References:

JoelA.Velasco1,2,MauricioA.Reynoso3,GermainPauluzzi3&JuliaBailey-Serres31:CenterforPlantCellBiologyREUSite:ResearchExperienceforUndergraduatesinNextGeneraKonPlantCellBiology.

DepartmentofBotanyandPlantSciences,UniversityofCaliforniaatRiverside2:DepartmentofBiologicalSciences,BoiseStateUniversity,Boise,ID

3:CenterforPlantCellBiology,DepartmentofBotanyandPlantSciences,UniversityofCaliforniaatRiverside

It’saTRAP!ConfirmingEfficiencyofRiceLinesDevelopedforCell-typeSpecificGeneExpressionAnalysis

(1)IntroducIon

(4)TRAPandINTACTricelines

●  INTACTandTRAPtechnologiescoupledwithbioinformaKcanalysistoolsallowforcell-typespecificgeneexpressionanalysis(Bailey-Serres,2013)

(5)Workflow

Thisworkissupportedby:-NSFREU-1461297-NSFDBI-PGRP-1238243

Bailey-Serres, J. (2013). Microgenomics: Genome-Scale, Cell-Specific Monitoring of Multiple Gene Regulation Tiers. Annual Review of Plant Biology 64, 293–325. Mustroph, A., Zanetti, M.E., Jang, C.J.H., Holtan, H.E., Repetti, P.P., Galbraith, D.W., Girke, T., and Bailey-Serres, J. (2009). Profiling translatomes of discrete cell populations resolves altered cellular priorities during hypoxia in Arabidopsis. Proc Natl Acad Sci U S A 106, 18843–18848. Deal, R.B., and Henikoff, S. (2010). A Simple Method for Gene Expression and Chromatin Profiling of Individual Cell Types within a Tissue. Developmental Cell 18, 1030–1040.

Yuxiu, Z., Zi, W., and Jin, X. (2007). Molecular mechanism of dehydrin in response to environmental stress in plant. Progress in Natural Science 17, 237–246. Ranathunge, K., Schreiber, L., Bi, Y.-M., and Rothstein, S.J. (2015). Ammonium-induced architectural and anatomical changes with altered suberin and lignin levels significantly change water and solute permeabilities of rice (Oryza sativa L.) roots. Planta 243, 231–249.

Contactinfo:-joelvelasco84@gmail.com

●  TransgenicricelinesdevelopedtoexpresstheINTACTandTRAPconstructsdrivenbycell-typespecificpromotersfacilitateaccesstonuclearorpolysomeassociatedmRNApopulaIonsfromspecificcell-types

●  HereweinterrogateapreliminaryNGS(nextgeneraKonsequencing)datasetgeneratedfromTRAPandINTACTriceplants(3week-old)inordertoevaluatecell-typespecificgeneexpressioninresponsetowaterstress

MeristemaKczone

QC

(3)TRAPandINTACTTechnologies IsolaIonofNucleiTaggedinSpecificCellTypes(INTACT)

•  IsolaIonofnucleifromspecificcelltypes

TranslaIngRibosomeAffinityPurificaIon(TRAP)

•  AccesstonuclearchromaInandpre-mRNA

•  AccesstopolysomalmRNA

•  INTACTconstructmakesthispossible

•  IsolaIonoftranslaIngribosomesfromspecificcelltypes

•  TRAPconstructmakesthispossible

ER

Pre-mRNA

ChromaKn

mRNA

AAAA…

AAAA…

Cytosol

BioKn-taggedproteinassociatedtonuclearenvelope(NTF)

Nucleartaggingfusion(NTF) INTACT

PolysomalmRNA

RPL18-FLAGLargeribosomalsubunitRPL18taggedwithFLAG

Y

mRNA

AAAA…

TRAP

●  InthispilotstudyweevaluatetheefficiencyofTRAPandINTACTricelinesdevelopedforcell-typespecificgeneexpressionanalysis.

WPP GFP BLRPPromoter

HF GFP OsRPL18Promoter

CMZ

(6)DatacollecIonandNGSdataanalysisusingbioinformaIctools

(7)HeatmapandIGVgeneexpressionanalysis

75µm

●  Rice(Oryzasa(va)isanimportantglobalcropspeciesthreatenedbytheunpredictabilityoffuturefloodsanddroughtsbroughtonbyglobalclimatechange

(2)Predictedchangeinfloodingfrequencyinthe21stcentury

EED

NatureClimateChangeHirabayashietal.,2013

E F

C DA

B

Epidermis

Sclerenchyma

Vasculature

Cortex

Endodermis

Pericycle

Exodermis

1.TRAPandINTACTtransgenicricelinedevelopment.GFPlocalizaKon inembryonicnodalroots(7day-oldseedlings).A-B:Cortex INTACT line.CloseupoftherootKp(focusedonthe lastcorKcalcelllayer) shows the localizaKonof theNTF (Nuclear Tagging Fusion) to thenuclearenvelope.C-D: TRAPRSS1 (RICE SALT SENSITIVE1) promoter totargetacKvelydividingcells.E-F:INTACTQHB1(QuiescentCenterSpecificHomeobox)promotertotargetquiescentcenter,GFPislocalizedinthequiescentcenter:Scale:A-D:50µmandE-F:25µm

(5)Transgenicricelinetreatments Waterlogging

Drought

Treatment

ControlCell type and technology

Shoot apical meristem (TRAP & INTACT)

Root cortex meristematic zone (TRAP)

Root endodermis meristematic zone (TRAP)

Root dividing cells (TRAP)

Root endodermis differentiated zone (TRAP)

Quiescent center (TRAP)

Endodermis/exodermis differentiated zone (TRAP)

Total mRNA (RNAseq)

Cell type and technology

Endodermis/exodermis differentiated zone (TRAP)

Total Drought mRNA (RNAseq)

Cell type and technology

Endodermis/exodermis differentiated zone (TRAP)

5days

5days

TissuecollecIon(cryopreserved)

TRAPandINTACTproceduresforRNAcapturefromspecificcell

types

IlluminacompaIblelibrarypreparaIon

SequencingonIlluminaHiSeq3000

1x50bpreads(7-10millionpersample)

Quality Control

Qualitycontrolledreads

fastqc module

Filtering and clipping

Filteredreads,oligosandsequencingadaptersremoved

fastq-grep fastx-clipper

Alignment

ReadsalignedtoreferenceOryzasa(vacv.Nipponbaregenome

Tophat2 alignment module

Counting, normalization, clustering, and

heatmap

100clusterheatmap&IGV(integraIvegenomicsviewer)filesgenerated

summarizeoverlaps function, PAM clustering, and

pheatmap package

TRAP Shoot apical meristem (Control)

INTACT shoot apical meristem (Control)

TRAP endo/exodermis (Control)

TRAP endo/exodermis (Waterlogged)

TRAP endo/exodermis (Drought)

Total mRNA (Drought)

TRAP endodermis meristematic zone (Control)

TRAP cortex meristematic zone (Control)

TRAP endodermis meristematic zone (Control)

TRAP quiescent center (Control)

TRAP dividing cells (Control)

Total mRNA (Control)

LOC_Os11g26790.1

Total drought

Total control

Endo/exodermis drought TRAP

Endo/exodermis waterlogged TRAP

Endo/exodermis control TRAP

15,343,000 bp 15,336,500 bp

LOC_Os11g26780.1 LOC_Os11g26760.1

15,328,500 bp

LOC_Os11g26750.1

15,321,000 bp

LOC_Os11g26570.1

15,224,000 bp

LOC_Os06g21590.2

12,453,000 bp 12,454,000 bp

(8)ConclusionsandfuturedirecIons

Total drought

Total control

Endo/exodermis drought TRAP

Endo/exodermis waterlogged TRAP

Endo/exodermis control TRAP

Shoot apical meristem TRAP

Shoot apical meristem INTACT

Endodermis differentiated control

TRAP

Endodermis meristematic control

TRAP

Cortex meristematic control TRAP

Dividing cells control TRAP

Quiescent center control TRAP

21,512 kb

LOC_Os08g34280.1

21,508 kb

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LOC_Os11g31090.1

18,095,000 bp 18,093,000 bp

Dehydrins: Water stress induced, water soluble proteins (Yuxiu et al., 2007)

Genes implicated in the production of lignin and suberin respectively (Ranathunge et al., 2015)

●  ThevisualizaKonofthesametranscriptinitspre-mRNAandribosomeassociatedmRNAformsupportstheideathatTRAPandINTACTtechnologiescanbeusedtoisolatedifferentRNApopulaKonswithinspecificcelltypes

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Total drought

Total control

Endo/exodermis drought TRAP

Endo/exodermis waterlogged TRAP

Endo/exodermis control TRAP

Shoot apical meristem TRAP

Shoot apical meristem INTACT

Endodermis differentiated control

TRAP

Endodermis meristematic control

TRAP

Cortex meristematic control TRAP

Dividing cells control TRAP

Quiescent center control TRAP

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●  ThispilotstudyconfirmstheefficiencyofTRAPandINTACTtechnologieswithinthesericelineswhichwillbeusedinfuture,morecomprehensiveanalyses

●  Waterstressinducedgenesvisualizedinbothtotalandendodermis/exodermisspecificsampleshelpconfirmtheefficiencyofthesetechnologies

Chlorophyll A-B binding protein