Professor Peter Gresshof - Australian Oilseeds Federation

Professor Peter Gresshof - Australian Oilseeds Federation

Soybean Biotechnology,
and End-User Benefits
Peter M. Gresshoff
www.cilr.uq.edu.au
p.gresshoff@uq.edu.au
Bundaberg March 2007
Thanks:
Arief Indrasumunar
Attila Kereszt
Ning Nontachaiyapoom
Qunyi Jiang
Yu-Hsiang Lin
Bernard Carroll
Akira Miyahara
Miki, Snow-Li, Dr Ning, Cuc, Aura, Sean, Satomi, Meng
Artem Men, Tripty Hirani, Pick-Kuen Chan
All other CILR colleagues, especially Uli Mathesius and
Michael Djordjevic (ANU)
ARC CoE funding, Qld Smart State Initiative, UQ Strategic Funds
FOOD
GRAIN
SEED
GRAIN
Industrial
Applications
Nutriceuticals
Embryo
Biofuels
Biopolymers
Flower
Carbon
Sequestration
Carbon
Sequestration
Feedstock
Leaf
Root
Nitrogen
Acquisition
Phosphorus
Acquisition
Water
Acquisition
Symbiotic
Symbiotic
Symbiotic
Mineral
Mineral
Mineral
Genome and Transcriptome Analysis
Proteome and Metabolome Analysis
Molecular Physiology Analysis
Use of Genetic and Genomic Models
Interactive data bases and
computational modeling
FEED
Noduleinduction
induction
Nodule
Colonisation
Colonisation
Roothair
hair
Root
deformation
deformation
andcurling
curling
and
Celldivision
division
Cell
Endosymbiosis
Endosymbiosis
Nitrogenfixation
fixation
Nitrogen
ProteinProteinandoil-rich
oil-richSeeds
Seeds
and
Biotechnology of
of soybean:
soybean:
Biotechnology
-The soybean
soybean genome
genome
-The
molecularmaps
maps
--molecular
molecularmarkers
markers
--molecular
Gene discovery
discovery
-- Gene
positionalcloning
cloning
--positional
TILLING
--TILLING
Gene expression
expression profiling
profiling
-- Gene
quantitativetarget
targetmethods
methods
--quantitative
genechips
chips(Affymetrix)
(Affymetrix)
--gene
-Gene transfer
transfer
-Gene
Agrobacterium-based
--Agrobacterium-based
lupin
groundnut
cowpea
phaseolus
soybean
Lotus
(Trifoleae)
Medicago
clover
alfalfa
pea
faba bean
lentil
lathyrus
(Vicieae)
Legume phylogeny
Legumes are closely related based on molecular markers
-30
-70
Hologalegoids
Papilionoids
after Wojciechowski M.F. (2003). Advances in Legume Systematics, part 10
-80
Myr bp
How Many Manhattan Phone Books Does It
Take to Print the Soybean Genome?
In small font, closely spaced
soybean -- 50 books
20 chromosomes
Human -160 phone
books
(1000 pages per phone book)
(22 million “letters” per phone book)
Fruit Fly -- 6 books
Roundworm -- 5 books
Yeast -- 1 book
E. coli -- ¼ book
HIV -- 1 page
The soybean genome?
soybean -- 50 books
• 20 chromosomes
• 1,100,000,000 base pairs (ATGC)
• about 36,000 genes
• many duplicated
• US DOE Soybean Genome Project
(JGI; 2006 onwards))
• Japanese Soybean Project
(2007 onwards)
How Many Genomes?
Genomes Published Since 1995
• 1995
– First genome published
(H. influenza)
• Today
Source for chart: Genomes Online Database
– Archaea: 26 complete, 26 draft/in progress
– Bacteria: >293 complete, 561 draft/in progress
– Eukaryotes: 20 complete, 204 draft/in progress
Legume Comparative Map
a1
e
3
l
E
8
g
G
2
I
T
S
5
A
7
3
T
T
c2
D
S
L
L
b2
T
S
8
S
S
L
g
S
L
L
j
3
L
S
M
c2
4
a2
4
III
Gm
5
4
m
VII
Lj
5
R
1
6
5
M
6
J
1
g
7
V
3
2
m
H
M
R
7
Mt
Ms
1
Ps
h
II
Ca
4
Vr
b1
4
Pv
Gm
M
R
R
D
F
j
f
3
H
A
E
S
L
S
S
L
c1
F
k
1
: Soybean, x=20, 1120Mbp
d2
k
P
T
8
b1
: Common bean, x=11, 620Mbp
c1
R
IV
2
Vr Pv Gm
: Mungbean, x=11, 520Mbp
6
2
C
: Lotus japonicus, x=6, 500Mbp
7
: Chickpea, x=8, 750Mbp
11
VI
: Pea, x=7, 5000Mbp
2
1
: Alfalfa, x=8, 1600Mbp
7/8
: M. truncatula, x=8, 500Mbp
6
P
S
K
Mt Ms Ps Ca Lj
6
S
5
c2
Basic Genome Info.
q
d1
b1
D
h
A
d1
i
Galegoid
Phaseoloid
454 Life Sciences “Pyro”Sequencing –
the latest in DNA sequencing
• Margulies et al. (2005) Genome
sequencing in microfabricated highdensity picolitre reactors. Nature 437,
376-380
• PCR amplification of single DNA
molecules and sequencing in picoliter
(10-12 litre) reactors
• 100-fold more efficient than
traditional automated sequencing
• no DNA cloning and plasmid
purification required
Now available at UQ Brisbane
Company
Format
Read Length
(bases)
Expected
Throughput
(million
bases/day)
454 Life Sciences
Parallel bead array
100
96
Agencourt Bioscience
Sequencing by litigation
50
200
Applied Biosystems
Capillary electrophoresis
1000
304
LI-COR Biosciences
Electronic microchip
20,000
14,000
Microchip Biotechnologies
Parallel bead array
850-1000
7
NimbleGen Systems
Map and survey microarray 30
100
Solexa
Parallel microchip
35
500
VisiGen Biotechnologies
Single-molecule array
NA
1000
Generation next. Companies racing for the $1000/human genome sequence
strive simultaneously for low cost, high accuracy, the ability to read long
stretches of DNA, and high throughput.
Use of Mutants to
Discover Genes
Forward Genetics
Wild type soybean root
Nodulation zone (was AON minus)
No-Nodulation zone (now AON plus)
Nodulation is internally regulated by
Autoregulation of Nodulation (AON) leading
to restricted nodule development
4
NODULATION
NODULATION
requiresthe
theRhizobiumRhizobiumrequires
producedNod
NodFactor
Factor
produced
Celldivision
division
Cell
Roothair
hair
Root
deformation
deformation
andcurling
curling
and
Infection
Infection
leaf
NARK
SDI
NODULE
PRIMORDIUM
Q
Maintenance of
Soybean CELL DIVISION
Induction of CELL DIVISION
LCO
LCO
Rhizobium
ROOT HAIR/EPIDERMIS
‘ACTIVATED STATE’
EMS
mutation
Wild type
nts1007
Nodule Autoregulation Receptor Kinase
PROMOTER
SP
PC
LRRs
Q106* K116*
=nts1007
Science 2003
TM
PC
K606*
kinase domain
intron
V837A Q920*
=nts382
The soybean
GmNARK promoter
Sureeporn Nontachaiyapoom
MPMI 2007 (in press)
GmNARK promoter analysis
•pGmNARK contains two
functional TATA boxes
•No control by inoculation
•Phloem Regulatory Region
was defined
1.7 kb
0.9 kb
Sureeporn Nontachaiyapoom
MPMI 2007 (in press)
GmNARK localisation
in transgenic soybean roots
• A: control (K599)
• D: pGmNARK::GFP
• I: pAtSUC2::GFP
• H: pGmNARK::GUS
Sureeporn Nontachaiyapoom
MPMI 2007 (in press)
GmNARK promoter model:
TF1 or TF3 may be LjKlavier, MtLSS or PsSYM28
Sureeporn Nontachaiyapoom
(CILR, Brisbane), MPMI (in press)
A complex loop
of control
leaf
NARK
SDI
NODULE
PRIMORDIUM
Q
Maintenance of
Soybean CELL DIVISION
Induction of CELL DIVISION
LCO
LCO
Rhizobium
ROOT HAIR/EPIDERMIS
‘ACTIVATED STATE’
The soybean drip system
Petiole Feeding of Liquid Extracts
Time = 0
8 – 10 hrs later
Blue food dye tracer
Source material from leaf of
nts1007
Nodule
Number
nts1007 (+Bj)
high
WT (- Bj)
high
WT (+Bj)
low
WT (+Bj) heat treated
low
Nod factors perception limits
nodule number
Non-Nodulation Mutants of Soybean
Bragg
Bragg
nod139
nod139
nod49
nod49
rj1
rj1
Clark
Clark
Non-nodulation
mutants made
with EMS
Nod+ +
Nod
Nod– –
Nod
Nod– –
Nod
Nod– –
Nod
Nod+ +
Nod
Nodulationmutants
mutants
Nodulation
areontogenically
ontogenicallyblocked
blockedin
in
are
differentplaces
places
different
nod139
nod49
x
x
Celldivision
division
Cell
x
nod49
nod139
Infection
Infection
Intracellular ‘Kinase’ Domain
LysM1 LysM2 LYSM3
Extra-cellular
Ancestral insertion
GmNFR5α
nod139
nn5
Intracellular ‘Kinase’ Domain
LysM1 LysM2 LYSM3
Extra-cellular
GmNFR5ß
nod139contains
containsaanaturally
naturallyinduced
inducedmutation
mutation(insertion)
(insertion)found
found
nod139
inmany
manyrelated
relatedcultivars
cultivarsbut
butnot
notancestral
ancestralgenotypes
genotypes
in
M
Gs
A
B
PI
A
B
B*
139
A B
A
49
B
A
Gs
: Glycine soja
PI
: PI437.654
B*
: Bragg wild type segregant
139
: nod139
49
: nod49
W
: Williams
NN5 : NN5
nts
: nts382, nts1007, nts1116
Cl
: Clark
rj1
: rj1
W
B
A
NN5
B A
nts
B A
B
nts
nts
A B
A B
Cl
rj1
A B A
B
Caution:
Caution:
Notall
allsoybean
soybeanlines
lines
Not
havethe
thesame
samebackground
background
have
GmNFR1 Mutant Alleles Detection
rj1
nod49
GmNFR1α
(4,690 bp)
LjNFR1
(5,687 bp)
GmNFR1β
(4,373 bp)
-374 bp
PI437654
NFR1α and ß receptor components of soybean
NFR1 receptors are Lys M receptor
kinases
Agrobacterium-based
Agrobacterium-based
complementation of
of the
the
complementation
nod49 mutant
mutant
nod49
nod49+35SGmNFR1α
Nodule
Nodule
Interval
Interval
A nod49+
Empty
Vector
nod49+nPGmNFR1α
GmNFR1α complements the nod49 mutation
GmNFR1α complements the nod49 mutation
and its over-expression increases nodule number
Nodulation of transgenic roots of different soybeans
GmNFR1α +Empty Vector
nod49
0
Bragg
97 ± 25
Clark
116 ± 9
*Number of nodules per root
± standard error; p< 0.05
+35S Promoter
279 ± 46
166 ± 30
236 ± 38
Improved nitrogen status of composite plants transformed
with an overexpressing GmNFR1α gene construct
Total N
mg/plant
nod49
+Empty
vector
4.2 ± 0.4 a
Bragg (WT) 54.5 ± 5.6 c
*numbers followed by the same letter for the same measured parameter
are not significantly different at P<0.05.
Plants were generated by A. rhizogenes K599 transformation.
nitrogen status
Improved
of composite plants
transformed with an over-expressing GmNFR1αgene construct
Total N
mg/plant
+Empty
vector
+35S
GmNFR1α
4.2 ± 0.4 a
126.5 ± 8.2b
Bragg (WT) 54.5 ± 5.6 c
74.2 ± 7.2c
nod49
*numbers followed by the same letter for the same measured parameter
are not significantly different at P<0.05. ± standard error
Plants were generated by A. rhizogenes K599 transformation.
Bradyrhizobium Response
Increased
(NN/plant)
of composite plants transformed with an over-expressing GmNFR1α
Empty vector
(no GmNFR1a)*
GmNFR1a + Native
3.4 kb promoter
102
0.0 a*
6.3 ± 3.6 b
134.0 ± 25.4 d
105
0.0 a
46.5 ± 9.5 c
570.0 ± 40.1 f
107
0.0 a
97.7 ± 25.4 d
565.3 ± 54.6 f
102
0.0 a*
2.0 ± 1.2 b
41.0 ± 15.3 c
105
0.0 a
151.3 ± 34.2 d
316.5 ± 28.5 e
107
0.0 a
143.0 ± 27.0 d
296.0 ± 35.8 e
B. japonicum
cfu.ml-1
GmNFR1a +
35S promoter
nod49
rj1
Bradyrhizobium Response
Increased
(NN/plant)
of composite plants transformed with an over-expressing GmNFR1α
Empty vector
(no GmNFR1a)*
GmNFR1a + Native
3.4 kb promoter
102
0.0 a*
6.3 ± 3.6 b
134.0 ± 25.4 d
105
0.0 a
46.5 ± 9.5 c
570.0 ± 40.1 f
107
0.0 a
97.7 ± 25.4 d
565.3 ± 54.6 f
102
0.0 a*
2.0 ± 1.2 b
41.0 ± 15.3 c
105
0.0 a
151.3 ± 34.2 d
316.5 ± 28.5 e
107
0.0 a
143.0 ± 27.0 d
296.0 ± 35.8 e
B. japonicum
cfu.ml-1
GmNFR1a +
35S promoter
nod49
rj1
Transcriptional
Profiling
The first Soybean EST Microarray (Maguire et al, 2002) 4092 genes
Affymetrix
Soybean
Microarray
# Transcripts
35,611 Soybean
15,421 Phytophthora
7,431 Heterodera
(cyst nematode)
Affymetrix
Soybean
Microarray
PM
MM
# Transcripts
35,611 Soybean
15,421 Phytophthora
7,431 Heterodera
(cyst nematode)
22 squares x 58,000 genes
Pto-like receptor kinase
Relative Expression
190
160
130
100
70
40
Relative Expression
Tryptophan synthase; hormone
nts+/-
20
16
12
8
4
0
0
1
2
3
4
5
WTWT+
Days Post Treatment
8
7
6
5
4
3
2
1
0.5
0.4
0.3
0.2
0.1
0.0
- A disease resistance gene
WT+/1,000 fold
nts+/0
- major effect
2
3
4
5
Days Post Treatment
n
o
rs
p
x
E
tiv
la
e
GmNARK-dependent Leaf
W T Inoculated
W T Uninoculated Transcriptome Profiling of
nts1007 Inoculated
Systemic Signal Effects
nts1007 Uninoculated
after Root Inoculation
R
n
m
re
tT
o
P
s
y
a
D
Expression of Gma.736
(T ryptophan Synthase) in Root
3.0
Relative Expression
.0
3
1
2.5
2.0
1.5
1.0
0.5
- no effect
0.0
0
1
2
3
Days Post Treatment
4
5
Mark Kinkema, CILR 2007
TILLING (Reverse Genetics)
Targeting
Induced
Local
Lesions
In
Genomes
Using molecular genetics without TRANSGENICS
The TILLING procedure
ENDO 1 is like Cel1: endonclease (patented); cuts single strand mismatch
GmClavata1A predicted protein domain structure and
amplicon location
SP
LRR Domain
TMD
Kinase Domain
Targeted Amplicon
The protein domains prediction was realized at http://smart.embl-heidelberg.de/
using the SMART program (Simple Modular Architecture Research Tool).
Position of the identified mutation within the
GmCLV1A amplicon (ps33 screen)
GmClavata1A and GmNARK
mutants
GmCLAVATA 1A
Individual
s17C2
s19H7
s22D6
s23E8
s23G7
s24F5
s7G8
s9E5
Sequence
g1021r
g903a
c306t
c365y
c403y
g516a
g821a
g883r
Effect
E765=
G726E
T527I
P547S
F559=
G597D
G699R
R719=
SIFT (IC)
0.01 (3.11)
0.25 (3.11)
0.00 (3.11)
0.24 (3.17)
0.00 (3.11)
Zygouscity DNA Stock
M2 Stock
Hetero
SB1208
3137
Homo
SB1407
301
Homo
SB1680
1172
Hetero
SB1765
1418
Hetero
SB1758
1204
3 equal sense mutants
Homo
SB1813 Outcome:
1885
Homo
SB511
868
5 missense mutants
Hetero
SB789
1392
0 non-sense mutants
GmNARK
Individual
s13B1
s13C3
s15D4
s15D4
s17B2
s18B5
Sequence
g1072r
c643y
c1177t
t1243g
g226a
g158a
Effect
M754I
N611=
H789=
H811Q
G472=
E450K
SIFT (IC)
0.03 (2.96)
s19B2
s19C3
s20A4
s5E7
c305t
g853r
c305y
g695r
P499S
A681=
P499S
D629N
0.00 (2.96)
0.00(2.97)
0.25 (2.95)
0.38 (2.95)
Zygouscity DNA Stock
Hetero
SB889
Hetero
SB908
Homo
SB1066
Homo
SB1066
Homo
SB1207
Homo
SB1305
Homo
Hetero
Hetero
Hetero
SB1345
SB1354
SB1454
SB405
3001
715
2175
Outcome:
2175
3136
3248
3189
3193
671
146
4 equal sense mutants
6 missense mutants
0 nonsense mutants
wt
SB889
M754I
GmCLV1A
GmNARK
Soybean TILLING Results
Conclusion/hypothesis:
CLAVATA1A in soybean
controls juvenile nodal integrity,
and
thus suppresses branching at
cotyledonary and unifoliate
nodes
NARK evolved after duplication.
CLV1A and NARK may have
other functions in the root
RNA interference-
Lipid transfer protein in soybean
Percentage expression relative to Actin
300.00
250.00
200.00
150.00
100.00
50.00
0.00
Controls
LTPRNAi
Comparison of LTP gene expression in LTPRNAi plants vs. controls
A
TILLING
Advantages of transgenics:
a) correct promoter
b) correct genome position
c) political sustainability
d) allelic series for structure : function
e) speedy if M2 DNA available and pooled
f) easily applied to existing seed collections
Conclusions
A
Soybean genomics allows structural
understanding and prediction
Nitrogen fixation and nodulation can be
increased by transgenic and
non-transgenic means
We know how to monitor soybean gene function
We can discover, isolate, change and transfer
soybean genes
A
End-user benefits
Increased nitrogen and phosphorus input
Improved flowering, rooting, plant structure
Altered products (phytoestrogens, taste)
Improved pest resistance
Altered protein to oil ratios
Biofuel (biodiesel)