Seeds are mutagenized in
the lab, then screened for
mutants in the ethylene
signaling pathway, based on
the “triple response”
phenotype.
The mutants that we
discover correspond to
mutated genes.
Why is this extremely
useful?
• Which of these seedlings is insensitive to the
plant hormone ethylene?
No
ethylene
+
ethylene
1. Seedling 1
2. Seedling 2
3. Seedling 3
1
2
3
• Which seedling is a “constitutive ethyleneresponse” mutant?
1.
2.
3.
4.
No
ethylene
+
ethylene
1
3
Seedling 1
Seedling 2
Seedling 3
Seedling 4
2
4
Ethylene insensitive mutants (“ein”)
Wild type
Mutant
*
+ ACC
No ACC
Constitutive ethylene response mutants (“ctr”)
Wild type
Mutant
+ ACC
*
No ACC
Ethylene responses in Arabidopsis
Ethylene-induced gene expression
“Triple
Response”
Inhibition of leaf
cell expansion
Senescence
A dormancy mutant called abi1-1
Wild type
ABA
Dormancy
(No germination)
NO ABA
NO Dormancy
(Seeds germinate)
abi1-1
ABA
NO Dormancy
(Seeds germinate!)
NO ABA
NO Dormancy
(Seeds germinate)
What kind of mutant is this abi1-1 mutant?
Wild type
ABA
Dormancy
(No germination)
NO ABA
NO Dormancy
(Seeds germinate)
1. ABA overproduction mutant
2. ABA constitutive response mutant
3. ABA insensitive mutant
abi1-1
ABA
NO Dormancy
(Seeds germinate!)
NO ABA
NO Dormancy
(Seeds germinate)
abi1-1 mutants are ABA-insensitive
in all their responses
Germination is
not inhibited on
ABA
Wild
abi1
type
Root growth is
not inhibited on
ABA
Guard cells are
not ABAresponsive
Leung, J., Bouvier-Durand, M., Morris, P., Guerrier, D., Chefdor, F., and Giraudat, J. (1994). Arabidopsis ABA response gene ABI1: features of a calcium-modulated protein phosphatase. Science 264
1448-1452; Meyer, K., Leube, M., and Grill, E. (1994). A protein phosphatase 2C involved in ABA signal transduction in Arabidopsis thaliana. Science 264: 1452-1455.
What would be the phenotype of an
ABA constitutive response mutant?
Wild type
ABA
Dormancy
(No germination)
NO ABA
NO Dormancy
(Seeds germinate)
abi1-1
ABA
Dormancy
(No germination)
NO ABA
Dormancy
(No germination)
Plant growth, development, and survival depend on appropriate responses to a diverse
array of constantly fluctuating external and internal signals
Ethylene-Response Mutants in Arabidopsis
Ethylene-insensitive mutants
etr1 etr2 ein4 (dominant)
ein2 ein3 ein5 (recessive)
ein6 ein7
C2H4
Constitutive-response mutants
ctr1 (recessive)
air
(eto1)
CHECK YOUR PLATES – Can you find any mutants?
Some of the proteins in the ethylene signaling pathway
ETR1
ETR2
An ethylene receptor
Ethylene
binding
domain
Signaling domain
An ethylene receptor
Ethylene
binding
domain
Signaling domain
A protein kinase
CTR1
Regulatory
domain
Kinase domain
EIN2
Membrane
domain
Soluble domain
A protein of unknown
function
Yeast two-hybrid assay shows interaction of ETR1 and ERS ethylene
receptors with the CTR1 protein kinase
Yeast colonies
Clark K L et al. PNAS 1998;95:5401-5406
Introduction to transcription activation
Inside the
NUCLEUS of the
yeast cell
Promoter
sequence
Y
X
DB
Coding sequence of a
gene
AD
Coding Sequence
DNA
UAS
transcription
mRNA
translation
DB
AD
= transcription
activator
Protein
Introduction to transcription activation
YAD
DB
DNA
Coding Sequence
UAS
Promoter sequence
Transcriptional activators have 2 domains
DB = DNA binding domain
AD = Activation domain
Underlying principle of the Yeast Two-Hybrid Assay
X
DB
UAS
HIS3 or lacZ
Promoter
Reporter Gene
Interaction of X and Y proteins
X
Reporter will be expressed
DB
UAS
HIS3 or lacZ
Promoter
Reporter Gene
Underlying principle of the Yeast Two-Hybrid Assay
“PREY”
“BAIT”
X
DB
UAS
HIS3 or lacZ
Promoter
Reporter Gene
The “BAIT” is defined as the protein fused to the DB
The “PREY” is defined as any protein fused to the AD
Underlying principle of the Yeast Two-Hybrid Assay
X
DB
UAS
X
HIS3 or lacZ
Promoter
z
Reporter Gene
No transcription
DB
UAS
HIS3 or lacZ
Promoter
Reporter Gene
Interaction of ETR1 and ERS ethylene receptors with the CTR1 protein kinase in
the yeast two-hybrid assay.
Clark K L et al. PNAS 1998;95:5401-5406
How do we get these proteins into yeast cells so that we can
test whether they interact?
X
DB
UAS
X
HIS3 or lacZ
Promoter
z
Reporter Gene
No transcription
DB
UAS
HIS3 or lacZ
Promoter
Reporter Gene
First we have to clone our bait and prey genes into yeast
plasmids to express the proteins fused to the DB and AD
Bait
*Transform the
plasmids into
yeast cells
Prey
Plasmids that
are constructed
in the lab
Resulting proteins
that are produced
by the yeast cells
LAB: Yeast 2-hybrid assays with ethylene signaling proteins
4 and 5
ETR1
An ethylene receptor
Ethylene
binding
domain
Signaling domain
2
ETR2
An ethylene receptor
Ethylene
binding
domain
CTR1
8
Signaling domain
7
A protein kinase
Regulatory
domain
Kinase domain
1
EIN2
Membrane
domain
Soluble domain
A protein of unknown
function
3 = empty prey plasmid; 6 = empty bait plasmid
RECORD YOUR DATA
Code
Bait clone
Prey clone
7+1
7+8
7+3
CTR1 Kinase (551-821)
CTR1 Kinase (551-821)
CTR1 Kinase (551-821)
EIN2 C-term (561-1924)
CTR1 N-term (53-568)
Empty prey vector
4+8
4+1
4+3
ETR1 (293-729)
ETR1 (293-729)
ETR1 (293-729)
CTR1 N-term (53-568)
EIN2 C-term (561-1924)
Empty prey vector
2+5
2+1
2+8
2+3
ETR2 (143-773)
ETR2 (143-773)
ETR2 (143-773)
ETR2 (143-773)
ETR1 (129-738)
EIN2-C-term (561-1924)
CTR1 N-term (53-568)
Empty prey vector
6+5
6+8
6+1
Empty bait vector
Empty bait vector
Empty bait vector
ETR1 (129-738)
CTR1 N-term (53-568)
EIN2 C-term (561-1924)
His reporter
Interaction?
(Yes/No)
Lac Z reporter
Interaction?
(Yes/No)
Ethylene signaling pathway
C2H4
Lumen
ER
ETR2
ETR1
Cu+
Cu+
5 4
2
EIN2
1
7
7
8
CTR1
Cytoplasm
Ethylene Responsive Gene
Expression
8
CTR1
Ethylene signaling pathway
C2H4
Lumen
ER
ETR2
ETR1
Cu+
Cu+
2
5 4
8
EIN2
1
7
CTR1
Cytoplasm
Ethylene Responsive Gene
Expression
Ethylene signaling pathway
C2H4
Lumen
ER
ETR2
ETR1
Cu+
Cu+
2
5 4
8
EIN2
1
7
CTR1
Cytoplasm
Ethylene Responsive Gene
Expression
RESULTS FROM CHANG LAB
++
++
no
no
++
++
no
++
++
no
+
+
?
?
no
no
no
no
no
no
no
no
no
no
no
no
Did
anyone
have
this
one
turn
blue?