SAR Signaling - MGH Molecular Biology - Genetics Webserver

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SAR Signaling - MGH Molecular Biology - Genetics Webserver

  1. 1. Plant Defense: A Glimpse By Wisuwat Songnuan
  2. 2. Outline <ul><li>Background </li></ul><ul><li>Systemic Acquired Resistance </li></ul><ul><li>NPR1-TGAs </li></ul><ul><li>That’s not all… </li></ul><ul><li>Future </li></ul>
  3. 3. Background
  4. 4. Background Outline <ul><li>Why study plant resistance? </li></ul><ul><li>Pathogen Recognition </li></ul><ul><li>Gene-for-gene interactions </li></ul><ul><li>Hypersensitive Response (HR) </li></ul><ul><li>Systemic Acquired Resistance (SAR) </li></ul>
  5. 5. Why study plant resistance? <ul><li>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). </li></ul><ul><li>We lose 12% of total crop yields to pathogen infection– equivalent to nine hundred million tons worldwide annually (Krimsky S. and Wrubel R., 1996). </li></ul>
  6. 6. Plants under attack <ul><li>Microorganisms: viruses, bacteria, fungi </li></ul><ul><li>Nematodes </li></ul><ul><li>Insects & a few others </li></ul><ul><li>Us? </li></ul>
  7. 7. What will YOU do? <ul><li>Lots of enemies, attacking from all sides </li></ul><ul><li>Huge body </li></ul><ul><li>Cannot escape </li></ul><ul><li>No “patrol” </li></ul><ul><li>(no NIH grant) </li></ul>
  8. 8. How THEY do it <ul><li>Right after plants are dead, they are rotten </li></ul><ul><li>No wasting energy for ‘just in case’ immunity </li></ul><ul><li>All through “signaling” </li></ul>
  9. 9. Pathogen recognition <ul><li>Gene-for-gene hypothesis : Upon infection by a particular avirulent pathogen, a corresponding R gene recognizes the avr product and triggers the defense mechanism. </li></ul><ul><ul><li>Why do pathogens still possess avr genes? </li></ul></ul><ul><li>Non-host resistance : Resistance of all members of a host species against all members of pathogen species </li></ul>
  10. 10. Resistance (R) Genes <ul><li>Dominant </li></ul><ul><li>Many ID so far </li></ul><ul><li>5 classes recognized </li></ul><ul><ul><li>NBS: Nucleotide binding site </li></ul></ul><ul><ul><li>Leucine-zipper and leucine-rich repeat (LRR) </li></ul></ul><ul><ul><li>Toll/IL-1R (TIR) </li></ul></ul><ul><ul><li>Protein kinase (PK), receptor-like kinase (RLKs) </li></ul></ul>
  11. 11. The popular ones… <ul><li>Maize Hm1 (1992): toxin reductase </li></ul><ul><li>Tomato Pto (1993): Ser/Thr kinase </li></ul><ul><li>Arabidopsis RPS2: </li></ul><ul><li>Tobacco N: </li></ul><ul><li>Tomato Cf9 </li></ul><ul><li>Flax L6 </li></ul><ul><li>Rice Xa21 </li></ul>
  12. 12. Hypersensitive Response (HR) <ul><li>Burst of oxygen reactive species around infection site </li></ul><ul><li>Synthesis of antimicrobial phytoalexins </li></ul><ul><li>Accumulation of Salicylic Acid (SA) </li></ul><ul><li>Directly kill and damage pathogens </li></ul><ul><li>Strengthen cell walls, and triggers apoptosis </li></ul><ul><li>Restrict pathogen from spreading </li></ul><ul><li>Rapid and local </li></ul>
  13. 13. Systemic Acquire Resistance (SAR) <ul><li>Secondary response </li></ul><ul><li>Systemic </li></ul><ul><li>Broad-range resistance </li></ul><ul><li>Leads to Pathogenesis-Related (PR) gene expression </li></ul><ul><li>Signals: SA, JA, ethylene </li></ul>
  14. 14. Systemic Acquired Resistance (SAR)
  15. 15. Salicylic Acid (SA) <ul><li>Accumulates in both local and systemic tissues (not the systemic signal) </li></ul><ul><li>Removal of SA (as in nahG plants) prevents induction of SAR </li></ul><ul><li>Analogs: INA or BTH </li></ul>COOH OH
  16. 16. Mutants affecting SA synthesis <ul><li>Elevated SA accumulation </li></ul><ul><ul><li>dnd1 ( defense, no death 1 ): increased SA, but reduced HR, DND1 gene encodes cyclic-nucleotide-gated ion channel </li></ul></ul><ul><ul><li>mpk4 : constitutive SA accumulation </li></ul></ul><ul><ul><li>edr1 ( enhanced disease resistance 1 ): defective MAPKKK </li></ul></ul>
  17. 17. Mutants affecting SA synthesis <ul><li>reduced SA accumulation </li></ul><ul><ul><li>eds1 ( enhanced disease susceptibility 1 ): lipase homolog </li></ul></ul><ul><ul><li>pad4 ( phytoalexin deficient 4 ): another lipase homolog </li></ul></ul><ul><ul><li>sid1 and sid2 ( salicylic acid induction-deficient ): defects in chorismate pathway </li></ul></ul>
  18. 18. Mutant Screen <ul><li>Aimed at identifying regulatory genes of SAR </li></ul><ul><li>Strategy: Transform Arabidopsis with GUS reporter driven by SA- and INA-responsive promotor from BGL2 gene </li></ul><ul><ul><li>npr1 (non-expresser of PR genes) mutant: reduced induction of reporter gene with or without SA, INA </li></ul></ul><ul><ul><li>cpr (constitutive expresser of PR genes) mutants: constitutively express reporter genes </li></ul></ul>
  19. 19. NPR1: non-expresser of PR genes <ul><li>Also known as NIM1 or SAI1 </li></ul><ul><li>Positive regulator of SAR </li></ul><ul><li>Downstream of SA, upstream of PR genes </li></ul><ul><li>npr1 mutants are susceptible to various pathogens </li></ul><ul><li>Overexpression of NPR1 generates broad-spectrum resistance </li></ul><ul><li>Unique, but similar to I κ -B (negative regulator of immunity in animals) </li></ul>
  20. 20. NPR1 overexpression
  21. 21. Pathogen-Related (PR) Genes <ul><li>Antimicrobial properties </li></ul><ul><li>Many identified </li></ul><ul><li>Categorized according to activity </li></ul><ul><li>Examples </li></ul><ul><ul><li>PR-2 : beta-1,3-glucanase </li></ul></ul><ul><ul><li>PR-3 : chitinase </li></ul></ul><ul><ul><li>PR-12: defensin </li></ul></ul>
  22. 22. SAR Avr R gene SA NPR1 PR-1 PR-2 PR-5 SAR
  23. 23. Structural features of NPR1 <ul><li>593 amino acids, 67 kD </li></ul><ul><li>Two protein-protein interaction domains: BTB/POZ and Ankyrin repeats </li></ul><ul><li>Contains NLS </li></ul><ul><li>Multiple phosphorylation sites </li></ul><ul><li>No DNA binding domain </li></ul>npr 1-1 BTB ARD S S NLS npr 1-2 nim 1-2
  24. 24. NPR1-GFP localizes in nucleus upon SAR induction MS MS - INA NPR1 - GFP GFP
  25. 25. TGA Factors <ul><li>Found to interact with NPR1 through yeast-two hybrid </li></ul><ul><li>bZIP transcription factors </li></ul><ul><li>Six members in Arabidopsis (TGA1-6) </li></ul><ul><li>Might be redundant </li></ul><ul><li>Bind to as-1 element </li></ul>
  26. 26. NPR1-TGA2 interaction <ul><li>Direct visualisation </li></ul>
  27. 28. TGA2 C-term interacts with NPR1
  28. 29. PR-1 expression reduced in TGA2CT lines Figure 2A, 2B
  29. 30. Reduced resistance to P.parasitica and tolerance to SA Figure 2C, D
  30. 31. DN effects depends on NPR1 Figure 3A, B
  31. 32. SA affects NPR1-TGA2 interaction Figure 3C, D
  32. 33. Chimera Reporter System Figure 4
  33. 34. TGA2-GAL4 is SA-responsive Figure 5A,B
  34. 35. TGA2-GAL4 as an activator Figure 5C
  35. 36. DNA binding dependent on NPR1 and enhanced by SA Figure 5D
  36. 37. Current model Figure 6
  37. 38. SAR Avr R gene SA NPR1 PR-1 PR-2 PR-5 SAR TGA2
  38. 39. NPR1-TGA5
  39. 40. Yeast-two hybrid Figure 1 a-d
  40. 41. Co-purification
  41. 42. TGA2 mRNA accumulation Figure 2 untreated P.parasitica INA
  42. 43. TGA5 mRNA accumulation Figure 3a untreated P.parasitica INA
  43. 44. Surprising accumulation of TGA5 in antisense lines Figure 3b untreated P.parasitica INA
  44. 45. PR-1 induction in TGA2 transformants Figure 4
  45. 46. Reduced PR-1 expression in lines with high TGA5 mRNA Figure 5
  46. 47. TGA5-antisense lines resistant to infection Figure 6 WT AS15 AS16
  47. 48. TGA5-antisense lines resistant to infection
  48. 49. AS15 resistance is independent of NIM1
  49. 50. SAR Avr R gene SA NPR1 PR-1 PR-2 PR-5 SAR TGA2 TGA5 SAR independent resistance
  50. 51. That’s not all…
  51. 52. A few others <ul><li>Ethylene-mediated response </li></ul><ul><li>Jasmonic acid-mediated response </li></ul><ul><li>Induced systemic resistance (ISR) </li></ul><ul><li>MAPK cascades </li></ul>
  52. 53. The future <ul><li>Still a lot to learn </li></ul><ul><li>2010 project </li></ul><ul><li>The golden era </li></ul>
  53. 54. Thank you!

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