Engineered resistance to tomato spotted wilt virus intransgenic peanut expressing the viral nucleocapsidgene

ZHIJIAN LI1 , ROBERT L. JARRET �

2 and JAMES W. DEMSKI1

1Department of Plant Pathology, Georgia Experiment Station, University of Georgia, 2USDA=ARS, PlantGenetic Resources, 1109 Experiment Street, Griffin, Georgia 30223 USA. (Fax: �1 770 229 3323)

Received 28 August 1996; revised 18 November 1996; accepted 10 December 1996

The nucleocapsid gene of tomato spotted wilt virus Hawaiian L isolate in a sense orientation, and the GUS andNPTII marker genes, were introduced into peanut (Arachis hypogaea cv. New Mexico Valencia A) usingAgrobacterium-mediated transformation. Modifications to a previously defined transformation protocol reduced thetime required for production of transformed peanut plants. Transgenes were stably integrated into the peanutgenome and transmitted to progeny. RNA expression and production of nucleocapsid protein in transgenic peanutwere observed. Progeny of transgenic peanut plants expressing the nucleocapsid gene showed a 10- to 15-day delayin symptom development after mechanical inoculations with the donor isolate of tomato spotted wilt virus. Alltransgenic plants were protected from systemic tomato spotted wilt virus infection. Inoculated non-transformedcontrol plants and plants transformed with a gene cassette not containing the nucleocapsid gene becamesystemically infected and displayed typical tomato spotted wilt virus symptoms. These results demonstrate thatprotection against tomato spotted wilt virus can be achieved in transgenic peanut plants by expression of the senseRNA of the tomato spotted wilt virus nucleocapsid gene.

Keywords: peanut; engineered virus resistance; tomato spotted wilt virus; transformation; nucleocapsid; geneexpression

Introduction

Tomato spotted wilt virus (TSWV) belongs to the genusTospovirus. The genome of this virus contains three linearnegative strand RNAs including; L RNA (8897 nt), M RNA(4821 nt) and S RNA (2961 nt). The virus genome isenclosed by the nucleocapsid (N) protein and the TSWVparticles are enveloped by a lipid membrane containingglycoproteins. The virus is mainly transmitted by thripsspecies (Thysanoptera) and is capable of infecting a widerange of hosts, including more than 600 species in 70 plantfamilies, many of which are crop plants of agricultural andeconomic importance (Peters and Goldbach, 1995).

Spotted wilt disease, caused by TSWV, has become amajor threat to peanut (A. hypogaea) production in thesoutheastern USA (Camann et al., 1995). TSWV-inducedpeanut yield reductions of up to 95% have been observed

in Texas since 1986. A recent survey of peanut fields inGeorgia revealed the occurrence of TSWV in up to 100%of the sample fields examined, in 10 counties (Camann etal., 1995). The 1995 Georgia Plant Disease LossEstimate indicates that TSWV in peanut resulted indamage to the crop valued at more than $44 million,approximately doubling the financial loss experienced byGeorgia farmers in 1994.

Various cultural and chemical control measures havebeen evaluated in recent years in efforts to reduce thedamage caused by spotted wilt in peanut. These measureshave been adopted primarily to control the TSWV vector(thrips) but generally have not substantially improvedyields in infected fields (Culbreath et al., 1994).Recently, the replacement of older and more susceptiblepeanut cultivars with improved varieties has resulted in areduced disease incidence which has led to a suppressionof spotted wilt epidemics in the major peanut growingregions (Culbreath et al., 1992; 1994). Nevertheless,

Transgenic Research 6, 297–305 (1997)

0962–8819 # 1997 Chapman & Hall

�To whom correspondence should be addressed.

efforts to screen the Arachis gene pool for sources ofTSWV resistance continue, as do efforts to incorporateincreased virus resistance characteristics into cultivatedpeanut using conventional breeding approaches.

The development of plant transformation technologyfor peanut, and the availability of virus-derived genesisolated from many peanut viruses including TSWV (DeHaan et al., 1990) may provide an alternative means toexpedite the development of virus resistant peanut, asdemonstrated with other plant species (Beachy et al.,1990). Gielen et al. (1991) demonstrated N gene-mediated resistance to TSWV in transgenic tobaccoplants expressing the N protein. Transgenic tobaccoplants that were asymptomatic after mechanical inocula-tion with the virus were also resistant followinginoculation by viruliferous vector thrips (De Haan etal., 1992). Additional studies, using transgenic plants ofdifferent species, established that either sense or antisensetranslatable and untranslatable N coding sequences arecapable of conferring resistance to the virus (Kim et al.,1994; Pang et al., 1993). The acquired TSWV resistancein transgenic plants is likely due to the presence of Ngene transcripts, while in other cases it is caused by theaccumulation of high levels of N protein (De Haan et al.,1992; Kim et al., 1994; MacKenzie and Ellis, 1992; Panget al., 1992; 1993; 1994).

In this study, we report the production of transgenicpeanut plants of cultivar New Mexico Valencia A (NMVal A) containing the nucleocapsid gene of TSWVHawaiian L isolate using modifications to the protocolfor Agrobacterium-mediated transformation previouslydescribed by Cheng et al.. (1996), and the successfulprotection of transgenic peanut plants expressing the Ngene against TSWV infection.

Materials and methods

Plant materials, chemicals and bacterial strains

Seed of peanut cultivar NM Val A (A. hypogaea) waspurchased from Borden Peanut Company, Portales, NM,USA.

The culture medium for peanut leaf tissues consisted ofMurashige and Skoog (1962) inorganic salts andGamborg (1966) vitamins that were purchased as apowdered mixture from Sigma Chemical Co., St. Louis,MO, USA (Cat. No. M-0404). All tissue culture mediawere adjusted to pH 5.8 and autoclaved for 20 min at121 8C. Agrobacterium tumefaciens strain EHA105 (Hoodet al., 1986) was provided by Dr G.C. Phillips, NewMexico State University, Las Cruces, NM, USA. Binaryvector pBI121 was purchased from Clontech Labora-tories, Palo Alto, CA. Cefotaxime was obtained asClaforan (Hoechst-Roussel Pharmaceutical Inc., Somer-ville, NJ, USA).

Construction of plant transformation vectors

A cDNA clone pBS-RP5.3 containing the N gene of theTSWV Hawaiian L (TSWV-L) isolate was provided by DrT.L. German, Department of Plant Pathology, Universityof Wisconsin, Madison, WI, USA. The clone was initiallygenerated by inserting a Pst I cDNA fragment into thePst I site of pBluescript=KS� and was subsequentlyconfirmed to contain the complete N gene ORF with 31bases at the 59 end and 95 bases at the 39 end (Kim et al.,1994). To engineer the N gene into the plant expressioncassette, pBS-RP5.3 was first linearized by HindIIIdigestion and blunt ends produced by treatment withKlenow fragment. Subsequent digestion with Xba Ireleased the N gene as a 0.9 kb fragment with a 59 Xba Iterminus and a blunt end 39 terminus. The N genefragment was then ligated into the Xba I-blunt-end adaptortermini at the multiple cloning site of a plant expressioncassette d35S-EC, developed in our laboratory. Theresulting vector, pGA302B, contained the sense N genecoding sequences under control of an enhanced doubleCaMV 35S promoter followed by the alfalfa mosaic virus(AMV) RNA4 non-translated leader sequence (50 bp) andthe tumor morphology large (tml) gene terminator andpolyadenylation signal sequences from A. tumefaciens.The entire N gene expression unit was subsequentlyisolated from pGA302B as a 1.9 kb HindIII fragment andinserted into the unique HindIII site of pBI121, resultingin the transformation vector pGA306 (Fig. 1). This vectorwas introduced into A. tumefaciens strain EHA105 by thefreeze-thaw method as described by Burow et al. (1990).

H HX E

N tmlamvd35S

35S GUS nos-tnos-t

HRB LB

nos-p NPTII pBI121

pGA302B

pGA306

Fig. 1. Physical map of transformation vectors containing the Ngene of TSWV-L isolate. The N gene coding sequence of TSWVHawaiian L isolate is represented by N. The arrow indicates thedirection of transcription. Other abbreviations include; d35S,enhanced double CaMV 35S RNA promoter; amv, alfalfa mosaicvirus RNA4 non-translated leader sequence; tml, tumour morphol-ogy large gene terminator; nos-p, nopaline synthase gene promoter;NPTII, neomycin phosphotransferase II; nos-t, nopaline synthaseterminator; 35S, CaMV 35S RNA promoter; RB, right border; LB,left border: H, HindIII; X, Xba I; and E, EcoRI.

298 Li et al.

Agrobacterium-mediated transformation

The Agrobacterium-mediated transformation method re-cently described by Cheng et al. (1996) was used toproduce fertile transgenic peanut plants. However, theprotocol was modified as follows: A. tumefaciens EHA105harbouring the binary vector pGA306 was culturedovernight to mid-log phase (OD600 � 0.8–1.0) in LBmedium supplemented with appropriate antibiotics; YEPmedium (pH 7.2) was used for inoculation of peanut leafexplants. During the cocultivation and subsequent selec-tion period, the use of Claforan was reduced from theoriginal concentration of 300 mg lÿ1 to 100 mg lÿ1.Putative transgenic shoots were recovered after two roundsof four-week selection on shooting medium containingkanamycin (100 mg lÿ1).

Southern blot analysis of T-DNA integration

Total DNA was isolated from mature peanut leavesessentially as described by Rogers and Bendich (1985)with a CTAB extraction solution modified to contain100 mM Na-EDTA. Ten ìg of DNA from each sample wasdigested with HindIII, fractionated on 0.8% agarose, andtransferred onto Nytran nylon membrane. The membranewas first probed with a 32P-labelled 0.9 kb fragmentcorresponding to the coding sequence of the TSWV Ngene. The membrane was stripped and rehybridized with a32P-labelled 1.8 kb fragment corresponding to the codingsequence of the â-glucuronidase (GUS) gene.

Northern blot analysis of the TSWV N gene transcripts

A method modified from Wadsworth et al. (1980) wasused to isolate total RNA from mature peanut leaves.From each sample 10 ìg of total RNA was denatured andfractionated on a 1% agarose gel containing formaldehyde.RNA was transferred onto Nytran nylon membrane andhybridized with a 32P-labelled TSWV N gene probe. Thesize of the hybridized RNA species was estimatedaccording to the RNA ladder markers separated on thesame gel.

Analysis of GUS expression

The analysis of GUS activity was performed using thefluorogenic assay procedure of Jefferson (1987). Equalamounts of leaf tissue from each sample plant wereground in GUS extraction buffer. For the GUS activityassay using the substrate 4-methyl umbelliferyl glucur-onide (MUG) 50 ìl of extract solution was used. RelativeGUS activity was determined by measuring the fluores-cence intensity of the enzymatic reaction product 4-methylumbelliferone (MU) using a TKO 100 Mini-Fluorometer (Hoefer Scientific Instrument, San Francisco,CA, USA) with an excitation wavelength of 365 nm andan emission wavelength of 460 nm.

Detection of TSWV N protein in transgenic peanut

The accumulation of the N protein in transgenic plantswas detected by using a double-antibody sandwichenzyme-linked immunosorbent assay (DAS-ELISA) andwestern blot analysis using polyclonal antibodies raisedagainst the TSWV-L N protein, according to proceduresdescribed by Pang et al. (1992).

Inoculation of transgenic peanut with TSWV

Ten-day-old seedlings grown in the greenhouse werechallenged with the TSWV Hawaiian L isolate. Inoculumwas prepared by grinding 1 g of TSWV-infected leaf tissuein 2 ml of inoculation buffer at 4 8C, into a slurry. Theinoculation buffer contained 0.01 M KPO4 buffer (pH 7.0),0.01 M each of Na2SO3 and diethyldithiocarbamic acid,and 0.01% (w=v) celite. The homogenate was immediatelyapplied, using a cotton swab, to both the abaxial andadaxial leaf surfaces previously dusted with siliconcarbide. Two leaves with a total of eight leaflets fromeach plant were inoculated. Treated plants were monitoredfor the appearance of visual symptoms and systemicinfections, on a daily basis. All the plants were treatedusing identical cultural practices.

Results

Production of transgenic peanut plants

Young leaf explants isolated from 10-day-old in vitroseedlings of NM Val A were inoculated with tobacco leafextract-treated A. tumefaciens EHA105 harbouring thebinary vector pGA306. Transgenic peanut plants wereidentified after selection of regenerated shoots forkanamycin resistance on selective medium and byhistochemical staining of leaf tissues of kanamycin-resistant shoots for GUS gene expression. This procedurewas described by Cheng et al. (1996) but was modifiedto utilize a reduced concentration of kanamycin andClaforan. These minor modifications resulted in asignificant reduction in the time period required fordevelopment and identification of transgenic shoots andresulted in a transformation frequency of 1.5% based onthe total number of inoculated leaf explants. Putativetransgenic shoots were removed from the in vitroselection regime and established in the greenhouse asearly as 90 days after explant inoculation. All plantsacclimated in the greenhouse were fertile and producedviable seeds within two months after transfer to the soil.The agronomic performance of both primary andprogency transgenic plants were similar to seed-derivedcontrol plants.

Molecular analysis of transgene integration

To confirm the integration of transgenes in transgenicpeanut plants, DNA analysis by Southern blot hybridiza-

Engineered resistance to tomato spotted wilt virus in transgenic peanut expressing the viral nucleocapsid gene 299

tion was performed using two randomly chosen transgenic(T0) lines containing the T-region of pGA306. The TSWVN gene probe hybridized to the intact total DNA fromVA306-1A (Fig. 2, lane 2) but not to the undigested DNAfrom a non-transformed NM Val A (lane 1). These dataindicate that the TSWV N gene sequences were integratedinto the peanut genome. All transgenic plants analysedproduced an expected 1.9 kb hybridization signal, corre-sponding to the intact expression unit of the TSWV Ngene, after restriction cleavage using HindIII (Fig. 1,Fig. 2 lanes 4 to 8).

Border analysis of transgene integration using a GUSprobe indicated that line VA306-1 contained threeindependent copies of the T-DNA insert of pGA306(Fig. 3, lanes 4 and 5), while line VA306-2 harboured asingle copy of the T-DNA insert (lanes 7 and 8). Thehybridization pattern of GUS-positive T1 progeny plantVA306-1A-1 was identical to its parent (lane 6 vs. lane4). In these hybridization experiments, two sister plantsfrom each independent line were analysed. Sister plantsrecovered from the same explant displayed identicalpatterns of transgene integration, indicating that a single

transformation event took place and that multipletransgenic shoots were subsequently regenerated fromthe single transformation event.

Expression of transgenes in transgenic peanut plants

To confirm RNA expression of the TSWV N gene intransgenic peanut plants, total RNA was isolated from anon-transformed NM Val A, transgenic plant VA121-12-4containing the T-DNA from pBI121 and transgenic plantVA306-1A containing the T-DNA of pGA306. A singlesignal was detected in the RNA from VA306-1 afterhybridization with a DNA probe corresponding to theentire N gene sequence, but not in the RNA from the othersamples (Fig. 4). The size of the hybridization signal wasestimated to be approximately 1.0 kb, corresponding to thecombined size of the mature RNA species derived fromthe 777 bp N gene ORF (Kim et al., 1994) and the 200 bppolyadenylation sequences.

A T1 population derived from self-pollinated transgenicplant VA306-1A was generated and utilized to examinethe stable transmission and expression of the transgenesin progeny plants. Quantitative analysis for GUS activityindicated that VA306-1A produced about one third of theGUS activity detected from a homozygous progeny(VA121-17-4) of the transgenic peanut VA121-17 trans-formed with pBI121 (Cheng et al., 1996). Among the 16

Fig. 2. Southern blot analysis of genomic DNA from transgenicpeanut plants containing the T-region of pGA306. Genomic DNAwas isolated from non-transformed control NM Val A; two sisterplants (A and C) from transgenic lines VA306-1, two sister plants(A and B) from transgenic line VA306-2; and a T1 progenyVA306-1A-1 derived from VA306-1A. DNA 10 ìg, either intact ordigested with HindIII (H), from each sample was electrophoresedin 0.8% agarose, transferred onto a Nytran nylon membrane andhybridized with a 32P-labelled TSWV N gene probe. Copy numbercontrols (lanes 9 and 10) were constructed using plasmid DNAfrom pGA306. The 1.9 kb fragment corresponds to the intactTSWV N gene expression cassette inserted into the unique HindIIIsite in pBI121.

Fig. 3. Border analysis of transgene integration in transgenicpeanut plants containing the T-region of pGA306. DNA blot inFig. 2 was stripped to remove the N gene probe and wasrehybridized with a 32P-labelled GUS probe. Since only oneHindIII site was located 59 upstream of the GUS gene expressionunit within the pGA306 T-region, the number of hybridizationbands corresponds to the number of T-DNA inserts. A 13 kbfragment corresponds to the size of the linearized plasmid pBI121.

300 Li et al.

progeny plants of VA306-1A, 14 showed levels of GUSactivity comparable to that of their parent, while twoprogeny plants (VA306-1A-8 and -16) did not displaydetectable levels of GUS activity (Fig. 5A). The segre-gation of the GUS gene in the progeny of VA306-1A didnot fit the expected 3:1 ratio of expressor vs. non-expressor for a single dominant gene, confirming theresults of our DNA integration analysis that VA306-1Acontained multiple copies of the T-DNA insert at morethan one locus in the genome.

All the plants subjected to GUS analysis were furthersubjected to DAS-ELISA analysis using antibodiesagainst the TSWV-L N protein (Fig. 5B). The TSWV-infected control plant produced a strong ELISA signal,while the healthy control and transgenic plant VA121-17-4 not containing the TSWV N gene, did not generate anELISA signal. These data indicate the specific serologicalreaction of the antibodies to the virus protein, but not toother proteins in the extract. Transgenic plant VA306-1Aand all GUS-positive T1 progeny plants exhibited positive

ELISA reactions to the antibodies against the N protein.No ELISA signal was detected in the two GUS-negativeprogeny plants. The level of the ELISA signal in eachprogeny plant was generally correlated with that of the

Fig. 4. Northern blot analysis of total RNA from transgenic peanutcontaining the T-region of pGA306. Total RNA was isolated from anon-transformed control NM Val A; transgenic plant VA121-112-4containing the T-DNA of pBI121; and transgenic plant VA306-1Acontaining the T-DNA of pGA306. Sample RNA 10 ìg wasdenatured and separated in a 1% agarose gel containing for-maldehyde. The size-fractionated RNA was transferred onto Nytrannylon membrane and hybridized to a 32P-labelled DNA probecontaining the entire N gene sequence. The expected 1.0 kbfragment of mature N gene transcript is indicated.

Fig. 5. Quantitative analysis of expression of the GUS and Ngenes in transgenic peanut VA306-1A and its T1 progeny. A.Fluorogenic assay for GUS activity. Leaf samples from TSWV-infected and healthy non-transformed control NM Val A;transgenic plant VA121-17-4 containing a homozygous form ofthe T-DNA of pBI121; transgenic plant VA306-1A and 16 of its T1

progeny after self-pollination were collected from the top third leafof each plant and analysed for GUS activity using the substrateMUG. Relative GUS activity was standardized based on samplefresh weight. B. Detection of accumulation of the N protein. Equalamounts of leaf tissue from the same group of test plants wereharvested from leaves at a similar growth stage and tested for theaccumulation of the N protein using DAS-ELISA with antibodiesagainst the N protein of TSWV Hawaiian L isolate. Absorbancewas taken within 30 min after the coated wells were exposed to thereaction substrate.

GUS Activity (pmol MU/min/mg tissue)

A

400

300

200

100

00.2 0.3

347.4

101.8

76.1 77

164.1

32.1

121.9

26.634.8

0.2

81.6 78.4

106.3

31.2

187.9

64.282.9

0.3

1A-1

6

1A-1

5

1A-1

4

1A-1

31A

-81A

-71A

-61A

-51A

-41A

-31A

-21A

-1

VA306-

1A

VAl121-

17-4

NM V

al A

VA-TSW

V1A

-9

1A-1

0

1A-11

1A-1

2

Test samples

0

0.1

0.2

0.3

0.4

0.5Absorbance at 410 nm

B

1A-11A

-21A

-31A

-41A

-51A

-61A

-71A

-81A

-9

1A-1

01A

-11

1A-1

2

1A-1

3

1A-1

4

1A-1

5

1A-1

6

VA306-

1A

VA121-

17-4

NM V

al A

VA-TSW

V

Test Samples

0.44

0 0

0.19

0.14

0.09

0.19

0.07

0.22

0.06

0.170.15

0

0.15

0.110.13

0.18

0.24

0.040

Engineered resistance to tomato spotted wilt virus in transgenic peanut expressing the viral nucleocapsid gene 301

GUS activity. These correlations demonstrated that theELISA signal detected in transgenic peanut plants wasindicative of the production of TSWV N protein as aresult of normal gene expression from the integrated Ngene in these plants. Consistent with the findings of theELISA analyses, transgenic plant VA306-1A and GUS-positive progeny plants produced a 29 kDa protein whichhybridized with antibodies produced against the TSWV-LN protein, in a western immunoblot hybridization assay(data not shown).

Protection of transgenic peanut to TSWV

In order to determine whether the expression of the Ngene confers resistance against virus infection, threegroups of peanut plants were challenged by the TSWV-Lisolate. These included nontransformed NM Val A, T2

transgenic progeny plants derived from the homozygousT1 progeny VA121-17-4 containing the T-DNA of pBI121,and T2 transgenic progeny plants derived from VA306-1Acontaining the T-DNA of pGA306. All transgenic plantsused in this experiment actively expressed the GUS geneas determined by histochemical staining (Jefferson, 1987).Plants were challenged once with TSWV inoculumprepared by grinding 1 g of infected peanut leaf tissuein 2 ml of buffer. No progeny plants of VA306-1Adeveloped visual symptoms or an increase in ELISAsignal strength one month after inoculation (data notshown). However, only 20% of the control plantsexpressed typical virus symptom during this same period,although use of the same inoculum resulted in 100%infection rate in tobacco plants. The low rate of infectionof inoculated peanut plants may have been attributable tothe low virus titre in the infected peanut leaf tissue.

In a second round of testing, ten-day-old seedlingswere mechanically inoculated three times within a four-day period, using infected peanut leaf tissue for the firstand second inoculations, and virus-infected tobacco leaftissue for the third inoculation, at a concentration of 1 ginfected leaf tissue per 2 ml of buffer. The appearance ofsymptoms, and the systemic symptom development thatresulted from this series of inoculations, are shown inFig. 6. Ten days after the first inoculation, 10 and 40%of non-transformed and VA121-17-4 progeny plants,respectively, displayed typical symptoms of TSWV,including chlorotic ring spots on the inoculated leaflets.All plants of both groups developed visible symptoms 17days after inoculation (Fig. 6A). A clear delay insymptom development was observed in the inoculatedprogeny plants of VA306-1A. Less than 20% of theseplants showed localized light green lesions around themid-vein of the inoculated leaflets 20 days afterinoculation. The number of symptomatic plants increasedto 60% by 30 days after inoculation (Fig. 6A). Symp-tomatic plants produced one or sometimes two slightlychlorotic spots on the inoculated leaflets, rather than the

numerous concentric yellow ringspots and necrotic spotsobserved in the symptomatic control plants.

All the plants were continuously monitored forsystemic symptom development. Within a period of 30days after inoculation, all the control plants includingnontransformed and transformed plants containing the T-DNA of pBI121, became systemically infected (Fig. 6B).These plants exhibited typical TSWV symptoms includ-

Fig. 6. Development of disease symptoms in transgenic peanutplants after inoculation with TSWV. Control group Val A containsseven nontransformed NM Val A plants. VA121-17-T2 includesfive GUS-positive T2 progeny plants derived from primarytransgenic plant VA121-17 harbouring a single copy of T-DNAof pBI121. VA306-1A-T2 contains six VA306-1A derived T2

progeny plants expressing the GUS and the N gene. Virusinoculation procedures were described in the text. The appearanceof disease symptoms (A) and systemic infection (B) in inoculatedplants of each group were visually scored after the firstinoculation.

0 10 15 20 25 30 35 400

20

40

60

80

100

Days after Inoculation

NM Val A

VA121-17-T2

VA306-1A-T2

Percentile of Plants Displaying Symptoms

A

0 10 15 20 25 30 35 400

20

40

60

80

100

Days after Inoculation

NM Val A

VA121-17-T2

VA306-1A-T2

Percentile of Plants Showing Systemic InfectionsB

302 Li et al.

ing; mottling, chlorosis, purple blotches on the adaxialleaf surface, leaf distortion and necrotic terminal buds. Incontrast, local lesions developed in N gene-containingtransgenic peanut were limited to only inoculated leaflets,while all other leaves remained asymptomatic. A com-parison of symptomatic leaves, and young leaves thatdeveloped subsequently from the same plant, is shown inFig. 7A. Forty days after inoculation, stunted plantdevelopment due to severe systemic TSWV infectionwas apparent in all the inoculated control plants, whiletransgenic plants containing the TSWV N gene grewnormally and were visually identical to the non-inoculated plants of the same cultivar (Fig. 7B). Although

there had been some thrip damage to the plants duringthe test period, no sign of virus transmission to the non-inoculated young leaves of the N gene-containing trans-genic plants, was observed.

Discussion

Introduction of the viral N gene into transgenic plants hasbeen a rapid and effective means to generate novelresistance against TSWV in crop plants. Our modifica-tions to a previously described protocol for Agrobacter-ium-mediated transformation of peanut cultivar NM Val A(Cheng et al., 1996) resulted in the successful productionof fertile transgenic peanut plants within a period of threemonths. Transgenic plants contained a low number (one tothree) of copies of intact T-DNA inserts. Based on ourresults of analyses of DNA integration and gene expres-sion, transgenes in transgenic peanut plants were faithfullytransmitted to the following generation and stably ex-pressed at the whole plant level. Transgenic peanut plantscontaining the N gene of TSWV-L isolate produced Ntranscripts and protein of the expected size as determinedby RNA hybridization, ELISA and western immunoblothybridization. Resistance to TSWV-L has been observedin transgenic peanut plants after mechanical inoculation inour greenhouse tests. A lower number of local lesions anda 10- to 15-day delay in symptom development wereobserved among the N gene-containing transgenic plantswhen compared with nontransformed control plants andtransgenic plants without the N gene. All the N gene-containing transgenic plants tested were highly protectedagainst systemic infection from TSWV, while all inocu-lated control plants developed severe systemic infectionsymptoms. These results indicate that nucleocapsidprotein-mediated resistance to TSWV can be achieved intransgenic peanut plants expressing the N gene, aspreviously described with other plant species.

A number of previous reports have indicated thattransgenic plants expressing the sense N gene of TSWVare protected against infection with the virus (Gielen etal., 1991; Kim et al., 1994; MacKenzie and Ellis, 1992;Pang et al., 1992, 1994). In general, a high level ofresistance to a homologous and closely related TSWVisolate from which the integrated N gene was derivedwas correlated with a lower level of N protein accu-mulation in the transgenic plants. A higher level of Nprotein accumulation was required for high levels ofresistance to heterologous isolates. Accordingly, differentmechanisms involving RNA and coat protein-mediatedresistance, respectively, were proposed to account for theresistance observed in transgenic plants (Pang et al.,1994).

ELISA analyses indicated that VA306-1 and themajority of progeny plants derived from it, accumulateda low level of N protein. However, the virus resistance

Fig. 7. Comparison of peanut leaves and plants after inoculationwith TSWV. Upper panel; symptoms of TSWV on leaves of non-transformed NM Val A (A), transgenic progeny plant VA121-17-T2containing the T-DNA of pBI121 (B), transgenic progeny plantVA306-1A-T2 containing the T-DNA of pGA306 (C), and healthyleaves of a non-inoculated NM Val A plant (D). Lower panel; plantdevelopment 40 days after inoculation with TSWV. Plants from leftto right are non-transformed NM Val A, transgenic progenyVA121-17-T2, transgenic progeny VA306-1A-T2, and a non-inoculated healthy NM Val A plant.

Engineered resistance to tomato spotted wilt virus in transgenic peanut expressing the viral nucleocapsid gene 303

observed in these transgenic peanut plants could bemediated by the presence of the N RNA transcripts.Previous studies suggested that the N gene RNA-mediated protection in transgenic plants is achievedthrough a process that inhibits viral replication (Pang etal., 1993, 1994). It has been demonstrated, by usingtobacco protoplasts, that the sense N gene RNA canhybridize to the genomic RNA of incoming virus andcompletely inhibit viral RNA replication after introduc-tion into plant cells (Pang et al., 1993). However,whether a high level of the N protein confers transgenicplant resistance to TSWV, awaits further investigationwith additional independent lines expressing the N geneat various levels.

In this study, GUS activity levels detected in transgenicplant VA306-1 and its progeny plants were about one-third of the level found in transgenic plant VA121-17-4containing the same GUS gene expression unit. Itremains to be determined whether the lower GUSexpression in VA306-1 plants is due to the presence ofmultiple copies of T-DNA inserts that results in sequencehomology-dependent transgene inactivation as describedpreviously (Flavell, 1994).

Resistance to TSWV in transgenic peanut plants hasbeen confirmed in our greenhouse experiments. Toascertain the usefulness of this newly acquired resistance,transgenic peanut plants are under field evaluation insouthern GA, at this time.

Acknowledgements

This work was supported by Peanut Cooperative ResearchSupport Program, United States Agency For InternationalDevelopment grant DAN-4048-G-00-0041-00, by theGeorgia Commodity Commission for Peanuts, by Stateand Hatch funds allocated to the University of Georgia,and by United States Department of Agriculture specificcooperative agreement No. 58-6435-3-126.

We thank T.L. German for cDNA clone pBS-RP5; J.L.Sherwood for antisera against TSWV-L N protein; G.C.Phillips for A. tumefaciens strain EHA105; C. Crawleyfor technical assistance; and R.H. Jarret for help inphotographic preparation. Special thanks to Dr M. Chengfor technical advice and assistance.

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