Genetics of homology-dependent gene silencing in Arabidopsis; a role for methylation

Ninety-eight independent transformed (T₁) Arabidopsis plants were generated, containing additional copies of the chalcone synthase (CHS) gene. Three T₂ generation families (A, B and C) were found that showed reduced anthocyanin biosynthesis, consistent with homology- dependent gene silencing of CHS. Clonal sectors of tissue showing CHS silencing were seen in the early generations. Affected individuals in family A showed only slight silencing, in family C such plants were almost completely silenced, and in family B affected individuals were intermediate. Plants homozygous for a single silencing insert were isolated from each family. Plants homozygous or hemizygous for insert A showed variable penetrance and expressivity of silencing. Self-fertilization of plants hemizygous for the B and C-inserts suggested that these CHS-silencing inserts each behave as single Mendelian dominant traits. The CHS mRNA of the C-insert homozygotes was reduced to undetectable levels. Outcrosses of B- and C-insert homozygotes to wild-type plants resulted in F₁ plants that were variegated. This variegation appears to be due to expression of the CHS allele from the wild-type parent, since use of a CHS mutant, tt4, as untransformed parent resulted in uniform green F₁ plants. Southern blots revealed a correlation between DNA methylation and CHS silencing. In addition, derivative plants were generated from C-insert homozygotes that had lost the silencing inserts, and these showed a partial reversion towards wild-type phenotype and methylation of the cellular CHS gene at the TT4 locus. This result suggests that the TT4 copy of CHS became methylated during the C-insert-induced silencing and retained methylation and partial silencing after the silencing T-DNA was lost.