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1Title1ImpairedexpressionofchloroplastHSP90Cchaperoneactivatesplant2defenseresponsesleadingtoadiseasesymptom-likephenotype34Runningtitle5ImpairedchloroplastHSP90supplyactivatesdefenseresponse67IslamShaikhul1,BhorSachinAshok1,TanakaKeisuke2,SakamotoHikaru3,8YaenoTakashi1,4,5,KayaHidetaka1,4,5andKobayashiKappei*,1,4,59101UnitedgraduateschoolofAgriculturalSciences,4GraduateSchoolofAgriculture,11and5ResearchUnitforCitromics,EhimeUniversity,Matsuyama,Ehime790-128566,Japan,2NODAIGenomeResearchCenter,TokyoUniversityofAgriculture,13Setagaya,Tokyo156-8502,Japan,and3FacultyofBio-Industry,TokyoUniversity14ofAgriculture,Abashiri,Hokkaido099-2493,Japan.
1516Emailaddresses17IS,islamshaikhul2014@outlook.
com;BSA,bhor.
sach@gmail.
com;18TK,kt205453@nodai.
ac.
jp;SH,h3sakamo@nodai.
ac.
jp;19YT,yaeno@agr.
ehime-u.
ac.
jp;KH,kaya.
hidetaka.
hu@ehime-u.
ac.
jp;20KK,kappei@ehime-u.
ac.
jp21*Correspondingauthor'stelephoneNo.
++81-79-946-98682223Tables,2;Figures,7.
24Supplementarymaterials:Supplementarytables,8;Supplementaryfigures,4.
2526.
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029116doi:bioRxivpreprint2Highlight27InducedsilencingofHSP90Cgenecausedtheupregulationofstress-responsive28genesandtheactivationofinnateimmuneresponse,whichresultedinthe29chlorosisdevelopmentaccompanyingcelldeath.
3031Abstract32RNA-seqanalysisofatransgenictobaccoplant,i-hpHSP90C,inwhich33chloroplastHSP90Cgenescanbesilencedinanartificiallyinduciblemanner34resultinginthedevelopmentofchlorosis,revealedtheup-anddown-regulation35of2746and3490genes,respectively.
GeneOntologyanalysisofthese36differentiallyexpressedgenesindicatedtheupregulationofROS-responsive37genes,theactivationoftheinnateimmunityandcelldeathpathways,andthe38downregulationofgenesinvolvedinphotosynthesis,plastidorganization,andcell39cycle.
Celldeathwasconfirmedbytrypanbluestainingandelectrolyteleakage40assayandtheH2O2productionbydiaminobenzidinestaining.
Theupregulation41ofERstress-responsivegenessuggestedtheinterplaybetweenERprotein42qualitycontrolandchloroplastorimmuneresponse.
Theresultscollectively43suggestthatthereducedlevelsofHSP90Cchaperoneleadstheplanttodevelop44chlorosisprimarilythroughtheglobaldownregulationofchloroplastand45photosynthesis-relatedgenesandadditionallythroughthelight-dependent46productionofROS,followedbytheactivationofimmuneresponsesincludingthe47celldeath.
4849KeyWords50Celldeath,Chlorosis,HSP90C,Immuneresponse,RNA-seq,Tobacco,51Transcriptome5253Abbreviations54CPRGs,Chloroplastandphotosynthesis-relatedgenes;DEGs,Differentially55expressedgenes;Dex,Dexamethasone;hp-RNA,HairpinRNA;HSP90C,56Chloroplastheatshockprotein90;ROS,Reactiveoxygenspecies;siRNA,Small57interferingRNA.
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029116doi:bioRxivpreprint3Introduction59Plantvirusdiseasesdeveloparangeofsymptomsresultingfromthe60morphologicalandphysiologicaldisturbancesofthehostcells.
Leafchlorosis,the61mostfrequentlyobservedsymptom,reducesplantproductivityandthus,leadsto62asignificantlossincropyield.
Virus-inducedchlorosisoftenaccompaniesthe63structuralchangeanddysfunctionofchloroplasts,includingthereductionin64chlorophyllcontentandtheexpressionofphotosyntheticgenes(Manfreetal.
,652011;MochizukiandOhki,2011;Mochizukietal.
,2014;Qiuetal.
,2018).
66Therefore,understandingthemechanismsofchloroplastdysfunctionwouldlead67ustotheestablishmentofcounter-measuresagainstcroploss.
Althoughthe68moleculareventsduringchlorosishavebeenextensivelydocumented,the69precisemechanismforthereducedchloroplastactivitieshadremainedtobe70elucidateduntilrecentpioneeringstudies,whichhaveshowntheinvolvementof71RNAsilencingofchloroplastproteingenesinthedevelopmentofchlorosisby72sub-viralRNAs.
Twogroupshaveindependentlyshownthatthebrightyellow73symptomsintobaccoplantsinfectedwithcucumbermosaicvirus(CMV)74harboringY-satelliteRNA(Y-sat)isattributedtotheRNAsilencingofmagnesium75protoporphyrinchelatasesubunitI(CHLI)involvedinchlorophyllbiosynthesis76mediatedbytheY-sat-derivedsmallinterferingRNA(siRNA)(Shimuraetal.
,772011;Smithetal.
,2011).
AnotherstudyhasshownthatsiRNAderivedfrom78Peachlatentmosaicviroid(PLMVd)directstheRNAsilencingofchloroplastheat-79shockprotein90(HSP90C)andconsequentlycausesseverechlorosisor80albinism(Navarroetal.
,2012).
Toanalyzethemolecularmechanismsof81chlorosisinducedbytheRNAsilencingofchloroplastproteins,wepreviously82establishedexperimentalsystemsusingchemicallyinduciblepromotertodrive83RNAstoinduceRNAsilencingofCHLIandHSP90Cintransgenictobaccoplants84(Bhoretal.
,2017a,b).
85HeatshockproteinsorHSPsareagroupofmultipleproteinfamilies,86whichareinducedupondifferentcellularstressesandhelpcellssurviveunder87stressedcondition(ParsellandLindquist,1994).
HSPsareevolutionarily88conservedthroughouttheprokaryotesandeukaryotesandhavebeenclassified89intodifferentfamilies,includingHSP100,HSP90,HSP70,HSP60,HSP40,and90.
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04.
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029116doi:bioRxivpreprint4HSP20(ParkandSeo,2015;Sableetal.
,2018).
Allofthesefamilieshelp91maintaincellularhomeostasisandplayrolesindifferentdevelopmental92processesthroughtheirmolecularchaperonefunction(ParkandSeo,2015;93Sableetal.
,2018).
Outofthesefamilies,HSP90familyproteinshavebeen94showntoplaypleiotropicrolesindiversebiologicalphenomena.
HSP90null95mutantsareknowntobelethal,andeventheheterozygotesforthemutation96showeddevelopmentalabnormalitiesinDrosophila(RutherfordandLindquist,971998).
ThepharmacologicalblockadeofHSP90functionresultedinphenotypic98variationsspecifictogeneticbackgrounds(Queitschetal.
,2002).
Thestudies99supportthatHSP90familyproteinstakewide-rangingclientproteinsfortheir100chaperoningfunctionindifferentsignaltransductionpathwaysandtranscriptional101regulatorynetworks(Echeverríaetal.
,2011;Wayneetal.
,2011).
102Inplants,theHSP90familycomprises7,9,and10members(Krishna103andGloor,2001;Mandaletal.
,2013)inArabidopsisthaliana,Oryzasativa,and104Populustrichocarpa,respectively(Zhangetal.
,2015).
OutofsevenArabidopsis105HSP90familyproteins,fourmembersexhibitnucleo-cytoplasmiclocalization,106whiletheotherthreelocalizetoeachofchloroplast,mitochondrion,and107endoplasmicreticulum(KrishnaandGloor,2001).
TheHSP90CorAtHSP90.
5,108whichlocalizestothechloroplast,wasfirstidentifiedasthecausalgenefora109chlorate-resistantmutation(Caoetal.
,2003).
Likecognateproteinsinthe110mitochondrion(Altierietal.
,2012;Altieri,2013)orendoplasmicreticulum111(Ishiguroetal.
,2002;Marzecetal.
,2012),HSP90Chasbeenproposedtohave112aroleinproteinfoldinginthechloroplast(SchrodaandMühlhaus,2009)and113showntobeessentialforthetransportofdifferentproteinsintochloroplasts114(Inoueetal.
,2013).
Althoughthechlorate-resistantmutant,cr88,whichhasa115singlemissensemutationinHSP90Ccodingsequence,wasviablealbeitwithan116impairedphotomorphogenesis(Caoetal.
,2003),nullmutantswithT-DNA117insertionsinHSP90Cgenewerelethal(Inoueetal.
,2013;Fengetal.
,2014),118indicatingitsessentialfunction.
ThesilencingofHSP90CinArabidopsisplants119resultedinvariegatedoralbinophenotype(Ohetal.
,2014).
Therefore,itisnot120surprisingthatthesilencingofHSP90Cbyviroid-derivedsiRNAresultedin121severechlorosisoralbinism(Navarroetal.
,2012).
122.
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029116doi:bioRxivpreprint5WepreviouslyshowedthattheinducedsilencingofHSP90Cin123transgenictobaccoresultednotonlyinvisiblechlorosiswithsignificantly124decreasedchlorophyllcontentsandthereducedexpressionofchloroplastprotein125genesbutalsointheinductionofsomepathogenesis-relatedgenes(Bhoretal.
,1262017b).
Theresultssuggestthatthechlorosisinthismodelsystemisattributed127notonlytotheimpairedchloroplastbiogenesisbutalsotoactiveplantresponses128totheimpairedchloroplastfunction.
Inthisreport,weemployedanRNA-seqto129examinethealterationingeneexpressionearlyaftertheinductionofHSP90C130silencing.
BycomparingthegeneexpressionpatternsbetweenHSP90Csilenced131andnon-silencedplants,wefoundupregulationofgenesrelatedtotheresponse132toreactiveoxygenspecies,celldeath,planthormonesignalingpathways,133defenseresponse,andinnateimmuneresponse.
Thedetectionofsporadiccell134deathsupportedthebiologicalsignificanceofthetranscriptomicchanges.
The135resultssuggestthatchlorosisdevelopmentwithimpairedHSP90Cexpression136involvestheactivationofcell-deathmediatedplantdefenseresponseinaddition137toasimplereductionofchloroplastfunction.
138139Materialsandmethods140Plantmaterials141Transgenictobaccolines,i-hpHSP90C6-1(H-6)andi-hpHSP90C4-5(H-1424),whichexpresshp-RNA(hairpinRNA)correspondingtotheHSP90C-specific143regionsunderthecontrolofadexamethasone-induciblepromoter,were144describedpreviously(Bhoretal.
,2017b).
TheseinducibleHSP90Csilencing145tobaccolinesandnon-transformanttobacco(Nicotianatabacumcv.
PetitHavana146SR1)wereusedinthisstudy.
Plantswereculturedinaplugtraycontaining147commercialsoilmix(SupermixA,SakataSeeds,Yokohama,Japan)foroneweek148at25°Cunder16/8hlight/darkcycleconditionwithanirradiationdoseofabout14960μMm-2s-1.
Theseedlingsweretransferredandgrownforadditional2weeksin150aplasticpot(6.
0cmindiameter)containingthemixture(1:1)ofvermiculiteand151SupermixA,withwateringeveryotherdaywith1000-timesdilutedHyponex6-15210-5(HyponexJapan,Osaka,Japan)solutiontwiceaweek.
Toinducethe153transgeneexpression,three-week-oldcontrolandtransgenictobaccoplants154.
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029116doi:bioRxivpreprint6weresprayedwithfreshlydiluted50Mdexamethasone(Dex)solution155containing0.
01%(v/v)Tween-20usingaspraybottle.
Controlplantsweremock-156treatedbysprayingwith0.
5%ethanolsolutioncontaining0.
01%(v/v)Tween-20.
157158ExtractionandsequencingofRNA159TheRNAwasextractedandtreatedwithRNase-freeDNaseasdescribed160previously(Waliullahetal.
,2014).
ForRNA-seqanalysis,RNAwasextracted161fromsixindividualplantseachDex-treatedanduntreated,controlandH-4(i-162hpHSP90C4-5)transgenicplantsat24hpost-Dextreatment.
Threesamples163eachfromplant/treatmentgroupswereselectedbytheintegrityofRNAassessed164using2100BioanalyzerwithRNA-nanochip(Agilent).
Librarieswereconstructed165usingtheTruSeqRNASamplePreparationv2kit(Illumina)accordingtothe166manufacturer'sprotocol.
100-bpsingle-endsequencingwascarriedoutusing167Hiseq2500atNodaiGenomeResearchCenter.
168169RNA-seqdataanalysis170Rawreadswereobtainedusingbcl2fastq2(Illumina)withtheadaptor171sequencesremoved.
Thereaddatawerefurthertrimmedusing172fastq_quality_trimmerwiththequalitycutoffat28andlengthcutoffat80.
The173cleanreaddatawereuploadedtothelocalGalaxyplatform(Boekeletal.
,2015),174andanalyzedthereinusingSalmon(Patroetal.
,2017)withNtab-TN90-AYMY-175SS_NGS.
mrna.
annot.
fnareferencetranscriptome(Sierroetal.
,2014);176downloadedfromSolGenomicsNetwork;177https://solgenomics.
net/organism/Nicotiana_tabacum/genome)fortranscript178abundanceestimationandexpressionvaluecomputation.
Wepreparedinthe179commandlinefromthefastafileabovetouseaTranscriptID-AGIcodetable180(SupplementarytableS1)tohavetheexpressionvalueswithAGIcodesbecause181theoriginalgeneIDsintheaccompanyinggff3filewerenotcompatiblewithour182GOenrichmentanalysis.
Thus,SalmongavetheTranscriptsPerMillion(TPM)183valuetoeachofthetranscriptswithAGIcodeannotation.
Of189,413mRNA184transcriptsintobaccoreferencetranscriptome,121,268transcripts(64.
02%)185wereannotatedwithAGIcodes,and68,145transcripts(35.
98%)wereomitted186.
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029116doi:bioRxivpreprint7fromtheanalysis(SupplementarytableS1).
187Thedetectionofdifferentiallyexpressedgenes(DEGs)wasperformed188usingDESeq2(AndersandHuber,2010)intheGalaxyplatform.
DESeq2was189runindifferentgroupings:Dex-treatedH-4vscontrolH-4;Dex-treatedH-4vs190Dex-treatedSR1;andDex-treatedH-4vsControlSR1.
Then,Venndiagrams191showingtheDEGsinvariouscombinationswerepreparedusingthewebprogram192Venny2.
1(http://bioinfogp.
cnb.
csic.
es/tools/venny/index.
html).
Thedifferentially193expressedgeneswereconsideredsignificantunderthefollowingcriteria:194correctedp-value(P(adj))lessthan0.
05andtheLog2(FC)valuesabove1or195below-1.
ForfunctionalannotationofDEGswithAGIcodes,weperformedthe196GOenrichmentanalysisusingthePANTHER(ProteinANalysisTHrough197EvolutionaryRelationships)ClassificationSystem(http://www.
pantherdb.
org/)198(Mietal.
,2019),whichimplementsone-sidedFisher'sexacttestwiththemultiple-199testingcorrectionmethodbeingsettoFDR.
GOswithFDRbelow0.
05were200consideredsignificant.
Heatmapforselectedup-regulatedgeneswasdrawn201usingggplot2functionintheheatmap2programintheGalaxyplatform.
The202relativenormalizedcountdatacalculatedintheExcelprogram(Microsoft)were203usedinsteadoftherawnormalizedcountdatafromDEseq2becausethelatters204werenotacceptedbytheprogram.
Theexpressionlevelsofparticulargenesin205theRNA-seqanalysiswerecomparedusingtherelativenormalizedcountdata206withstatisticalanalysisusingDESeq2.
207208QuantitativeRT-PCR209TotalRNAwasextractedusingtheISOSPINPlantRNAKit(NipponGene,Japan)210forqRT-PCRwithmoreplantsamples.
ThecDNAwassynthesizedusingtheM-211MLVRTase(NewEnglandBiolabsJapan)andsubjectedtothereal-timeqPCR212usingStepOnePlusReal-TimePCRsystem(AppliedBiosystems)withKAPA213SYBRFASTqPCRmastermix(KapaBiosystems).
TheqPCRconditionswere214asfollows:initialholdingat95°Cfor20s,40cyclesof95°Cfor3s,60°Cfor30215s,followedby95°Cfor15s,60°Cfor1min,95°Cfor15s.
Ameltingcurvewas216generatedtoconfirmthespecificityofthereactions.
Eachsamplewastestedin217triplicates.
Therelativeexpressionleveloftargetgeneswascalculatedby218.
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029116doi:bioRxivpreprint8comparativeCT(ΔΔCT)methodusingEF1αastheinternalreferenceanda219commonstandardsample(amixtureofRNAsfromuntreatedSR1)forthe220normalizationamongassayplates.
TheprimersusedforqRT-PCRanalysisare221listedinTableS2.
222223Determinationofcelldeath224CelldeathwasmeasuredusingtheTrypanBlueAssayasdescribedpreviously225(MorelandDangl,1999)withslightmodifications.
Three-week-oldtransgenicand226controlplantswereDex-orsolvent-treated,keptforsevendays,andobserved227forthephenotypicchanges.
TheAgroinfiltration–mediatedtransientexpression228ofTomatobushystuntvirus(TBSV)P19(Voinnetetal.
,2003)wereanalyzed2229dayspost-infiltrationasapositivecontrolforcelldeath(Scholthofetal.
,1995).
230Leafdisksof6mmindiameterwereheatedforthreeminutesintrypanblue231solution,cooledtoroomtemperature,de-colorizedusingchloralhydratesolution,232andmountedin50%glycerolformicroscopyanalysis.
Forthequantitative233determinationofcelldeath,electrolyteleakageassaywasconductedas234describedpreviously(Mackeyetal.
,2003)withslightmodifications.
Eightleaf235disksfromeachplantwerefloatedin9mlsterilizeddistilledwater(SDW)and236keptfor24hoursunderdarkconditions.
Theconductanceoftheleaked237electrolyteinSDWwasmeasuredusingaconductivitymeter(LAQUAtwin-EC-23811,HORIBAScientific,Japan).
Afterthemeasurement,leafdisksandSDWwere239recombined,boiledfor10minutes,andtheconductivityoftotalelectrolytewas240measured.
Thecelldeathwasevaluatedwiththerelativeconductivity,theratio241ofthoseforleakedtototalelectrolyteinthreetriplicateexperiments.
Statistical242analyses[Tukey'sHSD(honestlysignificantdifference)test]wereperformed243usingSPSS(Version17)andMicrosoftOfficeExcel2016.
244245DAB(3,3'-diaminobenzidine)stainingforhydrogenperoxidedetection246Forinsitudetectionofhydrogenperoxide(H2O2),weperformed3,3'-247diaminobenzidine(DAB)stainingasdescribedpreviously(DaudiandO'Brien,2482012)withslightmodifications.
Three-week-oldtransgenicandcontrolplants249wereDex-orsolvent-treatedandfurthergrownfor24hours.
Leaf-disksof6mm250.
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029116doi:bioRxivpreprint9indiameterwerevacuum-infiltratedwith1mg/mLDABsolutioncontaining0.
05%251Tween-20in50mLconicaltubes,keptonwetfilterpapersinPetridishes,and252incubatedunderlight(70-100molem-2s-1for30minutesfortestplantsand250-253300molem-2s-1for60minutesforpositivecontrolplants),andforadditional3254hours30minutesinthedark.
Theleafdiskswerede-colorizedbyboilingin99.
5%255ethanolfor5-10minandobserved.
256257Results258RNAsequencing,mapping,andidentificationofDEGs259WepreviouslyreportedthatthesilencingofHSP90Cintransgenic260tobaccolinesresultedinchlorosisandgrowthsuppression(Fig.
1A)261accompaniedbyanactivationofPRgenes(Bhoretal.
,2017b).
RNA-seq262analysiswasconductedtoelucidatethemechanismsunderlyingthe263developmentofchlorosisinthismodelsystem.
Three-week-oldi-hpHSP90C264transgeniclineH-4andnon-transformantSR1wereDex-orcontrol-treated,and265RNAwasextractedat24hpost-Dextreatmentfromfourindividualplantsfrom266eachplant/treatmentgroup.
ThreeeachofthemwereselectedforRNA-seq267basedontheirintegrityandpurity(datanotshown).
TheRNAsequencinggave26823Mreads/sampleonaveragefromthe12samples,80-90%ofwhichwere269mappedtothetobaccoreferencetranscriptome(SupplementarytableS3).
270Inourinitialanalysis,theoverallsimilaritywithinsampleswasevaluated271byaprincipalcomponentanalysis(PCA)(Fig.
1B).
Theresultshowedthattwo272samples,HD2andHD3,showedcleardifferencesfromthecontrolsamples273includingthenon-transformedSR1samplesregardlessofthetreatmentandDex-274untreatedi-hpHSP90Cplants.
Incontrast,oneoftheDex-treatedi-hpHSP90C275plants(HD4)showedtheleastdifferencesfromthosecontrolsamples(Fig.
1B).
276ThehighernormalizedHSP90CtranscriptcountsindicatedthattheHD4plant277hadnotefficientlybeensilencedinHSP90Cexpression(Fig.
2D),suggestingthat278thetranscriptomicchangesareobservedonlyafterasignificantHSP90Cdown-279regulation.
Totestthehypothesis,wecomparedtheexpressionlevelsbyqRT-280PCRofHSP90Candrepresentativegenesthatshowclearup-anddown-281regulationfoundintheRNA-seqanalysis(seebelow),theisochorismate282.
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029116doi:bioRxivpreprint10synthase1gene(ICS1)andalight-harvestingchlorophylla/b-bindingprotein283(LHCab),respectively.
ThegeneexpressionofICS1andLHCabcorrelatedwith284theHSP90Cexpressionnegativelyandpositively,respectively(Fig.
1CandD).
285Importantly,insomeplantsofthei-hpHSP90CH-4line,inwhichHSP90C286expressionlevelsweremorethanhalfofthecontrolplants,theup-regulationof287ICS1andthedown-regulationofLHCabwerenotveryprominentandwithinthe288variabilityofcontrol(Dex-untreated)plants.
IncontrasttotheH-4line,theH-6289lineofi-hpHSP90CtransgenicplantsshowedhighlyreproducibleHsp90C290silencingandtheup-anddown-regulationofICS1andLHCab,respectively291(SupplementaryFig.
S1).
TheSR1plantdidnotrespondtoDex-treatmentinthe292expressionofthegenesexamined.
Theseresultssupportthehypothesisabove,293andtherefore,weomittedtheHD4samplefromthedifferentialexpression294analysis.
295ThegeneexpressionofHD2andHD3samples(HD)werecompared296usingDESeq2withallthreegroups,untreatedlineH-4(HC),Dex-treatedSR1297(SD),anduntreatedSR1(SC),whichhavenevershownchlorosisinourrepeated298experiments.
DEGswerepickedupfromtheDESeq2databasedonthe299combinedcriteriaoflog2(FC)valuesbelow-1orabove1,andtheadjustedp-300valueslesserthan0.
05.
TheMAplotssupportthatthreecomparisonsidentified301aconsistentsetofDEGs(SupplementaryFig.
S2).
Theanalysesofdifferentially302expressedmRNAtranscriptsinthreedifferentcomparisonsabove—HDvsHC,303HDvsSD,andHDvsSC—identified7267(55.
9%)and8042(47.
4%)commonly304up-anddown-regulatedmRNAs,respectively(Fig.
3AandB).
Becausethe305mRNAIDsinthetobaccoreferencetranscriptomearenotreadilyusedfor306downstreamGOenrichmentanalysis,thoseannotatedwiththeArabidopsisAGI307codeswereselected.
OutofthedifferentiallyexpressedmRNAsabove,4896up-308regulatedmRNAshadannotationwith2746differentAGIcodescomprising30961.
4%ofDEGswithAGIannotationcommoninthreecomparisons,and6307310down-regulatedmRNAshad3490(59.
0%)AGIcodes(Fig.
3CandDand311SupplementarytablesS4&S5).
312313FunctionalclassificationofDEGs314.
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029116doi:bioRxivpreprint11TheAGIcodesgiventotheup-anddown-regulatedmRNAswereused315fortheGOenrichmentanalysis.
Tables1and2presenttheselectedGOterms316forbiologicalprocessesenrichedinthelistsofup-anddown-regulatedmRNAs,317respectively.
TheenrichedGOtermsintheup-regulatedgenesincludeinnate318immuneresponse,responsetowounding,responsetooxidativestress,response319tophytohormones[salicylicacid(SA),jasmonicacid(JA),andabscisic320acid(ABA)],andhypersensitivecelldeath(Table1&SupplementarytableS6).
In321accordancewiththeobservation,biosyntheticgenesofSAandJAwerefound322upregulated(Table1).
Theheatmap(Fig.
4)showstheexpressionchangesof323genesselectedfromthoseinGOterms,responsetoSA(GO:0009751),response324toJA(GO:0009753),responsetooxidativestress(GO:0006979)andcelldeath325(GO:0008219),whichareindicatedbydouble-headedarrowsontheright.
The326resultssuggestthatthereducedsupplyofHSP90Ctochloroplastelicitsdefense327responseinvolvingsomephytohormonepathways.
328Inadditiontotheactivationofdefenseresponse,genesinvolvedinthe329responsetoendoplasmicreticulum(ER)stresswereremarkablyupregulated330aftertheimpairedsupplyofHSP90C(Table1).
Thisobservationcouldbe331attributedtothenon-specificsilencingofER-localizingHSP90familyproteinby332theHSP90ChpRNA.
Therefore,weexaminedtheexpressionlevelsofsixoutof333sevenHSP90familyproteinsintheRNA-seqdatabecausenotobaccomRNAin334thereferencetranscriptomewasannotatedwithAT5G56010encodinganucleo-335cytosolicHSP90.
3.
ThetruetargetofhpRNA-mediatedsilencing,HSP90Cor336HSP90.
5,wassignificantlydownregulatedasshown(Fig.
2D),buttheexpression337ofcytosolicHSP90.
1andHSP90.
4,nucleo-cytosolicHSP90.
2,andmitochondrial338HSP90.
6wasnotaffectedbytheDex-treatment(Fig.
2A,B,C,andE).
Incontrast,339statisticallysignificantupregulationinDex-treatedHD2andHD3wasdetectedin340ER-localizingHSP90.
7(Fig.
2F).
Theresultscontradicttheoff-targetsilencingof341ER-localizingHSP90,andthus,suggesttheinterplaybetweentheproteinquality342controlsystemofERwiththatinplastidorimmuneresponses.
343TheenrichedGOterms(Biologicalprocess)inthedownregulatedgenes344includephotosynthesis,pigmentmetabolicprocess,andplastidorganization345(Table2&SupplementarytableS7),whichisconsistentwithcrucialrolesof346.
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029116doi:bioRxivpreprint12HS90Cinchloroplastbiogenesis.
Inaddition,someprimarymetabolismgenes347annotatedwithGOterms,carbohydratemetabolicprocess,lipidmetabolic348processandcellularaminoacidmetabolicprocess,othermetabolicgenes349annotatedwithcofactormetabolicprocessandvitaminmetabolicprocess,and350somecellularprocessgenesannotatedwithcellcycle,cellwallorganization,and351cellularhomeostasiswereshowntobedownregulated(Table2&Supplementary352tableS7),whichisconsistentwiththeupregulationofcelldeath-relatedgenes353(Table1&SupplementarytableS6).
Interestingly,threeGOterms,responseto354osmoticstress,responsetooxidativestress,andresponsetosaltstresswere355enrichedinthedownregulatedgenes.
However,theywerealsoenrichedinthe356upregulatedgenes(Tables1and2).
TheresultssuggestthatimpairedHSP90C357supplywouldinducedrasticswitchinginthesestress-responsivegenes358(SupplementaryFig.
S3).
359360DetectionofcelldeathandreactiveoxygenspeciesinHPS90C-silenced361plants362Thesignificantupregulationofthecelldeathpathwaypromptedusto363detectcelldeathini-hpHSP90Clines.
Becauselower(older)leavesshowed364severerchlorosisthantheupper(younger)leaves,weexaminedthecelldeathin365thoseleavesseparately.
Thetrypanbluestainingshowedlightbluestaininupper366leavesandmoreintensestaininginolderleaves,albeittoalesserextentthan367thepositivecontrol,ofDex-treatedH-4andH-6plants(Fig.
5J–LandP–R).
In368thepositivecontrolplants,inwhichTBSVP19hadtransientlybeenexpressed369fortwodays,someleafdiscswerecutouttoincludebothdeadandlivingparts370tomakethedifferenceinstainingofthosepartsclear(Fig.
5SandT).
Thestaining371oflowerleavesfromDex-treatedH-4andH-6plantswasnotuniform,suggesting372thecelldeathinductioninthoseleavesweresporadicwithintheleaftissue.
373Electrolyteleakageassayconfirmedthecelldeathinlowerleavesbutnotthatin374upperleaves(Fig.
5U).
Microscopicobservationoftrypanbluestainedleaftissue375indicatedthatdeadcellsinlowerleavesofDex-treatedH-4andH-6plantswere376barelyshrunken(Fig.
5handl),unlikethoseinthepositivecontrol(Figure5m).
377Theupperleavesexhibitedsmallpatchesofdeadcells,suggestingthesporadic378.
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029116doi:bioRxivpreprint13andage-dependentnaturesofcelldeathinDex-treatedH-4andH-6plants(Fig.
3795gandk).
AsmallfractionofcellsinlowerbutnotupperleavesofuntreatedH-6380showedbluestaining(Fig.
5j),suggestingthatthehpRNAtoHSP90Chadbeen381expressedinaleakyfashioninasmallnumberofcellsinuntreatedH-6lineand382thatthecelldeathobservedisage-dependent.
Dex-treatedanduntreatedcontrol383plants(SR1)anduntreatedH-4didnotshowanysignofcelldeath(Fig.
5a–d,e,384andf).
Theseresultscollectivelysuggestthatthedeathofcells,inwhichHSP90C385supplyisimpaired,isstochasticallyinitiatedandproceedssomehowslowly,386althoughtheRNA-seqsuggeststhatitisaplant-typehypersensitiveresponse387(Table1).
388Theupregulationofgenesinvolvedintheresponsetooxidativestress389(Table1andFig.
4)suggeststheproductionofreactiveoxygenspecies(ROS)390inDex-treatedH-4andH-6plants.
BecauseROSiswellstudiedasasignal391mediatorandanexecutorofplantcelldeath,wetriedtodetectH2O2asa392representativeofROS.
Positivecontrolwithintenselightresultedinthe393accumulationofbrownishDABprecipitateinchloroplasts(Fig.
6M).
IntenseDAB394stainingofchloroplastswasbarelyvisibleinDex-treatedanduntreatedcontrol395plants(SR1),anduntreatedH-4andH-6(Fig.
6A–D,E,F,IandJ).
EvidentDAB396stainingofchloroplastswasobservedinleavesofDex-treatedH-4andH-6plants397(Fig.
6G,H,K,andL).
Theresultssuggestthat,uponthelossofsufficientlevels398ofHSP90C,chloroplastsproduceROS,whichwouldtriggerthecelldeath399response.
AlthoughtheoverallDABpigmentationwasmoreintenseinlower400leaves,especiallyinH-4,pigmentationofeachchloroplastwascomparable401betweenupperandlowerleaves.
Theobservationsuggeststhattheextentsof402celldeathcorrelatewiththenumberofcellsproducingROSbutnotwiththe403magnitudeofROSproduction.
404405Discussion406WepreviouslyestablishedaninduciblesilencingsystemforHSP90Cin407tobaccoandconfirmedthatthesilencingofHSP90Calonecouldleadplantsto408chlorosis(Bhoretal.
,2017b).
Thesystemwouldhaveanadvantageoverthe409experimentalsystemswiththevirus-orviroid-infectedplantsintheanalysisof410.
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029116doi:bioRxivpreprint14mechanismsunderlyingthedevelopmentofdiseasesymptom-likephenotype411suchaschlorosis.
Itispossibleinthissystemtoanalyzeplantcellsthathave412committedtodevelopingbutnotexhibitedchlorosis.
Exploitingtheadvantagein413thepresentstudy,weexploredtheearlymolecularchangesleadingtothe414developmentofchlorosisusingRNA-seqanalysis.
Althoughstudieshaveshown415thetranscriptomechangesinvirus-andviroid-infectedplants(Mochizukietal.
,4162014;Xiaetal.
,2017;Zhengetal.
,2017),thestrengthofthepresentstudyis417thatwecoulddetectanychangesthatprecededetectablechlorosis.
418Fromtwoindependenttransgeniclines,H-4andH-6,whichshowmild419andseverechlorosisandgrowthsuppressionafterDextreatment,respectively,420weselectedtheformerfortheRNA-seqanalysis.
Inthetriplicateexperiment,an421individualDex-treatedofH-4plant(HD4inFig.
1BandFig.
2)showedamoderate422reductioninHSP90Cexpressionlevels(Fig.
2D)andthelackoftranscriptome423changesobservedinothertwoDex-treatedH4plants(Fig.
1B,Fig.
4,and424supplementaryFig.
S3).
BecausesuchavariationwithinindividualDex-treated425H-4plantswasconfirmedbyqRT-PCR(Fig.
1CandD),weomittedtheHD4data426fromourRNA-seqdataanalysis.
Althoughmorebiologicalreplicatesare427recommendedforreliableRNA-seqdataanalysisingeneral,DEseq2usedinthis428studyhasbeenshowntogivetheleastfalsepositiverate(Schurchetal.
,2016)429Therefore,mostofthedataanalysisresultsweretakenintoaccountinthisreport.
430IncontrasttoDex-treatedH-4plants,Dex-treatedH-6plantsshoweddrastic431downregulationofLHCabexpressionanddramaticinductionofICS1expression.
432Theresultssuggestamorerapidprogressionofmolecularchangestoward433chlorosisinH-6thaninH-4plants,whichmaybemoresuitablethanH-6for434analyzingtheearlymolecularchanges.
435WefoundseveralcharacteristictranscriptomechangesinHD2andHD3436plants,whichwebelievedtobeonthewaytodevelopchlorosis.
Firstly,the437downregulationwasobservedingenesinvolvedin(group1)photosynthesisand438plastidorganization,(group2)primaryandsecondarymetabolisms,and(group4393)cellandplantgrowth.
Secondly,thegenesinvolvedin(group4)immune440responseaccompanyingcelldeath,and(group5)ERstressresponse.
Finally,in441thegeneinvolvedinresponsetodifferentabioticstresses,asubsetwas442.
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029116doi:bioRxivpreprint15upregulated,andtheotherwasdownregulated.
443Amongthetranscriptomechangesabove,downregulationofchloroplast444andphotosynthesis-relatedgenes(CPRGs)orgroup1geneshasbeenwidely445reportedinsymptomatictissuesofdifferentcombinationsofhostplantsand446viruses(Satohetal.
,2010;PostnikovaandNemchinov,2012;Mochizukietal.
,4472014;Zanardoetal.
,2019).
AlthoughitisnaturalthattheexpressionofCPRGs448isdownregulatedinchlorotictissue,thepresentstudy,togetherwithourprevious449results(Waliullahetal.
,2014,2015;Bhoretal.
,2017a,b)stronglysuggestthat450theCPRGsdownregulationprecedingvisiblechlorosisistheprimarypathwayof451chlorosisdevelopment.
ThemechanismunderlyingtheCPRGsdownregulation452maydifferwithinpathosystems,butthepresentstudysuggestsapossible453involvementofretrogradesignaling(RS)inchlorosisdevelopmentinducedby454sub-viralpathogens.
Thedownregulationofgroups2and3geneswouldalsobe455attributedtotheRSactivation,whichreprogramstranscriptomefromgrowthand456differentiationstatetostressresponsestate(Crawfordetal.
,2018).
TheRNA-457seqdatasuggesttheactivationofRSpathwaysasmanifestedbytheincreased458expressionoftranscriptionfactorsinvolvedinRS-mediatedtranscriptome459changesanddecreasedexpressionofthoseinvolvedinchloroplastbiogenesis460(SupplementaryTableS8)(Leisteretal.
,2014).
GivenRSpathwaysareactivated461inthechlorosismodelofthepresentstudy,ROScouldbeaprimarysignalaswe462detectedH2O2productioninchloroplastsinadayaftertheinductionofHSP90C463silencing.
H2O2moleculesareassumedtomoveacrossthebiologicalmembrane464(HenzlerandSteudle,2000),andthus,wouldmovefromchloroplasttonucleo-465cytoplasmicspacetoactivatedifferentsignalingpathways.
Although466transcriptomeprofileandROSdetectionsupporttheRSactivationduring467chlorosisdevelopment,otherRSsignalingmoleculesshouldbeanalyzedto468confirmtheactivationofRSpathwaysinthepresentmodelsystem.
Itis469noteworthythatcyanobacterialHSP90,HtpG,interactswithandmodulatesthe470activityofuroporphyrinogendecarboxylaseandthusregulatingtetrapyrrole471biosynthesis(Saitoetal.
,2008).
472Amonggroup4orimmunitygenes,ICS1washighlyupregulatedinthe473chlorosismodel(Fig.
1C,SupplementaryFig.
S1,SupplementaryTablesS4,and474.
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04.
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029116doi:bioRxivpreprint16S6).
InadditiontopathogenattackandUVirradiation,ICS1expressionis475reportedlyupregulatedwithexcesslightandβ-cyclocitral,aretrogradesignaling476moleculeproducedbytheoxidationofβ-carotenewithsingletoxygen(Lvetal.
,4772015).
TheupregulationofICS1expressioninthepresentmodelsystemcould478alsoberegulatedbysomesignal(s)fromchloroplasttothenucleus.
OurRNA-479seqdatahasindicatedsignificantupregulationofthreetranscriptionfactors,480SARD1,CBP60g,WRKY28,whichhavesignificantrolesinICS1geneactivation481(Zhangetal.
,2010;vanVerketal.
,2011;Wangetal.
,2011;Sunetal.
,2015)482andtheirflg22-inducedexpressionreportedlydependsonchloroplast-localized483calciumsensor,CAS(Nomuraetal.
,2012).
Becausecytoplasmiccalcium-484dependentproteinkinaseshaveshowntoregulatethosetranscriptionfactors485(BoudsocqandSheen,2013;Poovaiahetal.
,2013),theimportanceof486chloroplastsignalinICS1inductionneedstobeclarifiedbyfurtherstudy.
487ICS1isthekeyenzymeofSAbiosynthesis,whichisrequiredforboth488localandsystemicacquiredresistance,whileSAsynthesizedthroughthis489pathwayseemstopotentiateplantcelldeath(NawrathandMétraux,1999;Gross490etal.
,2006;Strawnetal.
,2007;Garcionetal.
,2008;Chenetal.
,2009).
Itiswell491knownthatSAinducescelldeathinplantsasadefenseresponse(Brodersenet492al.
,2005;Radojiietal.
,2018).
WeobservedtheH2O2productioninthe493chloroplastofDex-treatedi-hpHSP90Cplants(Fig.
5)andtheupregulationof494ROS-responsivegenes(Fig.
4,SupplementaryTableS6).
ROSproducedinthe495chloroplast(Fig.
6)caninduceSAbiosynthesismostlikelythroughICS1gene496activation(Wildermuthetal.
,2001;Garcionetal.
,2008;Herrera-Vásquezetal.
,4972015).
Takentogether,thecelldeathweobservedinthechlorosismodelplants498(Fig.
5)issuggestedtobefirsttriggeredbytheROSproductionandthen499activatedthroughSA-ROSself-amplificationloop(Vlotetal.
,2009).
Althoughwith500theleastplantgrowth,ourpreliminaryexperimentofplantcultureinthedark501suggeststhechlorosisdevelopmentrequiresthelight-dependentROS502production(SupplementaryFig.
S4).
Studieshavereportedthatlight-dependent503ROSproductioninchloroplastleadstotheHR-likecelldeathandantiviral504resistance(Chandra-Shekaraetal.
,2006;Liuetal.
,2007;Chenetal.
,2015;505Hameletal.
,2016).
However,thesignificanceofSAincelldeathinthepresent506.
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029116doi:bioRxivpreprint17modelsystemneedstobestudiedfurther.
507Inadditiontodefensegenes,wefoundthatERstressresponsegenes508wereupregulatedinthechlorosismodel(Table1).
Althoughphysicalinteraction509betweenchloroplastsandERhasbeendemonstrated,noproteintransportwas510confirmedbetweentheseorganelles(Schattatetal.
,2012;Bartonetal.
,2018;511LiuandLi,2019).
Therefore,itisunlikelythatlossofproteinqualitycontrolin512chloroplastcoulddirectlyinducetheunfoldedproteinresponse(UPR).
The513indirectinductionofUPRbytheimpairedHPS90Csupplywouldinvolveeither514retrogradesignalingorstresshormoneresponse.
Aretrogradesignalmolecule,515methylerythritolcyclodiphosphate(MEcPP),hasbeenshowntoinduceUPRin516ER(Walleyetal.
,2015;Bennetal.
,2016)andSAhasbeenshowntoactivate517themajorIRE1-bZIP60pathwayofUPR(Nagashimaetal.
,2014;ParkandPark,5182019).
FurtherstudyofthemodeofinductionofUPRwouldprovideuswith519insightintoitsroleinchlorosisdevelopment.
520Wesummarizethemoleculareventsdiscussedabovethatleadtothe521developmentofchlorosisaftertheinducedsilencingoftheHSP90Cgene(Fig.
5227).
ReducedHSP90Csupplywouldimpairproteinqualitycontrolinthe523chloroplast,hampernormalchloroplastfunction,andthus,leadtotheproduction524ofROSinthechloroplast.
ROSwouldinduceSAproduction,andtheywould525developaself-amplifyingloop.
ROSwouldalsoactivatethechloroplast526retrogradesignaling,whichresultsintheupregulationofstress-responsivegenes,527includingpathogenesis-related(PR)genes,andthedownregulationofCPRGsin528thenucleus,whichismostlikelytobetheprimarycauseofchlorosis.
Inaddition,529theHR-likecelldeathpathwaywouldbeactivatedbyROS,SApathway,other530stresshormonepathways,and/orUPR.
Itiswellknownthatchlorosisinducedby531differentcueendsuptothedeathofplanttissue.
Therefore,theactivationofthe532celldeathresponsecouldhavearoleinchlorosisinductionoraccelerationofthe533processesleadingtovisiblechlorosis.
Althoughtheimportanceofcelldeath534inductioninchlorosisremainstoestablished,thepresentstudyshedslightonthe535importanceofprioractivationofthecelldeathpathwayinchlorosisdevelopment.
536Inthecaseofavirusorviroidinfection,theactivationoftheimmune537pathwaymustbeunfavorabletothepathogens.
Therefore,itisanacceptable538.
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04.
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029116doi:bioRxivpreprint18ideathatpathogenshavesomewaytoinhibitthedefenseresponseactivatedby539thechloroplaststress.
Indeed,PLMVdandsomeothervirusessuchasCMV540induceclearbleaching-typechlorosis,inwhichcelldeathisunlikely(Mochizuki541andOhki,2011;Navarroetal.
,2012).
ItisnotablethatPLMVdinfectionreportedly542producessiRNAstoseveralotherhostgenes,includingNB-LRRtypedisease543resistancegenes,inadditiontothosetoHSP90C(Navarroetal.
,2012;Chiumenti544etal.
,2018).
Theanalysisofpathogens'anti-defensestrategieswouldpavethe545wayforabetterunderstandingofmechanismsunderlyingthedevelopmentof546virusdiseasesymptoms.
547548Acknowledgments549ThisstudywassupportedinpartbyTheUnitedGraduateSchoolof550AgriculturalSciences,EhimeUniversity,andJSPSKAKENHIgrants26292026,55115K14664,and19K06055toKK.
RNA-seqanalysiswasconductedunderthe552supportoftheCooperativeResearchGrantoftheGenomeResearchfor553BioResource,NODAIGenomeResearchCenter,TokyoUniversityofAgriculture.
554IslamShaikhulwassupportedbytheMEXT.
555556.
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04.
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029116doi:bioRxivpreprint27FigureLegends806Fig.
1.
Phenotypeandtranscriptomechangeafterinducedsilencingof807HPS90intransgenictobaccoplants.
(A)Non-transformedSR1(SR1),i-808hpHSP90C-4(H-4)andi-hpHSP90C-6(H-6)plantsweregrownforthreeweeks,809sprayedwith0.
01%Tween-20containing0.
5%ethanol(Control)or50MDex810(Dex),andphotographedat7dayspost-treatment(dpt).
Scalebarsindicate1811cm.
(B)PrincipleComponentAnalysisofRNA-seqdata.
RNApreparationsfrom8121-dptcontrol-treatedSR1(SC2,3,and4)andDex-treatedSR1(SD1,3,and4),813control-treatedi-hpHSP90C-4(HC1,3,and4)andDex-treatedi-hpHSP90C-4814(HD2,3,and4)wereanalyzedusingDESeq2.
(CandD)VariationofHSP90C815silencinginindividualDex-treatedi-hpHSP90C-4plants.
Expressionlevelsof816ICS1(C)andLHCab(D),representativesofup-anddown-regulatedgenesfound817intheRNA-seqanalysis,respectively,wereplottedwiththoseofHSP90C.
The818expressionlevelsofthesegenesrelativetoafixedstandardsample(square)819werequantifiedineightindividualplantseachofcontrol-(circles)andDex-treated820(triangles)i-hpHSP90C-4plants.
821822Fig.
2.
ExpressionchangesofHSP90familygenesinRNA-seqsamples.
823RelativenormalizedreadcountsinallRNA-seqsamplesareshownfortobacco824homologsofArabidopsisHSP90familygenes:cytosolicHSP90.
1(A),nucleo-825cytosolicHSP90.
2(B),cytosolicHSP90.
4(C),chloroplastHSP90CorHSP90.
5826(D),mitochondrialHSP90.
6(E),ER-localizedHSP90.
7(F).
Statistical827significanceofthedifferenceinreadcountswasevaluatedbetweencontrol-828treated(HC1,3,and4)andDex-treated(HD2,and3)inDESeq2analysis.
ns,829notsignificant.
DatafromtheHD4samplewasomittedasmentionedinthetext.
830831Fig.
3.
Venndiagramsofdifferentiallyexpressedgenes.
Resultsfrom832differentialexpressionanalysiswithDESeq2weremadeinthreedifferent833comparisons:Dex-treatedH-4vscontrolH-4(HDvs.
HC),Dex-treatedH-4vs834Dex-treatedSR1(HDvs.
SD),andDex-treatedH-4vsControlSR1(HDvs.
SC).
835Thedifferentiallyexpressedgenes(DEGs)weredetectedusingDESeq2as836mentionedinMaterialsandmethods.
AandBshowDEGsdetectedinthe837.
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04.
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029116doi:bioRxivpreprint28analysiswithmRNAIDinthereferencetranscriptome,andCandDshowthose838annotatedwithAGIcodes.
AandCshownumbersofupregulatedgeneswhereas839BandDshowthoseofdownregulatedgenes.
840841Fig.
4.
HierarchicalclusteringofupregulatedDEGswithselected842annotations.
ThegenesannotatedwithGOterms,responsetoSA843(GO:0009751),responsetoJA(GO:0009753),responsetooxidativestress844(GO:0006979),andcelldeath(GO:0008219)wereselected(Supplementary845TableS9),andtheirrelative-normalizedcountdatawereusedtodrawaheatmap.
846Eachcolumnrepresentsasample,andeachrowrepresentsageneselected.
847Differencesintheexpressionareshownindifferentcolors,redandbluerepresent848theup-anddown-regulatedexpression,respectively.
849850Fig.
5.
Detectionofcelldeathinchloroticleaves.
Controlplants(SR1;A–F,851anda–d),i-hpHSP90Ctransgenicline4(H-4;G–Lande–h),andline6(H-6;M–852Randi–l)plantsweregrown,treatedwithcontrolorDexsolution.
Theywere853harvestedandphotographedat7dpt(A,DG,J,M,P,andS;representative854plantsfromtriplicateexperiments;scalebarsdenote1cm)followedbycelldeath855assays.
SR1leavestransientlyexpressingTBSVP19for2daysservedasa856positivecontrol(S,T,andm).
Celldeathassaywasperformedinupperleaves857(pointedbyarrows)andlowerleaves(pointedbyarrowheads)Leafdisksof6mm858indiameterwerestainedwithtrypanblueandphotographedatmacroscopic(B,859C,E,F,H,I,K,L,N,O,Q,R,andT)andmicroscopic(a–m;scalebarsdenote860100μm)levels.
U,quantificationofcelldeathbyanelectrolyteleakageassay.
861PC,positivecontrolwithtransientTBSVP19expression;SC,control-treated862SR1;SD,Dex-treatedSR1;H4C,control-treatedline4;H4D,Dex-treatedline4;863H6C,control-treatedline6;H6D,Dex-treatedline6;U,upperleaves;L,lower864leaves.
Errorbarsdenotestandarddeviationsintriplicateexperiments.
Different865lettersindicatestatisticallysignificantdifferencebetweentreatments(LSDtest,866PComparisonbetweentheexpressionofHSP90Cgene894andselectedDEGsinnon-transformedcontrolSR1andi-hpHSP90Cline6plants.
895X-axisshowsthelog2(FC)value(fromqRT-PCRanalysis)oftwoselectedgenes,896ICS1andLHCab,andY-axisindicatesthelog2(FC)valueofHSP90Cgene897relativetoacommonstandardsample.
898SupplementaryFig.
S2.
(A-C)MAplotshowingtheoverviewoftheDEGsintwo-899groupofcomparison(i.
e.
Dextreatedanduntreated).
900SupplementaryFig.
S3.
Heatmapsshowingtheoxidative,osmotic,andsalt901.
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029116doi:bioRxivpreprint30stress-responsivegenes.
Eachcolumnrepresentsasample,andeachrow902representsageneselected.
Differencesinexpressionareshownindifferent903colors,redandbluerepresenttheup-anddown-regulatedexpression,904respectively.
905SupplementaryFig.
S4.
Lackofchlorosisresponseindark-growni-hpHSP0C906(6-1)plants.
907SupplementaryTable1.
TranscriptIDandGeneIDmappingfile.
908SupplementaryTable2.
PrimersusedforqRT-PCRanalysisinthepresentstudy.
909SupplementaryTable3.
SummaryofRNA-Seqdataandmappingoftheclean910readstoN.
tabacumTN90referencetranscriptome.
S,SR1(ornon-911transformant);H,i-hpHSP90C;D,Dex-treated;C,Control.
912SupplementaryTable4.
ListofupregulatedDEGsinchlorosismodelplants913(commonlyupregulatedDEGsinallthreecomparisons).
914SupplementaryTable5.
ListofdownregulatedDEGsinchlorosismodelplants915(commonlydownregulatedDEGsinallthreecomparisons).
916SupplementaryTable6.
ListofGO(Biologicalprocess)enrichedwith917upregulatedDEGsofchlorosismodelplants.
918SupplementaryTable7.
ListofGO(Biologicalprocess)enrichedwith919downregulatedDEGsofchlorosismodelplants.
920SupplementaryTable8.
ExpressionlevelsofRS-relatedtranscriptionfactor921genes.
922.
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04.
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029116doi:bioRxivpreprint31Table1.
SelectedGOterms(Biologicalprocess)forupregulatedgenesini-hpHSP90CtransgeniclineafterDextreatment.
GOTermsTotalDEGsFoldenrich.
FDRImmunesystemprocess3721012.
73componentorganization25034541.
43<0.
01Plastidorganization2981363.
6<0.
01Responsetostimulus53279031.
34<0.
01Responsetoosmoticstress5451031.
490.
01Responsetooxidativestress392871.
75<0.
01Responsetosaltstress469911.
530.
01Responsetoauxin310721.
83<0.
01Responsetolightstimulus6801982.
29<0.
01Rhythmicprocess122432.
78<0.
01Circadianrhythm109423.
04<0.
01*AA=AminoAcid.
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04.
07.
029116doi:bioRxivpreprintFig.
1CBSR1ControlASR1H-6DexH-6H-4H-4-2-1.
5-1-0.
500.
511.
5-50510HSP90C(log2FC)ICS1(log2FC)-2-1.
5-1-0.
500.
511.
5-3-2-101HSP90C(log2FC)LHCab(log2FC)StandardControlDexStandardControlDexD1.
0cm-2.
5-1.
5-0.
50.
51.
52.
5SC2SC3SC4SD1SD3SD4HC1HC3HC4HD4HD2HD3-2.
5-1.
5-0.
50.
51.
52.
5SC2SC3SC4SD1SD3SD4HC1HC3HC4HD4HD2HD3ABHSP90.
1(Cytosol)HSP90.
2(Cytosol,Nucleus)-2.
5-1.
5-0.
50.
51.
52.
5SC2SC3SC4SD1SD3SD4HC1HC3HC4HD4HD2HD3-2.
5-1.
5-0.
50.
51.
52.
5SC2SC3SC4SD1SD3SD4HC1HC3HC4HD4HD2HD3HSP90.
4(Cytosol)HSP90C(Chloroplast)CD-2.
5-1.
5-0.
50.
51.
52.
5SC2SC3SC4SD1SD3SD4HC1HC3HC4HD4HD2HD3-2.
5-1.
5-0.
50.
51.
52.
5SC2SC3SC4SD1SD3SD4HC1HC3HC4HD4HD2HD3EFHSP90.
6(Mitochondrion)HSP90.
7(ER)Relativeexpression[Log2(FC)]nsP<0.
01nsnsnsP<0.
01Fig.
2CHDvsHCHDvsSD2746(61.
4%)377(8.
4%)390(8.
7%)289(6.
2%)184(4.
1%)282(6.
3%)214(4.
8%)HDvsSCHDvsHCHDvsSD3490(59.
0%)528(8.
9%)601(10.
2%)312(5.
3%)180(3.
0%)407(6.
9%)394(6.
7%)HDvsSCUpDownDAHDvsHCHDvsSD7267(55.
9%)1390(10.
7%)1209(9.
3%)832(6.
4%)570(4.
4%)1098(8.
4%)639(4.
9%)HDvsSCHDvsHCHDvsSD8042(47.
4%)2150(12.
7%)2183(10.
2%)1428(8.
4%)702(4.
1%)1249(7.
4%)1230(7.
2%)HDvsSCUpDownBFig.
3SAROSJACellDeathROSJA-202log2(FC)HD4HC1SD1SC4SD4HC3SC2SC3SD3HD3HD2Fig.
4H-6ControlDexUpperLowerUpperLowerSR1Positivecontrol(TBSVP19)H-6H-41cmH-4ControlUpperLowerUpperLowerSR1Positivecontrol(TBSVP19)100μmDexeabababaabababdabbabc020406080100Relativeconductivity(%)PCSC-USC-LSD-USD-LH6C-UH6C-LH6D-UH6D-LH4C-UH4C-LH4D-UH4D-LUFig.
5ADGJMPBECFHKILNQORSTadgjmbecfhkilFig.
6H-4H-6SR1ControlDexPositiveControlUpperLowerUpperLower100μmADGJMBECFHKILChloroplastReducedHSP90CSupplyLossofProteinQualityControlSASelf-amplifyingLoopROSLightPRGenesCPRGsNucleusActivationofCellDeathJA/ET/ABAPathwaysChlorosisRetrogradeSignalsHsp90ChpRNAFig.
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