An active role for endogenous beta-1,3-glucanase genes in transgene-mediated co-suppression in tobacco

Abstract

Post-transcriptional gene silencing (PTGS) is characterized by the accumulation of short interfering RNAs that are proposed to mediate sequence-specific degradation of cognate and secondary target mRNAs. In plants, it is unclear to what extent endogenous genes contribute to this process. Here, we address the role of the endogenous target genes in transgene-mediated PTGS of beta-1,3-glucanases in tobacco. We found that mRNA sequences of the endogenous glucanase glb gene with varying degrees of homology to the Nicotiana plumbaginifolia gn1 transgene are targeted by the silencing machinery, although less efficiently than corresponding transgene regions. Importantly, we show that endogene-specific nucleotides in the glb sequence provide specificity to the silencing process. Consistent with this finding, small sense and antisense 21- to 23-nucleotide RNAs homologous to the endogenous glb gene were detected. Combined, these data demonstrate that a co-suppressed endogenous glucan ase gene is involved in signal amplification and selection of homologous targets, and show that endogenous genes can actively participate in PTGS in plants. The findings are introduced as a further sophistication of the post-transciptional silencing model.

Fig. 1. Endogenous glb sequences are targeted less efficiently by the silencing mechanism than corresponding transgene gn1 sequences. (A) Schematic presentation of the gn1 and glb mRNAs. Exons are indicated. The glb test regions eK and eL, and the corresponding gn1 test regions tK and tL, are represented with solid lines. The two K regions both have a length of 351 nt, and share 77% homology with a maximal stretch of 32 nt of uninterrupted homology. The two L regions both have a length of 297 nt, and share 83% homology with a maximal stretch of 36 nt of uninterrupted homology. Plasmids carrying the test regions between the STNV leader and trailer (Jacobs et al., 1999) were linearized and in vitro transcribed to produce chimeric viral RNAs for delivery into protoplasts. (B) Northern blot analysis of total RNA extracted from protoplasts of hemizygous, expressing (He) and homozygous, silenced (Ho) T17 plants 20 h after delivery of TNV RNA and chimeric STNV RNA containing the test sequences shown in (A). ³²P-labeled RNA probes for detection of viral RNAs and rRNA were complementary to the (+) strand of the STNV trailer, the (+) strand of TNV and 18S rRNA sequences. For TNV and 18S rRNA probings, exposures of equal duration are shown for hemizygous and homozygous samples. For the STNV probing, a longer exposure is shown for homozygous samples as compared with hemizygous samples. (C) Relative accumulation of chimeric STNV RNAs in protoplasts of hemizygous versus homozygous plants (He/Ho ratio). In each case, the STNV signal was normalized to the 18S rRNA signal before calculation of the ratio. A higher He/Ho ratio indicates a higher silencing susceptibility. Results from two independent experiments are shown.

Fig. 2. Endogene-specific nucleotides contribute to sequence specificity of the RNA degradation step of PTGS. (A) Upper part: position of the X, Y and Z test subregions within the K test region. Lower part: display of the homology between the glb, gn1 and artificial test sequences in the X, Y and Z subregions. The gray boxes show the ‘consensus sequence’ between the transgenic, endogenous and artificial test sequences. Plasmids carrying the test regions between the STNV leader and trailer (Jacobs et al., 1999) were linearized and in vitro transcribed to produce chimeric viral RNAs for delivery into protoplasts. (B) Features of the subregions X, Y and Z. The length of the X, Y and Z test sequences is shown, as well as the overall similarity between endogenous, transgenic and artificial sequences. For each subregion, the largest stretch of uninterrupted homology between the endogenous, transgenic and artifical sequences is shown. (C) Northern blot analysis of total RNA extracted from protoplasts of He and Ho T17 plants 20 h after delivery of TNV RNA and chimeric STNV RNA containing transgenic, endogenous and artificial test sequences as shown in (A). The ³²P-labeled RNA probe was complementary to the (+) strand of the STNV trailer. (D) Relative accumulation of chimeric STNV RNAs in protoplasts of hemizygous versus homozygous plants (He/Ho ratio). Bars represent the average of two (Z region) or three (X and Y region) independent experiments. CAT: STNV-CAT; the He/Ho ratio for the silencing-insensitive STNV CAT RNA is indicated by the horizontal dashed line. Letter codes above columns indicate statistical significance of the differences between silencing susceptibilities within each region, as determined by repeated measures ANOVA. Columns marked a, b, c differ significantly at 95% confidence. The column marked a/b differs from those marked a and b at 88 and 80% confidence, respectively.

Fig. 3. Glucanase silencing correlates with accumulation of small sense and antisense RNAs homologous to gn1. (A) Nucleic acids from protoplasts and leaf tissue of hemizygous, expressing and homozygous, silenced T17 plants were enriched by PEG. Samples of 10 µg were separated on a 15% polyacrylamide gel and blotted to membranes. Filters were hybridized with ³²P-labeled RNA probes corresponding to the (+) or (–) strand of the gn1 cDNA. The arrows indicate the position of the small RNA species. Comparison to RNA size markers of 20, 22, 25 and 28 nt indicates that the small RNA species have a length of 21 and 23 nt. He, hemizygous, expressing plants; Ho, homozygous, silenced plants; pps, leaf protoplasts; tiss, leaf tissue.

Fig. 4. Transgene- and endogene-specific small RNAs accumulate in protoplasts of silenced T17 plants. Nucleic acids from protoplasts and leaf tissue of hemizygous, expressing and homozygous, silenced T17 plants were enriched by PEG. Samples of 45 µg and in vitro synthesized control RNAs were separated on a 15% polyacrylamide gel, blotted onto membranes and hybridized with ³²P-labeled RNA probes corresponding to the (+) or (–) strand of transgenic and endogenous X region. The arrowheads indicate the position of the small RNA. Control RNAs: sense tX, sense eX: in vitro synthesized sense RNAs of ∼70 nt corresponding to transgenic or endogenous region X; antisense tX, antisense eX: in vitro synthesized antisense RNAs of ∼70 nt, corresponding to transgenic and endogenous region X. Probes for eX detection do not cross-hybridize to in vitro synthesized tX RNA and vice versa.

Fig. 5. Model explaining the activation of endogenous genes in PTGS. It assumes that transgene-specific (light gray) and ‘common’ (black) siRNAs are produced from the inducing transgene. The small transgene-derived ‘common’ antisense siRNAs anneal to homologous regions (black boxes) in the endogenous mRNA (a). The resulting partial dsRNA–RNA hybrids are recognized by an RNA-dependent RNA polymerase (RdRP) for elongation (b) or by the RNA-induced silencing complex (RISC) for direct degradation (dashed arrow). The dsRNA molecules resulting from RdRP activity are processed further by a Dicer-like enzyme, leading to the accumulation of secondary, endogene-derived siRNAs, the sequence of which can be endogene specific (dark gray) or common (black) (c). The endogene-specific secondary siRNAs can directly tag secondary targets such as endogene-specific regions in the endogenous mRNA (d), which subsequently become substrates for RISC-related degradation or a second round of RdRP-mediated dsRNA production and Dicer cleavage (dashed arrows).