Plant Defense: A Glimpse�

By

SOMNATH ROY

Background Outline

Why study plant resistance?

Pathogen Recognition

Gene-for-gene interactions

Hypersensitive Response (HR)

Systemic Acquired Resistance (SAR)

Why study plant resistance?

• 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

• Microorganisms: viruses, bacteria, fungi
• Nematodes
• Insects & a few others
• Us?

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What will YOU do?

• Lots of enemies, attacking from all sides
• Huge body
• Cannot escape
• No “patrol”

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• (no NIH grant)

How THEY do it

• Right after plants are dead, they are rotten
• No wasting energy for ‘just in case’ immunity
• All through “signaling”

Pathogen recognition

• Gene-for-gene hypothesis: Upon infection by a particular avirulent pathogen, a corresponding R gene recognizes the avr product and triggers the defense mechanism.
• Why do pathogens still possess avr genes?
• Non-host resistance: Resistance of all members of a host species against all members of pathogen species

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Resistance (R) Genes

• Dominant
• Many ID so far
• 5 classes recognized
• NBS: Nucleotide binding site
• Leucine-zipper and leucine-rich repeat (LRR)
• Toll/IL-1R (TIR)
• Protein kinase (PK), receptor-like kinase (RLKs)

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The popular ones…

• Maize Hm1 (1992): toxin reductase
• Tomato Pto (1993): Ser/Thr kinase
• Arabidopsis RPS2:
• Tobacco N:
• Tomato Cf9
• Flax L6
• Rice Xa21

Hypersensitive Response (HR)

• 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

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Systemic Acquire Resistance (SAR)

• Secondary response
• Systemic
• Broad-range resistance
• Leads to Pathogenesis-Related (PR) gene expression
• Signals: SA, JA, ethylene

Systemic Acquired Resistance

(SAR)

Salicylic Acid (SA)

• 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

• Elevated SA accumulation
• dnd1 (defense, no death 1): increased SA, but reduced HR, DND1 gene encodes cyclic-nucleotide-gated ion channel
• mpk4: constitutive SA accumulation
• edr1 (enhanced disease resistance 1): defective MAPKKK

Mutants affecting SA synthesis

• reduced SA accumulation
• 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

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Mutant Screen

• Aimed at identifying regulatory genes of SAR
• Strategy: Transform Arabidopsis with GUS reporter driven by SA- and INA-responsive promotor from BGL2 gene
• 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

• 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 broad-spectrum resistance
• Unique, but similar to Iκ-B (negative regulator of immunity in animals)

NPR1 overexpression

Pathogen-Related (PR) Genes

• Antimicrobial properties
• Many identified
• Categorized according to activity
• Examples
• 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

• 593 amino acids, 67 kD
• Two protein-protein interaction domains: BTB/POZ and Ankyrin repeats
• Contains NLS
• Multiple phosphorylation sites
• No DNA binding domain

npr 1-1

BTB

ARD

S S

NLS

npr 1-2

nim 1-2

NPR1-GFP localizes in nucleus upon SAR induction

TGA Factors

• Found to interact with NPR1 through yeast-two hybrid
• bZIP transcription factors
• Six members in Arabidopsis (TGA1-6)
• Might be redundant
• Bind to as-1 element

NPR1-TGA2 interaction

• 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

NPR1

PR-1 PR-2 PR-5

SAR

TGA2

NPR1-TGA5

Yeast-two hybrid

Figure 1 a-d

Co-purification

TGA2 mRNA accumulation

Figure 2

untreated

P.parasitica

INA

TGA5 mRNA accumulation

Figure 3a

Surprising accumulation of TGA5 in antisense lines

Figure 3b

untreated

P.parasitica

INA

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

Figure 6

WT

AS15

AS16

TGA5-antisense lines resistant to infection

AS15 resistance is independent of NIM1

SAR

Avr

R gene

SA

NPR1

PR-1 PR-2 PR-5

SAR

TGA2

TGA5

SAR independent

resistance

That’s not all…

A few others

• Ethylene-mediated response
• Jasmonic acid-mediated response
• Induced systemic resistance (ISR)
• MAPK cascades

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The future

• Still a lot to learn
• 2010 project
• The golden era

Thank you!