IN VIVO AND IN VITRO MUTAGENESIS OF A BRASSICA NAPUS ACETOLACTATE SYNTHASE GENE FOR THE PRODUCTION OF HERBICIDE RESISTANCE

Paul A. WIERSMA, D.L. DURDA, I. BABIC, W.L. CROSByl, M.M. MOLONEY Dept. Biological Sciences, University of Calgary, 2500 University Dr. N.W., Calgary, AB, Canada T2N IN4 and 1) Molecular Genetics Section, National Research Council Canada, Plant Biotechnology Institute, 110 Gymnasium Road, Saskatoon, SK, Canada S7N OW9

INTRODUCTION Herbicide resistance in plants may occur as a result of point mutations in

the gene encoding the herbicide target. Such mutations have been well characterized for resistance to herbicides which affect photosynthesis (1) and amino-acid metabolism (2,3). A better understanding of how these mutant enzymes interact with various herbicides will aid the development of new herbicide/plant systems. The enzyme acetolactate synthase (ALS (or AHAS» has been the focus of several studies recently because it is the target for three classes of herbicides: sulfonylureas (4,5), imidazolinones (6) and triazolopyrimidines (7). Resistance to these herbicides has been produced, by mutagenesis of seeds or cultured tissure, for a number of plant species including: tobacco, Arabidopsis thaliana, oilseed rape, and sugar beet. Determining the structural basis of these mutations becomes more complex as crop species with multiple copies of ALS are used. For example, Brassica napus (oilseed rape) has 4 to 5 genes for ALS (8) and appears to regulate these sequences differentially. Characterization of new mutations requires isolation and sequencing of all ALS genes in that plant and then demonstration that the specific mutations found can cause a resistant phenotype upon re-introduction into plants. Since it is necessary to re-introduce these in vitro mutated genes into a suitable host to determine their phenotype, we sought to develop a bacterial expression system. This would enable us to take advantage of the convenience and simplicity of prokaryotic genetics and transformation. Expression of plant sequences in bacteria can be used in a number of ways, including: indentification of the function of unknown cDNA clones by complementing bacterial mutants; allowing production of plant enzymes and proteins which might be unstable or of very low abundance in the plant; and development of a system for mutagenesis where resultant clones can be rapidly analysed We describe here a method for efficiently expressing in bacteria a nuclear gene encoding an enzyme that is normally targetted to the plant chloroplast. The following steps describe a procedure for production of a herbicide resistant crop. It is first necessary to

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H. J. J. Nijkamp et al. (eds.), Progress in Plant Cellular and Molecular Biology© Kluwer Academic Publishers 1990

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isolate a wild-type herbicide-sensitive ALS gene and modify it by truncation to allow expression in bacteria. Mutations are introduced into the gene by in vitro mutagenesis, making changes that are known from other genes to produce the desired phenotype, or by in vivo mutagenesis of bacteria that harbor the plant gene. The effects of these mutations can be evaluated initially in the bacterial host. The mutated ALS genes are re-introduced into the best cultivars of various species using native or enhanced plant promoters for expression of herbicide resistance.

RESULTS AND DISCUSSION Characteristics of a genomic ALS gene from Brassica napus. Isolation of a 3.3 kb clone (pPAWl) from B. napus has been described previously (8). An uninterrupted coding region (no introns) encodes 637 amino acids with a strong similarity along most of its length to published sequences from other species. The N-terminal sequence however shows no similarity to the others which is consistent with this being a chloroplast transit peptide as discussed below.

Truncation of a chloroplast transit peptide allows efficient ALS complementation in bacteria. Salmonella typhimurium strain DU2603 (9) was selected as the host strain for the experiments described here. This strain has mutations in both genes encoding the large subunits of ALS and consequently shows no ALS activity. We sought to render this strain prototrophic by the use of plant ALS sequences cloned into the bacterial expression plasmid pKK233-2 (10). This plasmid was chosen for its strong chimaeric promoter trc (trp-Iac) and a Ncol site which not only contains the translational start codon (ATG) in the optimal context for bacterial expression, but also matches the restriction site enclosing the fIrst codon on the B. napus ALS gene used in these studies.

The coding regions of all the plant genes sequenced to date have 60 to 90 amino acids at their N-termini which are not represented in the bacterial sequences. These probably encode a chloroplast transit peptide which would be cleaved from the polypeptide to produce the functional protein. We therefore tested the idea that a functional enzyme could be produced in bacteria by expressing the plant ALS coding region with part or all of this transit peptide already removed. To do this a plasmid containing the plant gene was linearized with Pst I and Hind III, digested with exonuclease III and mung bean nuclease and the plasmid recircularized with a Ncol linker. This produced a library of randomly sized truncations into the transit peptide sequence with a Ncol site to give a new start of translation and an effIcient means of cloning into the bacterial expression vector. Members of this library were chosen at random and screened by DNA sequencing to identify a number of truncations which spanned the presumed processing site and which reproduced the correct reading frame for translation after the new ATG start codon. Several clones were selected for

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further complementation experiments and were assembled into the bacterial expression vector (Fig. 1).

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Figure 1 Construction of bacterial expression plasmids of the B. napus ALS coding region. The coding region fragment of ALS from pPA WI was inserted as a transcriptional fusion behind the strong trc promoter of pKK233-2. The capital letters above the bottom line d~signate the relative start sites of the complete coding region (A) and a number of truncation mutants (B-G). The values beneath the line indicate the growth rate, relative to wild-type bacteria, of ALS-deficient bacteria containing the construct of that size.

The presence of the complete transit peptide is inhibitory to expression of plant ALS in bacteria. This may be due to normal interaction of this amphihelical sequence with membrane and in this case with the bacterial membrane. If trapped on the membrane the protein may be more susceptible to degradation or it may be unable to assemble into an active multimeric form as is normally found in both bacterial and plant ALS. The inhibition of assembly could also be caused by the presence of extra amino acids in the soluble form of the enzyme, although a precise N-terminus does not appear to be required, as a window of approximately 20-25 amino acids including the start sites of constructs D and E can give efficient complementation. Addition of further amino acids decreases the functioning of the protein without eliminating it completely until past the site of C. The longer N-terminus may also interfere with the normal folding of the monomer or may block interaction of the protein with substrates or coenzymes.

This method should be generally applicable to other proteins with transit or signal peptides for which the precise processing site is not known. It would be useful to know the specificity of the processing enzymes in plants so that an

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accurate prediction of the mature protein could be made for this type of heterologous expression experiment.

Biochemical characteristics of plant ALS expressed in bacteria. Extracts of the expression system with various truncation constructs revealed different protein levels of the plant derived sequence. These protein levels did not correspond to the levels of complementation. It was also noted that use of E. coli hosts produced a different pattern of protein expression. Although we have not observed extrusion of these sequences into periplasm, the media, or into inclusion bodies it appears that some sort of differential processing/degradation may be occuring to sequences with slightly different start sites.

The level of ALS acitivity does correlate with the complementation observed for the different constructs. For constructs D and E the ALS activity is several fold higher than that of wild-type bacteria even though their growth rate is 70 % or less of wild-type. This may be explained by differences in the characteristics of these enzymes. The enzyme in plants has been isolated as a 440 kDa complex of which the exact subunit structure is unknown. This complex is feedback regulated by valine. The active plant gene product expressed in bacteria migrates at approximately 120 to 140 kDa and is not inhibited by valine. This is similar in size to the bacterial enzyme which has two large subunits (equivalent to the plant gene used here) and two small subunits which are thought to be involved in feedback regulation (11). It is uncertain if the plant product interacts with the bacterial small subunit to form the observed active complex but genetic and biochemical evaluation of this possiblity are underway. Although large amounts of protein are produced from some constructs, it appears that only a fraction of this is active enzyme as the bulk of protein does not copurify with the activity. This is consistent with the requirement of interaction with a limiting bacterial component, or with a very low probability of assembly without additional plant factors, or with a bacterial processing event that either inactivates the bulk of protein or activates a very low level of the protein. Purification and microsequencing of the active versus inactive peptides can answer this question.

Herbicide resistance of plant ALS mutants expressed in bacteria. A well documented mutation which gives chlorsulfuron resistance in plant ALS genes (Pro to Ser at amino acid 173 in pPA WI) (8) was introduced into the truncated B. nap us ALS gene by in vitro mutagenesis to test its efficacy in bacterially expressed plant ALS. As can be seen in the disc diffusion assay of Fig. 2 the bacteria expressing this construct (" 173S") does not show the inhibition of the wild-type plant gene ("68", which is equivalent to construct E). It is also possible to detect cross-resistance to different classes of herbicides caused by the mutation as observed here for chlorsulfuron and two imidazolinones. A more quantitative evaluation of these data is seen in the

Figure 2. Disc diffusion assay for herbicide inhibition. 100 III aliquots of overnight

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Table 1. Inhibition curve analysis of ALS activity in enzyme extracts

IsO, ~Ma

Source CS Pursuit Assert

E 20 30 400

173S >1000 55 5500

LT2 0.08 2000 2000

leaves 0.01 1 40

cultures of host bacteria containing the construct "E" (=68), the resistant mutant aExtracts from bacteria containing construct "173S", and the bacterial large subunit gene E (Fig. 1); or E modified by a Pro to Ser (ilv G) were poured in top agar onto mutation at amino acid 173 (173S); wild­minimal media plates containing type LT2; and B. napus leaves were pantothenate and ampicillin. Sterile 6 mm assayed for ALS activity in the presence of paper discs containing herbicides were increasing concentrations of herbicide. ISO applied to the surface and the plates designates the concentration of herbicide in incubated overnight. C, 40 Ilg which the enzyme activity is 50% of chlorsulfuron; A, 2 mg Assert; P, 40 Ilg uninhibited. CS designates chlorsulfuon. Pursuit.

inhibition assays of Table 1. The presence of the mutation causes at least a hundred-fold increase in resistance to chlorsulfuron, a ten-fold increase in Assert resistance and little effect with Pursuit. This demonstates that the bacterial expression system can model the effect mutations in the plant gene can have on herbicide sensitity.

Mutagenesis is now being done on the plant gene using standard in vivo bacterial techniques. Also methods in which the plasmid is mutated before reintroduction into the bacterial host are being evaluated. This latter method has the advantage of not perturbing the host genes and also of ensuring that all copies of the plasmid contain the mutation which contributes to the observed phenotype. This also greatly facilitates accurate isolation and sequencing of the mutant plasmid.

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Herbicide resistance phenotypes of plant ALS transformed into B. napus. Cotyledonary petiole ~xplants of B. napus cv Westar were transformed using Agrobacterium vectors as described by Moloney et al.(12). The vectors harbored within their T-DNA, constructs containing mutant ALS genes tested in the above bacterial expression system. Constructs included those containing the native promoter of plant ALS and also chimaeric genes using high level constitutive promoters such as CaMV 35S and its tandem repeat. Selections were performed both on kanamycin (15 mg L -1) and directly on the herbicide chlorsulfuron (10 nM). In both cases we obtained regenerating plants showing resistance to the selection agents.

Analysis of the ALS activity in the presence and absence of sulfonylureas shows a 1 to 2 order of magnitude shift from the normal (10 nM) 150 of the enzyme in transformed plants, depending on event. The progeny of these transformants are currently being evaluated for their resistance at the whole plant level.

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