Maize DIMBOA biosynthesis genes

Details Maize DIMBOA biosynthesis genes Maize DIMBOA biosynthesis genes

1998-03-13

2001-12-18

Nelson, Amy J. (Department: 1638)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

C800S279000, C800S283000, C800S284000, C800S289000, C800S290000, C800S295000, C800S298000, C800S300000, C800S300100, C800S301000, C800S302000, C800S312000, C800S314000, C800S317000, C800S317200, C800S317300, C800S008000, C800S320100, C435S069100, C435S440000, C435S419000, C435S418000, C536S063000, C536S023100, C536S063000

Reexamination Certificate

active

06331660

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the field of plant resistance to insects and pathogens. More specifically, it relates to plant genes of benzoxazinone biosynthesis.
2. Description of Related Art
Plants protect themselves against infestation with pathogens by a number of chemical defense mechanisms. In this regard, especially the secondary metabolites of plants can be mentioned. Among these chemically very heterogeneous compounds there are many substances which have a poisonous or deterrent effect on animals such as insects and on microorganisms. The synthesis of these defense substances can be regulated in different ways. It is often only induced in case of infestation with pathogens or in specific tissues of the plant or only during a specific developing stage of the plant.
Benzoxazinones are a class of secondary metabolites found almost exclusively in gramineae. DIMBOA (2,4-dihydroxy-7-methoxy-1,4benxoxazin-3-one) and DIBOA (2,4-dihydroxy-4-benzoxazin-3-one) are the primary benzoxazinones both found in maize. DIBOA is the main benzoxazinone occurring in rye, whereas DIMBOA is the main benzoxazinone occurring in wheat and maize (Walross et al., 1959). DIBOA and DIMBOA have been shown to have a broad spectrum of effects on insects and microorganisms. A positive correlation of the concentration of DIMBOA and resistance to (i) the fungus
Helminthosporium turcicum,
which causes of the Northern Leaf Blight disease (Couture et al., 1971), (ii) infestation with the maize plant louse
Rhophalosiphum maydis
(Long et al., 1977), (iii)
Diplodia maydis
(stalk rot), and (iv) especially to the European corn borer
Ostrinia nubilalis
(Campos et al., 1989), have been found in maize. The same applies to the resistance of wheat to the fungus
Puccinia graminis
(Long et al., 1977). In particular, the European corn borer causes great damage every year in all areas in which corn is cultivated. For example, in the USA the damage amounts to about 500 million dollars and can be controlled by pesticides only to a limited extent.
The effect of DIMBOA on larvae of the European corn borer has been the topic of a number of investigations. For instance, food containing a large amount of DIMBOA is refused by the corn borer larvae in favor of food without DIMBOA, whereas in the case of an exclusive DIMBOA diet, the amount of food taken is increased. The increase of the amount of food is necessary for the larvae, since DIMBOA leads to an obstruction of the proteolytic activity in the digestive tract of the larvae. Thus, plants containing DIMBOA are avoided by the European corn borer, or, if these plants are infested, the brood starves (Cuevas et al., 1990).
The accumulation of benzoxazinones in maize is absent in lines homozygous for a mutation known as bx1 (Hamilton, 1964). Homozygous bx1 plants grow normally, but are extremely susceptible to the above mentioned pathogen infections, further implicating DIMBOA and related compounds in a resistance mechanism. Furthermore, the mutant plants are not auxotrophs for tryptophan, a DIMBOA precursor, indicating that the mutation is specific for a gene involved in the DIMBOA biosynthesis pathway.
Genetic analysis with monosome lines and translocation lines has resulted in the localization of the Bx1 locus on the short chromosome arm of chromosome 4 near Rp4 (Simcox et al., 1985). Bx1 mapped with the Recombinant Inbred System to the short arm of chromosome 4, and within the limits of the method, was located at exactly the same map position as Bx2 (Burr et al., 1991). Further, genes which influence the benzoxazinone concentrations as drastically as the Bx1 genes are not known. Investigations of inbred lines led to an estimation of 7 additional loci associated with benzoxazinone biosynthesis, although the loci only condition the formed amount of benzoxazinone (Dunn et al., 1981). The available genetic data indicates that the Bx1 gene is a key enzyme in DIMBOA biosynthesis.
In one study, the Bx1 gene was reported to have been cloned (Frey et al., WO 93/2244). In this case, a cytochrome p450 gene was identified which genetically mapped near the Bx1 locus. The suggestion that this was Bx1 was based upon the close genetic linkage of the cloned gene to Bx1, and also because Bx1 was believed to be a cytochrome p450 gene. Upon further analysis of the reported bx1 mutant, however, it was realized that this was not the Bx1 locus and was instead a secondary gene involved in DIMBOA biosynthesis (Frey et al. 1995).
There has, therefore, been a failure in the art to identify the key gene in the biosynthesis of benzoxazinones in maize, Bx1. Still further, the art has failed in identifying methods and compositions for the production of transgenic plants having enhanced benzoxazinone biosynthesis as a result of having been transformed with one or more genes of the benzoxazinone biosynthetic pathway, for example, Bx1. Such plants are needed because, even in plants naturally producing DIMBOA or DIBOA, benzoxazinone levels are highest early in development and fall significantly as the plant ages. Thus DIMBOA mediated resistance to ECB is limited to the first brood of the insect, whereas infestation with the second brood takes place when the DIMBOA concentration in the plant has dropped (Niemeyer et al., 1988). Therefore, while the high levels of DIMBOA in young plants serve as an effective agent in minimizing the damage caused by insects and other deleterious agents, the limited levels of DIMBOA biosynthesis in older tissues diminishes its protective effects.
Elucidation of the DIMBOA biosynthetic pathway, as well as the cloning of genes involved in the pathway, would allow for the production of transgenic plants with enhanced profiles of DIMBOA biosynthesis. Such plants would have improved resistance to a broad spectrum of insects, chemicals and pathogens and represent a significant advance to agriculture. To date, however, reaching the goal of producing such plants has been severely limited by the general lack of information regarding the DIMBOA biosynthetic pathway and the genes which encode enzymes in the pathway.
SUMMARY OF THE INVENTION
In one aspect of the invention, transgenic plants are provided having enhanced levels of benzoxazinone biosynthesis, and concomitantly, resistance to various pests, pathogens and chemical agents. One aspect in achieving this goal, which is provided by the current invention, is an isolated Bx1 polypeptide. The Bx1 polypeptide may, in particular embodiments of the invention, be a maize polypeptide. The polypeptide may comprise a transit peptide having the amino acid sequence set forth as residue 1 to about residue 85-100 of SEQ ID NO:2. The polypeptide may further have the full length amino acid sequence as set forth in SEQ ID NO:2. The polypeptide may still further comprise a transit peptide heterologous to said Bx1 transit polypeptide. In particular embodiments of the invention, the transit peptide may be selected from the group consisting of chlorophyll a/b binding protein transit peptide, small subunit of ribulose bisphosphate carboxylase transit peptide, EPSPS transit peptide and dihydrodipocolinic acid synthase transit peptide.
In another aspect of the invention, an isolated nucleic acid encoding a Bx1 polypeptide is provided. The isolated nucleic acid encoding a Bx1 polypeptide may be a maize polypeptide. The isolated nucleic acid may further comprise a nucleic acid segment encoding a transit peptide having the amino acid sequence set forth as residue 1 to about residue 85-100 of SEQ ID NO:2. The isolated nucleic acid may have the nucleic acid sequence as set forth in SEQ ID NO:1.
In yet another aspect of the invention, an expression cassette is provided comprising a DNA sequence encoding a Bx1 polypeptide linked operably to a promoter functional in a plant cell. The expression cassette may encode a Bx1 polypeptide with an amino acid sequence as set forth in SEQ ID NO:2. In particular embodiments the expression cassette further comprising a selectable marker gene. The expres

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