Potential Application of Virus-Induced Gene Silencing (VIGS) in Flower Senescence Studies

Virus-induced gene silencing has been suggested as a powerful and rapid technique for analysis of gene function in plant growth and development (Baulcombe, 1999). Infecting Nicotiana benthamiana with Tobacco Rattle Virus (TRV) containing a fragment of the gene encoding phytoene desaturase, an essential enzyme in carotene synthesis, results in a photo-bleached phenotype in photosynthetic tissues. TRV has several interesting properties that could be utilized in studies of the function of candidate genes in floral senescence; it infects floral tissues, has only mild symptoms, and has a broad host range. We tested the effect of TRV containing PDS fragments on a range of host plants. In most cases, infection resulted in no apparent phenotype or local inoculation effects. In petunia, after a lag of several weeks, the characteristic photo-bleaching of upper portions of the plant indicated movement and silencing of the virus and of PDS. In preliminary tests with a TRV/CHS construct, the typical effects of post-transcriptional gene silencing of CHS in petunia – symmetrical and asymmetrical white patches on the dark purple petals – indicated the ability to use the system in petunia studies. INTRODUCTION Whole genome studies and gene sequencing from a wide variety of plant species have produced a massive amount of sequence information. It is now essential to develop rapid-throughput and large-scale functional genomic tools to decipher the biological meanings of these sequences. Virus-induced gene silencing (VIGS) offers an attractively quick method for loss of function assay that may reveal embryo-lethal sequences and avoids the need for timeconsuming transformation and regeneration processes (Baulcombe, 1999; Ratcliff et al., 2001). VIGS is a plant defense mechanism that limits the severity of virus infection (Baulcombe, 1999) by a process similar to post transcriptional gene silencing PTGS (Chicas and Macino, 2001). During replication of the virus, double stranded chimeric intermediates are produced. The plant cell recognizes these intermediates as foreign and cuts the double stranded RNA into small oliogonucleotides (siRNA) which serve as guides in an RNA-induced silencing complex to degrade anything with identical homology (Chicas and Macino, 2001). Many plant viruses have been used to develop VIGS vectors (Gossele et al., 2002; Ratcliff et al., 2001; Ruiz et al., 1998; Turnage et al., 2002). Among these tobacco rattle virus (TRV)-based vectors have been suggested to have potential for use with a wide range of plant species due to the wide host range of TRV (Ratcliff et al., 2001). The system has been shown to function effectively in Nicotiana benthamiana and tomato (Liu et al., 2002a; Liu et al., 2002b; Peart et al., 2002) using phytoene desaturase (PDS) as a marker gene. Infected plants show characteristic photo-bleaching symptoms resulting from the inhibition of carotene biosynthesis. Although TRV has a number of interesting properties; it infects floral tissues, has only mild symptoms, and has a broad host range; this powerful tool has not yet been applied to studies of gene function in ornamental Proc. VIII IS Postharvest Phys. Ornamentals Eds. N. Marissen et al. Acta Hort. 669, ISHS 2005 148 plants. We therefore decided to test TRV-based gene silencing technology in selected ornamentals. MATERIALS AND METHODS Plant Material Seedlings of a range of plant species (Nicotiana benthamiana), morning glory (Ipomoea purpurea), daffodil (Narcissus pseudonarcissus), viola (Viola × wittrockiana), daylily (Hemerocallis ‘Cradle Song’), four o’clocks (Mirabilis jalapa), snapdragon (Antirrhinum majus), delphinium (Delphinium cardinale, D. elatum), petunia (Petunia hybrida), salvia (Salvia splendens), Iceland poppy (Papaver nudicaule), Oriental poppy (Papaver orientale), marigold (Tagetes erecta) cosmos (Cosmos bipinnatus), love-in-amist (Nigella damascena), larkspur (Consolida ambigua), granny’s bonnet (Aquilegea ‘McKana Giant’), impatiens (Impatiens wallerana), English daisy (Bellis perennis), and zinnia (Zinnia grandiflora) were either obtained from commercial producers or produced in the greenhouse at the Department of Environmental Horticulture, University of California. Infection was carried out at the fourto sixtrue leaf stage. Constructs Initial testing was with a TRV construct obtained from Dinesh-Kumar, Yale University, comprising the TRV genome and an inserted fragment of the N. benthamiana PDS gene. Subsequent studies were with a construct containing a fragment of the Petunia chalcone synthase (CHS) gene. Agrobacterium-Mediated Infection Agrobacterium-mediated infection was used to inoculate N. benthamiana to generate virus particles and also to test the effect of different constructs on petunia subsequent to the host range experiments. The constructs, pTRV1 (TRV RNA1 construct) or pTRV2 (TRV RNA2 construct), and its derivatives were transformed into Agrobacterium strain GV3101 by electroporation. Infection of plants with the transformed Agrobacterium was performed as described in Liu et al. (2002b). The bacteria were cultured overnight at 28 °C in LB medium with appropriated antibiotics. The Agrobacterium cells were then harvested and re-suspended in inoculation buffer (10 mM MgCl2, 10 mM MES, 200 μM acetosyringone) to an O.D. of 2.0 and left at room temperature for 3 h. The bacteria containing pTRV1 and the bacteria containing pTRV2 were then mixed together in a 1:1 ratio. The leaves of N. benthamiana or petunia plants were infiltrated with approximately 0.1 ml of the mixed bacterial culture using a 1 ml disposable syringe without a needle. Virus Infection To test the host range of the TRV VIGS system, PDS-containing virus particles were extracted in water from 4-day post inoculated N. benthamiana leaves and mechanically transmitted using carborundum powder (to increase efficiency) on the leaves of the test plants. All inoculated plants were then transferred to the same greenhouse area and checked periodically for appearance of the photo-bleaching phenotype. RESULTS AND DISCUSSION Host Range of TRV-Induced Gene Silencing in Ornamentals Few of the tested plants showed PDS symptoms, and in those that did, it was usually near the infection site (Table I). Only two species, N. benthamiana and P. hybrida showed the photo bleaching in the apical tissues (Fig. 1) that indicated movement of the virus and its effectiveness in silencing the native PDS gene. Although TRV has been suggested to provide an excellent tool for gene silencing