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1222 VOLUME 21 NUMBER 10 OCTOBER 2003 NATURE BIOTECHNOLOGY

Transgenic crops are increasingly used in agriculture to controlinsect pests. The most common strategy is to produce an insecticidalBacillus thuringiensis (Bt) δ-endotoxin from an introduced gene,thus conferring resistance to feeding insects. However, the wide-spread deployment and prolonged use of Bt plants have raised someconcerns about the evolution of insect resistance1 and possible envi-ronmental risks1,2.

Photorhabdus luminescens is a Gram-negative bacterium thatforms symbioses with entomopathogenic Heterorhabditis spp. soilnematodes3,4. After a juvenile nematode invades an insect host, thebacteria are released into the insect hemocele, where they producetoxins and proteases that kill the insect host and render the cadaver arich source of nutrients for bacterial and nematode growth5. Severalprotein toxin complexes have been purified from P. luminescensstrain W14 and their corresponding genes cloned6. Earlier work hasshown that the fermentation broth of strain W14 contains at leasttwo potent toxins (toxins A and B) that independently contribute toinsecticidal activity7. Potencies (LD50 values (lethal dose for 50% ofinsects) in nanograms of protein per square centimeter of diet) ofelectrophoretically pure preparations of the two toxins differmarkedly in top-loaded artificial diet assays. LD50 values for toxin Aagainst southern corn rootworm (SCR; Diabrotica undecimpunctatahowardi), European corn borer (ECB; Ostrinia nubilalis) and tobaccohornworm (THW; Manduca sexta), respectively, are 4.7, 93 and 63,whereas the respective values for toxin B are 87, >5,000 and >2,500(ref. 8). Against sensitive insects, the potency of toxin A comparesfavorably with published values for Bt toxins9 (e.g., Cry1Aa, Cry1Aband Cry1Ac have LD50 values against M. sexta of 5–8 ng/cm2), andwe therefore chose to study toxin A further.

The toxin A protein (TcdA) of strain W14 is encoded by a singleopen reading frame (ORF), tcdA, of 7,548 bp10 and has a calculatedsize of 283 kDa. Native toxin A from bacterial broth exists in a large

complex (>860 kDa) consistent in size with a homotetramer7. Ourcharacterization of the proteins comprising the toxin A complexindicated that the N-terminal 88 amino acids (TcdAi) of the TcdAprimary gene product are removed (Fig. 1a) and the remaining pep-tide is cleaved into two large polypeptides, TcdAii and TcdAiii8.During this processing step, another 88 internal amino acids arelost. The order of these cleavage steps, and the significance, relativeto toxin activity, of the N-terminal and internal deletions have notbeen determined.

Here we report the use of a synthetic plant-codon-optimized vari-ant of tcdA, the gene encoding toxin A, for control of insect pests intransgenic Arabidopsis plants. In this study, we chose THW and SCRas test insects. Although THW is not a major agronomic pest, it is rel-atively easy to obtain and rear in the laboratory, and neonate larvaewill feed directly on young Arabidopsis leaves. Our results indicatethat high expression of tcdA in plants can confer high resistance toTHW feeding, and toxin A isolated from plants had a strong growth-inhibitory effect on SCR. In addition, we found that the 5′ and 3′UTR sequences of a tobacco osmotin gene substantially enhancedproduction of toxin A protein.

RESULTSExpression of toxin A from initial constructsFive plant vectors (Fig. 1b) containing derivatives of a plant-optimizedtcdA gene10 under the control of a constitutive cassava vein mosaicvirus (CsVMV) promoter11 were used to produce transformedArabidopsis plants. A kanamycin-resistance gene was included in thevectors to permit selection of transgene-bearing plants. Northernhybridization experiments indicated that 69% of the T0 plants exam-ined (39 of 56) showed a single toxin A RNA species of the expectedsize (about 8 kb; Fig. 1c). To our knowledge, the full-length tcdAtranscript is the largest active transgenic mRNA produced in plants

Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, Indiana 46268, USA. Correspondence should be addressed to D.L. (dliu@dow.com).

Published online 31 August 2003; doi:10.1038/nbt866

Insect resistance conferred by 283-kDa Photorhabdusluminescens protein TcdA in Arabidopsis thalianaDong Liu, Stephanie Burton, Todd Glancy, Ze-Sheng Li, Ronnie Hampton, Thomas Meade & Donald J Merlo

The tcdA gene of Photorhabdus luminescens encodes a 283-kDa protein, toxin A, that is highly toxic to a variety of insects,including some agriculturally important pests. We tested the efficacy of transgenic toxin A in Arabidopsis thaliana for control offeeding insects. Plants with toxin A expression above about 700 ng/mg of extractable protein were highly toxic to tobaccohornworm (Manduca sexta). Toxin A isolated from transgenic plants also strongly inhibited growth of the southern corn rootworm(Diabrotica undecimpunctata howardi). Addition of 5′ and 3′ untranslated regions of a tobacco osmotin gene (osm) increasedtoxin A production 10-fold and recovery of insect-resistant lines 12-fold. In the best line, high toxin A expression and insectresistance were maintained for at least five generations in all progeny. The intact tcdA mRNA represents the largest effectivetransgenic transcript produced in plants to date. These results may open a new route to transgenic pest control in agriculture.

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NATURE BIOTECHNOLOGY VOLUME 21 NUMBER 10 OCTOBER 2003 1223

to date. As expected, RNA expression varied from line to line (datanot shown).

We carried out immunoblot analysis to examine plant productionand processing of the toxin A proteins. Purified toxin A produced fromP. luminescens or a recombinant Escherichia coli strain produced threebands corresponding to TcdA, TcdAii and TcdAiii (Fig. 2a). Plantscarrying a full-length tcdA produced three protein bands that alignedwith these controls (Fig. 2a). Plants carrying any of the three TcdAiigene constructs, pDAB7033, pDAB7034 and pDAB7035, had singleTcdAii protein bands with the expected molecular sizes (Fig. 2b–d).No TcdAiii protein was detected in 32 plants transformed with con-struct pDAB7032, however, even though tcdAiii RNA was properlyproduced (Fig. 1c, lane 5). These results suggest that either the transla-tion efficiency of tcdAiii RNA is extremely low, the TcdAiii protein isvery unstable in Arabidopsis or the TcdAiii protein was not extractedby the methods used.

Insect toxicity of plants with initial constructsAccumulation of toxin A protein was quantified for primary trans-formants by ELISA (Table 1). All of the 78 positive pDAB7031 linescontained less than 200 p.p.m. toxin A protein except lines 7031-025(349 p.p.m.), 7031-043 (1,056 p.p.m.) and 7031-240 (788 p.p.m.).Bioassay results indicated that line 7031-240 had 100% mortalityagainst THW.

Thirty-two self-pollinated kanamycin-resistant T1 progeny fromthree pDAB7031 lines (7031-025, -043 and -240) were tested fortoxin A accumulation and toxicity to THW. Twenty-one T1 plantsfrom line 7031-240 retained high toxin A expression and 100%THW mortality, whereas insect mortality for control plants express-ing a CsVMV-GUS-ORF25 construct (pDAB7029) was 18.7%. Line7031-240 represents the first instance of heritable insect mortalityresulting from expression of a P. luminescens toxin gene in trans-genic plants.

These results indicated that toxin A accumulation above athreshold of about 700 p.p.m. could result in complete toxicity toTHW, but the frequency at which insect-resistant lines were recov-ered was very low (1/340, 0.3%). This was probably a result of theoverall low expression of the large tcdA gene from the constructpDAB7031, despite the use of a strong constitutive promoter andplant-optimized coding region.

Toxin A expression from improved constructsIn eukaryotic cells, sequences of 5′ and 3′ untranslated regions(UTR) are important for mRNA stability and translational effi-ciency12,13. The mRNA of the tobacco osmotin gene, osm, is very sta-ble in plant cells14,15, and therefore we used 5′ and 3′ UTR sequencesof this gene to enhance expression of tcdA in Arabidopsis. The UTRswere added to the corresponding ends of three tcdA coding regions:full-length tcdA (pDAB7026), tcdAii/∆C (pDAB7027) and tcdAiii(pDAB7028) (Fig. 1b). We conducted RNA analysis on 30 plants con-taining these three osm-tcdA constructs. Twenty plants showed a sin-gle species of toxin A mRNA of the expected sizes, similar to thosefrom non-osm tcdA constructs (Fig. 1c).

Concentrations of intact toxin A or toxin A subunits produced bythese osm-tcdA constructs were compared with their non-osm coun-terparts (Table 1). The osm-tcdA constructs resulted in a sixfoldgreater average total toxin A accumulation in the expressing plants(390 p.p.m. versus 67 p.p.m.) and a greater overall frequency of toxinA producers recovered (39% versus 23%), as compared to non-osmconstructs. When all transgenic plants examined (expressers andnonexpressers) were included in the statistical analysis, average toxin

A concentrations were tenfold higher in the osm-construct plants.There was also a difference in the number of high expressers (toxin Aprotein >700 p.p.m.) for each construct: the frequencies were 0.6%and 5.1%, respectively, for non-osm and osm constructs. For somehigh expressers (>2,000 p.p.m.), the large toxin A protein could beseen in a Coomassie blue–stained SDS-PAGE gel (Fig. 2f). Theseresults demonstrate that it is feasible to produce effective amounts ofthese large, processed insect toxins in plants.

Expression-enhancing effects of the osmotin UTRs were also seenin plants carrying constructs encoding the toxin A subunits. Of thenon-osm TcdAii construct (pDAB7033) plants, 58% produced thetruncated TcdAii protein (average of 251 p.p.m.; Table 1), whereas

TcdA; 283 kDa

TcdAi; 10 kDa TcdAiii; 63 kDaTcdAii; 208 kDa

a

bWith OSM UTRsWithout OSM UTRs

pDAB7032

pDAB7035

pDAB7034

pDAB7033

pDAB7031

pDAB7027

pDAB7026

pDAB7028

Not made

Not made

TcdAiii

TcdAii

TcdAii (∆C)

TcdAii (∆C+∆N)

TcdA

LB RBCsVMV kan RORF25

c

10/15 12/12 5/8 8/11 4/10

1 2 3 4 5MW (kb)

7.4

4.4

2.3

rRNA

9.5 Lane 1: pDAB3031 (tcdA)Lane 2. pDAB7035 (tcdAii)Lane 3: pDAB7033 (tcdAii )Lane 4: pDAB7034 (tcdAii ∆C+

∆C∆N)

Lane 5: pDAB7032 (tcdAiii)

Figure 1 Putative processing pathway of toxin A protein, tcdA constructsused for plant transformation, and RNA analysis of transformed plants. (a) The primary gene product, TcdA, is proteolytically cleaved after aminoacid 88 to release TcdAi. The remaining large peptide is cleaved internally to produce the large (TcdAii) and small (TcdAiii) subunits found in the toxin complex. Molecular sizes are calculated from the deduced amino acid sequence encoded by the tcdA gene. Filled box, N-terminal TcdAisegment; vertically striped box, 88 amino acids removed from the TcdAii C-terminus; inverted filled triangles, protease cleavage sites. (b) Eight tcdAgene derivatives were inserted between a CsVMV promoter and pTi-15955plasmid ORF25 3′ UTR sequences in binary vector pDAB1542. LB and RB,pTi-15955 T-DNA left and right borders, respectively; kanR, kanamycin-resistance gene for selection of transformed plants. The designations foreach tcdA fragment are shown within the box representing the coding region.Names at left are those of constructs made without tobacco osmotin 5′and 3′ UTR sequences; those at right are of constructs containing theosmotin UTRs. (c) Northern blot of RNA expression patterns of transgenicArabidopsis plants carrying five different tcdA constructs. The fraction ofplants with the expected RNA pattern (as shown in blot) versus the numberof examined plants for each construct is indicated below each lane. Arrowindicates nonspecific binding of the probe to ribosomal RNA.

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1224 VOLUME 21 NUMBER 10 OCTOBER 2003 NATURE BIOTECHNOLOGY

90% of the osm-construct (pDAB7027) plants produced TcdAii pro-tein, with average accumulation of 1,131 p.p.m. (4.5-fold increase).We did not detect TcdAiii protein in any of the 32 pDAB7032 (non-osm) plants examined (Table 1), but 40% of the plants carrying theosm construct (pDAB7028) produced a single band of TcdAiii pro-tein (Table 1 and Fig. 2e), although the overall accumulation wasnot high. These data clearly show that tobacco osmotin UTRsequences can greatly enhance tcdA gene expression in transgenicArabidopsis plants.

Insect toxicity of plants with improved constructBioassays of 234 T0 lines from construct pDAB7026 (osm-tcdA) iden-tified nine lines that produced 100% insect mortality (Table 2). Eightof the nine (line 7026-127 being the exception) had toxin A concen-trations greater than 1,000 p.p.m.

Bioactivity and high-level accumulation of toxin A protein werecoordinately transmitted to the next generation. We examined atleast 32 progeny from each of these 9 active lines as well as progeny ofline 7026-011, which also had abundant toxin A protein but was notbioassayed in the T0 generation (Table 2). For line 7026-011, all 71 T1plants showed 100% insect mortality, except for one that had 87.5%insect mortality (1 of 8 insects survived; mortality on the controlplant was 16.9%) (Table 2). In contrast, none of the T1 progeny ofline 7026-195 showed toxin A accumulation or insecticidal activity.For the other lines (Table 2), the percentage of progeny with hightoxin A accumulation and insecticidal activity ranged from 18.4% to

93.7%. In total, 214 of 333 tested T1 progeny of these ten linesretained a high level of toxin A protein, and 98% of the highexpressers (211 of 214) had high insecticidal activity (Table 2). Theseresults indicated that the high accumulation of toxin A protein wasresponsible for the plants’ insecticidal activity against THW.

An additional four T0 lines from construct pDAB7026 (osm-tcdA)(7026-101, -126, -268 and -298) were high expressers but caused nosignificant insect mortality. Thirty-two T1 progeny from each ofthese lines were further analyzed. None of the progeny of line 7026-268 showed toxin A protein or insect activity. Surprisingly, a total of11 T1 progeny of the other three lines showed high toxin A, and theaverage insect mortality was 98.2% (control mortality 14.0%). It isunknown if the lack of insect activity in the T0 generation was due toan aberration in the insect bioassay, or if the toxin A protein in thoseplants was truly inactive. These aberrant results underscore the needto examine transgenic progeny, rather than solely T0 plants, in assess-ing gene function.

To determine if the TcdAii polypeptide has insecticidal activity, weidentified 12 high expressers (>700 p.p.m.) from 146 T1 progeny rep-resenting all three tcdAii constructs. None showed substantiallyhigher insect mortality than the control plants.

Inheritance of insect activityThe stability of tcdA expression and associated insecticidal activitywere followed to the fifth generations (T4 plants) for T0 lines 7026-011 and 7026-057 (Table 3a,b). For line 7026-011 (Table 3a), both

a 1 2 3 4 5 6 7 8 9 10TcdA

TcdAii

TcdAiii

b 1 2 3 4 5 6 7

TcdATcdAi

TcdAii

f 1 2 3 4 5

TcdATcdAi

kDa

250150

10075

50

d 1 2 3 4 5 6

TcdATcdAii

TcdAiii

c 1 2 3 4 5 6 7

TcdATcdAi

TcdAii

e 1 2 3 4 5 6 7 8

TcdATcdAi

TcdAii

Figure 2 Analysis of toxin A protein production in transgenic Arabidopsis plants. Filled arrows indicate positions of toxin A proteins; bands below the TcdAiiiprotein (open arrows in a,c–e) indicate antibody-cross-reacting Arabidopsis proteins. Lane 1 in a–d and lane 5 in e contain purified toxin A protein producedfrom a recombinant E. coli strain expressing the native tcdA gene. The three bands, in decreasing size, represent proteins TcdA (283 kDa), TcdAii (208 kDa),and TcdAiii (63 kDa). Transgenic plants carrying a CsVMV-GUS-ORF25 construct (pDAB7029; see Methods) were used as negative controls (lane 2 in a, lane 7 in b–d and lane 5 in f). (a). Immunoblot analysis of full-length TcdA production. Lanes 3–10, transgenic plants with construct pDAB7031 (tcdA). (b) Immunoblot analysis of TcdAii production. Lanes 2–6, transgenic plants with construct pDAB7035 (tcdAii). (c) Immunoblot analysis of C-terminallytruncated TcdAii. Lanes 2–6, transgenic plants with construct pDAB7033 (tcdAii ∆C). (d) Immunoblot analysis of doubly truncated TcdAii. Lanes 2–6,transgenic plants with construct pDAB7034 (tcdAii ∆C+∆N). (e) Immunoblot analysis of Osm-TcdAiii. Lanes 1–4 and 6–8, transgenic plants with constructpDAB7028 (osm-tcdAiii). (f) SDS-PAGE gel analysis of TcdA protein expression in plants transformed with construct pDAB7026 (osm-tcdA). Lane 1, extractfrom a high expresser of tcdA; lanes 2–4, extracts from three low expressers of tcdA.

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phenotypes, high expression of toxin A and almost 100% insect mor-tality, were stably maintained in all progeny for five generations.Over the five generations of testing, only 2 of 2,704 insects survived.

In contrast, the heritability pattern for line 7026-057 progeny(Table 3b) was more unpredictable. As first seen by comparison ofthe T0 and T1 generations of this line (Table 2), there was no pre-dictability from one generation to the next regarding the frequencyor activity levels of the progeny. Similarly, T2 progeny of 6 of the 7active T1 lines (Table 3b) produced from 0 to 35% active plants.Progeny lines of select T2 and T3 lines also produced a wide range offrequencies of active plants. Lines with reasonably good activitycould have progeny with little or no activity.

In summary, from 274 pDAB7026 transgenic lines analyzed at theT1 generation, we identified a total of 12 lines with heritable hightoxin A production and insect activity (Table 1), although thedegree of heritability varied from 3% (line 7026-101) to 100% (line

7026-011). The enhanced accumulation oftoxin A protein mediated by the osmotinUTR sequences increased the recovery fre-quency of insect-resistant lines from 0.3%to 4.4% (Table 1).

THW susceptibility to toxin A duringdevelopmentWe also tested the activity of toxin A trans-genic plants against THW of different ages,and we found that sensitivity decreased withlarval development. Leaf materials from T4plants of line 7026-011 were fed to THWtaken at the beginning of each of the fiveinstars. For first- and second-instar larvae,greater mortality was caused by the toxinA–expressing plants (100% and 93.8%,respectively) than the control plants (12.5%and 9.4%, respectively). Growth of the sec-ond-instar survivors was inhibited by about60%. For third-instar larvae, no obvious dif-ference in mortality was seen between thetoxin A–expressing and control plants, but

toxin A plants caused about 40% growth inhibition. Neither highmortality nor growth inhibition was seen with fourth- and fifth-instar larvae.

Insect toxicity of plant-produced toxin AFinally, we tested the activity of plant-produced toxin A against SCR.Because Arabidopsis is not a natural host of SCR, we purified toxin Afrom plants of line 7026-11 and used this in a surface-treated dietassay to test its potency on SCR. In these assays, we fed neonate SCRseither a toxin A–treated diet or a water-treated diet and comparedthe weight of the insects. The purified toxin A had a strong inhibitoryeffect on SCR growth, with about 70% growth inhibition occurringat a concentration of 10 ng/cm2 (Fig. 3). We did not see insect deathuntil the toxin A concentration reached 500 ng/cm2. This contrastswith the activity of native toxin A, which has an LD50 of 5 ng/cm2

(ref. 7).

Table 1 Production of TcdA protein or subunit peptides and activity against tobacco hornworm in transgenic Arabidopsis carrying various TcdA constructs

Gene No. lines No. Average TcdA Average TcdA Highest No. high No. insect-construct examined expressing of expressers of all plants TcdA expressersa active lines

lines (p.p.m.) (p.p.m.) (p.p.m.)

pDAB7031 340b 78 (23%) 67 15 1,056 2 (0.6%) 1 (0.3%)

(tcdA)

pDAB7026 274c 106 (39%) 390 152 7,161 14 (5.1%) 12 (4.4%)

(osm-tcdA)

pDAB7033 41 24 (58%) 251 146 1,594 2 (4.8%) 0

(tcdAii/∆C)

pDAB7027 32 29 (90%) 1,131 1,058 6,074 10 (31%) 0

(osm-tcdAii/∆C)

pDAB7032 32 0 0 0 0 0 ND

(tcdAiii)

pDAB7028 25 10 (40%) 33 13 66 0 ND

(osm-tcdAiii)

pDAB7035 57 22 (38%) 71 19 799 1 (1.7%) 0

(tcdAii)

a>700 p.p.m. bBioassays were not performed on the first 15 T0 plants obtained, which included line 7031-043. cBioassayswere not performed on the first 40 T0 plants obtained, which included line 7026-011. ND, not determined.

Table 2 TcdA concentrations and tobacco hornworm bioassay results on T1 selfed progeny of ten transgenic Arabidopsis lines withconstruct pDAB7026 (osm-tcdA)

High expressersb Low expressers Control plantsc

7026 No. T1 No. insect- Number TcdA Average Average Average plant linea plants active plants (p.p.m.) insect insect insect

tested mortality (%) mortality (%) mortality (%)

-011 (7161) 71 71 (100%) 71 (100%) 761-7860 99.8 NA 16.9

-294 (1707) 32 30 (93.7%) 30 (93.7%) 914-2400 93.8 15.6 7.8

-293 (2133) 32 22 (68.7%) 22 (68.7%) 985-5120 100 12.5 7.8

-150 (2327) 32 20 (62.5 %) 20 (62.5%) 1219-3213 97.0 8.3 12.0

-190 (1505) 32 19 (59.3%) 19 (59.4%) 1254-2271 98.9 25.7 20.3

-286 (4265) 32 19 (59.3%) 19 (59.4%) 468-3657 93.8 15.6 12.0

-122 (1707) 32 15 (46.8%) 15 (46.9%) 472-2388 95.0 12.3 20.3

-127 (379) 32 8 (25.0%) 9 (28.1%) 486-1969 100 20.2 16.3

-057 (2725) 38 7 (18.4%) 9 (23.8%) 670-1844 100 17.1 12.5

-195 (1280) 32 0 0 (0.00%) NA 0 0 0

Total 333 211 (63.3%) 214 (64.3%) 97.2 15.9 14.4

aNumbers in parentheses are TcdA concentrations of T0 plants (p.p.m.). All these T0 plants except -011 (not tested) had 100% insect mortality. b>700 p.p.m. cTransformed with GUS gene constructs. At least eight control plants were used per test set.

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1226 VOLUME 21 NUMBER 10 OCTOBER 2003 NATURE BIOTECHNOLOGY

DISCUSSIONIn this work, we analyzed the expression of aplant-optimized version of the P. lumi-nescens gene tcdA, encoding toxin A, intransgenic Arabidopsis plants. The resultsprovide useful insights about the productionof toxin A protein in plants. (i) Plants trans-formed with a full-length tcdA gene can pro-duce toxin A protein whose final productsmimic those from the native P. luminescensstrain W14 and from a recombinant E. colistrain, indicating that toxin A protein isappropriately processed in plant cells. (ii) Theunusually large sizes of the tcdA coding reg-ion (7.5 kb) and gene product (over twicethe size of the Cry1 class of B. thuringiensisδ-endotoxins currently deployed for cropprotection9) do not preclude high-levelexpression and accumulation. (iii) High-level expression of the plant-optimized genein Arabidopsis, in the absence of any other P. luminescens genes, is sufficient for controlof THW and possibly SCR.

Further, we enhanced tcdA gene expressionby adding 5′ and 3′ UTR flanking sequencesfrom a tobacco osmotin gene. Structural fea-tures of the osmotin mRNA 5′ UTR (which ishighly AT rich, allowing the ribosome to eas-ily scan to the initial codon for translation)and 3′ UTR (which has strong secondarystructure, as determined by MFOLD func-tion of the Wisconsin Package, version 10.3;Accelrys) are consistent with the criteria fora stable, highly expressed plant mRNA12.Addition of these 5′ and 3′ UTR sequences tothe tcdA gene construct(s) boosted the over-all production of the TcdAii and TcdA pro-teins five- to tenfold. To our knowledge, thisrepresents the first report that thesesequences can enhance gene expression. Itwill be interesting to further investigate themechanism of expression enhancement by these UTR sequences andto determine if this strategy will increase production of other pro-teins of interest.

Most of the insect-resistant Arabidopsis lines that we generatedshowed a significant degree of gene silencing in the next generation.Within a given progeny population, tcdA expression in an individualplant ranged from high to nondetectable, and the degree of genesilencing at the next generation varied from line to line. No correla-tion was found between the degree of tcdA silencing and the T-DNAcopy number (data not shown). Apparently, the silencing is depend-ent on insertion position, and the chromosomal location of eachT-DNA locus affects tcdA expression and its heritability patterns.With the completion of the Arabidopsis genome project16, we candetermine the precise chromosomal locations of each integratedtransgene in these lines. Those results may contribute to furtherunderstanding of the role of chromosomal integration location ingene silencing mechanisms.

Most importantly, we demonstrated that production of the largetoxin A in transgenic Arabidopsis plants can render them highly toxicto feeding THW, and that toxin A purified from transgenic plants

had a strong inhibitory effect on the growth of SCR. We noticed,however, that the lethal dose for plant-produced toxin A is muchhigher than that of native toxin A. This discrepancy in insecticidalactivity is the focus of our continued investigation.

Many strains of Photorhabdus spp. and their close relativesXenorhabdus spp. produce multiple protein toxins with high activityagainst a wide variety of plant-feeding insects17,18. Our results indi-cate that the P. luminescens tcdA gene is an excellent candidate toevaluate further as a means for crop protection in agriculture. This,and related toxin genes, may open new routes for pest control viadeployment as primary insect control genes, or as alternatives to Btgenes for resistance management in a variety of crops.

METHODSDNA constructs. The 7,548-bp sequence of the tcdA ORF from P. luminescensstrain W14 was determined in this laboratory (GenBank accession numberAF188483.1). A 7,551-bp DNA sequence that encodes essentially the TcdAprotein was designed for optimal expression in monocots and dicots by adjust-ing the codon usage and removing putative RNA instability sequences, poten-tial intron splice signals and potential polyadenylation signal sequences10.Synthesis of gene fragments and coding region assembly were done by Operon

Table 3 Inheritance of TcdA expression and insecticidal activity against tobaccohornworm

a

Plant No. plants TcdA No. insects No. surviving Average mortality withgeneration tested (p.p.m.) assayed insects control plants (%)a

T0 1 7161 ND ND ND

T1 71 761-7860 568 1 16.9

T2 76 611-3662 608 1 14.0

T3 95 639-31211 760 0 7.2

T4 96 2000-10567 768 0 31.6

Total 309 2704 2 (0.07%) 17.2b

Inheritance of TcdA production and activity against THW in selfed progeny of Arabidopsis line 7026-011 (osm-tcdA)through five generations. aTransformed with GUS gene constructs. At least eight control plants were used per test set. ND, not determined. bAverage control mortality in the four tests.

b

T1 T2 T3 T4

7/38 (18.4%) Line 1: 0/13 (0.0%) ND ND

Line 2: 1/15 (6.7%) 1/16 (6.3%) ND

Line 3: 2/16 (12.5%) 0/16 (0.0%) ND

Line 4: 4/16 (25.0%) 4/16 (25.0%) ND

Line 5: 5/14 (35.7%) Line 5.1: 2/16 (12.5%) ND

Line 5.2: 3/16 (18.7%) ND

Line 5.3: 4/15 26.7%) ND

Line 5.4: 5/16 (31.2%) ND

Line 5.5: 9/16 (56.2%) ND

Line 6: 3/16 (18.7%) Line 6.1: 9/15 (60.0%) Line 6.1.1: 0/16 (0.0%)

Line 6.1.2: 0/16 (0.0%)

Line 6.1.3: 4/16 (25.0%)

Line 6.1.4: 5/16 (31.2%)

Line 6.1.5: 6/16 (37.5%)

Inheritance of insect activity against THW in selfed progeny of Arabidopsis T0 line 7026-057 (osm-tcdA) through fivegenerations. Numbers of active plants in each progeny line are indicated as n/N (active plants/total plants examined)and as the percentage of total plants examined (numbers in parentheses). ND, not determined. All the active plantswere high expressers (>700 p.p.m.) and induced 100% insect mortality. The generation number for each progenyfamily is indicated across the top. Insect activity of a line in one generation was not predictive of the activitymeasured in the next generation.

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Technologies. Unique 5′ NcoI and 3′ SacI sites were added to the coding region.Various derivatives with corresponding NcoI and SacI sites were generatedfrom the basic tcdA coding region by PCR. All tcdA fragments were placedunder the expression control of a constitutive CsVMV promoter11 and a 3′UTR-polyadenylation signal sequence derived from the intergenic regionbetween ORFs 25 and 26 of pTi-15955 (ref. 19). Each tcdA expression cassettewas cloned between the T-DNA borders of binary vector pDAB1542, whichcontains a kanamycin-resistance gene as a selectable marker for plant transfor-mation10. A control vector (pDAB7029) containing a CsVMV-GUS-ORF25expression cassette20 was also constructed.

The 40-bp 5′ UTR of a tobacco osmotin gene was assembled using a pair ofchemically synthesized complementary oligonucleotides on the basis of thepublished sequence15. (A G→T change was made at nucleotide 29 of the nativesequence remove a potential start codon.) The 3′ UTR of the same tobaccoosmotin gene was amplified by PCR from a cDNA clone15 (a kind gift fromR. Bressan, Purdue University). These elements were ligated 5′ and 3′, respec-tively, to the coding regions of three tcdA fragments (tcdA, tcdAii/∆C andtcdAiii), and expression constructs containing the osm-modified codingregions under control of the CsVMV promoter and ORF25–26 3′ UTR werecloned separately into pDAB1542.

Plant transformations. All plant transformation constructs were electropo-rated21 into Agrobacterium tumefaciens strain C58 (Z707)22. Arabidopsis plants(Columbia ecotype) were grown at 22 °C with a photocycle of 16 h light/8 hdark. Plant transformations were performed using the vacuum infiltrationmethod23. Transgenic seedlings were selected for kanamycin resistance.

Northern hybridization. Total RNA was extracted from 150 mg mature leaftissues using an RNeasy Mini Plant Kit (Qiagen), and 5 µg was loaded onto1.5% (wt/vol) agarose gels containing formaldehyde and processed for north-ern analysis24. Hybridizations were carried out (42 °C; 4 h) in ULTRAhyb solu-tion (Ambion), using a 32P-labeled 7.5 kb NcoI/SacI DNA fragment containingthe entire coding sequence of toxin A, prepared using a Ready-To-Go DNAlabeling kit (Amersham Biosciences). Membranes were washed twice with 2× SSPE, 0.5% (wt/vol) SDS for 15 min, then twice with 0.1× SSPE, 0.1%(wt/vol) SDS. The first three washes were at room temperature and the finalwash was at 42 °C.

Protein extraction. Leaf tissues were processed in a Costar 96-well cluster tubebox (Corning), sandwiched between 0.188-mm steel (on bottom) and 0.188-mmtungsten (on top) beads. Grinding was in 0.350 ml of extraction buffer (19 mM NaH2PO4, 31 mM Na2HPO4, 10 mM EDTA, 0.1% (vol/vol) Triton X-

100, 0.078% (vol/vol) β-mercaptoethanol and protease inhibitors (SigmaChemical). The cluster box was shaken for 2 min at the maximum setting in aKleco Model 2-96-A bead mill (Garcia Manufacturing) and then centrifuged(2500g; 4 °C), and the supernatants were used for protein analysis. Total solubleprotein in the leaf extracts was determined using a procedure for microtiter plates(Bio-Rad).

Immunoblot detection of toxin A. All antibodies were diluted in solution A(10% (wt/vol) milk, Tris-buffered saline, 0.05% (vol/vol) Tween 20). Rabbitanti–toxin A polyclonal antibody developed against recombinant toxin A pro-tein from E. coli was protein A purified and used at 1 µg/ml. Leaf extract pro-teins in sample buffer containing 0.1 M dithiothreitol were separated through4–20% Criterion polyacrylamide gels (Bio-Rad) and transferred to nitrocellu-lose membrane. The membrane was blocked with solution A for 1 h, the block-ing solution was removed and the membrane was probed with anti–toxin Aantibody for 1 h. The detection antibody was a goat anti-rabbit–horseradishperoxidase conjugate (Bio-Rad) used at a 1:1,000 dilution. Reacting proteinswere detected using the ECL substrate (Pierce) and X-ray film.

ELISAs. Goat polyclonal antibody was developed against recombinant toxin Afrom E. coli. The protein A–purified antibody was used as both the capture anddetection antibody. Nunc Maxisorb 96-well microtiter plates were coated withcapture antibody at 0.5 µg/ml, 100 µl/well, at 4 °C overnight. The plates werewashed in four well volumes of PBS wash buffer (Fisher Scientific) and 0.05%(vol/vol) Tween 20. Recombinant toxin A protein was used to generate a stan-dard curve (in duplicate) ranging from 1 ng/ml to 64 ng/ml on each 96-wellplate. Protein extract samples (diluted at 1:4–1:512) were added to the plates,which were incubated for 1 h and then washed as above. Detection antibodyconjugated to horseradish peroxidase was diluted to 0.75 µg/ml and used at100 µl/well. Plates were incubated for 1 h and washed as above, and then 100 µlof tetramethylbenzidine substrate was added to each well. The OD650 after10 min of incubation was measured and analyzed using SOFTmax Pro version2.6.1. software. Quadratic regression analysis on each 96-well plate was used tocompare the test samples to that plate’s standard curve and calculate the con-centration (in ng/ml) for each dilution tested. ELISA values reported here werederived from the test sample dilution that fit best the linear portion of thestandard curve. Toxin A protein concentrations are reported as parts per mil-lion (p.p.m.; 1 p.p.m. = 1 ng toxin A per milligram of extractable protein).

Bioassay of transgenic plants against THW. Tobacco hornworm eggs from theNorth Carolina State University insectary were incubated in lighted chambers(Percival) at 22 °C or 28 °C for 2–3 d in 90-mm-diameter Petri dishes contain-ing 2% (wt/vol) agar solution. Newly hatched larvae (<6 h old) were selectedthe morning of the bioassay. 128-well bioassay trays (CD International) wereprepared by placing 0.5 ml of a 2% (wt/vol) agar solution in each well. Foreach 5-week-old Arabidopsis plant, leaf tissue samples (∼ 1 cm2) were distrib-uted evenly among eight wells. A single neonate hornworm was placed intoeach well, which was then covered with a perforated sticky lid, and the insectswere allowed to feed in a chamber at 28 °C with a photoperiod of 16 h light/8 hdark for 72 h. Insect mortality and weight scores were recorded. Two controlplants with the GUS (β-glucuronidase) gene construct pDAB7029 wereincluded for every 16 toxin A transgenic plants tested.

Data analysis was done by comparing the percent insect mortality onexperimental versus control plants. Mortality scores were transformed and az-test was used. Plants that had moderate to high toxin A protein concentra-tions and significantly higher mortality than the controls (at P ≤ 0.05) wereconsidered ‘active’. To reveal any potential growth inhibition effects, individ-ual insect weights were analyzed by ANOVA, comparing transformed plantswith controls.

Purification of recombinant TcdA. 100 g of fresh Arabidopsis leaf tissue wasfrozen in liquid nitrogen and ground to a fine powder in a blender. Solubleproteins were extracted by blending the powder with 250 ml buffer containing50 mM Tris-HCl, pH 7.8, 1 mM dithiothreitol and 0.5% (vol/vol) Tween 20.After centrifugation at 40,000g, recombinant TcdA in the supernatant waspurified by Mono Q and Superose 6PG (Amersham Biosciences) columnchromatography was done as described7.

TcdA concentration (ng/cm2)

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Figure 3 Growth inhibition of southern corn rootworm larvae fed artificialdiets treated with toxin A extracted from transgenic Arabidopsis. Averageweights are expressed as a percentage of the average weight of larvae fedartificial diets treated with water.

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Bioassay of plant-produced toxin A against SCR. Purified TcdA was dilutedinto 50 mM sodium phosphate buffer, pH 7.8, with 5% (vol/vol) glycerol, andapplied directly in 50-µl aliquots to insect bioassay diets as described previ-ously7. SCR weight and mortality were scored in three independent assays.

ACKNOWLEDGMENTSWe thank Jim Hasler and Sean Russell for their technical assistance, and NickStorer for statistical analysis of bioassay data. We also thank Sam Reddy, Tim Heyand Amanda Schleper for helpful discussions and critical reading of themanuscript. The osmotin cDNA clone was kindly provided by Ray Bressan ofPurdue University.

COMPETING INTERESTS STATEMENTThe authors declare competing financial interests (see the Nature Biotechnologywebsite for details).

Received 27 March; accepted 17 July 2003Published online at http://www.nature.com/naturebiotechnology/

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