CLONING OF THE WOUND-INDUCIBLE PROTEIN CBP20 AND EXPRESSION IN SUSPENSION-CULTURES OF TOBACCO

One of the proteins secreted from suspension-cultured tobacco cells wa s identified as the wound-inducible protein CBP20. Four cDNA clones, C BP20/1-CBP20/4, containing fragments of different lengths of the corre sponding gene were isolated by immunoscreening two libraries prepared from tobacco leaves (CBP20/1-3) and the suspension culture line S-2 (C BP20/4). The clones, CBP20/1 and CBP20/4, with the largest inserts are identical and homologous to the clone cbp20-44 isolated by Ponstein e t al. [1] from a cDNA library prepared from tobacco (Nicotiana tabacum Samsun NN) leaves inoculated with tobacco mosaic virus. The protein C BP20 is constantly present in suspension cultures of tobacco during su bculturing in the presence of different combinations of plant growth r egulators. However, accumulation of CBP20 in the culture medium is sti mulated by 2,4 dichlorophenoxyacetic acid (2,4 D). The auxin induces a slight transient increase of the mRNA level indicating that the synth esis of CBP20 is at least partly regulated by the mRNA level. CBP20 is synthesized as a precursor of approximately 21 kDa. Maturation occurs over 3 h in a similar temporal manner to that of the vacuolar beta-1, 3-glucanase resulting in a protein of approximately 19 kDa. (C) 1997 E lsevier Science Ireland Ltd.