Transcript Yeast Two-Hybrid Screen

YUELIN ZHANG,WEIHUA FAN,MARK KINKEMA,XINLI, AND XINNIAN DONG
By Di Wu and Brian Kahnamelli
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Introduction
◦ What is SAR?
◦ What did we know about NPR1 Before This Paper?
◦ What did we want to learn about NPR1?
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Yeast Two-Hybrid Screen
◦ Mechanism of Action
◦ Limitations of YTH
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Why in Tomato?
Experiments
◦ Yeast Two-Hybrid Screens
◦ In Vitro Binding Affinity
◦ Gel Shift Assays
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Basic Leucine Zipper Transcription Factors
(bZIP)
Summary of Results
Impact and Implications
Future Research
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More than 70!
Rice Bacterial Blight
Rice Blast
Rice false smut
What is SAR?
Systemic Acquired Resistance
1, Broad-spectrum resistance
2, Relatively long-term resistance
3, “healthy” resistance
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Forward genetic screen.
Group
Screens
Mutants
obtained
The year
when
finished
Screening
strategy
Ryals, Novartis
Non-immunity
(NIM)
nim1-1 to
nim1-6
1995
Infection assay
after SAR elicitor
treatment
Dong, Duke
No PR-gene
expression
(NPR)
npr1-1
1994
BGL2::GUS
reporter system
In 1997, NPR1 gene was mapped and the protein sequence was
examined.
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What do you know about NPR1 protein?
**Wonderful time to improve your Participation Grade**
npr 1-2
nim 1-2 npr 1-1
NLS
SS
BTB
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ARD
593 amino acids, 67 kD
Two protein-protein interaction domains: BTB/POZ
and Ankyrin repeats
Contains NLS
Multiple phosphorylation sites
Multiple conserved cysteine residues
No DNA binding domain
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SA receptor?
◦ No SA binding activity
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Subcellular localization?
◦ Accumulation in nucleus after SAR induction
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Transcription factor?
◦ No bona fide DNA binding domain
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Screen for NPR1-interacting proteins (NIPs)
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Purpose
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Requirements
◦ To test protein-protein interactions
◦ Reporter construct of interest in yeast
◦ cDNA prey library with spliced activating domain
 Activating domain interacts with RNA pol to transcribe the
reporter gene
◦ cDNA bait construct with spliced Yeast DNA binding
domain (BD)
 Binding domain interacts with promoter region of the
reporter gene
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Preparation
◦ Transfect yeast cells with both plasmids and grow on
medium complementary to your reporter assay
Bait Construct:
•Plasmid Vector
•cDNA sequence of interest
•Binding domain
Prey Construct:
•Plasmid Vector
•cDNA sequence of interest
•Activating domain
Prey AD
RNA Pol
Bait
BD
Reporter Gene
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Can you think of any limitations of the Yeast
Two-Hybrid experiment?
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False Positive Results
◦ A) Bait already possess an Activating Domain
◦ B) Means of selection is error prone
◦ C) Prey possess a yeast binding domain – highly
unlikely
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False Negative Results
◦ A) Post Translational Modifications
 Ex. Phosphorylation
◦ B) Scaffold protein interaction
◦ C) Internal Yeast Error
 Protein Folding or Transcription error occurs
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Tomato Library was better characterized than
Arabidopsis Library
Tomato Library possessed a positive control
◦ PTO-PTI
◦ Remove some possibility of False Negative
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The quality of a yeast two-hybrid experiment
hinges on the quality of a library
◦ Is what you’re finding legitimate??
◦ Is what you’re not finding legitimate?
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Perform Yeast Two-Hybrid Experiment with
known Tomato homologue of NPR1
◦ Referred to as TomNPR1
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Use NPR1 homologue of NPR1as bait
◦ Transfect Yeast with gene, spliced to DNA binding
domain (BD)
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Screen cDNA Library for potential binding
(Prey)
◦ Transfect the same yeast with gene
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Use of Leucine Drop out Plates
◦ Reporter genes allow yeast to produce Leucine and
grow
Prey AD
TomNPR1
BD
RNA Pol
Leucine or β-gal Activity
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Researchers found NIF1 gene led to positive
Y2H result
◦ Leucine production, β-gal activity and colony
survival
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Test each component alone to test for
individual activity
◦ None found alone
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Sequence and Characterize NIF1
◦ Search Genebank for homologous structures in
Arabidopsis
◦ NIF1 codes for last 2/3 of a bZIP Transcription
Factor
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Found Three candidates: AHBP-1b (TGA2),
TGA6, and OBF5(TGA2)
◦ All are bZIP Transcription Factors
Figure 2. Sequence similarity
between NIF1 and associated
homologues
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Perform same experimentation in Arabidopsis
to confirm that Tomato homologues respond
under similar conditions
Only considering 3 possible candidates (bZIP
proteins)
◦ AHBP-1b, TGA-6, and OBF5 as prey
◦ NPR1 as Bait
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All three bZIP proteins were capable of
stimulating a Leucine and β-gal activity
indicating protein-protein interaction
Conclude: All three TFs show increased function when
compared to the negative control. NPR1 can potentially
interact with all of these TFs.
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Because Yeast Two-Hybrid is error prone,
confirmation by means of other
experimentation is required
Researchers aimed to confirm results by
performing an in vitro binding test
◦ Ni-NTA Resin Pull down Assay (Co-Purification)
Ni-NTA resin
NTA
Poly-histidine Tag
Scaffold
Ni-NTA resin
NPR1
His-tagged TGA
NTA
Poly-histidine Tag
Scaffold
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Aim to find regions of NPR1 involved in TGA
binding
Create truncated NPR1 gene constructs
Perform Yeast Two-Hybrid Screens with AHBP-1b
(TGA2) prey expressed along with truncated
NPR1 bait
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1-177aa – amino term, 1st exon
1-432aa – amino term, 1st exon, ankyrin repeat domain
178-593aa – ankyrin repeat domain, carboxyl term
1-593aa – Total Protein
Truncations along exon/intron boundries
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Active regions appear to be amino end in
combination with ankyrin domains
◦ 1-432 segment shows activity equal to WT
◦ N-terminus alone shows little activity
◦ Ankyrin Repeat alone shows little activity
Figure 4a. Specific regions of NPR1 appear to be more important in
regards to binding affinity
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Introduce point mutations into highly
conserved amino acids within the Ankyrin
repeat domains
◦ npr1-1 – histidine 334 to tyrosine
◦ npr1-2 – cysteine 150 to tyrosine
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Perform Yeast Two Hybrid Screens with these
mutants and observe activity
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Base Switches:
Histadine
Cysteine
Tyrosine
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Mutations into conserved amino acids lead to
complete abolishment of activity
Figure 4b. X-gal and Leucine
drop out plates showing loss
of activity in npr1-1 and npr12 mutants
Figure 4c. Immunoblot of NPR1
protein expression levels in WT
and mutant constructs
Primary structure of bZIP domain
Marc Jakoby et al. 2002
Hydrophobic interaction surface of the helices
O'Shea et al. 1991
MADS-box TFs
Marc Jakoby et al. 2002
Binding DNA sequences
with an ACGT core.
http://www.bmb.psu.edu/faculty/tan/lab/gallery_protdna.html
Recognizing the CArG-box
Plant bZIP transcription
factors are classified into
10 groups
Group D/ TGA/OBF family
Question:
What phenotypes would you expect if
the construct 35S::TGA2CT is
Clade II (TGA2/AHBP-1b, OBF5/ TGA5, TGA6)
introduced into col-0 background?
TGA2
NT: N-terminal region
bZIP: required for DNA binding
CT: responsible for NPR1 binding
 TGA factors bind specifically to variants of the palindrome TGACGTCA. Two of
these sequences separated by 4 bps are called an activation sequence-1 (as-1).
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Aimed to characterize the function of AHBP1b
PR-1 promoter region has as-1-like element
◦ Known to interact with bZIP proteins
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Test binding affinity of AHBP-1b to PR-1
promoter
◦ Radio-labeled promoter region
◦ Non-labeled promoter region
◦ Non-labeled as-1-like sequence
as-1-like element:
CTCTACGTCACTATTTTACTTACGTCATAGATG
Mutated version:
CTCTAttctACTATTTTACTTAttctATAGATG
Question:
How is TGA binding to PR1
promoter sequence possible
even without NPR1?
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Band shift observed when AHBP-1b was
present
Competitive binding seen between labeled
and non-labeled promoter region
Non-competitive binding seen between
labeled promoter region and mutated as-1like element
AHBP-1b can specifically bind the PR-1
promoter region in vitro
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TomNPR interacts with NIF1
NPR1 interacts with TGA transcription factors
via Ankyrin repeat and N-terminus domain
◦ Binding is specific and altered by single amino acid
changes
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TGA transcription factors interact with the
binding domains of PR genes
◦ TGA2 binds to promoter region of PR1 specifically
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Research helped to further connect SA to SAR
by connecting another link in the pathway
◦ Showed potential for redundancy as seen in three
bZIPs capable of binding to NPR1
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Added to body of knowledge regarding NPR1
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Examine bZIP and NPR1 in vivo
Loss of function and gain of function mutants
◦ Look at mutants lacking specific TGA TFs alone and
in combination
◦ What else can NPR1 and TGAs activate?
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Regulation
◦ How is NPR1 activity regulated?
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What else would you be interested in
knowing?
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TGA 2,5, and 6 have essential, redundant,
and overlapping roles
TGA 5 over expression is sufficient to
stimulate SAR
NPR1 regulation sensitive to redox changes
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Co-immunoprecipitation
In vivo protein fragment complementation assay
(PCA)
◦ Bimolecular fluorescence complementation (BiFC)
◦ Restoration of dihydrofolate reductase (DHFR) acitivity
Protein fragment Complementation Assay
(PCA) using DHFR enzyme
I’m a
protoplast.
No interaction
interaction
Subramaniam et al. 2001 Nat. Biotechnol.
Protein fragment Complementation Assay
(PCA) using DHFR enzyme
Subramaniam et al. 2001 Nat. Biotechnol.