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

? Signal transduction Response Signal plant cell The basic problem. Term coined in 1972. Gained popularity in 1980’s.

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 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

The seedling “triple response” Arabidopsis thaliana “Triple Response” Pea seedlings Neljubow (1901) Beih Bot Zentralbl 10, 128-139

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.

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)

*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: 6856-6860

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

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

EIN2 CTR1 ETR2 ETR1

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

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

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

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: 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 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

Introduction to transcription activation AD Y DB DNA 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 AD 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

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

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

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

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

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

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

Lab: Yeast two-hybrid assay

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?

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