International Journal o f Molecular Sciences Article Transcriptome Analysis Identifies a 140 kb Region of Chromosome 3B Containing Genes Specific to Fusarium Head Blight Resistance in Wheat XinLi1,2,†,ShengfuZhong1,†,WanquanChen2,*,SyedaAkashFatima1,QianglanHuang1, QingLi1,3,FeiquanTan1andPeigaoLuo1,2,* ID 1 ProvincialKeyLaboratoryofPlantBreedingandGenetics,SichuanAgriculturalUniversity,Chengdu611130, China;[email protected](X.L.);[email protected](S.Z.);[email protected](S.A.F.); [email protected](Q.H.);[email protected](Q.L.);[email protected](F.T.) 2 StateKeyLaboratoryforBiologyofPlantDiseasesandInsectPests,InstituteofPlantProtection, ChineseAcademyofAgriculturalSciences,Beijing100193,China 3 DepartmentofBiologyandChemistry,ChongqingIndustryandTradePolytechnicInstitute, FulingDistrictofChongqing,Chongqing408000,China * Correspondence:[email protected](W.C.);[email protected](P.L.); Tel:+86-28-86290978(P.L.);Fax:+86-28-8629-0870(P.L.) † Theseauthorscontributedequallytothiswork. Received:4February2018;Accepted:12March2018;Published:14March2018 Abstract: Fusariumheadblight(FHB),mainlycausedbyFusariumgraminearum,isoneofthemost destructive fungal diseases of wheat (Triticum aestivum L.). Because of the quantitative nature of FHBresistance,itsmechanismispoorlyunderstood. Weconductedacomparativetranscriptome analysis to identify genes that are differentially expressed in FHB-resistant and FHB-susceptible wheat lines grown under field conditions for various periods after F. graminearum infection and determinedthechromosomaldistributionofthedifferentiallyexpressedgenes(DEGs). Foreachline, theexpressioninthespike(whichexhibitssymptomsintheinfectedplants)wascomparedwiththat intheflagleaves(whichdonotexhibitsymptomsintheinfectedplants). Weidentifiedanislandof 53constitutiveDEGsina140kbregionwithhighhomologytotheFhbL693bregiononchromosome 3B. Of these genes, 13 were assigned to specific chloroplast-related pathways. Furthermore, onegeneencodedinositolmonophosphate(IMPa)andtwogenesencodedribulose-1,5-bisphosphate carboxylase/oxygenase(RuBisCO).Ourfindingssuggestthatthetemporarysusceptibilityinlocally infected spikes results from the cross-talk between RuBisCO and IMPa, which blocks secondary signaling pathways mediated by salicylic acid and induces a systemic acquired resistance in the distantleaftissue. Keywords: fusariumheadblight;geneisland;photosynthesis;transcriptome;wheat 1. Introduction Wheat (Triticum aestivum L.) is the most widely grown crop worldwide and accounts for approximately20%ofthecaloriesconsumedbyhumankind[1]. WheatFusariumheadblight(FHB), also called “wheat cancer” or “scab”, is an economically important fungal disease that is mainly causedbyFusariumgraminearum. FHBseriouslythreatenswheatproductionaroundtheworld[2]. TheaccumulationofmycotoxinsproducedbyFusarium, especiallydeoxynivalenol, inwheatand wheatproducts,causesacutefoodpoisoninginhumansandharmsanimalsthatconsumetheinfected grain[3,4]. Whilecropmanagementpracticesandchemicalapplicationsmayreducethedamagecaused byFHB,thedevelopmentofresistantcultivarsiscriticalforcombatingthisdisease[5,6]. Thelevel Int.J.Mol.Sci. 2018,19,852;doi:10.3390/ijms19030852 www.mdpi.com/journal/ijms Int.J.Mol.Sci. 2018,19,852 2of19 of resistance to FHB in wheat cultivars is low, even among cultivars that are less susceptible to FHB, such as Sumai 3 and Wangshuibai. To date, no genes have been characterized that confer completeresistancetoFHBinanycultivar,andthishasimpededthebreedingofresistantwheat[7]. The molecular mechanism underlying plant defense against F. graminearum infection is unknown. Frombotheconomicandhumanhealthperspectives,enhancingFHBresistanceinwheatiscriticalfor reducingyieldlossandmycotoxincontamination. FHB resistance is quantitative in nature, involving the additive effects of several genes [8]. ThegeneticfactorsunderlyingresistancetoFHBarealsohighlycomplex,andexperimentalerrorsmay havemaskeddifferencesintheresistancelevelsamonggenotypes[9]. Thedevelopmentofresistant cultivarshasbeenimpededbecauseofourpoorunderstandingofthegeneticmechanismsofFHB resistance. Geneexpressionprofilechangeshaveprovidedkeyinsightsintothegeneticmechanisms involvedinpathogeninvasion. Recently,sixputativeFHBresistancegeneswerelocalizedwithinthe Fhb2regionofchromosome6Busingtranscriptomeanalysis[10]. Thus,transcriptomeanalysisisa usefultoolforelucidatingthegeneticbasisofthiscomplextrait. Inadditiontocausingsymptomsinthespike,FHBseriouslyaffectsthedevelopingseeds.Sincethe developingseedsarethemostimportantcarbohydratesinks,andthereisadynamicbalancebetween carbohydratesources(e.g.,theleaves)andsinks[11,12],changesingeneexpressioninvarioustissues wouldbeexpectedtooccuruponplantpathogenattack[13]. Therefore,itwouldbeinterestingto characterizechangesingeneexpressioninboththeleavesandthespikesofwheatplantsduringFHB infectionandtousetheexpressionlevelsintheleavesasacontrolwhenconsideringgeneexpression changesinthespike. Genotypes with high-resistance and simple-resistance mechanisms provide a valuable pool of genes to improve crop resistance and are a prerequisite for fully understanding the resistance mechanism. In the past, studies of FHB resistance have focused on two Chinese wheat landraces, Wangshubai and Sumai 3, and their derived offspring. It is important to identify new sources of resistanceagainstFHBinwheat,bothtoelucidatetheresistancemechanismsandtoimprovecultivars. Inapreviousstudy,wedevelopedandidentifiedawheatlineL693fromF familiesofacross 6:7 betweenwheatcultivarMY11andthelineYU25,asthedonorofthreediseaseresistancegenes[14,15] that exhibit excellent resistance to stripe rust [14,16,17], powdery mildew [17], and FHB [17–19]. ThesisterlineL661,derivedfromthesameF families,wassusceptibletostriperustandFHB[17,18]. 6:7 FurthermoleculartestsrevealedthatL693andL661hadhighlysimilargeneticbackgroundsinwhich 41(2.4%)polymorphiclociweresharedamong1703lociamplifiedby781simplesequencerepeat(SSR) primers[16]. ThissegregationinresistanceshowedthatresistanceintheF andF populationswas 2 2:3 inheritedastwoMendelianfactors,FhbL693aandFhbL693b. Wethendetectedtwomajorquantitative traitloci(QTLs), Qfhs-2BandQfhs-3B,associatedwithFHBresistance[20]. Informationregarding pedigree, inheritance, resistanceresponse, chromosomallocation, andmarkerdiagnosisindicated thatQfhs-3BwasdifferentfromFhb1: itbehavedasasingleMendelianfactorandwasgiventhegene symbolFhbL693b[20]. L693isclearlyanimportantsourcefordiseaseresistanceinwheatbreedingprograms. However, despite the successful detection and mapping of QTLs for FHB resistance in L693, the underlying molecularbasisofFHBresistanceremainstobeelucidated. The objective of the present study was to explore the molecular basis of FHB resistance in wheat. Two wheat genotypes, consisting of the sister lines L693 (resistant) and L661 (susceptible) were inoculated with F. graminearum. As the differences caused by the genetic background were minimized in these lines, we could easily identify key resistance (R) genes and the chromosomal regionsresponsibleforFHBresistance. Theexpressionofseveralrandomlychosengenesinthespike (inoculatedtissue;localtissue)andleaf(non-inoculated;control,distanttissue)wasprofiledusing RNA-sequencing(RNA-Seq)andconfirmedusingquantitativereal-timePCR(qPCR).Differentially expressedgenes(DEGs)andtheirchromosomaldistributionswerecomparedinthetwogenotypes. Furthermore,wecomparedgeneexpressiondynamicsintheinoculatedspikeandnon-inoculatedleaf Int.J.Mol.Sci. 2018,19,852 3of19 ofthetwogenotypes,withtheaimofidentifyingthefunctionsandmetabolicpathwaysofgenesinthe FhbL6In9t3. Jb. Mcholr. oScmi. 2o0s1o8,m 19a, xl rFeOgRi PoEnERth RaEtVaIErWe d ifferentiallyexpressedinthespikesandleavesofthe3 towf 1o9 lines followingfungalinfection. in the FhbL693b chromosomal region that are differentially expressed in the spikes and leaves of the OurresultsprovideinsightintothegeneticbasisofFHBresistanceinL693andsuggestamolecular two lines following fungal infection. strategyforFHBresistancebasedonglobalexpressionprofiling. Furthermore,thisworkidentifies Our results provide insight into the genetic basis of FHB resistance in L693 and suggest a potentialgenesforbreedingapplications. molecular strategy for FHB resistance based on global expression profiling. Furthermore, this work identifies potential genes for breeding applications. 2. Results 2. Results 2.1. L693andL661ExhibitDifferentFHBResistanceLevel,butSimilarTranscriptomeSizes 2.1. L693 and L661 Exhibit Different FHB Resistance Level, but Similar Transcriptome Sizes WefoundthatL693exhibitedahighlevelofFHBresistance[20];onlytheinoculatedspikelet haddriedWoeu ftoaunndd dthiaetd La6t932 1exdhaiybisteadf tae rhiignho cleuvlaelt ioofn F(HDBA rIe)siwstiatnhceF .[2g0r]a;m oinnlyea trhuem in.oBcyulcaotendt rsapsitk,eilnetL 661, afterhthade dsarimede opuet rainodd doifedti mat e2,1 adlamyos satfttehr eineonctuilraetisopni (kDeAcIo) nwtaitihn Fin. ggrathmeinienaorucmu.l aBtye dcosnptriakset,l eint hLa66d1,d ried after the same period of time, almost the entire spike containing the inoculated spikelet had dried out outanddied(Figure1A).Furthermore,thegrainsofL693appearedfullyfilledat21DAI,asnormal, and died (Figure 1A). Furthermore, the grains of L693 appeared fully filled at 21 DAI, as normal, while the grains of L661 were shriveled (Figure 1B). The percentage of diseased spikes (PDS) was while the grains of L661 were shriveled (Figure 1B). The percentage of diseased spikes (PDS) was significantlyhigher(p<0.01),onthebasisofmultiplecomparisons,inL661thaninL693(Figure1C). significantly higher (p < 0.01), on the basis of multiple comparisons, in L661 than in L693 (Figure 1C). Figure 1. Comparisons of L693 and L661 in terms of Fusarium head blight (FHB) severity and Figure 1. Comparisons of L693 and L661 in terms of Fusarium head blight (FHB) severity and differentially expressed genes. (A) Photograph of spikes of L693 (left) and L661 (right), grown under differentiallyexpressedgenes.(A)PhotographofspikesofL693(left)andL661(right),grownunder greenhouse conditions, 21 days after inoculation with Fusarium graminearum in 2013. For L693, disease greenhouse conditions, 21 days after inoculation with Fusarium graminearum in 2013. For L693, symptoms are visible only in the inoculated spikelet, whereas, for L661, they are evident in the entire disease symptoms are visible only in the inoculated spikelet, whereas, for L661, they are evident spike. Bars, 1 cm. (B) Comparison of kernel health between the resistant line L693 (left) and the inthesuesncteipretibslpe ilkinee. BLa66rs1, (1rigcmht). i(nB 2)0C1o3.m Tphae rkiseornnelosf okf eLr6n9e3l wheerael tfhulblye tfwilleeden, wthheiler ethsiosstea noft Lli6n6e1 Lw6e9r3e (left) andtshherivsuelsecde pantidb lpeinlkiniseh.L B6a6r1s, (1r icgmht. )(Cin) M20u1lt3i.plTe hcoemkpearrniesolsn ooff Lth6e9 p3ewrceenretafguel loyf dfiilsleeads,edw shpiilkeetlehtos seof L661(wPDerSe) isnh r2i0v1e3l eadnda n20d14p.i nErkriosrh b.aBras risn,d1iccamte. t(hCe )sMtanudlatirpdl eercroorm opf aPrDisSo;.n Moefatnhse wpietrhc ethnet asgameeo fledttiesre ased spike(laebtsov(Pe DerSr)orin ba2r0)1 a3rea nndot2 s0i1g4n.ifEicrarnotrlyb adrifsfeinrednitc a(pt e> t0h.e01s)t aanndd awrdithe rdroifrfeorefnPtD leSt;t.erM aerea nssigwniiftihcatnhtleys ame letterd(iafbfeorvenete (rpr ≤o r0.b0a1r).) arenotsignificantlydifferent(p>0.01)andwithdifferentletteraresignificantly different(p≤0.01). Int.J.Mol.Sci. 2018,19,852 4of19 RInNt. JA. Msola. mScip. 2l0e1s8,w 19e, rxe FOtaRk PeEnERf rRoEmVIEsWp ik eandleaftissuesoftheresistantlineL693andthesu4 sofc 1e9p tible line L661 at three infection stages: 0, 24, and 72 h after inoculation (HAI) with F. graminearum, RNA samples were taken from spike and leaf tissues of the resistant line L693 and the susceptible andanalyzed(Table1). Genesexpressedinatleastthreeoutof12sampleswereconsideredexpressed line L661 at three infection stages: 0, 24, and 72 h after inoculation (HAI) with F. graminearum, and genesa.nIanlytzoetda l(,T1a1b2le, 418).4 Gaennneos teaxtpedresgseende isn aant dlea6s1t, 5th9r6eee xopurt eosfs e12d sgaemnpelsesw weerereo cbotnasiindeedredb yexgperneessreadl filter (cutogffenveasl.u Ine t≥ota1l), ;1t1o2,4g8e4t ahningohtlayterde lgieanbelse adnadt a6,1,a59c6u etxopffrevsaselud ege≥n2es wwaesrea opbptaliiended, abnyd g,en47er,4al4 f3iltreerl iable expre(scsuetdoffg evnaleusew ≥e 1re); fiton aglelyt hidigehnltyifi reedli.abOleu tdoaftat,h ae sceu4to7f,f4 4v3aleuxep ≥r2e swseads gaepnpelise,d2, 4a5n3d,,2 4170,34,4a3n rdel3ia9b9leg enes intheexLp6r9es3sesdp igkeenwese wreeruep friengaullyla itdeednctiofimedp. aOreudt owf itthhesteh e47L,464631 esxppirkees,sewdh geerneeass, 6244453,,7 25140,3a, nadnd3 73499g enes genes in the L693 spike were upregulated compared with the L661 spike, whereas 644, 754, and 374 weredownregulatedat0,24,and72HAI,respectively(Figure2A).However,inleaftissue,914genes genes were downregulated at 0, 24, and 72 HAI, respectively (Figure 2A). However, in leaf tissue, 914 were downregulated and 1192 and 375 genes were upregulated in L693, compared to 966 genes genes were downregulated and 1192 and 375 genes were upregulated in L693, compared to 966 genes that were upregulated, and 842 and 311 that were downregulated in L661 at 0, 24, and 72 HAI that were upregulated, and 842 and 311 that were downregulated in L661 at 0, 24, and 72 HAI (Figure (Figure2B),respectively. 2B), respectively. Figure 2. Comparison of differentially expressed genes in L693 and L661 following FHB infection. Figure2. ComparisonofdifferentiallyexpressedgenesinL693andL661followingFHBinfection. Number of upregulated genes (left, yellow circle), downregulated genes (right, blue circle), and genes Numberofupregulatedgenes(left,yellowcircle),downregulatedgenes(right,bluecircle),andgenes without differential expression (middle, purple circle) in L693 (resistant) compared to L661 withoutdifferentialexpression(middle,purplecircle)inL693(resistant)comparedtoL661(susceptible) (susceptible) at 0, 24, and 72 h after inoculation (hai) in spike (A) and leaf (B). at0,24,and72hafterinoculation(hai)inspike(A)andleaf(B). 2.2. Validation of the Differences in Gene Expression by RT-qPCR and Clustering Analysis 2.2. ValidationoftheDifferencesinGeneExpressionbyRT-qPCRandClusteringAnalysis To validate the differential gene expression of the 12 samples analyzed (i.e., two genotypes, two tissues, three time points), five genes were chosen randomly for RT-qPCR analysis (Table S1). The To validate the differential gene expression of the 12 samples analyzed (i.e., two genotypes, correlation between normalized mRNA-seq RPKM (Reads Per Kilobase per Million mapped reads) two tissues, three time points), five genes were chosen randomly for RT-qPCR analysis (Table S1). results and qRT-PCR expression values were high (R2 = 0.73) (Figure 3A, Supplementary Material 2: ThecorrelationbetweennormalizedmRNA-seqRPKM(ReadsPerKilobaseperMillionmappedreads) Table S1). In addition, 12 clustering samples based on Illumina read counts mapped against the resultsandqRT-PCRexpressionvalueswerehigh(R2=0.73)(Figure3A,SupplementaryMaterial2: Ensembl wheat genome sequence showed that the tissue type had the biggest effect on the changes Table S1). In addition, 12 clustering samples based on Illumina read counts mapped against the in gene expression, followed by the time after inoculation and the genotype of wheat used (Figure Ensem3Bb).l Twhhee calutsgteenrionmg reessueqltus eshnocweeshd othwaet dthteh datifftehreenticses uine gteynpee ehxapdretshsieonb ibgegtweseteenf ftehcet sopnikteh aencdh laenafg esin genewexasp greresasitoern t,hfaonl ltohwate bdetbwyetehne thtiem geenaofttyepreisn. oculationandthegenotypeofwheatused(Figure3B). Theclusteringresultsshowedthatthedifferenceingeneexpressionbetweenthespikeandleafwas greaterthanthatbetweenthegenotypes. Int.J.Mol.Sci. 2018,19,852 5of19 Table1.RNA-seqreadinformation. Tissue Samples RawData CleanReads Mapped Mapped% Multiple Multiple% Unique Unique% L661spikeat0HAI 48,619,708 44,064,636 31,413,442 71.3% 7,948,442 25.3% 23,472,782 74.7% L693spikeat0HAI 54,638,926 50,729,857 37,433,618 73.8% 15,223,441 40.7% 22,210,177 59.3% L661spikeat24HAI 80,401,044 14,261,236 6,799,856 47.7% 2,277,018 33.5% 4,522,838 66.5% spike L693spikeat24HAI 56,001,617 37,706,168 24,971,426 66.2% 7,770,208 31.1% 17,201,218 68.9% L661spikeat72HAI 64,589,048 47,444,396 29,577,848 62.3% 9,209,005 31.1% 20,368,843 68.9% L693spikeat72HAI 73,276,052 52,378,107 33,229,807 63.4% 9,061,381 27.3% 24,168,426 72.7% L661leafat0HAI 74,642,980 40,067,567 28,798,631 71.9% 9,266,948 32.2% 19,983,610 69.4% L693leafat0HAI 53,896,907 37,849,318 18,909,880 50.0% 8,498,338 44.9% 10,411,542 55.1% L661leafat24HAI 52,460,707 45,960,283 35,981,574 78.3% 20,792,164 57.8% 15,189,410 42.2% leaf L693leafat24HAI 54,132,118 45,690,464 32,199,399 70.5% 20,182,030 62.7% 12,017,369 37.3% L661leafat72HAI 50,556,369 26,746,381 18,315,598 68.5% 9,430,647 51.5% 6,319,347 34.5% L693leafat72HAI 49,560,263 26,728,939 18,240,700 68.2% 10,006,376 54.9% 8,234,324 45.1% HAI:Hoursafterinoculation. Int.J.Mol.Sci. 2018,19,852 6of19 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 6 of 19 Figure 3. The reliability of RNA-seq data, as demonstrated by qRT-PCR (Quantitative real time Figure 3. The reliability of RNA-seq data, as demonstrated by qRT-PCR (Quantitative real time polymerase chain reaction) and sample clustering. (A) Correlation between normalized mRNA-seq polymerasechainreaction)andsampleclustering.(A)CorrelationbetweennormalizedmRNA-seq RPRKPKMMr erseusultlstsa nanddq qRRTT-P-PCCRRe exxppreressssioionnv vaalluueess.. TThhee ssccaatttteerrpplloott sshhoowwss tthhee lloogg2 fofoldld cchhaanngge eoof fRRPPKKMM 2 and qRT-PCR expression values; a trend line is shown in red; (B) sample clustering based on counts andqRT-PCRexpressionvalues;atrendlineisshowninred;(B)sampleclusteringbasedoncounts of mapped Illumina reads. The dendrogram represents the hierarchical clustering of samples as of mapped Illumina reads. The dendrogram represents the hierarchical clustering of samples as determined by Euclidean distance. The heat map shows a false-color representation of the sample determinedbyEuclideandistance. Theheatmapshowsafalse-colorrepresentationofthesample correlation value. correlationvalue. 2.3. Differential Expression Analysis Revealed Tissue-Specific Expression Tendencies 2.3. DifferentialExpressionAnalysisRevealedTissue-SpecificExpressionTendencies Using the edgeR software [21], more DEGs were identified in the spike than in the leaf of both UsingtheedgeRsoftware[21],moreDEGswereidentifiedinthespikethanintheleafofboth L693 and L661 plants. Among the 3097 DEGs identified in the spike at 0 HAI, 14 were strongly L693 and L661 plants. Among the 3097 DEGs identified in the spike at 0 HAI, 14 were strongly upregulated (i.e., had logFC values greater than +12), but only two were strongly downregulated (i.e., upregulated (i.e., had logFC values greater than +12), but only two were strongly downregulated with logFC values lower than −12). High expression abundance (i.e., average logRPKM values greater (i.teh.,anw 3it)h wloags FfoCuvnadl uine s84lo owf tehret h2a45n3− u1p2r)e.gHuliagthede xgperneesss (ioshnoawbnu nind banlucee i(ni. eF.i,gauvreer 4aAge) cloomgRpPaKreMd tvoa tlhuee s gr6e4a4t edrothwannre3g)uwlaatsedfo ugenndeisn. A84t o2f4 thHeA2I4, 5t3heurpe rwegeurlea t2e8d57g eDnEeGs(ss hino wthne isnpbiklue,e iinnclFuigduinrge 41A3 )thcoatm wpaerree d tosttrhoen6g4ly4 udporwegnurelagtuelda taenddg oennleys .oAnet 2th4aHt AwIa,st hsterroenwgleyr edo28w5n7reDgEuGlasteidn itnh eLs6p9i3k ec,oimnpclaurdedin gto1 L36t6h1a t w(eFriegsutrreosn 2gAly aunpdr 4eBgu, Slautpepdleamndenotnalryy oMnaetethriaatl w2:a Tsasbtlreo Sn2g)l.y Adto 7w2 nHrAegI,u 7la7t3e DdEinGLs 6w9e3rec oombspearrveedd tion Lth6e6 1 (Fsipgiukreess o2fA thaen dtw4oB g,eSnuoptpypleems,e inntcalruydiMnga tseerviaeln2 t:hTaat bwleerSe2 s).trAontg7l2y HupArIe,g7u7l3atDedE Gansdw therreeeo tbhsaetr vweedrei n thsetrsopnigkleys doofwthnertewguolagteendo itny pLe6s9,3i nccolmudpianrgeds etvoe Ln6t6h1a (tFwigeurreesst r2oAn galnydu 4pCr,e gSuuplapteledmaenndtatrhyr eMeattheartiawl 2er: e stTroanbglel yS2d)o. wSenvreeng oufl attheed 3i7n4 Ld6o9w3ncroemguplaatreedd gtoenLe6s6 h1a(dF ihgiughreesr 2aAvearangde 4loCg,RSPuKpMpl evmaleunetsa rthyaMn athteer 3ia9l92 : TaubplereSg2u)l.ateSde vgeenneosf. the 374 downregulated genes had higher average logRPKM values than the 399upIrne gleualfa tteisdsugeesn, etsh.ere were 1880 DEGs at 0 HAI between the two genotypes; these included 11 stroInnglleya futpisrseugeusl,attheedr egweneerse a1n8d80 siDxE sGtrsonatg0lyH dAowIbnertewgeuelnattehde gtwenoegs einn oLt6y9p3e sc;otmhepsaeriendc ltuod Le6d6111. Fsitfrtoenegnl y uporfe tghuel a9t1e4d ugperneegsualantdeds ixgesntreosn hgalyd dhoigwhn erxegpurelassteiodng aebnuensdinanLc6e9 3cocmompapraerde dtot othLe6 96616. Fdioftweennreogfuthlaete9d1 4 upgreengeusl.a Atetd 2g4e nHesAhI,a dthheirgeh wexeprere 2ss0i3o4n DabEuGnsd ainn ctehceo mtwpoa rgeedntootythpees9,6 6indcoluwdninregg u1l0a ttehdatg ewneerse. Asttr2o4nHglAy I, thueprerewgeurleat2e0d3 4anDdE tGhrseien tthhaet twweoreg esntrootnygpleys ,dionwclnurdeignugla1t0etdh aint wL6e9r3e sctormonpgalryedu ptore Lg6u6la1t.e Adta 7n2d HthArIe,e 6t8h6a t wDerEeGstsr ownegrley ddeotwecnterdeg iunl athteed twinoL g6e9n3octoympepsa, roendlyto twL6o6 o1f. Awth7ic2hH wAeIr,e6 s8t6roDnEgGlys uwperreegduelatetecdte adnidn othnee towf o which was strongly downregulated (Figures 2B and 4F, Supplementary Material 2: Table S2). genotypes,onlytwoofwhichwerestronglyupregulatedandoneofwhichwasstronglydownregulated We identified some similar changes in gene expression between the spike and leaf tissues (Figure (Figures2Band4F,SupplementaryMaterial2:TableS2). S1). The number of upregulated genes in the spike compared to the corresponding leaf was greater We identified some similar changes in gene expression between the spike and leaf tissues than that of the downregulated genes at each time point in both L693 and L661 plants. The change in (Figure S1). The number of upregulated genes in the spike compared to the corresponding leaf gene expression in the L661 spike from 0 to 24 HAI was greater than that of L693 (Supplementary wasgreaterthanthatofthedownregulatedgenesateachtimepointinbothL693andL661plants. Material 1: Figure S2d,j). By contrast, there was a greater change in gene expression in the L693 spike The change in gene expression in the L661 spike from 0 to 24 HAI was greater than that of L693 compared to L661, both from 0 to 72 HAI and from 24 to 72 HAI (Supplementary Material 1: Figure (SupplementaryMaterial1: FigureS2d,j). Bycontrast,therewasagreaterchangeingeneexpression S2e,f,k,l). Many of the upregulated genes in the L661 spike from 0 to 24 HAI and from 0 to 72 HAI intheL693spikecomparedtoL661,bothfrom0to72HAIandfrom24to72HAI(Supplementary had larger logRPKM values than the corresponding downregulated genes (Supplementary Material Material1:FigureS2e,f,k,l).ManyoftheupregulatedgenesintheL661spikefrom0to24HAIandfrom 1: Figure S2d,e). A greater number of DEGs were seen in the L693 spike from 24 to 72 HAI than in the 0to72HAIhadlargerlogRPKMvaluesthanthecorrespondingdownregulatedgenes(Supplementary Int.J.Mol.Sci. 2018,19,852 7of19 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 7 of 19 Material1: FigureS2d,e). AgreaternumberofDEGswereseenintheL693spikefrom24to72HAI thLan66i1n tshpeikLe6 6(1Ssuppipkleem(Seunptparleym MenattaerryiaMl a1t:e rFiaiglu1:reF igSu2fr,el)S. 2Mf,lo).rMeoovreero, vienr ,iLn6L936,9 3t,htehreer ewweerree mmoorree dodwonwrnergeugluatlaetdedg egneensetsh tahnanu purpergeuglualtaetdedg genenese,s,w whhilielei ninL L666611t htheerreew weerreef feewweerr ddoowwnnrreegguullaatteedd ggeenneess thathnaunp urpegreuglautleadtegde gneense(sS u(SpupplepmleemnetanrtyarMy aMteartiearlia2l: 2T:a TblaebSle3 )S.3T).h Teheex pexrepsrseisosnioonf ogfe ngeesneins itnh ethleea lveeasveosf boothf bgoetnho gteynpoetsywpeesr ewseimre islaimr;itlhare; ltahreg leasrtgdeisftf edrieffnecreenbceet wbeetewneLe6n9 L36a9n3d anLd66 L16s6p1i kspeiskoecsc oucrcruerdrefrdo fmro0mt o0 24toH 2A4I ,HwAhIi,l wethhilaet tihnalte ianv elesawveass wfraosm fr2o4mt o247 2toH 7A2 IH(ASuI p(SpulepmpleenmtaernytaMrya tMeraiatelr1ia:lF 1ig: FuirgeuSr2e) S.2). FigFuigruer4e. L4o. g-Lfoolgd-fcohldan gceha(lnoggeF C()loaggFaCin)s taagvaeirnasgte laovgeRraPgKeM loingdRiPffKeMren ting endoiftfyepreesn.t (Ag–eFn)oDtyipffeesr.e n(tAia–lFly) exDprifefsesreedntgiaelnlye sexwpirtehssaefda lgseendeiss wcoivthe ray farlastee d(FisDcoRv)eoryf lreastse t(hFaDnR5) %of alensds tahlaong 5-f%ol adncdh aa nlogge-floalrdg ecrhtahnagne 2alraergheigr hthliagnh t2e adrien hriegdh.liIgnhetaecdh inp arnedel., Itnh eearcehd pdaontsela, bthoev eretdh eduoptsp aebrobvlue ethlien uep(lpoegrF bClu>e+ li2n)er e(lporgeFseCn >t t+h2e) uprreepgruelsaetnetd thgee nuepsrienguLl6a9t3edo rgtehneesd oinw Ln6r9e3g uolra tthede dgoenwensrienguLl6a6t1e;dt hgeenreesd idno Lts66b1lo; wthet hreedlo dwoetsr bblluowel itnhee (lolgoFwCer< b−lu2e) lrienper (elsoegnFtCt h<e −u2p) rreegpurelasetendt tgheen uesprinegLu6la6t1edor gtehneeds oinw Ln6re6g1 uolra ttehde dgeonwensrienguLl6a9t3e.dT gheenebslu ine doLts69r3e.p Trehsee nbtlugee ndeo(tss) rwepitrheseexnttr egmenee(vsa) lwueitsho efx−tr1e2m>e vloagluFeCs >of1 −21o2 r>w loitghFCan >a 1v2e roarg weiltohg RanP KavMer>ag3e. ThleoggRrePeKnMs y>m 3.b Tohlse grerpeernes seynmtbgoelnse rseporfeisnetnetr egsetniens othf iisntsetruedsty i;nr tehcitsa sntguldeyf;o rrecMtaSnTgRleG fo.2r4 M51S6T,RcGir.c2l4e5f1o6r, MSciTrcRlGe f.2o4r 5M51S,TtRriGan.2g4l5e5f1o,r tMriaSnTgRleG f.o2r4 5M5S2T. RG.24552. 2.4. DEGs Have a Biased Chromosomal Distribution 2.4. DEGsHaveaBiasedChromosomalDistribution The chromosomal distribution of DEGs was biased (Figure 5). In the spike, 36 (10.8%) of 333 ThechromosomaldistributionofDEGswasbiased(Figure5). Inthespike,36(10.8%)of333genes genes differentially co-expressed in L693 and L661 at the three time points examined were mapped differentially co-expressed in L693 and L661 at the three time points examined were mapped to to chromosome 3B. In the leaf, 33 (11.1%) of 298 genes differentially co-expressed in L693 and L661 chromosome3B.Intheleaf,33(11.1%)of298genesdifferentiallyco-expressedinL693andL661at at the three time points were mapped to chromosome 3B. In addition, there was a similar number of thethreetimepointsweremappedtochromosome3B.Inaddition,therewasasimilarnumberof total DEGs and DEGs on chromosome 3B between L693 and L661 at the three time points (Figure 5, totalDEGsandDEGsonchromosome3BbetweenL693andL661atthethreetimepoints(Figure5, Supplementary Material 2: Table S4). The genes that were differentially co-expressed in the spike and Supplementary Material 2: Table S4). The genes that were differentially co-expressed in the spike leaf at three points in both L693 and L661 exhibited no biased distribution on chromosome 3B andleafatthreepointsinbothL693andL661exhibitednobiaseddistributiononchromosome3B (Supplementary Material 1: Figure S3). In both L693 and L661 spikes, no biased distribution of co- (Supplementary Material 1: Figure S3). In both L693 and L661 spikes, no biased distribution of expressed DEGs (24 vs. 0, 72 vs. 0 and 72 vs. 24) on chromosome 3B was found, but the number of co-expressedDEGs(24vs. 0,72vs. 0and72vs. 24)onchromosome3Bwasfound,butthenumber co-expressed DEGs (24 vs. 0, 72 vs. 0 and 72 vs. 24) mapping to chromosome 3B in the leaves of L693 of co-expressed DEGs (24 vs. 0, 72 vs. 0 and 72 vs. 24) mapping to chromosome 3B in the leaves and L661 plants at the three time points was only 9 (2.8%) and 15 (3%) out of 317 and 496, respectively, ofL693andL661plantsatthethreetimepointswasonly9(2.8%)and15(3%)outof317and496, which is fewer than the chromosomal average value of about 5.2% (Supplementary Material 1: Figure respectively, which is fewer than the chromosomal average value of about 5.2% (Supplementary S4, Supplementary Material 2: Table S4). Material1: FigureS4,SupplementaryMaterial2: TableS4). Int.J.Mol.Sci. 2018,19,852 8of19 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 8 of 19 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 8 of 19 Figure 5. Venn diagram showing the number of differentially expressed genes shared by L693 and Figure5.VLe6n61n adt i0a gh r(aremd),s h24o hw (ignrgeetnh),e annud m72b he r(bolufed) iafffteerre innoticaulllaytioenx pforre sthsee dspgikeen (eAs) sahnadr leedaf b(By).L T6h9e3 andL661 at0h(redn),u2m4bhers( gwrietheonu)t, paanrednt7h2eshes (rbelpurees)enatf ttheer dinifofecreunltaiatilloyn exfporrestsheed sgpenikese a(cAro)ssa tnhed wlehaofle( gBe)n.oTmhee; numbers withoutpatrheen ntuhmebseerss rienp praersenetnhtestehse redpirfefesernetn tthiea ldliyffeerxepntriealslsye edxpgreesnseeds agecnroess sont hwehweaht cohlreomgeonsoommee 3;Bt.h enumbers inparenthesesrepresentthedifferentiallyexpressedgenesonwheatchromosome3B. 2.5. A 140 kb Differential Expression Island Exists on Chromosome 3B We then searched sequence data (Available online: http://plants.ensembl.org/Triticum_aestivum) 2.5. A140kbDFiigffuerree 5n. tViaelnnE dxipagrreasmsi oshnowIsilnagn tdheE nxuimsbtsero onf dCifhferroemntioaslloym exepr3eBssed genes shared by L693 and for genes on wheat chromosome 3B. We identified 8571 annotated genes with a total length of L661 at 0 h (red), 24 h (green), and 72 h (blue) after inoculation for the spike (A) and leaf (B). The 774,434,471 bp, excluding scaffolds, and an average length of 90.4 kb. Further analysis of the reference Wethensneuamrcbehres dwistheoquut peanrcenethdeasetsa r(eAprvesaeinlta tbhel edioffnerleinnteia:llhy tetxppr:e/s/sepd lgaennetss .aecnrosses mtheb wl.hoorlge /geTnroimtiec; um_aestivum) sequence revealed a gene island in the region between 181.40 and 181.54 Mb; this 140 kb sequence the numbers in parentheses represent the differentially expressed genes on wheat chromosome 3B. for genes on wheat chromosome 3B. We identified 8571 annotated genes with a total length of contained 73 annotated genes with an average density of 1.9 kb. Reliable transcription products of 774,434,47211.35b5. 0pA (, 11e64x.08 c%klbu) Doduiifftne orgef n8st5cia7al1 fE faxonplnrdoesstsa,itoaendn I dgsleaannneds E awxvieserters adogne teCechltereondmg wotshiotmho eaf n39B a0 v.e4rakgbe. dFenusrittyh eorf 5a7n3.a7l kybs,i wshoiflet h42e reference sequencer(5e7v.5e%a)l eoduta ofg e73n eaninsolatantedd ignentehse inr etghei o3nB bchertowmeoesonm1a8l 1re.4g0iona nfrdom1 81181.5.440 Mto b1;81t.h54is M1b4 0wkerbe sequence We then searched sequence data (Available online: http://plants.ensembl.org/Triticum_aestivum) transcribed. RNA-Seq data led to the identification of 4136 unannotated transcribed genes that may containedfo7r3 gaennens ootna twehdeagt ecnherosmwosiotmhea 3nB.a Wveer aidgeentidfieedn s8i5t7y1 oafnn1o.t9atkedb .geRneesl iwabithle at rtoatnals clernipgtthi oonf products encode unknown RNAs or proteins; these genes are hereafter referred to as novel transcribed genes of 1350 (1767.48,4%34),4o7u1 btpo, fex8cl5u7d1inga nscnafofotaldtse, dandg eann eavserwageer leendgetht eocf 9te0.d4 kwb. iFtuhrtahner aanvaelyrsaisg oef tdhee nresfeitryencoef 573.7 kb, (NTGs). A total of 12,707 annotated genes (including previously annotated genes and NTGs) were sequence revealed a gene island in the region between 181.40 and 181.54 Mb; this 140 kb sequence while42(5id7e.n5t%ifi)edo ount cohfro7m3oasonmneo 3tBa,t ewdithg aenn aevseriangeth geen3eB decnhsirtoy mof o60s.o9 mkba, wlrheilge i8o6n gefnroesm we1r8e 1fo.4u0ndt oon1 81.54Mb contained 73 annotated genes with an average density of 1.9 kb. Reliable transcription products of weretranscchrriobmeods.omRNe 3AB- Sfreoqm d1a8t1a.40le tdo t1o81t.h54e iMdbe,n wtiifithc aatni oanveorafg4e1 g3e6neu ndaennsnitoy taoft e1d.6 tkrab n(sFcigruibree d6).g enesthat 1350 (16.8%) out of 8571 annotated genes were detected with an average density of 573.7 kb, while 42 Furthermore, 2456 (19.3%) out of 12,707 genes on chromosome 3B exhibited reliable expression with mayencod(5e7.5u%n)k onuot wofn 73R aNnnAostatoerd pgreonetse iinn st;hteh 3eBs echgroemnoessomarael rheegrioena fftreomrr 1e8f1e.r4r0e tdo 1to81a.5s4 nMobv welerter anscribed an average gene density of 315.3 kb, while 53 (61.6%) out of 86 genes were reliably expressed on genes(NTtGranss)c.rAibetdo. tRaNlAof-S1eq2 ,d7a0t7a laedn ntoo tthaet eiddengtiefincaetsio(ni nocf l4u13d6i nungapnnroetvaiteodu tsrlaynsacrnibneodt gaetneeds gtheant emsaya ndNTGs) chromosome 3B from 181.40 to 181.54 Mb, with an average gene density of 2.6 kb. encode unknown RNAs or proteins; these genes are hereafter referred to as novel transcribed genes were identified on chromosome 3B, with an average gene density of 60.9 kb, while 86 genes were (NTGs). A total of 12,707 annotated genes (including previously annotated genes and NTGs) were foundonchromosome3Bfrom181.40to181.54Mb,withanaveragegenedensityof1.6kb(Figure6). identified on chromosome 3B, with an average gene density of 60.9 kb, while 86 genes were found on Furthermochrero,m2o4s5o6m(e1 93B.3 %fro)mo u1t81o.4f01 t2o, 710871.5g4e nMebs, ownithc harno mavoersaogme gee3nBe deexnhsiitbyi toefd 1.r6e lkiba b(lFeigeuxrep r6e).s sionwith an averagFeugrtehenremodreen, 2s4i5t6y (1o9f.33%1)5 o.u3t kofb 1,2w,70h7 igleen5es3 o(n6 c1h.r6o%m)osooumte o3Bf e8x6higbeitnede srelwiabelree exrperleiassbiolyn wexithp ressed on an average gene density of 315.3 kb, while 53 (61.6%) out of 86 genes were reliably expressed on chromosome3Bfrom181.40to181.54Mb,withanaveragegenedensityof2.6kb. chromosome 3B from 181.40 to 181.54 Mb, with an average gene density of 2.6 kb. Figure 6. Distribution of genes on wheat chromosome 3B. (A) Eighty-six genes annotated by the Genetics Diversity Ecophysiology of Cereals (GDEC) group at the French National Institute of Agronomic Research (INRA) located in region 181.40–181.54 Mb of wheat chromosome 3B. (B) Fifty- Figure 6. Distribution of genes on wheat chromosome 3B. (A) Eighty-six genes annotated by the Figure 6. Distribution of genes on wheat chromosome 3B. (A) Eighty-six genes annotated by Genetics Diversity Ecophysiology of Cereals (GDEC) group at the French National Institute of the GenetAicgsroDnoimveicr RsietsyeaErccho (IpNhRyAs)i ololcoagteyd ionf reCgieorne 1a8l1s.4(0G–1D81E.5C4 )Mgb roof uwpheaatt chthroemFosroemnec h3B.N (Ba)t Fioifntya-l Institute of Ag ronomic Research (INRA) located in region 181.40–181.54 Mb of wheat chromosome 3B. (B) Fifty-three expressed genes located in region 181.40–181.54 of chromosome 3B in this study. Theinsetsshowenlargementsoftheboxedregions. Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 9 of 19 Int.J.Molt.hSrceie. e2x01p8r,e1s9se,d85 2genes located in region 181.40–181.54 of chromosome 3B in this study. The insets 9of19 show enlargements of the boxed regions. 2.6.2G.6e. nGeesnines tihne t1h4e0 1k4b0 Ekbx pErxespsrieosnsiIosnla InsdlaEndx hEibxihtiHbitig HheigrhCeor nCsotintsuttiitvuetiEvxe pErxespsrieosnsiionnt ihne tFhHe FBHRBes RisetsainstcaenGcee notype Genotype Twenty-seven out of 53 genes had a higher expression level (i.e., −2 < logFC < 2) in the L693 spTikweenthtya-nseivnenth oeutL o6f6 513 sgpeinkees ahtad0 aH hAigIh,ewr hexilperensosiodnif lfeevreenl (cie.e.i,n −2g <e nloegeFxCp <r e2s) sinio tnhew La6s93o sbpsiekrev ed (i.et.,h−an2 i<n tlhoeg FLC66<1 s2p)ibkee tawt e0e HnAthI,e wshpiilkee nsoo dfiLff6e9re3nacne dinL g6e6n1e aetxp24reossrio7n2 HwaAsI o.bInsetrhveedle (ai.fe,.9, −,22 ,<a lnodgF1Co ut oft<h 2e)5 b3egtweneeens wtheer sepdikifefse roef nLt6ia9l3l yanedx pLr6e6s1s eadt 2in4 oLr6 9723 HanAdI.L I6n6 t1hea tle0a,f2, 49,, a2n, adn7d2 1h o,uret sopf etchtei v5e3l yg.eFnoesr all diffwereeren tdiaiflfleyreenxtpiarlelys seexdprgeessneeds ,inth Le69e3x panreds sLi6o6n1 laetv 0e,l 2i4n, aLn6d9 372w ha, srevsipseibctliyvehlyig. hFoerr athlla dnifftehraetntiinalLly6 61. Furetxhperremssoerde g,einnebs,o tthhe tehxeprsepsiskioena lnedvelle ianf ,La6l9l33 w6agse vniesisbleyx phirgehseser dthiann Lth6a9t3 ina nLd66L1.6 F6u1rathte0rmHoAreI, win ere both the spike and leaf, all 36 genes expressed in L693 and L661 at 0 HAI were upregulated in L661 upregulated in L661 from 0 to 24 HAI, while only 24 genes were upregulated in L693 during that from 0 to 24 HAI, while only 24 genes were upregulated in L693 during that period (Figure 7, period(Figure7,SupplementaryMaterial2: TableS5). Supplementary Material 2: Table S5). Figure 7. Distribution of gene expression levels on chromosome 3B in all 12 samples. The x-axis Figure 7. Distribution of gene expression levels on chromosome 3B in all 12 samples. The x-axis represents the chromosome range 0–774.43 Mb. The y-axis represents the normalized log (RPKM+1) representsthechromosomerange0–774.43Mb.They-axisrepresentsthenormalizedlog(RPKM+1) range 0–1. The region included in the dashed rectangle indicates the 181.40–181.54 Mb region. range 0–1. The region included in the dashed rectangle indicates the 181.40–181.54 Mb region. Distribution of gene expression level on chromosome 3B for six spike samples (A) and six leaf samples (B). Distribution of gene expression level in the gene island at region 181.40–181.54 Mb ofchromosome3Bforsixspikesamples(C)andsixleafsamples(D). Int.J.Mol.Sci. 2018,19,852 10of19 2.7. DEGsinthe140kbExpressionIslandAreMainlyInvolvedinChloroplastFunction Ofthe53DEGsidentified,42werepreviouslyannotatedand11(withtranscriptIDname“MSTRG” inTableS5)werenewlyannotatedinthisstudy. UsingtheBLASTprogram,DEGswereidentifiedthat hadhighlevelsofnucleotidesequencesimilaritywithannotatedgenesforchloroplast-relatedpathways (39;73.6%),mitochondrion-relatedpathways(3;5.7%),andgenesencodingputativeproteinswithout known functions (7; 13.2%). Four (7.5%) DEGs showed no similarity to any previously identified genes(TableS5). FurtherKyotoEncyclopediaofGenesandGenomes(KEGG)pathwayenrichment analysisrevealedthat13(24.5%)ofthe53DEGswereassignedtoaspecificpathway(Supplementary Material2:TableS5);ofthese,7(53.8%)wereinvolvedinoxidativephosphorylationpathways(Table2). SomeDEGswereinvolvedinotherknownpathways,includingpurinemetabolism,glyoxylateand dicarboxylate metabolism, biosynthesis of antibiotics, carbon fixation in photosynthetic organisms, thiaminemetabolism,pyrimidinemetabolism,galactosemetabolism,phenylpropanoidbiosynthesis, and starch and sucrose metabolism (Supplementary Material 2: Table S5). Thirteen DEGs showed wide pleiotropism; for example, MSTRG.24512, which encodes ec:3.6.1.3-adenylpyrophosphatase and ec:3.6.1.15-phosphatase, participates in four pathways including purine metabolism, thiamine metabolism, galactosemetabolism, andstarchandsucrosemetabolism(SupplementaryMaterial2: TableS5). Eleven(84.6%)ofthese13DEGsareinvolvedinchloroplast-relatedmetabolism,and2of theseDEGsareinvolvedinmitochondrion-relatedmetabolism. Furtheranalysisfoundthat9and4 ofthe13DEGsweredifferentiallyexpressedbetweenthetwogenotypesat0DAIinthespikeand leaf, respectively, with line L693 exhibiting higher constitutive expression than line L661. We paid specificattentiontothreeinterestingconstitutivelyexpressedgenesthatwereinduceduponinoculation with F. graminearum: one gene encoding inositol monophosphate (IMPa) and two genes encoding ribulose-1,5-bisphosphatecarboxylase/oxygenase(RuBisCO). Table2.Pathwaysassociatedwiththe13genesinthegeneislandatregion181.40–181.54Mbofwheat chromosome3B. Pathways Enzyme Definition SeqNu. Sequence ubiquinol-cytochromec ec:1.10.2.2—reductase reductasecytochrome MSTRG.24508 b/c1subunit MSTRG.24509, MSTRG.24524, MSTRG.24557, ec:1.6.99.3—dehydrogenase NADHdehydrogenase MSTRG.24558, MSTRG.24527, Oxidativephosphorylation 7 TRAES3BF007300290CFD_g cytochromecoxidase ec:1.9.3.1—oxidase MSTRG.24509 cbb3-typesubunitI MSTRG.24509, MSTRG.24524, NADH:ubiquinone ec:1.6.5.3—reductase MSTRG.24557, (H+-translocating) reductase MSTRG.24558, (H+-translocating) MSTRG.24527, TRAES3BF007300290CFD_g MSTRG.24512, ec:3.6.1.3—adenylpyrophosphatase adenosinetriphosphatase MSTRG.24554 MSTRG.24512, Purinemetabolism ec:3.6.1.15—phosphatase nucleoside-triphosphatase 3 MSTRG.24554 DNA-directedRNA ec:2.7.7.6—RNApolymerase MSTRG.24544 polymerasesubunitalpha Glyoxylateand MSTRG.24551, ec:4.1.1.39—carboxylase 2 dicarboxylatemetabolism MSTRG.24552 MSTRG.24551, Biosythesisofantibiotics ec:4.1.1.39—carboxylase 2 MSTRG.24552 Carbonfixationin MSTRG.24551, ec:4.1.1.39—carboxylase 2 photosyntheticorganisms MSTRG.24552