Og for group meeting 23march10

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Og for group meeting 23march10

  1. 1. Oligogalacturonide (OG) Signalling in Plant Innate Immunity 23 March 2010
  2. 2. Background <ul><li>Activation of plant innate immunity </li></ul><ul><ul><li>Specific detection of pathogen-associated molecular patterns (PAMPs) </li></ul></ul><ul><ul><li>Host encoded pattern-recognition receptors (PRRs) </li></ul></ul><ul><li>Perception of PAMPs </li></ul><ul><ul><li>signaling cascades </li></ul></ul><ul><ul><ul><li>activation of plant defenses and enhancement of basal immunity towards potential pathogens </li></ul></ul></ul>
  3. 3. Background <ul><li>Recognition of modified-self including damage-associated molecular patterns (DAMPs) </li></ul><ul><li>Plant cell-wall fragments </li></ul><ul><ul><li>Degrading enzymes secreted by the pathogen during the infection process </li></ul></ul><ul><ul><li>Recognised as DAMPs </li></ul></ul><ul><li>None of the hypothetical DAMP receptors have yet been identified </li></ul><ul><ul><li>Signaling processes not properly characterized. </li></ul></ul>
  4. 4. Background <ul><li>Pectin </li></ul><ul><ul><li>Central component in plant cell walls </li></ul></ul><ul><li>Release of oligogalacturonide (OG) fragments from plant cell walls </li></ul><ul><li>Many plant pathogens produce pectin degrading enzymes as crucial virulence factors </li></ul><ul><li>Recognition as DAMP </li></ul><ul><li>Activation of immune responses </li></ul>
  5. 5. Previous work in the group <ul><li>Activation tagging - Karen </li></ul><ul><li>35,000 BASTA resistant plants selected from 1 million seeds </li></ul><ul><li>1500 pools of 24 plants collected </li></ul><ul><li>Germinated on Whatman paper with water </li></ul><ul><li>Infiltrated with OGs </li></ul><ul><li>Selection for oligogalacturonide-tolerant seedlings </li></ul><ul><ul><ul><li>OGs of DP 2-6(8) : 300 putants </li></ul></ul></ul><ul><li>Reselection of putants (2x) : 30 ”mutants ” </li></ul>BAR 4 x 35S enh
  6. 6. Repeating Karen´s results <ul><li>On 6 and 12 well plates with MQ water </li></ul><ul><li>Problems: </li></ul><ul><ul><li>Seedling germination and growth rate </li></ul></ul><ul><ul><ul><li>Several lines very slow </li></ul></ul></ul><ul><ul><ul><li>Start to die before they are “ready” </li></ul></ul></ul>7 days old 9 days old
  7. 7. <ul><li>Solutions </li></ul><ul><ul><li>New “fresher” seeds </li></ul></ul><ul><ul><li>Grow in MS media from the start? </li></ul></ul><ul><ul><li>One line per plate? </li></ul></ul><ul><ul><ul><li>Infiltrate when “standard” size? </li></ul></ul></ul>
  8. 8. New screen <ul><li>Previous test to germinate on plates then move to liquid medium containing OGs. </li></ul><ul><li>Try to grow in 96-well plates. </li></ul><ul><li>Germinate in liquid ½ MS medium </li></ul><ul><ul><li>Low amount of liquid so seed can grow ”out” of liquid </li></ul></ul><ul><ul><li>After germination: add OGs </li></ul></ul><ul><ul><li>Selection for tolerant seedlings </li></ul></ul>
  9. 9. <ul><li>Problems </li></ul><ul><ul><li>Drying out at the edges of the plates </li></ul></ul><ul><ul><li>Time consuming to transfer seeds one by one </li></ul></ul><ul><ul><ul><li>Seed loader? </li></ul></ul></ul><ul><ul><li>Insufficient oxygen? </li></ul></ul>New screen 8 days old – Grown in different volumes of media ( μl indicated on the right side)
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