A SIMPLIFIED PROCEDURE FOR THE SUBTRACTIVE CDNA CLONING OF PHOTOASSIMILATE-RESPONDING GENES - ISOLATION OF CDNAS ENCODING A NEW CLASS OF PATHOGENESIS-RELATED PROTEINS

Transgenic tobacco plants (ppa-1) constitutively expressing Escherichi a coli pyrophosphatase behind the 35S CaMV promoter accumulate high le vels of soluble sugars in their leaves [27]. These plants were conside red a tool to study adaptation of leaves to photoassimilate accumulati on at the molecular level. By differential hybridization of a subtract ive library enriched for transcripts present in the transgenic plants 12 different cDNAs were isolated. By sequence analysis four cDNAs coul d be identified as 1-aminocyclopropane-1-carboxylate-oxidase and as th ree different pathogenesis-related proteins (PR-1b, PR-Q and SAR 8.2). Two cDNAs were homologous to a calmodulin-like protein from Arabidops is and a human ribosomal protein L19 while six cDNA clones remained un known. One of these clones (termed PAR-1 for photoassimilate-responsiv e) displayed features similar to pathogenesis-related proteins: Hybrid izing transcripts, 1.2 and 1.0 kb in length, were strongly inducible b y salicylate and accumulated in tobacco plants after infection with po tato virus Y (PVY) both in infected and uninfected systemic leaves. PA R-1 transcripts also accumulated in wildtype leaves upon floating on g lucose and sucrose whereas sorbitol and polyethylene glycol had no eff ect. Rescreening of the ppa-1 cDNA library with the PAR-1 cDNA as prob e resulted in 25 hybridizing cDNAs which by homology were found to fal l into three classes (PAR-1a, b, c). The cDNAs coding for PAR-1a and b were 90.6% homologous on the DNA level while both were less related t o the PAR-1c cDNA (70.5% and 75.2% homologous, respectively). One open reading frame was identified in all three PAR-1 cDNA classes. Transla tion would result in proteins with a theoretical molecular mass of abo ut 20 kDa. The N-terminal amino acid sequences resemble a signal pepti de which would direct the proteins to the secretory pathway. Using sel ective 3' hybridization probes of the three PAR-1 cDNAs it was possibl e to discriminate the different transcripts. Both PAR-1a and PAR-1c mR NAs are induced in plants treated with PVY.