Chapter
20
Transgenic Crop Varieties Resistant Downloaded via UNIV OF ARIZONA on July 28, 2018 at 18:57:48 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
to
Insects
Mark Vaeck, Arlette Reynaerts, Herman Höfte, and Herman Van Mellaert Plant Genetic Systems N.V., J. Plateaustraat 22, B-900 Ghent, Belgium The bt2 gene from Bacillus thuringiensis berliner 1715 encodes a 130 KDa protein that i s h i g h l y toxic to lepidopteran larvae. The active toxin i s a 60 KDa p o l y p e p t i d e , d e r i v e d from the NH2-terminal half of the molecule. Truncated genes encoding the a c t i v e t o x i n and gene fusions containing 5'-fragments of bt2 fused to the neomycin phosphotransferase gene (neo) of Tn5 were constructed. The l a t t e r encode stable fusion p r o t e i n s which are t o x i c and which e x h i b i t specific NPTII enzyme a c t i v i t y . Chimeric genes consisting of these modified toxin genes, flanked by regulatory sequences, were t r a n s f e r r e d to tomato, potato and tobacco p l a n t s , u s i n g Agrobacterium vectors. Transgenic plants expressed sufficiently high levels of active t o x i n to be protected from feeding damage caused by insect larvae. The insect resistance t r a i t was s t a b l y i n h e r i t e d i n subsequent generations.
Commercial preparations of B.t., obtained through conventional fermentation techniques, have been used for more than two decades as biological insecticides (I). They exhibit desirable properties such as high insect t o x i c i t y and environmental safety. B . t . does not affect n o n - t a r g e t i n s e c t s and i s completely nontoxic to vertebrates. Nevertheless i t s use has been limited due to high production costs, limited s t a b i l i t y i n field conditions and a too narrow i n s e c t i c i d a l spectrum. The i n s e c t i c i d a l a c t i v i t y of B . t . r e l i e s i n the c r y s t a l l i n e inclusions which are produced upon sporulation. The crystals contain i n s e c t i c i d a l proteins, delta-endotoxins, which affect the midgut epithelium of sensitive insects. The exact mechanism of t h e i r t o x i c a c t i v i t y i s s t i l l unknown. 0097-6156/88/0379-0280$06.00/0 * 1988 American Chemical Society
Hedin et al.; Biotechnology for Crop Protection ACS Symposium Series; American Chemical Society: Washington, DC, 1988.
20. V A E C K E T A L .
Crop Varieties Resistant to Insects
Most B . t . s t r a i n s are s e l e c t i v e l y t o x i c towards a v a r i e t y of l e p i d o p t e r a n l a r v a e . One B . t . s u b s p e c i e s , c a l l e d i s r a e l e n s i s , i s h i g h l y t o x i c t o d i p t e r a n l a r v a e (21 a n d i s u s e d f o r t h e c o n t r o l of m o s q u i t o e s and b l a c k f l i e s . R e c e n t l y , a B . t . s u b s p e c i e s tenebrionis. e x p r e s s i n g t o x i c i t y towards C o l e o p t e r a , has been i s o l a t e d (3). With t h e a d v e n t of b i o t e c h n o l o g y , i n t e r e s t i n B . t . h a s g r e a t l y i n c r e a s e d . U s i n g new p l a n t v e c t o r systems, f o r e i g n g e n e s can be t r a n s f e r r e d and e x p r e s s e d i n t o p l a n t s . I n t h i s p a p e r we p r e s e n t some d a t a on t h e s u c c e s f u l i n t r o d u c t i o n of B . t . g e n e s i n different crop species. The B . t . genes are e x p r e s s e d at i n s e c t c o n t r o l l i n g l e v e l s and p r o v i d e the t r a n s g e n i c p l a n t s w i t h a defense mechanism a g a i n s t d e v a s t a t i n g i n s e c t s . Results
and
Discussion
E x p r e s s i o n of B . t . G e n e s i n T r a n s g e n i c P l a n t s . We h a v e u s e d Agrobacterium mediated T-DNA t r a n s f e r t o e x p r e s s s e v e r a l m o d i f i e d genes, d e r i v e d from t h e L e p i d o p t e r a s p e c i f i c B t 2 g e n e i n tomato, t o b a c c o and p o t a t o . P a r t i c u l a r l y s u c c e s s f u l was t h e e x p r e s s i o n of genes t h a t c o n t a i n e d t h e N H 2 - t e r m i n a l h a l f o f b t 2 f u s e d t o t h e neo gene. These encode f u s i o n p r o t e i n s t h a t e x h i b i t b o t h i n s e c t t o x i c i t y and n e o m y c i n p h o s p h o t r a n s f e r a s e a c t i v i t y . T h e l a t t e r was u s e d t o s e l e c t , t h r o u g h s e l e c t i o n f o r h i g h kanamycin r e s i s t a n c e , c e l l s t h a t e x p r e s s e d s u b s t a n t i a l amounts of i n s e c t i c i d a l p r o t e i n (4). H i g h t o x i c i t y r e s u l t i n g i n 80-100% m o r t a l i t y o f M . s e x t a l a r v a e , was o b s e r v e d i n t o b a c c o p l a n t s e x p r e s s i n g f u s i o n p r o t e i n s or a truncated b t 2 gene ( T a b l e I ) . None of t h e p l a n t s transformed with the f u l l length bt2 gene p r o d u c e d i n s e c t k i l l i n g a c t i v i t y . A l s o tomato and p o t a t o p l a n t s t r a n s f o r m e d w i t h a f u s i o n gene o r a t r u n c a t e d bt2 gene e x h i b i t e d s i g n i f i c a n t i n s e c t i c i d a l a c t i v i t y . Greenhouse e x p e r i m e n t s r e v e a l e d t h a t t h e o b t a i n e d t r a n s g e n i c p l a n t s were p r o t e c t e d a g a i n s t i n s e c t f e e d i n g damage. They showed v e r y l i m i t e d damage, r e s t r i c t e d t o f e e d i n g a r e a s of a few mm , whereas n o n t r a n s f o r m e d c o n t r o l p l a n t s w e r e e n t i r e l y c o n s u m e d w i t h i n 10 d a y s . B . t . p r o t e i n l e v e l s i n t h e i n s e c t r e s i s t a n t p l a n t s r a n g e d from 7 t o 40 n g / m g t o t a l p r o t e i n c o n t e n t . The d a t a are summarized i n T a b l e I. I n h e r i t a n c e of t h e I n s e c t R e s i s t a n c e T r a i t . Copy numbers o r t h e T-DNA i n s e r t s i n t r a n s g e n i c p l a n t s were d e t e r m i n e d u s i n g Southern b l o t t i n g . P l a n t s e x p r e s s i n g highest l e v e l s of B . t . p r o t e i n c o n t a i n e d a r o u n d 5 c o p i e s of T - D N A . From one s u c h p l a n t , 15 F1 p r o g e n y were a s s a y e d and t h e y a l l e x h i b i t e d 100% i n s e c t killing. We a l s o a n a l y s e d i n h e r i t a n c e o f t h e new t r a i t i n p l a n t s w i t h one o r two T-DNA i n s e r t s . Km r e s i s t a n c e a n d i n s e c t i c i d a l a c t i v i t y were l i n k e d and the i n h e r i t a n c e p a t t e r n s were f u l l y compatible with simple Mendelian r u l e s for a dominant t r a i t . S e l e c t i o n of I n s e c t i c i d a l Genes. Although i n the p r e s e n t l y generated transgenic plants, B.t. protein levels are s u f f i c i e n t l y high to c o n t r o l M. sexta, other i n s e c t species,
Hedin et al.; Biotechnology for Crop Protection ACS Symposium Series; American Chemical Society: Washington, DC, 1988.
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BIOTECHNOLOGY FOR CROP PROTECTION
Table
Bt
I.
Insect k i l l i n g activity in transgenic plants expressing B.t.
gene
Ti plasmid
genes
P e r c e n t a g e of p l a n t s c a u s i n g a m o r t a l i t y of 0-40% 40-80% 80-100%
TOBACCO
BtrNPTII fusion Bt2 i n t a c t Bt2 t r u n c a t e d
p G S 1 1 52 p G S 1 1 61 pGS1163
24 1 00 37
20 3
18 54
30
56 0 60
0
POTATO
Bt:NPTII fusion Bt2 t r u n c a t e d
p G S 1 1 52 pGS1163
38
44 16
M o r t a l i t y i n f i r s t i n s t a r M. s e x t a l a r v a e i s d e t e r m i n e d day f e e d i n g assay
in a six
The r e c o m b i n a n t T i p l a s m i d s a r e d e s c r i b e d i n V a e c k e t
a l . , 1987.
w h i c h a r e l e s s s e n s i t i v e t o Bt2 t o x i n , may need h i g h e r l e v e l s o f e x p r e s s i o n t o be e f f e c t i v e l y c o n t r o l l e d . O r a l t e r n a t i v e l y , o n e may c o n s i d e r t h e u s e o f o t h e r B . t . t o x i n s more a c t i v e a g a i n s t such insects. Even within the l e p i d o p t e r a n p a t h o t y p e , l a r g e d i f f e r e n c e s i n the i n s e c t i c i d a l s p e c t r a of B . t . t o x i n s e x i s t . F o r example, s c r e e n i n g of a l a r g e c o l l e c t i o n o f B . t . s t r a i n s h a s l e a d t o t h e i d e n t i f i c a t i o n of s t r a i n s h i g h l y t o x i c t o S p o d o p t e r a l i t t o r a l i s , an i n s e c t w h i c h i s i n s e n s i t i v e t o most c o m m o n l y k n o w n B . t . strains. On t h e b a s i s o f i n s e c t i c i d a l s p e c t r u m a n d s i z e o f t h e t o x i c polypeptide fragment, we p r e s e n t l y d e f i n e d 3 t y p e s o f lepidopteran-active B.t. toxins. Bt2 i s a n e x a m p l e o f a t y p e 1 d e l t a - e n d o t o x i n . I t h a s a r e l a t i v e l y b r o a d spectrum of i n s e c t i c i d a l a c t i v i t y , b u t i s n e a r l y n o n t o x i c t o S. l i t t o r a l i s . A t y p e 2 t o x i n i s f o u n d i n p u r e form i n t h e c r y s t a l s of B . t . t h u r i n g i e n s i s s t r a i n 4412 a n d i s t o x i c t o P i e r i s b r a s s i c a e b u t n o t t o M . s e x t a a n d S. l i t t o r a l i s . A n example of a t y p e 3 t o x i n i s p r e s e n t t o g e t h e r w i t h a t y p e 1 t o x i n i n B . t . a i z a w a i and i s c h a r a c t e r i z e d b y h i g h t o x i c i t y t o S. l i t t o r a l i s .
Hedin et al.; Biotechnology for Crop Protection ACS Symposium Series; American Chemical Society: Washington, DC, 1988.
20. VAECKETAL,
Crop Varieties Resistant to Insects
Specific monoclonal antibodies were generated against these three types of delta-endotoxins. These antibodies provide an efficient tool for very rapid immunological screening of large numbers of B . t . s t r a i n s . In a random set of 28 B.t. s t r a i n s tested with monoclonal antibodies specific for type 3 toxin, only 8 showed positive reaction. In bioassay only the same 8 strains, and only these, exhibited toxicity to S. l i t t o r a l i s . Thus a perfect correlation exists between the antigenic structure of B.t. toxins and t h e i r i n s e c t i c i d a l spectrum. This approach is now further refined and w i l l be extended to the selection and cloning of various new B.t. genes. This will be an essential step in the process towards the engineering of resistance against whole insect complexes in a g r i c u l t u r a l l y important crop species. L i t e r a t u r e Cited 1. Dulmage, H.T. Insecticidal activity of HD-1, a new isolate of Bacillus thuringiensis var. a l e s t i . J . I n v e r t e b r . P a t h o l . 1970, 15, 232-239. 2. Goldberg, L . J . and M a r g a l i t , J . A b a c t e r i a l spore demonstrating rapid l a r v i c i d a l a c t i v i t y against Anopheles sergentii, Uranotaenia unguiculata, Culex univitattus, Aedes aegypti, Culex pipiens. Mosquito News., 1977, 37, 355-358 3. Krieg, A.; Huger, A.M.; Langenbruch, G.A.; Schnetter, W. Bacillus thuringiensis var. tenebrionis : ein neuer gegenüber Larven von Coleopteren wirksamer Pathotyp, Z. Ang. Ent., 1983, 96, 500-508. 4. Vaeck, M.; Reynaerts, A.; Höfte, H.; Janssens, S.; De Beuckeleer, M.; Dean, C.; Zabeau, M.; Van Montagu, M.; Leemans, J. Transgenic plants protected from insect attack, Nature, 1987, 327, 33-37. RECEIVED April 1, 1988
Hedin et al.; Biotechnology for Crop Protection ACS Symposium Series; American Chemical Society: Washington, DC, 1988.
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