CLONING OF TOBACCO GENES THAT ELICIT THE HYPERSENSITIVE RESPONSE

We used a functional screening method to isolate genes whose products elicit the hypersensitive response (HR) pathway of defense against pla nt pathogens. A cDNA library derived from tobacco leaves undergoing th e HR was cloned into a tobacco mosaic virus (TMV)-based expression vec tor. Infectious transcripts were generated and used to inoculate tobac co plants lacking the N resistance gene (genotype Xanthi nn). Approxim ately 1/1000 of the infectious transcripts produced local lesions, and may thus elicit the HR. The cDNA inserts from 50 lesion-forming clone s were recovered by RT-PCR, and 12 unique clones were sequenced. Compa risons with protein databases revealed homologies to (a) ubiquitin, (b ) tobacco tumor-related protein, similar to Kunitz-type trypsin inhibi tors and (c) ribosomal protein S14. The remaining nine clones revealed no homology to known proteins and are thus considered novel. Five clo nes were able to induce the expression of PR2, a gene which is specifi cally activated in the tobacco HR. Northern and western blot analyses of leaves infected by the clone encoding ubiquitin strongly suggest th at the infection produced a co-suppression response; the endogenous le vels of ubiquitin mRNA and protein in infected leaves are ca. 50% less than those found in healthy leaves. This observation supports a previ ous report on the involvement of the ubiquitin system in the tobacco H R [2], and validates the utility of the functional cloning method.