which of these seedlings is insensitive to the plant hormone ethylene? seedling 1 seedling 2

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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?. - PowerPoint PPT Presentation

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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?

1Which of these seedlings is insensitive to the plant hormone ethylene?

Seedling 1Seedling 2Seedling 3

1 2 3

Noethylene+ ethyleneWhich seedling is a constitutive ethylene-response mutant?

Seedling 1Seedling 2Seedling 3Seedling 4

1 2 3 4

Noethylene+ ethylene+ ACCNo ACCWild typeMutant

Ethylene insensitive mutants (ein)*+ ACCNo ACCWild typeMutantConstitutive ethylene response mutants (ctr)

*Ethylene responses in Arabidopsis

TripleResponse

Inhibition of leaf cell expansion

Senescence

Ethylene-induced gene expression6There are other phenotypes in arabidopsis that are easily scored

A dormancy mutant called abi1-1

Wild typeabi1-1Dormancy(No germination)

ABA NO Dormancy (Seeds germinate)NO ABA NO Dormancy (Seeds germinate)NO ABAABA NO Dormancy (Seeds germinate!)What kind of mutant is this abi1-1 mutant?

Wild typeabi1-1Dormancy(No germination)

ABA NO Dormancy (Seeds germinate)NO ABA NO Dormancy (Seeds germinate)NO ABAABA NO Dormancy (Seeds germinate!)ABA overproduction mutant ABA constitutive response mutant ABA insensitive mutantabi1-1 mutants are ABA-insensitive in all their responsesLeung, 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 ABAGuard cells are not ABA-responsiveWildtypeabi1

Germination is not inhibited on ABA

Wild typeabi1-1Dormancy(No germination)

ABA NO Dormancy (Seeds germinate)NO ABA Dormancy (No germination)NO ABAABA Dormancy (No germination)What would be the phenotype of an ABA constitutive response mutant? What would be the phenotype if the receptor is disrupted?10

Plant growth, development, and survival depend on appropriate responses to a diverse array of constantly fluctuating external and internal signals

11ctr1 (recessive)

(eto1)ein2 ein3 ein5 (recessive)ein6 ein7Constitutive-response mutantsEthylene-insensitive mutantsetr1 etr2 ein4 (dominant)Ethylene-Response Mutants in Arabidopsis

air

C2H412CHECK YOUR PLATES Can you find any mutants?Some of the proteins in the ethylene signaling pathwayCTR1EIN2Kinase domainSoluble domainMembrane domainRegulatory domainETR1Signaling domainEthylene binding domainAn ethylene receptorETR2Signaling domainEthylene binding domainAn ethylene receptorA protein kinaseA protein of unknown functionYeast 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

Introduction to transcription activationDBAD X YUASPromoter sequence Coding SequenceCoding sequence of a gene Inside the NUCLEUS of the yeast cellDNA

transcriptionmRNAtranslation ProteinDBAD= transcription activator YUASPromoter sequence DNATranscriptional activators have 2 domainsDB = DNA binding domainAD = Activation domainDBADIntroduction to transcription activationCoding SequenceUnderlying principle of the Yeast Two-Hybrid AssayDBAD YHIS3 or lacZUAS XPromoter Reporter GeneReporter will be expressed XAD YInteraction of X and Y proteinsDBHIS3 or lacZUASPromoter Reporter GeneUnderlying principle of the Yeast Two-Hybrid AssayDBAD YHIS3 or lacZUAS XPromoter Reporter GeneBAITPREYThe BAIT is defined as the protein fused to the DB

The PREY is defined as any protein fused to the ADUnderlying principle of the Yeast Two-Hybrid Assay XDBHIS3 or lacZUASPromoter Reporter Gene XAD zNo transcription YHIS3 or lacZUAS Promoter Reporter GeneAD YDB XInteraction 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? XDBHIS3 or lacZUASPromoter Reporter Gene XAD zNo transcription YHIS3 or lacZUAS Promoter Reporter GeneAD YDB X

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 cellsBaitPreyPlasmids that are constructed in the labResulting proteins that are produced by the yeast cells

LAB: Yeast 2-hybrid assays with ethylene signaling proteinsCTR1EIN2Kinase domainSoluble domainMembrane domainRegulatory domainETR1Signaling domainEthylene binding domainAn ethylene receptorETR2Signaling domainEthylene binding domainAn ethylene receptorA protein kinaseA protein of unknown function4 and 581723 = empty prey plasmid; 6 = empty bait plasmid

RECORD YOUR DATA26Ethylene Responsive Gene ExpressionETR1Nucleus-CytoplasmEREIN2NLumenCETR2Ethylene signaling pathwayNC2H4NCu+Cu+1CTR178542CTR17827Ethylene Responsive Gene ExpressionETR1Nucleus-CytoplasmEREIN2NLumenCETR2Ethylene signaling pathwayNC2H4NCu+Cu+154278CTR128Ethylene Responsive Gene ExpressionETR1Nucleus-ERLumenEthylene signaling pathwayNC2H4NCu+EIN2NC14ETR2Cu+2Cytoplasm78CTR1529

RESULTS FROM CHANG LAB++++++++++++++??nonononononononononononononononoDid anyone have this one turn blue?30CodeBait clonePrey cloneHis reporter Interaction? (Yes/No)Lac Z reporterInteraction? (Yes/No)

7+1CTR1 Kinase (551-821)EIN2 C-term (561-1924)

7+8CTR1 Kinase (551-821)CTR1 N-term (53-568)

7+3CTR1 Kinase (551-821)Empty prey vector

4+8ETR1 (293-729)CTR1 N-term (53-568)

4+1ETR1 (293-729)EIN2 C-term (561-1924)

4+3ETR1 (293-729)Empty prey vector

2+5ETR2 (143-773)ETR1 (129-738)

2+1ETR2 (143-773)EIN2-C-term (561-1924)

2+8ETR2 (143-773)CTR1 N-term (53-568)

2+3ETR2 (143-773)Empty prey vector

6+5Empty bait vectorETR1 (129-738)

6+8Empty bait vectorCTR1 N-term (53-568)

6+1Empty bait vectorEIN2 C-term (561-1924)

CodeBait clonePrey cloneHis reporter Interaction? (Yes/No)Lac Z reporterInteraction? (Yes/No)

7+1CTR1 Kinase (551-821)EIN2 C-term (561-1924)

7+8CTR1 Kinase (551-821)CTR1 N-term (53-568)

7+3CTR1 Kinase (551-821)Empty prey vector

4+8ETR1 (293-729)CTR1 N-term (53-568)

4+1ETR1 (293-729)EIN2 C-term (561-1924)

4+3ETR1 (293-729)Empty prey vector

2+5ETR2 (143-773)ETR1 (129-738)

2+1ETR2 (143-773)EIN2-C-term (561-1924)

2+8ETR2 (143-773)CTR1 N-term (53-568)

2+3ETR2 (143-773)Empty prey vector

6+5Empty bait vectorETR1 (129-738)

6+8Empty bait vectorCTR1 N-term (53-568)

6+1Empty bait vectorEIN2 C-term (561-1924)