Transcript Y2H Lecture 2013

Ethylene responses
Developmental processes
Responses to abiotic and biotic stress
Fruit ripening - ethylene is essential
Promotion of seed germination
Root initiation
Bud dormancy release
Inhibition/promotion of flowering
Sex shifts in flowers
Senescence of leaves, flowers
Abscission of leaves, flowers, fruits
Epinasty of leaves
Inhibition/promotion of cell division/elongation
Altered geotropism in roots, stems
Induction of phytoalexins/disease resistance
Aerenchyma formation
Signal transduction
Signal
?
Response
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
The seedling “triple response”
Arabidopsis thaliana
Pea seedlings
Neljubow (1901) Beih Bot Zentralbl 10,
128-139
“Triple
Response”
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
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)
Molecular markers provide
a link between genetic loci
and physical DNA
Chang et al. (1988) PNAS 85: 6856-6860
*A genetic map
of molecular
markers on the
chromosome
allows one to
clone any gene
for which there is
a mutant
phenotype
Generating a mapping population
mut
mut
X
Landsberg
Columbia
hand-pollinate
heterozygous for mut
F1
self-pollinate
Recombinant genotypes
F2
1
2
3
4
Mapping population
5
.....
Example of mapping with molecular markers
Mapping population
Marker A
Marker B
EIN2
ETR2
ETR1
CTR1
Ethylene signaling proteins
ETR1
ETR2
An ethylene receptor
Ethylene
binding
domain
Signaling domain
An ethylene receptor
Ethylene
binding
domain
Signaling domain
CTR1
A protein kinase
Regulatory
domain
Kinase domain
EIN2
Membrane
domain
Soluble domain
A protein of
unknown function
Cloned the genes, but now look at:
1. Subcellular localization of the proteins
2. Protein-protein interactions
Ethylene signaling pathway
Cu+
Golgi
C2H4
RAN1
Cu+
Lumen
ETR2
ER
ETR1 EIN2
Cu+
Cu+
ETP1/2
Degradation by
26S proteasome
CTR1
C
Cytoplasm
EIN3/EIL1
Ethylene Responsive Gene
Expression
EBP1/2
Degradation by
26S proteasome
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
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 b-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
Inside the
NUCLEUS of the
yeast cell
Promoter
sequence
Y
X
DNA
DB
AD
Coding sequence of a
gene
Coding Sequence
UAS
transcription
mRNA
translation
DB
AD
= transcription
activator
Protein
Introduction to transcription activation
AD
Y
DNA
DB
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
But 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
ETR2
Ethylene
binding
domain
Ethylene
binding
domain
8
An ethylene receptor
Signaling domain
2
An ethylene receptor
Signaling domain
7
A protein kinase
CTR1
Regulatory
domain
Kinase domain
1
EIN2
Membrane
domain
Soluble domain
A protein of
unknown function
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