Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part
Reexamination Certificate
1999-11-16
2002-09-10
Fox, David T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
Higher plant, seedling, plant seed, or plant part
C435S468000, C435S471000, C435S411000, C435S413000, C435S414000, C435S415000, C435S416000, C435S419000, C435S418000, C435S422000, C435S417000, C435S427000, C435S069700, C435S069800, C435S193000, C800S278000, C800S287000, C800S298000, C800S288000
Reexamination Certificate
active
06448476
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates in general to herbicide resistance in plants, and more particularly to a new class of phosphonate metabolizing genes and methods of using these genes for improving plant tolerance to phosphonate herbicides.
DESCRIPTION OF THE PRIOR ART
Phosphorous containing organic molecules can be naturally occurring or synthetically derived. Organic molecules containing phosphorous-carbon (C—P) bonds are also found naturally or as synthetic compounds, and are often not rapidly degraded, if at all, by natural enzymatic pathways. Synthetic organophosphonates and phosphinates, compounds that contain a direct carbon-phosphorous (C—P) bond in place of the better known carbon-oxygen-phosphorous linkage of phosphate esters (Metcalf et al., Gene 129:27-32, 1993), have thus been widely used as insecticides, antibiotics, and as herbicides (Chen et al., J. Biol. Chem. 265:4461-4471, 1990; Hilderbrand et al.,
The role of phosphonates in living systems
, Hilderbrand, R. L., ed, pp. 5-29, CRC Press, Inc., Boca Raton, Fla., 1983). Phosphonates are ubiquitous in nature, and are found alone and in a diversity of macromolecular structures in a variety of organisms (Jiang et al., J. Bacteriol. 177:6411-6421, 1995). Degradation of phosphonate molecules proceeds through a number of known routes, a C—P lyase pathway, a phosphonatase pathway, and a C—N hydrolysis pathway (Wanner, Biodegradation 5:175-184, 1994; Barry et al., U.S. Pat. No. 5,463,175, 1995). Bacterial isolates capable of carrying out these steps have been characterized (Shinabarger et al., J. Bacteriol. 168:702-707, 1986; Kishore et al., J. Biol. Chem. 262:12,164-12,168, 1987; Pipke et al., Appl. Environ. Microbiol. 54:1293-1296,1987; Jacob et al., Appl. Environ. Microbiol. 54:2953-2958, 1988; Lee et al., J. Bacteriol. 174:2501-2510, 1992; Dumora et al., Biochim. Biophys. Acta 997:193-198, 1989; Lacoste et al., J. Gen. Microbiol. 138:1283-1287, 1992). However, with the exception of phosphonatase and glyphosate oxidase (GOX), other enzymes capable of carrying out these reactions have not been characterized.
Several studies have focused on the identification of genes required for C—P lyase degradation of phosphonates. Wackett et al. (J. Bacteriol. 169:710-717, 1987) disclosed broad substrate specificity toward phosphonate degradation by
Agrobacterium radiobacter
and specific utilization of glyphosate as a sole phosphate source. Shinabarger et al. and Kishore et al. disclosed C—P lyase degradation of the phosphonate herbicide, glyphosate, to glycine and inorganic phosphate through a sarcosine intermediate by Pseudomonas species.
E. coli
B strains had previously been shown to be capable of phosphonate utilization (Chen et al.), whereas
E. coli
K-12 strains were incapable of phosphonate degradation. However, K-12 strains were subsequently shown to contain a complete, though cryptic, set of genes (psiD or phn) capable of phosphonate utilization (Makino et al.), as mutants were easily selected by growth on low phosphate media containing methyl- or ethyl-phosphonate as sole phosphorous sources. Such K-12 strains adapted for growth on methyl- or ethylphosphonate were subsequently shown to be able to utilize other phosphonates as sole phosphorous sources (Wackett et al., J. Bacteriol. 169:1753-1756, 1987).
Avila et al. (J. Am. Chem. Soc. 109:6758-6764, 1987) were interested in the mechanistic appraisal of biodegradative and detoxifying processes as related to aminomethyl-phosphonates, including elucidating the intermediates, products, and mechanisms of the degradative dephosphorylation process. Avila et al. studied the formation of dephosphorylated biodegradation products from a variety of aminophosphonate substrates in
E. coli
K-12 cultures previously adapted to growth on ethylphosphonate. Furthermore, Avila et al. utilized N-acetyl-AMPA (N-acetyl-amino-methyl-phosphonate) as a sole phosphate source in some of their studies in order to show that acetylated AMPA was not inhibitory to C—P bond cleavage. In addition, Avila et al. noted that N-acetyl-AMPA was able to serve as a sole phosphate source during
E. coli
K-12 growth, however, they did not observe N-acetyl-AMPA formation when AMPA was used as a sole phosphate source. Their results indicated that AMPA was not a substrate for acetylation in
E. coli.
Chen et al. identified a functional psiD locus from
E. coli
B by complementation cloning into an
E. coli
K-12 strain deficient for phosphonate utilization, which enabled the K-12 strain to utilize phosphonate as a sole phosphate source (J. Biol. Chem. 265:4461-4471, 1990). Chen et al. thus disclosed the DNA sequence of the psiD complementing locus, identified on a 15.5 kb BamHI fragment containing 17 open reading frames designated phnA-phnQ, comprising the
E. coli
B phn operon. The cryptic phn (psiD) operon from
E. coli
K-12 was subsequently found to contain an 8-base pair insertion in phnE. The resulting frameshift in phnE not only results in defective phnE gene product, but also apparently causes polar effects on the expression of downstream genes within the operon, which prevent phosphonate utilization (Makino et al., J. Bacteriol. 173:2665-2672, 1991). The operon has been more accurately described to contain the genes phnC-phnP by the work of Makino et al. Further research has been directed to understanding the nature of the function of each of the genes within this operon (Chen et al., J. Biol. Chem. 265:4461-4471, 1990; Makino et al., J. Bacteriol. 173:2665-2672, 1991; Wanner et al., FEMS Microbiol. Lett. 100:133-140, 1992; Metcalf et al., Gene 129:27-32, 1993; Ohtaki et al., Actinomyceteol. 8:66-68, 1994). In all of these efforts, the phnO gene has been implicated as a regulatory protein based on its similarity to other nucleotide binding proteins containing structural helix-turn-helix motifs. Furthermore, mutagenesis of genes in the phn operon demonstrated that phnO was not required for phosphonate utilization, further supporting the proposed regulatory function for this gene (Metcalf et al., J. Bacteriol. 173:587-600, 1991), at least for the phosphonates tested. Homologous phn sequences have been identified from other bacteria, including a gene substantially similar to
E. coli
phnO, isolated from
S. griseus
, using nucleotide sequences deduced from those in the
E. coli
phnO gene (Jiang et al., J. Bacteriol. 177:6411-6421, (1995); McGrath et al., Eur. J. Biochem. 234:225-230, (1995); Ohtaki et al., Actinomyceteol. 8:66-68, (1994)). However, no function other than as a regulatory factor has been proposed for phnO. A regulatory role for phnO in the CP lyase operon has been cited again in a recent review (Berlyn, Microbiol. Molec. Biol. Rev. 62:814-984, 1998).
Advances in molecular biology, and in particular in plant sciences in combination with recombinant DNA technology, have enabled the construction of recombinant plants which contain nonnative genes of agronomic importance. Furthermore, when incorporated into and expressed in a plant, such genes desirably confer some beneficial trait or characteristic to the recombinant plant. One such trait is herbicide resistance. A recombinant plant capable of growth in the presence of a herbicide has a tremendous advantage over herbicide-susceptible species. In addition, herbicide tolerant plants provide a more cost effective means for agronomic production by reducing the need for tillage to control weeds and volunteers.
Chemical herbicides have been used for decades to inhibit plant metabolism, particularly for agronomic purposes as a means for controlling weeds or volunteer plants in fields of crop plants. A class of herbicides which have proven to be particularly effective for these purposes are known as phosphonates or phosphonic acid herbicides. Perhaps the most agronomically successful phosphonate herbicide is glyphosate (N-phosphono-methyl-glycine).
Recombinant plants have been constructed which are tolerant to the phosphonate herbicide glyphosate. When applied to plants, glyphosate is absorbed into the plant tissues and inhibit
Ball Timothy K.
Fox David T.
Hoerner, Jr. Dennis R.
Kruse David H
Monsanto Technology LLC
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