Silencing of a plant gene by transcriptional interference

Abstract

Integration of foreign DNA into eukaryotic genomes results frequently in a total or partial loss of gene function, caused by the interruption of indispensable structures of the gene itself. Using T-DNA insertions in Arabidopsis we screened for mutants with deregulated chlorophyll precursor accumulation in etiolated seedlings. A mutant designated rfd1 (red fluorescent in darkness) with increased protochlorophyllide accumulation showed a fluorescent phenotype that was associated with a lack of transcript initiation from the AtRibA1 promoter situated downstream of the integrated T-DNA. Complementation experiments confirmed rfd1 to be a knockout phenotype. Comparison with two SALK insertion lines bearing T-DNA integrations in the 5'UTR of AtRibA1 demonstrated that the insertion event in rfd1 itself does not explain the complete lack of transcript initiation. A 35S tetrameric enhancer sequence present on the rfd1 T-DNA causes the overaccumulation of a large polycistronic transcript originating inside the T-DNA. This 5.5-kb RNA runs over the downstream situated AtRibA1 promoter, which was shown by 5'RACE analyses to be consequently silenced. Hence, a transcription process that starts upstream and overlaps AtRibA1 blocks the initiation at the AtRibA1 promoter in rfd1. This regulatory mechanism has recently been introduced in yeast as transcriptional interference and is described here for the first time in a plant system.

Figure 1.

(A) Phenotype of etiolated rfd1 seedlings. Plants grew 6 days in the dark and were analyzed by fluorescence microscopy as described in ‘Materials and Methods’ section. (B) A. thaliana wild-type seedlings under the conditions described in (A). (C) Etiolated seedlings of line rfd1 in the presence of 5 µM riboflavin. (D) Nine-day-old light-grown seedlings of line rfd1. (E) Scheme of the pWA5 T-DNA insertion upstream of At5g64300 (AtRibA1) in rfd1 DNA. Exons 1–7 are indicated by red arrows, the mRNA sequence by a gray box. pWA5-derived T-DNA is inserted upstream of the AtRibA1 5′ end. The inserted sequence contains a 35S enhancer tetramer next to the right border (RB) sequence. The phosphinotricine resistance marker gene (pat) is controlled by a CaMV 35S promoter (35S prom.). Primers used in (F) are indicated by blue arrows. Since the schemes of AtRibA1 and the inserted T-DNA are scaled differently, sizes of both DNA stretches are given in parentheses. (F) Southern blot analysis of DNA from single heterozygous rfd1 plants cut by restriction endonucleases DraI, BspHI and NdeI (lanes 1–3, respectively). Hybridization was performed using a LB-specific PCR product of pWA5. Sizes of DNA fragments are given on the right. (G) Detection of T-DNA in individual etiolated non-fluorescent (lanes 1–4) or red fluorescent seedlings (5–7) of line rfd1. The Primer pair F1/R1 specifically amplified the wild-type allele (upper panel), whereas primer combination F1/LB1 detected AtRibA1-alleles containing integrated pWA5 T-DNA (lower panel). Arabidopsis wild-type and nontemplate controls are shown in lanes 8 and 9, respectively.

Figure 2.

(A) Semi-quantitative RT-PCR of etiolated seedlings. Homozygous (rfd1, homoz.) as well as nonfluorescent (rfd1, mixed) rfd1 seedlings were collected and compared to A. thaliana wild-type seedlings (Wt). Accumulation of AtRibA1 (RibA1, upper panel) and alpha-tubulin (tua, lower panel) transcripts was investigated using primer pairs F3/R3 (see Figure 2D) and tuaF/tuaR, respectively. Reverse-transcribed total RNA measuring 2 ng, 6 ng and 18 ng (from left to right) were used as template. Arrowheads on the right side of each panel indicate the migration of 0.4-kb and 0.3-kb marker fragments. Note that, due to segregation, the nonfluorescent sample (rfd1, mixed) contained heterozygous rfd1 as well as wild-type seedlings. (B) Northern blot of RNA isolated from heterozygous rfd1 mutants (rfd1) and A. thaliana wild type (Wt) was hybridized to AtRibA1 cDNA. 25S rRNA (3.3 kb) and 18S rRNA (1.7 kb) positions as well as the estimated sizes of specific bands are indicated. The lower panel depicts an ethidium bromide stain of RNA before blotting. (C) Hybridization of RNA blots of heterozygous rfd1 mutants and A. thaliana wild type (Wt) with probes I, II and III, respectively. (D) Localization of DNA fragments I, II and III (blue boxes) used as probes in (C); positions of primers for sq RT-PCR of AtRibA1 (F3, R3) are indicated by blue arrows. The transcription start site of the 5.5-kb T-DNA:AtRibA1 fusion transcript identified in line rfd1 is indicated by a red arrow. (E) 5′ RACE of the pat gene in homozygous (rfd1) or heterozygous (het.) rfd1 mutant plants, compared to A. thaliana wild type (Wt) and an alternative T-DNA insertion line carrying the pWA5 plasmid (rfd4). Primary RACE PCR (upper panel) was performed with 35 cycles using primers Ad1 and pat_RACE351. The secondary, nested amplification used Ad2 and pat_RACE113 and 25 additional cycles (lower panel). A non-template control is shown in lane NC. Fragment lengths are indicated by arrowheads at the right margin.

Figure 3.

(A) 5′ RACE reactions to detect AtRibA1 transcript ends in Arabidopsis wild type (Wt), SALK 036891 (S91), SALK 094736 (S36) and homozygous rfd1 seedlings (rfd1). Analysis of a heterozygous rfd1 plant (het.) and a nontemplate control (NC) are included. The DNA marker lane (M) contains a 100-bp DNA ladder (New England Biolabs, Ipswich). Red arrows depict the two major transcription start sites TSS1 and TSS2 detectable in A. thaliana wild type. (B) Northern blot of A. thaliana wild type and SALK lines 036891 and 094736, respectively. The filter was hybridized to an AtRibA1 cDNA fragment. 25S rRNA and 18S rRNA positions are indicated. The lower panel depicts the ethidium bromide stain of RNA before blotting. (C) Overview of the AtRibA1 upstream sequence with transcription start sites mapped by 5′ RACE (arrows) and T-DNA insertion sites. The 5′ UTR (highlighted in gray) in wild-type RNA results from transcription starting at TSS1 and TSS2 (red arrows). Altered transcript 5′ ends in the two SALK lines are designated TSS S36 and TSS S91. Insertions of T-DNA resulted in losses of genomic DNA stretches (colored residues) of different lengths. Insertions of pWA5 T-DNA in rfd1 (blue) and pROK2 in SALK 094736 (green) and 036891 (brown), respectively, are indicated. Nucleotide numbers are given as distances from the AtRibA1 start codon at the left margin.