Gene silencing in transgenic tobacco hybrids: frequency of the event and visualization of somatic inactivation pattern.

We have investigated the stability of the expression of different T-DNA-borne genes in hybrid tobacco lines. These lines were constructed to rescue rolC-induced male sterility in kanamycin-resistant P35s-rolC transgenic tobacco plants by expression of rolC antisense genes. Using five different tester lines, a total of 158 hybrids was obtained. We observed inactivation of transgene expression in 20% of the F1 progeny and in 35% of the backcrossed F2 progeny, as indicated by the loss of kanamycin resistance. In 3% of all crosses complete loss of antibiotic resistance was noted, while in most affected hybrid progeny only part of the population became kanamycin sensitive. Single genes could be selectively inactivated on T-DNAs harboring several genes. Gene inactivation was not restricted to one of the two T-DNAs examined. Somatic silencing, visualized by a cell-specific 35SGUSINT marker gene, occurred in a random fashion or exhibited an inherited specific pattern. The type of somatic silencing pattern observed indicated developmental control of the process. Two phenotypic classes could be distinguished with respect to frequency and timing of the inactivation process. Rapid gene inactivation, occurring within a few weeks after germination of hybrid seedlings, was characterized by complete methylation of restriction sites in the promoter of the silenced gene, resetting of gene expression during meiosis, heredity of the developmentally controlled program of gene silencing in subsequent generations, and rapid reactivation of gene expression after genetic separation of the different T-DNAs. In contrast, a slow type of gene inactivation was of a more stochastic nature and was recognized only in hybrids of the backcrossed F2 generation. In this case the degree of promoter methylation, which could extend beyond the T-DNA borders, was not correlated with the reduction in steady-state poly(A)+ mRNA levels, the silenced state was transmitted through meiosis and reactivation lasted several generations. The implications of the observations for our understanding of the gene inactivation process are discussed.