating that, in this plant model, Asa is the main source of Oxa [26]. Other studies showed that Oxa itself is involved in pro- grammedcelldeath(PCD)andisresponsiblefortheincreaseofROSin plant. However, when ROS production was inhibited, theapoptotic-like-cell death induced by Oxa does not occur [27].Accordingtothese fi ndingsGlp, CaOxandOxaare involvedin CaOxcrystaldissolutionandsubsequentH 2 O 2 formation,andimplicated,inonehand,inplantresistanceprocess(actingattheearlystagesof the interaction) or, on the other hand, contribute to pathogen lifecycle in the susceptible variety (at later stages of the disease). It isinteresting to note that even  M. perniciosa  also produces CaOxcrystals[28],theseprobablyarenotinvolvedintheearlieststagesof  disease, but contribute, in the susceptible variety, to the develop-mentofthepathogen.ThelowexpressionofDharinthesusceptiblevariety explains the differences found in the behavior of ascorbateand oxalate between the contrasting varieties. In conclusion, theincreased H 2 O 2  levels in the two cacao varieties present distincttemporal and functional patterns. Once produced at the beginningof the infection by the resistant variety, it contributes to the infec-tion control and to plant resistance. In advanced stages of thedisease in the susceptible variety, it promotes the pathogen devel-opment and the  fi nalization of its life cycle. 4. Material and methods 4.1. Plant material Plantletsof  T.cacao L.susceptible(Catongovariety)andresistant(TSH1188variety)to M. perniciosa werecultivatedingreenhouseatCEPEC/CEPLAC (Centro de Pesquisas do Cacau da Comissão Execu-tivadoPlanodaLavouraCacaueira,Ilhéus,BahiaState,Brasil)undernatural light and 90% of relative humidity as described by [29].Apical meristems of 6-week old-plantlets were inoculated by thespraying method [30] using a 10 6 .ml  1 basidiospore suspensionfrom the  M. perniciosa  Cp1441 CEPEC/CEPLAC strain. After inocu-lation, plantlets were kept during 24 h at 25   C   2   C in a water-saturated atmosphere to allow  M. perniciosa  spore germination,penetration and infection [30]. A spore viability testwas conducted in the humid chamber (25   C) 24 h after inoculation and comparedto spore viability obtained before inoculation. Apical meristems of three individual inoculated plantlets of each variety (biologicalreplicates) were collected before inoculation (0), at 24 h, 48 h, 72 hafter inoculation (hai) and at 15, 30, 45 and 60 days after inocula-tion (dai). In the time course disease, 24 e 72 hai corresponded tothe early stages of the infection, 30 dai to wilt and necrosis of theyoung leaves, and 45 dai to hypertrophy of the stem ( “ greenbroom ” ) and  fi rst microscopic necrosis symptoms [10]. At 60 dai, the infected plant presented macroscopic symptoms called  “ drybroom ”  and at 90 dai the plant was considered as completelynecrotic. Plantlets inoculated with water, and maintained andharvested in the same condition as the inoculated ones, were usedas control. Inoculated and control samples were frozen in liquidnitrogen, ground to a  fi ne powder and stored at  80   C. 4.2. Detection of H   2 O  2  by DAB staining methodand visualization of CaOx crystals Apical meristems of TSH 1188 and Catongo varieties harvestedat 24, 48 and 72 hai were immersed in 1 mg/ml of 3 e 3 0 Fig. 3.  Expression pattern of Apx, Glp and Dhar in cacao varieties inoculated with M. perniciosa . A. Resistant inoculated variety. B. Susceptible inoculated variety. Resultsare the average of three biological replicates  standard deviation.  t  -test was made bycomparison to 0 hai for each gene. ns: non signi fi cant (  p  >  0.05); **: signi fi cant at  p  <  0.001; *: signi fi cant at  p  <  0.05. Dark gray bars: Apx; light gray bars: Glp; whitebars: Dhar. C.V. Dias et al. / Plant Physiology and Biochemistry 49 (2011) 917  e 922 920  diaminobenzidene (DAB)  e  HCl, pH 3.8 (Sigma) and in fi ltratedundervacuumfor4h,asdescribedby[31].Sampleswereclearedinboiling ethanol (96%) for 20 min, then hand-sectioned with a razorblade, mounted in 50% glycerol and examined using an opticalmicroscope (Olympus CX41). Images were obtained using a digitalcamera Olympus C-7070. H 2 O 2  was visualized as a reddish-browncoloration. Control samples were immersed and in fi ltrated withdistilled water.For CaOx crystal visualization, apical meristems were cleared inboiling ethanol (96%) for 20 min, then hand-sectioned with a razorblade, mounted in 50% glycerol and examined using opticalmicroscope with dark  fi eld (Olympus CX41). Crystals were visual-ized asbrightspots at lowmagni fi cationandperfect facetedcrystalat high magni fi cation. 4.3. Quanti  fi cation of ascorbic and oxalic acid by HPLC  Organic acid extraction and quanti fi cation was carried out asdescribed by [13] with some modi fi cations. In 2 ml tubes, 6 mg of frozen material were mixed with 1 ml of extraction buffer (4 mMH 2 SO 4 , 5 mMDTT,PVPPadded toa concentration of 10 mg/ml). Thesample was vortexed for 15 min and centrifuged at 26,200  g   for20 min at room temperature. One hundred microliters of   fi lteredsupernatant (0.45  m m) was injected into the HPLC (ÄKTAbasic  ,GE-Health Care). Asa and Oxa were separated in a Bio-Rad AminexHPX-87H column (300  7.8 mm) at a fl ow rate of 0.7 ml/min, withdetection of Oxa at 210 nm (Rt  ¼  8 min) and Asa at 243 nm(Rt  ¼  10 min). The chromatogram peaks were integrated andanalyzed using the UNICORN   version 5.0 software. Oxa and Asaconcentrationsincacaosampleswereobtainedbycomparisonwitha calibration curve obtained by separation in the HPLC of knownamounts of pure standards (Sigma e Aldrich). Three biologicalreplicates(3meristemsforeachcacaovarietyatagivenpointofthetimecourse)andthreeexperimentalreplicates(3quanti fi cationsof Asa and Oxa for each meristem) were performed. The quanti fi ca-tion of Oxa e Asa were made concomitantly, in the same run andfrom the same sample to minimize bias in the analysis. Statisticalanalysis was performed with the software SASM  e  Agri whichtested the experiments as a completely randomised design. Anal-ysis of variance (ANOVA) was applied and for means comparisonDuncan ’ s test was employed, with a critical value of   P  ¼ 0.05. CaOxquanti fi cation was not performed since its formation/dissolutionpattern was well established by [10] in both varieties. 4.4. RNA extraction and RT-qPCR analyses Total RNA from frozen inoculated and control tissues was iso-lated as described by [32], and cleaned using the Rneasy Plant Mini kitasdescribedbythemanufacturer(Qiagen).TheRNAwastreatedwith DNaseRNase-free according tothe manufacturer(Fermentas),and then separated on 1% DEPC-treated agarose gel and stainedwith ethidium bromide to con fi rm RNA integrityand the estimatedRNA concentration. The  fi rst strand cDNA was synthesized in 10  m lfrom 1  m g of total RNA using the SuperScript II kit (Invitrogen)according tothe manufacturer ’ s instructions. RT-qPCR analysis wascarried out for the germin-like oxalate oxidase (Glp), ascorbateperoxidase (Apx) and dehydroascorbate reductase (Dhar) cacaogenes [29]. The tubulin gene (Tub) from cacao was used as refer- ence (endogenous control) in the qPCR experiments (Supplemen-tary material 1). The qPCR reaction containing 150 ng of cDNA,0.2 mM of each primer and 10  m l of Applied Mix was performedusingtheSyber GreenKit PCRmastermixontheApplied 7500 RealTime PCR (Applied Biosystems). The expression of each gene wasobtained in comparison to the expression level of the Tub gene(relative quanti fi cation/2 ∆∆ CT ). Cycling parameters were as follows:95   C for 10 min and 45 cycles of 95   C for 15 s and 60   C for 45 s.Automated gene expression analysis was performed using theequipment ’ s software (Applied Biosystems 7500 System SDS Soft-ware v1.3.1.21).  Acknowledgements This research was supported by the  Moniliophthora perniciosa Proteomic Network granted by the Financiadora de Estudos eProjetos (FINEP; project n  01.07.0074-00) and the Fundação deAmparo à Pesquisa da Bahia (FAPESB; project n  1431080017116).TheworkofC.VillelaDiasandJ.SalesMendeswassupportedbytheFAPESBandtheConselhoNacionaldeDesenvolvimentoCientí fi coeTecnológico do Brasil (CNPq). We thank Dr. Marcio G.C. Costa(UESC) for advises about statistical analysis and Dr. Claudia FortesFerreira (Embrapa) for critical reading of the manuscript. Wededicate this work to Julio Cezar de Mattos Cascardo: the seedsplanted in our minds will make his presence will always remainalive in our hearts.  Appendix. Supplementary data Supplementary data associated with this article can be found inthe online version, at doi:10.1016/j.plaphy.2011.05.004. References [1] L.-J. Quan, B. Zhang, W.-W. Shi, H.-Y. Li, Hydrogen peroxide in plants:a versatile molecule of the reactive oxygen species network, J. Integr. PlantBiol. 50 (2008) 2 e 18.[2] E.M. Govrin, A. Levine, The hypersensitive response facilitates plant infectionby the necrotrophic pathogen  Botrytis cinerea , Curr. Biol. 10 (2000) 751 e 757.[3] J.A.L. van Kan, Licensed to kill: the lifestyle of a necrotrophic plant pathogen,Trends Plant Sci. 11 (2006) 247 e 253.[4] N. Temme, P. 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