ethylene responses developmental processes fruit ripening - ethylene is essential promotion of seed...
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Ethylene responses
Developmental processesFruit ripening - ethylene is essential Promotion of seed germinationRoot initiationBud dormancy release Inhibition/promotion of floweringSex shifts in flowers Senescence of leaves, flowers
Responses to abiotic and biotic stress Abscission of leaves, flowers, fruitsEpinasty of leaves Inhibition/promotion of cell division/elongationAltered geotropism in roots, stems Induction of phytoalexins/disease resistanceAerenchyma formation
Signal transduction
Response
Signal
plant cell
?
WHAT CONSTITUTES AN UNDERSTANDING OF SIGNALING PATHWAYS?
HOW CAN RESEARCHERS ELUCIDATE SIGNALING PATHWAYS?
“Genetic Dissection” of the Ethylene Signaling
Pathway
How to genetically dissect a pathway
1. Identify a phenotype that is specific to the process you are interested in
2. Design appropriate screen for isolating mutants based on this phenotype
3. Clone the corresponding gene by map-based cloning
4. Investigate the function of the corresponding protein at cell biological and biochemical levels
Pea seedlings
Neljubow (1901) Beih Bot Zentralbl 10, 128-139
The seedling “triple response”
“Triple Response”
Arabidopsis thaliana
Seeds are mutagenized in the lab and then screened for mutants in the ethylene signaling pathway, based on the “triple response” phenotype.
The mutants that we discover correspond to mutated genes.
Bleecker et al. (1988) Science 241, 1086–1089
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
Molecular markers provide a link between genetic loci and physical DNA
*A genetic map of molecular markers on the chromosome allows one to clone any gene for which there is a mutant phenotype
Chang et al. (1988) PNAS 85: 6856-6860
X
Landsberg Columbia
F1
F2
1 2 3 4 5 . . . . .
mutmut
heterozygous for mut
Recombinant genotypes
Generating a mapping population
Mapping population
self-pollinate
hand-pollinate
Example of mapping with molecular markers
Mapping population
Marker B
Marker A
ETR1
CTR1
EIN2
ETR2
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
Cloned the genes, but now look at:
1.Subcellular localization of the proteins 2. Protein-protein interactions
Ethylene Responsive Gene Expression
CTR1
ETR1
Nucleus
-
C
Cytoplasm
ER
EIN2
N
Lumen
C
EIN3/EIL1
ETR2
ETP1/2
Degradation by 26S proteasome
Ethylene signaling pathway
EBP1/2
Degradation by 26S proteasome
RAN1
Golgi
N
C2H4N
N
Cu+
Cu+
Cu+Cu+
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
The yeast two-hybrid assay utilizes two different reporter genes:
1.HIS gene encodes a protein that synthesizes the amino acid histidine
• When the gene is present in the yeast, then the yeast can grow on medium lacking histidine
2. lacZ encodes the -galactosidase enzyme, which turns the X-gal substrate into a blue pigment
• When the gene is present in the yeast, then the yeast turn blue when X-gal is put into the growth medium
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
Y
UAS
Promoter sequence
DNA
Transcriptional activators have 2 domainsDB = DNA binding domainAD = Activation domain
DB
AD
Introduction to transcription activation
Coding Sequence
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
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
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
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
But 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
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
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
Lab: Yeast two-hybrid assay
1. What is a “reporter gene”, and what are the reporter genes in this assay?
2. What are “-LW” and “-LWH” plates? What is each type of plate used for?
3. Which plate should be used for the lac Z assay and why?
4. In terms of your results, should there be a correlation between the growth of transformants on -LWH plates and the blue color in the lacZ assay? Why?
5. What is a negative control, and why is it important in the yeast two-hybrid assay? 6. In your experiment, which yeast transformants are the negative controls? 7. Suppose you have a known protein that serves as your bait protein, and you want to find a protein that interacts with this bait. Can you think of how the yeast two-hybrid assay be used to find an interacting protein?
A single bait can tested for interaction with many different preys
