https://ndsu.edu/pubweb/~mcclean/plsc731/homework/papers/buschges et al - the barley mlo gene - a novel control element of plant pathogen resistance.pdf

The Barley Mlo Gene: A Novel Control Element of Plant Pathogen Resistance

Herbicides that target the enzyme acetolactate synthase (ALS) are among the most widely used in the world. Unfortunately, these herbicides are also notorious for their ability to select resistant (R) weed populations. Now, there are more weed species that are resistant to ALS-inhibiting herbicides than to any other herbicide group. In most cases, resistance to ALS-inhibiting herbicides is caused by an altered ALS enzyme. The frequent occurrence of weed populations resistant to ALS inhibitors can be attributed to the widespread usage of these herbicides, how they have been used, the strong selection pressure they exert, and the resistance mechanism. In several cropping systems, ALS-inhibiting herbicides were used repeatedly as the primary mechanism of weed control. These herbicides exert strong selection pressure because of their high activity on sensitive biotypes at the rates used and because of their soil residual activity. Several point mutations within the gene encoding ALS can result in a herbicide-resistant ALS. From investigations of numerous R weed biotypes, five conserved amino acids have been identified in ALS that, on substitution, can confer resistance to ALS inhibitors. Substitutions of at least 12 additional ALS amino acids can also confer herbicide resistance in plants and other organisms but, to date, have not been found in R weed populations. Mutations in ALS conferring herbicide resistance are at least partially dominant, and because the gene is nuclear inherited, it is transmitted by both seed and pollen. Furthermore, in many cases there is apparently a negligible fitness cost of the resistance gene in the absence of herbicide selection. Although resistance to ALS-inhibiting herbicides has been a bane to weed management, it has spurred many advances within and beyond the weed science discipline. As examples, resistance to ALS-inhibiting herbicides has been exploited in the development of herbicide-resistant crops, studies of weed population dynamics, and in developing protocols for targeted gene modification. Resistance to ALS-inhibiting herbicides has greatly affected weed science by influencing how we view the sustainability of our weed management practices, what we consider when developing and marketing new herbicides, and how we train new weed scientists.

https://bioone.org/journals/Weed-Science/volume-50/issue-6/0043-1745(2002)050[0700:RROWTA]2.0.CO;2/Resistance-of-weeds-to-ALS-inhibiting-herbicides--what-have/10.1614/0043-1745(2002)050[0700:RROWTA]2.0.CO;2.pdf

Resistance of weeds to ALS-inhibiting herbicides: what have we learned?

Imidazolinone herbicides, which include imazapyr, imazapic, imazethapyr, imazamox, imazamethabenz and imazaquin, control weeds by inhibiting the enzyme acetohydroxyacid synthase (AHAS), also called acetolactate synthase (ALS). AHAS is a critical enzyme for the biosynthesis of branched-chain amino acids in plants. Several variant AHAS genes conferring imidazolinone tolerance were discovered in plants through mutagenesis and selection, and were used to create imidazolinone-tolerant maize (Zea mays L), wheat (Triticum aestivum L), rice (Oryza sativa L), oilseed rape (Brassica napus L) and sunflower (Helianthus annuus L). These crops were developed using conventional breeding methods and commercialized as Clearfield* crops from 1992 to the present. Imidazolinone herbicides control a broad spectrum of grass and broadleaf weeds in imidazolinone-tolerant crops, including weeds that are closely related to the crop itself and some key parasitic weeds. Imidazolinone-tolerant crops may also prevent rotational crop injury and injury caused by interaction between AHAS-inhibiting herbicides and insecticides. A single target-site mutation in the AHAS gene may confer tolerance to AHAS-inhibiting herbicides, so that it is technically possible to develop the imidazolinone-tolerance trait in many crops. Activities are currently directed toward the continued improvement of imidazolinone tolerance and development of new Clearfield* crops. Management of herbicide-resistant weeds and gene flow from crops to weeds are issues that must be considered with the development of any herbicide-resistant crop. Thus extensive stewardship programs have been developed to address these issues for Clearfield* crops.

Imidazolinone-tolerant Crops: History, Current Status and Future

Disclosed herein are structure-based modelling methods for the preparation of acetohydroxy acid synthase (AHAS) variants, including those that exhibit selectively increased resistance to herbicides such as imidazoline herbicides and AHAS inhibiting herbicides. The invention encompasses isolated DNAs encoding such variants, vectors that include the DNAs, and methods for producing the variant polypeptides and herbicide resistant plants containing specific AHAS gene mutations. Methods for weed control in crops are also provided.

Structure-based designed herbicide resistant products

In Arabidopsis and other Brassicaceae, the enzyme myrosinase (beta-thioglucoside glucohydrolase, TGG) degrades glucosinolates to produce toxins that deter herbivory. A broadly applicable selection for meiotic recombination between tightly linked T-DNA insertions was developed to generate Arabidopsis tgg1tgg2 double mutants and study myrosinase function. Glucosinolate breakdown in crushed leaves of tgg1 or tgg2 single mutants was comparable to that of wild-type, indicating redundant enzyme function. In contrast, leaf extracts of tgg1tgg2 double mutants had undetectable myrosinase activity in vitro, and damage-induced breakdown of endogenous glucosinolates was apparently absent for aliphatic and greatly slowed for indole glucosinolates. Maturing leaves of myrosinase mutants had significantly increased glucosinolate levels. However, developmental decreases in glucosinolate content during senescence and germination were unaffected, showing that these processes occur independently of TGG1 and TGG2. Insect herbivores with different host plant preferences and feeding styles varied in their responses to myrosinase mutations. Weight gain of two Lepidoptera, the generalist Trichoplusia ni and the facultative Solanaceae-specialist Manduca sexta, was significantly increased on tgg1tgg2 double mutants. Two crucifer-specialist Lepidoptera had differing responses. Whereas Plutella xylostella was unaffected by myrosinase mutations, Pieris rapae performed better on wild-type, perhaps due to reduced feeding stimulants in tgg1tgg2 mutants. Reproduction of two Homoptera, Myzus persicae and Brevicoryne brassicae, was unaffected by myrosinase mutations.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-313X.2006.02716.x

Arabidopsis myrosinases TGG1 and TGG2 have redundant function in glucosinolate breakdown and insect defense.

Acetohydroxy acid synthase (AHAS) is an essential enzyme for many organisms as it catalyzes the first step in the biosynthesis of the branched-chain amino acids valine, isoleucine, and leucine. The enzyme is under allosteric control by these amino acids. It is also inhibited by several classes of herbicides, such as the sulfonylureas, imidazolinones and triazolopyrimidines, that are believed to bind to a relic quinone-binding site. In this study, a mutant allele of AHAS3 responsible for sulfonylurea resistance in a Brassica napus cell line was isolated. Sequence analyses predicted a single amino acid change (557 Trp→Leu) within a conserved region of AHAS. Expression in transgenic plants conferred strong resistance to the three classes of herbicides, revealing a single site essential for the binding of all the herbicide classes. The mutation did not appear to affect feedback inhibition by the branched-chain amino acids in plants.

An acetohydroxy acid synthase mutant reveals a single site involved in multiple herbicide resistance

We have isolated a triazolopyrimidine-resistant mutant csrl-2, of Arabidopsis thaliana (L.) Heynh. Here, we compare csrl-2 with the previously isolated mutants csrl and csr1-1, and with wild-type Arabidopsis for responses to members of four classes of herbicides, namely, sulfonylureas, triazolopyrimidines, imidazolinones, and pyrimidyl-oxy-benzoates. Two separable herbicide binding sites have been identified previously on the protein of acetolactate synthase (ALS). Here, the mutation giving rise to csrl, originating in a coding sequence towards the 5′ end of the ALS gene, and that in csrl-2, affected the inhibitory action on growth and ALS activity of sulfonylurea and triazolopyrimidine herbicides but not that of the imidazolinones or pyrimidyl-oxybenzoates. The other mutation, in csrl-1, originating in a coding sequence towards the 3′ end of the ALS gene, affected the inhibitory action of imidazolinones and pyrimidyl-oxy-benzoates but not that of the sulfonylureas or triazolopyrimidines. Additional, stimulatory effects of some of these herbicides on growth of seedlings was unrelated to their effect on their primary target, ALS. The conclusion from these observations is that one of the two previously identified herbicide-binding sites may bind sulfonylureas and triazolopyrimidines while the other may bind imidazolinones and pyrimidyl-oxy-benzoates within a herbicide-binding domain on the ALS enzyme. Such a comparative study using near-isogenic mutants from the same species allows not only the further definition of the domain of herbicide binding on ALS but also could aid investigation of the relationship between herbicide-, substrate-, and allosteric-binding sites on this enzyme. This research was supported by an Operating Grant from the Natural Sciences and Engineering Research Council of Canada to J.K.

Effect of four classes of herbicides on growth and acetolactate-synthase activity in several variants of Arabidopsis thaliana.

Four classes of herbicides are known to inhibit plant acetolactate synthase (ALS). In Arabidopsis, ALS is encoded by a single gene, CSR1. The dominant csr1-1 allele encodes an ALS resistant to chlorsulfuron and triazolopyrimidine sulfonamide while the dominant csr1-2 allele encodes an ALS resistant to imazapyr and pyrimidyl-oxy-benzoate. The molecular distance between the point mutations in csr1-1 and csr1-2 is 1369 bp. Here we used multiherbicide resistance as a stringent selection to measure the intragenic recombination frequency between these two point mutations. We found this frequency to be 0.008 +/- 0.0028. The recombinant multiherbicide-resistant allele, csr1-4, provides an ideal marker for plant genetic transformation.

Intragenic recombination in the CSR1 locus of Arabidopsis.

A valine-resistant mutant line, VAL-2, of Arabidopsis thaliana (L.) Heynh. was identified by screening M 2 populations of ethylmethane-sulfonate-mutagenized seeds. The resistance was found to be due to a single, dominant, nuclear gene mutation. Assay of acetolactate synthase (ALS) indicated that the valine resistance in this mutant is caused by decreased sensitivity of ALS to the branched-chain amino acids, valine, leucine and isoleucine. A two fold decrease in apparent Km value for pyruvate of the mutant ALS enzyme was detected compared with that of the wild type. The sensitivity of the ALS enzyme to sulfonylurea, imidazolinone and triazolopyrimidine herbicides was not altered in the mutant. At the plant growth level the mutant was also resistant to valine plus leucine, but was sensitive to leucine or isoleucine alone. The mutant gene, var1, maps, or is very closely linked, to CSR1, the gene encoding acetolactate synthase in Arabidopsis.

A valine-resistant mutant of Arabidopsis thaliana displays an acetolactate synthase with altered feedback control

Threonine dehydratase/deaminase (TD), the first enzyme in the isoleucine biosynthetic pathway, is feedback inhibited by isoleucine. By screening M, populations of ethyl methane sulfonatetreated Arabidopsis thaliana Columbia wild-type seeds, we isolated five independent mutants that were resistant to i-O-methylthreonine, an isoleucine structural analog. Crowth in the mutants was 50- to 600-fold more resistant to i-Omethylthreonine than in the wild type. lhe resistance was dueto a single, dominant nuclear gene that was denoted omrl and was mapped to chromosome 3 in CM11 b, the mutant line exhibiting the highest level of resistance. Biochemical characteristics (specific activities, K,, V,,,, and pH optimum) of TD in extracts from the wild type and CMllb were similar except for the inhibition constant of isoleucine, which was 50-fold higher in CMllb than in the wild type. Levels of free isoleucine were 20-fold higher in extracts from CMllb than in extracts from wild type. Therefore, isoleucine feedback insensitivity in GMll b is due to a mutant form of the TD enzyme encoded by omrl. lhe mutant allele Omrl of the line CMllb could provide a new selectable marker for plant genetic transformation.

George Mourad

Papers

An acetohydroxy acid synthase mutant reveals a single site involved in multiple herbicide resistance

Effect of four classes of herbicides on growth and acetolactate-synthase activity in several variants of Arabidopsis thaliana.

Intragenic recombination in the CSR1 locus of Arabidopsis.

A valine-resistant mutant of Arabidopsis thaliana displays an acetolactate synthase with altered feedback control

L- O- Me t h y I t h reo n i n e- Resi s t an t M u tan t of A ra bidopsis Defective in lsoleucine Feedback Regulation'