Virus-induced gene silencing as a tool for comparative functional studies in Thalictrum

Abstract

Perennial woodland herbs in the genus Thalictrum exhibit high diversity of floral morphology, including four breeding and two pollination systems. Their phylogenetic position, in the early-diverging eudicots, makes them especially suitable for exploring the evolution of floral traits and the fate of gene paralogs that may have shaped the radiation of the eudicots. A current limitation in evolution of plant development studies is the lack of genetic tools for conducting functional assays in key taxa spanning the angiosperm phylogeny. We first show that virus-induced gene silencing (VIGS) of a PHYTOENE DESATURASE ortholog (TdPDS) can be achieved in Thalictrum dioicum with an efficiency of 42% and a survival rate of 97%, using tobacco rattle virus (TRV) vectors. The photobleached leaf phenotype of silenced plants significantly correlates with the down-regulation of endogenous TdPDS (P<0.05), as compared to controls. Floral silencing of PDS was achieved in the faster flowering spring ephemeral T. thalictroides. In its close relative, T. clavatum, silencing of the floral MADS box gene AGAMOUS (AG) resulted in strong homeotic conversions of floral organs. In conclusion, we set forth our optimized protocol for VIGS by vacuum-infiltration of Thalictrum seedlings or dormant tubers as a reference for the research community. The three species reported here span the range of floral morphologies and pollination syndromes present in Thalictrum. The evidence presented on floral silencing of orthologs of the marker gene PDS and the floral homeotic gene AG will enable a comparative approach to the study of the evolution of flower development in this group.

Figure 1. VIGS of Thalictrum dioicum PHYTOENE DESATURASE ortholog TdPDS results in varying degrees of leaf photobleaching.

A: Untreated T. dioicum plant. B–F: Distribution of photobleaching in TRV2-TdPDS treated plants. G: Leaflet showing signs of silencing along the vascular tissue. H: Detail of partially photobleached leaflet. I: Typical range of silencing in TRV2-TdPDS treated leaflets. Scale bar  = 1 cm.

Figure 2. Downregulation of TdPDS and detection of TRV transcripts in VIGS photobleached leaves of Thalictrum dioicum.

A: Expression of TdACTIN control, native TdPDS and viral transcripts in leaves by Reverse Transcriptase (RT)-PCR. Untreated and mock-treated (empty TRV2) T. dioicum plants are compared to TRV2-TdPDS treated plants showing photobleached (white), variegated (green/white) and green leaf tissue. RT-PCR was performed with locus-specific primers to the housekeeping gene ACTIN (loading control), to endogenous TdPDS and to the viral transcripts TRV1/TRV2. Approximate band size indicated for TRV2: larger band results from the presence of the TdPDS insert, smaller band from an empty TRV2 (mock control). B: Comparative expression of TdPDS normalized with TdACTIN among treatments and resulting phenotypes of Thalictrum dioicum . Values based on quantification of RT-PCR gel bands in part A using ImageJ (see text for details). Different letters indicate statistical significance in a one-way ANOVA followed by Tuckey test (p<0.05), same letters indicate no statistical difference. Average and standard error bars are shown. Sample sizes are n = 4 for untreated and mock-treated, n = 6 for treated bleached and n = 3 for treated variegated or green.

Figure 3. Virus-induced gene silencing of TtPDS causes photobleaching in leaves and flowers of Thalictrum thalictroides.

A: Flower and leaf TtPDS silencing phenotypes compared to controls. Ai: Untreated flower of T. thalictroides, note green leaflets and green/yellow floral center; Aii: TRV2-TPDS treated plant, showing partial photobleaching of leaflets that appear variegated; Aiii: Detail of varying degrees of photobleaching in leaflets; Aiv: Detail of untreated flower, note that carpels and young stamens are normally photosynthetic (green); Av: Detail of treated flower showing silencing in stamens and carpels, three older stamens are not photobleached and therefore look green (asterisks), a patch of necrotic tissue (a viral effect) is indicated with an arrow; Avi: empty TRV2 mock-control flower showing background viral effects: arrow points to reduced sepal with patch of necrotic tissue. Scale bar  = 1 mm. B: Comparative expression of TtPDS in leaves and flowers of T. thalictroides plants treated with TRV2- TPDS , relative to controls. Untreated and mock-treated (empty TRV2) plants are compared to TRV2-TPDS treated plants showing photobleached leaves (left panels) or flowers (right panels). Reverse-transcriptase PCR was performed with locus-specific primers to the housekeeping gene TtACTIN (loading control), to endogenous TtPDS and to the viral transcripts TRV1/TRV2. For TRV2: larger band results from the presence of insert, smaller band from an empty TRV2 (mock control).

Figure 4. Virus-Induced Gene Silencing of Thalictrum clavatum AGAMOUS ortholog TcAG-1 results in homeotic floral phenotypes.

A: Flower silencing phenotypes of TcAG-1 , relative to controls. Ai, Untreated flower of T. clavatum showing sepals (se), stamens (st) and carpels (ca); Aii: strongly silenced flower in TRV2-TAG-1 treated plant, showing an array of sepals and no stamens nor carpels, all reproductive organs have been homeotically converted to sepals; Aiii: intermediate phenotype with partial conversion of organs and some normal ones; Aiv: detail of dissected organs in an untreated flower (sepal, stamen, carpel, from left to right); Av: detail of all sepaloid dissected organs from a strong TcAG-1 silencing phenotype (from the outside to the inside of the flower, left to right); Avi: detail of sample chimeric organs, arrows point to anther tissue on the edges of an internal “sepal”. Scale bar = 1 mm. B: Gene expression by Reverse Transcriptase (RT)-PCR in TcAG-1 silenced plants compared to controls. Untreated and mock-treated (empty TRV2) plants are compared to TRV2-TAG-1 treated plants showing strong homeotic conversions (Aii, Av). RT-PCR was performed with locus-specific primers to the housekeeping gene ACTIN (loading control); to the MADS box gene TcAG-1 and to the viral transcripts TRV1/TRV2. For TRV2: larger bands result from the presence of insert, the smaller band from an empty TRV2 (mock control).