1. Ethylene responses Developmental processes
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
Responses to abiotic and biotic stress 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

2. ? Signal transduction Response Signal plant cell The basic problem.
Term coined in Gained popularity in 1980’s.

3. WHAT CONSTITUTES AN UNDERSTANDING OF SIGNALING PATHWAYS?
HOW CAN RESEARCHERS ELUCIDATE SIGNALING PATHWAYS?

4. “Genetic Dissection” of the Ethylene Signaling Pathway

5. How to genetically dissect a pathway
Identify a phenotype that is specific to the process you are interested in
Design appropriate screen for isolating mutants based on this phenotype
Clone the corresponding gene by map-based cloning
Investigate the function of the corresponding protein at cell biological and biochemical levels

6. The seedling “triple response”
Arabidopsis thaliana
“Triple Response”
Pea seedlings
Neljubow (1901) Beih Bot Zentralbl 10,

7. Bleecker et al. (1988) Science 241, 1086–1089
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.

8. Ethylene-Response Mutants in Arabidopsis
Ethylene-insensitive mutants
etr1 etr2 ein4 (dominant)
ein2 ein3 ein5 (recessive)
ein6 ein7
C2H4
Constitutive-response mutants
air
ctr1 (recessive)
(eto1)

9. *A genetic map of molecular markers on the chromosome allows one to clone any gene for which there is a mutant phenotype
Molecular markers provide
a link between genetic loci and physical DNA
Chang et al. (1988) PNAS 85:

10. Generating a mapping population
mut
mut X
Landsberg
Columbia
hand-pollinate
heterozygous for mut F1
self-pollinate
Recombinant genotypes F2 1 2 3 4 5
Mapping population

11. Mapping population Marker A Marker B
Example of mapping with molecular markers
Mapping population
Marker B
Marker A

12. EIN2
CTR1
ETR2
ETR1

13. A protein of unknown function EIN2
Ethylene signaling proteins
An ethylene receptor
ETR1
Ethylene binding domain
Signaling domain
An ethylene receptor
ETR2
Ethylene binding domain
Signaling domain
CTR1
A protein kinase
Regulatory domain
Kinase domain
A protein of unknown function
EIN2
Membrane domain
Soluble domain

14. Cloned the genes, but now look at:
Subcellular localization of the proteins
2. Protein-protein interactions

15. Ethylene Responsive Gene Expression
Ethylene signaling pathway
Cu+
Golgi
RAN1
C2H4 N
Cu+
Lumen
ETR2
ETR1
EIN2 N N ER
Cu+
Cu+ N
ETP1/2 C
Degradation by
26S proteasome -
CTR1 C
Cytoplasm
EIN3/EIL1
EBP1/2
Degradation by
26S proteasome
Nucleus
Ethylene Responsive Gene Expression

16. 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:

17. The yeast two-hybrid assay utilizes two different reporter genes:
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

19. Introduction to transcription activation Coding sequence of a gene
Inside the NUCLEUS of the yeast cell
Promoter sequence
Coding sequence of a gene X Y DB AD
DNA
Coding Sequence
UAS
transcription
mRNA
translation
Protein DB AD
= transcription
activator

20. Introduction to transcription activationAD Y DB
DNA
Coding Sequence
UAS
Promoter sequence
Transcriptional activators have 2 domains
DB = DNA binding domain
AD = Activation domain

21. Underlying principle of the Yeast Two-Hybrid AssayAD Y X DB
HIS3 or lacZ
UAS
Promoter
Reporter Gene AD Y
Interaction of X and Y proteins X
Reporter will be expressed DB
HIS3 or lacZ
UAS
Promoter
Reporter Gene

22. Underlying principle of the Yeast Two-Hybrid Assay
“PREY”
“BAIT” AD Y 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

23. Underlying principle of the Yeast Two-Hybrid Assay
HIS3 or lacZ
UAS
Promoter
Reporter Gene AD DB X AD z X X
No transcription DB
HIS3 or lacZ
UAS
Promoter
Reporter Gene

24. 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:

25. Promoter Reporter Gene No transcription Promoter Reporter Gene
But how do we get these proteins into yeast cells so that we can test whether they interact? Y
HIS3 or lacZ
UAS
Promoter
Reporter Gene AD DB X AD z X X
No transcription DB
HIS3 or lacZ
UAS
Promoter
Reporter Gene

26. 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

27. A protein of unknown function
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

28. Lab: Yeast two-hybrid assay

29. What is a “reporter gene”, and what are the reporter genes in this assay?
What are “-LW” and “-LWH” plates? What is each type of plate used for?
Which plate should be used for the lac Z assay and why?
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?

30. 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?

31. A single bait can tested for interaction with many different preys