moss systems biology for translational research ralf reski
TRANSCRIPT
Moss Systems Biology for
Translational Research
Ralf Reski
Moss Physcomitrella patens: A New Model Plant
Small
Few cell types
Cell lineage
Tightly controlled growth conditions
Predominant generation haploid
Fully sequenced genome
Efficient homologous recombination
targeted gene knockout /
replacement
unique to higher eukaryotes
Modelling Plant Development 1: Light Microscope
Modelling Plant Development 2: Pattern Recognition
Modelling Plant Development 3: Simulation
branching
type of branching
F-type branching,
left
F-type branching,
right
Y-type of branching
Growth!
no
yes
miRNAs Regulate Gene Expression Qickly
micro RNAs:
not translated
more efficient than synthesis of regulatory proteins (e. g. repressors)
faster regulatory circuits
Biogenesis and Function of micro RNAs
AGO
RISC
CAPAAAAA
Dicer
CAPAAAAA
CAPAAAAA
CAPAAAAA
CAPAAAAA
Nucleus
Cytosol
HASTY
HEN
HYL
AGO
RISC
pri-miRNA
pre-miRNA
Post-transcriptional regulation of gene expression
Animals and Plants: Different Sets of DICER
PLA
NTS
AN
IMA
LS
Targeted knockout of DCL1a: Drastic Effects
0.5 cm 0.5 cm 0.5 cm
WTWT
RT-PCR analysis of miRNAsRT-PCR analysis of miRNAs
DCL1aDCL1aKO1KO1
0.5 cm 0.5 cm 0.5 cm
DCL1aDCL1aKO2KO2
0.5 cm 0.5 cm 0.5 cm
WTWT KO1KO1 KO2KO2
miRNA156miRNA156
miRNA160miRNA160
miRNA166miRNA166
miRNA390miRNA390
RT-PCR analysis of target transcriptsRT-PCR analysis of target transcripts
0
0,001
0,002
0,003
0,004
0,005
0,006
0,007
SBP3 ARF1 PpC3HDZIP1 PpHB10 TAS1PpSBP3PpSBP3 PpARFPpARF PpHDZIPPpHDZIPPpHB10PpHB10 PpTAS1PpTAS1
77
66
55
44
33
22
11
00Rel.
Tra
nscri
pt
Level
Rel.
Tra
nscri
pt
Level
WTWT
KO1KO1
KO2KO2
No DCL1a: No miRNAs, enhanced TF levels,
serverely affected growth
Targeted knockout of DCL1b: Less Drastic Effects
DCL1bDCL1b
200 µm
100 µm
Wild TypeWild Type
WTWT 11 22 33 44PpDCL1b KOPpDCL1b KO
miRNA156miRNA156
miRNA160miRNA160
miRNA166miRNA166
miRNA390miRNA390
miRNA535miRNA535
miRNA538miRNA538
U6 snRNAU6 snRNA
miRNA ExpressionmiRNA Expression
miRNAs still present BUT developmental arrest
DCL1b: miRNAs Cannot Cleave Target mRNAs
Control PpGNT1Control PpGNT1(no miRNA target)(no miRNA target)
WTWTKOKO11
KOKO22
KOKO33
KOKO44
PpSBP3 (Squamosa Promoter Binding Protein)PpSBP3 (Squamosa Promoter Binding Protein)PpSBP3 5’ U 3’ GUGCUC CUCUCUUCUGUCA CACGAG GAGAGAAGACAGUmiR156 3’ U 5’
PpC3HDZIP (HD leucine zipper)PpC3HDZIP (HD leucine zipper)PpC3HDZIP1 5’CU U 3’ GG AUGAAGCCUGGUCCGG CC UACUUCGGACCAGGCUmiR165 3’CC C 5’
PpHB10 (PpHB10 (HD leucine zipperHD leucine zipper))PpHB10 5’CU U 3’ GG AUGAAGCCUGGUCCGG CC UACUUCGGACCAGGCUmiR165 3’CC C 5’
miRNA Target Genes Silenced in DCL1b Mutant
miRNAs cannot cleave target mRNAs in the mutant,
nevertheless transcript levels of target genes reduced.
Novel feed-back loop: Transcriptional control by miRNAs
miRNA target transcriptsmiRNA target transcripts
Controls (no miRNA targets)Controls (no miRNA targets)EF1EF1
PpHDZIP1PpHDZIP1
PpHB10PpHB10
PpARFPpARF
PpGNT1PpGNT1
WTWT 11 22 33 44
PpDCL1b-MutantsPpDCL1b-Mutants
PpSBP3PpSBP3
• Arabidopsis: 21 ARF-Gene
• Reis: 24 ARF-Gene
• Physcomitrella: 15 ARF-Gen
Gruppe 2
Gruppe 1
Gruppe 3
PpARF3-1
PpARF3-2 (Tandemduplikation)
PpARF1-1
PpARF1-2
PpARF1-3
PpARF1-4
PpARF1-5
PpARF1-6
PpARF1-7
PpARF1-8
PpARF1-9
PpARF1-10
PpARF1-11
PpARF1-12
Phypa_61245
Group 3
PpARF3-1PpARF3-2Tandem duplication
• Arabidopsis: 21 ARF Genes
• Rice: 24 ARF-Genes
• Physcomitrella: 15 ARF Genes
PpARF3-1
PpARF1-1
PpARF1-2
PpARF1-3
PpARF1-4
PpARF1-5
PpARF1-6
PpARF1-7
PpARF1-8PpARF1-
9
PpARF1-10
PpARF1-11
PpARF1-12
Phypa_61245
Gro
up
1G
rou
p 3
Gro
up
2
Tandem duplication
PpARF3-2
miRNA160 regulates ARF Transcription Factors
miRNA160: Part of Multilevel Regulatory Network
Wild type ARFm Mutant
ARFARFmiRNAmiRNA160160
AuxinAuxin
GH3-2GH3-2
ARFARFmiRNAmiRNA160160
AuxinAuxin
GH3-2GH3-2
Precise Manipulation of miRNA160 Network
Targeted base-specific mutation of Targeted base-specific mutation of endogenous gene!endogenous gene!
t ggc atg cag ggg gcc agg ca G M Q G A R
t ggc atg caa ggc gcg cga ca
Pau I
G M Q G A R
Abolish miRNA160 binding site Abolish miRNA160 binding site without changing amino acids in the transcription factorwithout changing amino acids in the transcription factor
Dynamic Modelling of 4-Level Regulatory Network
EndogenousEndogenousauxin synthesisauxin synthesis
Impact of GH3-2Impact of GH3-2on [Auxin]on [Auxin]AuxinAuxin
applicationapplication
Auxin:Auxin:
miR160:miR160:
Activation of miR160Activation of miR160by auxinby auxin
Activation of miR160Activation of miR160by ARFby ARF
GH3-2:GH3-2:
Activation of GH3-2Activation of GH3-2by ARFby ARF
ARF:ARF:
Activation of ARFActivation of ARFby auxinby auxin Inhibition of ARFInhibition of ARF
by miR160by miR160
33=0=0in thein the
ARFARFm mutantm mutant
Expectations from Dynamic Modelling
ARFMutant
2.02.0
1.51.5
1.01.0
0.50.5
001010 2020 3030 4040 5050
[Exp
ressio
n]
[Exp
ressio
n]
[Time][Time]
GH3-2GH3-2
GH3-2GH3-2miRNAmiRNA
miRNAmiRNA
ARFARF
ARFARF
Steady-Steady-StateState
AuxinAuxinapplicationapplication
Wildtype
Mutant: Elevated ARF mRNA levels
ARF transcript levels respond to auxinARF transcript levels respond to auxin
WT ARFm Mutant
0
0,2
0,4
0,6
0,8
1
1,2
1,4
1,6
Control 1h 4h 8h 12h 24h
Auxin treatment
Rela
tive E
xp
ressio
n
Elevated Transcript Levels of Downstream Gene
GH3 conjugates (= inactivates) auxinGH3 conjugates (= inactivates) auxin
GH3-2GH3-2 mRNA levels enhanced by auxin mRNA levels enhanced by auxinR
ela
tive E
xp
ressio
n
0
0,5
1
1,5
2
2,5
control 1h 4h 8h 12h 24h
Auxin treatment
WT ARFm Mutant
Elevated Levels of Conjugated (= Inactive) Auxin
AuxinAuxin
0
0,5
1
1,5
2
2,5
control 1h 4h 8h 12h 24h
NAA treatments
rela
tive
exp
ress
ion
WT Mutant
GH3-2:Auxin conjugating enzyme
Free & conjugated Auxin
Free
Conjugated
WT ARFMutant
NA
A +
IA
A [
ng
/g F
resh
Weig
ht]
Mutant is Hyposensitive to Auxin
WT
ARFmMutant
[Auxin][Auxin]00 0.1 µM0.1 µM 0.5 µM0.5 µM 1 µM1 µM
ARFARFm mutant confirms m mutant confirms prediction.prediction.
AuxinAuxin
Extended Model to Include Diurnal Changes
Current Modelling Approaches
The Players
Gotelinde Seumel
Basel Kraiwesh
Asif Arif
Erika Lang
Philipp Rödel
Marta Tomek
Jutta Ludwig-Müller
Olaf Rönneberger
Stefan Jansen
Andreas Schlosser
Daniel Lang
Marco Vervliet-Scheebaum
Wolfgang Frank
Jens Timmer