vigs (virus-induced gene silencing) works in all important...

INTRODUCTION:Plant virus-based vectors carrying sequences homologous to endogenous genes trigger silencing through a homology-dependent RNAdegradation mechanism. This phenomenon, called virus-induced gene silencing (VIGS), has been shown to be of great potential in plantreverse genetics. Advantage of VIGS over other approaches, such as T-DNA or transposon tagging, include the circumvention of planttransformation, methodological simplicity, robustness, and speedy results. These features make VIGS an attractive alternativeinstrument in functional genomics. In cereals like wheat and barley, modified Barley Stripe Mosaic Virus (BSMV) have been used tosilence genes expressed in leaf tissue of young plants only. Applicability of this technique at later growth stages and in imporatnttissue types is not yet known.

OBJECTIVE:To check the expression of VIGS vector (modified BSMV) in roots, older leaves, floral tissue and seed by using green fluorescentpigment (GFP) and phytoene desaturase (PDS) as markers.

VIGS (Virus-Induced Gene Silencing) Works in All Important Tissues of WheatHarvinder S. Bennypaul, Upinder S. Gill, Sridhar Jarugula and Kulvinder S. Gill

Washington State University, Pullman,WA

MATERIALS AND METHODS:Plant Material and Growth Conditions:Spring wheat cultivar Zak was grown in 6-inch pots using peat mix, with 14g Nutricote 14-14-14 100-day timed release per 1L of peatmix. Plants were grown at 20oC-25oC with 23-50% RH and 16 hours of light (approximately 6000 lumens/m2 light intensity). Solublefertilizer 20-20-20 (Plantco Inc.) was applied twice a week at 100 ppm.

Inoculation Protocol:The plasmids used in these experiments are described by Holzberg et al. (2002).Capped in vitro transcripts were prepared fromlinearized plasmids (that contain the tripartite BSMV genome) using mMessage mMachine T7 in vitro transcription kit (Ambion, Austin,TX. Transcripts of each of the BSMV genomes were mixed in 1:1:1 ratio. A 7.5µl transcription mix was combined with 45µl of FES(abrasive material)and directly applied with two light strokes to the leaf. Inoculations were done at two leaf stage and boot stage.

Fig.4. GFP expression in root tissue

Control 7 Days after Inoculation(in phloem tissue)

17 Days after inoculation(spreading to cortex)

Fig.5. PDS silencing phenotype in wheat seeds

Inoculated Control

Fig. 1. GFP expression in phloem tissue of leaf

Control 7 Days after Inoculation

Fig.3. PDS silencing phenotype in wheat head

Inoculated Control

Fig.2. PDS silencing at flag leaf stage

RESULTS AND DISCUSSION: GFP expression was observed in phloem tissue of leaf (Fig.1) and root

(Fig.4) as early as 7 days after inoculation. After 17 days expressionspread to other tissues of root.This suggests that VIGS will also beuseful in functional genomics of roots.

Inoculation at 2 leaf stage produced photobleaching due to silencing ofPDS gene. Bleaching phenotype was at maximum in 4th or 5th leaf thereafter it went down to mere specks on flag leaf. Inoculation at bootstage produced a robust bleaching phenotype of flag leaf, lemma, palea,awns (Fig.3) and seeds (Fig.5). Thus, inoculation at boot stage is better for studying genes expressing in reproductive tissue.

GFP expression

GFP expression

FUTURE WORK: To corroborate these preliminary findings,Waxy gene (which expresses in

developing grains)and Tacoi1, a gene which expresses in roots, would betargeted for silencing.

REFERENCES:Holzberg S, Brosio P, Gross C, Pogue GP (2002) Barleystripe mosaic virus-induced gene silencing in amonocotplant. Plant J 30:315-327