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![Page 1: Seeds are mutagenized in the lab, then screened for mutants in the ethylene signaling pathway, based on the “triple response” phenotype. The mutants that.](https://reader038.fdocuments.us/reader038/viewer/2022110100/56649e455503460f94b394bb/html5/thumbnails/1.jpg)
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?
![Page 2: Seeds are mutagenized in the lab, then screened for mutants in the ethylene signaling pathway, based on the “triple response” phenotype. The mutants that.](https://reader038.fdocuments.us/reader038/viewer/2022110100/56649e455503460f94b394bb/html5/thumbnails/2.jpg)
• Which of these seedlings is insensitive to the plant hormone ethylene?
1. Seedling 12. Seedling 23. Seedling 3
1 2 3
Noethylene
+ ethylene
![Page 3: Seeds are mutagenized in the lab, then screened for mutants in the ethylene signaling pathway, based on the “triple response” phenotype. The mutants that.](https://reader038.fdocuments.us/reader038/viewer/2022110100/56649e455503460f94b394bb/html5/thumbnails/3.jpg)
• Which seedling is a “constitutive ethylene-response” mutant?
1. Seedling 12. Seedling 23. Seedling 34. Seedling 4
1 2 3 4
Noethylene
+ ethylene
![Page 4: Seeds are mutagenized in the lab, then screened for mutants in the ethylene signaling pathway, based on the “triple response” phenotype. The mutants that.](https://reader038.fdocuments.us/reader038/viewer/2022110100/56649e455503460f94b394bb/html5/thumbnails/4.jpg)
+ ACC
No ACC
Wild type Mutant
Ethylene insensitive mutants (“ein”)
*
![Page 5: Seeds are mutagenized in the lab, then screened for mutants in the ethylene signaling pathway, based on the “triple response” phenotype. The mutants that.](https://reader038.fdocuments.us/reader038/viewer/2022110100/56649e455503460f94b394bb/html5/thumbnails/5.jpg)
+ ACC
No ACC
Wild type Mutant
Constitutive ethylene response mutants (“ctr”)
*
![Page 6: Seeds are mutagenized in the lab, then screened for mutants in the ethylene signaling pathway, based on the “triple response” phenotype. The mutants that.](https://reader038.fdocuments.us/reader038/viewer/2022110100/56649e455503460f94b394bb/html5/thumbnails/6.jpg)
Ethylene responses in Arabidopsis
“TripleResponse”
Inhibition of leaf cell expansion
Senescence
Ethylene-induced gene expression
![Page 7: Seeds are mutagenized in the lab, then screened for mutants in the ethylene signaling pathway, based on the “triple response” phenotype. The mutants that.](https://reader038.fdocuments.us/reader038/viewer/2022110100/56649e455503460f94b394bb/html5/thumbnails/7.jpg)
A dormancy mutant called abi1-1Wild type abi1-1
Dormancy(No germination)
ABA
NO Dormancy (Seeds germinate)
NO ABA
NO Dormancy (Seeds germinate)
NO ABA
ABA
NO Dormancy (Seeds germinate!)
![Page 8: Seeds are mutagenized in the lab, then screened for mutants in the ethylene signaling pathway, based on the “triple response” phenotype. The mutants that.](https://reader038.fdocuments.us/reader038/viewer/2022110100/56649e455503460f94b394bb/html5/thumbnails/8.jpg)
What kind of mutant is this abi1-1 mutant?
Wild type abi1-1
Dormancy(No germination)
ABA
NO Dormancy (Seeds germinate)
NO ABA
NO Dormancy (Seeds germinate)
NO ABA
ABA
NO Dormancy (Seeds germinate!)
1. ABA overproduction mutant 2. ABA constitutive response mutant 3. ABA insensitive mutant
![Page 9: Seeds are mutagenized in the lab, then screened for mutants in the ethylene signaling pathway, based on the “triple response” phenotype. The mutants that.](https://reader038.fdocuments.us/reader038/viewer/2022110100/56649e455503460f94b394bb/html5/thumbnails/9.jpg)
abi1-1 mutants are ABA-insensitive in all their responses
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.
Root growth is not inhibited on
ABA
Guard cells are not ABA-
responsiveWildtype abi1
Germination is not inhibited on
ABA
![Page 10: Seeds are mutagenized in the lab, then screened for mutants in the ethylene signaling pathway, based on the “triple response” phenotype. The mutants that.](https://reader038.fdocuments.us/reader038/viewer/2022110100/56649e455503460f94b394bb/html5/thumbnails/10.jpg)
Wild type abi1-1
Dormancy(No germination)
ABA
NO Dormancy (Seeds germinate)
NO ABA
Dormancy (No germination)
NO ABA
ABA
Dormancy (No germination)
What would be the phenotype of an ABA constitutive response mutant?
![Page 11: Seeds are mutagenized in the lab, then screened for mutants in the ethylene signaling pathway, based on the “triple response” phenotype. The mutants that.](https://reader038.fdocuments.us/reader038/viewer/2022110100/56649e455503460f94b394bb/html5/thumbnails/11.jpg)
Plant growth, development, and survival depend on appropriate responses to a diverse array of constantly fluctuating external and internal signals
![Page 12: Seeds are mutagenized in the lab, then screened for mutants in the ethylene signaling pathway, based on the “triple response” phenotype. The mutants that.](https://reader038.fdocuments.us/reader038/viewer/2022110100/56649e455503460f94b394bb/html5/thumbnails/12.jpg)
ctr1 (recessive)
(eto1)
ein2 ein3 ein5 (recessive)ein6 ein7
Constitutive-response mutants
Ethylene-insensitive mutants
etr1 etr2 ein4 (dominant)
Ethylene-Response Mutants in Arabidopsis
air
C2H4
![Page 13: Seeds are mutagenized in the lab, then screened for mutants in the ethylene signaling pathway, based on the “triple response” phenotype. The mutants that.](https://reader038.fdocuments.us/reader038/viewer/2022110100/56649e455503460f94b394bb/html5/thumbnails/13.jpg)
CHECK YOUR PLATES – Can you find any mutants?
![Page 14: Seeds are mutagenized in the lab, then screened for mutants in the ethylene signaling pathway, based on the “triple response” phenotype. The mutants that.](https://reader038.fdocuments.us/reader038/viewer/2022110100/56649e455503460f94b394bb/html5/thumbnails/14.jpg)
Some of the proteins in the ethylene signaling pathway
CTR1
EIN2
Kinase domain
Soluble domainMembrane domain
Regulatory domain
ETR1Signaling domainEthylen
e binding domain
An ethylene receptor
ETR2Signaling domainEthylen
e binding domain
An ethylene receptor
A protein kinase
A protein of unknown function
![Page 15: Seeds are mutagenized in the lab, then screened for mutants in the ethylene signaling pathway, based on the “triple response” phenotype. The mutants that.](https://reader038.fdocuments.us/reader038/viewer/2022110100/56649e455503460f94b394bb/html5/thumbnails/15.jpg)
Yeast two-hybrid assay shows interaction of ETR1 and ERS ethylene receptors with the CTR1 protein kinase
Clark K L et al. PNAS 1998;95:5401-5406
Yeast colonies
![Page 16: Seeds are mutagenized in the lab, then screened for mutants in the ethylene signaling pathway, based on the “triple response” phenotype. The mutants that.](https://reader038.fdocuments.us/reader038/viewer/2022110100/56649e455503460f94b394bb/html5/thumbnails/16.jpg)
Introduction to transcription activation
DBAD
X Y
UAS
Promoter sequence
Coding Sequence
Coding sequence of a gene
Inside the NUCLEUS of the
yeast cell
DNA
transcription
mRNA
translation
ProteinDB
AD = transcription activator
![Page 17: Seeds are mutagenized in the lab, then screened for mutants in the ethylene signaling pathway, based on the “triple response” phenotype. The mutants that.](https://reader038.fdocuments.us/reader038/viewer/2022110100/56649e455503460f94b394bb/html5/thumbnails/17.jpg)
Y
UAS
Promoter sequence
DNA
Transcriptional activators have 2 domainsDB = DNA binding domainAD = Activation domain
DB
AD
Introduction to transcription activation
Coding Sequence
![Page 18: Seeds are mutagenized in the lab, then screened for mutants in the ethylene signaling pathway, based on the “triple response” phenotype. The mutants that.](https://reader038.fdocuments.us/reader038/viewer/2022110100/56649e455503460f94b394bb/html5/thumbnails/18.jpg)
Underlying principle of the Yeast Two-Hybrid Assay
DB
AD Y
HIS3 or lacZUAS
X
Promoter Reporter Gene
Reporter will be expressed X
AD
Y
Interaction of X and Y proteins
DBHIS3 or lacZ
UAS
Promoter Reporter Gene
![Page 19: Seeds are mutagenized in the lab, then screened for mutants in the ethylene signaling pathway, based on the “triple response” phenotype. The mutants that.](https://reader038.fdocuments.us/reader038/viewer/2022110100/56649e455503460f94b394bb/html5/thumbnails/19.jpg)
Underlying principle of the Yeast Two-Hybrid Assay
DB
AD Y
HIS3 or lacZUAS
X
Promoter Reporter Gene
“BAIT”
“PREY”
The “BAIT” is defined as the protein fused to the DB
The “PREY” is defined as any protein fused to the AD
![Page 20: Seeds are mutagenized in the lab, then screened for mutants in the ethylene signaling pathway, based on the “triple response” phenotype. The mutants that.](https://reader038.fdocuments.us/reader038/viewer/2022110100/56649e455503460f94b394bb/html5/thumbnails/20.jpg)
Underlying principle of the Yeast Two-Hybrid Assay
X
DBHIS3 or lacZ
UAS
Promoter Reporter Gene
X
AD
z No transcription
Y
HIS3 or lacZUAS
Promoter Reporter Gene
AD Y
DB
X
![Page 21: Seeds are mutagenized in the lab, then screened for mutants in the ethylene signaling pathway, based on the “triple response” phenotype. The mutants that.](https://reader038.fdocuments.us/reader038/viewer/2022110100/56649e455503460f94b394bb/html5/thumbnails/21.jpg)
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
![Page 22: Seeds are mutagenized in the lab, then screened for mutants in the ethylene signaling pathway, based on the “triple response” phenotype. The mutants that.](https://reader038.fdocuments.us/reader038/viewer/2022110100/56649e455503460f94b394bb/html5/thumbnails/22.jpg)
How do we get these proteins into yeast cells so that we can test whether they interact?
X
DBHIS3 or lacZ
UAS
Promoter Reporter Gene
X
AD
z No transcription
Y
HIS3 or lacZUAS
Promoter Reporter Gene
AD Y
DB
X
![Page 23: Seeds are mutagenized in the lab, then screened for mutants in the ethylene signaling pathway, based on the “triple response” phenotype. The mutants that.](https://reader038.fdocuments.us/reader038/viewer/2022110100/56649e455503460f94b394bb/html5/thumbnails/23.jpg)
First we have to clone our bait and prey genes into yeast plasmids to express the proteins fused to the DB and AD
*Transform the plasmids into yeast cells
Bait
Prey
Plasmids that are constructed in the lab
Resulting proteins that are produced by the yeast cells
![Page 24: Seeds are mutagenized in the lab, then screened for mutants in the ethylene signaling pathway, based on the “triple response” phenotype. The mutants that.](https://reader038.fdocuments.us/reader038/viewer/2022110100/56649e455503460f94b394bb/html5/thumbnails/24.jpg)
![Page 25: Seeds are mutagenized in the lab, then screened for mutants in the ethylene signaling pathway, based on the “triple response” phenotype. The mutants that.](https://reader038.fdocuments.us/reader038/viewer/2022110100/56649e455503460f94b394bb/html5/thumbnails/25.jpg)
LAB: Yeast 2-hybrid assays with ethylene signaling proteins
CTR1
EIN2
Kinase domain
Soluble domainMembrane domain
Regulatory domain
ETR1Signaling domainEthylen
e binding domain
An ethylene receptor
ETR2Signaling domainEthylen
e binding domain
An ethylene receptor
A protein kinase
A protein of unknown function
4 and 5
8
1
7
2
3 = empty prey plasmid; 6 = empty bait plasmid
![Page 26: Seeds are mutagenized in the lab, then screened for mutants in the ethylene signaling pathway, based on the “triple response” phenotype. The mutants that.](https://reader038.fdocuments.us/reader038/viewer/2022110100/56649e455503460f94b394bb/html5/thumbnails/26.jpg)
RECORD YOUR DATA
![Page 27: Seeds are mutagenized in the lab, then screened for mutants in the ethylene signaling pathway, based on the “triple response” phenotype. The mutants that.](https://reader038.fdocuments.us/reader038/viewer/2022110100/56649e455503460f94b394bb/html5/thumbnails/27.jpg)
Ethylene Responsive Gene Expression
ETR1
Nucleus
-
Cytoplasm
ER
EIN2
N
Lumen
C
ETR2
Ethylene signaling pathway
N
C2H4
N
Cu+
Cu+
1 CTR1
785 42
CTR1
78
![Page 28: Seeds are mutagenized in the lab, then screened for mutants in the ethylene signaling pathway, based on the “triple response” phenotype. The mutants that.](https://reader038.fdocuments.us/reader038/viewer/2022110100/56649e455503460f94b394bb/html5/thumbnails/28.jpg)
Ethylene Responsive Gene Expression
ETR1
Nucleus
-
Cytoplasm
ER
EIN2
N
Lumen
C
ETR2
Ethylene signaling pathway
N
C2H4
N
Cu+
Cu+
15 42 78CTR1
![Page 29: Seeds are mutagenized in the lab, then screened for mutants in the ethylene signaling pathway, based on the “triple response” phenotype. The mutants that.](https://reader038.fdocuments.us/reader038/viewer/2022110100/56649e455503460f94b394bb/html5/thumbnails/29.jpg)
Ethylene Responsive Gene Expression
ETR1
Nucleus
-
ER
Lumen
Ethylene signaling pathway
N
C2H4
N
Cu+
EIN2
N
C14
ETR2Cu+
2
Cytoplasm
78CTR1
5
![Page 30: Seeds are mutagenized in the lab, then screened for mutants in the ethylene signaling pathway, based on the “triple response” phenotype. The mutants that.](https://reader038.fdocuments.us/reader038/viewer/2022110100/56649e455503460f94b394bb/html5/thumbnails/30.jpg)
RESULTS FROM CHANG LAB
++ ++
++++++ ++
+ +
? ?
nonono
nonono
nonono
nonono
no no
nono
Did anyone have this one turn blue?