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E)E) GeneGene ExpressionExpression AnalysisAnalysis
?? RTRT--PCRPCR ofof samplessamples collectedcollected atat 33h,h, 99h,h, andand 2020dd waswas conductedconducted usingusing thethe
primersprimers forfor PeroxidasePeroxidase andand Mat/samMat/sam withwith ActinActin asas loadingloading controlcontrol..
?? AtAt 99h,h, 1010dd andand 2020dd ATTRXATTRX11,, PORA,PORA, PMonPRPMonPR1010,, PsylSupOxDis,PsylSupOxDis, VvinTh,VvinTh,
PsylApPsylAp11,, PLDAlfhaPLDAlfha11,, EE22FF11,, PP450450,, PRPR44,, SEPSEP11,, glychase,glychase, HSPHSP6060,, MSDMSD11,, PRPR55,,
PsylGAPDH,PsylGAPDH, PtaeMSynPtaeMSyn withwith 1818SS asas internalinternal controlcontrol..
D)D) cDNAcDNA synthesissynthesis andand RTRT--PCRPCR
?? cDNAcDNA synthesissynthesis (Applied(Applied Biosystems)Biosystems);;
?? RTRT--PCRPCR
MATERIALS AND METHODSMATERIALS AND METHODS
RESULTS AND DISCUSSIONRESULTS AND DISCUSSION
Acknowledgments:Acknowledgments:
Autoridade Florestal Nacional, Dr. HajmeAutoridade Florestal Nacional, Dr. Hajme
Kosaka, Dr, Takuya Aikawa, Dr. Manuel MotaKosaka, Dr, Takuya Aikawa, Dr. Manuel Mota
References:References:
((11)) Zhao,Zhao, BoBo GuangGuang etet alal,, 20082008,, PinePine WiltWilt Disease,Disease,
SpringerSpringer ISBNISBN:: 978978--44--431431--7565475654--55
((22)) Fraedrich,Fraedrich, BruceBruce RR..,, 19991999,, PinewoodPinewood NematodeNematode
(Pine(Pine--WiltWilt Disease)Disease):: TechnicalTechnical Report,Report, BartlettBartlett
TreeTree ResearchResearch Lab,Lab, CharlotteCharlotte NCNC
A) CulturesA) Cultures
ii.. Nematode culture and extractionNematode culture and extraction
?? BotrytisBotrytis cinereacinerea (Fig(Fig.. 11));;
?? VirulentVirulent (HF)(HF) oror nonnon--virulentvirulent isolatesisolates (C(C1414--55)) (Fig(Fig.. 22..));;
iiii.. PinePine seedlingsseedlings
?? EightEight monthmonth oldold PP.. pineapinea andand PP.. pinasterpinaster (Fig(Fig.. 33 andand 44))..
B)B) InoculationInoculation TrialTrial
?? PlantsPlants werewere inoculatedinoculated withwith 20002000
nematodes/mlnematodes/ml andand waterwater (control)(control) andand
samplessamples werewere collectedcollected atat 00h,h, 33h,h, 99h,h, 1010dd
andand 2020dd..
C) RNA Extraction and PurificationC) RNA Extraction and Purification
?? RNARNA waswas clearedcleared ofof DNADNA withwith TurboTurbo--DNADNA (Ambion)(Ambion);;
?? Quantification/QCQuantification/QC:: NanophotometerNanophotometer (Implen)(Implen) andand gelgel electrophoresiselectrophoresis..
Fig.5Fig.5 Primer design workflowPrimer design workflow
Fig.1Fig.1 Botrytis cinereaBotrytis cinerea growing ongrowing on barleybarley
Fig.2Fig.2 FungusFungus--depleteddepleted
nematode culturenematode culture
Fig.3Fig.3 P. pinaster and P. pinae seedlings pre- inoculation Fig.4Fig.4 InoculatedInoculated P. pinaeP. pinae seedlingsseedlings
NematodeNematode
inoculationinoculation
Table 1. Gene expression at 9 hours, 10 days and 20 days in P. pinaster and P. pinea with
different condicions (0, water, virulent and non-virulent), 18S was used as internal control.
BursaphelenchusBursaphelenchus xylophilusxylophilus,, alsoalso knownknown asas thethe PinePine WoodWood NematodeNematode (PWN),(PWN), isis responsibleresponsible forfor thethe yearlyyearly lossloss ofof millionsmillions ofof pinepine treestrees worldwideworldwide
duedue toto thethe diseasedisease itit causes,causes, thethe PinePine WiltWilt DiseaseDisease (PWD)(PWD)((11)).. HavingHaving originatedoriginated inin NorthNorth America,America, thethe diseasedisease hashas spreadspread toto PortugalPortugal((11)),, seriouslyseriously
threateningthreatening EuropeanEuropean nativenative pinepine forestsforests andand economyeconomy.. SymptomsSymptoms areare wiltingwilting ofof needles,needles, reddishreddish brownbrown foliagefoliage andand lacklack ofof resinresin exudationexudation.. DiseaseDisease
developmentdevelopment cancan bebe rapid,rapid, withwith treetree deathdeath occurringoccurring inin aboutabout threethree monthsmonths dependingdepending onon environmentalenvironmental conditionsconditions((11,,22))..
FieldField observationsobservations ofof areasareas wherewhere PP.. pinaepinae andand PP.. pinasterpinaster coexistcoexist producedproduced thethe hypothesishypothesis thatthat thethe firstfirst isis resistantresistant toto thethe infectioninfection withwith PWNPWN..
InIn orderorder toto testtest thisthis hypothesishypothesis wewe havehave conductedconducted inoculationinoculation trialstrials withwith virulentvirulent andand nonnon--virulentvirulent nematodesnematodes inin oneone yearyear oldold seedlings,seedlings, andand
determineddetermined resistanceresistance toto thethe diseasedisease byby symptomsymptom observationobservation andand genegene expressionexpression studiesstudies..
WeWe proposepropose thethe identificationidentification ofof genesgenes responsibleresponsible forfor thethe reportedreported naturalnatural resistanceresistance ofof PP.. pinaepinae inin orderorder toto betterbetter understandunderstand thethe
molecularmolecular mechanismsmechanisms ofof treetree susceptibilitysusceptibility andand resistanceresistance..
ABSTRACTABSTRACT
?? PeroxidasePeroxidase expressionexpression (Table(Table 22)) waswas onlyonly observedobserved 33hh andand 99hh afterafter inoculation,inoculation,
whichwhich maymay indicateindicate thethe genegene isis expressedexpressed onon thethe initialinitial stepssteps ofof thethe infectioninfection..
However,However, itit waswas notnot detecteddetected onon PP.. pinasterpinaster inoculatedinoculated plantsplants withwith virulentvirulent strain,strain,
suggestingsuggesting thatthat thethe presencepresence ofof differentdifferent NWPNWP virulentvirulent strainstrain isis sufficientsufficient toto induceinduce
aa stressstress responseresponse byby thethe plantplant;;
?? OtherOther stressstress inducedinduced genesgenes werewere detecteddetected onon bothboth plantsplants (Table(Table 22),), suchsuch asas genesgenes
expressedexpressed duringduring plantplant cellcell deathdeath ((Mat/samMat/sam)) andand oxidativeoxidative stressstress ((ATTRXATTRX11))..
However,However, inin PP.. pineapinea plantsplants afterafter 2020 daysdays ofof inoculation,inoculation, thethe genesgenes ceasedceased toto bebe
expressedexpressed.. Also,Also, inin PP.. pinasterpinaster plantsplants withwith nonnon--virulentvirulent NWPNWP strain,strain, ATTRXATTRX11
expressionexpression stoppedstopped..
?? ATTXR1ATTXR1 (Table 1)(Table 1),, an important gene in oxidative stress response, was expressed in both species at all time points,an important gene in oxidative stress response, was expressed in both species at all time points,
except with C14except with C14--5 (non5 (non--virulent strain) in the last two time points;virulent strain) in the last two time points;
?? There was no expression ofThere was no expression of PORAPORA (photosynthetic factor) in(photosynthetic factor) in P. pinasterP. pinaster, but in, but in P. pineaP. pinea it was expressed at 9h withit was expressed at 9h with
water, and at 10d when plants were inoculated with C14water, and at 10d when plants were inoculated with C14--5;5;
?? PLDAlpha1PLDAlpha1, a gene activated by abcisic acid as a response to plant stress which generates the acidification of plant, a gene activated by abcisic acid as a response to plant stress which generates the acidification of plant
cells, was only detected 10 days after inoculation in allcells, was only detected 10 days after inoculation in all P. pineaP. pinea treatments. No expression was observed intreatments. No expression was observed in P. pinasterP. pinaster
plants, indicating that plant species may have a differentiated stress response to the same pathogenic attack;plants, indicating that plant species may have a differentiated stress response to the same pathogenic attack;
?? InIn P. pinaster,P. pinaster, PsylAP1PsylAP1 gene (cell death) only expresses at 9h with HF. Moreover,gene (cell death) only expresses at 9h with HF. Moreover, P. pinaeP. pinae at 9h doesn't express withat 9h doesn't express with
HF;HF;
?? PsylSupOxDisPsylSupOxDis, (gene associated with oxidative stress factors) had no expression in, (gene associated with oxidative stress factors) had no expression in P. pinasterP. pinaster at 9h. Inat 9h. In P. pinaeP. pinae itit
doesn��t express at 9h and 20d with C14doesn��t express at 9h and 20d with C14--5;5;
?? The variable expression of several stress related genes during the time course (The variable expression of several stress related genes during the time course (PsylSupOxDisPsylSupOxDis ,, PMonPR10PMonPR10, and, and PsylAP1PsylAP1) may indicate that the plant generated) may indicate that the plant generated aa
cascaded signallingcascaded signalling response over time;response over time;
??Also,Also, P. pinasterP. pinaster andand P.P. pineapinea may have different stress induced mechanisms, as suggested by the presented results.may have different stress induced mechanisms, as suggested by the presented results.
?? SSH and 454SSH and 454 PyrosequencingPyrosequencing studies are underway to collect data that should shed light on the defence mechanisms underlying species specific resistancestudies are underway to collect data that should shed light on the defence mechanisms underlying species specific resistance toto the PWD.the PWD.
?? MetabolomicMetabolomic and proteomic studies will follow, providing a wider perspective on the disease response model.and proteomic studies will follow, providing a wider perspective on the disease response model.
Table 2. Gene expression at 3 hours, 9 hours, 10 dayss and 20 days in P.pinaster and P. pinea with different condicions: Control (O),
Water (H2O), Virulent NWP (HF) and Non-virulent NWP (C14-5). Actin was used as internal control.
Biotechnological strategies for resistance
induction to the pinewood nematode
(PWN) in Pinus pinaster
PlantPlant
inoculationinoculation
Rui Rodrigues, Andreia Reis, Albina Franco, Carla Santos, Maciej Markowski, Marta W. Vasconcelos
Escola Superior de Biotecnologia -Universidade Cat��lica Portuguesa
CONCLUSIONS AND FUTURE WORKCONCLUSIONS AND FUTURE WORK
RT-PCR

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