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Chapter 2

Molecular Biology of Resistance to Sulfonylurea Herbicides Downloaded via UNIV OF CALIFORNIA SANTA BARBARA on July 20, 2018 at 15:19:03 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

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Julie K. Smith , C. Jeffry Mauvais, Susan Knowlton, and Barbara J. Mazur Agricultural Biotechnology Division, Experimental Station, Ε. I. du Pont de Nemours and Company, Wilmington, DE 19898 The sulfonylureas, an extremely potent class of herbicides, act by inhibiting acetolactate synthase (ALS), which is the first common enzyme in the biosynthetic pathways leading to the branched chain amino acids. Two other unrelated classes of herbicides also act by interfering with this enzyme. We have cloned and characterized the genes encoding ALS from several higher plants. The ALS genes isolated from herbicide sensitive and herbicide resistant plants have been compared, and several mutations which confer the herbicide resistant phenotype have been identified. Cloned herbicide resistant ALS genes have been used to transform both homologous and heterologous plant species. ALS genes can be modified in vitro in order to achieve selective resistance toward broad or narrow classes of inhibitors. The modified genes can be introduced into a variety of commercial crops.

The s u l f o n y l u r e a h e r b i c i d e s a r e a r e l a t i v e l y new c l a s s o f c r o p p r o t e c t i o n c h e m i c a l s w h i c h a r e n o t a b l e f o r t h e i r low a p p l i c a t i o n r a t e s ( t y p i c a l l y grams/hectare) and low mammalian t o x i c i t y . I n a d d i t i o n many a r e s e l e c t i v e , h a v i n g t h e a b i l i t y t o k i l l weeds without i n j u r i n g the t a r g e t crop. B a c t e r i a , f u n g i , and p l a n t s have a l l been shown t o be n a t u r a l l y s e n s i t i v e t o t h e s e compounds. The mode o f a c t i o n o f t h e s u l f o n y l u r e a s was i n i t i a l l y d i s c o v e r e d i n a m i c r o b i a l system. C e r t a i n s t r a i n s o f b a c t e r i a were a b l e t o t o l e r a t e the s u l f o n y l u r e a s when grown on r i c h media b u t n o t when grown on m i n i m a l media ( 1 ) . By s t u d y i n g t h e response o f v a r i o u s b a c t e r i a t o s u l f o n y l u r e a s i n media supplemented w i t h v a r i o u s n u t r i e n t s , i t was d e t e r m i n e d t h a t t h e s e h e r b i c i d e s a c t by i n t e r f e r i n g w i t h t h e b r a n c h e d c h a i n amino a c i d b i o s y n t h e t i c pathways. The t a r g e t enzyme was found t o be a c e t o l a c t a t e s y n t h a s e (ALS, EC 4.1.3.18) w h i c h i s the f i r s t common enzyme i n t h e pathways l e a d i n g t o l e u c i n e , Current address: Biology Department, Philadelphia College of Pharmacy and Science, Philadelphia, PA 19104 0097-6156/88/0379-0025$06.00/0 « 1988 American Chemical Society

Hedin et al.; Biotechnology for Crop Protection ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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BIOTECHNOLOGY FOR CROP PROTECTION

i s o l e u c i n e , and v a l i n e . ALS was s u b s e q u e n t l y shown t o be t h e t a r g e t o f t h e s u l f o n y l u r e a s i n y e a s t (2) and i n h i g h e r p l a n t s ( 3 , 4 ) . A n i m a l s do n o t u t i l i z e t h i s pathway, and must i n g e s t t h e b r a n c h e d c h a i n amino a c i d s i n t h e i r d i e t s . T h i s presumably c o n t r i b u t e s t o the low mammalian t o x i c i t y o f t h e s u l f o n y l u r e a s . ALS i s a l s o i n h i b i t e d by a number o f compounds w h i c h a r e s t r u c t u r a l l y u n r e l a t e d t o t h e s u l f o n y l u r e a s . These i n c l u d e two o t h e r c l a s s e s o f h e r b i c i d e s : t h e i m i d a z o l i n o n e s (5) and t h e t r i a z o l o p y r i m i d i n e s (Hawkes, T.R.; Howard, J . L . ; P o n t i n , S.E. I n H e r b i c i d e s and P l a n t M e t a b o l i s m , i n p r e s s ) . LaRossa e t a l . have s p e c u l a t e d on why ALS i s such an e f f e c t i v e t a r g e t f o r so many i n h i b i t o r s ( 6 ) . B l o c k i n g ALS l e a d s t o t h e b u i l d u p o f t h e t o x i c substrate a-ketobutyrate. The e l e v a t e d l e v e l s o f t h i s m e t a b o l i t e combined w i t h t h e reduced l e v e l s o f t h e b r a n c h e d c h a i n amino a c i d s appear t o make t h e i n h i b i t i o n o f ALS a p a r t i c u l a r l y l e t h a l event. T o l e r a n c e toward t h e s u l f o n y l u r e a s i s known t o o c c u r n a t u r a l l y due e i t h e r t o t h e p r e s e n c e o f a form o f ALS t h a t i s i n s e n s i t i v e t o the i n h i b i t o r s (7) o r t o a mechanism f o r d e t o x i f i c a t i o n o f t h e i n h i b i t o r s ( 8 ) . A n o t h e r mechanism t h a t c o u l d i n p r i n c i p l e l e a d t o t o l e r a n c e i s t h e o v e r p r o d u c t i o n o f t h e t a r g e t (ALS) enzyme. We a r e i n t e r e s t e d i n engineering h e r b i c i d e tolerance i n crop p l a n t s i n order to increase the margin o f s a f e t y f o r the a p p l i c a t i o n o f e x i s t i n g s e l e c t i v e c h e m i c a l s , t o a c h i e v e s e l e c t i v i t y i n c r o p s where s e l e c t i v e c h e m i c a l s do n o t c u r r e n t l y e x i s t , and t o reduce damage i n r o t a t e d c r o p s w h i c h i s due t o t h e p r e s e n c e o f h e r b i c i d e r e s i d u e s . As a f i r s t s t e p toward t h i s g o a l we have c h a r a c t e r i z e d t h e ALS genes from s e v e r a l h i g h e r p l a n t s , i n c l u d i n g A r a b i d o p s i s t h a i i a n a and N i c o t i a n a tabacum ( t o b a c c o ) . ALS genes have been c l o n e d from b o t h w i l d t y p e p l a n t s and from l i n e s w h i c h were s e l e c t e d t o be h e r b i c i d e r e s i s t a n t . The c l o n e d ALS genes a r e now b e i n g e n g i n e e r e d i n v i t r o and a r e b e i n g r e i n t r o d u c e d i n t o s e v e r a l c r o p s p e c i e s . C l o n i n g and C h a r a c t e r i z a t i o n o f P l a n t ALS Genes The genes e n c o d i n g each o f t h e t h r e e known isozymes o f ALS i n Ε. c o l i have been c l o n e d and sequenced (9-15). The s i n g l e ALS gene i n the y e a s t Saccharomyces c e r e v i s i a e has a l s o been c l o n e d ( 2 ) , and i t s sequence has been d e t e r m i n e d ( T 6 ) . The deduced amino a c i d sequences o f y e a s t and b a c t e r i a l ALS p r o t e i n s have been compared ( 1 6 ) . There are t h r e e b l o c k s o f h i g h sequence homology i n t e r s p e r s e d w i t h b l o c k s o f sequence t h a t a r e n o t w e l l c o n s e r v e d . The degree o f sequence c o n s e r v a t i o n i n t h e ALS enzymes from t h e s e d i v e r s e organisms s u g g e s t e d t h a t t h e ALS genes from h i g h e r p l a n t s c o u l d be i s o l a t e d u s i n g t h e method o f h e t e r o l o g o u s h y b r i d i z a t i o n . The y e a s t ALS gene was u s e d t o probe genomic l i b r a r i e s from t h e b l u e - g r e e n a l g a Anabaena and from t h e h i g h e r p l a n t s A r a b i d o p s i s and t o b a c c o . H y b r i d i z a t i o n s were c a r r i e d o u t under c o n d i t i o n s o f reduced s t r i n g e n c y , and phage c o n t a i n i n g p u t a t i v e ALS genes were i s o l a t e d i n each case ( 1 7 ) . The genes from the A r a b i d o p s i s and tobacco l i b r a r i e s were s u b c l o n e d i n p l a s m i d v e c t o r s , mapped and sequenced, and t h e i r deduced amino a c i d sequences were d e t e r m i n e d (Mazur, B . J . ; C h u i , C.-F.; Smith, J.K. P l a n t P h y s i o l . , i n p r e s s ) . N e i t h e r p l a n t gene has i n t r o n s . The genes encode p r o t e i n s o f 667 ( t o b a c c o ) o r 670 ( A r a b i d o p s i s ) amino a c i d s , w i t h p r e d i c t e d m o l e c u l a r w e i g h t s o f a p p r o x i m a t e l y 73,000 d a l t o n s . F i g u r e 1 shows a


SMITH ET AL.

Resistance to Sulfonylurea Herbicides

10 30 50 70 MAAAA..PSPSSS.AFSKTLSPSSSTSSTLLPRSTFPFPHHPHKTTPPPLHLTHTHIHIHSQRRRFTISNVISTNQKVSQ INI III II Mill I I II I I I I MAAATTTTTTSSSISFSTKPSPSSSKSPLPISRFSLPFSI1IPNKSSSSSRRRGIKSSSPSSISAVLNTTTNVTTTPSPTK I I II I I MIRQSTLKNFAIKRCFQHIAYRNTPAMRSVALAQRFYSSSSRYYSASPLPASKRPEPAPSFNVDPLEQPAEPSKL 90 110 130 150 TEKTETFVSRFAPDEPRKGSDVLVEALEREGVTDVFAYPGGASMEIHQALTRSSIIRNVLPRHEQGGVFAAEGYARATGF I III l l l l l l M i l I l l l l l l l II III I M M 11 III M M III 111111111111111111111 I PTKPETFISRFAPDQPRKGADILVEALERQGVETVFAYPGGASMEIHQALTRSSSIRNVLPRHEQGGVFAAEGYARSSGK I I I I II I III M i l l I I III M M llllll Ml AKKUUVEPDMDTSFVGLTGGQIFNEMMSRQ^DTVFGYPGGAILPVYDAIHNSDKFNFVLPKHEQGAGHMAEGYARASGK I II I I I III l l l l l l l I I llllll I I II II MASSGTTSTRKRFTGAEFIVHFLEQQGIKIVTGIPGGSILPVYDALSQSTQIRHILARHEQGAGFIAQGN^ 170 190 210 230 PGVCIATSGPGATNLVSGIADALLDSVPIVAITGQVPRRNIGTDAFQETPIVEVTRSITKHNYLVMDVEDIPRVVREAFF M 11111111111111 1111111111 I 111 M 11111II1111 M 111 M M 11 1111 11111 I M IIII I M M PGICIATSGPGATNLVSGLADALLDSVPLVAITGQVPRRMIGTDAFQETPIVEVTRSITKHNYLVMDVEDIPRIIEEAFF M f 1111111 I III I I I Mill llllllll I II II I I II I I III PGWLVTSGPGATNVVTPMADAFADGIPMWFTGQVPTSAIGTDAFQEADWGISRSCTKWNVMVKSVEELPLRINEAFE I I l l l l l l l II III I II M i l l I l l l l l l l l I III II I I MM II PAVCNACSGPGATNLVTAIADARLOSIPLICITGQVPASMIGTDAFQEVDTYGISIPITKHNYLVRHIEELPQVMSDAFR 250 270 290 310 LARSGRPGPI LI DVPKDIQQQLVIPDWDQPMR LPGYMSRLPKLPNEMLLEQIVRLISESKKPVLYVGGGCSQSSE M llllll I llllllllll l l l l l l l l l l l l l II I III111 1111 1111111111II II LATSGRPGPVLVDVPKDIQQQLAIPNWEQAMR LPGYMSRMPKPPEDSHLEQIVRLISESKKPVLYVGGGCLNSSD M M I I I I I I I I III I II II l l l l l l l l I II I IATSGRPGPVLVDLPKDVTAAILRNPIPTKTTLPSNALNQLTSRAQDEFVMQSINKAADLINLAKKPVLYVGAGILNHAD M lllllll I MM I I I II II II II M M I I IAQSGRPGPVWIDIPKDVQTAVFEIETQ PAMAEKAAAPAFSEESIRDAAAMINAAKRPVLYLGGG. . .VIN 330 350 370 390 DLRRFVEL.. .TGIPVASTIltGLGAFPTGDELSIâHI/SMHGTVYANYAVDSSDIXIAFGVRFDDRVTGKLEAFASRAKIV I Mill 1111111111111 I M i l l 11111111111111 11111111111111 M I M 1111111111 ELGRFVEL...TGIPVASTLMGLGSYPCDDELSLHMLGMHGTVYANYAVEHSDLLLAFGVRFDDRVTGKLEAFASRAKIV M III II 1111 I II l l l l l l II II M II l l l l l l l l II I GPRLLKEIâDRAQXPVTTTLQGLGSFDQEDPKSLDM^ I II II I I II II I II l l l l l l I I III I l l l l l l l I III APARVREIAEKAQU>TTMTI2fAI^MIJ>KAHPI£ 410 430 450 470 HI DI DS AEIGKNKQPHVSI CADI KLALQGLNSILESKEGKLKLDFS AWRQELTEQKVKHPLNFKTF GDAIP 1111111111111 1111 I I l l l l l l I II Mill II II II I II 1111 I III HIDIDSAEIGKNKTPHVSVCGDVKIALQGMNKVLENRAEELKL^^ GEAIP I I I I I I I AAEGRGGIIHFEVSPKNINKWQTQIAVEGDATTNLGKMMSKIFFVIŒRSEWF I I I I HVDIDRAQLGKIKQPHVAIQADVDDVLAQLIPLVEAQPRAEWHQLVADLQREFPCPIPKA CDPLS 490 510 530 550 PQYAIQVLDELTNGNAIISTGVGQHQMWAAQYYKYRKPRQWLTSGGI/^MGFGLPAAIGAAVGRPDEVVVDIDGDGSFIM M i l l l l l l l l I 111IIIIIIIII II II I I l l l l l l 11111111 M 1111111 I II llllllllllll PQYAIKVLDELTDGKAIISTGVGQHQMWAAQFYNYKKPRQWLSSGGI/JAMGFGLPAAIGASVANPDAIVVDIDGDGSFIM I I llllllllllll I M i l l II l l l l l l l l II I I M i l l II I VIKKLSKVANDTGRHVIVTTGVGQHQHWAAQHWIVRNPHTFITSGGI/STMGYGLPAAIGAQV DI DGDASFNM 11 I II l l l l l l l II I IMMIIII l l l l l l l l I I I II I I H YGLINAVAACVDDN AI ITTDVGQHQMWTAQAY PLNRPRQWLTSGGLGTMGFGLPAAIGAALANPDRKVLCFSGDGS LMM 570 590 610 630 KVQEIATIKVENU^IMIJJiNQHLGMWQWEDRFYKANRAHTYLGNPSNEAEIFPNMI^ llllllll lllllll l l l l l l l l l l 11111111111111 II I I l l l l l l l II III l l l l l l Ml NVQEIATIRVENLPVKVUJJINQHIXSHVMQWEDRFYKANR II III I III III II II II I I I I II I I TLTELSS AVQAGTPVKI LI LNNEEQGMVTQWQS LFYEHRYSHTHQL NPDFIKLAEAMGLKGLRVKKQEELDA I I III III III llllll I I II I NIQEMATASENQLDVKIILMNNEALGLVHQQQSLFYEQGAFVATYP GKINFMQIAAGFGLETCDLNMEADPQA 650 670 AIQKMLDTPGPYLLDVIVPHQEHVLPMIPSGGAFKDVITEGDGRSSY TOBACCO ALS III 1111111111111 l l l l l l l l l l l l l l I I M M I I I I I AIQTMLDTPGPYLLDVICPHQEHVLPMIPSGGTFMDVITEGDGRIKY ARABIDOPSIS ALS I II II I Mil I I KUMVAGGSGIJ)EFINFDPEVERG^EIJ«KRTGGKH YEAST ALS II II I I I I III I I SLQE11NRPGPALIHVRIDAEEKVYPMVPPGAANTEMVGE E. COLI ALS

F i g u r e 1. ALS sequences. Deduced amino a c i d sequences o f ALS enzymes from p l a n t s , y e a s t ( 1 6 ) , a n d c o l i (large subunit o f ALS I , 9, 1 5 ) . V e r t i c a l l i n e s i n d i c a t e i d e n t i c a l r e s i d u e s .


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c o m p a r i s o n o f the deduced amino a c i d sequences o f ALS p r o t e i n s from t o b a c c o , A r a b i d o p s i s , y e a s t , and b a c t e r i a . T h i s diagram i l l u s t r a t e s the p r e s e n c e o f b l o c k s o f h i g h sequence c o n s e r v a t i o n w h i c h a r e s e p a r a t e d by r e g i o n s where the v a r i o u s p r o t e i n s have d i v e r g e d . The p r e s e n c e o f the s t r o n g l y c o n s e r v e d r e g i o n s e x p l a i n s why the p l a n t genes c o u l d be c l o n e d u s i n g the y e a s t gene as a h y b r i d i z a t i o n probe. The two p l a n t p r o t e i n s a r e h i g h l y c o n s e r v e d w i t h r e s p e c t t o each o t h e r , even i n r e g i o n s where the y e a s t and c o l i p r o t e i n s have d i v e r g e d . A p p r o x i m a t e l y 75% o f the n u c l e o t i d e s and 85% o f the amino a c i d s a r e i d e n t i c a l between the ALS genes and p r o t e i n s from the the two p l a n t s . T h i s l e v e l o f c o n s e r v a t i o n s u g g e s t e d t h a t ALS genes i s o l a t e d from one p l a n t s p e c i e s would l i k e l y be f u n c t i o n a l when introduced i n t o a heterologous species. The o n l y r e g i o n where the two p l a n t p r o t e i n s a r e n o t w e l l c o n s e r v e d i s i n the N - t e r m i n a l r e g i o n , w h i c h encodes the c h l o r o p l a s t t r a n s i t peptides. I n t h i s r e g i o n o n l y about 23% o f the amino a c i d r e s i d u e s and 40% o f the n u c l e o t i d e s are i d e n t i c a l . I n p l a n t s ALS i s encoded i n the n u c l e u s y e t l o c a l i z e d i n c h l o r o p l a s t s (18-20). The t r a n s i t p e p t i d e i s thought t o d i r e c t the n a s c e n t p r o t e i n p o s t - t r a n s l a t i o n a l l y i n t o the c h l o r o p l a s t s . The t r a n s i t p e p t i d e i s t h e n c l e a v e d t o y i e l d the mature ALS p r o t e i n . T h i s p r o c e s s can be s t u d i e d i n a model system c o n t a i n i n g i s o l a t e d c h l o r o p l a s t s and ALS p r o t e i n w h i c h has been t r a n s c r i b e d and t r a n s l a t e d i n v i t r o (Bascomb, N.; G u t t e r i d g e , S.; L e t o , K.; Smith, J.K., s u b m i t t e d f o r p u b l i c a t i o n ) . The p u t a t i v e ALS t r a n s i t p e p t i d e s o f t o b a c c o and A r a b i d o p s i s show l i t t l e homology when compared w i t h each o t h e r o r w i t h y e a s t , w h i c h has a t r a n s i t p e p t i d e t h a t d i r e c t s ALS i n t o the m i t o c h o n d r i a (21, 22). The two p l a n t ALS t r a n s i t sequences a l s o show l i t t l e homology w i t h the t r a n s i t sequences w h i c h have been determined f o r other c h l o r o p l a s t - l o c a l i z e d p r o t e i n s (23). S t r u c t u r a l s i m i l a r i t i e s can be seen, however, when the h y d r o p a t h y p r o f i l e s o f the tobacco and A r a b i d o p s i s ALS t r a n s i t p e p t i d e s are compared (not shown). T h i s s u g g e s t s t h a t a f u n c t i o n a l t r a n s i t sequence depends more on secondary o r h i g h e r o r d e r s t r u c t u r a l c o n s t r a i n t s t h a n on p r i m a r y sequence i n f o r m a t i o n . The i n v i t r o uptake system d e s c r i b e d above can be u s e d t o f u r t h e r i n v e s t i g a t e the t r a n s i t p e p t i d e domain. The c l o n e d p l a n t ALS genes have been used t o s t u d y the o r g a n i z a t i o n and r e g u l a t i o n o f ALS i n p l a n t s . By h y b r i d i z a t i o n a n a l y s i s , i t was shown t h a t A r a b i d o p s i s has o n l y a s i n g l e ALS gene (Mazur, B.J.; C h u i , C.-F.; S m i t h , J.K. P l a n t P h y s i o l . , i n p r e s s ) . Tobacco was shown t o have two ALS genes, c o n s i s t e n t w i t h i t b e i n g an a l l o t e t r a p l o i d ( 2 4 ) , and w i t h e a r l i e r g e n e t i c d a t a s u g g e s t i n g two ALS l o c i ( 1 8 ) . A t the RNA l e v e l , each o f the two t o b a c c o genes i s t r a n s c r i b e d , a l t h o u g h one i s c o n s i s t e n t l y e x p r e s s e d a t h i g h e r l e v e l s t h a n the o t h e r ( M a r t i n , S.; Mazur, B.J.; Smith, J.K., m a n u s c r i p t i n p r e p a r a t i o n ) . The h i g h e s t l e v e l s o f ALS message a r e seen i n f l o w e r s and i n the youngest l e a v e s , w h i l e e x p r e s s i o n i s b a r e l y d e t e c t a b l e i n r o o t s and i n the o l d e r t i s s u e s . These c l o n e d A r a b i d o p s i s and t o b a c c o ALS genes have been used as h y b r i d i z a t i o n probes t o i s o l a t e ALS genes from o t h e r crop s p e c i e s and t o i s o l a t e ALS genes from p l a n t s s e l e c t e d f o r r e s i s t a n c e t o sulfonylurea herbicides.


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C l o n i n g and C h a r a c t e r i z a t i o n o f Mutant ALS

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Genes

Two s e l e c t i o n s t r a t e g i e s have been used to o b t a i n p l a n t s r e s i s t a n t to s u l f o n y l u r e a h e r b i c i d e s . Tobacco M u t a n t s . C h a l e f f and Ray (18) p l a t e d c a l l u s c u l t u r e s from h a p l o i d N i c o t i a n a tabacum on media c o n t a i n i n g 2 ppb ( a p p r o x i m a t e l y 6 nM) c h l o r s u l f u r o n o r s u l f o m e t u r o n m e t h y l . R e s i s t a n t c e l l l i n e s were r e g e n e r a t e d and d i p l o i d p l a n t s were c h a r a c t e r i z e d g e n e t i c a l l y . The h e r b i c i d e r e s i s t a n c e m u t a t i o n s f e l l i n t o two c l a s s e s , r e p r e s e n t e d by mutant l i n e s C3 and S4, w h i c h d e f i n e d two semidominant n u c l e a r l o c i , SuRA and SuRB. Homozygous mutant p l a n t s o f the S4 t y p e were a b l e to t o l e r a t e a t l e a s t 1 0 0 - f o l d more h e r b i c i d e t h a n were t h e i r w i l d type progenitors. C a l l u s i n i t i a t e d from homozygous S4 p l a n t s was s u b s e q u e n t l y exposed t o even h i g h e r l e v e l s o f h e r b i c i d e (200 ppb), and r e s i s t a n t l i n e s were r e g e n e r a t e d i n t o p l a n t s ( 2 5 ) . One such l i n e , d e s i g n a t e d Hra, was a b l e t o t o l e r a t e c o n c e n t r a t i o n s o f h e r b i c i d e 1 0 0 0 - f o l d g r e a t e r t h a n t h a t r e q u i r e d t o i n h i b i t w i l d type l i n e s . The Hra m u t a t i o n was shown t o be g e n e t i c a l l y l i n k e d t o the S4 m u t a t i o n . These t h r e e mutant tobacco l i n e s (C3, S4, and Hra) a l l had an a l t e r e d form o f ALS w h i c h was l e s s s e n s i t i v e t o i n h i b i t i o n by the s u l f o n y l u r e a h e r b i c i d e s t h a n was enzyme e x t r a c t e d from w i l d type p l a n t s . T h i s s u g g e s t e d t h a t c l o n e d mutant ALS genes c o u l d be used t o t r a n s f o r m o t h e r p l a n t s t o h e r b i c i d e r e s i s t a n c e . A l t h o u g h the t o b a c c o ALS gene d e s c r i b e d above was i s o l a t e d from an S4 genomic l i b r a r y , t r a n s f o r m a t i o n o f t h i s gene i n t o t o b a c c o c e l l s showed t h a t i t encoded a h e r b i c i d e - s e n s i t i v e form o f ALS. T h i s gene was t h e r e f o r e u s e d as a h y b r i d i z a t i o n probe t o i s o l a t e the ALS genes from genomic l i b r a r i e s made from the C3 and the Hra mutant l i n e s (Lee, K.Y.; Townsend, J . ; Tepperman, J . ; B l a c k , M.; C h u i , C.-F.; Dunsmuir, P.; Mazur, B.J.; Bedbrook, J . , s u b m i t t e d f o r p u b l i c a t i o n ) . By i n d e p e n d e n t l y i n t r o d u c i n g each c l o n e d gene i n t o s e n s i t i v e tobacco c e l l s and t h e n a s s a y i n g t r a n s f o r m a n t s f o r r e s i s t a n c e , mutant and w i l d type genes were d i s t i n g u i s h e d . Each gene was sequenced and the m u t a t i o n s r e s p o n s i b l e f o r the h e r b i c i d e r e s i s t a n t phenotype were identified. The C3 gene has a s i n g l e m u t a t i o n w h i l e the Hra gene has a l t e r a t i o n s l e a d i n g t o two amino a c i d s u b s t i t u t i o n s . The Hra gene has been used t o t r a n s f o r m a v a r i e t y o f c r o p s t o h e r b i c i d e r e s i s t a n c e as d e s c r i b e d below. A r a b i d o p s i s M u t a n t s . Haughn and S o m e r v i l l e (26) o b t a i n e d h e r b i c i d e r e s i s t a n t A r a b i d o p s i s l i n e s by s c r e e n i n g a mutagenized seed population. A r a b i d o p s i s seeds were exposed t o the mutagen EMS and t h e n grown t o m a t u r i t y . Seeds c o l l e c t e d from the r e s u l t i n g p l a n t s were p l a t e d on media c o n t a i n i n g 200 nM c h l o r s u l f u r o n (a commercial s u l f o n y l u r e a h e r b i c i d e ) . Of 300,000 seeds s c r e e n e d , f o u r s e e d l i n g s germinated. One l i n e (GH50) was f u r t h e r c h a r a c t e r i z e d . T h i s l i n e was homozygous w i t h r e s p e c t t o the r e s i s t a n t phenotype, d e f i n i n g a s i n g l e dominant n u c l e a r m u t a t i o n d e s i g n a t e d C s r - l . I t was a b l e to t o l e r a t e approximately 300-fold higher l e v e l s of h e r b i c i d e than c o u l d w i l d type A r a b i d o p s i s . ALS a c t i v i t y i n e x t r a c t s from mutant p l a n t s was l e s s s e n s i t i v e t o i n h i b i t i o n by s u l f o n y l u r e a s t h a n was enzyme from w i l d type p l a n t e x t r a c t s . The c l o n e d w i l d type A r a b i d o p s i s gene d e s c r i b e d above was used t o i s o l a t e the ALS gene from a l i b r a r y made from the h e r b i c i d e r e s i s t a n t l i n e (Haughn, G.;


30


S m i t h , J.K.; Mazur, B.J.; S o m e r v i l l e , C. Mol. Gen. Genet., i n p r e s s ) . The mutant gene was sequenced and compared t o the w i l d type gene. A s i n g l e m u t a t i o n , w h i c h changed p r o l i n e 197 t o a s e r i n e , was i d e n t i f i e d . A m u t a t i o n a t t h i s p o s i t i o n ( t o s e r i n e ) has a l s o been d e s c r i b e d i n yeast s e l e c t e d f o r h e r b i c i d e r e s i s t a n c e (27). The c l o n e d mutant A r a b i d o p s i s ALS gene has been used i n t r a n s f o r m a t i o n e x p e r i m e n t s by Haughn e t a l . (Haughn, G.; Smith, J.Κ.; Mazur, B.J.; S o m e r v i l l e , C. Mol. Gen. Genet., i n p r e s s ) , and as d e s c r i b e d below. I n t r o d u c t i o n o f Mutant ALS

Genes i n t o P l a n t s

The A r a b i d o p s i s Gene. Genes e n c o d i n g b o t h s u l f o n y l u r e a s e n s i t i v e and s u l f o n y l u r e a r e s i s t a n t forms o f ALS were c l o n e d i n t o T i plasmid-derived p l a n t transformation vectors which a l s o contained a gene c o n f e r r i n g r e s i s t a n c e t o the a n t i b i o t i c kanamycin. A f t e r t r a n s f e r i n t o A g r o b a c t e r i u m , each c o n s t r u c t i o n was u s e d t o t r a n s f o r m a s e n s i t i v e tobacco l i n e , u s i n g a m o d i f i e d l e a f - d i s k t r a n s f o r m a t i o n p r o t o c o l . F o l l o w i n g s e l e c t i o n on kanamycin, t r a n s f o r m e d s h o o t s were e x c i s e d and p l a c e d on r o o t i n g media. S e v e r a l s m a l l l e a v e s from each s h o o t were a l s o p l a c e d on c a l l u s i n d u c t i o n media c o n t a i n i n g kanamycin, h e r b i c i d e , or n e i t h e r compound. Seventeen o f 19 p l a n t s t r a n s f o r m e d w i t h the mutant A r a b i d o p s i s ALS gene were a b l e t o form c a l l u s on media c o n t a i n i n g 10 ppb c h l o r s u l f u r o n , w h i l e none o f the p l a n t s t r a n s f o r m e d w i t h the w i l d type gene were a b l e t o form c a l l u s on the same media. W i t h few e x c e p t i o n s , c a l l u s c a r r y i n g the mutant ALS gene was a b l e t o grow i n the p r e s e n c e o f b o t h kanamycin and h e r b i c i d e . T h i s was as e x p e c t e d , s i n c e the two genes were l i n k e d i n the t r a n s f o r m a t i o n v e c t o r . The kanamycin r e s i s t a n t c a l l u s c u l t u r e s from s e v e r a l t r a n s f o r m a n t s were s u b c u l t u r e d i n o r d e r t o g e n e r a t e large q u a n t i t i e s of healthy c a l l u s t i s s u e f o r f u r t h e r t e s t i n g . C a l l u s l i n e s d e r i v e d from t r a n s f o r m a n t s w h i c h had r e c e i v e d the mutant ALS gene grew much b e t t e r i n the p r e s e n c e o f 10 ppb c h l o r s u l f u r o n t h a n d i d l i n e s d e r i v e d from t r a n s f o r m a n t s w h i c h r e c e i v e d the w i l d type gene, as shown i n F i g u r e 2. A l s o shown i n F i g u r e 2 are the r e s u l t s o f ALS assays p e r f o r m e d on e x t r a c t s o f t r a n s f o r m e d p l a n t s . A t 100 ppb c h l o r s u l f u r o n the enzyme a c t i v i t y from p l a n t s c o n t a i n i n g the s e n s i t i v e ALS gene was a l m o s t c o m p l e t e l y i n h i b i t e d , w h i l e a c t i v i t y i n p l a n t s c o n t a i n i n g the r e s i s t a n t gene was i n h i b i t e d o n l y 30-60%. Each o f the r e s i s t a n t t r a n s f o r m a n t s was f o r c e d t o s e l f - p o l l i n a t e , and progeny t e s t s were performed. These t e s t s i n d i c a t e d t h a t each t r a n s f o r m a n t had r e c e i v e d a s i n g l e h e r b i c i d e r e s i s t a n t ALS gene w h i c h was s u b s e q u e n t l y i n h e r i t e d i n a simple Mendelian fashion. The Tobacco Gene. The Hra l i n e o f t o b a c c o i s the p l a n t mutant w h i c h has shown the h i g h e s t l e v e l o f r e s i s t a n c e t o the s u l f o n y l u r e a s . For t h i s r e a s o n the c l o n e d Hra gene has been used i n t r a n s f o r m a t i o n e x p e r i m e n t s . I n one s e t o f e x p e r i m e n t s the Hra gene was u s e d t o t r a n s f o r m commercial t o b a c c o c u l t i v a r s . F i g u r e 3 shows enzyme a s s a y s r u n on e x t r a c t s from some o f the t r a n s f o r m a n t s and on two w i l d type p l a n t s . ALS a c t i v i t y i n the t r a n s f o r m a n t s was c l e a r l y more r e s i s t a n t t o h e r b i c i d e t h a n was the a c t i v i t y from c o n t r o l p l a n t s . T o l e r a n c e t o h e r b i c i d e was a l s o a s s a y e d by m e a s u r i n g the a b i l i t y t o form c a l l u s i n the p r e s e n c e o f h e r b i c i d e , by m e a s u r i n g the a b i l i t y o f progeny seeds t o germinate i n the p r e s e n c e o f


SMITH ET AL.

31


ο

• ο

% Uninhibited Activity

F i g u r e 2. Assays o f tobacco t r a n s f o r m e d w i t h A r a b i d o p s i s ALS genes. Tobacco c o n t a i n i n g e i t h e r the h e r b i c i d e s e n s i t i v e (S) o r r e s i s t a n t (R) ALS gene was t e s t e d f o r the a b i l i t y o f t r a n s f o r m e d c a l l u s t o grow i n the p r e s e n c e o f h e r b i c i d e ( s l a s h e d boxes) and the h e r b i c i d e r e s i s t a n c e o f enzyme a c t i v i t y i n p l a n t e x t r a c t s ( s o l i d b o x e s ) . Each measurement i s e x p r e s s e d as a p e r c e n t a g e o f t h e v a l u e t h a t was o b t a i n e d i n the absence o f herbicide.

ο

2.

1

K3 WT2 WT4 7

11

31 40

41

1!II!I 9c

27 10c 32c 29 Plant

10

53

54

42

54a

F i g u r e 3. A s s a y s o f t o b a c c o t r a n s f o r m e d w i t h a mutant t o b a c c o ALS gene. Enzyme a c t i v i t y i n the p r e s e n c e o f h e r b i c i d e was measured i n l e a v e s o f commercial t o b a c c o c u l t i v a r s t r a n s f o r m e d w i t h the HRA gene ( p l a n t s #7-54), and i n u n t r a n s f o r m e d p l a n t s (WT2, WT4). A c t i v i t y i s e x p r e s s e d as a p e r c e n t a g e o f the a c t i v i t y measured i n the absence o f h e r b i c i d e .


32


i n c r e a s i n g c o n c e n t r a t i o n s o f h e r b i c i d e , and by m e a s u r i n g p h y t o t o x i c i t y f o l l o w i n g f o l i a r a p p l i c a t i o n s of various herbicides. T r a n s f o r m a n t s were found t o be more r e s i s t a n t t h a n w i l d type p l a n t s by a l l o f t h e s e c r i t e r i a , a l t h o u g h q u a n t i t a t i v e d i f f e r e n c e s p o i n t e d t o the n e c e s s i t y o f a s s a y i n g t r a n s f o r m a n t s by more t h a n one method. S e g r e g a t i o n a n a l y s e s o f progeny p l a n t s and b l o t h y b r i d i z a t i o n s were u s e d t o d e t e r m i n e the number o f r e s i s t a n t ALS l o c i s e g r e g a t i n g i n each t r a n s f o r m e d l i n e . Most o f the t r a n s f o r m a n t s had o n l y one or two c o p i e s o f the mutant gene. L i n e #7, w h i c h showed one o f the h i g h e s t l e v e l s o f r e s i s t a n c e o f any o f the t r a n s f o r m a n t s (see F i g u r e 3) had f o u r c o p i e s o f the mutant a l l e l e . For b r e e d i n g programs i t i s d e s i r a b l e t o use a l i n e w h i c h has a h i g h l e v e l o f r e s i s t a n c e d e r i v e d from a s i n g l e mutant a l l e l e . Gene copy number as w e l l as the p o s i t i o n a t w h i c h the mutant genes a r e i n t e g r a t e d i n t o the p l a n t genome are e x p e c t e d t o i n f l u e n c e the degree o f h e r b i c i d e r e s i s t a n c e of a given l i n e . S e v e r a l o f the p l a n t s d e s c r i b e d i n F i g u r e 3 a r e c u r r e n t l y b e i n g e v a l u a t e d i n the f i e l d . F i g u r e 4 shows an e a r l y r e s u l t from t h i s work. W i l d t y p e and t r a n s f o r m e d tobacco p l a n t s were t e s t e d f o r t h e i r a b i l i t y t o t o l e r a t e a 32 grams/hectare f o l i a r a p p l i c a t i o n o f a s u l f o n y l u r e a h e r b i c i d e . T h i s dose was a p p r o x i m a t e l y f o u r times the t y p i c a l f i e l d a p p l i c a t i o n r a t e . Untransformed tobacco i s extremely s e n s i t i v e t o t h i s h e r b i c i d e , as can be seen by the p h y t o t o x i c e f f e c t s d i s p l a y e d by the the w i l d type p l a n t s i n the f i g u r e . The t r a n s f o r m a n t s were u n a f f e c t e d by t h i s l e v e l o f h e r b i c i d e a p p l i c a t i o n , d e m o n s t r a t i n g t h a t the c l o n e d Hra gene i s a b l e t o confer u s e f u l l e v e l s of herbicide resistance i n transgenic plants grown under f i e l d c o n d i t i o n s . The Hra gene has a l s o been used t o t r a n s f o r m a number o f h e t e r o l o g o u s s p e c i e s , and s e l e c t a b l e l e v e l s o f r e s i s t a n c e have been o b t a i n e d i n each c a s e . The Hra gene can p r o b a b l y be used t o c o n f e r u s e f u l l e v e l s o f h e r b i c i d e r e s i s t a n c e i n most p l a n t s p e c i e s . I n a d d i t i o n t o b e i n g u s e f u l i n the f i e l d , the h e r b i c i d e r e s i s t a n c e phenotype c o n f e r r e d by mutated ALS genes i s a u s e f u l s e l e c t a b l e marker i n the l a b o r a t o r y . Engineering

o f New

Mutant ALS

Genes

A l t e r i n g C l o n e d P l a n t ALS Genes. The mutant A r a b i d o p s i s and tobacco genes d e s c r i b e d above have been shown t o c o n f e r the h e r b i c i d e r e s i s t a n t phenotype when i n t r o d u c e d i n t o b o t h homologous and h e t e r o l o g o u s s p e c i e s . The c l o n e d ALS genes can a l s o be m o d i f i e d i n v i t r o i n o r d e r t o modulate the l e v e l and/or s p e c i f i c i t y o f h e r b i c i d e r e s i s t a n c e i n t r a n s g e n i c p l a n t s . S e v e r a l s t r a t e g i e s can be u s e d t o a c h i e v e t h i s g o a l . The a l t e r a t i o n o f r e g u l a t o r y elements such as promoters and enhancers can be used t o i n c r e a s e / d e c r e a s e mRNA l e v e l s , o r t o cause the message t o be e x p r e s s e d i n a t i s s u e - s p e c i f i c , d e v e l o p m e n t a l l y - s p e c i f i c , or i n d u c i b l e manner. M u t a t i o n s can a l s o be i n c o r p o r a t e d i n t o the ALS gene i n o r d e r t o a l t e r the s p e c i f i c i n t e r a c t i o n s between the enzyme and i t s inhibitors. S i t e d i r e c t e d mutagenesis has been u s e d t o i n c o r p o r a t e m u t a t i o n s i n t o c l o n e d p l a n t ALS genes b a s e d b o t h on p r e v i o u s l y c h a r a c t e r i z e d mutants and on computer m o d e l i n g i n f o r m a t i o n . These a l t e r e d genes have been t r a n s f o r m e d i n t o p l a n t s , and the t r a n s g e n i c p l a n t s have been t e s t e d f o r t h e i r s e n s i t i v i t y t o a v a r i e t y o f


2.

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h e r b i c i d e s . T r a n s g e n i c p l a n t s can c o n t a i n mutated c o p i e s o f t h e i r n a t i v e ALS genes, o r mutant ALS genes from h e t e r o l o g o u s s o u r c e s . The former may be p r e f e r a b l e i n o r d e r t o e x p e d i t e r e g u l a t o r y a p p r o v a l f o r commercial r e l e a s e s . I n a d d i t i o n , n a t i v e ALS genes may f u n c t i o n b e t t e r i n some h o s t s t h e n h e t e r o l o g o u s ones. E x p r e s s i o n o f P l a n t ALS Genes i n M i c r o o r g a n i s m s . S i t e d i r e c t e d mutagenesis has been u s e d t o i n c o r p o r a t e a v a r i e t y o f m u t a t i o n s i n t o s e v e r a l c l o n e d ALS genes, as d e s c r i b e d above. However, the t r a n s f o r m a t i o n , r e g e n e r a t i o n , and progeny t e s t i n g are l a b o r i o u s processes. I n o r d e r t o t a k e advantage o f the power o f m i c r o b i a l g e n e t i c s t o s t u d y p l a n t ALS genes, a b a c t e r i a l e x p r e s s i o n system was d e v e l o p e d (Smith, J.K.; S c h l o s s , J.V.; Mazur, B . J . , s u b m i t t e d f o r p u b l i c a t i o n ) . ALS genes ( i n c l u d i n g the c h l o r o p l a s t t r a n s i t p e p t i d e c o d i n g r e g i o n ) were c l o n e d i n t o e x p r e s s i o n v e c t o r s under the c o n t r o l o f b a c t e r i a l r e g u l a t o r y s i g n a l s . The p l a n t ALS genes were f u n c t i o n a l l y e x p r e s s e d i n E^ c o l i , and the p l a n t p r o t e i n was a b l e t o complement a b r a n c h e d c h a i n amino a c i d a u x o t r o p h y o f the b a c t e r i a . T h i s system has been u s e d t o d e t e r m i n e the h e r b i c i d e s e n s i t i v i t y o f new mutant ALS p r o t e i n s , as demonstrated i n F i g u r e 5. Bacteria e x p r e s s i n g a w i l d t y p e o r mutant p l a n t ALS gene were p l a t e d on m i n i m a l media, and f i l t e r paper d i s k s impregnated w i t h the a c t i v e i n g r e d i e n t s from two commercial h e r b i c i d e s were p l a c e d on the p l a t e s u r f a c e . A r a d i a l h e r b i c i d e c o n c e n t r a t i o n g r a d i e n t formed and, a f t e r a l l o w i n g f o r b a c t e r i a l growth, the zones o f i n h i b i t i o n were compared. The mutant shown i n F i g u r e 5 was more r e s i s t a n t t o the s u l f o n y l u r e a C l a s s i c t h a n was the w i l d type b u t i t r e t a i n e d i t s s e n s i t i v i t y t o the i m i d a z o l i n o n e S c e p t e r . T h i s mutant t h u s e x h i b i t s s e l e c t i v e h e r b i c i d e r e s i s t a n c e . U l t i m a t e l y , i n t e r e s t i n g mutations i d e n t i f i e d i n t h i s system must be i n c o r p o r a t e d i n t o p l a n t s f o r f u r t h e r t e s t i n g . P r e l i m i n a r y work has shown t h a t t h e r e i s a good c o r r e l a t i o n between the phenotypes r e s u l t i n g from the mutant genes e x p r e s s e d i n b a c t e r i a and the same mutant genes e x p r e s s e d i n p l a n t s . The E^ c o l i e x p r e s s i o n system d e s c r i b e d h e r e has a l s o p r o v e n u s e f u l f o r p u r i f y i n g p l a n t ALS enzymes. Because i t i s p r e s e n t i n such s m a l l amounts, ALS has been d i f f i c u l t t o p u r i f y from p l a n t sources. P u r i f y i n g the p l a n t enzymes e x p r e s s e d i n b a c t e r i a has p r o v i d e d m a t e r i a l f o r use i n e n z y m a t i c and s t r u c t u r a l s t u d i e s as w e l l as f o r the g e n e r a t i o n o f i m m u n o l o g i c a l r e a g e n t s . Future

Prospects

We have d e s c r i b e d the c l o n i n g , m o l e c u l a r c h a r a c t e r i z a t i o n , e x p r e s s i o n , and r e i n t r o d u c t i o n i n t o p l a n t s o f w i l d t y p e and mutant ALS genes. E f f o r t s t o use i n f o r m a t i o n about the ALS t a r g e t p r o t e i n i n o r d e r t o m a n i p u l a t e h e r b i c i d e s e n s i t i v i t y i n c r o p s p e c i e s can p r o c e e d i n two d i r e c t i o n s . I n p h y s i c a l s t u d i e s , the ALS p r o t e i n i t s e l f can be c h a r a c t e r i z e d . I n f o r m a t i o n about how the enzyme f u n c t i o n s and how i t s p e c i f i c a l l y i n t e r a c t s w i t h i n h i b i t o r s can c o n t r i b u t e t o mutagenesis programs and t o a more r a t i o n a l d e s i g n o f new h e r b i c i d e c a n d i d a t e s . Most c u r r e n t l y used commercial h e r b i c i d e s were d i s c o v e r e d i n l a r g e s y n t h e s i s / s c r e e n i n g programs, b u t t h a t "shotgun" approach i s becoming i n c r e a s i n g l y u n w i e l d y due t o the enormous number o f compounds t h a t need t o be t e s t e d b e f o r e a s i n g l e s e l e c t i v e h e r b i c i d e i s found. I n b i o l o g i c a l s t u d i e s , new mutants


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F i g u r e 4. F i e l d t e s t s o f t r a n s f o r m e d t o b a c c o . W i l d type t o b a c c o (WT) and t o b a c c o t r a n s f o r m e d w i t h t h e HRA gene (SUR) were s p r a y e d a t 4X t h e t y p i c a l f i e l d a p p l i c a t i o n r a t e w i t h a commercial sulfonylurea herbicide preparation.

F i g u r e 5. D i s k a s s a y s o f p l a n t ALS genes e x p r e s s e d i n b a c t e r i a . E. c o l i e x p r e s s i n g a w i l d type o r a h e r b i c i d e r e s i s t a n t ALS gene were t e s t e d f o r t h e i r a b i l i t y t o grow i n t h e p r e s e n c e o f t h e a c t i v e i n g r e d i e n t s from two commercial h e r b i c i d e s .


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can be c h a r a c t e r i z e d , m u t a t i o n s can be i n c o r p o r a t e d i n t o ALS genes from a v a r i e t y o f s p e c i e s , and mutants can be t e s t e d f o r t h e a b i l i t y to t o l e r a t e new o r e x i s t i n g compounds. The b i o l o g i c a l and p h y s i c a l approaches a r e complementary, and t o g e t h e r w i l l f u r t h e r t h e development o f new e f f i c a c i o u s c r o p p r o t e c t i o n s t r a t e g i e s . Acknowledgments We g r a t e f u l l y acknowledge our many c o l l e a g u e s a t Du Pont who have c o n t r i b u t e d b o t h d i r e c t l y and i n d i r e c t l y t o the e x p e r i m e n t s d e s c r i b e d h e r e . S p e c i a l thanks a r e due t o Tony G u i d a , Chok-Fun C h u i , and Sharon M a r t i n f o r m o l e c u l a r a n a l y s e s o f t h e p l a n t ALS genes, and t o Todd Houser and C h r i s Kostow f o r p l a n t t r a n s f o r m a t i o n e x p e r i m e n t s . We have e n j o y e d f r u i t f u l c o l l a b o r a t i o n s w i t h George Haughn and C h r i s S o m e r v i l l e a t M i c h i g a n S t a t e U n i v e r s i t y on t h e i s o l a t i o n and c h a r a c t e r i z a t i o n o f t h e mutant A r a b i d o p s i s ALS gene, and w i t h J o h n Bedbrook, K a t h y Lee, J e f f Townsend, and Pamela Dunsmuir a t Advanced G e n e t i c S c i e n c e s on t h e c l o n i n g and c h a r a c t e r i z a t i o n o f mutant tobacco ALS genes.

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16. Falco, S. C.; Dumas, K. D.; Livak, K. J. Nucleic Acids Res. 1985, 13, 4011-4027. 17. Mazur, Β. J.; Chui, C.-F.; Falco, S. C.; Mauvais, C. J.; Chaleff, R. S. In The World Biotech Report 1985; Online International: New York, 1985; pp 97-108. 18. Chaleff, R. S.; Ray, T. B. Science 1984, 223, 1148-1151. 19. Jones, Α. V.; Young, R. M.; Leto, K. J. Plant Physiol. 1985, 77, S293. 20. Miflin, B. J. Plant Physiol. 1974, 75, 827-831. 21. Magee, P. T.; Robichon-Szulmajster, H. de Eur. J. Biochem. 1968, 3, 502-506. 22. Ryan, E. D.; Kohlhaw, G. Β. J. Bacteriol. 1974, 120, 631-637. 23. Karlin-Neumann, G. Α.; Tobin, E. M. EMBO J. 1986, 5, 9-13. 24. Smith, H. In Nicotiana: Procedures for Experimental Use; Durbin, R. D., Ed.: US Dept. of Agriculture Technical Bulletin 1586; Academic Press: New York, 1979; ρ 3. 25. Chaleff, R. S.; Sebastian, S. Α.; Creason, G. L.; Mazur, B. J.; Falco, S. C.; Ray, T. B.; Mauvais, C. J.; Yadav, N. S. In Molecular Strategies for Crop Protection; Alan R. Liss: New York, 1987; pp 415-425. 26. Haughn, G. W.; Somerville, C. R. Mol. Gen. Genet. 1986, 204, 430-434. 27. Yadav, N. S.; McDevitt, R. E.; Benard, S.; Falco, S. C. Proc Nat. Acad. Sci. USA 1986, 83, 4418-4422. RECEIVED February 25, 1988


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