MAPPING THE TOMATO ROOT GENE EXPRESSION IN DROUGHT...
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0h 1h 2h 4h 6h 0.01 0.1 1 10 100 1000 0h 1h 2h 4h 6h 8h 0h 1h 2h 4h 6h 8h MAPPING THE TOMATO ROOT GENE EXPRESSION IN DROUGHT AND WATERLOGGING KAISA KAJALA 1,2 ,DONNELLY WEST 1 , MAGGIE WOODHOUSE,JIAWEN ZENG 1,2 ,NIBA NIRMAL 1,2 ,REINA MARIE SANZ 1,2 , AYUMI GOTHBERG 1,2 ,KRISTINA ZUMSTEIN 1 ,NEELIMA SINHA 1 ,SIOBHAN BRADY 1,2 1 DEPARTMENT OF PLANT BIOLOGY, UC DAVIS, USA. 2 GENOME CENTER, UC DAVIS, USA. Solanum pennellii “PENN” DROUGHT-TOLERANT! Solanum lycopersicum var. M82 CULTIVATED A B C D M82 M82 M82 CONTROL NO WATER WATERLOG M82 CONTROL B M82 NO WATER B SUBERIN STAINING WITH FLUOROL YELLOW SHOWS SUBERIN ACCUMULATION IN EXODERMIS AFTER NO WATER TREATMENT IN M82, WHILE EXODERMAL SUBERIN IS CONSTITUTIVE IN S. PENNELLII. @kaisakajala @bradylabs Tomato has • a sequenced genome • drought-tolerant inter- crossable species with developmental differences We are inves]ga]ng the responses to drought and flooding in • Specific cell types • Different regulatory levels of gene expression • Roots and shoots • Mul]ple different species Cross-sec]on of M82 root Cross-sec]on of PENN root We tested the effect of long-term drought and waterlogging on roots. Plants grown on clay media. 21 days well watered + 12 days treatment • control (well watered) • no water • waterlogging (rootball submerged) Samples taken in 6 cm segments. Flooding Control 1 2 3 4 5 1 2 3 4 5 6 7 Epidermis Cortex Middle Cortex Endodermis Pericycle Phloem Procambium Xylem Exodermis HYPOCOTYL-DERIVED ADVENTITOUS ROOTS (AR) INCREASE IN WATERLOGGING. ARS HAVE MORE CORTEX LAYERS WITH MORE EXTRACELLULAR SPACE AFTER WATERLOGGING. M82 CONTROL AR M82 WATERLOGGED AR * p = 0.0013 INTACT: ISOLATION OF NUCLEI TAGGED IN CELL TYPES 1) Use a promoter to express a nuclear bio]n tag. 2) Pull down the nuclei with streptavidin beads. 3) Subsequent analyses: ATAC-seq (Assay for Transposon Accessible Chroma]n) comparable to DNase-seq but easier! (Buenrostro et al., 2013) RNA-seq – we established a pipeline to sequence nuclear mRNA. Nuclei have low amounts of polyA mRNA, so total nuclear RNA is subjected to rRNA degrada]on. TRAP: TRANSLATING RIBOSOME AFFINITY PURIFICATION 1) Use a promoter to express a ribosome flag tag. 2) Pull down the ribosomes with α-flag beads. 3) Subsequent analyses: RNA-seq – rapid random-primed libraries (Townlsey et al., 2015) are generated from polysomal mRNA to query which transcripts are associated with polysomes Ribo-seq – RNA immunoprecipita]on allows iden]fica]on of exact ribosome loca]ons on mRNA. Chromatin TRAP Translating Ribosome Affinity Purification INTACT Isolation of Nuclei TAgged in Cell Types mRNA Polysomal mRNA AAAA… nucleus AtPEP:INTACT SlCO2:INTACT SlCO2:TRAP Cortex Meristema]c cortex S. lycopersicum M82 S. pennellii 35S:INTACT S. lycopersicum cv. M82 S. pennellii S. lycopersicum S. pennellii SlRPL11C: INTACT Dividing cells (meristema]c) SlRPL11C: INTACT SlWOX5:INTACT Quiescent center & ini]als SlWOX5:INTACT TRANSGENIC MARKER LINES TO STUDY UNDERLYING GENE EXPRESSION TRANSGENIC MARKER LINES USED IN PROFILING MULTIPLE LEVELS OF GENE EXPRESSION S. lycopersicum Hairy root culture 35S:TRAP 35S:TRAP 35S:TRAP 35S:INTACT 35S:INTACT S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum Endodermis & QC Vasculature Phloem Xylem Near cons]tu]ve Lateral root cap, epidermis & exodermis S. lycopersicum S. lycopersicum S32:TRAP S18:INTACT SlSHR:INTACT SlSCR:TRAP AtWER:TRAP AtWER:INTACT SlACT2:TRAP S. lycopersicum Cortex (no exodermis) AtPEP:TRAP SlPEP:TRAP S. lycopersicum M82 & S. pennellii: 7 days on ver]cal plates Horizontal sealed control plates Horizontal submerged plates EXPERIMENTAL SET-UP & TIMECOURSE OF RESPONSE HAIRY ROOT CULTURES • induced by Agrobacterium rhizogenes • a useful tool for genera]ng rapid transgenic tomato roots to study root development (Ron et al., 2014) Hairy root cultures: 9 days of growth on horizontal plates ADH Rela]ve transcript level Time (h) S. lycopersicum cv. M82 primary root S. lycopersicum cv. M82 hairy roots S. pennellii primary root Submergence Control GENE EXPRESSION PATTERNS CAN DIFFER BETWEEN LEVELS OF REGULATION HAIRY ROOT SUBMERGENCE RESPONSE IS DISTINCT FROM PRIMARY ROOT RESPONSE S. lycopersicum S. pennellii MDS plot of uniquely mapped reads from TOTAL mRNA libraries UNLIKE PRIMARY ROOTS, HAIRY ROOT SUBMERGENCE- RESPONSIVE GENES ARE ENRICHED IN • transcrip]on factors • lateral root ini]a]on related genes NEXT UP: DIFFERENTIALLY ACCESSIBLE CHROMATIN REGIONS! COMPARISON OF DIFFERENT REGULATORY LEVELS! COMPARISONS ACROSS TOMATOES, MEDICAGO & RICE! Submergence responsive genes – TOTAL mRNA Primary root Hairy root Submergence responsive genes – Nuclear mRNA (INTACT) Primary root Hairy root Hairy root submergence responsive genes Polysomal mRNA (TRAP) Nuclear RNA (INTACT) Total RNA CONTROL HAIRY ROOT SUBMERGED HAIRY ROOT CONTROL PRIMARY ROOT SUBMERGED PRIMARY ROOT CHROMATIN ACCESSIBILITY OF TRANSPOSABLE ELEMENTS IS GLOBALLY HIGH IN CONTROL HAIRY ROOTS, BUT IS REDUCED TO PRIMARY ROOT LEVEL IN SUBMERGENCE TRANSPOSABLE ELEMENT LOCATIONS MAPPING REFERENCE LOCATIONS ATAC-SEQ READS READ COUNT VS. BACKGROUND RED (HIGH) à BLUE (LOW) ATAC-SEQ READS READ COUNT VS. BACKGROUND RED (HIGH) à BLUE (LOW) TRANSPOSABLE ELEMENT LOCATIONS MAPPING REFERENCE LOCATIONS ATAC-SEQ READS READ COUNT VS. BACKGROUND RED (HIGH) à BLUE (LOW) ATAC-SEQ READS READ COUNT VS. BACKGROUND RED (HIGH) à BLUE (LOW)
Transcript of MAPPING THE TOMATO ROOT GENE EXPRESSION IN DROUGHT...
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MAPPINGTHETOMATOROOTGENEEXPRESSIONINDROUGHTANDWATERLOGGINGKAISAKAJALA1,2,DONNELLYWEST1, MAGGIEWOODHOUSE,JIAWENZENG1,2,NIBANIRMAL1,2,REINAMARIESANZ1,2,AYUMIGOTHBERG1,2 ,KRISTINAZUMSTEIN1,NEELIMASINHA1,SIOBHANBRADY1,21DEPARTMENTOFPLANTBIOLOGY,UCDAVIS,USA.2GENOMECENTER,UCDAVIS,USA.
Solanumpennellii“PENN”
DROUGHT-TOLERANT!
Solanumlycopersicumvar.M82
CULTIVATED
0"
50"
100"
150"
Control' Drought' Waterlogging'
'Dry'W
eight'(mg)'
A"B"C"D"
M82 M82 M82CONTROL NOWATER WATERLOG
M82CONTROLB M82NOWATERB
SUBERINSTAININGWITHFLUOROLYELLOWSHOWSSUBERINACCUMULATIONINEXODERMISAFTERNOWATERTREATMENTINM82,WHILEEXODERMALSUBERINISCONSTITUTIVEINS.PENNELLII.
@kaisakajala@bradylabs
Tomatohas
• asequencedgenome
• drought-tolerantinter-crossablespecieswithdevelopmentaldifferences
Weareinves]ga]ngtheresponsestodroughtandfloodingin
• Specificcelltypes
• Differentregulatorylevelsofgeneexpression
• Rootsandshoots
• Mul]pledifferentspeciesCross-sec]onof
M82root
Cross-sec]onofPENNroot
Wetestedtheeffectoflong-termdroughtandwaterloggingonroots.
Plantsgrownonclaymedia.21dayswellwatered+12daystreatment
• control(wellwatered)• nowater• waterlogging(rootballsubmerged)
Samplestakenin6cmsegments.
FloodingControl
1234
5
1234567
Epidermis
CortexMiddleCortexEndodermisPericyclePhloemProcambiumXylem
Exodermis
HYPOCOTYL-DERIVEDADVENTITOUSROOTS(AR)INCREASEINWATERLOGGING.ARSHAVEMORECORTEXLAYERSWITHMOREEXTRACELLULARSPACEAFTERWATERLOGGING.
M82CONTROLAR M82WATERLOGGEDAR
*p=0.0013
INTACT:ISOLATIONOFNUCLEITAGGEDINCELLTYPES1) Useapromotertoexpressanuclearbio]ntag.
2) Pulldownthenucleiwithstreptavidinbeads.3) Subsequentanalyses:ATAC-seq(AssayforTransposonAccessibleChroma]n)comparabletoDNase-seqbuteasier!(Buenrostroetal.,2013)
RNA-seq–weestablishedapipelinetosequencenuclearmRNA.NucleihavelowamountsofpolyAmRNA,sototalnuclearRNAissubjectedtorRNAdegrada]on.
TRAP:TRANSLATINGRIBOSOMEAFFINITYPURIFICATION1) Useapromotertoexpressaribosomeflagtag.
2) Pulldowntheribosomeswithα-flagbeads.
3) Subsequentanalyses:RNA-seq–rapidrandom-primedlibraries(Townlseyetal.,2015)
aregeneratedfrompolysomalmRNAtoquerywhichtranscriptsareassociatedwithpolysomes
Ribo-seq–RNAimmunoprecipita]onallowsiden]fica]onofexactribosomeloca]onsonmRNA.
Chromatin
TRAP Translating Ribosome
Affinity Purification
INTACT Isolation of Nuclei
TAgged in Cell Types
mRNA
Polysomal mRNA
AAAA…
nucleus
AtPEP:INTACT
SlCO2:INTACT SlCO2:TRAP
Cortex
Meristem
a]c
cortex
S.lycopersicumM82 S.pennellii
35S:INTACT
S.lycopersicumcv.M82
S.pennellii
S.lycopersicum S.pennellii
SlRPL11C:INTACT
Dividingcells(meristema]c)
SlRPL11C:INTACT
SlWOX5:INTACT
Quiescentcenter&ini]als
SlWOX5:INTACT
TRANSGENICMARKERLINESTOSTUDYUNDERLYINGGENEEXPRESSION
TRANSGENICMARKERLINESUSEDINPROFILINGMULTIPLELEVELSOFGENEEXPRESSION
S.lycopersicumHairyrootculture
35S:TRAP 35S:TRAP 35S:TRAP
35S:INTACT 35S:INTACT
S.lycopersicum S.lycopersicum S.lycopersicum S.lycopersicum S.lycopersicum
Endodermis&QC Vasculature Phloem Xylem Nearcons]tu]ve Lateralrootcap,epidermis&exodermis
S.lycopersicum S.lycopersicum
S32:TRAP S18:INTACTSlSHR:INTACTSlSCR:TRAP AtWER:TRAP AtWER:INTACTSlACT2:TRAP
S.lycopersicum
Cortex(noexodermis)
AtPEP:TRAP
SlPEP:TRAP
S.lycopersicumM82&S.pennellii:7daysonver]calplates
Horizontalsealedcontrolplates
Horizontalsubmergedplates
EXPERIMENTALSET-UP&TIMECOURSEOFRESPONSE HAIRYROOTCULTURES• inducedbyAgrobacteriumrhizogenes
• ausefultoolforgenera]ngrapidtransgenictomatorootstostudyrootdevelopment(Ronetal.,2014)Hairyrootcultures:
9daysofgrowthonhorizontalplates
ADH
Rela]vetranscrip
tlevel
Time(h)
S.lycopersicumcv.M82primaryroot
S.lycopersicumcv.M82hairyroots
S.pennelliiprimaryroot
SubmergenceControl
GENEEXPRESSIONPATTERNSCANDIFFERBETWEENLEVELSOFREGULATION
HAIRYROOTSUBMERGENCERESPONSEISDISTINCTFROMPRIMARYROOTRESPONSE
S.lycopersicum S.pennellii
MDSplotofuniquelymappedreadsfromTOTALmRNAlibraries
UNLIKEPRIMARYROOTS,HAIRYROOTSUBMERGENCE-RESPONSIVEGENESAREENRICHEDIN
• transcrip]onfactors• lateralrootini]a]onrelatedgenes
NEXTUP:DIFFERENTIALLYACCESSIBLECHROMATINREGIONS!COMPARISONOFDIFFERENTREGULATORYLEVELS!COMPARISONSACROSSTOMATOES,MEDICAGO&RICE!
Submergenceresponsivegenes–TOTALmRNA
Primaryroot Hairyroot
Submergenceresponsivegenes–NuclearmRNA(INTACT)
Primaryroot Hairyroot
Hairyrootsubmergenceresponsivegenes
PolysomalmRNA(TRAP)
NuclearRNA(INTACT)
TotalRNA CONTROL
HAIRYROOT
SUBMERGEDHAIRYROOT
CONTROLPRIMARYROOT
SUBMERGEDPRIMARYROOT
CHROMATINACCESSIBILITYOFTRANSPOSABLEELEMENTSISGLOBALLYHIGHINCONTROLHAIRYROOTS,BUTISREDUCEDTOPRIMARYROOTLEVELINSUBMERGENCE
TRANSPOSABLEELEMENTLOCATIONSMAPPINGREFERENCELOCATIONS
ATAC-SEQREADSREADCOUNTVS.BACKGROUNDRED(HIGH)àBLUE(LOW)ATAC-SEQREADSREADCOUNTVS.BACKGROUNDRED(HIGH)àBLUE(LOW)
TRANSPOSABLEELEMENTLOCATIONSMAPPINGREFERENCELOCATIONS
ATAC-SEQREADSREADCOUNTVS.BACKGROUNDRED(HIGH)àBLUE(LOW)ATAC-SEQREADSREADCOUNTVS.BACKGROUNDRED(HIGH)àBLUE(LOW)
