ISB NEWS REPORT JULY 2003

TRANSGENIC POTATOES RESISTANT TO COLEOPTERAN AND LEPIDOPTERAN PESTS

THROUGH THE USE OF SINGLE HYBRID BACILLUS THURINGIENSIS CRY GENE
Ruud A. de Maagd and Samir Naimov

Bacillus thuringiensis (Bt) is a bacterium commonly found in soil and on plant surfaces. What makes the
bacterium interesting for agricultural research is that many of its strains kill insect pests. It does so primarily
because during the formation of its spores, which help the bacterium to survive adverse conditions, it forms one
or more insecticidal proteins enclosed in a crystal. The crystal (Cry) proteins, when ingested by insect larvae,
dissolve in the insect’s midgut fluid, undergo a process of activation by gut enzymes, and subsequently bind to
receptors on the cells lining the gut. This is followed by the formation of pores in the membranes of those cells,
killing them, which eventually leads to the death of the insect1. Sprays based on Bt crystals and spores have a
long history of safe use in agriculture. More recently, transgenic crops such as corn, cotton, and potato
expresing a Cry protein (Bt crops), rendering them resistant to one or several pests, have been commercialized.

One of the big advantages of Cry proteins is sometimes also a drawback: specificity. Cry proteins form a large
family of proteins, similar in overall structure, but differing in details that determine their activity for particular
insect species. Each Cry protein is active against only one or a few pest species, usually of the same insect
order (Lepidoptera: caterpillars; Diptera: larvae of flies and mosquitoes; Coleoptera: larvae of beetles and
weevils). While this helps to reduce non-target effects of sprays or Bt crops, it limits the utility of such an
application. Additionally, the use of a single yet effective toxin in a transgenic crop leads to a high selection
pressure on the insect population that, it is feared, may then rapidly develop resistance to the toxin. Although
this resistance has not yet been seen in the field since the introduction of Bt crops, strategies for delaying or
preventing the occurrence of resistance (resistance management) are deemed necessary. One of these strategies
is the simultaneous expression of two toxins that recognize different receptors, so that the insect population
would have to lose two, not one, receptors to become resistant to both toxins. In our laboratories, we are
studying the mode of action of Cry proteins and the factors that determine specificity, and applying this knowl-
edge to the production of toxins that are more effective or have a broader spectrum, or may be used for resis-
tance management strategies.

Essential for the understanding of specificity is the relation between structure and function of the Cry proteins.
The Cry proteins used in our study probably all have the same overall three-dimensional structure shown in
Fig. 1. The proteins consist of three structural domains, of which the first domain is thought to be responsible
for forming the pores in insect cell membranes. The second and third domains function in receptor recognition
and are thereby in a large part responsible for determining specificity. How these two domains act together in
receptor recognition is not well understood, but several groups, including ourselves, recognize that “swapping”
single domains between toxins having different activity or specificity may result in “hybrid” or “chimeric”
toxins with improved activity or target spectrum. As screening of Bt strains for new Cry proteins proceeds and
different toxins are found, it is becoming increasingly clear that recombination between cry genes resulting in a
domain swap is in fact likely to be one of the mechanisms that generated the diversity of toxins in Nature. In
the laboratory, we are mimicking this process by inducing recombination between genes of our choice and
screening for hybrid toxins with interesting properties.

In more recent research, we focused our attention on toxins that are active against Colorado potato beetle
(CPB), a coleopteran pest of potatoes in both North America and (particularly Eastern) Europe. The
mostactive known Cry protein for CPB is Cry3Aa, which has been utilized against this pest in sprays as well
as in transgenic plants (e.g., NewLeaf potatoes from Monsanto Company). Two proteins of the Cry1 subfam-
ily, Cry1Ba and Cry1Ia, which are relatively unrelated to Cry3Aa, are primarily active against Lepidopterans
and have been shown to have some, albeit low, activity against CPB (see Fig. 2). To test our hypothesis that a
combination of domains from two weakly active toxins, in this case Cry1Ba and Cry1Ia, may form a more
active toxin, we swapped restriction enzyme fragments of the two genes corresponding to the parts encoding

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Figure. 1. Schematical representation of the three-dimensional structure of a typical Bacillus thuringiensis Cry protein, showing its three
structural domains (I-III).

domain III of the active toxins2. Toxins were expressed in Escherichia coli, which is easier to handle in the
laboratory. Typical of such experiments, some of the hybrid genes did not express much protein or gave
proteins that were unstable, yet some could be purified in sufficient amounts for bioassays on CPB larvae using
potato leaves dipped in toxin solution. We determined the concentrations of Cry3Aa, the parental Cry1 toxins,
and the hybrid toxins giving 50% mortality (LC50) in newly hatched CPB larvae. One of the hybrid proteins,
SN15, consisting of domains I and II of Cry1Ia and domain III of Cry1Ba, had considerably higher activity
(lower LC50) than both parent toxins (Fig. 2). SN15 was not expressed very efficiently, suggesting that its
stability is not optimal. In an attempt to improve this, we replaced the first domain of SN15 with that of
Cry1Ba as well. The resulting mosaic toxin SN19 not only was expressed at higher levels, but also had even
higher activity against CPB than SN15. On a molar basis, SN19 activity against CPB larvae is near (42%) to
that of the most active known toxin, Cry3Aa. This result showed that domain swappingmight improve toxin

Figure 2. Domain compositions and activity against neonate CPB larvae of wild type and hybrid toxins, with Cry3Aa shown for comparison.
The three structural domains of the active toxin as shown in Fig. 1 are depicted in light gray shades. The dark gray areas are parts of the so-
called protoxin but are removed during activation. LC50 is the concentration (µg per ml of leaf dipping solution) giving 50% mortality. Relative
toxicity is on per mol basis, with Cry3Aa activity set at 100.

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activity, not only for lepidopterans as shown before, but also for coleopterans like CPB.

To test the actual utility of SN19 for CPB control, we transferred the encoding gene back to Bt cells as well as
used it for transformation of potato plants. Results of the latter experiments were reported earlier this year3.
Cry genes, of obviously bacterial origin, are not expressed very well in transgenic plants without extensive
modifications both in the transcription regulation sequences (promoter and terminator) as well as in the protein
coding sequence. Starting from a synthetic, plant-optimized cry1Ba gene, we replaced the domain II encoding
region with that of Cry1Ia, either with or without extra modification for expression in plants. For transcription
signals we used a chrysanthemum Rubisco small subunit-promoter and terminator isolated in our laboratory,
which had been shown previously to give high expression levels in green tissues of various plant species4.
Potato cultivar Desiree plants were transformed with one of the two gene constructs, or an empty vector as a
negative control, via Agrobacterium tumefaciens-mediated gene transfer. Not surprisingly, without modifica-
tion of the domain II encoding part, there was no detectable expression of SN19 in transgenic plants. With the
modifications, several transgenic plants with expression levels up to 0.25% of total soluble leaf proteins were
obtained. Detached leaves of transgenic plants were infested with 10 neonate CPB larvae to test their resis-
tance. All plants expressing SN19 at 0.2% or higher were fully resistant to CPB larvae, giving 100% mortality
and no detectable damage on the leaves. Not only CPB larvae, but also the adults are voracious pests on potato,
and older larvae or adults usually require much higher doses of Bt toxins to be killed. Our transgenic SN19-
potato leaves with the high expression levels were also fully resistant to attack by adult CPB; however, not all
CPB adults were killed during the timeframe of the experiment. The insect simply would barely feed on the
leaves, resulting in minimal damage, and not grow as a result.

As mentioned earlier, the parental toxins of SN19, Cry1Ba and Cry1Ia, are known more for their activity
against lepidopterans (caterpillars, larvae of moths and butterflies) including, for both toxins, European corn
borer (ECB) and potato tuber moth larvae (PTM). Although the name implies differently, ECB can be an
occasional pest on potato in North America, where it damages the plant by boring into the stems. PTM is a
more tropical pest, infesting and spoiling stored potato tubers as well as infesting plants in the fields where the
larvae make tunnels inside the leaf tissue. We speculated that a hybrid of theseproteins might well have retained
these properties and tested SN19-potato and control leaves with ECB and PTM larvae. SN19 in transgenic
leaves gave full protection and 100% mortality for both species. Thus, we have produced the first transgenic Bt
crop with resistance to insects from two different orders, Lepidoptera and Coleoptera, conferred by a single gene.

Potato may rarely, if ever, encounter all three mentioned pests in the same field, but two out of three of these
are not uncommon. SN19, with good activity against CPB as shown in our studies, may well be a good alterna-
tive for (or beside) Cry3Aa against CPB in resistance management strategies. This presumption, however,
awaits further experimental confirmation that SN19 and Cry3Aa recognize different receptors in this insect.
Similarly, SN19 could be an alternative for Cry1Ab in corn, currently in use for ECB control. The use of SN19
in a Bt spray, preferably with other active toxins like Cry1Ab and Cry3Aa, could yield a product with a broad
activity spectrum as well as built-in resistance management. We are currently testing the utility of SN19 in Bt
spore/crystal-mixtures. We conclude from this and previous work that, in the future, domain swapping may be
an effective approach to increase the utility of Bt toxins in agriculture, whether in transgenic crops or in sprays.

References
1. de Maagd RA, Bravo A, and Crickmore N. (2001) How Bacillus thuringiensis has evolved specific toxins to
colonize the insect world. Trends Genet 17: 193-199.

2. Naimov S, Weemen-Hendriks M, Dukiandjiev S, and de Maagd RA. (2001) Bacillus thuringiensis delta-endotoxin
Cry1 hybrid proteins with increased activity against the Colorado potato beetle. Appl Environ Microbiol 67: 5328-5330.

3. Naimov S, Dukiandjiev S, and de Maagd RA. (2003) A hybrid Bacillus thuringiensis delta-endotoxin gene gives
resistance against a coleopteran and a lepidopteran pest in transgenic potato. Plant Biotechnol J 1: 51-57.

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4. Outchkourov NS, Peters J, de Jong J, Rademakers W, and Jongsma MA. (2003) Novel rubisco small subunit
promoter from chrysanthemum (Dendranthema grandiflora L) yields very high foreign gene expression levels in
plants. Planta 21: 1003-1012.

Ruud A. de Maagd Samir Naimov

Business Unit Bioscience Dept of Plant Phys and Mol Biol
Plant Research Intl Univ of Plovdiv
The Netherlands Bulgaria
ruud.demaagd@wur.nl naimov0@pu.acad.bg

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