Download A light-dependent selection marker system in plants

A light-dependent selection marker system in plants
Ingyu Hwang and Jae Sun Moon
Seoul National University
Korea Research Institute of Bioscience and Biotechnology
January 15, 2012
PAG meeting
Burkholderia glumae:
The causal agent of rice panicle blight (bacterial rice grain rot) and bacterial wilt
Burkholderia glumae
• Infects roots, stems, and flowers.
• Produces toxoflavin and N-octanoyl-L-homoserine lactone.
• Optimum growth temperature is 37oC.
• Possesses 2-4 polar flagella.
C4
CH3
N
Toxoflavin
C6
C8
syn
thet
ic
pur
ified
O
365 nm
N
N
N
H3C
N
O
CH3
N
O
N
N
N
H3C
N
O
O
H
N
O
N
N
H
O
N
H
N
H3C
O
N-octanoyl-L-homoserine lactone (C8-HSL)
N
O
Toxoflavin is a photosensitizer.
Production of hydrogen peroxide
H2O2
light
O2
Reduction
O
MeN
5
O
N
O
Oxidation
N
MeN
4
8
1
N
N
Me
Toxoflavin
Oxidized type
Reduction
H
R C
O
H
N
4
8
N
H
N
N
Me
4,8-Dihydrotoxoflavin
Reduced type
Oxidation
OH
H
Alcohols
Substrate
R CHO
Aldehydes
H2
Substrate
T. Nagamatsu, Y. Hashiguchi, Y. Sakuma, and F. Yoneda, Chem. Lett., 1982, 1309.
Toxoflavin is a key pathogenicity factor.
Disease severity
5
5
3.5
40 μg/ml
20 μg/ml
10 μg/ml
2
5 μg/ml
0.6
0
2.5 μg/ml
SDW
Milyang 23
10 days after treatment
Regulation of toxoflavin biosynthesis by quorum sensing
Practical Application: Toxin degradation
1.
2.
Developing disease resistant lines
A novel selection marker for plant transformation
Kim J. et. al. Mol. Microbiol. 54: 921-934 (2004)
Isolation and identification of toxoflavin-degrading
bacteria from natural environment
Plant material
LB plate
(40 μg/ml
toxoflavin)
Soil
Other sources
incubation
Rice seedling
Identification:
16s rDNA sequencing
FAME analysis
Biolog analysis
Pure culture of
survivors
Isolation of Paenibacillus polymyxa strain JH2 from rice seeds
Deletion analysis of pJ9
Toxoflavin
degradation
activiy
1kb
pJ9
pJ91
pJ90
(E)
H
H
H
B
E
B
E B H HE
(H)
H
H E
H
H
tflA
1kb
+
+
+
E. coli DH5α carrying pJ9 degrades toxoflavin.
2
JH
5
DH
α
a
1) n
)
)
9)
1
J
4
yx
6
J
J
J
vi
(p
(p
a
(p
(p
ym
l
l
α
5
5α xof
5α
5α
po
H
H
.
H
H
to
D
D
P
D
D
UV 265 nm
Toxoflavin
After 16 hr in LB broth (Toxoflavin 40 μg/ml)
TflA is similar to ring-cleavage extradiol dioxygenases.
1
2
3
4
5
6
7
8
9
1
2
3
4
5
6
7
8
9
1
2
3
4
5
6
7
8
9
Degradation of toxoflavin by TflA depends on Mn++ and DTT.
100µM toxoflavin plus
1: CK (10µM MnCl2)
2: Purified His-TflA (10µM MnCl2)
3: CK (5mM DTT)
4: Purified His-TflA (5mM DTT)
5: CK (5mM DTT/10µM MnCl2)
6: Purified His-TflA
(5mM DTT/10µM MnCl2)
1
2
3
4
5
6
Comparison of TflA with other extradiol dioxygenases
TflA
NahC
NahH
ThnC
BphC
Specific
activity
(units/mg)
Metal
0.04
MnCl2
25oC
MnCl2
25oC
FeCl2
25oC
FeCl2
30oC
MnCl2
25oC
1.5
7
2
0.65
Temperature
pH
KM
Substrate
(μM)
6.5
7.5
7.5
6.8
8.0
69.72
Toxoflavin
400
2,3dihydroxybiphenyl
0.25
2,3dihydroxybiphenyl
18.6
1,2dihydroxybiphenyl
7
2,3dihydroxybiphenyl
Purified TflA degrades toxoflavin derivatives.
Toxoflavin
3-Methyl
toxoflavin
3-Phenyl
toxoflavin
4,8-Dihydro
toxoflavin
3-Methyl
4,8-Dihydro
toxoflavin
+++
+++
_
Reumycin
3-Methyl
reumycin
3-Phenyl
reumycin
+
+++
_
+++
Fervenulin
+
++
5-Deazaflavin
_
Degradation activity:-0~20% degradation; +,20~50%; ++, 50~80%; +++, 80~100%
Toxoflavin derivatives with phenyl group on 3 carbon were not degraded by TflA.
O
O
CH3 H3C
N
H3C
N
O
N
N
H
N
H
N
H
N
H3C
N
H
3-Methyltoxoflavin
N
N
N
N
H
O
O
N
O
O
H3C
N
N
3
N
N
N
N
O
CH3
N
O
N
H
N
H3C
5-Deazaflavin
O
N
Reumycin
CH3
N
H3C
N
N
N
N
N
CH3
O
N
N
H
N
Toxoflavin
O
O
H3C
CH3
3-Phenyltoxoflavin
H3C
H
N
N
N
O
N
O
N
Fervenulin
3'-Methyl 4,8-Dihydrotoxoflavin
O
H3C
N
CH 3
CH3
4,8-Dihydrotoxoflavin
N
H
H
N
N
CH3
CH3
H3C
CH3
N
N
N
O
O
O
N
3-Methylreumycin
N
O
N
H
N
3-Phenylreumycin
Extradiol dioxygenases
ThnC
TflA
O
OH
N
H3C
H
N
N
O
N
Mn2+, DTT
N
?
O
O2
COOH
OH
Fe2+
1,2-dihydroxytetralin
CH3
Toxoflavin
CM-2
COO-
OH
BphC
COO-
O2
O2
CHO
Mn2+
COOH
OH
OH
3,4-dihydroxyphenylacetate
OH
Mn2+
OH
OH
2,3-dihydroxybiphenyl
O
COOH
OH
Nature Biotechnology (2006) 20:575-580
Generation of transgenic rice plants expressing tflA
XhoI
XhoI
SacI
XbaI
HindIII
LB
RB
HygromycinR
NOS
35S
tflA
MCS
35S
genomic DNA
genomic DNA
Fig. 1. Genetic construction of pJ904(pCamLA::tflA).
A
B
D
I
E
C
F
G
H
Shoot
Root
Dt 1-2
T4 plants
AT rich repeat region
10,021bp
3,404bp
32003573~32003575
(Chromosome 3)
Muscle M-line assembly protein unc-89
(putative gypsy-type retrotransposon protein)
Hypothetical protein
L
B
water
Dongjin wild-type
T-DNA
Dt1-2
water
Dongjin
wild-type
Dt1-2
0
0.6
3.7
Disease degree (1~5)
0.1
10
20
30
40
50
(%)
60
0 Healthy panicle
1 Panicle 0-20% discolored
2 Panicle 21-40% discolored
3 Panicle 41-60% discolored
4 Panicle 61-80% discolored
5 Panicle 81-100% discolored
70
80
90
100
Comparison of selection markers in rice and Arabidopsis
Koh et.al. Plant Biotech. J. (2010), pp.1-11
Efficiency of rice transformation
Marker
pCamLA
(hpt)
No. of calli
inoculated
No. of
resistant calli
No. of putative
regenerated plants
PCR positive
independent plantsa
Transformation
frequency (%) b
300
80
20
16
80
300
134
43
38
88.37
300
84
28
25
89.28
300
192
26
21
80.76
pTflA
a
Independent plants : plants coming from different calli
b Number of PCR-positive plants/number of putative regenerated plants
Seed selection with toxoflavin (7mg/L)
Dongjin wild-type
(Hygromycin;30mg/L)
Dongjin wild-type
Dt27-4-1
Dt28-2-5
The photograph was taken 10 days after seeding.
Dt7-3-5
Dt40-9-3
Spray screening of transgenic rice plants
WT
Dt2-7-2
Dt4-1-4
Toxoflavin (mg/L)
0
10
50
The photograph was taken after 48h after spray.
Dt27-4-1
Dt40-9-1
Light-dependent sensitivity of various crops to toxoflavin
Light
Toxoflavin (mg/L)
0
1
2
4
6
8 10 20
Dark
Toxoflavin (mg/L)
0
1
2
4
6
8 10 20
Rice
Barley
Corn
Arabidopsis
Hot pepper
Soybean
Tomato
Tobacco
Cucumber
Watermelon
Melon
Zucchini
Koh et.al. Plant Biotech. J. (2010), pp.1-11
Acknowledgments
• Graduate Students
Hongsup Kim, Eunhye Goo
• Former Students and Postdocs
Jinwoo Kim, Okhee Choi,
Yun-Jung Kim
• Funding:
- Crop Functional Genomic Center (MEST)
- The Creative Research Initiatives (NRF)
• Collaborators
- Tomohisa Nagamatsu (Okayama Univ.)
- Jae Sun Moon, Serry Koh (KRIBB)
- Nam-Soo Jwa (Sejong Univ.)