www.czu.cz ; www.raupp.info

Current GM crops are thus far the almost exclusive domain of herbicide and
insect resistance traits. The Bt toxins used for insect control have a
narrow specificity against lepidopteran and coleopteran pests only. Yet,
aphids and thrips are highly important pests worldwide, causing severe
direct losses and transmitting devastating viruses such as Tomato Spotted
Wilt Virus (TSWV) November 2004 by Maarten Jongsma .


So far, few useful traits against aphids or thrips have been reported. The
ideal of an insecticide-free culture of GM crops like cotton or potato is,
therefore, currently compromised by the continued need in those crops to
fight sucking pests using chemical means.

At Plant Research International in Wageningen, The Netherlands, we have
identified two new types of genes to fight sucking pests. The first involves
protease inhibitors and the other involves mono- and sesquiterpene synthase
genes. Protease inhibitors interfere with protein digestion, causing stunted
growth, increased mortality, and reduced fecundity. Mono- and sesquiterpenes
act primarily as cues emitted by plants in response to insect attack. They
determine food choices and call in the help of predators and parasites to
fight the herbivore. We found that manipulation of these traits can be a
successful way of controlling major sucking insect pests such as western
flower thrips and aphids.

Protease inhibitors
Protease inhibitors were successfully applied for the first time in
transgenic tobacco in 1987 against Heliothis zea. This initiated a rush of
research to employ these commonly found genes in plants as insect resistance
traits. However, it became quite evident that overexpression of most plant
protease inhibitors was quite ineffective and was resulting at the most in a
minor slowdown of growth rate. In 1995 it was demonstrated that in response
to dietary inhibitors the insects were able to induce protease genes that
were insensitive to them. Recently, we published a detailed analysis of how
these resistant enzymes evolved from their sensitive ancestral genes. It was
clear that there was a need to find inhibitors still effective against such
"resistant" enzymes. To find a source of such inhibitors, two approaches
were proposed. In the first approach, synthetic libraries of inhibitor
variants were selected using phage display in order to generate novel
structures1. In the other approach, inhibitors from mainly the animal
kingdom were tested against the insect proteases(2,3).

The phage display method, although elegant in principle, suffered from a
lack of sufficient quantities of resistant enzymes to be used in the
selection experiments. Also, it became evident that the tertiary protein
fold of the inhibitors was more crucial than the primary amino acid sequence
in blocking inhibitors from entering the active site. Changing the folds of
proteins was not a realistic option, and, thus, the successes using phage
display remained few. Nevertheless, Ceci et al.(1) demonstrated that they
could select a chymotrypsin inhibitor (Chy8), which was five times more
effective against pea and peach aphid than the parent trypsin inhibitor
molecule MTI-2. For pea aphid the IC50 and LC50 were both around 75 ug/ml,
which translates into an expression level in plants of 0.5 ? 1% of total
protein.

The use of inhibitors from the animal kingdom proved to be an easier way of
finding novel molecules with potency against insect pests. A large range of
known cysteine and aspartic protease inhibitors was tested against aphids
and thrips. Several inhibitors appeared to be potentially useful against
these insects and, in the case of western flower thrips, were investigated
in detail. Particularly effective was a dual inhibitor from sea anemone,
called equistatin. This inhibitor represented a new class of protease
inhibitors with a novel fold that was very good at blocking both cysteine
and aspartic gut proteases of many insects and had good results in in vitro
bioassays. Upon overexpression in some plants like potato, this inhibitor,
however, was quite susceptible to cleavage by asparagine-specific plant
proteases called legumains. The combination of equistatin with a number of
different cystatins (which also act as legumain inhibitors) in the form of
fusion proteins of four to seven independent domains prevented degradation,
and in addition, proved to be much more effective against thrips than any of
the single domains. Greenhouse trials, which monitored the survival of adult
insects and the number of offspring produced during the first 14 days,
demonstrated that the multidomain transgenic potato and chrysanthemum plants
had fewer adults and 80% less offspring. From the data it was predicted that
the population would eventually die out3. In vitro assays had only found
effects on fecundity and not on adult mortality. Choice assays had, however,
indicated that protease inhibitors not only reduce the growth of larvae and
fecundity of adults, but are also strongly deterrent to adult insects in a
dose dependent fashion2. So the disappearance of the adults from their cages
in the greenhouse was explained as a result of deterrence and not mortality.
If insects even try to escape their only food source in a no-choice
situation, deterrence or repellence may prove an effective, additional way
of protecting plants against herbivores. Volatile organic compounds emitted
by plants are an interesting second strategy in that respect.

Terpene synthases
Volatile organic compounds emitted by plants are known to provide strong
cues to predators and parasites of herbivores to locate their prey. We
recently published4 that the herbivore itself is affected by these
compounds. In the article, we demonstrate that in choice assays aphids are
deterred from Arabidopsis plants that constitutively produce high levels of
linalool. Recent unpublished data further corroborate these results on other
plant species such as potato and chrysanthemum. On those plants, the
deterrence was higher, with 75% of aphids and 90% of adult thrips preferring
the control over the transgenic plants. In the next year, greenhouse and
field trials will be carried out to measure the effects of overexpression of
linalool in a realistic situation and on other insects. In the future, the
challenge will be to find the right balance between cost for the plant and
effect in terms of resistance. These aspects will also hinge on our ability
to find the most active volatiles and to manage their expression in the
right tissue and with the proper timing, just like plants have done over
millions of years.

Conclusion
Recently, the ecological roles of both monoterpenes such as linalool and
protease inhibitors on oviposition success of moths and seed set success of
plants were demonstrated in field experiments using wild tobacco species by
the group of Ian Baldwin. This has improved our understanding of the
decisive role of these genes for the survival of plants in a natural
setting. We have demonstrated that engineering these traits to make them
more effective by the selection of more active inhibitors or by promoting
the emission of higher levels of specific volatile organic compounds can
make an even stronger difference to the success of sucking insect pests on
transgenic plants.

References

1. Ceci LR, Volpicella M, Conti S, Gallerani R, Beekwilder MJ, Jongsma M.A.
(2003) Selection by phage display of a mustard chymotrypsin inhibitor toxic
to pea aphid. Plant Journal 33: 557-566.

2. Outchkourov NS, de Kogel WJ, Schuurman-de Bruin A, Abrahamson M, Jongsma
MA (2004a) Specific cysteine protease inhibitors act as deterrents of
Western flower thrips Frankliniella occidentalis (Pergande) in transgenic
potato. Plant Biotechnology Journal 2: 439-448.

3. Outchkourov NS, de Kogel WJ, Wiegers GL, Abrahamson M, and Jongsma MA.
(2004b) Engineered multidomain cysteine protease inhibitors yield resistance
against western flower thrips (Frankliniella occidentalis) in greenhouse
trials. Plant Biotechnology Journal 2: 449-458.

4. Aharoni A et al. (2003) Terpenoid metabolism in wild-type and transgenic
Arabidopsis thaliana plants. Plant Cell 15: 2866-2884.

[www.plant.wur.nl]

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