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Potential Application of Virus-Induced Gene Silencing (VIGS) in Flower Senescence Studies Jen-Chih Chen1, Felicity Johnson1, David G. Clark2, Tim Gookin1 and Michael S. Reid1 1Department of Environmental Horticulture, University of California, One Shields Ave., Davis, CA 95616, USA

2Department of Environmental Horticulture, University of Florida, 1545 Fifield Hall, Gainesville, FL 32611, USA

Keywords: CHS (chalcone synthase), PDS (phytoene desaturase), PTGS (post-

transcriptional gene silencing), VIGS (virus-induced gene silencing), TRV (tobacco rattle virus)

Abstract

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 time-consuming 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. VIIIth IS Postharvest Phys. OrnamentalsEds. N. Marissen et al. Acta Hort. 669, ISHS 2005

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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-a-mist (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 four- to six- true 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

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studies because of its wide host range, our studies with a range of ornamental species suggests that its utility may be more limited. In a wide range of ornamentals we saw silencing of PDS only in two - both members of the Solanaceae, namely N. benthamiana (the source of the PDS fragment) and P. hybrida. This result may be due to the lack of homology between the N. benthamiana PDS and the PDS sequences in the other species tested. However, the lack of any obvious systemic viral symptoms in most of the plants tested suggests that slow movement of the virus may be more responsible for the lack of symptoms than a lack of homology of the inserted gene. Silencing of Chalcone Synthases in P. hybrida

Four weeks after infection, P. hybrida plants infected with TRV whose RNA2 contained a 820 bp fragment of the P. hybrida CHS gene showed a CHS gene silencing phenotype in the floral tissues, with a range of symptoms from white spots or streaks through symmetrical or asymmetrical white sectors, and even entirely white corollas (Fig. 2).

Although TRV-based vectors did not work on most of the test species, the vectors thus generated striking silencing phenotypes in petunias. The pattern of silencing of anthocyanin biosynthesis (Fig. 2) bears a striking resemblance to that reported from co-suppression of chalcone synthase in petunia (Napoli et al., 1990). This observation supports the hypothesis that co-suppression and virus-induced gene silencing operate by a common mechanism. The patterning of silencing in the petals presumably relates to the pattern of movement of the virus through the plant, and this is supported by the observation that silencing becomes more complete in later-produced flowers (unpublished observations). This method offers exciting potential for functional genomic studies on petunia, in particular to test the role of a wide range of senescence-associated genes being isolated from this species (Clark, unpublished). It appears likely that several genes may be included in the viral construct and will be silenced simultaneously. This offers the exciting prospect that a tandem construct combining chalcone synthase and a target gene could be used in functional genomics studies. In such a system, silencing of the genes in the floral tissues would be indicated by the lack of anthocyanins. Those tissues would then be examined for the effect of silencing the unknown genes on senescence. We are presently examining the feasibility of this system for rapid throughput screening of senescence-associated genes in petunia. ACKNOWLEDGEMENTS

We appreciate the assistance of Dr. Dinesh-Kumar Savithramma, Yale University, in providing the TRV constructs, and the supply of V26 Petunia seeds by Dr. Loverine P. Taylor, Washington State University. This study was partially supported by a grant to MSR from the American Floral Endowment. Literature Cited Baulcombe, D.C. 1999. Fast forward genetics based on virus-induced gene silencing.

Current Opinion in Plant Biology 2: 109-113. Chicas, A. and Macino, G. 2001. Characteristics of post-transcriptional gene silencing.

EMBO Reports 2: 992-996. Gossele, V., Fache, I., Meulewaeter, F., Cornelissen, M. and Metzlaff, M. 2002. SVISS –

a novel transient gene silencing system for gene function discovery and validation in tobacco plants. Plant Journal 32: 859-866.

Liu, Y., Schiff, M., Marathe, R. and Dinesh-Kumar, S.P. 2002a. Tobacco Rar1, EDS1 and NPR1/NIM1 like genes are required for N-mediated resistance to tobacco mosaic virus. Plant Journal 30: 415-429.

Liu, Y., Schiff, M. and Dinesh-Kumar, S.P. 2002b. Virus-induced gene silencing in tomato. Plant Journal 31:777-786.

Napoli, C., Lemieux, C. and Jorgensen, R. 1990 Introduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes

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in trans. Plant Cell 2: 279-289 Peart, J.R., Cook, G., Feys, B.J., Parker, J.E. and Baulcombe D.C. 2002. An EDS1

orthologue is required for N-mediated resistance against tobacco mosaic virus. Plant Journal 29: 569-579.

Ratcliff, F., Martin-Hernandez, A.M. and Baulcombe, D.C. 2001. Tobacco rattle virus as a vector for analysis of gene function by silencing. Plant Journal 25: 237-245.

Ruiz, M.T., Voinnet, O. and Baulcombe, D.C. 1998. Initiation and maintenance of virus-induced gene silencing. Plant Cell 10: 937-946.

Turnage, M.A., Muangsan, N., Peele, C.G. and Robertson, D. 2002. Geminivirus-based vectors for gene silencing in Arabidopsis. Plant Journal 30: 107-114.

Tables Table 1. The plant species tested in this study. No clear phenotypic change Systemic photo bleached phenotype Antirrhinum majus Nicotiana benthamiana Aquilegia spp. Petunia × hybrida Bellis perennis Consolida ambigua Cosmos bipinnatus Dahlia hybrida, Delphinium cardinale Delphinium elatum Hemerocallis hybria Impatiens wallerana Ipomoea purpurea Mirabilis jalapa Narcissus pseudonarcissus Nigella damascena Papaver nudicaule Papaver orientale Salvia splendens Tagetes erecta Viola × wittrockiana Zinnia grandiflora

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Figures Fig. 1. Virus induced silencing of phytoene desaturase. Petunia and N. benthamiana plant

were infected with Agrobacterium strains containing TRV vector alone (a,c) or TRV vector containing a portion of the N. benthamiana phytoene desaturase sequence (TRV-NbPDS) (b,d). Infection with TRV-NbPDS) resulted in a characteristic photo-bleaching phenotype on petunia and N. benthamiana upper leaves.

a bTRV TRV-NbPDS

c d

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Fig. 2. Virus-induced silencing of chalcone synthase in petunia petals. Petunia plants (cvs

V26 (a,b) and a commercial hybrid (c,d) were infected with Agrobacterium strains containing TRV vector alone (a,c) or TRV vector containing a portion of the petunia chalcone synthase sequence (TRV-CHSJ) (b,d). Infection with TRV- PhCHSJ) caused striking white spots or sectors on the normally purple flowers as a result of silencing of chalcone synthase.

TRV-PhCHSJ

V26

Hybrid

TRV

d

b a

c