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Proc. Natl. Acad. Sci . USA
Vol. 96, pp. 1840–1845, March 1999
Agricultural Sciences
Overexpression of the Bacillus thuringiensis (Bt) Cry2Aa2
protein in chloroplasts confers resistance to plants against
susceptible and Bt-resistant insects
(chloroplast transformation
y
insecticide resistance
y
resistance management)
MADHURI KOTA*, HENRY DANIELL†‡,SAM VARMA†,STEPHEN F. GARCZYNSKI*, FRED GOULD§,
AND WILLIAM J. MOAR*¶
Departments of *Entomology and †Molecular Genetics Program, Botany and Microbiology, Auburn University, Auburn, AL 36849; and §Department of
Entomology, North Carolina State University, Raleigh, NC 27695
Communicated by William S. Bowers, Universit y of Arizona, Tucson, AZ, December 22, 1998 (received for review August 4, 1998)
ABSTR ACT Evolving levels of resistance in insects to the
bioinsecticide Bacillus thuringiensis (Bt) can be dramatically
reduced through the genetic engineering of chloroplasts in
plants. When transgenic tobacco leaves expressing Cry2Aa2
protoxin in chloroplasts were fed to susceptible, Cry1A-
resistant (20,000- to 40,000-fold) and Cry2Aa2-resistant (330-
to 393-fold) tobacco budworm Heliothis virescens, cotton boll-
worm Helicoverpa zea, and the beet armyworm Spodoptera
exigua, 100% mortality was observed against all insect species
and strains. Cry2Aa2 was chosen for this study because of its
toxicity to many economically important insect pests, rela-
tively low levels of cross-resistance against Cry1A-resistant
insects, and its expression as a protoxin instead of a tox in
because of its relatively small size (65 kDa). Southern blot
analysis confirmed stable integration of cry2Aa2 into all of the
chloroplast genomes (5,000–10,000 copies per cell) of trans-
genic plants. Transformed tobacco leaves expressed Cry2Aa2
protoxin at levels between 2% and 3% of total soluble protein,
20- to 30-fold higher levels than current commercial nuclear
transgenic plants. These results suggest that plants expressing
high levels of a nonhomologous Bt protein should be able to
overcome or at the very least, significantly delay, broad
spectrum Bt-resistance development in the field.
The use of commercial, nuclear transgenic crops expressing
Bacillus thuringiensis (Bt) toxins has escalated in recent years
because of their advantages over traditional chemical insecti-
cides. However, in crops with several target pests with varying
degrees of susceptibility to Bt (e.g., cotton), there is concern
regarding the suboptimal production of toxin, resulting in
reduced efficacy and increased risk of Bt resistance (1, 2).
Additionally, reliance on a single (or similar) Bt protein(s) for
insect control increases the likelihood of Bt-resistance devel-
opment (3). Plant-specific recommendations to reduce Bt-
resistance development include increasing Bt expression levels
(high-dose strategy), expressing multiple toxins (gene pyra-
miding), or expressing the protein only in tissues highly
sensitive to damage (tissue-specific expression) (4).
Expression of economically important genes via the chlo-
roplast has been reported for insect (Bt Cry1Ac) and herbicide
(glyphosate) resistance with much higher expression levels
than nuclear transgenic plants (5, 6). For a recent historical
overview of chloroplast transformation see Daniell et al. (6).
Besides extremely high protein levels, chloroplast gene expres-
sion also results in tissue specificity occurring predominantly
where functional plastids are present. Chloroplast transforma-
tion uses two flanking sequences that, through homologous
recombination, insert foreign DNA into the spacer region
between the functional genes of the chloroplast genome, thus
targeting the foreign genes to a precise location. Such precise
targeting eliminates the ‘‘position effect’’ frequently observed
in nuclear transgenic plants. The maternal inheritance of the
chloroplast genome in most crops also reduces the potential for
outcrossing of foreign genes to other plants (especially weedy
species) (6).
Most current commercial transgenic plants in the United
States that target lepidopteran pests contain either Cry1Ab
(corn) or Cry1Ac (cotton) (7, 8). Bt corn is targeted primarily
against European corn borer Ostrinia nubilalis (Hu¨bner),
although other pests such as the corn earworm Helicoverpa zea
(Boddie) also may be affected. Bt cotton is targeted primarily
against the tobacco budworm Heliothis virescens (F.), pink
bollworm Pectinophora gossypiella (Saunders), and H. zea.
Especially with cotton, other pests such as Spodoptera spp. also
can be economically damaging, but have only limited suscep-
tibility to Cry1Ac. Use of single Bt proteins to control insects
such as H. virescens and H. zea could lead to relatively rapid
Bt-resistance development (4, 9). Additionally, because
Cry1Ab and Cry1Ac share more than 90% protein homology
(10) resistance to one Cry1A protein most likely would confer
resistance to another Cry1A protein as seen in H. virescens (9,
11). Nowhere is this threat of resistance more of a concern than
with H. zea, which usually feeds on corn in the spring and early
summer, then migrates over to cotton to complete several
more generations (4). Clearly, different Bt proteins are needed
to decrease the development of resistance.
Another class of Bt proteins that are toxic to many lepi-
doptera such as O. nubilalis and H. virescens but exhibit limited
homology to Cry1A proteins are the Cry2A proteins (10,
12–15). The atomic structure, ion channel formation, and
binding of Cry2A is different from Cry1 toxins (16), and
Cry2A is already under commercial development (nuclear
transformation) (17). Although, one strain of Cr y1Ac-resistant
H. virescens (Cp73) exhibited a high level of cross-resistance to
Cry2A (9), another strain of Cry1Ac-resistant H. virescens
(YHD2) exhibited only slight cross-resistance to Cry2A, and it
was suggested that the major genetic and biochemical mech-
anisms responsible for resistance to Cry1Ac in these two
strains are likely different (11).
We report here the overexpression of the Cry2Aa2 (18)
protoxin in tobacco chloroplasts and a possible solution to the
evolution of resistance to Bt observed in the field. Cry2Aa2
The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked ‘‘advertisement’’ in
accordance with 18 U.S.C. §1734 solely to indicate this fact.
PNAS is available online at www.pnas.org.
Abbreviations: Bt, Bacillus thuringiensis; RR, resistant ratio; FL,
fiducial limits.
‡Present address: Department of Molecular Biology and Microbiol-
ogy, BIO 306, University of Central Florida, Orlando, FL 32816-2360.
¶To whom reprint requests should be addressed at: Department of
Entomology, 301 Funchess Hall, Auburn University, Auburn, AL
36849. e-mail: wmoar@acesag.auburn.edu.
1840
was chosen because of its high toxicity against many insect
pests and small size of the protoxin (65 kDa) compared with
the Cry1A protoxins (130–135 kDa). Although Bt protoxins
are assumed to be more environmentally stable than toxins,
most current transgenic corn and cotton contain essentially the
Bt toxin because gene size can be a limiting factor for optimal
expression in plants (7, 8). Further, through the use of pro-
toxin, the collateral damage to nontarget insects is minimized
(19).
MATERIALS AND METHODS
Construction of a Chloroplast Expression Vector and Plant
Transformation. The tobacco chloroplast expression vector
pZS-KM-cry2A was constructed by inserting a 2.2-kb
SmaI
y
HincII fragment from pMTS-6 containing cry2Aa2 into
a Klenow-filled and dephosphorylated SpeI-digested pZS-197
(15, 20). This plasmid was amplified in Escherichia coli XL1-
Blue (Stratagene). Generation of chloroplast transgenic to-
bacco (Nicotiana tabacum var. Petit Havana) was carried out
as described (6, 21). Green calli and shoot formation were
observed after about 5–8 weeks of selection. Leaf segments
were allowed to grow and produce additional leaflets under
continued selection for another 4–5 weeks. About six shoots
that were still in physical contact with the selection medium
were selected and transferred to bottles or test tubes with
rooting medium (21). Only those shoots with intact root
primordia developed roots under selection. Shoots with suf-
ficient leaves and roots were transferred to autoclaved potted
soil and grown in growth chambers.
Primer Construction and PCR Analysis. To distinguish
Cry2Aa2-chloroplast transgenics from mutants, PCR was per-
formed on total DNA extracted from transformed plants. PCR
primers were designed by using OMEGA (IntelliGenetics).
Primer oligomers corresponding to rbcL59(59-CAAGTGTT-
GGATTCAAAGCTGGTGT-39), rbcL39(59-GGACATCCT-
TGGGGTATGCGC-39), and aadA(59-AATGGTGACTTC-
TACAGCGCGGAGA-39) were obtained from Genosys (The
Woodlands, TX). Total leaf DNA from unbombarded and
putative transgenic plants was isolated (22). PCRs were per-
formed as suggested by the manufacturer by using a GeneAmp
PCR system 2400 (Perkin–Elmer). Samples were carried
through 30 cycles by using the following temperature se-
quence: 94°C for 1 min, 55°C for 1.5 min, and 72°C for 2 min.
Cycles were preceded by denaturation for 5 min at 94°C and
a final extension cycle at 72°C for 7 min. PCR products were
electrophoresed through a 0.7% agarose gel.
Southern Blot Analysis. Integration of foreign genes into the
chloroplast genome and cell copy number was determined by
Southern blot analysis. Total plant DNA from untransformed
and transgenic plants was isolated by using the cetyltrimeth-
ylammonium bromide procedure (23). Ten micrograms of
DNA per sample was digested with EcoRV and separated on
a 0.7% agarose gel, then transferred to a nylon membrane. This
process was repeated, generating a total of six blots. Two
different probes were used, one to determine foreign gene
integration (chloroplast border sequences) and the other to
estimate gene copy number (cry2Aa2). Hybridization was
performed by using 18 310
6
mCurie
32
P-labeled DNA (24).
Immunoblot Analysis. To confirm cry2Aa2 expression in
leaf tissue, Western blot analysis was done. Proteins were
extracted from 100 mg of leaf material (excluding veins and
leaf midrib). Fresh tissue was homogenized in extraction buffer
(50 mM TriszHCl, pH 8.0
y
2% SDS
y
2mM
b
-mercaptoetha-
nol
y
0.1 mg/ml phenylmethylsulfonyl fluoride). To determine
expression levels, solubilized Cry2Aa2 (0.0002–2.0
m
g), ex-
pressed and purified from E. coli, was used for comparisons
(15). Protein concentrations were determined by using the
Bradford assay with BSA as the protein standard (Bio-Rad)
(25). Ten micrograms of total soluble protein was loaded per
lane and electrophoresed in a 10% SDS
y
PAGE gel. Proteins
were transferred to a nylon membrane and incubated with
Cry2A polyclonal antibodies (1:25,000 dilution) (26). Alkaline
phosphatase-conjugated goat anti-rabbit IgG (Bio-Rad) was
used (1:10,000 dilution). The Cry2Aa2 concentration from leaf
material was determined by comparing the intensity of 65-kDa
bands by using QUANTISCAN gel scanning software (Biosoft,
Milltown, NJ).
Insect Bioassays. Bioassays were conducted either against
resistant and susceptible H. virescens or against H. zea and
Spodoptera exigua (Hu¨bner). All H. virescens were obtained
from the laboratory of F.G. and H. zea and S. exigua from the
laboratory of W.J.M. H. virescens colonies were: YDK (sus-
ceptible); YHD
2
1000MVP, a H. virescens strain (YHD2) that
has been further selected for resistance to Cry1Ac (20,000- to
40,000-fold) (ref. 11; F.G., unpublished data); and CxC
1000IIA, a Cry1Ac-resistant H. virescens strain that was se-
lected for Cry2Aa2 cross-resistance and exhibits up to a
393-fold resistance to Cry2Aa2 (ref. 9; data presented below).
The CxC 1000IIA line was derived from the CP73 strain (9).
The CP73 strain was crossed to a susceptible strain (CPN) and
then selected on artificial diet containing Cry2Aa2 protoxin.
At the time of experiments reported here the CxC strain had
been selected on Cry2Aa2 for more than 24 generations. The
current selection regime involved placing neonate larvae on
diets containing 200 or 1,000
m
g
y
ml of Cry2Aa2 (15). Larvae
surviving after 5–7 days always were transferred to nontoxic
diets for further rearing. Bioassays conducted with Cry2Aa2 in
August 1998 in F.G.’s laboratory showed a resistant ratio (RR)
of 393 between YDK and CxC 1000IIA [LC
50
(
m
g
y
ml) for
YDK 52.15 (95%FL 51.23–3.06) slope 53.30; LC
50
(
m
g
y
ml)
for CxC 1000IIA 5843 (95%FL 5535-1960) slope 51.87].
Similar bioassays with Cry1Ac gave a RR of 104 between YDK
andCXC1000IIA [LC
50
(
m
g
y
ml) for YDK 50.13 (95%FL 5
0.08–0.22) slope 51.76; LC
50
(
m
g
y
ml) for CxC 1000IIA 5
13.4 (95%FL 57.38–23.06) slope 51.14], where FL is fiducial
limits.
H. virescens were subjected to artificial diet-incorporation
bioassays (27) concurrently with leaf-disc bioassays to confirm
susceptibility and resistance. Cry1Ac and Cry2Aa2 were pro-
duced as single proteins in E. coli (28). Protein concentrations
used against H. virescens were: YDK, Cry1Ac (1
m
g
y
g) and
Cry2Aa2 (1.0, 2.0, 2.5, 5.0, 10.0, and 25.0
m
g
y
g); YHD
2
1000MVP, Cry1Ac (1.0, 5.0, 10.0, 25.0, and 50.0
m
g
y
g), and
Cry2Aa2 (1.0, 2.0, 3.5, 5.0, 10.0, 25.0, 50.0, 100.0, 200.0, and
400.0
m
g
y
g); CxC 1000IIA, Cry1Ac (1.0, 5.0, 10.0, 25.0, and
50.0
m
g
y
g), and Cry2Aa2 (25.0, 50.0, 100.0, 250.0, 500.0, and
1,000.0
m
g
y
g). Mortality was assessed after 5 days. Bioassays
were conducted at least twice for all treatments. Data were
analyzed by using POLO (29). RR’s were generated by dividing
the LC
50
’s of the resistant colony by the LC
50
of YDK. When
the highest concentration tested resulted in less than 50%
mortality, the highest concentration tested was then used as
the LC
50
for generating RR.
Leaf bioassays were conducted on about 2-cm
2
excised leaf
material and placed on distilled water-soaked cardboard lids in
50 312-mm plastic Petri dishes with tight-fitting lids. Five to
10 neonates were assayed per sample, with two samples per
treatment, and evaluated daily for mortality for 5 days. Treat-
ments were replicated at least twice, but 4 –5 times in most
cases. Preliminary leaf-disc bioassays also were performed by
using transformed leaf tissue containing the aadA gene prod-
uct, but without Cry2Aa2, to confirm that no insecticidal
activity could be attributed to the transformed plant in the
absence of Cry2Aa2.
RESULTS
Construction of a Chloroplast Expression Vector and Plant
Transformation. The plasmid pZS-197 contains the chimeric
Agricultural Sciences: Kota et al.Proc. Natl. Acad. Sci. USA 96 (1999) 1841
aminoglycoside 39-adenyltransferase (aadA) gene that confers
resistance to spectinomycin-streptomycin and integrates
gene(s) of interest into the large single copy region of the
tobacco chloroplast genome at the intergenic spacer region
between rbcL and accD (20). Integration occurs through two
homologous recombination events between the chloroplast
border sequences and the corresponding homologous se-
quences of the chloroplast genome (20). The aadA and
cry2Aa2 genes in pZS-KM-cry2A are driven by the chloroplast
promoter Prrn, and transcription is terminated by psbA39
untranslated region. Prrn is a constitutive promoter of the
rRNA operon, and psbA39untranslated region functions as a
strong terminator. Because of similar protein synthetic ma-
chinery between chloroplasts and E. coli (30), cry2Aa2 expres-
sion in pZS-KM-cry2A was analyzed by Western blots using
Cry2A antibodies. This analysis showed the presence of the
65-kDa Cry2Aa2 protein (data not shown). Tobacco leaves
then were bombarded with pZS-KM-cry2A DNA-coated tung-
sten particles and placed on RMOP medium (21), with no
antibiotic selection pressure, for 2 days. This procedure was to
allow for aminoglycoside 39-adenyltransferase accumulation in
tissues so as to exhibit phenotypic resistance when placed on
RMOP medium containing spectinomycin dihydrochloride
(500 mg
y
liter), after day 2. The 5-mm
2
bombarded leaf pieces
placed on the selection medium enlarged in size and were
bleached of all color. Green calli (spectinomycin resistant)
emerged from underneath (bombarded surface) the bleached
calli. Green calli later grew into green shoots with leaf lets.
Thirteen putative transformants were obtained from 13 bom-
barded leaves in experiment 1 and three from experiment 2.
Foreign Gene Integration. The strategy used to determine
integration of the foreign gene specifically into the chloroplast
genome was two-tiered. Initially putative transgenics were
screened by using PCR. Primers were designed to confirm
incorporation of the gene cassette into the chloroplast genome.
The primer on the plus strand, designated rbcL59, landed on
the chloroplast genome upstream of the chloroplast border
used for homologous recombination (refer to Fig. 1A). The
rbcL59box represents the primer sequence, and the perpen-
dicular dotted line is the region on the native chloroplast
genome where this primer lands. The minus strand primer
lands on aadA. In Fig. 1Alanding of this primer is represented
by the aadA box (the perpendicular dotted line represents the
region on aadA where the primer lands). The rationale for this
strategy is that there can be no 2.1-kb PCR product unless
there is site-specific integration between rbcL and accD (Fig.
1A, the thick dotted lines show the site of integration in the
tobacco chloroplast genome). Therefore, the presence of a
2.1-kb PCR product in three of the 16 putative transgenics
confirmed the site-specific integration of the heterologous
gene cassette. To investigate the possibility of random priming,
the homology of rbcL59and aadA primers to all known gene
sequences was checked in all existing databases; no homology
was observed for the aadA primer, and homology to the rbcL
primers was observed only in the chloroplast genome. There-
fore, no random PCR product was predicted by this analysis,
as confirmed by the lack of PCR products in the untrans-
formed plants.
The three positive clones were further analyzed with South-
ern hybridization to further confirm the site-specific integra-
tion of cry2Aa2 and to establish copy number. In the chloro-
plast genome, EcoRV sites flank the chloroplast border se-
quence 59of rbcL and 39of accD (Fig. 1A), which generates a
3-kb fragment when digested with EcoRV. A transformed
chloroplast genome has the foreign gene cassette inserted
between rbcL and accD, which increases the size of the
EcoRV-digested fragment to 6 kb (Fig. 1, the thick dotted lines
represent the site of integration).
Total DNA from each clone and untransformed tobacco
were digested with EcoRV, and two blots were made from
these samples (Fig. 2). The blot shown in Fig. 2Awas probed
with a
32
P random primer-labeled 2.9-kb rbcL-aacD DNA
fragment. The probe hybridized with the control as expected,
lighting up a 3-kb fragment, characteristic of all untransformed
chloroplast genomes. For clones 2 and 5, only a 6-kb fragment
was generated, showing that all plastid genomes had the gene
cassette inserted between rbcL and accD. The number of
chloroplast genomes per cell is between 5,000 and 10,000. By
establishing that digestion of the chloroplast genomes of clones
2 and 5 generated only a 6-kb fragment, we confirmed
homoplasmy (identical plastid genomes) and simultaneously
determined the cry2Aa2 copy number to be between 5,000 and
10,000. This observation explains the high levels of protoxin
observed in our transgenic tobacco plants. In clone 7 the probe
hybridized with a 3-kb and an anomalous smaller fragment,
indicating heteroplasmy and rearrangement, resulting in par-
tial deletion of the expression cassette. The blot shown in Fig.
2Bwas probed with a
32
P random primer-labeled BglII–
HindIII gene fragment from cry2Aa2. The control plant
showed no hybridizing fragments (cry2Aa2 not present in
tobacco), nor did clone 7 (confirming deletion). Clones 2 and
5 showed presence of the gene. This observation was supported
by subsequent leaf bioassays where clones 2 and 5, but not
clone 7, were toxic to H. virescens.
Protein Expression. To confirm and quantify cry2Aa2 ex-
pression, total soluble protein obtained from transformed and
nontransformed leaves was subjected to Western blot analysis.
From the results obtained by using titration curves of E.
coli-expressed Cry2Aa2 and gel scanning, Cry2Aa2 is ex-
pressed between 2% and 3% of total soluble leaf protein
(Fig. 3).
FIG.1. (A) Structure of the chloroplast genome with the site of
foreign integration represented by a shaded dotted line. (B) PCR
analysis to screen for chloroplast transformants. Primers used: rbcL59
region and aadA. Lanes 1 and 8, 1-kb ladder; template DNA from lane
2, nontransformed tobacco, lanes 3– 6, clones 2, 5, 7 and 8, respectively;
and lane 7, plasmid pZS-KM-cry2A.
1842 Agricultural Sciences: Kota et al.Proc. Natl. Acad. Sci. USA 96 (1999)
Insect Bioassays. To test the toxicity of transformed tissue
to insects, preliminary leaf-disc bioassays were conducted with
leaflets collected from first- and second-generation clones.
Clones 2 and 5, but not clone 7, were toxic to lepidopteran
larvae, (in agreement with Southern blot analysis, data not
shown). Additionally, leaves containing pZS-197, (pZS-KM-
cry2A minus cry2Aa2) were nontoxic to lepidoptera.
Diet-incorporation bioassays were conducted concurrently
to confirm the susceptibility reported for the various insect
species and strains and to ensure that Bt-resistant genotypes
also were phenotypically resistant to Bt. Because insects and Bt
proteins were limited in availability, methodologies for some
bioassays were reduced. Bioassays conducted against H. vire-
scens confirmed that YDK was susceptible to Cry1Ac and
Cry2Aa2 at concentrations similar to those previously re-
ported [100% mortality observed by using Cry1Ac (1
m
g
y
ml);
Cry2Aa2 LC
50
(
m
g
y
ml) 53.03 (95%FL 50.8–5.41) slope 5
1.42 (SEM 50.235) n5240]; YHD2 1000MVP was highly
resistant to Cry1Ac and marginally resistant to Cry2Aa2 (0%
mortality observed at 50
m
g
y
ml Cry1Ac; Cry2Aa2 LC
50
(
m
g
y
ml) 5138 (95%FL 5N
y
A) slope 51.64 (SEM 50.251)
n5241]; and CxC 1000IIA was highly resistant to both Cry1Ac
and Cry2Aa2 [0% mortality observed at 50
m
g
y
ml Cry1Ac;
0% mortality obser ved at 1,000
m
g
y
ml Cry2Aa2) (Tables 1 and
2). Based on these results, RR’s were generated for both
YHD2 andCXC1000IIA (Tables 1 and 2).
Plant Tests. H. virescens. There was 100% mortality of
neonate YDK feeding on clone 2 leaves, and the leaf pieces
were essentially intact, whereas the control leaf pieces were
completely devoured (Fig. 4). Similar results were obtained
with YHD
2
1000MVP, and CxC 1000IIA (Fig. 4.).
Bioassays also were conducted by using YDK and CxC
1000IIA that were reared on control leaves or artificial diet for
5 days (about 2nd–3rd instar), and then moved to transgenic
leaves. Even these older larvae that are more tolerant than
neonates (31) showed 100% mortality (data not shown).
Other insects tested. When leaves from clone 2 were fed to H.
zea and S. exigua, 100% mortality was observed, whereas there
was no mortality observed in the control, and the entire leaf
piece was eaten (Fig. 5). Although there was no detectable
feeding damage on clone 2 with H. zea, there was some leaf
damage observed with S. exigua, indicative of the high toler-
ance of this insect species to Cry2Aa2 (15, 17).
DISCUSSION
This study reports high expression levels of Cry2Aa2 protoxin
in tobacco chloroplasts. Several reasons should have contrib-
uted for such high levels of expression in comparison to those
observed by using nuclear expression. Chloroplasts are pro-
karyotic compartments inside eukaryotic plant cells, the pro-
karyotic codon composition of cry2Aa2, high copy number of
cry2Aa2 genes per cell, and the small size of the protoxin gene
(24). This level could be further increased with the doubling of
gene dosage by inserting cry2Aa2 into the inverted repeat
region of the chloroplast genome instead of the single copy
region. It is not clear whether the high cry2Aa2 expression
levels observed in chloroplasts resulted in the formation of
Table 1. Toxicity of Cry1Ac protoxins against H. virescens using
artificial diet incorporation bioassays
Insect,
H. virescens
Cry1Ac LC
50
,
m
gyml Resistance ratio
Reported* Observed Reported* Observed
YDK 0.13 ,1†NyANyA
YHD
2
1000MVP 20,000– 40,000 .50 20,000–40,000 .100
CxC 1000IIA 13.4 .50 104 .100
NyA, not available.
*See text for references.
†100% mortality observed at 1
m
gyml.
FIG.2. (A) Southern blot analysis to confirm homoplasmy. Hy-
bridization of EcoRV-digested total genomic DNA, with EcoRV
y
SstI-
digested chloroplast border flanking sequences. DNA from lane 1,
nontransformed tobacco and lanes 2– 4, DNA from clones 2, 5, and 7,
respectively. (B) Southern blot analysis to confirm chloroplast inte-
gration of cry2Aa2. Hybridization of EcoRV-digested total genomic
DNA, with BglII
y
HindIII-digested cry2Aa2 probe. Lanes 1 and 7, 1-kb
ladder; DNA from lane 2, plasmid pZS-KM-cry2A; lane 3, nontrans-
formed tobacco, and lanes 4 –6, clones 2, 5, and 7, respectively.
FIG. 3. Immunoblot analysis. Lane A, molecular weight marker;
lanes B and C, 10
m
g of total soluble leaf proteins from clone 2 and
nontransformed tobacco, respectively; lanes D-H, dilutions (0.0002,
0.002, 0.02, 0.2, and 2.0
m
g, respectively) of solubilized Cry2Aa2
protoxin.
Agricultural Sciences: Kota et al.Proc. Natl. Acad. Sci. USA 96 (1999) 1843
crystals. Studies in E. coli suggest that ORFs upstream of
cry2Aa2 (within the cry2Aa2 operon) are required for folding
Cry2Aa2 proteins to form crystals (32, 33). If crystals are
desired for enhanced stability, the entire cry2Aa2 operon
should be expressed in chloroplasts, because chloroplasts
routinely express and process polycistrons (33). Yet another
advantage of expressing insecticidal proteins in chloroplasts is
tissue specificity. Most caterpillars feed on green tissues that
are rich in chloroplasts, thereby consuming the highest level of
insecticidal protein. Several chloroplast genes are light regu-
lated and hence chloroplasts express high levels of proteins
compared with plastids in nongreen tissues.
With the successful introduction of cry2Aa2 into the chlo-
roplast genome, the high-dose strategy should be attainable for
insects such as H. virescens,H. zea, and S. exigua. This study
shows 100% mortality of both Bt-susceptible and Cry1Ac-
resistant and Cry2Aa2-resistant H. virescens. We show that
neonate insects, highly resistant to Bt, were killed by using
Bt-transgenic leaf material even though H. virescens is less
sensitive to Cry2Aa2 than Cry1Ac (1, 15). These results also
are promising when related to reports showing marginal to
high levels of cross-resistance to Cry2Aa2 (9, 11). This study
also shows 100% mortality of neonate H. zea that contrasts
with Bt cotton (Cr y1Ac) efficacy against H. zea. The inefficient
control of H. zea also might result in faster development of Bt
resistance because a moderate level of suppression (25–50%
mortality) can increase the probability of resistance develop-
ment (4, 34). In this context, plants expressing cry2Aa2 through
the chloroplast either singly, or as part of a gene-pyramid with
other insect proteins (preferably non-Bt proteins with different
modes of action), could become an invaluable tool for resis-
tance management.
Bilang and Potrykus (35) recently have discussed several
requirements for transforming chloroplasts of useful crops
(35). One of the major limitations is the lack of knowledge of
chloroplast genome sequences to locate spacer regions and
transcriptional units to target site-specific integration of for-
eign genes. For example, it is important to transform cotton
chloroplasts with cry2Aa2 for insect control. However, not
much is known about the cotton chloroplast genome. To
overcome this limitation, Daniell et al. (6) recently have
developed a universal vector that can transform any chloro-
plast genome because it integrates into a highly conserved
region.
Another limitation is the ability to regenerate plants only
from embryonic tissues in cereals and not from mesophyll cells.
Cells from embryonic tissues contain only proplastids and not
FIG. 4. Leaf bioassay of control (Left) and Cry2Aa2 chloroplast
transgenic tobacco leaves (Right) assayed against various H. virescens
strains. YDK, susceptible (Top), YHD
2
1000MVP, Cry1Ac-resistant
(Middle), and CxC 1000IIA, Cry2Aa2-resistant (Bottom). Photographs
were taken on day 4 of the assay.
FIG. 5. Leaf bioassay of control (Left) and Cry2Aa2 chloroplast
transgenic tobacco leaves (Right) assayed against H. zea (Upper) and
S. exigua (Lower). Photographs were taken on day 4 of the assay.
Table 2. Toxicity of Cry2Aa2 protoxins against H. virescens using artificial diet incorporation bioassays
Insect,
H. virescens Toxin
Cry2ALC
50
,
m
gyml Resistance ratio
Reported* Observed Reported* Observed
YDK Solubilized NyA 14.5 NyANyA
YDK Inclusion bodies 0.5–5 3.0 NyANyA
YHD
2
1000MVP Solubilized NyA 138.0 3–25 9.5
CxC 1000IIA Inclusion bodies 843 0% mortality at
1,000
m
gyml
200–393 .330
NyA, not available.
*See text for references.
1844 Agricultural Sciences: Kota et al.Proc. Natl. Acad. Sci. USA 96 (1999)
mature plastids. It has been suggested that these plastids are
smaller than the size of microprojectiles used for DNA delivery
and therefore may pose problems in transformation experi-
ments. Successful expression of chloramphenicol acetyl trans-
ferase in proplastids of NT1 cells (36) and
b
-glucuronidase in
proplastids of wheat embryos (37) via particle bombardment
suggest that particle size may not be a problem in transforming
proplastids. Some of the challenges in transforming agronom-
ically useful crops include optimization of tissue culture tech-
niques and the selection process to obtain transgenic plants via
particle bombardment, especially from nongreen tissues. Even
if homoplasmy is not obtained in the first generation, it could
be accomplished in subsequent generations by germination of
F
1
seeds under appropriate selection. Furthermore, high levels
of expression of cry2Aa2 in transgenic tobacco have not
affected growth rates, photosynthesis, chlorophyll content,
flowering, or seed setting as observed in the laboratory.
However, long-term tests using agronomically important crops
grown under field conditions are needed before validation of
this potential new methodology can be obtained.
We are grateful to P. Maliga (Rutgers University) for providing
pZS-197 and S. Clarke and C. Guda (Auburn University) for technical
assistance. This study was supported in part by the U.S. Department
of Agriculture-National Research Initiative Competitive Grants Pro-
gram Grants 93-37311, 95-02770, and 98-01853 to H.D.
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