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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?.
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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? • Seedling 1 • Seedling 2 • Seedling 3 No ethylene + ethylene 1 2 3
Which seedling is a “constitutive ethylene-response” mutant? • Seedling 1 • Seedling 2 • Seedling 3 • Seedling 4 No ethylene + ethylene 1 2 3 4
Wild type Mutant +ACC No ACC Ethylene insensitive mutants (“ein”) *
Wild type Mutant +ACC No ACC Constitutive ethylene response mutants (“ctr”) *
Inhibition of leaf cell expansion Ethylene responses in Arabidopsis Ethylene-induced gene expression “TripleResponse” Senescence
A dormancy mutant called abi1-1 Wild type abi1-1 ABA ABA NO Dormancy (Seeds germinate!) Dormancy (No germination) NO ABA NO ABA NO Dormancy (Seeds germinate) NO Dormancy (Seeds germinate)
What kind of mutant is this abi1-1mutant? Wild type abi1-1 ABA ABA NO Dormancy (Seeds germinate!) Dormancy (No germination) NO ABA NO ABA NO Dormancy (Seeds germinate) NO Dormancy (Seeds germinate) ABA overproduction mutant ABA constitutive response mutant ABA insensitive mutant
abi1-1 mutants are ABA-insensitive in all their responses Germination is not inhibited on ABA Root growth is not inhibited on ABA Guard cells are not ABA-responsive Wild type abi1 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 abi1-1 ABA ABA Dormancy (No germination) Dormancy (No germination) NO ABA NO ABA NO Dormancy (Seeds germinate) Dormancy (No germination)
Plant growth, development, and survival depend on appropriate responses to a diverse array of constantly fluctuating external and internal signals
air Ethylene-Response Mutants in Arabidopsis Ethylene-insensitive mutants etr1 etr2 ein4 (dominant) ein2 ein3 ein5 (recessive) ein6 ein7 C2H4 Constitutive-response mutants ctr1 (recessive) (eto1)
Some of the proteins in the ethylene signaling pathway ETR1 An ethylene receptor Ethylene binding domain Signaling domain ETR2 An ethylene receptor Ethylene binding domain Signaling domain CTR1 A protein kinase Regulatory domain Kinase domain EIN2 A protein of unknown function Membrane domain Soluble domain
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 Coding sequence of a gene Y X AD AD DB DB Coding Sequence DNA UAS transcription mRNA translation = transcription activator Protein
Introduction to transcription activation AD Y DB Coding Sequence DNA UAS Promoter sequence Transcriptional activators have 2 domains DB = DNA binding domain AD = Activation domain
Underlying principle of the Yeast Two-Hybrid Assay AD Y X DB HIS3 or lacZ UAS Promoter Reporter Gene Interaction of X and Y proteins AD X Y Reporter will be expressed DB HIS3 or lacZ UAS Promoter Reporter Gene
Underlying principle of the Yeast Two-Hybrid Assay “PREY” AD Y “BAIT” X DB HIS3 or lacZ UAS 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 Y AD DB HIS3 or lacZ Y UAS Promoter Reporter Gene AD z X X No transcription DB HIS3 or lacZ UAS 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 Y AD DB HIS3 or lacZ Y UAS Promoter Reporter Gene AD z X X No transcription DB HIS3 or lacZ UAS 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 Resulting proteins that are produced by the yeast cells Plasmids that are constructed in the lab
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 Signaling domain 8 7 CTR1 A protein kinase Regulatory domain Kinase domain 1 EIN2 A protein of unknown function Membrane domain Soluble domain 3 = empty prey plasmid; 6 = empty bait plasmid
Ethylene signaling pathway C2H4 Lumen ETR2 ETR1 EIN2 N N ER Cu+ Cu+ N CTR1 CTR1 C 5 4 2 8 8 1 7 7 - Nucleus Cytoplasm Ethylene Responsive Gene Expression
Ethylene signaling pathway C2H4 Lumen ETR2 ETR1 EIN2 N N ER Cu+ Cu+ N C 5 2 7 1 4 8 - CTR1 Nucleus Cytoplasm Ethylene Responsive Gene Expression
Ethylene signaling pathway C2H4 Lumen ETR2 ETR1 EIN2 N N ER Cu+ Cu+ N C 7 2 5 1 4 8 - CTR1 Nucleus Cytoplasm Ethylene Responsive Gene Expression
RESULTS FROM CHANG LAB ++ ++ Did anyone have this one turn blue? ? ? no no ++ ++ ++ ++ no no + + no no no no no no no no no no no no