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Library of Leaf Tissue Under Mixed Elicitor Stress

Library: cLET
TIGR ID: TME
Authors: Tishina Subrahmanyam and Gregory Martin
Date made: 07/99
Species: Lycopersicon esculentum
Accession: Rio Grande PtoR
Tissue: leaf
Developmental stage: 4-6 week old plants
Vector: pBluescript SK(+/-)
Host: SOLR
Primary pfu: 1.0 x 106
Number mass excised: 1.0 x 108
Average insert length: 1.0 Kb
Cloning sites: 5' EcoRI, 3' XhoI
Antibiotic: ampicillin
Primers: M13F and M13R
Comments: The EcoRI site was destroyed during cloning. Use any enzyme to the left of it along with XhoI to cut out the insert. Also note that the library was amplified prior to mass excision. 11,520 clones were picked into 30 384-well plates. In the following table, BTH stands for the chemical benzothiadiazole, which induces SAR in many dicot species. EIX is ethylene inducing xylanase from the fungus Trichoderma. JA is jasmonic acid.
Biotic/abiotic elicitor Method of treatment Concentration Time points (hours) Has the treatment worked?
INA (2,6 dichloroisonicotinic acid) (1)foliar spray application (2)Petiole immersion - treat in solution for 30 minutes, then transfer to water 0.5 mM 4, 24 RNA gel blot analysis - probe with osmotin
BTH (1)foliar spray application (2)Petiole immersion - treat in solution for 30 minutes, then transfer to water 1.5 mM 4, 24 RNA gel blot analysis - probe with basic glucanase
JA petiole immersion in closed chamber incubation 100 uM 4, 12 RNA gel blot analysis - probe with pin gene
ethylene closed chamber incubation 50 ppm of gas injected into chamber 2, 12 plants showed epinasty - leaf curl and bending downwards
fenthion vacuum infiltration 0.001% Silwet 4, 12 fenthion cell death observed 11 hours after infiltration
EIX vacuum infiltration 1 ug/ml solution 8, 48 small lesions visible 48 hours after infiltration
okadaic acid transpiration stream for 45 minutes, then transfer to dd water 1 uM 4, 12 wilting
systemin petiole immersion, then transfer to dd water 90 ul of 1 nM solution in sodium phosphate buffer 4, 12 RNA gel blot analysis - probe with pin gene