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Plant Defense: A Glimpse
By
Wisuwat Songnuan
Outline
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Background
Systemic Acquired Resistance
NPR1-TGAs
That’s not all…
Future
Background
Background Outline
Why study plant resistance?
Pathogen Recognition
Gene-for-gene interactions
Hypersensitive Response (HR)
Systemic Acquired Resistance (SAR)
Why study plant resistance?
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80% of total calories consumed by human
population come from only six crops: wheat,
rice, maize, potatoes, sweet potatoes, and
manioc (Raven, P.H. et al, 1999).
We lose 12% of total crop yields to pathogen
infection– equivalent to nine hundred million
tons worldwide annually (Krimsky S. and
Wrubel R., 1996).
Plants under attack
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Microorganisms: viruses, bacteria, fungi
Nematodes
Insects & a few others
Us?
What will YOU do?
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Lots of enemies, attacking from all sides
Huge body
Cannot escape
No “patrol”
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(no NIH grant)
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How THEY do it
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Right after plants are dead, they are rotten
No wasting energy for ‘just in case’ immunity
All through “signaling”
Pathogen recognition
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Gene-for-gene hypothesis: Upon infection by a
particular avirulent pathogen, a corresponding
R gene recognizes the avr product and triggers
the defense mechanism.
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Why do pathogens still possess avr genes?
Non-host resistance: Resistance of all
members of a host species against all members
of pathogen species
Resistance (R) Genes
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Dominant
Many ID so far
5 classes recognized
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NBS: Nucleotide binding site
Leucine-zipper and leucine-rich repeat (LRR)
Toll/IL-1R (TIR)
Protein kinase (PK), receptor-like kinase (RLKs)
The popular ones…
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Maize Hm1 (1992): toxin reductase
Tomato Pto (1993): Ser/Thr kinase
Arabidopsis RPS2:
Tobacco N:
Tomato Cf9
Flax L6
Rice Xa21
Hypersensitive Response (HR)
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Burst of oxygen reactive species around
infection site
Synthesis of antimicrobial phytoalexins
Accumulation of Salicylic Acid (SA)
Directly kill and damage pathogens
Strengthen cell walls, and triggers apoptosis
Restrict pathogen from spreading
Rapid and local
Systemic Acquire Resistance (SAR)
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Secondary response
Systemic
Broad-range resistance
Leads to Pathogenesis-Related (PR) gene
expression
Signals: SA, JA, ethylene
Systemic Acquired
Resistance
(SAR)
Salicylic Acid (SA)
COOH
OH
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Accumulates in both local and systemic tissues
(not the systemic signal)
Removal of SA (as in nahG plants) prevents
induction of SAR
Analogs: INA or BTH
Mutants affecting SA synthesis
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Elevated SA accumulation
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dnd1 (defense, no death 1): increased SA, but
reduced HR, DND1 gene encodes cyclicnucleotide-gated ion channel
mpk4: constitutive SA accumulation
edr1 (enhanced disease resistance 1): defective
MAPKKK
Mutants affecting SA synthesis
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reduced SA accumulation
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eds1 (enhanced disease susceptibility 1): lipase
homolog
pad4 (phytoalexin deficient 4): another lipase
homolog
sid1 and sid2 (salicylic acid induction-deficient):
defects in chorismate pathway
Mutant Screen
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Aimed at identifying regulatory genes of SAR
Strategy: Transform Arabidopsis with GUS
reporter driven by SA- and INA-responsive
promotor from BGL2 gene
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npr1 (non-expresser of PR genes) mutant: reduced
induction of reporter gene with or without SA, INA
cpr (constitutive expresser of PR genes) mutants:
constitutively express reporter genes
NPR1: non-expresser of PR genes
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Also known as NIM1 or SAI1
Positive regulator of SAR
Downstream of SA, upstream of PR genes
npr1 mutants are susceptible to various
pathogens
Overexpression of NPR1 generates broadspectrum resistance
Unique, but similar to Iκ-B (negative regulator
of immunity in animals)
NPR1 overexpression
Pathogen-Related (PR) Genes
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Antimicrobial properties
Many identified
Categorized according to activity
Examples
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PR-2 : beta-1,3-glucanase
PR-3 : chitinase
PR-12: defensin
SAR
Avr
R gene
SA
NPR1
PR-1 PR-2 PR-5
SAR
Structural features of NPR1
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
No DNA binding domain
NPR1-GFP localizes in nucleus upon
SAR induction
GFP
NPR1-GFP
MS
MS-INA
TGA Factors
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Found to interact with NPR1 through yeasttwo hybrid
bZIP transcription factors
Six members in Arabidopsis (TGA1-6)
Might be redundant
Bind to as-1 element
NPR1-TGA2 interaction
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Direct visualisation
TGA2 C-term interacts with NPR1
PR-1 expression reduced in
TGA2CT lines
Figure 2A, 2B
Reduced resistance to
P.parasitica and tolerance to SA
Figure 2C, D
DN effects depends on NPR1
Figure 3A, B
SA affects NPR1-TGA2
interaction
Figure 3C, D
Chimera Reporter System
Figure 4
TGA2-GAL4 is SA-responsive
Figure 5A,B
TGA2-GAL4 as an activator
Figure 5C
DNA binding dependent on
NPR1 and enhanced by SA
Figure 5D
Current model
Figure 6
SAR
Avr
R gene
SA
TGA2
NPR1
PR-1 PR-2 PR-5
SAR
NPR1-TGA5
Yeast-two hybrid
Figure 1 a-d
Co-purification
TGA2 mRNA accumulation
untreated
P.parasitica
INA
Figure 2
TGA5 mRNA accumulation
untreated
P.parasitica
INA
Figure 3a
Surprising accumulation of
TGA5 in antisense lines
untreated
P.parasitica
INA
Figure 3b
PR-1 induction in TGA2
transformants
Figure 4
Reduced PR-1 expression in lines
with high TGA5 mRNA
Figure 5
TGA5-antisense lines resistant to
infection
WT
AS15
AS16
Figure 6
TGA5-antisense lines resistant to
infection
AS15 resistance is independent of
NIM1
SAR
Avr
R gene
SA
TGA2
NPR1
TGA5
PR-1 PR-2 PR-5
SAR
SAR independent
resistance
That’s not all…
A few others
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Ethylene-mediated response
Jasmonic acid-mediated response
Induced systemic resistance (ISR)
MAPK cascades
The future
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Still a lot to learn
2010 project
The golden era
Thank you!