1. Plant Defense: A GlimpseBy
Wisuwat Songnuan

2. Outline Background Systemic Acquired Resistance NPR1-TGAs
That’s not all…
Future

3. Background

4. Background Outline Why study plant resistance? Pathogen Recognition
Gene-for-gene interactions
Hypersensitive Response (HR)
Systemic Acquired Resistance (SAR)

5. 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).

6. Plants under attack Microorganisms: viruses, bacteria, fungi Nematodes
Insects & a few others
Us?

7. What will YOU do? Lots of enemies, attacking from all sides Huge body
Cannot escape
No “patrol”
(no NIH grant)

8. How THEY do it Right after plants are dead, they are rotten
No wasting energy for ‘just in case’ immunity
All through “signaling”

9. 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

10. 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)

11. The popular ones… Maize Hm1 (1992): toxin reductase
Tomato Pto (1993): Ser/Thr kinase
Arabidopsis RPS2:
Tobacco N:
Tomato Cf9
Flax L6
Rice Xa21

12. 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

13. Systemic Acquire Resistance (SAR)
Secondary response
Systemic
Broad-range resistance
Leads to Pathogenesis-Related (PR) gene expression
Signals: SA, JA, ethylene

14. Systemic Acquired Resistance
(SAR)

15. Salicylic Acid (SA)
COOH OH
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

16. 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

17. 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

18. 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

19. 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)

20. NPR1 overexpression

21. 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

22. SAR
Avr
R gene SA
NPR1
PR-1 PR-2 PR-5
SAR

23. Structural features of NPR1
nim 1-2
npr 1-1
NLS
S S
BTB
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

24. NPR1-GFP localizes in nucleus upon SAR inductionMS
MS-INA
NPR1-GFP
GFP

25. 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

26. NPR1-TGA2 interaction
Direct visualisation

28. TGA2 C-term interacts with NPR1

29. PR-1 expression reduced in TGA2CT lines
Figure 2A, 2B

30. Reduced resistance to P.parasitica and tolerance to SA
Figure 2C, D

31. DN effects depends on NPR1
Figure 3A, B

32. SA affects NPR1-TGA2 interaction
Figure 3C, D

33. Chimera Reporter System
Figure 4

34. TGA2-GAL4 is SA-responsive
Figure 5A,B

35. TGA2-GAL4 as an activator
Figure 5C

36. DNA binding dependent on NPR1 and enhanced by SA
Figure 5D

37. Current model
Figure 6

38. SAR
Avr
R gene SA
TGA2
NPR1
PR-1 PR-2 PR-5
SAR

39. NPR1-TGA5

40. Yeast-two hybrid
Figure 1 a-d

41. Co-purification

42. TGA2 mRNA accumulation
untreated
P.parasitica
INA
Figure 2

43. TGA5 mRNA accumulation
untreated
P.parasitica
INA
Figure 3a

44. Surprising accumulation of TGA5 in antisense lines
untreated
P.parasitica
INA
Figure 3b

45. PR-1 induction in TGA2 transformants
Figure 4

46. Reduced PR-1 expression in lines with high TGA5 mRNA
Figure 5

47. TGA5-antisense lines resistant to infectionWT
AS15
AS16
Figure 6

48. TGA5-antisense lines resistant to infection

49. AS15 resistance is independent of NIM1

50. SAR Avr R gene SA TGA2 NPR1 TGA5 PR-1 PR-2 PR-5 SAR SAR independent
resistance

51. That’s not all…

52. A few others Ethylene-mediated response
Jasmonic acid-mediated response
Induced systemic resistance (ISR)
MAPK cascades

53. The future
Still a lot to learn
2010 project
The golden era

54. Thank you!