One monomeric and three oligomeric potato spindle tuber viroid (PSTVd) cDNA units were introduced into the tobacco genome via the Agrobacterium-mediated leaf-disc transformation. Southern analysisof the integrates revealed that only their PSTVd-specific sequences become fully methylated, whereas the flanking T-DNA and the genomic plant DNA remain unaltered. Viroid cDNA methylation could only be observed after autonomous viroid RNA-RNA replication had taken place in these plants. These findings demonstrate that a mechanism of de novo methylation of genes might exist that can be induced and targeted in a sequence-specific manner by their own mRNA. lndroduction

Despite the abundance of information about the relationship between DNA methylation and its effects on various biological functions (for reviews see Doerfler, 1983; Razin and Cedar, 1991) the regulation of DNA methylation itself is practically unknown. It has been found that in certain tissues, the general methylation pattern is largely maintained during DNA replication (Gruenbaum et al., 1982). On the other hand, gross alteration of methylation of distinct sequences and changes of the overall methylation pattern of the entire genomic DNA have been detected in the course of cell development and differentiation (for reviews see Cedar and Razin, 1990; Kafri et al., 1992). Despite all these observations, little progress has been made in the elucidation of the molecular mechanisms that initiate, specify, and regulate de novo hyper-and hypomethylation. Our present report on viroid replication in transgenic plants provides direct experimental evidence for viroid RNA-targeted induction of hypermethylation of genome-integrated viroid-specific cDNA sequences. Viroids are autonomously replicating circular, singlestranded, and nonencapsidated plant pathogenic RNA molecules whose replication proceeds via the RNA-RNA pathway (for review see Sanger, 1987). In the course of our studies on replication of the 359 nt long potato spindle tuber viroid (PSTVd), we have introduced one monomeric and three oligomeric PSTVd cDNA units into the tobacco genome. Southern analysis of the four T-DNAs with methylation-sensitive restriction enzymes revealed that the PSTVd cDNAs become fully methylated whenever PSTVd RNA-RNA replication has taken place in these plants. In plants in which no PSTVd replication was detectable,