Library of Leaves From Pseudomonas Resistant Variety

Library: cLER
TIGR ID: TPR
Authors: Xiaohua He, Mark D'Ascenzo, Jamie Lyman, and Greg Martin
Date made: 01/99
Species: Lycopersicon esculentum
Accession: tomato line R11-12 (35S: Pto in Rio Grande x Money Maker)
Resistance: tomato line is resistant to Pseudomonas syringae pv. tomato strain T1 that expresses the avrPto gene
Tissue: leaf
Developmental stage: 4 week old plants
Vector: pBluescript SK(+/-)
Host: SOLR
Primary pfu: 1.0 x 106
Number mass excised: 1.0 x 107
Average insert length: 1.0 Kb
Cloning sites: 5' EcoRI, 3' XhoI
Antibiotic: ampicillin
Primers: M13F and M13R
Comments: For this library four-week-old plants were inoculated with the Pst(avrPto) strain and the leaves were harvested. Two plants were used for each time-point as follows. High inoculation level: 108 cfumL of Pst(avrPto). Equal amounts of leaves were harvested at 0, 2, 4, 6, and 8 hours after inoculation. Low inoculation level: 105 cfumL of Pst(avrPto). Equal amounts of leaves were harvested at 0, 12, 24, 36, and 48 hours after inoculation. High bacterial titre gives no HR on R11-12, but results in disease symptoms beginning at about 48 hours after inoculation. Low bacterial titre gives no HR on R11-12, but results in disease symptoms beginning at about 48 hours after inoculation. Keeping leaves from each line separate, equal amounts of leaves from each time-point were pooled and used to extract polyA RNA using Promega's polyATract mRNA isolation system. Stratagene's Zap-cDNA synthesis kit was used to prepare cDNA. The cDNA was cloned into Uni-ZAP XR vector using the EcoRI site at the 5' end and the XhoI site at the 3' end. When a sample of each library was plated on NZY agar plates containing IPTG and X-gal, the white:blue colony ratio was about 100:1. After mass excision, twenty-five clones from each library were picked randomly and characterized. All 50 clones contained inserts with sizes ranging from 0.5 Kb to more than 3 Kb (avg=1 Kb). The 5' ends of 14 random clones were sequenced (7 from each library). The EcoRI site was altered/missing in 11 of the clones. We expect the R11-12 library will contain cDNAs corresponding to genes that are induced by the hypersensitive response and during "field" resistance. The R11-13 library is expected to contain cDNAs corresponding to genes that are induced during the disease susceptibility response.