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Molecular Plant Pathology

A Comprehensive Subtractive cDNA Cloning Approach to Identify Nematode-Induced Transcripts in Tomato. Mark A. Wilson, Department of Nematology and Graduate Genetics Group, University of California, Riverside 92521; David McK. Bird(2), and Esther van der Knaap(3). (2)(3)Department of Nematology and Graduate Genetics Group, University of California, Riverside 92521; (3)Current address: MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing 48824-1312. Phytopathology 84:299-303. Accepted for publication 15 December 1993. Copyright 1994 The American Phytopathological Society. DOI: 10.1094/Phyto-84-299.

Using a cDNA cloning technique that incorporated polymerase chain reaction amplification of cDNA and subtraction in a single-strand phagemid vector, we constructed a library of transcripts exhibiting up regulation in tomato cells infected with the parasitic nematode Meloidogyne incognita. Starting with 51 mg of dissected, nematode-induced giant cells, we constructed a primary cDNA library of 2.2 106 recombinants. Subtraction against uninfected tomato roots gave a 4,860-fold enrichment. Analysis of the library as a whole indicated that contamination by nematode sequences was less than 1%. Transcripts from high copy number genes accounted for 14% of the clones; the remaining 244 recombinants appeared to be derived from distinct, unique genes. Partial DNA sequence analysis of one cDNA revealed homology with the RB7 gene from tobacco, the only gene previously known to be up regulated in giant cells. In addition to permitting a transcriptional analysis of giant cells, our cloning technique should be well suited for isolating genes from other pathogen-infected plant cells.