Virus-induced gene silencing (VIGS) in Cysticapnos vesicaria, a zygomorphic-flowered Papaveraceae (Ranunculales, basal eudicots)

Abstract

Background and aims: Studies of evolutionary diversification in the basal eudicot family Papaveraceae, such as the transition from actinomorphy to zygomorphy, are hampered by the lack of comparative functional studies. So far, gene silencing methods are only available in the actinomorphic species Eschscholzia californica and Papaver somniferum. This study addresses the amenability of Cysticapnos vesicaria, a derived fumitory with zygomorphic flowers, to virus-induced gene silencing (VIGS), and describes vegetative and reproductive traits in this species.

Methods: VIGS-mediated downregulation of the C. vesicaria PHYTOENE DESATURASE gene (CvPDS) and of the FLORICAULA gene CvFLO was carried out using Agrobacterium tumefaciens transfer of Tobacco rattle virus (TRV)-based vectors. Wild-type and vector-treated plants were characterized using reverse transcription-PCR (RT-PCR), in situ hybridization, and macroscopic and scanning electron microscopic imaging.

Key results: Cysticapnos vesicaria germinates rapidly, can be grown at high density, has a short life cycle and is self-compatible. Inoculation of C. vesicaria with a CvPDS-VIGS vector resulted in strong photobleaching of green parts and reduction of endogenous CvPDS transcript levels. Gene silencing persisted during inflorescence development until fruit set. Inoculation of plants with CvFLO-VIGS affected floral phyllotaxis, symmetry and floral organ identities.

Conclusions: The high penetrance, severity and stability of pTRV-mediated silencing, including the induction of meristem-related phenotypes, make C. vesicaria a very promising new focus species for evolutionary-developmental (evo-devo) studies in the Papaveraceae. This now enables comparative studies of flower symmetry, inflorescence determinacy and other traits that diversified in the Papaveraceae.

Fig. 1.

Heteroblastic leaf series of wild-type Cysticapnos vesicaria. (A) Some characteristics of leaves along a primary axis, corresponding to the series of leaf silhouettes illustrated in (B). Units for lengths are in cm and for area in cm². (B) Some leaves are labelled: Co, cotyledons; 1, primary leaf; 5, leaf 5 with maximum area; 7, first leaf with tendrils; 11, leaf with maximum length and dissection; 19, leaf preceding terminal inflorescence; Br, inflorescence bracts subtending flowers. Scale bar = 1 cm.

Fig. 2.

Morphology and ontogeny of reproductive structures in Cysticapnos vesicaria. (A) Branching architecture and naming system for inflorescence position. Inflorescences are circled in blue. (B, C) Details of the inflorescence. (C) Black, last foliage leaf preceding the terminal inflorescence; dark grey, terminal inflorescence, corresponding to ‘T’ in (A). Three flowers are subtended by bracts b1–b3. The pin-like inflorescence apex is marked with a blue arrowhead; light grey, sympodial shoot and inflorescence arising from the foliage leaf axil, corresponding to ‘A’ in (A). (D–G) SEM micrographs of inflorescence ontogeny. (D) Pin-like ending of the mature inflorescence axis that may be preceded by an empty bract. (E) Two-flowered inflorescence; subtending bracts b1 and b2 removed. Perianth organs are labelled. (F) Terminal inflorescence with bracts b1 and b2 subtending flowers. Bract three is empty; inflorescence apex not visible. Sympodial inflorescence in the axil of the last foliage leaf is framed and shown enlarged in (G). (G) Early-stage inflorescence with three flower primordia. The inflorescence apex is still meristematic. Abbreviations: b1–b3, consecutive bracts; fp1–fp3, consecutive flower primordia; ia, inflorescence apex; ip, inner petal; op, outer petal; pe, pedicel; s, sepal. Scale bars: (D, F) = 500 µm, (E) = 250 µm, (G) = 200 µm.

Fig. 3.

CvFLO-VIGS phenotypes (A) Control plants inoculated with pTRV2-E showed, from left to right, wild-type flowers that were zygomorphic and had two sepals, an abaxial and a spurred adaxial outer petal, two inner petals, two stamen bundles (adaxial faces shown) and a gynoecium. (B) A pTRV2-CvFLO flower with altered floral phyllotaxis and symmetry. Dissection of this flower revealed four sepals, one sepaloid organ, three organs with mixed outer and inner petal identities and a composite organ with carpel, stamen and inner petal characteristics. Abbreviations: ad op, adaxial outer petal; ab op, abaxial outer petal; gy, gynoecium; ip, inner petals; s, sepals; st, stamen bundles.

Fig. 4.

CvPDS silencing symptoms in vegetative and reproductive parts of Cysticapnos vesicaria. (A) Young plant showing beginning of photobleaching. (B) Young inflorescence of a CvPDS-VIGS plant with complete photobleaching. (C) Mature reproductive shoots of a control (pTRV2-empty; left) and a CvPDS-VIGS plant (right). (D) Capsules of a control (left) and a CvPDS-VIGS plant (right). (E) RT–PCR showing the strong reduction of CvPDS transcripts in C. vesicaria plants infected with pTRV2-CvPDS (n = 6), compared with a pTRV2-empty control (n = 2). Expression levels are shown relative to GAPDH. Asterisks indicate a significant difference compared with the control group at 99 % confidence intervals after ANOVA. Scale bars = 1 cm.

Fig. 5.

Degree of CvPDS silencing. (A) Intensity of leaf and fruit bleaching symptoms in the primary axis. ‘Last leaf’ = last leaf before inflorescence. Abbreviations: W, white; MW, mostly white; MG, mostly green; G, green. Sample sizes are indicated above each column. Note that CvPDS silencing has a negative effect on plant robustness and survival, leading to an over-representation of partially silenced plants. (B) Different intensities of CvPDS silencing are reflected in a gradient from almost completely green (left) to completely white (right).