Note: Descriptions are shown in the official language in which they were submitted.
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TITLE
GENETICALLY TRANSFORMED PLANTS DEMONSTRATING
RESISTANCE TO PORPHYRINOGEN
BIOSYNTHESIS-INHIBITING HERBICIDES
FIELD OF INVENTION
The present invention relates to the field of molecular biology and the
genetic transformation of plants with foreign gene fragments. More
specifically
the invention provides a transformed plant demonstrating resistance to
porphyrinogen biosynthesis inhibiting (PBI) herbicides and genes encoding PBI
resistant protoporphyrinogen oxidase (PROTOX) enzymes.
BACKGROUND
Rational design of useful herbicides is often dependent on knowledge of
plant metabolic enzymatic pathways. One of the most important of these is the
plant porphyrin pathway responsible for the synthesis of chlorophyll, heme,
and
other pigments vital to plant metabolism. To date, thousands of compounds have
been developed that exert phytotoxicity through the disruption of the plant
porphyrin pathway. These compounds are commonly known as porphyrin
biosynthesis inhibiting (PBI) herbicides and represent a significant portion
of the
herbicide market.
The early steps of porphyrin biosynthesis (Figure 1 ) occur in plastids,
producing the porphyrin intermediate protoporphyrinogen (PROTOGEN).
PROTOGEN is converted into protoporphyrin (PROTO) by the enzyme
protoporphyrinogen oxidase (PROTOX). PROTOX isozymes are located in both
the plastid and the mitochondrion. The PROTOGEN that serves as the substrate
for PROTOX in the mitochondrion is transported from the plastid. The vast
majority of PBI compounds act as inhibitors of the plastid and mitochondriai
PROTOX enzymes (Duke et al., ACS Symp. Ser. (1994), 559(Porphyric
Pesticides, I91-204). In the presence of light and molecular oxygen, blockage
of
the porphyrin pathway at the PROTOX enzyme results in the production of toxic
oxygen species including singlet oxygen, superoxide, peroxide and hydroxyl
radicals. These toxic species trigger peroxidation of polyunsaturated fatty
acid
moieties in cell lipid membranes resulting in plant cell death (Komives et
al., ACS
Symp. Ser. (1994), 559(Porphyric Pesticides), 177-90). This production of
toxic
oxygen species is believed to be caused by the movement of PROTOGEN to the
plasma membrane following blockage of the plastid PROTOX. In the plasma
membrane there exists a third form of PROTOX which, unlike the plastid and
mitochondrial forms, is resistant to PBI compounds. This PROTOX converts the
PROTOGEN into PROTO within the plasma membrane. The plasma membrane
lacks enzymes to efficiently further metabolize the PROTO, resulting in its
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accumulation. In the presence of light and oxygen, PROTO photooxidizes
producing the toxic oxygen intermediates.
Although PBI herbicides are useful, they affect an enzymatic pathway
common to all plants and therefore are generally indiscriminate in their
action.
Consequently, care must be exercised in the application of these compounds
around crop plants. Crop plants resistant to PBI herbicide compounds would
represent a useful companion to the use of PBI compounds in the field. Because
it
is the site of action of the majority of the PBI herbicides, the PROTOX
enzyme,
promises to play an important role in the development of such plants.
PROTOX is a ubiquitous enzyme and has been isolated from plants
(Jacobs et al., Biochem J. 244, 219, ( 1987)), yeast (Labbe-Bois et al., (
1990) in
Biosynthesis of I-Ieme and Chlorophylls, Dailey, H.A. ed., pp 235-285, McGraw-
Hill Publishing Co. New York), bacteria (Klemm et al., J. Bacteriol., 169,
5209,
(1987)), and mammalian species (bailey et al., Biochem., 26, 2697, (1987);
Proulx
et al., Protein Sci., 1, 801, (1992); Siepker et al., Biochem. Biophys. Acta.,
913,
349 ( 1987}), PROTOX deficiencies have also been reported in a variety of
species including man (Kappas et al., in The Metabolic Basis Of Inherited
Disease, Stanbury et al., ed. (1983) McGraw-Hill, New York, pp 1301-1348) and
bacteria (Sasarman et al., J. Gen. Microbiol., 113, 297, (1979)).
Although the enzymatic site and mechanism of action of PBI compounds
is known, little progress has been made in developing plants resistant to
these
herbicides. PBI resistance has been shown in plant tissue culture, but not in
whole
plants. For example, Pornprom et al. (Weed Res., 3apan, 39, 102, ( 1994) have
reported the selection of soybean cell lines resistant to several PBI
compounds
including oxyfluorfen. Cells were made resistant using suspension cultures
grown
in increasing concentrations of PBI compound. Cell lines developed from this
selection process maintained PBI resistance for six months. No seeds or plants
were generated from any cell line.
PBI resistance has also been reported in green algae (Shibata et al., Res.
Photosynth., Proc. Int. Congr. Photosynth., 9th (1992), Volume 3, 567-70.
Editor(s): Murata, Norio. Publisher: Kluwer, Dordrecht, Netherlands); Sato et
al.,
ACS Symp. Ser. (1994), 559(Porphyric Pesticides), 91-104) where strains of
Chlamydomonas reinhardtii have been isolated with resistance to certain PBI
photo-bleaching herbicides including acifluofenethyl, oxyfluorfen and
oxadiazon.
These organisms derive their resistance from a PBI-resistant PROTOX produced
by a mutant gene.
Finally, WO 9534659 describes the isolation of plant and yeast PROTOX
sequences and suggests that the overexpression of those sequences or mutated
versions of them in plant tissues will result in resistance to PBI compounds.
2
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WO 98133927 PCTIUS98/01622
WO 9534659 does not teach the transformation of plant tissue with prokaryotic
genes that encode a PROTOX resistant to PBI compounds, and they do not
demonstrate PBI resistance in any transformed plant.
The hemG gene, encoding the wildtype PROTOX gene in E. toll, has been
isolated and sequenced (Sasarman et al., J. Gen. Microbiol., 113, 297, (
1979);
Sasarman et al., Can. J. Microbiol., 39, 1155, (1993)). The E. toll PROTOX has
been found to be resistant to PBI-inhibiting compounds (Jacobs et al., Arch.
Biochem. Biophys., 280, 369, (1990).
The problem to be overcome, therefore, is how to modify the PROTOX
enzyme to develop plants resistant to PBI herbicides. Applicants have solved
this
problem by cloning an E. toll gene encoding a PBI-resistant PROTOX enzyme
into a unique vector and transforming suitable plant tissue with the gene.
Transformed plants demonstrate significant PBI resistance. Seed derived from
transformed plants give rise to plants carrying the PBI-resistant phenotype,
demonstrating the trait is heritable.
The hemG PROTOX from E. toll differs from the other previously
characterized PROTOX enzymes from plant sources not only in its sensitivity to
PBI herbicides, but also in its size, sequence and cofactor requirements.
These
significant differences make the functional expression of the E. toll gene in
plants
less than certain. For example, the E. toll hemG gene codes for a protein of
21 kDa (Sasarman et al., Can. J. Microbiol., 39, 1155, (1993)), while the
three
complete PROTOX genes characterized from plants (two from Arabidopsis
thaliana [Narita,S., genbank ID g1183006 and WO 9534659] and one from maize
[WO 9534659]) encode proteins of 55-59 kDa. Furthermore, homology
comparisons using the GCG Bestfit program [Program Manual for the Wisconsin
Package, Version 8, September 1994, Genetics Computer Group, 575 Science
Drive, Madison, Wisconsin, USA 53711 ] between the hemG gene of E. toll and
one of the Arabidopsis genes (genbank ID g 1183006) demonstrate only 22%
identity over the 181 amino acids of the shorter hemG protein. The E. toll
PROTOX is inactivated by detergent solubilization, whereas the plant enzyme
can
be detergent-extracted and retain activity (Jacobs et al., Arch. Biochem.
Biophys.,
229, 312 (1984)). Based on this plus the fact that prokaryotic forms of PROTOX
are sensitive to the respiratory inhibitor cyanide whereas plant enzymes are
not, it
has been suggested that the PROTOX from E. toll and other Prokaryotes is
obligatorily coupled to the cell's respiratory chain whereas plant enzymes can
use
molecular oxygen as the terminal electron acceptor (Jacobs et al., Arch.
Biochem.
Biophys., 211, 305 (1981); (Jacobs et al., Arch. Biochem. Biophys., 229, 312
(1984)). Moreover, the environment in which the E. toll PROTOX normally
functions in the bacterial cytosol is markedly different than in the plant
chlorplast.
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Adding to the uncertainty of hemG expression in plants are reports of the
problematic expression of bacterial proteins in eukaryotic species and plants
in
particular. For example, the expression of a full-length lepidopteran-specific
Bacillus thuringiensis toxin has been reported to be unsuccessful in yielding
insecticidal levels of expression in some plant species (Vaeck et al., Nature
(1987)
vol. 328, pp. 33-37). Similarly, it has also been reported that the full
length gene
from Bacillus thuringiensis kurstaki HD-73 gave some insecticidal effect in
tobacco (Adang et al., UCLA Symp. Mol. Cell. Biol. ( 1987) vol. 48, pp. 345-3
53 ).
However, the Bacillus thuringiensis mRNA detected in these plants was only
1.7 kb compared to the expected 3.7 kb indicating improper expression of the
gene.
Finally, if the E. coli PROTOX enzyme is expressed in the plant and is
able to function in the chloroplast, there is still an uncertainty as to the
effect of
ectopically expressing a foreign PROTOX enzyme at high, constitutive levels.
The plant porphyrin pathway is highly regulated both by allosteric and genetic
controls (S. I. Beale and J. D. Weinstein, New Comprehensive Biochemistry
(1991) vol. 19, pp. 155-235) suggesting that the proper flux through the
pathway
is critical to the health of the plant. Altering the activity of PROTOX by
introducing the E. coli PROTOX gene might disrupt the regulation of the
porphyrin pathway and lead to highly deleterious effects on the plant.
Duke et al. (Second International Weed Control Congress, ( 1996)
pp. 775-780, Protoporphyrinogen oxidase inhibitors - their current and future
roles) have considered the possibility of engineering resistance to PBI
herbicides
using a bacterial PROTOX gene. They concluded that this is unlikely to succeed
because inhibition of the native plastid PROTOX would still lead to
accumulation
of PROTO and integration of the bacterial PROTOX into the normal plastid
porphyrin biosynthesis will be difficult.
Considering the differences between the bacterial and plant PROTOX
genes, the lack of success in the literature in the expression of Bacillus
genes in
plant tissue, and the potential for deleterious effects of expression of a
foreign
PROTOX enzyme, Applicants' success in the expression of active bacterial
PROTOX in plant tissue without deleterious effects is highly unexpected and
unusual.
SUMMARY OF THE INVENTION
The present invention provides a plant which is resistant to porphyrin
biosynthesis-inhibiting herbicides, the plant containing a chimeric gene
encoding
a herbicide-resistant protoporphyrinogen oxidase activity and wherein the
porphyrin biosynthesis-inhibiting herbicides to which the plant is resistant
are
according to the formula:
4
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J-G
I
wherein
G is
R5
f
R2
R3 ~ R3 , R3
R1 R1 R1
G-1 G-2 G-3
R6 R6\ Y
4
N Y p R 5 \N R4
~R
p ~ X R5
R1 R1 R1
G-4 G-5 G-6
D7 0\\ ~~ R$ 0 0
R5 N \5//
\ 'Rq
/~\F
R~
3 3 or X1
R1 R1 R1
G-7 G-8 G-9
and wherein J is
S
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WO 98/33927 PCTIUS98l01622
O 0 0
R9 R11\
r ~n N N
Z I ~N- Z N- N-
/ .
0 m R10~N/ R12 N
O
J-1 J-2 J-3
R13
Q Q
Rlq N Q R N R16~N
Z ~ I N - Z JJ~~~ ~' '''~~ _~\N -
W N\ , R10~N~ . R17 -m
\'\1O Q
Q
J-4 J-5 J-6
Q 0
Rll Q
\N~ O~ R18\
N- N- N
/ N-
R12 ' R18 N ' N.~ /
N
Q
J-7 J-B J-9
R19 R20
N-
N
R9Z Q R1 R9 1
Z ~ Q
R10~N~N~ ~ ~ R10~N~ .
m ~' I( ''\,m
Q Q
J-10 J-11 J-12
N N - R23
R21\N~ R9 ~ R9
1 ~' N
Q Z Q1. Z
R22~N ' R10~N/ ' R10~N\N/
Q
J-13 J-14 J-15
6
__.-.._...-. ......... w._~_..___._. .___~.~-.~ .~.T.
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29 R23
R
~N~C\NH/ R ~'N~C~NH/
Z
R25/N~N~ ~ R10Z~ /OR26 ~ R10~ /OR26
0 O
J-16 J-17 J-18
23
R R R20 0 R 9 OR21
Z N- Rlg
R10 m w N/ N- O
0 \\ , N~
0
J-19 J-20 J-21
R19 R14 N QR21 R9 ~,T Q
\ /N
Z ~-
W N~ , or R10~N~N~
Q
J-22 J-23 J-29
wherein the dashed line in J-5, J-6, J-12 and J-24 indicates that the left-
hand
ring contains only single bonds or one bond in the ring is a
carbon-carbon double bond;
XisOorS;
YisOorS;
R~ is hydrogen or halogen;
R2 is H; C1-Cg alkyl; Cl-Cg haloalkyl; halogen; OH; OR2~; SH; S(O)PR27;
COR27; C02R2~; C(O)SR27; C(O)NR29R3o; CHO; CR29=NOR36;
CH=CR37C02R27; CH2CHR3~C02R2~; C02N=CR31R32; vitro;
cyano; NHS02R33; NHSO~NHR33; NR27R3g; NHZ; or phenyl
optionally substituted with at least one member independently selected
from C1-C4 alkyl;
p is 0; 1; or 2;
R3 is C~-CZ alkyl; Cl-C2 haloalkyl; OCH3; SCH3; OCHF2; halogen; cyano
or vitro;
R4 is H; Cl-C3 alkyl; C~-C3 haloalkyl; or halogen;
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R5 is H; C1-C3 alkyl; halogen; C1-C3 haloalkyl; cyclopropyl; vinyl; C2
alkynyl; cyano; C(O)R3g; C02R3g; C(O)NR3gR39; CR34R35CN;
CR34R35C(O}R38; CR34R35C02R38; CR34R35C(O)NR38R39;
CHR340H; CHR340C(O)R3g; or OCHR340C(O)NR3gR39; or
when G is G-2 or G-6, then R4 and R5 can be taken together with the carbon
to which they are attached to form C=O;
R6 is C ~-C6 alkyl; C 1-C6 haloalkyl; C2-C6 alkoxyalkyl; C;-C6 alkenyl; or
C3-C6 alkynyl;
XI is a direct bond; O; S; NH; N(C~-C3 alkyl); N(CI-C3 haloalkyl); or
N(allyl);
R~ is H; Cl-C6 alkyl; C~-C6 haloalkyl; halogen; S(O)~(C~-C6 alkyl); or
C(=O)R4o;
Rg is H; Cl-Cg alkyl; C3-Cg cycloalkyl; C3-Cg alkenyl; C3-Cg alkynyl;
CI-Cg haloalkyl; C~-Cg alkoxyalkyl; C3-Cg alkoxyalkoxyalkyl; C3-Cg
haloalkynyl; C3-Cg haloalkenyl; C~-Cg alkylsulfonyl; C~-Cg
haloalkylsulfonyl; C3-Cg alkoxycarbonylalkyl; S(O)2NH(C~-Cg
alkyl); C(O)R4 ~ ; or benzyl optionally substituted on the phenyl ring
with R42;
n and m are each independently 0; 1; 2; or 3; provided that m + n is 2 or 3;
Z is CR9R~o; O; S; S(O); S(O}2; or N(C1-C4 alkyl);
each R9 is independently H; C 1-C3 alkyl; halogen; hydroxy; C ~-C6 alkoxy;
C~-C6 haloalkyl; C1-C6 haloalkoxy; C2-C6 alkylcarbonyloxy; or
C2-C6 haloalkylcarbonyloxy;
each Rio is independently H; C1-C3 alkyl; hydroxy; or halogen;
R11 and R12 are each independently H; halogen; C~-C6 alkyl; C3-C6
alkenyl; or C1-C6 haloalkyl;
R13 is H; C ~-C6 alkyl; C I-C6 haloalkyl; C3-C6 alkenyl; C3-C6 haloalkenyl;
C3-C6 alkynyl; C3-C6 haloalkynyl; HC(=O); (C~-C4 alkyl)C(=O); or
NH2;
R14 is Ci-C6 alkyl; C~-C6 alkylthio; C1-C6 haloalkyl; or N(CH3)2;
W is N or CR15;
R~5 is H; C1-C6 alkyl; halogen; or phenyl optionally substituted with Cl-C6
alkyl, 1-2 halogen, C1-C6 alkoxy, or CF3;
each Q is independently O or S;
Q ~ is O or S;
Z1 is CR16R»; O; S; S(O); S(O)2; orN(Cl-C4 alkyl);
each R16 is independently H; halogen; hydroxy; C1-C6 alkoxy; C1-C6
haloalkyl; CI-C6 haloalkoxy; C2-C6 alkylcarbonyloxy; or C2-C6
haloalkylcarbonyloxy;
8
___~..~.._ _. ..__ . _~......._ T ~
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WO 98/33927 PCT/US98/O1G22
each R1~ is independently H; hydroxy; or halogen; or
when R16 and R» are bonded to adjacent atoms they can be taken together
with the carbons to which they are attached to form
optionally substituted with at least one member selected from 1-2
halogen and 1-2 CI-C3 alkyl;
R~8 is C~-C6 alkyl; halogen; or C~-C~ haloalkyl;
R19 and R2~ are each independently H; C1-C6 alkyl; or C~-C6 haloalkyl;
R21 and R22 are each independently Cl-C6 alkyl; C1-C6 haloalkyl; C3-C6
alkenyl; C3-C6 haloalkenyl; C3-C6 alkynyl; or C;-C6 haloalkynyl;
R23 is H; halogen; or cyano;
R24 is C~-C6 alkylsulfonyl; C1-C6 alkyl; C1-C6 haloalkyl; C3-C6 alkenyl;
C3-C6 alkynyl; C~-Cg alkoxy; CI-C6 haloalkoxy; or halogen;
R25 is C1-C6 alkyl; C~-C6 haloalkyl; C3-C6 alkenyl; or C3-C6 alkynyl;
R26 is C ~-C6 alkyl; C 1-C6 haloalkyl; or phenyl optionally substituted with
C1-C6 alkyl, 1-2 halogen, 1-2 nitro, C1-C6 alkoxy, or CF3;
R2~ is C ~-Cg alkyl; C3-Cg cycloalkyl; C3-Cg alkenyl; C3-Cg alkynyl; C ~-C~
haloalkyl; C2-Cg alkoxyalkyl; C2-Cg alkylthioalkyl; C2-Cg
alkylsulfinylalkyl; C2-Cg alkylsulfonylalkyl; C ~-Cg alkylsulfonyl;
phenylsulfonyl optionally substituted on the phenyl ring with at least
one substituent selected from the group halogen and C 1-C4 alkyl;
C4-Cg alkoxyalkoxyalkyl; C4-Cg cycloalkylalkyl; C6-Cg
cycloalkoxyalkyl; C4-Cg alkenyloxyalkyl; C4-Cg alkynyloxyalkyl;
C3-Cg haloalkoxyalkyl; C4-Cg haloalkenyloxyalkyl; C4-Cg
haloalkynyloxyalkyl; C6-Cg cycloalkylthioalkyl; C4-Cg
alkenylthioalkyl; C4-Cg alkynylthioalkyl; C1-C4 alkyl substituted with
phenoxy or benzyloxy, each ring optionally substituted with at least
one substituent selected from the group halogen, Cl-C3 alkyl and
C 1-C3 haloalkyl; C4-Cg trialkylsilylalkyl; C3-Cg cyanoalkyl; C;-Cg
halocycloalkyl; C3-Cg haloalkenyl; CS-Cg alkoxyalkenyl; CS-Cg
haloalkoxyalkenyl; CS-Cg alkylthioalkenyl; C3-Cg haloalkynyl; CS-Cg
alkoxyalkynyl; C5-Cg haloalkoxyalkynyl; CS-Cg alkylthioalkynyl;
C2-Cg alkylcarbonyl; benzyl optionally substituted with at least one
substituent selected from the group halogen, Cl-C3 alkyl and C1-C3
haloalkyl; CHR34COR28; CHR34CO~R28; CHR34P(O)(OR28)2;
CHR34P(S)(OR28)2; CHR34C(O)NR29R3o; or CHR34C(O)NH2;
R2g is C 1-C6 alkyl; C2-C6 alkenyl; C2-C6 alkynyl; or tetrahydrofuranyl;
9
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WO 98/33927 PCT/US98/01622
R29 and R3 ~ are independently hydrogen or C 1-C4 alkyl;
R3~ and R32 are independently C~-C4 alkyl or phenyl optionally substituted
with at least one substituent selected from the group halogen, CI-C3
alkyl, and C~-C3 haloalkyl; or
R29 and R3~ can be taken together to form -(CH~)5-, -(CH2)4- or
-CH2CH20CH2CHz-, each ring thus formed optionally substituted
with a substituent selected from the group C~-C; alkyl, phenyl and
benzyl; or
R3 ~ and R32 can be taken together with the carbon to which they are
attached to form C3-Cg cycloalkyl;
R33 is C1-C4 alkyl; C~-C4 haloalkyl; or C~-C6 alkenyl;
R34 and R35 are independently H or C~-C4 alkyl;
R36 is H; C~-C6 alkyl; C3-C6 alkenyl; or C3-C6 alkynyl;
R37 is H; Cl-C4 alkyl; or halogen;
R38 is H; C ~-C6 alkyl; C3-C6 cycloalkyl; C3-C6 alkenyl; C3-C6 alkynyl;
C2-C6 alkoxyalkyl; C1-C6 haloalkyl; phenyl optionally substituted
with at least one substituent selected from the group halogen, C~-C4
alkyl, and C~-C4 alkoxy; -CH2C02(C1-C4 alkyl); or
-CH(CH3)C02(C~-C4 alkyl);
R39 is H; Cl-C2 alkyl; or C(O)O(C~-C4 alkyl);
R4~ is H; C~-C6 alkyl; C1-C6 alkoxy; or NH(C~-C6 alkyl);
R41 is CI-C6 alkyl; CI-C6 haloalkyl; C~-C6 alkoxy; NH(Ci-C6 alkyl);
phenyl optionally substituted with R42; benzyl; or C2-Cg
dialkylamino; and
R42 is Cl-C6 alkyl; 1-2 halogen; C~-C6 alkoxy; or CFA.
The invention also provides stably transformed plants expressing a
chimeric gene where the chimeric gene comprises:
(i) a nucleic acid fragment encoding protoporphyrinogen oxidase
enzyme which is resistant to inhibition by porphyrin biosynthesis inhibiting
herbicides; and
(ii) a plant regulatory sequence,
wherein the nucleic acid fragment encoding protoporphyrinogen oxidase enzyme
is operably linked to the regulatory sequence.
The chimeric gene may optionally further contain various constitutive and
inducible plant promoters and plant organelle targeting sequences useful for
the
expression of the gene and in the translocation of the gene into suitable
organelles.
BRIEF DESCRIPTION OF THE FIGURES.
BIOLOGICAL DEPOSITS AND SEQUENCE LISTING
Figure 1 is a schematic of the porphyrin biosynthetic pathways in plants.
_._~r._._ . _.r T ... . _
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Figure 2 illustrates leaf damage produced in tobacco transformants treated
with 300 ~M PBI-1.
Figure 3 illustrates ion leakage from leaves of tobacco transformants
incubated in 500 qM PBI-1.
Figure 4 illustrates tobacco leaf spotting damage on transformants in
response to varying concentrations of PBI-1.
Figure 5 is a plasmid map of the p35S-PROTOX binary transformation
vector which contains a plant nptII selectable marker, right and left T-DNA
border
fragments, and the hemG expression cassette derived from pHGV4.
Figure 6 is a plasmid map of pHGV4 which contains the hemG expression
cassette including the hemG gene under the control of the 3 5 S cauliflower
mosaic
virus promoter, a cab 5' leader, a chloroplast targeting sequence, and a nos
3'
terminator sequence.
Figure 7 is a plasmid map of pBT455, used in the construction of pHGV4,
containing the dapA gene under the control of the 355 cauliflower mosaic virus
promoter, a cab 5' leader,a chloroplast targeting sequence, and a nos 3 ~
terminator
sequence.
Figure 8 is a plasmid map of pZS 199, used in the construction of the
binary plasmid p35S-PROTOX, containing a chimeric gene nopaline
synthase/neomycin phosphotransferase, the left and right borders of the T-DNA
of
the Ti plasmid, the E. coli IacZ alpha-complementing segment with unique
restriction endonuclease sites for EcoRI, KpnI, BamHI, HinDIII, and SaII, the
bacterial replication origin from the Pseudomonas plasmid pVSI, and the
bacterial neomycin phosphotransferase gene from TnS.
Figure 9 illustrates PBI-1-induced ion leakage damage from leaf disks of
tobacco transformants.
Figures l0a-f illustrate leaf spotting assays on primary tobacco
transformants with six diverse PBI compounds, including PBI-1, PBI-2, PBI-3,
PBI-4, PBI-5 and PBI-6.
Figure 11 illustrates a leaf spotting assay of sensitivity of PROTOX-24
and Binary Control-2 to varying concentrations of PBI-1.
The plasmid pHGV4 containing the dapA gene under the control of the
3 5 S cauliflower mosaic virus promoter, a cab 5' leader, a chloroplast
targeting
sequence and a nos 3' terminator sequence was deposited on 7 August 1996 with
the American Type Culture Collection international depository (12301 Parklawn
Drive, Rockville, MD 10852 U.S.A.) under the terms of the Budapest Treaty and
is identified by the designation ATCC 97675.
The plasmid p35S-Protox containing a plant nptII selectable marker, right
and left T-DNA border fragments, and the hemG expression cassette was
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deposited on 7 August 1996 with the American Type Culture Collection
international depository (12301 Parklawn Drive, Rockville, MD 20852 U.S.A.)
under the terms of the Budapest Treaty and is identified by the designation
ATCC 97674.
Applicants have provided 7 sequences in conformity with "Rules for the
Standard Representation of Nucleotide and Amino Acid Sequences in Patent
Applications" (Annexes I and II to the Decision of the President of the EPO,
published in Supplement No. 2 to OJ EPO, 12/1992) and with
37 C.F.R. 1.821-1.825 and Appendices A and B ("Requirements for Application
Disclosures Containing Nucleotides and/or Amino Acid Sequences").
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a chimeric gene encoding a PBI herbicide-
resistant PROTOX enzyme. The chimeric gene is linked to a plant chloroplast
transit sequence and a constitutive regulatory sequence. Plants transformed
with
the instant chimerical gene express an active PBI-resistant PROTOX enzyme and
are resistant to the toxic effects of PBI herbicides. Seeds produced from
transformed plants gave rise to mature plants having the PBI-resistant
phenotype.
In the context of this disclosure, the following terms have the meaning set
out below.
The term "homologous to" refers to the similarity between the nucleotide
sequence of two nucleic acid molecules or between the amino acid sequences of
two protein molecules. Estimates of such homology are provided by either
DNA-DNA or DNA-RNA hybridization under conditions of stringency as is well
understood by those skilled in the art [as described in Hames and Higgins
(eds.)
Nucleic Acid Hybridisation, IRL Press, Oxford, U.K.); or by the comparison of
sequence similarity between two nucleic acids or proteins.
"Gene" refers to a nucleic acid fragment that expresses a specific protein,
including regulatory sequences preceding {5' non-coding) and following (3' non-
coding) the coding region. "Native" gene refers to the gene as found in nature
with its own regulatory sequences.
A "chimeric" gene refers to a gene comprising heterogeneous regulatory
and coding sequences.
An "endogenous" gene refers to the native gene normally found in its
natural location in the genome.
A "foreign" gene refers to a gene not normally found in the host organism
but that is introduced by gene transfer.
A "coding sequence" refers to a DNA sequence that codes for a specific
protein and excludes the non-coding sequences.
12
CA 02274502 1999-06-11
WO 98133927 PCT/US98/01622
An "initiation codon" and "termination codon" refer to a unit of three
adjacent nucleotides in a coding sequence that specifies initiation and chain
termination, respectively, of protein synthesis (mRNA translation).
An "open reading frame" refers to the amino acid sequence encoded
between translation initiation and termination codons of a coding sequence.
"Suitable regulatory sequences" refer to nucleotide sequences located
upstream (5'), within, and/or downstream (3') to a coding sequence, which
control
the transcription and/or expression of the coding sequences, potentially in
conjunction with the protein biosynthetic apparatus of the cell. These
regulatory
sequences include promoters, translation leader sequences, transcription
termination sequences, and polyadenylation sequences.
An "enhancer" is a DNA sequence which can stimulate promoter activity.
It may be an innate element of the promoter or a heterologous element inserted
to
enhance the level and/or tissue-specificity of a promoter.
The term "promoter" refers to a DNA sequence in a gene, usually
upstream (S') to its coding sequence, which controls the expression of the
coding
sequence by providing the recognition for RNA polymerase and other factors
required for proper transcription. A promoter may also contain DNA sequences
that are involved in the binding of protein factors which control the
effectiveness
of transcription initiation in response to physiological or developmental
conditions. It may also contain enhancer elements.
"Constitutive promoters" refers to those that direct gene expression in all
tissues and at all times. "Organ-specific" or "development-specific" promoters
as
referred to herein are those that direct gene expression almost exclusively in
specific organs, such as leaves or seeds, or at specific development stages in
an
organ, such as in early or late embryogenesis, respectively.
"Inducible promoters" are promoters induced to activity by specific
triggers such as light or particular chemical compounds. Examples include
light-
inducible promoters, ABA inducible promoters, benzenesulfonamide-inducible
promoters, and methyl jasmonate-inducible promoters.
The term "operably linked" refers to nucleic acid sequences on a single
nucleic acid molecule which are associated so that the function of one is
affected
by the other. For example, a promoter is operably linked with a structural
gene
when it is capable of affecting the expression of that structural gene.
The term "expression", as used herein, is intended to mean the production
of the protein product encoded by a gene. "Overexpression" refers to the
production of a gene product in transgenic organisms that exceeds levels of
production in normal or non-transformed organisms.
13
CA 02274502 1999-06-11
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The "3' non-coding sequences" refers to the DNA sequence portion of a
gene that contains a polyadenylation signal and any other regulatory signal
capable of affecting mRNA processing or gene expression. The polyadenylation
signal is usually characterized by affecting the addition of polyadenylic acid
tracts
to the 3' end of the mRNA precursor.
The "translation leader sequence" refers to that DNA sequence portion of a
gene between the promoter and coding sequence that is transcribed into RNA and
is present in the fully processed mRNA upstream (5') of the translation start
codon. The translation leader sequence may affect processing of the primary
transcript to mRNA, mRNA stability or translation efficiency.
A "chloroplast targeting signal" or "chloroplast targeting sequence" is an
amino acid sequence which is translated in conjunction with a protein and
specifically directs the protein to the chloroplast.
A "mitochondria) targeting signal" or "mitochondria) targeting sequence"
is an amino acid sequence which is translated in conjunction with a protein
and
specifically directs the protein to the mitochondria.
"Chloroplast transit sequence" refers to a nucleotide sequence that encodes
a chloroplast targeting signal.
"Transformation" herein refers to the transfer of a foreign gene into the
genome of a host organism and its genetically stable inheritance. Examples of
methods of plant transformation include Agrobacterium-mediated transformation
and particle-accelerated or "gene gun" transformation technology as described
in
U.S. Patent No. 5,204,253.
The term "transformants" refer to plants which have been through the
transformation process and contain a foreign gene integrated into their
genome.
The term "primary transformants" or the "Tl generation" are of the same
genetic generation as the tissue which was initially transformed, i.e., not
having
gone through meiosis and fertilization since the transformation.
The term "secondary transformants" or the "T2, T3, T4, etc. generations"
are derived from primary transformants through one or more meiotic and
fertilization cycles. They may be derived by self fertilizations of primary or
secondary transformants or crosses of primary or secondary transformants with
other transformed or untransformed plants.
The term "tolerance" means the heritable ability of a plant to sustain less
damage than other individuals of a given species in the presence of an
injurious
concentration of a toxin or pathogen. "Resistance" refers to a special case of
tolerance in which there is a heritable ability to survive (with agronomically
acceptable injury) a concentration of toxin or pathogen that is normally
lethal or
severely injurious to individuals of given species.
14
.._._.... __.~._.__ T. T _.~......._.. . .
CA 02274502 1999-06-11
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The term "hemG" will refer to a bacterial gene encoding a PBI herbicide
resistant protoporphyrinogen oxidase enzyme.
The term "PROTOX" will refer to protoporphyrinogen oxidase. an enzyme
responsible for the conversion of protoporphyrinogen to protoporphyrin.
The terms "PROTOGEN" or "PROTOGEN IX" will refer to compound
protoporphyrinogen IX, an intermediate in the porphyrin biosynthetic pathway
and the substrate for PROTOX.
The terms "PROTO" or "PROTO IX" will refer to compound
protoporphyrin IX, an intermediate in the porphyrin biosynthetic pathway and
the
product of PROTOX.
The term "PBI herbicide" or "PBI compounds" will refer to herbicides that
inhibit the plant porphyrin biosynthetic pathway at the level of PROTOX.
Typical
PBI compounds fall into six general classes of compounds consisting of the
triazolones , cyclic imides (e.g. N-(4-chloro-5(cyclopropenyloxy) -2-
fluorophenyl)tetrahydro-2-phthalimide), thiadiazoles , pyrazoles , uracils and
diphenylethers (e.g., acifluorfen, nitrofen and oxyfluorfen).
Isolation of genes encoding PBI resistant PROTOX enzymes
Both the E. coli hemG (Sasarman et al., supra) and the Bacillus hemY
(bailey et al., J. Biol. Chem. (1994), 269(2), 813-15) genes encode PBI-
resistant
PROTOX enzymes. Recently mutations in eukaryotic forms of the PROTOX
genes (e.g., Arabidopsis, maize, and yeast, WO 9534659) have been isolated. It
is
contemplated that any of these genes will be suitable in the present invention
and
may be isolated from native sources by methods well known in the art.
The sequence of the hemG gene, encoding the PBI resistant PROTOX
from E. coli is known. The gene may be obtained by a variety of methods, the
most direct being by the use of polymerase chain reaction (PCR) using suitable
primers. In the present application genomic DNA was amplified using standard
PCR protocols [Sambrook, J. et al., Molecular Cloning: A Laboratory Manual,
Second Edition, Cold Spring Harbor Laboratory Press ( 1989)], and isolated and
purified by gel electrophoresis. Purified amplification product was then
restricted
with HaeIII and Kpnl to allow for insertion into the appropriate
transformation
vector.
Construction of chimeric genes for the expression of hemG in plants
The expression of foreign genes in plants is well-established [De Blaere et
al. (1987) Meth. Enrymol. 143:277-291]. Proper level of expression of the hemG
mRNAs may require the use of different chimeric genes utilizing different
promoters. Such chimeric genes can be transferred into host plants either
together
in a single expression vector or sequentially using more than one vector.
CA 02274502 1999-06-11
WO 98/33927 PCT/US98/01622
A preferred class of heterologous hosts for the expression of the coding
sequence of the hemG gene are eukaryotic hosts, particularly the cells of
higher
plants. Particularly preferred among the higher plants and the seeds derived
from
them are soybean, rapeseed (Brassica napus, B. campestris), sunflower
(Helianthus annus), cotton (Gossypium hirsutum), corn (Zea mays), tobacco
(Nicotiana tabacum), alfalfa (Medicago sativa), wheat (Triticum sp), barley
(Hordeum vulgate), oats (Avena sativa, L}, sorghum (Sorghum bicolor), rice
(Oryza sativa), Arabidopsis, cruciferous vegetables (broccoli, cauliflower,
cabbage, parsnips, etc.}, melons, carrots, celery, parsley, tomatoes,
potatoes,
strawberries, peanuts, grapes, grass seed crops, hardwood trees, softwood
trees,
and forage grasses. Expression in plants will use regulatory sequences
functional
in such plants.
The origin of the promoter chosen to drive the expression of the coding
sequence is not critical as long as it has sufficient transcriptional activity
to
accomplish the invention by expressing translatable mRNA for the hemG gene in
the desired host tissue. Preferred promoters for expression in all plant
organs, and
especially for expression in leaves include those directing the 195 and 35S
transcripts in Cauliflower mosaic virus [Odell et al.( 1985) Nature 313:810-
812;
Hull et al. (1987} Virology 86:482-493], small subunit of ribulose
1,5-bisphosphate carboxylase [Morelli et al.(1985) Nature 315:200; Broglie et
al.
( 1984) Science 224: 83 8; Hererra-Estrella et al.( 1984) Nature 3 I 0:115;
Coruzzi et
al.( 1984) EMBO J. 3:1671; Faciotti et al.( 1985) BiolTechnolo~ 3:241 ], and
chlorophyll a/b binding protein [Lampa et al.(1986) Nature 316:750-752],
ferredoxin promoter [Caspar and Quail ( 1993) Plant J. 3 :161 ], actin
promoters
[Park et al., J. Plant Biol. (1995), 38(4), 365-71], ubiquitin promoters
[Garbarino,
et al., Plant Mol. Biol. (1994), 24(1), 119-27], and opine promoters [von
Lintig et
al., J. Bacteriol. {1994), 176(2), 495-503].
Depending upon the application, it may be desirable to select promoters
that are specific for expression in one or more organs of the plant. Examples
include the light-inducible promoters of the small subunit of ribulose
1,5-bisphosphate carboxylase if the expression is desired in photosynthetic
organs,
or promoters active specifically in roots {e.g., subdomains of the CaMV 35S
promoter [Benfey et al. (1990) EMBO J. 9:1677]}. Other inducible promoters
that may prove useful include those sensitive to various chemical agents such
as
those induced to an activity by benzenesulfonamides [WO 9513389; U.S. Patent
No. 5364780], abscisic acid [Devic et al., Plant J. (1996), 9(2), 205-15],and
methyl jasmonate [Xu et al., Plant Mol. Biol. (1993), 22(4), 573-88].
It is envisioned that the introduction of enhancers or enhancer-like
elements into other promoter constructs will also provide increased levels of
16
CA 02274502 1999-06-11
WO 98/33927 PCT/US98101622
primary transcription for the hemG gene to accomplish the invention. These
would include viral enhancers such as that found in the 3 5 S promoter [Odell
et al.
(1988) Plant Mol. Biol. 10:263-272], enhancers from the opine genes [Fromm et
al. ( 1989) Plant Cell 1:977-984], or enhancers from any other source that
result in
increased transcription when placed into a promoter operably linked to the
nucleic
acid fragment of the invention.
Any 3' non-coding region capable of providing a polyadenylation signal
and other regulatory sequences that may be required for the proper expression
of
the hemG coding regions can be used to accomplish the invention. This would
include the 3' end from viral genes such as the 3' end of the 35S or the 19S
cauliflower mosaic virus transcripts, the 3' end from the opine synthesis
genes, the
3' ends of ribulose 1,5-bisphosphate carboxylase or chlorophyll a/b binding
protein, or 3' end sequences from any source such that the sequence employed
provides the necessary regulatory information within its nucleic acid sequence
to
result in the proper expression of the promoter/hemG coding region combination
to which it is operably linked. There are numerous examples in the art that
teach
the usefulness of different 3' non-coding regions [for example, see
Ingelbrecht et
al. ( 1989) Plant Cell 1:671-680].
DNA sequences coding for intracellular localization sequences may be
added to the hemG coding sequence if required for the proper expression of the
proteins to accomplish the invention. One of the plant PROTOX isozymes is
localized in the chloroplasts and therefore must be synthesized with a
chloroplast
targeting signal. Bacterial proteins such as the E. toll PROTOX enzyme have no
such signal. A chloroplast transit sequence could, therefore, be fused to the
hemG
coding sequences. Preferred chloroplast transit sequences are those of the
small
subunit of ribulose 1,5-bisphosphate carboxylase, e.g. from soybean [Berry-
Lowe
et al. ( 1982) J. Mol. Appl. Genet. 1:483-498] for use in dicotyledonous
plants and
from corn [Lebrun et al. (1987) Nucleic Acids Res. 15:4360] for use in
monocotyledonous plants.
It is contemplated that the hemG gene may be integrated in the chloroplast
genome. Methods of incorporating foreign DNA into plant plastid genomes are
known [Golds et al., BiolTechnolog~ ( 1993 ), 11 ( 1 ), 95-7]. Transformation
of the
plastid genome requires a method for the translocation of the foreign DNA
across
the plastid double membrane and subsequent integration of the DNA into the
plastid genome. Suitable methods for the introduction of the foreign DNA into
the plastid include biolistic bombardment, treatment of the plant tissue with
polyethylene glycol and Agrobacterium vector transfection. Suitable vectors
for
plastid genome transformation have been developed [Svab et al., Proc. Natl.
Acad.
Sci. U. S. A. (1993), 90(3), 913-17]. Vectors will typically include the
foreign
17
CA 02274502 1999-06-11
WO 98/33927 PCT/US98/01622
DNA to be incorporated into the plastid genome flanked 5' by a suitable
plastid
promoter (often ribosomal RNA operon promoters) and 3' by other regulatory
sequences.
In similar fashion the hemG gene product may be tageted to the
mitochondrion by fusing the hemG gene to a mitochondria) targeting sequence
such as that found in the F 1-ATPase .beta. subunit of Nicotiana
plumbaginifolia[Chaumont et al., Plant Molecular Biology 24:631-641 (1994)].
Alternatively, the hemG construct could be integrated into and expressed from
the
mitochondria) genome.
Transformation of the mitochondria) genome requires a method for the
translocation of the foreign DNA across the mitochondria) membrane, and
integration of the DNA into the mitochondria) genome. Suitable methods for the
introduction of the foreign DNA into the mitochondria) genome include
biolistic
bombardment, treatment of the plant tissue with polyethylene glycol and
Agrobacterium vector transfection. Vectors will typically include the foreign
DNA to be incorporated into the mitochondria) genome flanked 5' by a suitable
mitochondria) promoter and 3' by other regulatory sequences that are effective
for
the expression of the desired foreign gene.
Expression of hemG Chimeric Genes in Plants
Various methods of introducing a DNA sequence (i.e., of transforming)
into eukaryotic cells of higher plants are available to those skilled in the
art (see
EPO publications 0 295 959 A2 and 0 13 8 341 A 1 ). Such methods include those
based on transformation vectors based on the Ti and Ri plasmids of
Agrobacterium spp. It is particularly preferred to use the binary type of
these
vectors. Ti-derived vectors transform a wide variety of higher plants,
including
monocotyledonous and dicotyledonous plants, such as soybean, cotton, rape,
tobacco, and rice [Pacciotti et al. (1985) BiolTechnology 3:241; Byrne et al.
(1987) Plant Cell, Tissue and Organ Culture 8:3; Sukhapinda et al. (1987)
Plant
Mol. Biol. 8:209-216; Lorz et al. (1985) Mol. Gen. Genet. 199:178; Potrykus
(1985) Mol. Gen. Genet. 199:183; Park et al., J. Plant Biol. (1995), 38(4),
365-71;
I-iiei et aL, PIantJ. (1994), 6:271-282].
For introduction into plants, the chimeric genes of the invention can be
inserted into binary vectors as described in Example 3.
Other transformation methods are available to those skilled in the art, such
as direct uptake of foreign DNA constructs [see EPO publication 0 295 959 A2],
techniques of electroporation [see Fromm et al. ( 1986) Nature (London)
319:791 ]
or high-velocity ballistic bombardment with metal particles coated with the
nucleic acid constructs [see Kline et al. ( 1987) Nature (London) 327:70, and
see
18
CA 02274502 1999-06-11
WO 98/33927 PCT/US98/01622
U.S. Patent No. 4,945,050]. Once transformed, the cells can be regenerated by
those skilled in the art.
Of particular relevance are the recently described methods to transform
foreign genes into commercially important crops, such as rapeseed [see De
Block
et al. ( 1989) Plant Physiol. 91:694-701 ], sunflower [Everett et al. ( 1987)
BiolTechnology 5:1201 ], soybean [MeCabe et al. ( 1988) BiolTechnology 6:923;
Hinchee et al. (1988) BiolTechnology 6:915; Chee et al. (1989) Plant Physiol.
91:1212-1218; Christou et al. ( 1989) Proc. Natl. Acad Sci USA 86:7500-7504;
EPO Publication 0 301 749 A2], rice [Hiei et al., Plant J. ( 1994), 6:271-
282], and
corn [cordon-Kamm et al. ( 1990) Plant Cell 2:603-618; Fromm et al. ( 1990)
Biotechnology 8:833-839].
Assay methods
To assay for expression of the chimeric genes in leaves or seeds of the
transformed plants, PROTOX enzyme can be extracted, detected and quantitated
enzymatically and/or immunologically or visually by methods known to those
skilled in the art. In this way lines producing high levels of expressed
protein can
be easily identified. Levels of active PROTOX in plant tissue may be measured
in
a variety of ways as described for example in Wang et al., (Biosci.,
Biotechnol.,
Biochem. (1993), 57(12), 2205-6). Preferred is the method of Camadro, J-M. et
al., [(1993), Fluorometric assay ofprotoporphyrinogen oxidase in chloroplasts
and in plant, yeast, and mammalian mitochondria. In, Target Assays for Modern
Herbicides and Related Phytotoxic Compounds. P. Boger and G. Sandmann, eds.,
Lewis Publishers, Boca Raton, FL, pp 29-34] more fully described in the
GENERAL METHODS.
Methods for determining the resistance of plants to various herbicides are
common and well known in the art. Typical methods include leaf disk assays and
ion leakage assays such as are described by; Koch et al., [Bull. Environ.
Contam.
Toxicol. (1995), 54(4), 606-13]; and Whitlow et al., [Plant Physiol. (1992),
98(1),
198-205]. Ion leakage and leaf spotting assays are more fully described in the
GENERAL METHODS.
Porphyrin Biosynthesis-Inhibiting compounds
Porphyrin Biosynthesis-Inhibiting (PBI) compounds are common and have
been commercially available since the 1960's. Many common PBI compounds
fall into six general classes of compounds consisting of the triazolones ,
cyclic
imides (e.g. N-(4-chloro-5-(cyclopropenyloxy) -2-fluorophenyl)tetrahydro-2-
phthalimide), thiadiazoles , pyrazoles , uracils and diphenylethers (e.g.,
acifluorfen, nitrofen and oxyfluorfen). Methods for synthesis of PBI compounds
are common and well known in the art (see, for example, DE 3905916 for the
preparation of cyclic imides, U.S. Patent No. 5,446,197 for the preparation of
19
CA 02274502 1999-06-11
WO 98/33927 PCT/US98/01622
dipheylethers and FR 2222378 for the preparation of oxadiazoles, JP 05097848
for the preparation of flumioxazin, EP 698604 for the preparation of
fluthiacetmethy, and U.S. Patent No. 5,176,735 for the preparation of uracils
such
as flupropacil).
The instant PBI resistant plants are expected to be tolerant to a wide
variety of PBI compounds including but not limited to 1 H-Isoindole-I,3(2H)-
dione, 2-[4-chloro-5-(cyclopentyloxy)-2- fluorophenyl]-4,5,6,7-tetrahydro- {
};
Benzoic acid, 2-chloro-5-[2-chloro-4-(trifluoromethyl)phenoxy]-, 2-ethoxy-I-
methyl-2-oxoethyl ester {HC 252}; 2H-1,4-Benzoxazin-3(4H)-one, 6-[(6,7-
dihydro-6,6-dimethyl-3H,SH-pyrrolo[2,1-c] [ 1,2,4]thiadiazol-3-ylidene)amino]-
7-
fluoro-4-(2-propynyl){SN 124085}; Benzoic acid, 2-chloro-5-[3,6-dihydro-3-
methyl-2,6-dioxo-4- (trifluoromethyl)-I(2H)-pyrimidinyl]-, 1-methylethyl ester
{ UCC-C 4243 } ; Acetic acid, [[2-chloro-4-fluoro-5-[(tetrahydro-3-oxo-1 H,3H
[1,3,4]thiadiazolo[3,4-a]pyridazin-1 ylidene)amino]phenyl]thio]-, methyl ester
{KIH 9201 }; 2,4-Oxazolidinedione, 3-[4-chloro-5-(cyclopentyloxy)-2
fluorophenyl]- 5-( I -methylethylidene) { B W 4 } ; 2,4-Oxazolidinedione, 3-[4-
chloro-
2-fluoro-5-[( I -methyl-2-propynyl)oxy]phenyl]-5-( I -methylethylidene) { BW 3
} ;
Acetic acid, [[[ 1-[5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitrophenyl]-2-
methoxyethylidene]amino]oxy]-, methyl ester {AKH 7088}; 1 H-Isoindole-
1,3(2H)-dione, 2-[7-fluoro-3,4-dihydro-3-oxo-4-(2-propynyl)-2H-1,4-benzoxazin-
6-yl]-4,5,6,7-tetrahydro-{Flumioxazin; S 53482}; Acetic acid, [2-chloro-4-
fluoro-
5-(1,3,4,5,6,7-hexahydro-1,3-dioxo- {S 23031 };1H-Isoindole-1,3(2H}-dione, 2-
[4-
chloro-2-fluoro-5-[( I -methyl-2-propynyl)oxy]phenyl]-4,5,6,7-tetrahydro-
{Flumipropyn}; Furan, 3-[5-[2-chloro-4-(trifluoromethyl)phenoxy]-2
nitrophenoxy]tetrahydro-2H-isoindol-2-yl)phenoxy]-, pentyl ester
{Furyloxyfen};
Benzoic acid, 5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitro-,2-ethoxy-2
oxoethyl ester {RH 0265}; Benzoic acid, 5-[2-chloro-4-{trifluoro-
methyl)phenoxy]-2-nitro-,2-ethoxy-1-methyl-2-oxoethyl ester {PPG 844};
Benzenamine, 2-chloro-6-nitro-3-phenoxy-{ CME I27}; Benzamide, 5-[2-chloro-
4-(trifluoromethyl)phenoxy]-N-(methylsulfonyl)-2-nitro- {Fomesafen};
Methanone, (2,4-dichlorophenyl)[1,3-dimethyl-5-[[(4-methylphenyl)sulfonyl]-
oxy]-IH-pyrazol-4-yl]- {H468T}; Benzoic acid, 5-[2-chloro-4-(trifluoromethyl)-
phenoxy]-2-nitro- {Acifluorfen};Benzene, 2-chloro-I-(3-ethoxy-4-nitrophenoxy)-
4-(trifluoromethyl)- {Oxyfluorfen; RH 2915 };Benzene, 2-chloro-1-(4-nitro-
phenoxy}-4-(trifluoromethyl)- {RH 2512}; Benzoic acid, 5-(2,4-dichloro-
phenoxy}-2-nitro-, methyl ester {MC 4379}; 1,3,4-Oxadiazol-2(3H)-one, 3-[2,4-
dichloro-5-(2-propynyloxy)phenyl]-5-( I ,1-dimethylethyl)-; Benzene, 2,4-
dichloro-1-(3-methoxy-4-nitrophenoxy)- {PI 3468} ; 1,3,4-Oxadiazol-2(3H)-one,
3-[2,4-di chloro-5-( I -methylethoxy)phenyi]-5-{ l , l -dimethylethyl)- { RP
17623 } ;
_...__ T_. __~______.
CA 02274502 1999-06-11
WO 98/33927 PCT/US9810162Z
Benzene, 2-nitro-1-(4-nitrophenoxy)-4-(trifluoromethyl)-{C 6989}; Benzene, 1,5-
dichloro-3-fluoro-2-(4-nitrophenoxy)-{FluoronitroJen}; Benzene, 1,3,5-
trichloro-
2-(4-nitrophenoxy)-{CNP 1032}; Benzoic acid, 5-[2-chloro-4-
(trifluoromethyl)phenoxy]-2-nitro- carboxymethyl ester {Fluoroglycofen}; and
Benzene, 2,4-dichloro-1-(4-nitrophenoxy)-{FW 925}.
Preferred within the context of the present invention are compounds that
correspond to the formula:
J-G
I
wherein
G is
R5
F
R2
R3 ~ R3 , R3
R1 R1 R1
G-1 G-2 G-3
6
R' R R\ Y
\4
N Y p~ 5 N R9
R
O ~ X R5
R1 R1 R1
G-4 G-5 G-6
o R~ o\S~ .~R6 R ~ ~~II
R5 N N-S
R4 ' R4
R3 or ~ xl R
R1 R1 R1
G-7 G-8 G-9
21
CA 02274502 1999-06-11
WO 98/33927 PCT/US98/01622
and wherein J is
0 0
~n N N
R9 R9 R11\
Z I \N- Z N-- N-
R m R10~N/ ~ R12~N/
O
J-1 J-2 J-3
R13
Q Q
Rlq N Q Rg N // R16~N
Z\J~Cy. '~'~ ~ -~\N- Z JJ~~~_~. ''''~_ ~~J(~N-
W N~ . R10~N~ . R17 m
1,\\O Q
Q
J-4 J-5 J-6
Q O
R11 ~ Q
\ N O R18
N- N- \N~
N-
R12 ' R18 N ' N~N~ '
Q
J-7 J-8 J-9
R19 R20 R19 R20 N -
R Z Q R18 Q R9 1
N
Z I Q
R10~N~N~ . ~ R10~N~ .
m I I ~ N ~\'m
Q Q Q
J-10 J-11 J-12
N N - R23
R21\W~ R9 ~ R9
1 ~' N /
Q z Ql z
R22~N ~ R10~N~ ' R10~N~~
Q
J-13 J-14 J-15
22
CA 02274502 1999-06-11
WO 98133927 PCTIUS98/01622
R R23 Rg
~N/C~NH/ R ~"'N/C\NH/
Z
26 . 26
R25~N'~N/ R10Z~C/OR R10%~C/OR
O O
J-16 J-17 J-18
23
R R R20 O R g OR21
Z N- Rlg
R10 m w N/ N- 0
O , N~
0
J-19 J-20 J-21
R19 R19 N QR21 Rg N Q
N n /
Z
O , W N~ , or R10~N~N/
N
Q
J-22 J-23 J-24
wherein the dashed line in J-5, J-6, J-12 and J-24 indicates that the left-
hand
ring contains only single bonds or one bond in the ring is a
carbon-carbon double bond;
XisOorS;
YisOorS;
R1 is hydrogen or halogen;
R2 is H; C1-Cg alkyl; C~-Cg haloalkyl; halogen; OH; OR2~; SH; S(O)pR2y
COR27; C02R27; C(O)SR27; C(O)NR29R3o; CHO; CR29=NOR36;
CH=CR3~C02R27; CH2CHR3~C02R27; CON=CR3~R32; nitro;
cyano; NHS02R33; NHS02NHR33; NR2~R38; NH2; or phenyl
optionally substituted with at least one member independently selected
from C~-C4 alkyl;
p is 0; 1; or 2;
R3 is C~-C2 alkyl; C1-C2 haloalkyl; OCH3; SCH3; OCHF2; halogen; cyano
or nitro;
R4 is H; C1-C3 alkyl; C1-C3 haloalkyl; or halogen;
23
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WO 98/33927 PCT/US98/01622
R5 is H; C 1-C3 alkyl; halogen; C 1-C3 haloalkyl; cyclopropyl; vinyl; CZ
alkynyl; cyano; C(O)R38; C02R3g; C(O)NR38R~9; CR34R35CN;
CR34R35C(O)R3s; CR34R35C02R38; CR34R3sC(O)NR38R39;
CHR340H; CHR340C(O)R3g; or OCHR340C(O)NR3gR39; or
when G is G-2 or G-6, then R4 and R5 can be taken together with the carbon
to which they are attached to form C=O;
R6 is C1-C6 alkyl; C~-C6 haloalkyl; C2-C6 alkoxyalkyl; C3-C6 alkenyl; or
C3-C6 alkynyl;
X~ is a direct bond; O; S; NH; N(C1-C3 alkyl); N(CI-C3 haloalkyl); or
N(allyl);
R~ is H; C1-C6 alkyl; C~-C6 haloalkyl; halogen; S(O)~(C~-C6 alkyl); or
C(=O)R4o;
Rg is H; C~-Cg alkyl; C3-Cg cycloalkyl; C3-Cg alkenyl; C3-Cg alkynyl;
C1-Cg haloalkyl; C2-Cg alkoxyalkyl; C3-Cg alkoxyalkoxyalkyl; C3-Cg
haloalkynyl; C3-Cg haloalkenyl; C~-Cg alkylsulfonyl; CI-Cg
haloalkylsulfonyl; C3-Cg alkoxycarbonylalkyl;
S(O)2NH(C ~-Cg alkyl); C(O}R4 ~ ; or benzyl optionally substituted on
the phenyl ring with R42;
n and m are each independently 0; l; 2; or 3; provided that m + n is 2 or 3;
Z is CR9Rlo; O; S; S(O); S(O)2; orN(C~-C4 alkyl);
each R9 is independently H; C 1-C3 alkyl; halogen; hydroxy; C ~ -C6 alkoxy;
Ci-C6 haloalkyl; C~-C6 haloalkoxy; C2-C6 alkylcarbonyloxy; or
C2-C6 haloalkylcarbonyloxy;
each Rio is independently H; C1-C3 alkyl; hydroxy; or halogen;
R~ 1 and R12 are each independently H; halogen; C ~-C6 alkyl; C3-C6
alkenyl; or Cl-C6 haloalkyl;
R I 3 is H; C ~ -C6 alkyl; C ~ -C6 haloalkyl; C3-C6 alkenyl; C3-C6
haloalkenyl;
C3-C6 alkynyl; C3-C6 haloalkynyl; HC(=O); (C~-C4 alkyl)C(=O); or
NHZ;
R14 is CI-C6 alkyl; C1-C6 alkylthio; C1-C6 haloalkyl; orN(CH3)2;
W is N or CR15;
R15 is H; C 1-C6 alkyl; halogen; or phenyl optionally substituted with C 1-C6
alkyl, 1-2 halogen, C1-C6 alkoxy, or CF3;
each Q is independently O or S;
Q1 is O or S;
Z~ is CR~6R1~; O; S; S(O); S(O)2; or N(C1-C4 alkyl);
each R16 is independently H; halogen; hydroxy; C1-C6 alkoxy; C1-C6
haloalkyl; C1-C6 haloalkoxy; C2-C6 alkylcarbonyloxy; or C2-C6
haloalkylcarbonyloxy;
24
_ __ _..~ .._.. T T
CA 02274502 1999-06-11
WO 98133927 PCT/US98/01622
each R» is independently H; hydroxy; or halogen; or
when R16 and R1~ are bonded to adjacent atoms they can be taken together
- Q~-
with the carbons to which they are attached to form
optionally substituted with at least one member selected from 1-2
halogen and 1-2 C1-C3 alkyl;
R~g is C1-C6 alkyl; halogen; or CI-C6 haloalkyl;
R19 and R2~ are each independently H; Cl-C6 alkyl; or C~-C6 haloalkyl;
R2~ and R22 are each independently C 1-C6 alkyl; C 1-C6 haloalkyl; C3-C6
alkenyl; C3-C6 haloalkenyl; C3-C6 alkynyl; or C3-C6 haloalkynyl;
R23 is H; halogen; or cyano;
R24 is C ~-C6 alkylsulfonyl; C ~-C6 alkyl; C ~-C6 haloaikyl; C3-C6 alkenyl;
C3-C6 alkynyl; C~-C6 alkoxy; C~-C6 haloalkoxy; or halogen;
Rzs is C~-C6 alkyl; C1-C6 haloalkyl; C3-C6 alkenyl; or C3-C6 alkynyl;
R26 is C ~-C6 alkyl; C 1-C6 haloalkyl; or phenyl optionally substituted with
C~-C6 alkyl, 1-2 halogen, 1-2 nitro, C1-C6 alkoxy, or CF3;
R2~ is C ~-Cg alkyl; C3-Cg cycloalkyl; C3-Cg alkenyl; C3-Cg alkynyl; C 1-Cg
haloalkyl; C2-Cg alkoxyalkyl; CZ-Cg alkylthioalkyl; C2-Cg
alkylsulfinylalkyl; CZ-Cg alkylsulfonylalkyl; C ~-Cg alkylsulfonyl;
phenylsulfonyl optionally substituted on the phenyl ring with at least
one substituent selected from the group halogen and CI-C4 alkyl;
C4-Cg alkoxyalkoxyalkyl; C4-Cg cycloalkylalkyl; C6-Cg
cycloalkoxyalkyl; C4-Cg alkenyloxyalkyl; C4-Cg alkynyloxyalkyl;
C3-Cg haloalkoxyalkyl; C4-Cg haloalkenyloxyalkyl; C4-Cg
haloalkynyloxyalkyl; C6-Cg cycloalkylthioalkyl; C4-Cg
alkenylthioalkyl; C4-Cg aikynylthioalkyl; C ~ -C4 alkyl substituted with
phenoxy or benzyloxy, each ring optionally substituted with at least
one substituent selected from the group halogen, C 1-C3 alkyl and
C~-C3 haloalkyl; C4-Cg trialkylsilylalkyl; C3-Cg cyanoalkyl; C3-Cg
halocycloalkyl; C3-Cg haloalkenyl; Cg-Cg alkoxyalkenyl; C5-Cg
haloalkoxyaikenyl; CS-Cg alkylthioalkenyl; C3-Cg haloalkynyl; C5-Cg
alkoxyalkynyl; CS-Cg haloalkoxyalkynyl; CS-Cg alkylthioalkynyl;
C2-Cg alkylcarbonyl; benzyl optionally substituted with at least one
substituent selected from the group halogen, C~-C3 alkyl and C1-Cg
haloalkyl; CHR34COR28; CHR34COZR2g; CHR34P(O)(OR2g)2;
CHR~4P(S)(OR2g)2; CHR34C(O)NR29R3~; or CHR34C(O)NH2;
R2g is C1-C6 alkyl; C2-C6 alkenyl; C2-C6 alkynyl; or tetrahydrofuranyl;
CA 02274502 1999-06-11
WO 98/33927 PCT/US98/01622
R29 and R3 ~ are independently hydrogen or C ~ -C4 alkyl;
R3~ and R32 are independently Cl-C4 alkyl or phenyl optionally substituted
with at least one substituent selected from the group halogen, C ~ -C;
alkyl, and CI-C3 haloalkyl; or
R29 and R3~ can be taken together to form -(CH2)5-, -(CH2)4- or
-CHZCH20CH2CH2-, each ring thus formed optionally substituted
with a substituent selected from the group C ~ -C; alkyl, phenyl and
benzyl; or
R31 and R32 can be taken together with the carbon to which they are
IO attached to form C3-Cg cycloalkyl;
R33 is C1-C4 alkyl; C~-C4 haloalkyl; or C2-C6 alkenyl;
R34 and R35 are independently H or Cl-C4 alkyl;
R36 is H; C 1-C6 alkyl; C3-C6 alkenyl; or C3-C6 alkynyl;
R3~ is H; C~-C4 alkyl; or halogen;
R3g is H; C~-C6 alkyl; C3-C6 cycloalkyl; C3-C6 alkenyl; C3-C6 alkynyl;
C2-C6 alkoxyalkyl; C~-C6 haloalkyl; phenyl optionally substituted
with at least one substituent selected from the group halogen, C 1-C4
alkyl, and Ci-C4 alkoxy; -CH~C02(C~-C4 alkyl); or
-CH(CH3)C02(C~-C4 alkyl);
R39 is H; C 1-CZ alkyl; or C(O)O(C 1-C4 alkyl);
R4~ is H; C 1-C6 alkyl; C ~-C6 alkoxy; or NH(C 1-C6 alkyl);
R41 is C1-C6 alkyl; C1-C6 haloalkyl; C~-C6 alkoxy; NH(CI-C6 alkyl);
phenyl optionally substituted with R42; benzyl; or CZ-Cg
dialkylamino; and
R42 is C1-C6 alkyl; 1-2 halogen; C1-C6 alkoxy; or CF;.
Within the context of the present invention the compounds six PBI
compounds are preferred as described below:
PBI-1 is described by the formula:
o C1
N
N ~ ~ C1
~ N/
0
PBI-2 is described by the formula:
26
__._-__ __ T ~
CA 02274502 1999-06-11
WO 98!33927 PCT/US98/01622
0 F
j ~N \ / 0
a N
0
PBI-3 is described by the formula:
F
N ~ ~ C1
N
S S~
N ~ C02CH3
O
PBI-4 is described by the formula:
ci F
FZHCO
'N~~C1
-:~//N
H3C
PBI-5 is described by the formula:
CH3
CF3 N' /O
~I/N
0
C1
C02-i-Pr
PBI-6 is described by the formula:
27
CA 02274502 1999-06-11
WO 98133927 PCT/US98t01622
C1
o
o_y+ \ ~ \ ~ F
II F ~F
O
O O
Exyression of the hemG eene and demonstration of PBI compound resistance in
transformed plants
Plant tissue from soybean and tobacco were transformed either by high-
velocity biolistic bombardment with metal particles coated with the nucleic
acid
constructs containing the hemG gene or using an Agrobacterium tumefaciens
containing a binary plasmid. Callus and mature plants were regenerated from
the
transformed cells and assayed for production of active E. toll PROTOX enzyme
and resistance to PBI compounds.
Embryonic suspension cultures of soybean tissue, biolisticaliy transformed
with the plasmid pHGV4 and expressing the E. toll hemG gene were analyzed.
Extracts from soybean cultures expressing hemG and untransformed cultures were
assayed for PROTOX levels in the presence and absence of 3 ~M PBI-1. Direct
comparisons of the activity from the hemG and untransformed cultures in the
absence of PBI-1 was somewhat compromised by the fact that transformed
soybean tissue was somewhat sick. This is frequently the case with transformed
soybean tissue grown under these conditions, irrespective of the identity of
the
foreign DNA. However, there was sufficient PROTOX activity in both the hemG
transformed and untransformed cultures to determine their sensitivity to PBI-
1.
Control cultures showed no PROTOX activity after exposure to 3 ~M PBI-1
whereas cultures expressing the hemG gene retained 67% of normal PROTOX
levels (Table 1, Example 2), indicating that the hemG construct expressed a
functional PROTOX in plant cells.
Tobacco transformants containing the binary p35S-protox vector
demonstrated clear resistance of about 30-fold over controls to the compound
PBI-1 at levels up to 1000 ~M in leaf spotting assays (Figures 2, 4, and 11).
Similar results were seen in ion leakage assays where leaf disks from
transformants which were exposed to between 0 and 1000 p.M PBI-1 showed
about a 100-fold increase in resistance over controls (Figures 3 and 9).
Tobacco transformants were also tested for PBI resistance against a broad
range of PBI herbicide compounds including PBI-1, PBI-2, PBI-3, PBI-4, PBI-5,
and PBI-6, all defined above. As shown in Figure 10 and Table 2, Example 5
28
CA 02274502 1999-06-11
WO 98133927 PCT/US98t01622
tobacco transformants demonstrated some resistance in leaf spotting assays to
all
compounds tested with levels of resistance as compared to controls ranging
from
3-fold to 300-fold depending on the compound tested.
These data clearly indicate that the expression of the bacterial hemG gene
is able to confer resistance, both in vivo and in vitro to a broad range of
PBI
herbicides.
The present invention is further defined in the following Examples. It
should be understood that these Examples, while indicating preferred
embodiments of the invention, are given by way of illustration only. From the
above discussion and these Examples, one skilled in the art can ascertain the
essential characteristics of this invention, and without departing from the
spirit
and scope thereof, can make various changes and modifications of the invention
to
adapt it to various usages and conditions.
EXAMPLES
Restriction enzyme digestions, phosphorylations, ligations and
transformations were done as described in Sambrook, J. et al., Molecular
Cloning:
A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press
(1989). Restriction enzymes were obtained from New England Biolabs (Boston,
MA), GIBCO/BRL (Gaithersburg, MD), or Promega (Madison, WI). Taq
polymerase was obtained from Perkin Elrner (Branchburg, NJ). Growth media
was obtained from GIBCO/BRL (Gaithersburg, MD).
The meaning of abbreviations is as follows: "h" means hour(s), "min"
means minute(s), "sec" means second(s), and "d" means day(s).
GENERAL METHODS
Growing Seeds and Seedlings:
Seedling Growth on Petri plates:
Unless otherwise noted, all seeds grown in Petri plates were sterilized in
50% bleach with 0.1% Tween-20 for 7-10 min and then washed in sterile water
3-5 times before plating. Seeds were then placed onto sterile media containing
1 /2
strength Murashige-Skoog salts (Gibco # I 1117-066) plus 0.7% agar and 1
sucrose. Kanamycin was prepared as a 50 mg/mL stock in water, sterilized by
passage through a 0.2 pm filter, and added to the media after it had been
autoclaved and cooled to 60°C. Plates containing kanamycin were stored
at 4°C
and used within one month of preparation. For testing transformants for
antibiotic
sensitivity, 20-100 seeds per 57 cm2 Petri plate were used. Plates were
incubated
in a growth chamber at 23°C with illumination from fluorescent tubes of
about
60 pmol/m2/sec photosynthetically active radiation and a 14 h photoperiod.
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WO 98/33927 PCT/US98/01622
Growth in soil:
Plants were grown in commercial soil mixes (metro mix or others) in
growth chambers at 20-25°C with fluorescent and incandescent
illumination of
100-300 ~.mol/m2/sec photosynthetically active radiation and a photoperiod
ranging from 12 h to continuous illumination or in greenhouses at 23-
28°C on a
natural daylength supplemented with artificial lighting.
Herbicide Stocks:
All porphyrin biosynthesis-inhibiting compounds were obtained from the
DuPont chemical library and included representatives of the three major
classes of
PBI compounds. Compounds used in the following examples included PBI
compounds PBI-1 through PBI-6 as described above.
Compounds were dissolved at 10 mg active ingredient/mL in DMSO
except for PBI-6 which was dissolved at 2.5 mg active ingredientlmL in 25%
DMSO, 75% water, 0.19% X-77~, a mixture of nonionic surfactants
(alkylarylpolyoxyethylene glycols), free fatty acids, isopropanol, and water
was
obtained from Loveland (Loveland Industries Inc., Greeley, CO 80632-289). All
herbicide stocks were used immediately or stored at -20°C until use.
Protoporphyrinogen Oxidase Assay:
Protoporphyrinogen oxidase (PROTOX) was assayed fluorometrically
essentially as described in the literature with slight modifications [Camadro
et al.,
( 1993 ), FI uorometric assay of protoporphyrinogen oxidase in chl oropl asts
and in
plant, yeast, and mammalian mitochondria. In, Target Assays for Modern
Herbicides and Related Phytotoxic Compounds. P. Boger and G. Sandmann, eds.,
Lewis Publishers, Boca Raton, FL, pp 29-34.]
Protoporphyrinogen (PROTOGEN) was obtained by chemical reduction of
protoporphyrin (PROTO) (Porphyrin Products, Logan, UT) with 3 percent Na
amalgam [Jacobs et al., Enzyme 28, 206, ( 1982)]. The amalgam was prepared by
melting Na spheres (Aldrich Chemical Company, Milwaukee, WI) under a
nitrogen stream using a heat gun and then adding Hg (triple distilled, J.T.
Baker,
Inc., Phillipsburg, NJ). PROTO was dissolved in 10 mM KOH, 20 percent
ethanol, with the balance water, to make a 1 mM solution. The PROTO stock was
diluted 1:1 with 10 mM KOH, 10 mL placed into a 100 mL Pyrex test tube,
150 ~L of a 1:100 (v/v) dilution of antifoam A emulsion (Sigma Chemical
Company, St. Louis, MO) added, and the solution saturated with argon.
Laboratory lights were dimmed and freshly crushed amalgam (approximately
18 grams) was added to the tube which was then vortexed under argon for 4 min.
Residual amalgam and particles of unreduced PROTO were removed by passing
the solution through a 0.2 pm syringe filter. The filtered solution was
sparged
with argon, DTT was added (0.0154 grams dissolved in 1 mL of 1 M MOPS), and
__._ _ ~T..__~.... _.....
CA 02274502 1999-06-11
WO 98/33927 PCT/US98/01622
the PROTOGEN solution adjusted to approximately pH 8 with 1 M MOPS
saturated with nitrogen gas. Aliquots were placed in 1.5 mL amber vials
(Wheaton, Millville, NJ), overlaid with mineral oil (J.T. Baker, Phillipsburg,
NJ),
closed with septa-containing caps, and stored at -80°C until use. A
portion of the
PROTOGEN was oxidized to PROTO with rat mitochondria to determine the
concentration of PROTOGEN.
PROTOX activity was determined in an assay mixture consisting of
buffer A (i00 mM HEPES pH 7.5, 1 mM EGTA, 5 mM EDTA, 2 mM DTT,
percent glycerol, 0.03 percent Tween-80), 250 pL of tissue extract, 3.8 ~M
10 PROTOGEN and 1 percent DMSO with or without 3 ~M PBI-1 in a total volume
of 1 mL. (DMSO was used to dissolve the PBI-1.) The assay was started by
addition of PROTOGEN. The oxidation of PROTOGEN to PROTO was
followed with a Millipore CytoFluor 2300 multiwell fluorescence plate reader
using an excitation filter of 395 nm with a 25 nm bandwidth and emission
filter of
620 nm with a 40 nm bandwidth with no temperature control. Assays were
conducted in Corning 24 well plates, PBI-1 was added at least 1 min before the
assay was started, and the change in fluorescence was monitored for S min. The
rate of non-enzymatic oxidation of PROTOGEN to PROTO was determined using
extracts that were heat killed by boiling for 5 min. The assay volume with the
heat-killed enzyme was a total of 500 p,L with only 125 ~L of extract, due to
insufficient plant material for a 1 mL assay. Other assay components were
halved
so that the final concentration of substrate, etc., remained the same.
Leaf Spotting_Tests:
Responses to herbicide damage were assessed by measuring the damage
produced by spotting the compounds onto attached leaves of plants grown in
soil
in a greenhouse. Test compounds were dissolved in DMSO and then diluted into
0.25% X-77 to the appropriate concentration. The DMSO concentrations of the
samples spotted onto the leaves was always 3% or below. Preliminary
experiments (not shown) demonstrated that 10% DMSO in 0.25% X-77 (with no
herbicide) had no affect on tobacco in this assay. Test compounds were applied
to
leaves in rows of 5 to 10, 1 p.L drops for each concentration. Four to 7 rows
were
placed on each leaf. Damage to the area of the leaf surrounding the drop was
scored visually on a 0 (no injury) to 10 (complete death of region surrounding
the
drop) following spotting. For a given concentration of herbicide, the rate of
production of damaged tissue and the final degree of damage depended on the
age
of the plant and environmental conditions. All experiments were conducted
using
control plants which lacked the E toll PROTOX gene (hemG), but were treated in
exactly the same manner as the PROTOX transformants.
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WO 98/33927 PCT/US98/01622
Ion leakage assay:
Responses to herbicide damage were also assessed by measuring
electrolyte leakage from tobacco leaf discs treated with herbicides. Leaf
discs
(5 mm) were cut from expanded leaves and washed twice in deionized water for
1-2 h. Leaf discs were placed into 20 mL of deionized water and transferred to
the
dark. Herbicides were dissolved in DMSO and added to the indicated
concentration. [Controls with no herbicide contained the same concentration of
DMSO (3.4%) as found in the highest tested herbicide concentration and this
amount of DMSO did not affect ion leakage (results not shown).] Plates were
incubated in the dark at 25°C for 12-18 h. The plates were then placed
in the light
(220 pmol/m2/s photosynthetically active radiation) at 25°C. The
conductivity of
the bathing solution was measured just before the plates were illuminated
(time
zero) and at intervals afterward using a Cole-Parmer Conductivity meter (model
1481-60). Conductivity results arc presented as the increase in conductivity
over
that at time zero. Preliminary experiments (not presented) indicated that ion
leakage caused by PBI treatment in the light was not observed in discs which
were
kept in the dark. In addition, PBI-induced ion leakage in the light was
inhibited
by treatment with either 1 mM gabaculine or 1 mM dioxoheptanoic acid which
inhibit the early steps of porphyrin synthesis. The results from these
preliminary
experiments confirm that the ion leakage caused by the PBI treatment is indeed
due to inhibition of PROTOX.
EXAMPLE 1
CONSTRUCTION OF A GENE EXPRESSION CASSETTE FOR THE
EXPRESSION OF E. COLI hemG IN PLANT TISSUE
Construction of parent vector
A unique gene expression cassette was used for construction of chimeric
genes for expression of the E. coli hemG gene in plants. This cassette was
developed for the expression of E. coli dapA in plant chloroplasts (WO
9515392}.
To create vectors for expressing E. coli PROTOX, the E. coli hemG gene was
ligated into the vector in place of the dapA.
The dapA leaf expression cassette is inserted into the vector pGEM9Z
giving pBT455 (Figure 7). The cassette is composed of the 35S promoter of
cauliflower mosaic virus [Odell et al. (1985) Nature 313:810812; Hull et al.
(1987) Virology 86:482493], the translation leader from the chlorophyll
binding
protein (cab} gene, [Dunsmuir ( 1985) Nucleic Acids Res. 13:2503-2518], the
chloroplast transit sequence (cts) of the small subunit of ribulose 1,5-
bisphosphate
carboxylase from soybean [Berry-Lowe et al. (1982) J. Mol. Appl. Genet.
1:483-498], the E. coli dapA coding sequence, and 3' transcription termination
region from the nopaline synthase (nos) gene [Depicker et al. ( 1982) J. Mol.
Appl.
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CA 02274502 1999-06-11
WO 98/33927 PCT/US98/01622
Genet. 1:561-570]. The entire cassette is flanked by SaII sites; there is also
a
BaInHI site upstream of the cassette.
Isolation of the E. toll hemGgene and construction of a hemG expression vector
The hemG gene of E. toll has been cloned and sequenced (Sasarman et al.,
J. Gen. Microbiol., 113, 297, (1979); Sasarman et al., Can. J. Microbiol., 39,
1155, (1993), and was amplified from genomic E. toll (strain BAR1091
[Rasmussen et al. ( 1985) J. Bact. 164:665-673]) DNA using PCR with primers
SEQ ID NOS:1, 2. These primers were designed to amplify the entire open
reading frame of the hemG gene plus they contain additional sequences at the
5'
ends which add restrictions endonuclease sites (HaeIII/StuI for SEQ ID NO:1
and
KpnI for SEQ ID N0:2 at the sites indicated by the ~ symbol).
5' CTGCAGG~CCTCGGTGAAAACATTAATTC 3' (SEQ ID NO:1)
5' GACGTGGTAC~CATTATTTCAGCGTCGG 3' (SEQ ID N0:2)
Amplification was accomplished using the UlTma~ DNA polymerase
(Perkin-Elmer, Branchburg, NJ) using the conditions suggested by the supplier.
UlTma~ DNA polymerase was used for its lower error rate relative to Taq~
Polymerase. In order to further reduce the chance of PCR-induced mutations
occurring in the final hemG vector, four amplifications were done in parallel
and
the products from these reactions were kept separate (but treated identically)
throughout all of the following steps. Ultimately, unique hemG vectors from
three
of these four PCR products were identified and characterized. The amplified
570
by hemG fragment was gel-purified and then digested with HaeIII and KpnI. The
resultant 552 by fragment was gel-purified and used for subsequent ligations.
The transformation vector pBT455 (see above) was modified by replacing
the dapA gene with the PCR amplified hemG product. The dapA gene was
excised from the pBT455 vector using NruI and KpnI in order to create
compatible ends for ligation with the hemG fragment. NruI cuts 22 by
downstream of the junction between the rbcS chloroplast targetting sequence
and
the beginning of the dapA coding sequence. KpnI cuts at the junction between
the
end of the dapA coding sequence and the nos 3' terminator. The vector was also
digested with BstEII (which cuts inside the dapA sequence) to reduce the
number
of pBT455 parent vectors recovered due to incomplete digestion at the Nrul and
KpnI sites or religation.
The HaeIII/KpnI digested hemG fragment was ligated with the
NruI/KpnI/BstEII digested pBT455. (Both HaeIII and NruI produce blunt ends
which were ligated together.) The ligation produces an in-frame fusion of the
rbcS chloroplast targetting signal, the dapA coding sequence, and the hemG
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CA 02274502 1999-06-11
WO 98133927 PCT/US98/01622
coding sequence. The NruI site is 22 by downstream from the dapA start codon
and, therefore seven amino acids from the dapA gene are attached to the
N-terminus of the hemG protein encoded by this construct. Two additional amino
acids, derived from the linker region of primer SEQ ID NO:1, are also in the
chimeric hemG protein encoded by this construct between the dapA and the hemG
sequences.
E. coli DHSoc competent cells (Gibco-BRL) were transformed via heat
shock treatment with the pBT455/hemG ligation mixture. Transformed cells
containing the desired hemG construct were differentiated from the pBT455
parent vector by PCR analysis using SEQ ID NOS:3 and 4 and restriction
digests.
SEQ ID NOS:3 and 4 are shown below.
5'-CATGGTCACGGGAAG-3' (SEQ ID N0:3)
5'-TCAGAAACTTGCGCG-3' (SEQ ID N0:4)
The PCR primers were designed to anneal upstream of the 5' ligation site
in both the parent and the ligated vector (SEQ ID N0:3) and in the middle of
the
hemG gene (SEQ ID N0:4).
PCR products were analyzed by gel electrophoresis and confirmed by
restriction with EcoRI. Clones giving correct PCR products were confirmed by
restriction digests of plasmid DNA. Three clones (pHGV2, pHGV3, and
pHGV4), one each from three of the four original, independent PCR reactions,
were determined to have the correct restriction digests. Sequence analysis of
these
clones confirmed their identity and indicated that each contains one or more
mutations induced by the PCR within the hemG sequence. pHGV4 was chosen
for further use. The DNA sequence of the translated part of chimeric hemG
protein, including the chloroplast targeting sequence, dapA residues, linker
region
and hemG coding sequence is shown in SEQ ID N0:6. Within the hemG region,
pHGV4 contains a conservative mutation of the threonine at residue 67 of the
chimeric hemG protein to serine. It also contains a deletion of 1 base pair
within
the original stop codon which results in the addition 3 amino acids (tyrosine -
glycine - threonine) at the carboxy terminus of the protein.
EXAMPLE 2
Transformation of Soybean and Expression of
E coli hemG confernng resistance to PBI-1
Soybean was transformed by biolistic bombardment using the pHGV4
plasmid described in Example 1. Embryogenic cultures of soybean were used as
the recipient tissue for bombardment by DNA-coated particles. These cultures
were initiated according to methods described by Finer and Nagasawa [Finer et
34
CA 02274502 1999-06-11
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al., (1988) Development of an embryogenic suspension culture of soybean
(Glycine max Merrill) Plant Cell Tissue Organ Cult. 15:125-136] by placing
immature zygotic cotyledons about 2 to 4 mm in length on agarose-solidified MS
medium containing 2,4-dichlorophenoxyacetic acid. After about 6 weeks of
incubation in the light ( 16 h daylength, 30 pEinsteins) at 28°C,
masses of globular
stage somatic embryos form on the surface of the cotyledons. These somatic
embryos are excised and transferred to liquid medium containing modified MS
medium as described by Finer et al. (see above) with 10 ~g/mL 2,4-dichloro-
phenoxyacetic acid. The tissue proliferates in this medium and is subcultured
by
transferring green, globular stage somatic embryos to fresh medium every two
weeks. This tissue can be used for transformation using modifications of
published procedures [Parrott et al., ( 1994), Recovery and evaluation of
soybean
(Glycine max ~L.J Merr.) plants transgenic for a Bacillus thuringiensis var.
kurstaki insecticidal gene, In Vitro Cell. Dev. Biol. 30P:144-149; Finer et
al.,
( 1991 ), Transformation of soybean via particle bombardment of embryogenic
suspension culture tissue In Vitro Cell. Dev. Biol. 27P:175-182.]
Gold particles (1 pm in diameter) (Bio-Rad Labs, Hercules, CA )were
coated with DNA using the following technique. pHGV4 plasmid DNA was co-
precipitated with a plasmid containing a hygromycin phosphotransferase gene
(HPTII) for use in selecting transformed cells. The HPTII protein inactivates
the
antibiotic hygromyicn and acts as a selectable marker for plant transformation
(Waldron et al., (1985), Resistance to hygromycin B: a new marker for plant
transformation studies, Plant Mol. Biol. 18:189-200). Any of a number of
plasmids containing a hygromycin phosphotransferase gene suitably engineered
for high level expression in soybean tissue cultured cells can be used as the
selective plasmid. The actual selective piasmid used was pML 151, which
harbors
the hygromycin phosphotransferase gene [Gritz et al., (1983) Gene 25:179-188]
under the control of the 35S promoter [Odell (1985) Nature 313:810812] and the
nos 3' end (Depicker et al., ( 1982), J. Mol. Appl. Genet. , 1:561-574). pML
151 was
made by deleting the ampicillin resistance gene from the plasmid pSP72
(Promega
Biotech, Madison, WI) and inserting the HPTII cassette described above.
Plasmid
DNA (I p.g of pML151 and 9 Pg of the pHGV4 plasmid) were added to 50 p,L of
a suspension of gold particles (60 mg/mL). Calcium chloride (50 p,L of a 2.5 M
solution) and spermidine free base (20 ~L of a 1.0 M solution) were added to
the
particles. The suspension was vortexed during the addition of these solutions.
After 10 min, the tubes were briefly centrifuged and the supernatant removed.
The particles were then rinsed with 200 p,L of 100% ethanol, the ethanol rinse
was
performed again and the particles resuspended in a final volume of 30 p,L of
CA 02274502 1999-06-11
WO 98/33927 PCT/US98/01622
ethanol. An aliquot (5 pL) of the DNA-coated particles was placed in the
center
of a KaptonT'" flying disc (Bio-Rad Labs, Hercules, CA).
For bombardment, about 0.5 g of suspension culture (covering an area of
about 3 cm2) was placed in the center of a petri dish. The tissue was
bombarded
3 times with gold particles 1-p,m in diameter using a Bio-Rad Biolistic(TM)
gene
gun (Model #PDS-1000/He). The tissue was placed about 5 cm from the stopping
screen and bombarded under a vacuum of 28 in Hg. The particles were
accelerated using a flying disc propelled by a shock wave generated with a
1100 psi rupture disc.
Following bombardment, the tissue was transferred to liquid medium and
incubated on a rotary shaker for 10 d. Hygromycin was then added to the media
at
a concentration of 50 mg/L. Fresh hygromycin-containing medium was then
added at weekly intervals for 6 weeks. After about 4 to 6 weeks, sectors of
surviving green tissue was transferred to fresh medium without hygromycin. The
transgenic soybean tissue was proliferated in SB55 medium.
Soybean tissue was transformed, as described above, with the pHGV4
construct. DNA was isolated (Edwards et al., Nucl. Acids Res. 19:1349) from
part
of this tissue or from control, untransformed tissue-culture grown tissue and
used
as template in PCR assays using primers specific for pHGU~ (SEQ ID NOS:3 and
5) and standard PCR conditions (Sambrook, supra) with a 43°C annealing
temperature. When DNA from the transformed tissue was used as template, a
single strong product of about 600 by was amplified as predicted from the
sequence of the pHGV4 plasmid. A second product of about 700 by of much
lower abundance was also produced. Products of the same size and relative
abundance were also produced when the PCR was conducted with the pHGV2
plasmid. No products were produced from DNA made from control,
untransformed soybean tissue. These results indicate that the soybean tissue
had
been successfully transformed with the pHGV4 plasmid.
Transformed Tissue Synthesizes a PROTOX Resistant
to the PBI Compound PBI-1
Protein extracts were made from both the transformed and control soybean
tissues and were tested for PROTOX activity. Callus was removed from agar
growth media, frozen at -78°C, and transferred tn a glass homogenizer
containing
1 mL of buffer A ( 100 mM HEPES pH 7.5, 1 mM EGTA, 5 mM EDTA, 2 mM
DTT, 10 percent glycerol, 0.03 percent Tween-80) on ice. The plant tissue was
homogenized on ice and then poured through one layer of miracloth. PROTOX
activity was determined as described in the GENERAL METHODS. PROTOX
activity was determined both in the presence and absence of 3 pM PBI-1. This
concentration of PBI-1 is approximately 1000-fold above the level needed to
36
_.... _.._ T T ._~..~.~....w._..._ _~__._.._. _._.
CA 02274502 1999-06-11
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inhibit 50% of the activity of a corn chloroplast PROTOX (Maxwell et al.,
unpublished results). The results (Table 1 ) show that in the absence of
inhibitor
both lines had substantial PROTOX activity. However, the transformed line had
only about 40% of the PROTOX activity of the untransformed control, probably
due to the poor vigor of the pHGV4 transformed tissue. Soybean tissue grown in
these types of tissue culture conditions is frequently sick, irrespective of
the
presence or absence of inserted foreign DNA. In the presence of 3 pM PBI-1,
the
untransformed control tissue had no activity above the background found in the
heat-killed control samples indicating that this concentration of PBI-1
completely
inhibits normal soybean PROTOX activity. By contrast, the transformant
retained
about 67% of its activity in the presence of the inhibitor. These results
demonstrate that the pHGV4 construct directs the production of a protein in
plant
cells which has PROTOX activity and, moreover, this activity is resistant to
high
levels of PBI-1.
TABLE 1
PROTOX activity
in transformed
and control soybean
tissue
PROTOX Activity
Sam le Treatment Units/sec
Contro 1 none 19. 8
pHGV4 Transformant none 8.0
Control 3 ~M PBI-1 1.0
pHGV4 Transformant 3 p,M PBI-1 5.4
Control Heat denatured 1.1
pHGV4 Transformant Heat denatured 0.2
Tissue-culture grown soybean tissue that had been transformed with the
pHGV4 construct or untransformed control tissue was assayed for PROTOX
activity. Activity was measured in the absence of any treatment (total
activity), in
the presence of 3 ~M PBI-1 {resistant activity) and after heat denaturation of
the
extracts (background activity).
EXAMPLE 3
Construction of an Agrobacterium tumefaciens Binary
Plasmid p35S-PROTOX for Plant Transformation
A vector for transformation of the chimeric hemG construction described
above into plants using Agrobacterium tumefaciens was produced by constructing
a binary Ti plasmid vector [Bevan et al., (1984) Nucl. Acids Res., 12:8711-
8720].
The starting vector used for this work (pZS 199, Figure 8) is based on a
vector
37
CA 02274502 1999-06-11
WO 98/33927 PCT/US98/01622
which contains: ( 1 ) the chimeric gene nopaline synthase/neomycin
phosphotransferase as a selectable marker for transformed plant cells [Bevan
et
al., (1984) Nature, 304:184-186], (2) the left and right borders of the T-DNA
of
the Ti plasmid [Bevan et al., (1984) Nucl. Acids Res., 12:8711-8720], (3) the
E. coli lacZ a-complementing segment (Vieria and Messing ( 1982), Gene,
19:259-267) with unique restriction endonuclease sites for Eco RI, Kpn I,
Bam HI, Hin DIII, and Sal I, (4) the bacterial replication origin from the
Pseudomonas plasmid pVSI (Itoh et al., (1984) Plasmid 11:206-220), and (5) the
bacterial neomycin phosphotransferase gene from Tn5 (Berg et al., (1975) Proc.
Natnl. Acad. Sci. U.S.A., 72:3628-3632) as a selectable marker for transformed
A.
tumefaciens. The nopaline synthase promoter in the plant selectable marker was
replaced by the 35S promoter (Odell et al. (1985) Nature, 313:810-813) by a
standard restriction endonuclease digestion and ligation strategy. The 35S
promoter is required for efficient tobacco transformation as described below.
pZS 199 was digested with XbaI and SaII. The hemG chimeric gene from
pHGV4 (i.e., 35S promoter, cab leader, rbcS chloroplast transit sequence,
residual
dapA sequences, hemG coding region, and nos 3' terminator) was excised by
digestion with XbaI and SaII. The hemG fragment was ligated with the XbaI/SaII
digested pZS199 vector yielding p35S-PROTOX (Figure 5). The ligation mixture
was transformed into E. coli and candidate plasmids were confirmed by
restriction
analysis. The p35S-protox plasmid was introduced by tri-parental mating
[Ruvkin
et al., ( 1981 ), Nature, 289:8588] to Agrobacterium strain LBA4404/pAL4404
[Hockema et al., (I983) Nature, 303:179180 (1983)) using the E coli helper
strain
PRK2013 and selected for kanamycin resistance.
EXAMPLE 4
Transformation of Tobacco with p35S-protox and
Resistance of Transformants to PBI-1
Cultures ofAgrobacterium containing the binary vector p35S-protox were
used to transform tobacco (cultivarXanthi) leaf disks [Horsch et al., (1985)
Science 227:12291231 ]. Forty-eight independently transformed tobacco plants
were generated and are termed PROTOX-1 through PROTOX-48. In addition, the
pZS 199 binary vector was also used for parallel transformation experiments
giving three control lines termed Binary Control 1 through Binary Control 3.
These control lines were treated in the same manner as the PROTOX
transformants and contain the same exogenous DNA except they lack the hemG
transgene.
The sensitivity of the PROTOX and Binary Control primary transforrnants
to PBI-1 were tested using the leaf spotting and ion leakage assays described
above and the results are given in Figures 2, 3, 4, 9, and 11.
38
_... _ .. _ _... ~ _.r. . ........T.r....-.~. __....
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Figure 2 shows the results from a leaf spotting experiment using 1 ~L
drops of 300 pM PBI-1. The damage produced S d after the herbicide was spotted
onto the leaves of the tobacco transformants is plotted. The results indicate
that
many of the hemG-containing lines are more resistant to 300 ~M PBI-1 than the
S controls. Other lines have a similar level of sensitivity as the controls.
Similar
results were also observed when 30 pM PBI-1 was spotted onto these leaves
(results not shown).
Figure 3 shows the results from an ion leakage experiment in which the
conductivity increase for 50 leaf discs incubated in 500 p.M PBI-1 for 29
hours in
the light is plotted. These results confirm that, by this second criteria,
many of the
lines are more resistant to 500 pM PBI-1 than the controls. Other hemG-
containing lines have a similar level of sensitivity as the controls. Similar
results
were also obtained when leaf discs were incubated in 10 pM PBI-1 (results not
shown).
Based on these experiments, smaller subsets of PROTOX plants, which
appeared most resistant to PBI-1, were chosen for further characterization
using
the leaf spotting and ion leakage assays as described below.
Concentrations of PBI-1, ranging from 0.3 to 900 p.M were spotted onto
leaves of 2 Binary Control and 13 PROTOX transformants. (The PROTOX lines
were not spotted at the 0.3, 0.9 and 3 ~M concentrations.) PBI-1-induced
damage
was scored visually after 10 d. The results are illustrated in Figure 4 which
shows
the average of the scores of the two Binary Controls and of PROTOX-23. From
this analysis, PROTOX-23 appears about 30-fold more resistant to PBI-1 than
the
control lines.
Figure 11 shows the results of a third leaf spotting experiment in which
PBI-1 was spotted onto Binary Control-2 and PROTOX-24 leaves. Five, 1 ~L
drops of PBI-1 of the indicated concentrations were spotted onto each leaf at
the
positions marked by the black dots. After 12 d of growth in the greenhouse,
visual observations of the damage produced were made and photographs were
taken. Only very minor damage was produced in PROTOX-24 by 900 p.M PBI-1,
the highest concentration tested. By contrast, severe damage was produced in
the
Binary Control-2 leaf at concentrations as low as 30 ~,M. Based on these
results,
PROTOX-24 is at least 30-fold more resistant to PBI-1 than the control plant.
Concentrations of PBI-1, ranging from 0-1000 ~.M, were used in an ion
leakage experiment using leaf disks from 2 Binary Control and 5 PROTOX
primary tobacco transformants. PBI-1-induced ion leakage was measured after
incubation of the disks for 17 h in the dark and 25 h in the light and the
results are
illustrated in Figure 9 which shows the average scores of the two Binary
Controls
and of PROTOX-22. Under the conditions of this experiment, PROTOX-22
39
CA 02274502 1999-06-11
WO 98/33927 PCT/LTS98/01622
shows no consistent PBI-1-induced ion leakage in the light even at the highest
tested concentration, 1000 p,M. By contrast, ion leakage in the Binary
Controls is
clearly detectable at 10 pM. From this analysis, PROTOX-22 appears at least
100-fold more resistant to PBI-1 than the control lines.
S EXAMPLE 5
Resistance of Transformants to PBI compounds
PBI-2. PBI-3, PBI-4, PBI-5, PBI-6
In order to determine whether the PROTOX transformants are resistant to
all PBIs, a representative set of diverse PBIs was tested at a range of
concentrations on Binary Control and PROTOX primary tobacco transformants
using the leaf spotting procedure. PBI compounds tested were PBI-1, PBI-2,
PBI-3, PBI-4, PBI-5 and PBI-6 and are fully described in the details of the
invention.
Leaves were spotted with five, 1 pL drops of the indicated PBI herbicides
at the concentrations indicated. Plants were maintained in a greenhouse and
the
damage produced by the herbicides was scored visually 4 d (6 d for the slower
acting PBI-6) after spotting. The results with one of the most resistant
lines,
PROTOX-23 are shown in Figure 10. In this figure, the results labeled Binary
Control represent the average of those from the Binary Control-2 and Binary
Control-3 plants. Overlapping data points in this figure have been offset by
0.05
vertical units to improve visual clarity.
As seen in Figure 10, the PROTOX-23 line showed no response to 5 of the
6 PBIs, whereas the Binary Control plants were strongly affected by all of the
compounds. Because of the lack of response for PROTOX-23, even at the highest
concentrations, only a minimum level of resistance for most of these compounds
can be determined (Table 2). The actual level of resistance is likely to be
higher
than these estimates.
TABLE 2
Resistance to
a varie of PBIs
in tobacco PROTOX-23
transformant
PBI Test CompoundFold Increase in Resistance in PROTOX-23
PBI-1 >30-fold
PBI-2 >300-fold
PBI-3 >3-fold
PBI-4 >3-fold
PBI-5 >300-fold
PBI-6 15-fold
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The fold increase in resistance of the PROTOX-23 line, relative to the
Binary Control-2 and -3 lines was calculated using the leaf spotting assay
with the
results shown in Figure 10. For PBI-6, the estimate is based on the response
at
840 ~M in PROTOX-23 compared with the rate that would be required to produce
a similar level of damage in the Binary Control by interpolating the dose
response
curve shown in Figure 10. For the other PBIs, since PROTOX-23 did not show
any response to even the highest level of PBI tested, the fold increase in
resistance
is a minimal estimate based on the lowest concentration that produced a
response
in the Binary Controls, compared to the highest dose tested in PROTOX-23.
EXAMPLE 6
Characterization of Trans~ene Loci Conferrine Resistance to PBI Herbicides
In order to determine the number of transgene loci present in each tobacco
transformant, progeny from the primary transformants were analyzed for their
resistance to kanamycin, since the T-DNA also carries the nptII gene as a
transformation marker. Segregation data indicated that 70% of the PROTOX
tobacco transformants and 4 of the 5 selected for more detailed study (Table
3)
segregated approximately 75% kanamycin resistant to 25% kanamycin sensitive,
indicative of a single transgene locus. The fifth line (PROTOX-18) segregated
with a ratio indicative of 2 transgene loci.
Genomic DNA was isolated [see Reiter et al. ( 1992), Proc. Nat. Acad. Sci.
89:1477] from leaves of progeny of the same 5 Protox lines and restricted with
either BamHI or EcoRI. DNA was blotted onto Hybond N+ (Amersham,
Arlington Heights, Illinois) in 0.4 N NaOH and probed with a DNA fragment
containing the hemG coding region using procedures recommended by the
manufacturer. Based on these results (Table 3 ), three of the five lines
(PROTOX-23, 24, and 36) contain 2 apparently intact copies of the T-DNA. A
fourth line (PROTOX-18) contains 4 apparently intact copies and the fifth line
(PROTOX-26) contains 1 or 2 apparently rearranged copies of the T-DNA.
The combined Southern and kanamycin segregation results indicate that
only a small number of chimeric hemG genes (from 2 to 4) are sufficient to
produce the high levels of PBI resistance observed in these tobacco
transformants.
41
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TABLE 3
Number
of genetically
defined
T-DNA
loci
and T-DNA
inserts
in tobacco
Protox
transformants
Line # Kanr# Kans Kanr/Kans# loci Transgene copy
#
PROTOX- 114 9 12.7 2 4
I 8
PROTOX-23 280 82 3.4 1 2
PROTOX-24 142 60 2.4 1 2
PROTOX-26 322 72 4.5 I 1 or 2
PROTOX-36 108 36 3.0 1 2
Kanamycin resistance was determined on progeny of primary
transformants by scoring growth of seedlings on standard media supplemented
with 200 ~g/mL kanamycin sulfate. Transgene copy number in each line was
determined by Southern blotting of DNA from 1 or 2 progeny from each primay
transformant. The T-DNA was rearranged in Protox 26 so the copy number could
not be determined precisely.
42
_. TT
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) ADDRESSEE: E. I. DU PONT DE NEMOURS AND COMPANY
(B) STREET: 1007 MARKET STREET
(C) CITY: WILMINGTON
(D) STATE: DELAWARE
(E) COUNTRY: U.S.A.
(F) ZIP: 19898
(G) TELEPHONE: 302-892-7229
(H) TELEFAX: 302-773-0164
(I) TELEX: 6717325
(ii) TITLE OF INVENTION: GENETICALLY TRANSFORMED PLANTS
DEMONSTRATING RESISTANCE TO
PORPHYRINOGEN BIOSYNTHESIS
INHIBITING HERBICIDES
(iii) NUMBER OF SEQUENCES: 7
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: DISKETTE, 3.5 INCH
(B) COMPUTER: IBM PC COMPATIBLE
(C) OPERATING SYSTEM: MICROSOFT FOR WINDOWS '95
(D) SOFTWARE: MICROSOFT WORD 7.0
(v) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 60/036,793
(B) FILING DATE: JANUARY 31, 1997
(vii) ATTORNEY/AGENT INFORMATION:
(A) NAME: KING, KAREN K.
(B) REGISTRATION NO.: 39,850
(C) REFERENCE/DOCKET NUMBER: CR-9854
43
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(2) INFORMATION FOR SEQ ID N0:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B} TYPE: nucleic acid
(C} STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
CTGCAGGCCT CGGTGAAAAC ATTAATTC 2g
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
GACGTGGTAC CATTATTTCA GCGTCGG 27
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
CATGGTCACG GGAAG 15
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
TCAGAAACTT GCGCG 15
44
_.. _.__ .__ T.. ~,__.~.._... _..__ _
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(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
TTGGATCTCA CTATT 15
(2) INFORMATION FOR SEQ ID N0:6:
(iy SEQUENCE CHARACTERISTICS:
(A) LENGTH: 747 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
ATGGCTTCCT CAATGATCTC CTCCCCAGCT GTTACCACCG TCAACCGTGC CGGTGCCGGC 60
ATGGTTGCTC CATTCACCGG CCTCAAATCC ATGGCTGGCT TCCCCACGAG GAAGACCAAC 120
AATGACATTA CCTCCATTGC TAGCAACGGT GGAAGAGTAC AATGCATGGT CACGGGAAGT 180
ATTGTCGCCT CGGTGAAATC ATTAATTCTT TTCTCAACAA GGGACGGACA AACGCGCGAG 240
ATTGCCTCCT ACCTGGCTTC GGAACTGAAA GAACTGGGGA TCCAGGCGGA TGTCGCCAAT 300
GTGCACCGCA TTGAAGAACC ACAGTGGGAA AACTATGACC GTGTGGTCAT TGGTGCTTCT 360
ATTCGCTATG GTCACTACCA TTCAGCGTTC CAGGAATTTG TCAAAAAACA TGCGACGCGG 420
CTGAATTCGA TGCCGAGCGC CTTTTACTCC GTGAATCTGG TGGCGCGCAA ACCGGAGAAG 980
CGTACTCCAC AGACCAACAG CTACGCGCGC AAGTTTCTGA TGAACTCGCA ATGGCGTCCC 540
GATCGCTGCG CGGTCATTGC CGGGGCGCTG CGTTACCCAC GTTATCGCTG GTACGACCGT 600
TTTATGATCA AGCTGATTAT GAAGATGTCA GGCGGTGAAA CGGATACGCG CAAAGAAGTT 660
GTCTATACCG ATTGGGAGCA GGTGGCGAAT TTCGCCCGAG AAATCGCCCA TTTAACCGAC 720
AAACCGACGC TGAAATATGG TACCTAA 747
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 184 amino acids
(B) TYPE: amino acid
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(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
Val Lys Ser Leu Ile Leu Phe Ser Thr Arg Asp Gly Gln Thr Arg Glu
1 5 10 15
Ile Ala Ser Tyr Leu Ala Ser Glu Leu Lys Glu Leu Gly Ile Gln Ala
20 25 30
Asp Val Ala Asn Val His Arg Ile Glu Glu Pro Gln Trp Glu Asn Tyr
35 40 45
Asp Arg Val Val Ile Gly Ala Ser Ile Arg Tyr Gly His Tyr His Ser
50 55 60
Ala Phe Gln Glu Phe Val Lys Lys His Ala Thr Arg Leu Asn Ser Met
65 70 75 80
Pro Ser Ala Phe Tyr Ser Val Asn Leu Val Ala Arg Lys Pro Glu Lys
85 90 95
Arg Thr Pro Gln Thr Asn Ser Tyr Ala Arg Lys Phe Leu Met Asn Ser
100 105 110
Gln Trp Arg Pro Asp Arg Cys Ala Val Ile Ala Gly Ala Leu Arg Tyr
115 120 125
Pro Arg Tyr Arg Trp Tyr Asp Arg Phe Met Ile Lys Leu Ile Met Lys
130 135 140
Met Ser Gly Gly Glu Thr Asp Thr Arg Lys Glu Val Val Tyr Thr Asp
145 150 155 160
Trp Glu Gln Val Ala Asn Phe Ala Arg Glu Ile Ala His Leu Thr Asp
165 170 175
Lys Pro Thr Leu Lys Tyr Gly Thr
180
46
__ _._._T. T __
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INDICATIONS RELATING TO A DEPOSITED MICROORGANISM
(PCT Rule 136is)
A. The indications made below relate
to the microorganism referred
to in the description
on page 11 , line 'i4-~7
B. IDENTIFICATION OF DEPOSIT Further
deposits are identified on an
additional shcct
Name of depositary institution
AMERICAN TYPE CULTURE COLLECTION
Address of depositary institution
(including postal code and country)
12301 Parklawn Drive
Rockville, Maryland 20852
US
Dale of deposit Accession Number
7 August 1996 ATCC97675
C. ADDITIONAL INDICATIONS (leave
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in which a European patent is
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a sample of the deposited microorganism
will be made available until
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of the grant of the European patent
or
until the'date on which the application
has been refused or withdrawn
or is deemed to be withdrawn, only
by the issue of such a sample
to an
expert nominated by the person
requesting the sample. (Rule 28(4)
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Bureau later (specify the general
nature ofthe indications e.~..
'flccession
Nunrber ojDepasiY)
~~ For receiving O use only For International Bureau use only
Q-~Ftfs s~eet w~ec~d wit~~interna~onal application a This sheet was received
by the International Bureau on:
Authorized o~'('tcer ~v ~ ~~ ~~, .-~~.~" Authorized officer
form PCT/RO/134 (luly 1992)
47
CA 02274502 1999-06-11
WO 98133927 PCT/US98/01622
INDICATIONS RELATING TO A DEPOSITED MICROORGANISM
(PCT Rule l3bis)
A. 'hhe indications made below
relate to the microorganism referred
to in the description
on page 12 ( lint 1-5
B. IDENTIFICATION OF DEPOSIT Further
deposits are identified on an
additional sheet
Name of depositary institution
AMERICAN TYPE CULTURE COLLECTION
Address of depositary institution
(including postal code and country)
12301 Parklawn Drive
Rockville) Maryland 20852
US
Date of deposit Accession Number
7 August 1996 ATCC97674
C. ADDITIONAL INDICATIONS (leave
blank ijnot applicable) This information
is continued on an additional
sheet
In respect of those designations
in which a European patent is
sought,
a sample of the deposited microorganism
will be made available until
the publication of the mention
of the grant of the European patent
or
until the-date on which the application
has been refused or withdrawn
or is deemed to be withdrawn, only
by the issue of such a sample
to an
expert nominated by the person
requesting the sample. (Rule 28(4)
EPC)
D. DESIGNATED STATES FOR WHICH
INDICATIONS ARE MADE (ijthe indications
are not jor all designated States)
E. SEPARATE FURNISHING OF INDICATIONS
(leave blank ijnot applicable)
The indications listed below will
be submitted to the lntemational
Bureau later (spec~thegeneral
nature ojlhe indication e.F..
'Accession
Numher njDepasit')
For receiving Office use only For International Bureau use only
This~leet v~re~ved w~t the in~'rtational application a This sheet was received
by the International Bureau on:
Authorized officer ~ // // ~ r- /~ ~ ~ Authorized officer
form PCT/R0/134 (luly 1992)
48
