A COMPREHENSIVE SUBTRACTIVE CDNA CLONING APPROACH TO IDENTIFY NEMATODE-INDUCED TRANSCRIPTS IN TOMATO

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 x 10(6) 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.