Exploring Plant Defense Mechanisms: Insights into Systemic Acquired Resistance

1. Plant Defense: A Glimpse By 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. COOH OH 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

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 npr 1-2 nim 1-2 npr 1-1 NLS • 593 amino acids, 67 kD • Two protein-protein interaction domains: BTB/POZ and Ankyrin repeats • Contains NLS • Multiple phosphorylation sites • No DNA binding domain S S BTB ARD

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

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. untreated P.parasitica INA TGA5 mRNA accumulation 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. WT AS15 AS16 TGA5-antisense lines resistant to infection 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 independent resistance SAR