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

Using a cDNA cloning technique that incorporated polymerase chain reac tion amplification of cDNA and subtraction in a single-strand phagemid vector, we constructed a library of transcripts exhibiting up regulat ion in tomato cells infected with the parasitic nematode Meloidogyne i ncognita. Starting with 51 mg of dissected, nematode-induced giant cel ls, we constructed a primary cDNA library of 2.2 x 10(6) recombinants. Subtraction against uninfected tomato roots gave a 4,860-fold enrichm ent. Analysis of the library as a whole indicated that contamination b y nematode sequences was less than 1%. Transcripts from high copy numb er genes accounted for 14% of the clones; the remaining 244 recombinan ts appeared to be derived from distinct, unique genes. Partial DNA seq uence analysis of one cDNA revealed homology with the RB7 gene from to bacco, the only gene previously known to be up regulated in giant cell s. 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.