HERBICIDE TOLERANT PLANTS

Abstract
The present invention relates, inter alia, a method of selectively controlling weeds at a locus comprising crop plants and weeds, wherein the method comprises application to the locus of a weed controlling amount of a pesticide composition comprising an homogentisate solanesyltransferase (HST) inhibiting herbicide and/or hydroxyphenyl pyruvate dioxygenase (HPPD) inhibiting herbicide, wherein the crop plants comprise at least one recombinant polynucleotide which comprises a region which encodes an HST; to a method of selectively controlling weeds at a locus comprising crop plants and weeds, wherein the method comprises application to the locus of a weed controlling amount of a pesticide composition comprising an homogentisate solanesyltransferase (HST) inhibiting herbicide, wherein the crop plants comprise at least one recombinant polynucleotide which comprises a region which encodes a HPPD enzyme and to recombinant polynucleotides and vectors for utilised in the methods. The present invention further relates to a herbicidal composition comprising a HPPD-inhibiting herbicide and a HST-inhibiting herbicide.
Description

The present invention relates to methods for selectively controlling weeds at a locus. The invention further relates to recombinant DNA technology, and in particular to the production of transgenic plants which exhibit substantial resistance or substantial tolerance to herbicides when compared with non transgenic like plants. Plants which are substantially “tolerant” to a herbicide when they are subjected to it provide a dose/response curve which is shifted to the right when compared with that provided by similarly subjected non tolerant like plants. Such dose/response curves have “dose” plotted on the x-axis and “percentage kill”, “herbicidal effect” etc. plotted on the y-axis. Tolerant plants will typically require at least twice as much herbicide as non tolerant like plants in order to produce a given herbicidal effect. Plants which are substantially “resistant” to the herbicide exhibit few, if any, necrotic, lytic, chlorotic or other lesions when subjected to the herbicide at concentrations and rates which are typically employed by the agricultural community to kill weeds in the field.


More particularly, the present invention relates to the production of plants that are resistant to herbicides that inhibit hydroxyphenyl pyruvate dioxygenase (HPPD) and/or herbicides that inhibit the subsequent, homogentisate solanesyl transferase (HST) step in the pathway to plastoquinone.


Herbicides that act by inhibiting HPPD are well known in the art. Inhibition of HPPD blocks the biosynthesis of plastoquinone (PQ) from tyrosine. PQ is an essential cofactor in the biosynthesis of carotenoid pigments which are essential for photoprotection of the photosynthetic centres. HPPD-inhibiting herbicides are phloem-mobile bleachers which cause the light-exposed new meristems and leaves to emerge white where, in the absence of carotenoids, chlorophyll is photo-destroyed and becomes itself an agent of photo-destruction via the photo-generation of singlet oxygen. Methods for production of transgenic plants which exhibit substantial resistance or substantial tolerance to HPPD-inhibiting herbicides have been reported—for example WO02/46387.


The enzyme catalysing the following step from HPPD in the plastoquinone biosynthesis pathway is HST. The HST enzyme is a prenyl tranferase that both decarboxylates homogentisate and also transfers to it the solanesyl group from solanesyl diphosphate and thus forms 2-methyl-6-solanesyl-1,4-benzoquinol (MSBQ), an intermediate along the biosynthetic pathway to plastoquinone. HST enzymes are membrane bound and the genes that encode them include a plastid targeting sequence. Methods for assaying HST have recently been disclosed.


Over expression of HST in transgenic plants has been reported—and said plants are said to exhibit slightly higher concentrations of α-tocopherol. However, it has not hitherto been recognised that HST is the target site for certain classes of herbicidal compounds—which act wholly or in part by inhibiting HST. Furthermore, it has now been found, inter alia, that over expression of HST in a transgenic plant provides tolerance to HST-inhibiting and/or HPPD-inhibiting herbicides.


Thus, according to the present invention there is provided a method of selectively controlling weeds at a locus comprising crop plants and weeds, wherein the method comprises application to the locus of a weed controlling amount of a pesticide composition comprising an homogentisate solanesyltransferase (HST) inhibiting herbicide and/or hydroxyphenyl pyruvate dioxygenase (HPPD) inhibiting herbicide, wherein the crop plants comprise at least one heterologous polynucleotide which comprises a region which encodes an HST. In a preferred embodiment of the method the crop plants further comprise an additional heterologous polynucleotide which comprises a region which encodes a hydroxyphenyl pyruvate dioxygenase (HPPD).


The invention still further provides a method of selectively controlling weeds at a locus comprising crop plants and weeds, wherein the method comprises application to the locus of a weed controlling amount of a pesticide composition comprising an HST-inhibiting herbicide, wherein the crop plants comprise at least one heterologous polynucleotide which comprises a region which encodes a HPPD enzyme.


In a preferred embodiment the pesticide composition referred to in the aforementioned methods comprises both an HST-inhibiting herbicide and an HPPD-inhibiting herbicide


For the purposes of the present invention—an HST inhibiting herbicide is one which itself, or as a procide generates a molecule that inhibits Arabidopsis HST exhibits an IC50 less than 150 ppm, preferably less than 60 ppm using the “total extract” assay method as set out herein. It should be appreciated that the HST inhibiting herbicides may also act as a HPPD inhibitors (possible to identify using, for example, HPPD enzyme assays and/or the differential responses of HPPD or HST over expressing transgenic plant lines) and, therefore, as shown below, self-synergise the effect of their inhibition of HST. Preferably the HST inhibiting herbicide is selected from the group consisting of a compound of formula (IIa)




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wherein


R1, R2, R3 and R4 are independently hydrogen or halogen; provided that at least three of R1, R2, R3 and R4 are halogen; or salts thereof;


a compound of formula (IIb)




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wherein


R1 and R2 are independently hydrogen, C1-C4alkyl, C1-C4haloalkyl, halo, cyano, hydroxy, C1-C4alkoxy, C1-C4alkylthio, aryl or aryl substituted by one to five R6, which may be the same or different, or heteroaryl or heteroaryl substituted by one to five R6, which may be the same or different;


R3 is hydrogen, C1-C10alkyl, C2-C10alkenyl, C2-C10alkynyl, C3-C10cycloalkyl, C3-C10cycloalkyl-C1-C6alkyl-, C1-C10alkoxy-C1-C6alkyl-, C1-C10cyanoalkyl-, C1-C10alkoxycarbonyl-C1-C6alkyl-, N—C1-C3alkyl-aminocarbonyl-C1-C6alkyl-, N,N-di-(C1-C3alkyl)-aminocarbonyl-C1-C6alkyl-, aryl-C1-C6alkyl- or aryl-C1-C6alkyl- wherein the aryl moiety is substituted by one to three R7, which may be the same or different, or heterocyclyl-C1-C6alkyl- or heterocyclyl-C1-C6alkyl- wherein the heterocyclyl moiety is substituted by one to three R7, which may be the same or different;


R4 is aryl or aryl substituted by one to five R8, which may be the same or different, or heteroaryl or heteroaryl substituted by one to four R8, which may be the same or different;


R5 is hydroxy, R9-oxy-, R10-carbonyloxy-, tri-R11-silyloxy- or R12-sulfonyloxy-, each R6, R7 and R8 is independently halo, cyano, nitro, C1-C10alkyl, C1-C4haloalkyl, C2-C10alkenyl, C2-C10alkynyl, hydroxy, C1-C10alkoxy, C1-C4haloalkoxy, C1-C10alkoxy-C1-C4alkyl-, C3-C7cycloalkyl, C3-C7cycloalkoxy, C3-C7cycloalkyl-C1-C4alkyl-, C3-C7cycloalkyl-C1-C4alkoxy-, C1-C6alkylcarbonyl-, formyl, C1-C4alkoxy-carbonyl-, C1-C4alkylcarbonyloxy-, C1-C10alkylthio-, C1-C4haloalkylthio-, C1-C10alkylsulfinyl-, C1-C4haloalkylsulfinyl-, C1-C10alkylsulfonyl-, C1-C4haloalkylsulfonyl-, amino, C1-C10alkylamino-, di-C1-C10alkylamino-, C1-C10alkylcarbonylamino-, aryl or aryl substituted by one to three R13, which may be the same or different, heteroaryl or heteroaryl substituted by one to three R13, which may be the same or different, aryl-C1-C4alkyl- or aryl-C1-C4alkyl- wherein the aryl moiety is substituted by one to three R13, which may be the same or different, heteroaryl-C1-C4alkyl- or heteroaryl-C1-C4alkyl- wherein the heteroaryl moiety is substituted by one to three R13, which may be the same or different, aryloxy- or aryloxy-substituted by one to three R13, which may be the same or different, heteroaryloxy- or heteroaryloxy-substituted by one to three R13, which may be the same or different, arylthio- or arylthio-substituted by one to three R13, which may be the same or different, or heteroarylthio- or heteroarylthio-substituted by one to three R13, which may be the same or different;


R9 is C1-C10alkyl, C2-C10alkenyl, C2-C10alkynyl or aryl-C1-C4alkyl- or aryl-C1-C4alkyl- wherein the aryl moiety is substituted by one to five substituents independently selected from halo, cyano, nitro, C1-C6alkyl, C1-C6haloalkyl or C1-C6alkoxy;


R10 is C1-C10alkyl, C3-C10cycloalkyl, C3-C10cycloalkyl-C1-C10alkyl-, C1-C10haloalkyl, C2-C10alkenyl, C2-C10alkynyl, C1-C4alkoxy-C1-C10alkyl-, C1-C4alkylthio-C1-C4alkyl-, C1-C10alkoxy, C2-C10alkenyloxy, C2-C10alkynyloxy, C1-C10alkylthio-, N—C1-C4alkyl-amino-, N,N-di-(C1-C4alkyl)-amino-, aryl or aryl substituted by one to three R14, which may be the same or different, heteroaryl or heteroaryl substituted by one to three R14, which may be the same or different, aryl-C1-C4alkyl- or aryl-C1-C4alkyl- wherein the aryl moiety is substituted by one to three R14, which may be the same or different, heteroaryl-C1-C4alkyl- or heteroaryl-C1-C4alkyl- wherein the heteroaryl moiety is substituted by one to three R14, which may be the same or different, aryloxy- or aryloxy-substituted by one to three R14, which may be the same or different, heteroaryloxy- or heteroaryloxy-substituted by one to three R14, which may be the same or different, arylthio- or arylthio-substituted by one to three R14, which may be the same or different, or heteroarylthio- or heteroarylthio-substituted by one to three R14, which may be the same or different;


each R11 is independently C1-C10alkyl or phenyl or phenyl substituted by one to five substituents independently selected from halo, cyano, nitro, C1-C6alkyl, C1-C6haloalkyl or C1-C6alkoxy;


R12 is C1-C10alkyl or phenyl or phenyl substituted by one to five substituents independently selected from halo, cyano, nitro, C1-C6alkyl, C1-C6haloalkyl or C1-C6alkoxy;


each R13 is independently halo, cyano, nitro, C1-C6alkyl, C1-C6haloalkyl or C1-C6alkoxy; and


each R14 is independently halo, cyano, nitro, C1-C10alkyl, C1-C4haloalkyl, C1-C10alkoxy, C1-C4alkoxycarbonyl-, C1-C4haloalkoxy, C1-C10alkylthio-, C1-C4haloalkylthio-, C1-C10alkylsulfinyl-, C1-C4haloalkylsulfinyl-, C1-C10alkylsulfonyl-, C1-C4haloalkylsulfonyl-, aryl or aryl substituted by one to five substituents independently selected from halo, cyano, nitro, C1-C6alkyl, C1-C6haloalkyl or C1-C6alkoxy, or heteroaryl or heteroaryl substituted by one to four substituents independently selected from halo, cyano, nitro, C1-C6alkyl, C1-C6haloalkyl or C1-C6alkoxy; or salts or N-oxides thereof;


a compound of formula (IIc)




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wherein


R1 and R2 are independently hydrogen, C1-C4alkyl, C1-C4haloalkyl, halo, cyano, hydroxy, C1-C4alkoxy, C1-C4alkylthio, aryl or aryl substituted by one to five R6, which may be the same or different, or heteroaryl or heteroaryl substituted by one to five R6, which may be the same or different;


R3 is C1-C4haloalkyl, C2-C4haloalkenyl or C2-C4haloalkynyl;


R4 is aryl or aryl substituted by one to five R8, which may be the same or different, or heteroaryl or heteroaryl substituted by one to four R8, which may be the same or different;


R5 is hydroxy or a group which can be metabolised to the hydroxy group;


each R6 and R8 is independently halo, cyano, nitro, C1-C10alkyl, C1-C4haloalkyl, C2-C10alkenyl, C2-C10alkynyl, hydroxy, C1-C10alkoxy, C1-C4haloalkoxy, C1-C10alkoxy-C1-C4alkyl-, C3-C7cycloalkyl, C3-C7cycloalkoxy, C3-C7cycloalkyl-C1-C4alkyl-, C3-C7cycloalkyl-C1-C4alkoxy-, C1-C6alkylcarbonyl-, formyl, C1-C4alkoxycarbonyl-, C1-C4alkylcarbonyloxy-, C1-C10alkylthio-, C1-C4haloalkylthio-, C1-C10alkylsulfinyl-, C1-C4haloalkylsulfinyl-, C1-C10alkylsulfonyl-, C1-C4haloalkylsulfonyl-, amino, C1-C10alkylamino-, di-C1-C10alkylamino-, C1-C10alkylcarbonylamino-, aryl or aryl substituted by one to three R13, which may be the same or different, heteroaryl or heteroaryl substituted by one to three R13, which may be the same or different, aryl-C1-C4alkyl- or aryl-C1-C4alkyl- wherein the aryl moiety is substituted by one to three R13, which may be the same or different, heteroaryl-C1-C4alkyl- or heteroaryl-C1-C4alkyl- wherein the heteroaryl moiety is substituted by one to three R13, which may be the same or different, aryloxy- or aryloxy-substituted by one to three R13, which may be the same or different, heteroaryloxy- or heteroaryloxy-substituted by one to three R13, which may be the same or different, arylthio- or arylthio-substituted by one to three R13, which may be the same or different, or heteroarylthio- or heteroarylthio-substituted by one to three R13, which may be the same or different; and


each R13 is independently halo, cyano, nitro, C1-C6alkyl, C1-C6haloalkyl or C1-C6alkoxy; or a salt or N-oxide thereof;


a compound of formula (IId)




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wherein


R1 and R2 are independently hydrogen, C1-C4alkyl, C1-C4haloalkyl, halo, cyano, hydroxy, C1-C4alkoxy, C1-C4alkylthio, aryl or aryl substituted by one to five R6, which may be the same or different, or heteroaryl or heteroaryl substituted by one to five R6, which may be the same or different;


R3 is hydrogen, C1-C10alkyl, C1-C4haloalkyl, C2-C10alkenyl, C2-C4haloalkenyl, C2-C10alkynyl, C2-C4haloalkynyl, C3-C10cycloalkyl, C3-C10cycloalkyl-C1-C6alkyl-, C1-C10alkoxy-C1-C6alkyl-, C1-C10cyanoalkyl-, C1-C10alkoxycarbonyl-C1-C6alkyl-, N—C1-C3alkyl-aminocarbonyl-C1-C6alkyl-, N,N-di-(C1-C3alkyl)-aminocarbonyl-C1-C6alkyl-, aryl-C1-C6alkyl- or aryl-C1-C6alkyl- wherein the aryl moiety is substituted by one to three R7, which may be the same or different, or heterocyclyl-C1-C6alkyl- or heterocyclyl-C1-C6alkyl- wherein the heterocyclyl moiety is substituted by one to three R7, which may be the same or different;


R4 is aryl or aryl substituted by one to five R8, which may be the same or different, or heteroaryl or heteroaryl substituted by one to four R8, which may be the same or different;


R5 is hydroxy or a group which can be metabolised to the hydroxy group;


each R6, R7 and R8 is independently halo, cyano, nitro, C1-C10alkyl, C1-C4haloalkyl, C2-C10alkenyl, C2-C10alkynyl, hydroxy, C1-C10alkoxy, C1-C4haloalkoxy, C1-C10alkoxy-C1-C4alkyl-, C3-C7cycloalkyl, C3-C7cycloalkoxy, C3-C7cycloalkyl-C1-C4alkyl-, C3-C7cycloalkyl-C1-C4alkoxy-, C1-C6alkylcarbonyl-, formyl, C1-C4alkoxy-carbonyl-, C1-C4alkylcarbonyloxy-, C1-C10alkylthio-, C1-C4haloalkylthio-, C1-C10alkylsulfonyl-, C1-C4haloalkylsulfonyl-, amino, C1-C10alkylamino-, C1-C10alkylcarbonylamino-, aryl or aryl substituted by one to three R13, which may be the same or different, heteroaryl or heteroaryl substituted by one to three R13, which may be the same or different, aryl-C1-C4alkyl- or aryl-C1-C4alkyl- wherein the aryl moiety is substituted by one to three R13, which may be the same or different, heteroaryl-C1-C4alkyl- or heteroaryl-C1-C4alkyl- wherein the heteroaryl moiety is substituted by one to three R13, which may be the same or different, aryloxy- or aryloxy-substituted by one to three R13, which may be the same or different, heteroaryloxy- or heteroaryloxy-substituted by one to three R13, which may be the same or different, arylthio- or arylthio-substituted by one to three R13, which may be the same or different, or heteroarylthio- or heteroarylthio-substituted by one to three R13, which may be the same or different; and


each R13 is independently halo, cyano, nitro, C1-C6alkyl, C1-C6haloalkyl or C1-C6alkoxy; or a salt or N-oxide thereof;


IIe) a compound of formula (IIe)




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wherein


A1, A2, A3 and A4 are independently C—R1 or N, provided at least one of A1, A2, A3 and A4 is N, and provided that if A1 and A4 are both N, A2 and A3 are not both C—R1;


each R1 is independently hydrogen, C1-C4alkyl, C1-C4haloalkyl, halo, cyano, hydroxy, C1-C4alkoxy, C1-C4alkylthio, aryl or aryl substituted by one to five R6, which may be the same or different, or heteroaryl or heteroaryl substituted by one to five R6, which may be the same or different;


R3 is hydrogen, C1-C10alkyl, C1-C4haloalkyl, C2-C10alkenyl, C2-C4haloalkenyl, C2-C10alkynyl, C2-C4haloalkynyl, C3-C10cycloalkyl, C3-C10cycloalkyl-C1-C6alkyl-, C1-C10alkoxy-C1-C6alkyl-, C1-C10cyanoalkyl-, C1-C10alkoxycarbonyl-C1-C6alkyl-, N—C1-C3alkyl-aminocarbonyl-C1-C6alkyl-, N,N-di-(C1-C3alkyl)-aminocarbonyl-C1-C6alkyl-, aryl-C1-C6alkyl- or aryl-C1-C6alkyl- wherein the aryl moiety is substituted by one to three R7, which may be the same or different, or heterocyclyl-C1-C6alkyl- or heterocyclyl-C1-C6alkyl- wherein the heterocyclyl moiety is substituted by one to three R7, which may be the same or different;


R4 is aryl or aryl substituted by one to five R8, which may be the same or different, or heteroaryl or heteroaryl substituted by one to four R8, which may be the same or different;


R5 is hydroxy or a group which can be metabolised to a hydroxy group;


each R6, R7 and R8 is independently halo, cyano, nitro, C1-C10alkyl, C1-C4haloalkyl, C2-C10alkenyl, C2-C10alkynyl, hydroxy, C1-C10alkoxy, C1-C4haloalkoxy, C1-C10alkoxy-C1-C4alkyl-, C3-C7cycloalkyl, C3-C7cycloalkoxy, C3-C7cycloalkyl-C1-C4alkyl-, C3-C7cycloalkyl-C1-C4alkoxy-, C1-C6alkylcarbonyl-, formyl, C1-C4alkoxy-carbonyl-, C1-C4alkylcarbonyloxy-, C1-C10alkylthio-, C1-C4haloalkylthio-, C1-C10alkylsulfinyl-, C1-C4haloalkylsulfinyl-, C1-C10alkylsulfonyl-, C1-C4haloalkylsulfonyl-, amino, C1-C10alkylamino-, di-C1-C10alkylamino-, C1-C10alkylcarbonylamino-, aryl or aryl substituted by one to three R13, which may be the same or different, heteroaryl or heteroaryl substituted by one to three R13, which may be the same or different, aryl-C1-C4alkyl- or aryl-C1-C4alkyl- wherein the aryl moiety is substituted by one to three R13, which may be the same or different, heteroaryl-C1-C4alkyl- or heteroaryl-C1-C4alkyl- wherein the heteroaryl moiety is substituted by one to three R13, which may be the same or different, aryloxy- or aryloxy-substituted by one to three R13, which may be the same or different, heteroaryloxy- or heteroaryloxy-substituted by one to three R13, which may be the same or different, arylthio- or arylthio-substituted by one to three R13, which may be the same or different, or heteroarylthio- or heteroarylthio-substituted by one to three R13, which may be the same or different; and


each R13 is independently halo, cyano, nitro, C1-C6alkyl, C1-C6haloalkyl or C1-C6alkoxy; or a salt or N-oxide thereof; and


a compound of formula (IIf)




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wherein


R1 is C1-C6 alkyl or C1-C6alkyloxy-C1-C6alkyl;


R2 is hydrogen or C1-C6alkyl;


G is a hydrogen, —(C=L)R3, —(SO2)R4, or —(P=L)R5R6, wherein


L is oxygen or sulfur;


R3 is C1-C6alkyl, C3-C8cycloalkyl, C2-C6alkenyl, C2-C6alkynyl, C6-C10aryl, C6-C10aryl-C1-C6alkyl-, C1-C6alkyloxy, C3-C8cycloalkyloxy, C2-C6alkenyloxy, C2-C6alkynyloxy, C6-C10aryloxy, C6-C10aryl-C1-C6alkyloxy-, amino, C1-C6alkylamino, C2-C6alkenylamino, C6-C10arylamino, di(C1-C6alkyl)amino, di(C2-C6alkenyl)amino, (C1-C6alkyl)(C6-C10aryl)amino or a three- to eight-membered nitrogen containing heterocyclic ring,


R4 is C1-C6alkyl, C6-C10aryl, C1-C6alkylamino group or di(C1-C6 alkyl)amino; and


R5 and R6 may be same or different and are independently C1-C6alkyl, C3-8cycloalkyl, C2-C6alkenyl, C6-C10aryl, C1-C6alkyloxy, C3-C8cycloalkyloxy, C6-C10aryloxy, C6-C10aryl-C1-C6alkyloxy, C1-C6alkylthio, C1-C6alkylamino or di(C1-C6alkyl)amino, whereby any R3, R4, R5 and R6 group may be substituted with halogen, C3-C8cycloalkyl, C6-C10aryl, C6-C10aryl-C1-C6alkyl-, C3-C8cycloalkyloxy, C6-C10aryloxy, C6-C10aryl-C1-C6alkyloxy-, C6-C10arylamino, (C1-C6 alkyl)(C6-C10aryl)amino and a three- to eight-membered nitrogen containing heterocyclic ring which may be substituted with at least one C1-C6alkyl;


Z1 is C1-C6alkyl;


Z2 is C1-C6alkyl;


n is 0, 1, 2, 3 or 4;


and each of Z2 may be same or different when n represents an integer of 2 or more, and a sum of the number of carbon atoms in the group represented by Z1 and that in the group represented by Z2 is equal to 2 or more.


The HST inhibitors of formula (IIa) are known, for example haloxydine and pyriclor. The HST inhibitors of formula (IIb) are known from, for example WO 2008/009908. The HST inhibitors of formula (IIc) are known from, for example WO 2008/071918. The HST inhibitors of formula (IId) are known from, for example WO 2009/063180. The HST inhibitors of formula (IIe) are known from, for example WO2009/090401 and WO2009/090402. The HST inhibitors of formula (IIf) are known from, for example WO 2007/119434.


Preferred are the compounds of formula (IIa)




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wherein


R1, R2, R3 and R4 are independently hydrogen, bromo, chloro or fluoro; provided that at least three of R1, R2, R3 and R4 are either bromo, chloro or fluoro, most preferred is the compound of formula (IIa) wherein R1 and R4 are fluoro and R2 and R3 are chloro (haloxydine) or wherein R1, R2 and R3 are chloro and R4 is hydrogen (pyriclor).


The term “HPPD inhibiting herbicide” refers to herbicides that act either directly or as procides to inhibit HPPD and that, in their active form, exhibit a Ki value of less than 5 nM, preferably 1 nM versus Arabidopsis HPPD when assayed using the on and off rate methods described in WO 02/46387. Within the context of the present invention the terms hydroxy phenyl pyruvate (or pyruvic acid) dioxygenase (HPPD), 4-hydroxy phenyl pyruvate (or pyruvic acid) dioxygenase (4-HPPD) and p-hydroxy phenyl pyruvate (or pyruvic acid) dioxygenase (p-HPPD) are synonymous.


Preferably, the HPPD-inhibiting herbicide is selected from the group consisting of


a compound of formula (Ia)




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wherein R1 and R2 are hydrogen or together form an ethylene bridge;


R3 is hydroxy or phenylthio-; R4 is halogen, nitro, C1-C4alkyl, C1-C4alkoxy-C1-C4alkyl-, C1-C4alkoxy-C1-C4alkoxy-C1-C4alkyl-;


X is methine, nitrogen, or C—R5 wherein R5 is hydrogen, C1-C4haloalkoxy-C1-C4alkyl-, or a group




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and


R6 is C1-C4alkylsulfonyl- or C1-C4haloalkyl;


a compound of formula (Ib)




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R1 and R2 are independently C1-C4alkyl; and the free acids thereof;


a compound of formula (Ic)




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wherein R1 is hydroxy, phenylcarbonyl-C1-C4alkoxy- or phenylcarbonyl-C1-C4alkoxy- wherein the phenyl moiety is substituted in para-position by halogen or C1-C4alkyl, or phenylsulfonyloxy- or phenylsulfonyloxy- wherein the phenyl moiety is substituted in para-position by halogen or C1-C4alkyl;


R2 is C1-C4alkyl;


R3 is hydrogen or C1-C4alkyl; R4 and R6 are independently halogen, C1-C4alkyl, C1-C4haloalkyl, or C1-C4alkylsulfonyl-; and


R5 is hydrogen, C1-C4alkyl, C1-C4alkoxy-C1-C4alkoxy-, or a group




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a compound of formula (Id)




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wherein R1 is hydroxy;


R2 is C1-C4alkyl;


R3 is hydrogen; and R4, R5 and R6 are independently C1-C4alkyl;


a compound of formula (Ie)




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wherein R1 is cyclopropyl;


R2 and R4 are independently halogen, C1-C4haloalkyl, or C1-C4alkylsulfonyl-; and R3 is hydrogen; and


a compound of formula (If)




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wherein R1 is cyclopropyl;


R2 and R4 are independently halogen, C1-C4haloalkyl, or C1-C4alkylsulfonyl-; and


R3 is hydrogen.


Example HPPD-inhibitors are also disclosed in WO2009/016841. In a preferred embodiment the HPPD inhibitor is selected from the group consisting of benzobicyclon, mesotrione, sulcotrione, tefuryltrione, tembotrione, 4-hydroxy-3-[[2-(2-methoxyethoxy)methyl]-6-(trifluoromethyl)-3-pyridinylicarbonyl]-bicyclo[3.2.1]-oct-3-en-2-one (bicyclopyrone), ketospiradox or the free acid thereof, benzofenap, pyrasulfotole, pyrazolynate, pyrazoxyfen, topramezone, [2-chloro-3-(2-methoxyethoxy)-4-(methylsulfonyl)phenyl](1-ethyl-5-hydroxy-1H-pyrazol-4-yl)-methanone, (2,3-dihydro-3,3,4-trimethyl-1,1-dioxidobenzo[b]thien-5-yl)(5-hydroxy-1-methyl-1H-pyrazol-4-yl)-methanone, isoxachlortole, isoxaflutole, α-(cyclopropylcarbonyl)-2-(methylsulfonyl)-β-oxo-4-chloro-benzenepropanenitrile, and α-(cyclopropylcarbonyl)-2-(methylsulfonyl)-β-oxo-4-(trifluoromethyl)-benzenepropanenitrile.


These HPPD inhibitors are known and have the following Chemical Abstracts registration numbers: benzobicyclon (CAS RN156963-66-5), mesotrione (CAS RN 104206-82-8), sulcotrione (CAS RN 99105-77-8), tefuryltrione (CAS RN 473278-76-1), tembotrione (CAS RN 335104-84-2), 4-hydroxy-3-[[2-(2-methoxyethoxy)methyl]-6-(trifluoromethyl)-3-pyridinyl]carbonyl]-bicyclo[3.2.1]oct-3-en-2-one (CAS RN 352010-68-5), ketospiradox (CAS RN 192708-91-1) or its free acid (CAS RN 187270-87-7), benzofenap (CAS RN 82692-44-2), pyrasulfotole (CAS RN 365400-11-9), pyrazolynate (CAS RN 58011-68-0), pyrazoxyfen (CAS RN 71561-11-0), topramezone (CAS RN 210631-68-8), [2-chloro-3-(2-methoxyethoxy)-4-(methylsulfonyl)phenyl](1-ethyl-5-hydroxy-1H-pyrazol-4-yl)-methanone (CAS RN 128133-27-7), (2,3-dihydro-3,3,4-trimethyl-1,1-dioxidobenzo[b]thien-5-yl)(5-hydroxy-1-methyl-1H-pyrazol-4-yl)-methanone (CAS RN 345363-97-5), isoxachlortole (CAS RN 141112-06-3), isoxaflutole (CAS RN 141112-29-0), α-(cyclopropylcarbonyl)-2-(methylsulfonyl)-β-oxo-4-chloro-benzenepropanenitrile (CAS RN 143701-66-0), and α-(cyclopropylcarbonyl)-2-(methylsulfonyl)-β-oxo-4-(trifluoromethyl)-benzenepropanenitrile (CAS RN 143701-75-1).


The following definitions apply to those terms used in respect of Formula I and Formula II.


Alkyl moiety (either alone or as part of a larger group, such as alkoxy, alkoxy-carbonyl, alkylcarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl) is a straight or branched chain and is, for example, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl or neo-pentyl. The alkyl groups are preferably C1 to C6 alkyl groups, more preferably C1-C4 and most preferably methyl groups.


Alkenyl and alkynyl moieties (either alone or as part of a larger group, such as alkenyloxy or alkynyloxy) can be in the form of straight or branched chains, and the alkenyl moieties, where appropriate, can be of either the (E)- or (Z)-configuration. Examples are vinyl, allyl and propargyl. The alkenyl and alkynyl groups are preferably C2 to C6 alkenyl or alkynyl groups, more preferably C2-C4 and most preferably C2-C3 alkenyl or alkynyl groups.


Alkoxyalkyl groups preferably have a chain length of from 2 to 8 carbon or oxygen atoms. An example of an alkoxyalkyl group is 2-methoxy-ethyl-.


Halogen is generally fluorine, chlorine, bromine or iodine, preferably fluorine or chlorine. The same is true of halogen in conjunction with other meanings, such as haloalkyl.


Haloalkyl groups preferably have a chain length of from 1 to 4 carbon atoms. Haloalkyl is, for example, fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 2-fluoroethyl, 2-chloroethyl, pentafluoroethyl, 1,1-difluoro-2,2,2-trichloroethyl, 2,2,3,3-tetrafluoroethyl or 2,2,2-trichloroethyl; preferably trichloromethyl, difluorochloromethyl, difluoromethyl, trifluoromethyl or dichlorofluoromethyl.


Haloalkoxyalkyl groups preferably have a chain length of from 2 to 8 carbon or oxygen atoms. An example of an alkoxyalkyl group is 2,2,2-trifluoroethoxymethyl-Alkoxyalkoxy groups preferably have a chain length of from 2 to 8 carbon or oxygen atoms. Examples of alkoxyalkoxy are: methoxymethoxy, 2-methoxy-ethoxy, methoxypropoxy, ethoxymethoxy, ethoxyethoxy, propoxymethoxy and butoxybutoxy.


Alkoxyalkyl groups have a chain length of preferably from 1 to 6 carbon atoms. Alkoxyalkyl is, for example, methoxymethyl, methoxyethyl, ethoxymethyl, ethoxyethyl, n-propoxymethyl, n-propoxyethyl, isopropoxymethyl or isopropoxyethyl. Alkoxyalkoxyalkyl groups preferably have a chain length of from 3 to 8 carbon or oxygen atoms. Examples of alkoxy-alkoxy-alkyl are: methoxymethoxymethyl, methoxyethoxymethyl, ethoxymethoxymethyl and methoxyethoxyethyl.


Cyanoalkyl groups are alkyl groups which are substituted with one or more cyano groups, for example, cyanomethyl or 1,3-dicyanopropyl.


Cycloalkyl groups can be in mono- or bi-cyclic form and may optionally be substituted by one or more methyl groups. The cycloalkyl groups preferably contain 3 to 8 carbon atoms, more preferably 3 to 6 carbon atoms. Examples of monocyclic cycloalkyl groups are cyclopropyl, 1-methylcyclopropyl, 2-methylcyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.


In the context of the present specification the term “aryl” refers to a ring system which may be mono-, bi- or tricyclic. Examples of such rings include phenyl, naphthalenyl, anthracenyl, indenyl or phenanthrenyl. A preferred aryl group is phenyl.


The term “heteroaryl” refers to an aromatic ring system containing at least one heteroatom and consisting either of a single ring or of two or more fused rings. Preferably, single rings will contain up to three and bicyclic systems up to four heteroatoms which will preferably be chosen from nitrogen, oxygen and sulfur. Examples of such groups include pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, furanyl, thiophenyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyrrolyl, pyrazolyl, imidazolyl, triazolyl and tetrazolyl. A preferred heteroaryl group is pyridine. Examples of bicyclic groups are benzothiophenyl, benzimidazolyl, benzothiadiazolyl, quinolinyl, cinnolinyl, quinoxalinyl and pyrazolo[1,5-a]pyrimidinyl.


The term “heterocyclyl” is defined to include heteroaryl and in addition their unsaturated or partially unsaturated analogues such as 4,5,6,7-tetrahydro-benzothiophenyl, chromen-4-onyl, 9H-fluorenyl, 3,4-dihydro-2H-benzo-1,4-dioxepinyl, 2,3-dihydro-benzofuranyl, piperidinyl, 1,3-dioxolanyl, 1,3-dioxanyl, 4,5-dihydro-isoxazolyl, tetrahydrofuranyl and morpholinyl.


It should be understood that in the aforementioned methods the herbicide composition may be applied to the locus pre-emergence of the crop and/or post-emergence of the crop. In a preferred embodiment the herbicide composition is applied post-emergence of the crop—a so-called “over-the-top” application. Single or indeed multiple applications may be applied as necessary to obtain the desired weed control.


The term “weeds” relates to any unwanted vegetation and includes, for example, carry-over or “rogue” or “volunteer” crop plants in a field of soybean crop plants.


Typically, the heterologous polynucleotide will comprise (i) a plant operable promoter operably linked to (ii) the region encoding the HST enzyme and (iii) a transcription terminator. Typically, the heterologous polynucleotide will further comprise a region which encodes a polypeptide capable of targeting the HST enzyme to subcellular organelles such as the chloroplast or mitochondria—preferably the chloroplast. The heterologous polynucleotide may further comprise, for example, transcriptional enhancers. Furthermore, the region encoding the HST enzyme can be “codon-optimised” depending on plant host in which expression of the HST enzyme is desired. The skilled person is well aware of plant operable promoters, transcriptional terminators, chloroplast transit peptides, enhancers etc that have utility with the context of the present invention.


The HST may be a “wild type” enzyme or it may be one which has been modified in order to afford preferential kinetic properties with regard to provision of herbicide tolerant plants. In a preferred embodiment the HST is characterised in that it comprises one or more of the following polypeptide motifs:—


W-(R/K)-F-L-R-P-H-T-I-R-G-T; and/or


N-G-(Y/F)-I-V-G-I-N-Q-I-(Y/F)-D; and/or


I-A-I-T-K-D-L-P; and/or


Y-(R/Q)-(F/W)-(I/V)-W-N-L-F-Y.

Suitable HSTs are derived from Arabidopsis thaliana, Glycine max, Oryza sativa or Chlatnydomonas reinhardtii. In an even more preferred embodiment the HST is selected from the group consisting of SEQ ID NO:1 to SEQ ID NO. 10. It should be noted that amino acid sequences provided in SEQ ID NOS:1 to 10 are examples of HST amino acid sequences that include a region encoding a chloroplast transit peptide.


SEQ ID NOS 11-20 correspond to DNA sequences encoding the HSTs depicted as SEQ ID NO. 1-10 while SEQ ID NOS 21-24 are examples of DNA sequences encoding truncated mature HST sequences without the transit peptide region.


Amino acid sequences provided in SEQ ID NOS 25-28 are examples of HPPD amino acid sequences and SEQ ID NOS 29-32 are examples of DNA sequences encoding them. HPPDs suitable for providing tolerance to HPPD-inhibiting herbicides are well known to the skilled person—e.g WO 02/46387. SEQ ID No 33 provides the DNA sequence of the TMV translational enhancer and SEQ ID No 34 provides the DNA sequence of the TMV translational enhancer fused 5′ to the DNA sequence encoding Arabidopsis HST.


It should be further understood that the crop plant used in said method may further comprise a further heterologous polynucleotide encoding a further herbicide tolerance enzyme. Examples of further herbicide tolerance enzymes include, for example, herbicide tolerance enzymes selected from the group consisting of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), Glyphosate acetyl transferase (GAT), Cytochrome P450, phosphinothricin acetyltransferase (PAT), Acetolactate synthase (ALS), Protoporphyrinogen oxidase (PPGO), Phytoene desaturase (PD), dicamba degrading enzymes (e.g WO 02/068607), and aryloxy herbicide degrading enzymes as taught in WO2007/053482 & WO2005/107437.


The pesticide composition used in the aforementioned methods may further comprise one or more additional pesticides—in particular herbicides—to which the crop plant is naturally tolerant, or to which it is resistant via expression of one or more additional transgenes as mentioned herein. In a preferred embodiment the one or more additional herbicides are selected from the group consisting of glyphosate (including agrochemically acceptable salts thereof); glufosinate (including agrochemically acceptable salts thereof); chloroacetanilides e.g alachlor, acetochlor, metolachlor, S-metholachlor; photo system II inhibitors e.g triazines such as ametryn, atrazine, cyanazine and terbuthylazine, triazinones such as hexazinone and metribuzin, ureas such as chlorotoluron, diuron, isoproturon, linuron and terbuthiuron; ALS-inhibitors e.g sulfonyl ureas such as amidosulfuron, chlorsulfuron, flupyrsulfuron, halosulfuron, nicosulfuron, primisulfuron, prosulfuron, rimsulfuron, triasulfuron, trifloxysulfuron and tritosulfuron; diphenyl ethers e.g acifluorofen and fomesafen.


The present invention further provides a recombinant polynucleotide which comprises a region which encodes an HST-enzyme operably linked to a plant operable promoter, wherein the region which encodes the HST-enzyme does not include the polynucleotide sequence depicted in SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 14 or SEQ ID NO. 15. In a preferred embodiment the HST-enzyme is selected from the group consisting of SEQ ID NO. 3, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 10.


The present invention still further provides a recombinant polynucleotide comprising (i) a region which encodes a HST enzyme operably linked to a plant operable promoter and (ii) at least one additional heterologous polynucleotide, which comprises a region which encodes an additional herbicide tolerance enzyme, operably linked to a plant operable promoter. The additional herbicide tolerance enzyme is, for example, selected from the group consisting of hydroxyphenyl pyruvate dioxygenase (HPPD), 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), Glyphosate acetyl transferase (GAT), Cytochrome P450, phosphinothricin acetyltransferase (PAT), Acetolactate synthase (ALS), Protoporphyrinogen oxidase (PPGO), Phytoene desaturase (PD) and dicamba degrading enzymes as taught in WO 02/068607.


Preferably the recombinant polynucleotide comprises (i) a region which encodes a HST operably linked to a plant operable promoter and (ii) a region which encodes an HPPD operably linked to a plant operable promoter. It is also possible for the recombinant polynucleotide to comprise at least two, three, or more additional regions each encoding a herbicide tolerance enzyme for example as defined previously. Thus, in another preferred embodiment the recombinant polynucleotide comprises (i) a region which encodes a HST enzyme, (ii) a region which encodes a HPPD enzyme and (iii) a region which encodes a glyphosate tolerance enzyme.


The present invention further provides a vector comprising a recombinant polynucleotide according to the present invention.


The present invention further relates to transformed plants over expressing an HST enzyme which exhibit substantial resistance or substantial tolerance to HST-inhibiting herbicides and/or HPPD-inhibiting herbicides when compared with non transgenic like plants. It should also be appreciated that the transformed plants of the present invention typically exhibit enhanced stress tolerance including heat and drought tolerance.


Thus, the present invention further provides a plant cell which exhibits substantial resistance or substantial tolerance to HST-inhibiting herbicides and/or HPPD-inhibiting herbicides when compared with non transgenic like plant cell—said plant cell comprising the recombinant polynucleotide of the present invention as herein described. It should be appreciated that the region encoding the HST and any region encoding one or more additional herbicide tolerance enzymes may be provided on the same (“linked”) or indeed separate transforming recombinant polynucleotide molecules.


The plant cell may further comprise further transgenic traits, for example heterologous polynucleotides providing resistance to insects, fungi and/or nematodes.


The present invention further provides morphologically normal fertile HST-inhibitor tolerant plants, plant cells, tissues and seeds which comprise a plant cell according to the present invention.


Plants or plant cells transformed include but are not limited to, field crops, fruits and vegetables such as canola, sunflower, tobacco, sugar beet, cotton, maize, wheat, barley, rice, sorghum, tomato, mango, peach, apple, pear, strawberry, banana, melon, mangelworzel, potato, carrot, lettuce, cabbage, onion, etc. Particularly preferred genetically modified plants are soya spp, sugar cane, pea, field beans, poplar, grape, citrus, alfalfa, rye, oats, turf and forage grasses, flax and oilseed rape, and nut producing plants insofar as they are not already specifically mentioned. In a particularly preferred embodiment of the method the said plant is a dicot, preferably selected from the group consisting of canola, sunflower, tobacco, sugar beet, soybean, cotton, sorghum, tomato, mango, peach, apple, pear, strawberry, banana, melon, potato, carrot, lettuce, cabbage, onion, and is particularly preferably soybean. In further preferred embodiments the said plant is maize or rice. Preferably the plant of the invention is soybean, rice or maize. The invention also includes the progeny of the plant of the preceding sentence, and the seeds or other propagating material of such plants and progeny.


In a particularly preferred aspect, the recombinant polynucleotide of the present invention is used to protect soybean crops from the herbicidal injury of HPPD inhibitor herbicides of the classes of HPPD chemistry selected from the group consisting of the compounds of formula Ia or Ig. In a further embodiment the HPPD inhibitor herbicide is selected from sulcotrione, mesotrione, tembotrione and compounds of formula Ia where X is nitrogen and R4 is CF3, CF2H or CFH2 and/or where R1 and R2 together form an ethylene bridge.


The present invention still further provides a method of providing a transgenic plant which is tolerant to HST-inhibiting and/or HPPD-inhibiting herbicides which comprises transformation of plant material with a recombinant polynucleotide(s) which comprises a region which encodes an HST enzyme, selection of the transformed plant material using an HST-inhibiting herbicide and/or HPPD-inhibiting herbicide, and regeneration of that material into a morphological normal fertile plant. In a preferred embodiment the transformed plant material is selected using a HST-inhibiting herbicide alone or in combination with a HPPD-inhibiting herbicide.


The present invention further relates to the use of polynucleotide which comprises a region which encodes an HST enzyme as a selectable marker in plant transformation and to the use of a polynucleotide comprising a region which encodes an HST enzyme in the production of plants which are tolerant to herbicides which act wholly or in part by inhibiting HST.


The present invention still further relates to the use of HST inhibitors as selection agents in plant transformation and to the use of a recombinant HST enzyme in in vitro screening of potential herbicides.


The present invention still further provides a herbicidal composition, preferably a synergistic herbicide composition, comprising an HPPD-inhibiting herbicide (as defined herein) and a HST-inhibiting herbicide (as defined herein). The ratio of the HPPD-inhibiting herbicide to the HST-inhibiting herbicide in the composition is any suitable ratio—typically from 100:1 to 1:100, preferably from 1:10 to 1:100, even more preferably from 1:1 to 1:20. The skilled person will recognise that the optimal ratio will depend on the relative potencies and spectrum of the two herbicides which can be derived as a matter of routine experimental optimisation.


The herbicidal composition may further comprise one or more additional pesticidal ingredient(s). The additional pesticides may include, for example, herbicides, fungicides or insecticides (such as thiomethoxam)—however herbicides are preferred. Thus, the additional herbicide is preferably selected from the group consisting of glyphosate (including agrochemically acceptable salts thereof); glufosinate (including agrochemically acceptable salts thereof); chloroacetanilides e.g alachlor, acetochlor, metolachlor, S-metholachlor; photo system II (PS-II) inhibitors e.g triazines such as ametryn, atrazine, cyanazine and terbuthylazine, triazinones such as hexazinone and metribuzin, and ureas such as chlorotoluron, diuron, isoproturon, linuron and terbuthiuron; ALS-inhibitors e.g sulfonyl ureas such as amidosulfuron, chlorsulfuron, flupyrsulfuron, halosulfuron, nicosulfuron, primisulfuron, prosulfuron, rimsulfuron, triasulfuron, trifloxysulfuron and tritosulfuron; diphenyl ethers e.g acifluorofen and fomesafen. PS-II herbicides are a particularly preferred as such mixtures exhibit particularly good efficacy.


Thus, the present invention still further provides a method of selectively controlling weeds at a locus comprising crop plants and weeds comprising applying to the locus a weed controlling amount of a synergistic herbicidal composition as previously defined. In a further embodiment of the current invention HPPD and HST inhibiting herbicides are sprayed sequentially rather than at the same time as a mixture. Thus for example, in a programme of weed control of the current invention, the HST herbicide can be advantageously applied over a crop locus to which an HPPD herbicide has already been previously applied. Normally this would be in the same season but, especially in the case of more persistent HPPD herbicides, there would also be an advantage in using the HST herbicide in the following season. In addition HST inhibiting herbicides are advantageously used as part of a programme of weed control wherein an HPPD inhibiting herbicide is applied earlier in the season or even in the preceding season.


The HPPD-inhibiting herbicide may be applied to the locus at any suitable rate—for example from 1 to 1000 g/ha, more preferably from 2 to 200 g/ha. Likewise, the HST-inhibiting herbicide may be applied at any suitable rate—for example from 10 to 2000 g/ha, more preferably from 50 to 400 g/ha.


In another embodiment, the HPPD-inhibiting herbicide is applied to the locus at a rate which is sub-lethal to the weeds were the HPPD-inhibiting herbicide to be applied in the absence of other herbicides. The actual sub-lethal rate will depend on weed species present and the actual HPPD inhibitor but will typically be less than 50 g/ha—more preferably less than 10 g/ha. Thus, the present invention further provides the use of a sub-lethal application of an HPPD-inhibiting herbicide to increase the weed controlling efficacy of an HST-inhibiting herbicide.


The invention will be further apparent from the following non-limiting examples and sequence listings.









SEQUENCE LISTING


ARABIDOPSIS HST AMINO ACID SEQUENCE


SEQ ID NO. 1


melsisqsprvrfsslaprflaashhhrpsvhlagkfislprdvrfts


lstsrmrskfvstnyrkisiracsqvgaaesddpvldriarfqnacwr


flrphtirgtalgstalvtralienthlikwslvlkalsgllalicgn


gyivginqiydigidkvnkpylpiaagdlsvqsawllviffaiagllv


vgfnfgpfitslyslglflgtiysvpplrmkrfpvaafliiatvrgfl


lnfgvyhatraalglpfqwsapvafitsfvtlfalviaitkdlpdveg


drkfqistlatklgvrniaflgsglllvnyvsaislafympqvfrgsl


mipahvilasglifqtwvlekanytkeaisgyyrfiwnlfyaeyllfp


fl





RICE HST AMINO ACID SEQUENCE


SEQ ID NO. 2


maslaspplpcraaatasrsgrpaprllgpppppaspllssasarfpr


apcnaarwsrrdavrvcsqagaagpaplsktlsdlkdscwrflrphti


rgtalgsmslvaralienpqlinwwlvfkafyglvalicgngyivgin


qiydiridkvnkpylpiaagdlsvqtawllvvlfaaagfsivvtnfgp


fitslyclglflgtiysvppfrlkrypvaafliiatvrgfllnfgvyy


atraalgltfqwsspvafitcfvtlfalviaitkdlpdvegdrkyqis


tlatklgvrniaflgsgllianyvaaiavaflmpqafrrtvmvpvhaa


lavgiifqtwvleqakytkdaisqyyrfiwnlfyaeyiffpli





RICE HST VARIANT AMINO ACID SEQUENCE


SEQ ID NO. 3


maslaspplpcraaatasrsgrpaprllgpppppaspllssasarfpr


apcnaarwsrrdavrvcsqagaagpaplsktlsdlkdscwrflrphti


rgtalgsialvaralienpqlinwwlvfkafyglvalicgngyivgin


qiydiridkvnkpylpiaagdlsvqtawllvvlfaaagfsivvtnfgp


fitslyclglflgtiysvppfrlkrypvaafliiatvrgfllnfgvyy


atraalgltfqwsspvafitcfvtlfalviaitkdlpdvegdrkyqis


tlatklgvrniaflgsgllianyvaaiavaflmpqafrrtvmvpvhaa


lavgiifqtwvleqakytkdaisqyyrfiwnlfyaeyiffpli





SOYA HST AMINO ACID SEQUENCE


SEQ ID NO. 4


melslsptshrvpstiptlnsaklsstkatksqqplflgfskhfnsig


lhhhsyrccsnavperpqrpssiractgvgasgsdrplaerlldlkda


cwrflrphtirgtalgsfalvaralientnlikwslffkafcglfali


cgngyivginqiydisidkvnkpylpiaagdlsvqsawflviffaaag


lsiaglnfgpfifslytlglflgtiysvpplrmkrfpvaafliiatvr


gfllnfgvyyatraslglafewsspvvfittfvtffalviaitkdlpd


vegdrkyqistfatklgvrniaflgsgillvnyivsvlaaiympqafr


rwllipahtifaisliyqarileqanytkdaisgfyrfiwnlfyaeya


ifpfi





CHLAMYDOMONAS HST AMINO ACID SEQUENCE


SEQ ID NO. 5


mdlcsstgrgaclspastsrpcpapvhlrgrrlafspaqpagrrhlpv


lssaavpaplpnggndesfaqklanfpnafwkflrphtirgtilgtta


vtakvlmenpgcidwallpkallglvallcgngyivginqiydvdidv


vnkpflpvasgelspalawglclslaaagagivaanfgnlitslytfg


lflgtvysvpplrlkqyavpafmiiatvrgfllnfgvysatraalglp


fewspavsfitvfvtlfatviaitkdlpdvegdqannistfatrmgvr


nvallaigllmanylgaialaltystafnvplmagahailaatlalrt


lklhaasysreavasfyrwiwnlfyaeyallpfl





NICOTINIA HST AMINO ACID SEQUENCE


SEQ ID NO. 6


melacsscsslrfssvlthqdtaasryrklpptspsckaanfvlkssk


nlsssaglhigytnfsktvsyrkyrhisiracsqvgtagsepvldkls


qfkdafwrflrphtirgtalgslslvtralienpnlirwslamkafsg


lialicgngyivginqiydigidkvnkpylpiaagdlsvqsawflvll


famagllivginfgpfitslyclglflgtiysvppfrmkrfavvafli


iatvrgfllnygvyyattaalglsfqwsspvafittfvtlfalviait


kdlpdvegdrkfqistlatklgvrniaflgsglllanyigavvaaiym


pqafrsslmipvhailalclvfqawllekanytkeaisayyqfiwnff


yaeylifpfi





AQUILEGIA HST AMINO ACID SEQUENCE


SEQ ID NO. 7


lcfsspsisipphcsttthyrkipinstfkstnflskasnnlttfgfs


rnkkysrsilsrksrhfsiwassqvgaagsddpllkkipdfkdavwrf


lrphtirgtalgsialvsralienthlikwsllfkaicgvfalmcgng


yivginqiydigidkvnkpylpiaagdlsvqsawslvtffavagvciv


afnfgpfitslyclglflgtiysvpplrmkrypvaafliiatvrgfll


nfgvyhatraalgltfewsypvafittfvtmfalviaitkdlpdvegd


rkfqistlatklgvrniallgtglllanyigaivaaiylpqafrrnlm


ipahtilalglvfqawaleqakyskeaildfyrfvwnlfyseyflfp


fi





BRASSICA NAPUS HST AMINO ACID SEQUENCE


SEQ ID NO. 8


melsishspclrfssssprflaasshhyrpsvhlagkllsrskdadlt


slssscmrskfvstnyrkisirassqvgaagsdpvldrlarfqnacwr


flrphtirgtalgstalvtralienthlikwslvlkalsgllalicgn


gyivginqiydigidkvnkpylpiaagdlsvqsawllviffaiagltv


vgfnfgpfitclyslglflgtiysvppfrmkrfpvaafliiatvrgfl


lnfgvyhatraalglsfqwsapvafitsfvtlfalviaitkdlpdveg


drkfqistlatklgvrniaflgsglllvnyisaislafympqvfrgsl


mipahmilasclvfqtwvlekanytkeaiagyyrfiwnlfyaeyllfp


ff





VITIS VINIFERA HST AMINO ACID SEQUENCE


SEQ ID NO. 9


mkvdavqastqvgaagsdpplnkfsvfkdacwrflrphtirgtalgst


alvaralienpnlikwsllfkafsgllalicgngyivginqiydisid


kvnkpylpiaagdlsvqsawflvlffavagvlivgsnfgsfitslycl


glvlgtiysvppfrmkrfpvaafliiatvrgfllnfgvyyatraalgl


pfmwsapvvfittfvtlfalviaitkdlpdvegdrkyqistlatklgv


rniaflgsgllivnyigsilaaiympqafrlslmipahailaaglifg


arvleqanytkeaisdfyrfiwnlfyveyiifpfi





PHYSCOMITRELLA PATENS HST SEQUENCE


SEQ ID NO. 10


mgltaivvdvagassssvalsqgrgatrrlpgglalgdafkglrkrey


aqglqcrvrreggcasearvwkvrcssdsagslggdlpasqpqqsevs


girdpaaasaasfaplpqrialfydafwrflrphtirgtflgtsalvt


rallenptlinwallpkalrgllallcgngfivginqifdsgidkvnk


pflpiaagdlsvpaawalvgglaalgvglvatnfgplittlytfglfl


gtiysvpplrlkqypvpafmiiatvrgfllnfgvyyatraalglsyew


spsvmfitifvtlfatviaitkdlpdiegdkkfnistfatnlgvrkis


flgaglllvnyigaivaafylpqafktkimvtghavlglsliyqtwll


dtakyskeaisnfyrfiwnlfyseyalfpfi





ARABIDOPSIS HST (DNA)


SEQ ID NO. 11


atggagctctcgatctcacaatcaccgcgtgttcggttctcgtctctg


gcgcctcgtttcttagcagcttctcatcatcatcgtccttctgtgcat


ttagctgggaagtttataagcctccctcgagatgttcgcttcacgagc


ttatcaacttcaagaatgcggtccaaatttgtttcaaccaattataga


aaaatctcaatccgggcatgttctcaggttggtgctgctgagtctgat


gatccagtgctggatagaattgcccggttccaaaatgcttgctggaga


tttcttagaccccatacaatccgcggaacagctttaggatccactgcc


ttggtgacaagagctttgatagagaacactcatttgatcaaatggagt


cttgtactaaaggcactttcaggtcttcttgctcttatttgtgggaat


ggttatatagtcggcatcaatcagatctacgacattggaatcgacaaa


gtgaacaaaccatacttgccaatagcagcaggagatctatcagtgcag


tctgcttggttgttagtgatattttttgcgatagcagggcttttagtt


gtcggatttaactttggtccattcattacaagcctatactctcttggc


ctttttctgggaaccatctattctgttccacccctcagaatgaaaaga


ttcccagttgcagcatttcttattattgccacggtacgaggtttcctt


cttaactttggtgtgtaccatgctacaagagctgctcttggacttcca


tttcagtggagtgcacctgtggcgttcatcacatcttttgtgacactg


tttgcactggtcattgctattacaaaggaccttcctgatgttgaagga


gatcgaaagttccaaatatcaaccctggcaacaaaacttggagtgaga


aacattgcattcctcggttctggacttctgctagtaaattatgtttca


gccatatcactagctttctacatgcctcaggtttttagaggtagcttg


atgattcctgcacatgtgatcttggcttcaggcttaattttccagaca


tgggtactagaaaaagcaaactacaccaaggaagctatctcaggatat


tatcggtttatatggaatctcttctacgcagagtatctgttattcccc


ttcctctag





RICE HST (DNA)


SEQ ID NO. 12


atggcttccctcgcctcccctcctctcccctgccgcgccgccgccacc


gccagccgcagcgggcgtcctgctccgcgcctcctcggccctccgccg


ccgcccgcttcccctctcctctcctccgcttcggcgcgcttcccgcgt


gccccctgcaacgccgcacgctggagccggcgcgacgccgtgcgggtt


tgctctcaagctggtgcagctggaccagccccattatcgaagacattg


tcagacctcaaggattcctgctggagatttttacggccacatacaatt


cgaggaactgcattgggatccatgtcattagttgctagagctttgata


gagaacccccaactgataaattggtggttggtattcaaagcgttctat


gggctcgtggcgttaatctgtggcaatggttacatcgttgggatcaat


cagatctatgacattagaatcgataaggtaaacaagccatatttacca


attgctgccggtgatctctcagttcagacagcatggttattggtggta


ttatttgcagctgcgggattttcaattgttgtgacaaactttggacct


ttcattacctctctatattgccttggtctatttcttggcaccatatac


tctgttcctccattcagacttaagagatatcctgttgctgcttttctt


atcattgcaacggtccgtggttttcttctcaactttggtgtgtactat


gctactagagcagcactgggtcttacattccaatggagctcgcctgtt


gctttcattacatgcttcgtgactttatttgctttggtcattgctata


accaaagatctcccagatgttgaaggggatcggaagtatcaaatatca


actttggcgacaaagctcggtgtcagaaacattgcatttcttggctct


ggtttattgatagcaaattatgttgctgctattgctgtagattttctc


atgcctcaggctttcaggcgcactgtaatggtgcctgtgcatgctgcc


cttgccgttggtataattttccagacatgggttctggagcaagcaaaa


tatactaaggatgctatttcacagtactaccggttcatttggaatctc


ttctatgctgaatacatcttcttcccgttgata





RICE VARIANT HST (DNA)


SEQ ID NO. 13


atggcttccctcgcctcccctcctctcccctgccgcgccgccgccacc


gccagccgcagcgggcgtcctgctccgcgcctcctcggccctccgccg


ccgcccgcttcccctctcctctcctccgcttcggcgcgcttcccgcgt


gccccctgcaacgccgcacgctggagccggcgcgacgccgtgcgggtt


tgctctcaagctggtgcagctggaccagccccattatcgaagacattg


tcagacctcaaggattcctgctggagatttttacggccacatacaatt


cgaggaactgccttgggatccatagcattagttgctagagctttgata


gagaacccccaactgataaattggtggttggtattcaaagcgttctat


gggctcgtggcgttaatctgtggcaatggttacatcgttgggatcaat


cagatctatgacattagaatcgataaggtaaacaagccatatttacca


attgctgccggtgatctctcagttcagacagcatggttattggtggta


ttatttgcagctgcgggattttcaattgttgtgacaaactttggacct


ttcattacctctctatattgccttggtctatttcttggcaccatatac


tctgttcctccattcagacttaagagatatcctgttgctgcttttctt


atcattgcaacggtccgtggttttcttctcaactttggtgtgtactat


gctactagagcagcactgggtcttacattccaatggagctcgcctgtt


gctttcattacatgcttcgtgactttatttgctttggtcattgctata


accaaagatctcccagatgttgaaggggatcggaagtatcaaatatca


actttggcgacaaagctcggtgtcagaaacattgcatttcttggctct


ggtttattgatagcaaattatgttgctgctattgctgtagattttctc


atgcctcaggctttcaggcgcactgtaatggtgcctgtgcatgctgcc


cttgccgttggtataattttccagacatgggttctggagcaagcaaaa


tatactaaggatgctatttcacagtactaccggttcatttggaatctc


ttctatgctgaatacatcttcttcccgttgatatag





SOYA HST (DNA)


SEQ ID NO. 14


tctgctaaattatcttctactaaagctactaaatctcaacaaccttta


tttttaggattttctaaacattttaattctattggattacatcatcat


tcttatagatgttgttctaatgctgtacctgaaagacctcaaagacct


tcttctattagagcttgtactggagtaggagcttctggatctgataga


cctttagctgaaagattattagatttaaaagatgcttgttggagattt


ttaagacctcatactattagaggaactgctttaggatcttttgcttta


gtagctagagatttaattgaaaatactaatttaattaaatggtcttta


ttttttaaagctttttgtggattatttgctttaatttgtggaaatgga


tatattgtaggaattaatcaaatttatgatatttctattgataaagta


aataaaccttatttacctattgctgctggagatttatctgtacaatct


gcttggtttttagtaattttttttgctgctgctggattatctattgct


ggattaaattttggaccttttattttttctttatatactttaggatta


tttttaggaactatttattctgtacctcctttaagaatgaaaagattt


cctgtagctgcttttttaattattgctactgtaagaggatttttatta


aattttggagtatattatgctactagagcttctttaggattagctttt


gaatggtcttctcctgtagtatttattactacttttgtaacttttttt


gctttagtaattgctattactaaagatttacctgatgtagaaggagat


agaaaatatcaaatttctacttttgctactaaattaggagtaagaaat


attgcttttttaggatctggaattttattagtaaattatattgtatct


gtattagctgctatttatatgcctcaagcttttagaagatggttatta


attcctgctcatactatttttgctatttctttaatttatcaagctaga


attttagaacaagctaattatactaaagatgctatttctggattttat


agatttatttggaatttattttatgctgaatatgctatttttcctttt


att





CHLAMYDOMONAS HST (DNA)


SEQ ID NO. 15


atggacctttgcagctcaactggaagaggagcatgcctttcgccggca


tccacgtcgcggccgtgcccagcaccagtgcatttgcgcggccgacgc


ctggctttctctccggctcagcctgctggacggcgccacttgccggtg


ctctcatctgcagcggtccccgctcccctcccaaatggtggaaacgac


gagagcttcgcacaaaaactggctaactttccaaacgccttctggaag


ttcctgcggccacacaccatccgggggactatcctgggcaccacagct


gtgaccgccaaggtccttatggagaaccccggctgcatagactgggca


ctgctgccgaaggcgctgctcggcctggtggcgctgctgtgcggcaac


ggctacattgtgggcatcaaccaaatctacgacgtcgacattgacgtg


gtcaacaagccattcctccccgtggcgtcgggcgagctgtcgccggcg


ctggcgtggggcctgtgtctgtcgctggcggctgcgggcgcgggcatc


gtagccgccaacttcggcaacctcatcaccagcctctacacctttggc


ctcttcctgggcaccgtgtacagtgtgcctcccctgcgcctgaagcag


tacgcggtgccggccttcatgatcatcgccacggtgcgcggcttcctg


ctcaacttcggcgtgtacagcgccacgcgggcggcactgggactgccc


ttcgagtggagcccggccgtcagcttcatcacggtgtttgtgacgctg


tttgccactgtgatcgccatcaccaaggacctgccggacgtggagggc


gaccaggccaacaacatctccaccttcgccacgcgcatgggcgtgcgc


aacgtggcactgctggccatcggccttctcatggccaactacctgggt


gccatcgcgctggcactcacctactccaccgccttcaacgtgccgctc


atggcgggcgcgcacgccatcctggccgccacgctggcgctgcgcacg


ctcaagctgcacgccgccagctacagccgggaggcggtggcgtccttc


taccgctggatctggaacctgttctacgccgagtacgcgctgctgccg


ttcctgtag





NICOTINIA HST DNA


SEQ ID NO. 16


atggaattagcttgttcttcttgttcttctttaagattttcttctgta


ttaactcatcaagatactgctgcttctagatatagaaaattacctcct


acttctccttcttgtaaagctgctaattttgtattaaaatcttctaaa


aatttatcttcttctgctggattacatattggatatactaatttttct


aaaactgtatcttatagaaaatatagacatatttctattagagcttgt


tctcaagtaggaactgctggatctgaacctgtattagataaattatct


caatttaaagatgctttttggagatttttaagacctcatactattaga


ggaactgctttaggatctttatctttagtaactagagctttaattgaa


aatcctaatttaattagatggtctttagctatgaaagctttttctgga


ttaattgctttaatttgtggaaatggatatattgtaggaattaatcaa


atttatgatattggaattgataaagtaaataaaccttatttacctatt


gctgctggagatttatctgtacaatctgcttggtttttagtattatta


tttgctatggctggattattaattgtaggaattaattttggacctttt


attacttctttatattgtttaggattatttttaggaactatttattct


gtacctccttttagaatgaaaagatttgctgtagtagcttttttaatt


attgctactgtaagaggatttttattaaattatggagtatattatgct


actactgctgctttaggattatcttttcaatggtcttctcctgtagct


tttattactacttttgtaactttatttgctttagtaattgctattact


aaagatttacctgatgtagaaggagatagaaaatttcaaatttctact


ttagctactaaattaggagtaagaaatattgcttttttaggatctgga


ttattattagctaattatattggagctgtagtagctgctatttatatg


cctcaagcttttagatcttctttaatgattcctgtacatgctatttta


gctttatgtttagtatttcaagcttggttattagaaaaagctaattat


actaaagaagctatttctgcttattatcaatttatttggaattttttt


tatgctgaatatttaatttttccttttatt





AQUILEGIA HST DNA


SEQ ID NO. 17


atgttatgtttttcttctccttctatttctattcctcctcattgttct


actactactcattatagaaaaattcctattaattctacttttaaatct


actaattttttatctaaagcttctaataatttaactacttttggattt


tctagaaataaaaaatattctagatctattttatctagaaaatctaga


catttttctatttgggcttcttctcaagtaggagctgctggatctgat


gatcctttattaaaaaaaattcctgattttaaagatgctgtatggaga


tttttaagacctcatactattagaggaactgctttaggatctattgct


ttagtatctagagctttaattgaaaatactcatttaattaaatggtct


ttattatttaaagctatttgtggagtatttgctttaatgtgtggaaat


ggatatattgtaggaattaatcaaatttatgatattggaattgataaa


gtaaataaaccttatttacctattgctgctggagatttatctgtacaa


tctgcttggtctttagtaactttttttgctgtagctggagtatgtatt


gtagcttttaattttggaccttttattacttctttatattgtttagga


ttatttttaggaactatttattctgtacctcctttaagaatgaaaaga


tatcctgtagctgcttttttaattattgctactgtaagaggattttta


ttaaattttggagtatatcatgctactagagctgctttaggattaact


tttgaatggtcttatcctgtagcttttattactacttttgtaactatg


tttgctttagtaattgctattactaaagatttacctgatgtagaagga


gatagaaaatttcaaatttctactttagctactaaattaggagtaaga


aatattgctttattaggaactggattattattagctaattatattgga


gctattgtagctgctatttatttacctcaagcttttagaagaaattta


atgattcctgctcatactattttagctttaggattagtatttcaagct


tgggctttagaacaagctaaatattctaaagaagctattttagatttt


tatagatttgtatggaatttattttattctgaatattttttatttcct


tttatt





BRASSICA NAPUS HST DNA


SEQ ID NO. 18


ttagctgcttcttctcatcattatagaccttctgtacatttagctgga


aaattattatctagatctaaagatgctgatttaacttctttatcttct


tcttgtatgagatctaaatttgtatctactaattatagaaaaatttct


attagagcttcttctcaagtaggagctgctggatctgatcctgtatta


gatagattagctagatttcaaaatgcttgttggagatttttaagacct


catactattagaggaactgctttaggatctactgctttagtaactaga


gctttaattgaaaatactcatttaattaaatggtctttagtattaaaa


gctttatctggattattagctttaatttgtggaaatggatatattgta


ggaattaatcaaatttatgatattggaattgataaagtaaataaacct


tatttacctattgctgctggagatttatctgtacaatctgcttggtta


ttagtaattttttttgctattgctggattaactgtagtaggatttaat


tttggaccttttattacttgtttatattctttaggattatttttagga


actatttattctgtacctccttttagaatgaaaagatttcctgtagct


gcttttttaattattgctactgtaagaggatttttattaaattttgga


gtatatcatgctactagagctgctttaggattatcttttcaatggtct


gctcctgtagcttttattacttcttttgtaactttatttgctttagta


attgctattactaaagatttacctgatgtagaaggagatagaaaattt


caaatttctactttagctactaaattaggagtaagaaatattgctttt


ttaggatctggattattattagtaaattatatttctgctatttcttta


gctttttatatgcctcaagtatttagaggatctttaatgattcctgct


catatgattttagcttcttgtttagtatttcaaacttgggtattagaa


aaagctaattatactaaagaagctattgctggatattatagatttatt


tggaatttattttatgctgaatatttattatttccttttttt





VITIS VINIFERA HST DNA


SEQ ID NO. 19


atgaaagtagatgctgtacaagcttctactcaagtaggagctgctgga


tctgatcctcctttaaataaattttctgtatttaaagatgcttgttgg


agatttttaagacctcatactattagaggaactgctttaggatctact


gctttagtagctagagctttaattgaaaatcctaatttaattaaatgg


tctttattatttaaagctttttctggattattagctttaatttgtgga


aatggatatattgtaggaattaatcaaatttatgatatttctattgat


aaagtaaataaaccttatttacctattgctgctggagatttatctgta


caatctgcttggtttttagtattattttttgctgtagctggagtatta


attgtaggatctaattttggatcttttattacttctttatattgttta


ggattagtattaggaactatttattctgtacctccttttagaatgaaa


agatttcctgtagctgcttttttaattattgctactgtaagaggattt


ttattaaattttggagtatattatgctactagagctgctttaggatta


ccttttatgtggtctgctcctgtagtatttattactacttttgtaact


ttatttgctttagtaattgctattactaaagatttacctgatgtagaa


ggagatagaaaatatcaaatttctactttagctactaaattaggagta


agaaatattgcttttttaggatctggattattattagtaaattatatt


ggatctattttagctgctatttatatgcctcaagcttttagattatct


ttaatgattcctgctcatgctattttagctgctggattaatttttcaa


gctagagtattagaacaagctaattatactaaagaagctatttctgat


ttttatagatttatttggaatttattttatgtagaatatattattttt


ccttttatt





PHYSCOMITRELLA PATENS HST DNA


SEQ ID NO. 20


atgggattaactgctattgtagtagatgtagctcaagcttcttcttct


tctgtagctttatctcaaggaagaggagctactagaagattacctgga


ggattagctttaggagatgcttttaaaggattaagaaaaagagaatat


gctcaaggattacaatgtagagtaagaagagaaggaggatgtgcttct


gaagctagagtatggaaagtaagatgttcttctgattctgctggatct


ttaggaggagatttacctgcttctcaacctcaacaatctgaagtatct


ggaattagagatcctgctgctgcttctgctgcttcttttgctccttta


cctcaaagaattgctttattttatgatgctttttggagatttttaaga


cctcatactattagaggaacttttttaggaacttctgctttagtaact


agagctttattagaaaatcctactttaattaattgggctttattacct


aaagctttaagaggattattagctttattatgtggaaatggatttatt


gtaggaattaatcaaatttttgattctggaattgataaagtaaataaa


ccttttttacctattgctgctggagatttatctgtacctgctgcttgg


gctttagtaggaggattagctgctttaggagtaggattagtagctact


aattttggacctttaattactactttatatacttttggattattttta


ggaactatttattctgtacctcctttaagattaaaacaatatcctgta


cctgcttttatgattattgctactgtaagaggatttttattaaatttt


ggagtatattatgctactagagctgctttaggattatcttatgaatgg


tctccttctgtaatgtttattactatttttgtaactttatttgctact


gtaattgctattactaaagatttacctgatattgaaggagataaaaaa


tttaatatttctacttttgctactaatttaggagtaagaaaaatttct


tttttaggagctggattattattagtaaattatattggagctattgta


gctgctttttatttacctcaagcttttaaaactaaaattatggtaact


ggacatgctgtattaggattatctttaatttatcaaacttggttatta


gatactgctaaatattctaaagaagctatttctaatttttatagattt


atttggaatttattttattctgaatatgctttatttccttttatt





DNA ENCODING MATURE ARABIDOPIS HST


SEQ ID NO. 21


agaaaaatctcaatccgggcatgttctcaggttggtgctgctgagtct


gatgatccagtgctggatagaattgcccggttccaaaatgcttgctgg


agatttcttagaccccatacaatccgcggaacagctttaggatccact


gccttggtgacaagagctttgatagagaacactcatttgatcaaatgg


agtcttgtactaaaggcactttcaggtcttcttgctcttatttgtggg


aatggttatatagtcggcatcaatcagatctacgacattggaatcgac


aaagtgaacaaaccatacttgccaatagcagcaggagatctatcagtg


cagtctgcttggttgttagtgatattttttgcgatagcagggctttta


gttgtcggatttaactttggtccattcattacaagcctatactctctt


ggcctttttctgggaaccatctattctgttccacccctcagaatgaaa


agattcccagttgcagcatttcttattattgccacggtacgaggtttc


cttcttaactttggtgtgtaccatgctacaagagctgctcttggactt


ccatttcagtggagtgcacctgtggcgttcatcacatcttttgtgaca


ctgtttgcactggtcattgctattacaaaggaccttcctgatgttgaa


ggagatcgaaagttccaaatatcaaccctggcaacaaaacttggagtg


agaaacattgcattcctcggttctggacttctgctagtaaattatgtt


tcagccatatcactagctttctacatgcctcaggtttttagaggtagc


ttgatgattcctgcacatgtgatcttggcttcaggcttaattttccag


acatgggtactagaaaaagcaaactacaccaaggaagctatctcagga


tattatcggtttatatggaatctcttctacgcagagtatctgttattc


cccttcctcta





DNA ENCODING MATURE RICE HST


SEQ ID NO. 22


cggcgcgacgccgtgcgggtttgctctcaagctggtgcagctggacca


gccccattatcgaagacattgtcagacctcaaggattcctgctggaga


tttttacggccacatacaattcgaggaactgccttgggatccatgtca


ttagttgctagagctttgatagagaacccccaactgataaattggtgg


ttggtattcaaagcgttctatgggctcgtggcgttaatctgtggcaat


ggttacatcgttgggatcaatcagatctatgacattagaatcgataag


gtaaacaagccatatttaccaattgctgccggtgatctctcagttcag


acagcatggttattggtggtattatttgcagctgcgggattttcaatt


gttgtgacaaactttggacctttcattacctctctatattgccttggt


ctatttcttggcaccatatactctgttcctccattcagacttaagaga


tatcctgttgctgcttttcttatcattgcaacggtccgtggttttctt


ctcaactttggtgtgtactatgctactagagcagcactgggtcttaca


ttccaatggagctcgcctgttgctttcattacatgcttcgtgacttta


tttgctttggtcattgctataaccaaagatctcccagatgttgaaggg


gatcggaagtatcaaatatcaactttggcgacaaagctcggtgtcaga


aacattgcatttcttggctctggtttattgatagcaaattatgttgct


gctattgctgtagcttttctcatgcctcaggctttcaggcgcactgta


atggtgcctgtgcatgctgcccttgccgttggtataattttccagaca


tgggttctggagcaagcaaaatatactaaggatgctatttcacagtac


taccggttcatttggaatctcttctatgctgaatacatcttcttcccg


ttgatatag





DNA ENCODING MATURE RICE VARIANT HST


SEQ ID NO. 23


cggcgcgacgccgtgcgggtttgctctcaagctggtgcagctggacca


gccccattatcgaagacattgtcagacctcaaggattcctgctggaga


tttttacggccacatacaattcgaggaactgccttgggatccatagca


ttagttgctagagctttgatagagaacccccaactgataaattggtgg


ttggtattcaaagcgttctatgggctcgtggcgttaatctgtggcaat


ggttacatcgttgggatcaatcagatctatgacattagaatcgataag


gtaaacaagccatatttaccaattgctgccggtgatctctcagttcag


acagcatggttattggtggtattatttgcagctgcgggattttcaatt


gttgtgacaaactttggacctttcattacctctctatattgccttggt


ctatttcttggcaccatatactctgttcctccattcagacttaagaga


tatcctgttgctgcttttcttatcattgcaacggtccgtggttttctt


ctcaactttggtgtgtactatgctactagagcagcactgggtcttaca


ttccaatggagctcgcctgttgctttcattacatgcttcgtgacttta


tttgctttggtcattgctataaccaaagatctcccagatgttgaaggg


gatcggaagtatcaaatatcaactttggcgacaaagctcggtgtcaga


aacattgcatttcttggctctggtttattgatagcaaattatgttgct


gctattgctgtagcttttctcatgcctcaggctttcaggcgcactgta


atggtgcctgtgcatgctgcccttgccgttggtataattttccagaca


tgggttctggagcaagcaaaatatactaaggatgctatttcacagtac


taccggttcatttggaatctcttctatgctgaatacatcttcttcccg


ttgatatag





DNA Encoding Insect Cell Codon Optimized


Mature Arabidopsis HST


SEQ ID NO. 24


gggatccctcgtgcttgctcccaggtcggcgctgctgagtccgacgac


cccgtgctggaccgtatcgctcgtttccagaacgcttgctggcgtttc


ctgcgtccccacaccatccgtggcaccgctctgggttccaccgccctg


gtgacccgtgctctgatcgagaacacccacctgatcaagtggtccctg


gtgctgaaggctctgtccggtctgctggctctgatctgcggtaacggt


tacatcgtgggtatcaaccagatctacgacatcggtatcgacaaggtg


aacaagccctacctgcccatcgctgctggtgacctgtccgtgcagtcc


gcttggctgctggtcatcttcttcgctatcgctggtctgctggtcgtg


ggtttcaacttcggtcccttcatcacttccctgtactccctgggcctg


ttcctgggcaccatctactccgtgccccccctgcgtatgaagcgtttc


cccgtggctgctttcctgatcatcgctaccgtgcgtggtttcctgctg


aacttcggtgtctaccacgctacccgtgctgctctgggtctgcccttc


cagtggtccgctcccgtggctttcatcaccagcttcgtgaccctgttc


gctctggtgatcgctatcaccaaggacctgcccgacgtggagggtgac


cgtaagttccagatctccaccctggctaccaagctgggtgtgcgtaac


atcgctttcctcggttccggcctgctgctcgtgaactacgtgtccgct


atctccctggctttctacatgccccaggtgttccgtggttccctgatg


atccccgctcacgtgatcctggcttccggtctgatcttccagacctgg


gtgctcgagaaggctaactacaccaaggaagctatctccggttactac


cgcttcatctggaacctgttctacgctgagtacctgctgttccccttc


ctgtaa





HPPD a/a sequence from Pseudomonas



fluorescens strain 87-79



SEQ ID No. 25


madqyenpmglmgfefiefasptpgtlepifeimgftkvathrsknvh


lyrqgeinlilnnqpdslasyfaaehgpsvcgmafrvkdsqqaynral


elgaqpihietgpmelnlpaikgiggaplylidrfgegssiydidfvy


legvdrnpvgaglkvidhlthnvyrgrmaywanfyeklfnfrearyfd


ikgeytgltskamsapdgmiriplneesskgagqieeflmqfngegiq


hvafltedlvktwdalkkigmrfmtappdtyyemlegrlpnhgepvdq


lqargilldgssiegdkrlllqifsetlmgpvffefiqrkgddgfgeg


nfkalfesierdqvrrgvlttd





HPPD a/a sequence from Avenasativa


SEQ ID NO. 26


mpptpatatgaaaaavtpehaarsfprvvrvnprsdrfpvlsfhhvel


wcadaasaagrfsfalgaplaarsdlstgnsahaslllrsgalaflft


apyapppqeaataaatasipsfsadaartfaaahglavrsvgvrvada


aeafrvsvaggarpafapadlghgfglaevelygdvvlrfvsypdetd


lpflpgfervsspgavdygltrfdhvvgnvpemapvidymkgflgfhe


faeftaedvgttesglnsvvlannseavllplnepvhgtkrrsqiqty


leyhggpgvqhialasndvlrtlremrartpmggfefmappqakyyeg


vrriagdvlseeqikecqelgvlvdrddqgvllqiftkpvgdrptffl


emiqrigcmekdevgqeyqkggcggfgkgnfselfksiedyekslevk


qsvvaqks





HPPD a/a sequence from wheat


SEQ ID NO. 27


mpptpttpaatgaaavtpeharprrmvrfnprsdrfhtlafhhvefwc


adaasaagrfafalgaplaarsdlstgnsvhasqllrsgnlaflftap


yangcdaataslpsfsadaarqfsadhglavrsialrvadaaeafras


vdggarpafspvdlgrgfgfaevelygdvvlrfvshpdgrdvpflpgf


egvsnpdavdygltrfdhvvgnvpelapaaayvagftgfhefaeftte


dvgtaesglnsmvlannsegvllplnepvhgtkrrsqiqtflehhggs


gvqhiavassdvlrtlremrarsamggfdflppplpkyyegvrriagd


vlseaqikecqelgvlvdrddqgvllqiftkpvgdrptlflemiqrig


cmekdergeeyqkggcggfgkgnfselfksiedyeksleakqsaavq


gs





HPPD a/a sequence from Shewanellacoliwelliana


SEQ ID NO. 28


Maseqnplgllgieftefatpdldfmhkvfidfgfsklkkhkqkdivy


ykqndinfllnnekqgfsaqfakthgpaissmgwrvedanfafegava


rgakpaadevkdlpypaiygigdsliyfidtfgddnniytsdfealde


piitqekgfievdhltnnvhkgtmeywsnfykdifgftevryfdikgs


qtalisyalrspdgsfcipinegkgddrnqideylkeydgpgvqhlaf


rsrdivasldamegssiqtldiipeyydtifeklpqvtedrdrikhhq


ilvdgdedgyllqiftknlfgpifieiiqrknnlgfgegnfkalfesi


erdqvrrgvl





HPPD DNA sequence from Pseudomonas



fluorescens strain 87-79



SEQ ID NO: 29


atggccgaccaatacgaaaacccaatgggcctgatgggctttgaattt


attgaattcgcatcgccgactccgggcaccctggagccgatcttcgag


atcatgggcttcaccaaagtcgcgacccaccgctccaagaatgtgcac


ctgtaccgccagggcgagatcaacctgatcctcaacaaccagcccgac


agcctggcctcgtacttcgccgccgaacacggcccttcggtgtgcggc


atggcgttccgggtcaaagactcgcagcaggcttacaaccgcgcgttg


gaactgggcgcccagccgattcatatcgaaaccggcccgatggaactc


aacctgccggccatcaagggcatcggcggtgcgccgctgtacctgatc


gaccgcttcggtgaaggcagctcgatatatgacatcgacttcgtgtac


ctcgaaggtgtcgaccgcaacccggtaggcgcgggcctcaaggtcatc


gaccacctgacccacaacgtgtatcgcggccgcatggcctactgggcc


aacttctacgagaaactgttcaacttccgtgaagcacgctacttcgat


atcaagggcgaatacaccggccttacgtccaaggccatgagtgccccg


gacggcatgatccgcatcccgctgaacgaggaatcgtccaagggcgcc


ggccagatcgaagagttcctgatgcagttcaacggcgagggcatccag


cacgtggcgttcctcaccgaagacctggtcaagacctgggatgcgttg


aagaagatcggcatgcgcttcatgaccgcgccgccggacacctactac


gaaatgctcgaaggccgcctgccaaaccacggcgagccggtggaccaa


ctgcaggcgcgcggtattttgctggacggctcctcgatcgagggcgac


aagcgcctgctgctgcagatcttctcggaaaccctgatgggcccggtg


ttcttcgaattcatccagcgcaaaggcgacgatgggtttggcgagggc


aacttcaaggcgctgttcgagtcgatcgagcgcgaccaggtacgtcgc


ggtgtactgaccaccgac





HPPD DNA sequence from Avenasativa


SEQ ID NO: 30


atgccgcccacccccgccaccgccaccggcgccgccgcggccgccgtg


actccagagcacgcggcccggagctttccccgagtggtccgcgtcaac


ccgcgcagcgaccgcttccccgtgctctccttccaccacgtcgagctc


tggtgcgccgacgccgcctcagcggccggacgcttctccttcgcgctc


ggcgcgccgctcgccgcccggtccgacctctccacggggaactccgcg


cacgcctccctcctgctccgctcgggcgccctcgccttcctcttcacg


gcgccctacgcgccgccgccgcaggaggccgccacggccgcagccacc


gcctccatcccctccttctccgccgacgccgcgcggacgttcgccgcc


gcccacggcctcgcggtgcgctccgtcggggtccgcgtcgctgacgcc


gccgaggccttccgcgtcagcgtagccggcggcgctcgcccggccttc


gccccagccgacctcggccatggcttcggcctcgccgaggtcgagctc


tacggcgacgtcgtgctacgcttcgtcagctacccggacgagacagac


ctgccattcctgccagggttcgagcgcgtgagcagccccggcgccgtg


gactacggcctcacgcggttcgaccacgtcgtgggcaacgtcccggag


atggccccggtcatagactacatgaaaggcttcttggggttccacgag


ttcgccgagttcaccgccgaggacgtgggcacgaccgagagcgggctc


aactcggtggtgctcgccaacaactccgaggccgtgctgctgccgctc


aacgagcccgtgcacggcacaaagcgacggagccagatacagacgtac


ctggagtatcacggcgggcccggcgtgcagcacatcgcgctcgccaga


aacgacgtgctcaggacgctcagggagatgcgggcgcgcacgcccatg


ggcggcttcgagttcatggcgccaccgcaggcgaaatactatgaaggc


gtgcggcgcatcgcaggtgacgtgctctcggaagagcagatcaaggaa


tgccaggagctgggggtgctagtcgacagggatgatcaaggggtgttg


ctccaaatcttcaccaagccagtaggggacaggccaacgtttttcctg


gagatgatccaaagaatcgggtgcatggagaaggacgaggtcgggcaa


gagtaccagaagggtggctgcggcgggtttggcaagggcaatttctcc


gagctgttcaagtccattgaggactatgagaaatcccttgaggtcaag


caatctgttgtagctcagaaatcctag





HPPD cDNA sequence from Wheat


SEQ ID No. 31


atgccgcccacccccaccacccccgcagccaccggcgccgccgcggtg


acgccggagcacgcgcggccgcgccgaatggtccgcttcaacccgcgc


agcgaccgcttccacacgctcgccttccaccacgtcgagttctggtgc


gcggacgccgcctccgccgccggccgcttcgccttcgcgctcggcgcg


ccgctcgccgccaggtccgacctctccacggggaactccgtgcacgcc


tcccagctgctccgctcgggcaacctcgccttcctcttcacggccccc


tacgccaacggctgcgacgccgccaccgcctccctgccctccttctcc


gccgacgccgcgcgccagttctccgcggaccacggcctcgcggtgcgc


tccatagcgctgcgcgtcgcggacgctgccgaggccttccgcgccagc


gtcgacgggggcgcgcgcccggccttcagccctgtggacctcggccgc


ggcttcggcttcgcggaggtcgagctctacggcgacgtcgtgctccgc


ttcgtcagccacccggacggcagggacgtgcccttcttgccggggttc


gagggcgtgagcaacccagacgccgtggactacggcctgacgcggttc


gaccacgtcgtcggcaacgtcccggagcttgcccccgccgcggcctac


gtcgccgggttcacggggttccacgagttcgccgagttcacgacggag


gacgtgggcacggccgagagcgggctcaactcgatggtgctcgccaac


aactcggagggcgtgctgctgccgctcaacgagccggtgcacggcacc


aagcgccggagccagatacagacgttcctggaacaccacggcggctcg


ggcgtgcagcacatcgcggtggccagcagcgacgtgctcaggacgctc


agggagatgcgtgcgcgctccgccatgggcggcttcgacttcctgcca


cccccgctgccgaagtactacgaaggcgtgcggcgcatcgccggggat


gtgctctcggaggcgcagctacaaatcttcaccaagccagtaggggac


aggccgacgttgttcctggagatgatccagaggatcgggtgcatggag


aaggacgagagaggggaagagtaccagaagggtggctgcggcgggttc


ggcaaaggcaacttctccgagctgttcaagtccattgaagattacgag


aagtcccttgaagccaagcaatctgctgcagttcagggatcatag





HPPD DNA sequence from Shewanellacollwelliana


SEQ ID NO: 32


gactttatgagtcgcacaggtatcgaagcgggctacatgaccttacat


caaaaaggcgtgccgcatggaccacaacctggtcgtactgaagcctca


gtgggcaaaactgaaacctatgagtatgcagtaatggtggacaccttt


gcaccactgcaactgacccagcatgtcaatgcgtgcatgagcaaagat


tacaaccgttcctggctagaagagtaaaagcgttcagccagtgctgaa


catctaataaatataacaccagaggtgacaccgaagagtgcccttggt


tgcaataagttgaaagaggataattacatggcaagcgaacaaaaccca


ctgggtctacttggtatcgaattcactgaatttgctacaccagatcta


gattttatgcataaagtttttatcgactttggtttctcaaaacttaaa


aaacacaagcagaaagatattgtttactataaacaaaatgatattaac


tttttactcaacaatgaaaaacagggcttttcagcccagtttgccaaa


acgcatggcccagccattagttctatgggctggcgtgtagaagatgcc


aactttgcctttgaaggtgctgtagcccgtggggctaaacccgcagca


gatgaggtgaaagatcttccctatcccgctatctatggcattggtgac


agccttatctactttatcgatacgtttggcgatgacaacaatatctac


acttctgattttgaagcgttagatgagcctatcatcacccaagagaaa


ggcttcattgaggtcgaccatctcaccaataatgtccataagggcacc


atggaatattggtcaaacttctacaaagacatttttggctttacagaa


gtgcgttacttcgacattaagggctcacaaacagctcttatctattac


gccctgcgctcgccagatggtagtttctgcattccaattaacgaaggc


aaaggcgatgatcgtaaccaaattgatgagtacttaaaagagtacgat


ggcccaggtgtccaacacttagcgttccgtagccgcgacatagttgcc


tcactggatgccatggaaggaagctccattcaaaccttggacataatt


ccagagtattacgacactatctttgaaaagctgcctcaagtcactgaa


gacagagatcgcatcaagcatcatcaaatcctggtagatggcgatgaa


gatggctacttactgcaaattttcaccaaaaatctatttggtccaatt


tttatcgaaatcatccagcgtaaaaacaatctcggttttggcgaaggt


aattttaaagccctatttgaatcgattgagcgtgatcaggtgcgtcgc


ggcgtactctaacaatcacccagtgatccaacctcaaaaaaccagcat


cgcgctggtttttttattgcagcacaacaataaacctctacactagca





TMV translational enhancer nucleotide


sequence


SEQ ID NO. 33


tatttttacaacaattaccaacaacaacaaacaacaaacaacattaca


attactatttacaattacac





Fusion of TMV and Arabidopsis HST coding


sequence


SEQ ID NO. 34


tatttttacaacaattaccaacaacaacaaacaacaaacaacattaca


attactatttacaattacacatatggagctctcgatctcacaatcacc


gcgtgttcggttctcgtctctggcgcctcgtttcttagcagcttctca


tcatcatcgtccttatgtgcatttagctgggaagtttataagcctccc


acgagatgttcgcttcacgagcttatcaacttcaagaatgcggtccaa


atttgtttcaaccaattatagaaaaatctcaatccgggcatgttctca


ggttggtgctgctgagtctgatgatccagtgctggatagaattgcccg


gttccaaaatgcttgctggagatttcttagaccccatacaatccgcgg


aacagctttaggatccactgccttggtgacaagagctttgatagagaa


cactcatttgatcaaatggagtcttgtactaaaggcactttcaggtct


tcttgctcttatttgtgggaatggttatatagtcggcatcaatcagat


ctacgacattggaatcgacaaagtgaacaaaccatacttgccaatagc


agcaggagatctatcagtgcagtctgcttggttgttagtgatattttt


tgcgatagcagggcttttagttgtcggatttaactttggtccattcat


tacaagcctatactctcttggcctttttctgggaaccatctattctgt


tccacccctcagaatgaaaagattcccagttgcagcatttcttattat


tgccacggtacgaggtttccttcttaactttggtgtgtaccatgctac


aagagctgctcttggacttccatttcagtggagtgcacctgtggcgtt


catcacatcttttgtgacactgtttgcactggtcattgctattacaaa


ggaccttcctgatgttgaaggagatcgaaagttccaaatatcaaccct


ggcaacaaaacttggagtgagaaacattgcattcctcggttctggact


tctgctagtaaattatgtttcagccatatcactagctttctacatgcc


tcaggtttttagaggtagcttgatgattcctgcacatgtgatcttggc


ttcaggcttaattttccagacatgggtactagaaaaagcaaactacac


caaggaagctatctcaggatattatcggtttatatggaatctcttcta


cgcagagtatctgttattccccttcctctag





HST Polypeptide Motif 1.


SEQ ID NO. 35


W(R/K)FLRPHTIRGT





HST Polypeptide Motif 2.


SEQ ID NO. 36


NG(Y/F)IVGINQI(Y/F)D





HST Polypeptide Motif 3.


SEQ ID NO. 37


IAITKDLP





HST Polypeptide Motif 4.


SEQ ID NO. 38


Y(R/Q)(F/W)(I/V)WNLFY











EXAMPLES

The average and distribution of herbicide tolerance or resistance levels of a range of primary plant transformation events are evaluated in the normal manner based upon plant damage, meristematic bleaching symptoms etc. at a range of different concentrations of herbicides. These data can be expressed in terms of, for example, GR50 values derived from dose/response curves having “dose” plotted on the x-axis and “percentage kill”, “herbicidal effect”, “numbers of emerging green plants” etc. plotted on the y-axis where increased GR50 values may, for example, correspond to increased levels of inherent inhibitor-tolerance (e.g increased Ki×kcat./KmHPP value) and/or level of expression of the expressed HPPD and/or HST.


The following experiments are conducted using a variety of HST-inhibiting herbicides which are described in Tables A-F.









TABLE A







Compounds 1.1-1.4.




embedded image
















Compound
R1
R2
R3
R4





1.1
F
Cl
Cl
F


1.2
Cl
Cl
Cl
Cl


1.3
F
F
Br
F


1.4
F
Br
Br
F
















TABLE B







Compounds 2.1-2.34.




embedded image




















Compound
A1
R1
R2
R3
R4
R5
R6
R7


















2.1
N
F
H
H
Br
Cl
H
OH


2.2
N
—CH3
H
—CH3
H
—CH3
—CH3
OH


2.3
N
F
H
H
F
Cl
—CH2CF2H
OH


2.4
N
F
H
H
F
Cl
CH3
OH


2.5
N
F
H
H
Br
Cl
CH3
OH


2.6
N
H
H
H
H
—OCF3
H
OH


2.7
N
F
H
H
F
Cl
H
OH


2.8
N
F
H
H
Br
Cl
—CH2CF2H
OH


2.9
N
H
CF3
H
H
Cl
—CH2CF2H
OH


2.10
N
—CH2CH3
H
—CH2CH3
H
—CH2CH3
H
OH


2.11
N
—CH3
H
—CH3
H
—CH3
—CH2CF2H
OH


2.12
N
H
H
H
H
—OCF3
H
OH


2.13
CH
F
H
H
F
Cl
—CH2CF2H
OH


2.14
N
Cl
Cl
H
H
Cl
—CH2CF2H
OH


2.15
CH
F
H
H
Br
Cl
—CH2CF2H
OH


2.16
N
Cl
H
H
H
—CF3
—CH3
OH


2.17
N
F
H
H
F
Cl
—CH2CF3
OH


2.18
N
F
H
H
F
Cl
—C≡CH
OH


2.19
N
H
Cl
H
H
CF3
—CH2CF2H
OH


2.20
N
Cl
H
H
H
CF3
—CH2CF2H
OH


2.21
N
F
H
H
Cl
Cl
—CH2CF2H
OH


2.22
CH
Cl
H
H
Br
Cl
—CH2CF2H
OH


2.23
N
F
H
H
Br
Cl
H
—O(C═)-Et


2.24
N
—CH3
H
—CH3
H
—CH3
—CH3
—O(C═O)-iPr


2.25
N
F
H
H
Cl
Cl
—CH2CF2H
—O(C═O)-tBu


2.26
CH
F
H
H
F
Cl
CH3
—O(C═O)-tBu


2.27
N
F
H
H
Br
Cl
CH3
—O(C═O)-iPr


2.28
N
Cl
H
H
H
—CF3
—CH2CF2H
—O(C═O)-iPr


2.29
N
F
H
H
F
Cl
H
—O(C═O)-tBu


2.30
N
F
H
H
Br
Cl
—CH2CF2H
—O(C═O)-iPr


2.31
N
Cl
H
H
H
CF3
—CH2CF2H
—O(C═O)-iPr


2.32
N
—CH2CH3
H
—CH2CH3
H
—CH2CH3
H
—O(C═O)-iPr


2.33
N
—CH3
H
—CH3
H
—CH3
—CH2CF2H
—O(C═O)-Et


2.34
N
H
H
H
H
—OCF3
H
—O(C═O)-iPr
















TABLE C







Compounds 3.1-3.6.




embedded image




















Com-










pound
A1
R1
R2
R3
R4
R5
R6
R7





3.1
N
CF3
H
H
H
Cl
—CH2CH3
OH


3.2
N
H
H
Cl
H
Cl
—CH3
OH


3.3
N
Cl
H
H
H
CF3
—CH2CF2H
OH


3.4
N
Cl
H
H
Cl
Cl
—CH2CF2H
OH


3.5
N
H
H
Br
H
CF3
—CH2CF2H
OH


3.6
N
CF3
H
H
Cl
H
—CH2CF2H
—O(C═O)-iPr
















TABLE D







Compound 4.1




embedded image


















Compound
R1
R2
R3
R4
R5
R6





4.1
F
H
H
F
Cl
CH3
















TABLE E







Compound 5.1




embedded image




















Compound
A1
R1
R2
R4
R5
R6







5.1
N
Cl
H
H
Cl
—CH2CF2H

















TABLE F







Compounds 6.1




embedded image

















Compound
R1
R2
R3
R4
R5





6.1
—CH3
—CH3
—CH3
—CH3
—CH3









Example 1
Cloning and Expressing Plant HST Enzymes in Insect Cells and in E. coli

The full length HST coding sequences (minus the ATG start codon) are amplified, with flanking EcoRI sites, from Arabidopsis (SEQ ID 11) and Rice (SEQ ID12 or SEQ ID 13) from cDNA libraries or made synthetically. Both these full length and also the truncated coding sequences (encoding the mature sequences starting from ARG 64) Arabidopsis SEQ ID 21, rice SEQ ID 22 and rice SEQ ID 23) were cloned into the EcoRI site of the pAcG3X vector (BD Biosciences Cat. No. 21415P) transformed into and then expressed in Sf9 (Spodoptera fugiperda) insect cells (as described below) as a N-terminal GST fusion proteins having a factor Xa cleavage site. Similarly, SEQ ID 24, the insect cell codon optimized DNA sequence encoding the alternative truncated mature (ARG 69) Arabidopsis HST is cloned into the EcoRI site of pAcG3X and expressed in Sf9 cells as a N-terminal GST fusion protein. The Arabidopsis HST SWISSPROT accession number (protein) is Q1ACB3 and the Arabidopsis HST EMBL accession number (DNA): DQ231060.


Alternatively, Arabidopsis and Chlamydomonas mature HST coding sequences are cloned as GST N-terminal fusion enzymes and expressed in E. coli.


Example 2
Growth of Cells and Preparation of HST Enzyme Extracts


E. coli. BL21A1 cells expressing mature Arabidopsis or Chlamydonionas HST as a GST N-terminal fusion proteins are grown, harvested, broken and membrane fractions expressing HST produced.


For example, 1 ng of recombinant DNA is used to transform BL21DE3 cells to obtain a plateful of individual colonies. One of these colonies is picked and used to inoculate an overnight culture of 100 ml of Luria Broth (LB) supplemented with 50 ug/ml of kanamycin at final concentration, grown at 37° C. with shaking at 220 rpm. Next morning, 10 mls of the overnight culture is used to inoculate 11 of fresh sterile LB supplemented with 50 ug/ml of kanamycin at final concentration, grown at 37° C. with shaking at 220 rpm until the OD reached 0.6 at 600 nm, induced with the addition of 0.1 mM IPTG and left to induce at 15° C. overnight. The cells are harvested by centrifugation at 4600 rpm for 10 min at 4° C. and the pellet stored at −80° C. For example it is found that one litre of cells yields approximately 5 g of wet cell pellet. E. coli cell pellet is then resuspended in 25 ml of 50 mM Tris, pH 7.5 supplemented with Roche EDTA-free protease inhibitor tablet (one tablet in 200 mls of buffer). 10 ml of cells are lysed by sonication on ice. The resultant lysed cells are centrifuged at 3000 g for 10 min to pellet the cell nuclei/debris etc. 10 mls of supernatant is aspirated and centrifuged at 150,000 g for 60 min at 4° C. The pellet containing the membranes is resuspended in 2 ml of the above buffer. These samples are stored as 100 ul aliquots at −80° C., after being diluted with addition of glycerol to 50% v/v.


The HST expression pAcG3X—derived transfer vectors (described above) are independently co-transformed into Sf9 suspension cells with FlashBac (Oxford Expression Technologies) parental baculovirus vector. Baculovirus amplification and HST protein expression is performed in accordance with the manufacturer's instructions


SD Suspension cell cultures are subcultured at a density of 1.0 EXP 6 cells/ml in 140 ml Sf900II medium (Invitrogen Cat No. 10902) in 500 ml Erlenmeyer flasks. After 24 hours culture at 27° C. shaking at 120 rpm the cell density is measured and readjusted to 2.0 EXP 6 cells/ml in 140 ml. Volumes of amplified virus stock of known titre are added to prepared suspension flasks to give a multiplicity of infection of 10. Flasks are sealed and incubated at 27° C. shaking at 125 rpm for 72 hours to allow adequate protein expression without cell lysis. Cells are harvested by dividing flask contents evenly between three 50 ml Falcon tubes and centrifuging at 900 rpm for 4 minutes. Medium is discarded leaving a 3 ml cell pellet which is snap frozen in liquid nitrogen and maintained at −80° C.


The pellet from 25 mls of Sf9 cells (after induction of expression for 4 days) is resuspended in 10 mls of 50 mM Tris, pH 7.5 supplemented with Roche-EDTA free protease inhibitor tablet (1 tablet in 200 mls of buffer) and homogenised using a hand held homogeniser. The resultant lysed cells are centrifuged at 3000 g for 10 min to pellet the cell nuclei/debris etc. 10 mls of supernatant is aspirated and centrifuged at 150,000 g for 60 min at 4° C. The pellet containing the membranes is resuspended in 1 ml of above buffer and samples are stored as 100 ul aliquots at −80° C., after first being diluted with addition of glycerol to 50% v/v.


Western blotting to monitor expression is with anti-GST HRP conjugated Ab (GE Healthcare, 1:5000 working dilution) incubation followed by ECL (GE Healthcare).


HST enzyme preparations for assay are also prepared directly from fresh plant material. For example HST enzyme preparations are from spinach. In the first step intact spinach chloroplasts are prepared from two lots of 500 g of fresh baby spinach leaves (e.g from the salad section of the local supermarket). Prepacked spinach is usually already washed, but if buying loose leaves these must be rinsed in water before proceeding. Stalks, large leaves and mid-ribs are removed. Each 500 g lot of leaves is added to 1.5 l of ‘Grinding medium’ in a 2 L plastic beaker. Grinding medium is cold (4° C.) 50 mM Tricine/NaOH buffer at pH 7.1 containing 330 mM glucose, 2 mM sodium isoascorbate, 5 mM MgCl2 and 0.1% bovine serum albumen The beaker, kept at 4° C., is placed under a Polytron 6000 blender, fitted with a 1.5″cutting probe and the mixture blended in short bursts of 5-8 sec up to 8-10K rpm until all the leaves are macerated. The homogenate is filtered into a 5 L beaker (embedded in an ice bucket) through four layers of muslin, and two layers of 50μ mesh nylon cloth. The filtrate is transferred to 250 ml buckets of a Beckman GS-6 centrifuge and spun at 200×g (3020 rpm) for 2 min at 4° C. The supernatant is drained away and discarded to leave a sediment of chloroplasts. Chloroplasts are resuspended in a few ml of cold resuspension medium by gentle swirling and gentle use of a quill brush soaked in resuspension medium. Resuspension medium is 50 mM Hepes/KOH pH 7.8 containing 330 mM sorbitol, 2 mM EDTA, 5 mM KH2PO4, 2 mM MgCl2 and 0.1% bovine serum albumen at 4° C. The chloroplasts are resuspended in 5-10 ml of resuspension buffer, recentrifuged down and resuspended again in order to wash them. The chloroplasts are then once again centrifuged down and then broken by resuspension in about 5 ml of 50 mM Tricine-NaOH pH 7.8 to a protein concentration of about 40 mg/ml. The solution is stored frozen at −80° C. in aliquots. This resuspension is defrosted and used directly in HST activity assays. Alternatively, chloroplasts are prepared resuspended in 50 mM Tris/HCl buffer at pH 7.8 containing 330 mM sorbitol (alternative resuspension buffer) and layered on top of a percol gradient (comprising the same buffer containing 45% percol), spun down, the intact chloroplast fraction taken and washed 2 or 3 times in the alternative resuspension buffer and then spun down again, resuspended in breaking buffer (without sorbitol), flash frozen and stored in aliquots at −80° C.


Example 3
Assay of HST Enzymes

Prenyltransferase (HST) activities are measured by determining the prenylation rates of [U—14C]homogentisate using farnesyl diphosphate (FDP) as prenyl donor. 14C homogentisate is prepared from 14C tyrosine using L amino acid oxidase and HPPD. For inhibitor testing, compounds are dissolved in dimethylsulfoxide (DMSO). DMSO added at up to 2% v/v has no effect on assays. Control assays contain DMSO at the same concentration as in inhibitor containing assays.


Assays using spinach chloroplast extracts (100 μl final volume) contain up to 2 mg of chloroplast protein, 50 mM Tricine-NaOH pH 8.5, 50 mM MgCl2, 200 μM farnesyl diphosphate (FDP) and 26 μM 14C-homogentisate (167 dpm/pmol). Assays are run for about an hour at 28° C. For inhibitor studies, haloxydine at a final concentration of 500 ppm is found to completely inhibit the reaction. Alternatively, stopping the reaction and carrying out solvent extraction at zero time also provides a 100% inhibition baseline reference. Lipophilic reactions products are extracted and analyzed essentially as described in the literature.


The recombinant Chlamydomonas HST expressed in E. coli membranes is assayed in standard reaction mixtures with 200 μM FDP and 100 μM 14C-homogentisate (40 dpm/pmol) in 50 mM Tricine-NaOH pH 8.5, 20 mM MgCl2. Assays are started with the addition of enzyme and run for ˜20 min at 28° C.


Recombinant Arabidopsis and Rice HSTs are expressed in insect cells. Assays are run as for Chlamydomonas HST except that assay temperature is 27° C. Assays are stopped with 300 ul of solvent mix (1:2, Chloroform:Methanol) and 100 ul of 0.5% NaCl, agitated/mixed and spun at 13,000 rpm in a benchtop eppendorf centrifuge for 5 minutes. 80 ul of the lower phase extract is loaded onto a TLC plate (silica Gel 60, 20 cm×20 cm) FLA3000 system and run for 35 minutes in dichloromethane. The radioactivity is quantified using a Fuji Phosphoimager and band intensity integrated as quantitative measure of product amount. The bands corresponding to oxidised and reduced 2-methyl-6-farnesyl-1,4-benzoquinol (MFBQ) are identified and the total of the two (oxidised and reduced) band intensities is calculated in order to estimate the total amount of MFBQ product formation. Specific activities of 8 pmol MFBQ min−1 mg−1 protein (23 pmol) and 7 pmol MFBQ min−1 mg−1 protein (14 pmol) are, for example, estimated for the GST-fusion truncated Arabidopsis HST gene (SEQ ID # 3) expressed in membranes from insect cells 4 days and 5 days after transfection respectively. Similar results are noted from past literature on E. coli expressed Arabidopsis HST. Activity from the insect cell expressed GST-fusion truncated rice HST (SEQ ID# 4) is similar. Expression of the non-truncated HST coding sequences as GST fusions also gives HST activity. Expression of the insect cell optimised GST-fusion truncated Arabidopsis HST (SEQ ID#23) gives, for example, an approximately 3-10 fold increase in specific activity over the non-optimized genes.


Using the above assays percentage inhibition of the amount of MFBQ formed at a range of doses of inhibitors relative to the control amount obtained with no inhibitor present is reported (Table 1). The better inhibitors give greater percentage inhibition at lower doses.


It is found that for all of the HST enzyme preparations assayed under the prescribed reaction conditions formation of MFBQ is by no means the only reaction catalysed by HST from 14C homogentisate (HGA). In fact the major (˜90%) radiolabelled products from the 14C HGA are not MFBQ. These unknown other products, are also extracted into chloroform/methanol but, in dichloromethane TLC, are found to stay on or chromatograph near the base line. Four apparent 14C-labelled bands (presumably corresponding to two quinone/quinol pairs) are partly resolved in a second dimension of TLC using 12:3:5:0.5, dichloromethane:hexane:acetonitrile:forinic acid. No such bands are seen in the absence of FPP (or indeed when FPP is replaced with pyrophosphate) and, presumptively, the bands correspond to products formed due to farnesylation and decarboxylation not being tightly coupled; .i,e. farnesylation in the absence of decarboxylation (giving rise to carboxylated MFBQ) and decarboxylation in the absence of farnesylation (giving rise to the methyl quinol/quinone). Whatever their identity it is quite clear that these products more polar than MFBQ are all bone fide enzyme reaction products since, in the absence of FDP or using like non-transgenic (non-HST expressing) membranes, they are not formed. In addition it is found that inhibitors such as haloxydine inhibit the formation of these other products in a way that, as dose is varied, is apparently co-linear with inhibition of the formation of MFBQ. Thus 500 ppm haloxydine about completely inhibits the HST enzyme reaction and neither MFBQ nor any of the other products are formed.


Thus in an improved, more sensitive and convenient version of the above assay, the TLC step is dispensed with, treatment with 500 ppm haloxydine (or other inhibitor at a suitable concentration) is used as the 100% inhibition ‘control’ and a portion of the chloroform/methanol extract is taken directly into a scintillation vial and counted.


These assays are routinely run at 100 μM 14C HGA, 25° C. for <20 min (i.e over a period for which the control assay rate remains linear) and at a range of test inhibitor concentrations used so that IC50s can be derived by curve fitting to the Hill equation (allowing the value of the slope, n, to vary).


Results.









TABLE 1







Observed percentage inhibition of the HST reaction (relative to controls) at various


concentrations of various compounds using HST from various sources (as labelled).


Assays based on TLC and estimated amount of MFBQ formed.















Spinach






Chlamydomonas.

chloroplast




Chlamydomonas.


Chlamydomonas. HST

HST
extract


Compound
HST % I at 10 ppm
% I at 25 ppm
% I at 100 ppm
% I at 25 ppm





Amitrole
 0

 0



Chlorsulfuron

 0

 0


1.1
70
80
95
70


1.2

70

80


1.3

55

65


1.4

65

75


2.1
10

40



2.2
70

95



2.3
40

85



2.4
 5

45



2.5
70

90



2.6
 5

20



2.7
 5

20



2.8

85

70


2.9

90

90


2.10

50

 0


2.11

80

30


2.12

 0

 0


2.13

60

40


2.14

85

80


3.1

20

20


3.2

 0

15


3.3

20

35


3.4

50

40


4.1
 0

 0



5.1

20

10
















TABLE 2







Estimated IC50 values for inhibition of Arabidopsis HST based on


total extraction (non TLC) assay.










Estimated



Compound
IC50 (ppm)
95% Confidence Limits












2.3
 19*



2.4
211*


2.5
 48*


2.8
12
10.5-14  


2.9
 6
4.5-8  


2.11
 31*


2.13
 54*


2.14
 9
  8-9.5


2.15
12
9.6-14 


2.16
78
 51-118


2.17
38
21-68


2.18
 6
4.3-7.5


2.19
 6
4.5-8  


2.20
16
11-25


2.21
64
52-78


3.5
42
28-62


6.1
63
 39-102





*These estimates were from 3 point dose curves only conducted at 50 μM HPP.






Example 4
Preparation of Stable Transgenic Plants Lines Expressing a Heterologous HST Enzyme

For example the Arabidopsis HST SEQ ID # 11 is cloned behind a double 35s CMV promoter sequence and a TMV translational enhancer sequence and in front of the 3′ terminator from the nos gene. This expression cassette is ligated into pMJB1 (described in WO98/20144) and then into pBIN19 and then transformed into Agrobacterium tuniqfaciens strains LBA4404 prior to plant transformation.


For example, the full length Arabidopsis HST seq ID # 11 is fused to the TMV translational enhancer sequence (SEQ ID #33) by overlapping PCR and, at the same time, 5′ XhoI site and a 3′KpnI site are added by PCR. Site-directed mutagenesis is performed to remove an internal XhoI site. The TMV/HPPD fusion is removed from the pBIN19 by digestion with XhoI/KpnI and is replaced by the TMV/HST fusion (SEQ ID #34). The TMV/HST fusion is now cloned behind a double 35s promoter and in front of the 3′ terminator from the nos gene. Again the modified pBIN19 vector (‘pBinAT HST’) is then transformed into Agrobacterium tumefaciens strain LBA4404.


Likewise, vectors for plant transformation are constructed to comprise DNA sequences any HST and, for example, SEQ Ids nos. 12-20.


Alternatively vectors comprise DNA encoding HSTs from photosynthetic protozoans, higher and lower plants the sequences of which are derived from cDNA libraries using methods known to the skilled man. For example total RNA is prepared from 5-20-day-old plant seedlings using the method of Tri-Zol extraction (Life Technologies). mRNA is obtained, for example, from Avena sativa using the Oligotex mRNA purification system (Qiagen). The 5′ end of, for example, the A. sativa HST gene is identified using 5′ RACE, performed using the Gene Racer kit (Invitrogen) with internal HST gene specific primers (based on HST consensus regions e.g SEQ No. 35, 36, 37 and 38). The 3′ end of the gene is identified by 3′ RACE, performed using Themoscript RT (Life Technologies) with appropriate oligo dT primer and an appropriate internal HST gene primer, followed by PCR All methodologies are performed according to protocols provided by the various stated manufacturers. Products obtained from the 5′ and 3′ RACE reactions are cloned into pCR 2.1 TOPO (invitrogen) and the cloned products sequenced using universal M13 forward and reverse primers with an automated ABI377 DNA sequencer. Primers are then designed to the translation initiation and termination codons of the HST gene respectively. Both primers are used in conjunction with the One-step RTPCR kit (Qiagen or Invitrogen) to obtain full length coding sequences. Products obtained are cloned into pCR 2.1 TOPO, sequenced, and identified as HST by comparison with sequences known in the art (and for example the HST sequences herein).


A master plate of Agrobacterium tumefaciens containing the binary vector pBinAT HST (described above) or analogous binary vector comprising a different HST is used to inoculate 10 ml LB containing 100 mg/1 Rifampicin plus 50 mg/1 Kanamycin using a single bacterial colony. This is incubated overnight at 28° C. shaking at 200 rpm. This entire overnight culture is used to inoculate a 50 ml volume of LA (plus antibiotics). Again this is cultured overnight at 28° C. shaking at 200 rpm. The Agrobacterium cells are pelleted by centrifuging at 3000 rpm for 15 minutes then resuspended in MS medium with 30 g/1 sucrose, pH 5.9 to an OD (600 nM)=0.6. This suspension is dispensed in 25 ml aliquots into petri dishes.


Clonal micro-propagated tobacco shoot cultures are used to excise young (not yet fully expanded) leaves. The mid rib and outer leaf margins are removed and discarded, the remaining lamina is cut into 1 cm squares. These are transferred to the Agrobacterium suspension for 20 minutes. Explants are then removed, dabbed on sterile filter paper to remove excess suspension then transferred to NBM medium (MS medium, 30 g/1 sucrose, 1 mg/1 BAP, 0.1 mg/1 NAA, pH 5.9, solidified with 8 g/1 Plantagar), with the abaxial surface of each explant in contact with the medium. Approximately 7 explants are transferred per plate, which are then sealed then maintained in a lit incubator at 25° C., 16 hour photoperiod for 3 days.


Explants are then transferred to NBM medium containing 100 mg/1 Kanamycin plus antibiotics to prevent further growth of Agrobacterium (200 mg/1 timentin with 250 mg/1 carbenicillin). Further subculture on to this same medium is then performed every 2 weeks.


As shoots start to regenerate from the callusing leaf explants these are removed to Shoot elongation medium (MS medium, 30 g/1 sucrose, 8 g/1 Plantagar, 100 mg/1 Kanamycin, 200 mg/1 timentin, 250 mg/1 carbenicillin, pH 5.9). Stable transgenic plants readily root within 2 weeks. To provide multiple plants per event to ultimately allow more than one herbicide test per transgenic plant all rooting shoots are micropropagated to generate 3 or more rooted clones.


Putative transgenic plants that are rooting and show vigorous shoot growth on the medium incorporating Kanamycin are analysed by PCR using primers that amplified a 500 bp fragment within the Arabidopsis HST transgene. Evaluation of this same primer set on untransformed tobacco showed conclusively that these primers would not amplify sequences from the native tobacco HST gene.


To roughly evaluate comparative levels of ectopic HST expression independent PCR positive tobacco shoots have young leaves removed, cut into 1 cm squares and plated onto NBM medium incorporating 1 mg/1 haloxydine. These leaf explants produce callus and ultimately regenerated shoots. Those explants over expressing HST regenerated green callus and shoots. Untransformed explants or those from transformants with limited HST expression produced bleached callus and stunted bleached shoots that ultimately died.


It is found that PCR positive events correspond with callus which yields green shoot proliferation (scores>=3) in the presence of haloxydine and many more exhibit some level of tolerance that is greater than the untransformed control material.


Rooted transgenic T0 plantlets are transferred from agar and potted into 50% peat, 50% John Innes soil no 3 or, for example, MetroMix® 380 soil (Sun Gro Horticulture, Bellevue, Wash.) with slow-release fertilizer in 3 inch round or 4 inch square pots and left regularly watered to establish for 8-12 d in the glass house. Glass house conditions are about 24-27° C. day; 18-21° C. night and approximately a 14 h (or longer in UK summer) photoperiod. Humidity is ˜65% and light levels up to 2000 μmol/m2 at bench level. Once new tissue emerges and plants they have reached the 2-4 leaf stage some of the clones from each event are sprayed with test chemicals dissolved in water with 0.2-0.25% X-77 surfactant and sprayed from a boom on a suitable track sprayer moving at 2 mph in a DeVries spray chamber with the nozzle about 2 inches from the plant tops. Spray volume is suitably 25 gallons per acre or, for example, 2001/ha.


Test chemicals are, for example, compound 2.3 at 500 g/ha. At the same time that transgenic plants are sprayed so too are w/t Samsun tobacco plants grown from seed as well as non-transgenic plants regenerated from tissue culture and non-transgenic tissue culture escapes. Damage is assessed versus unsprayed control plants of like size and development.









TABLE 3








Arabidopsis HST transgenic tobacco plants assessed 11 DAT with



compound 2.3 (designated compound 3 in the table).













Compound 3

Compound 3



EVENT
at 500 g/ha
EVENT
at 500 g/ha
















A1
0
E5
1



A12
1.5
E6
2.5



A4
0
E8
0



A6
0
F12
4



A9
0.5
F2
4



B1
2
F3
1



B3
2.5
F4
1



B8
5
F5
2



C11
2.5
F7
0



C12
0
G1
4



C4
2
G4
0



C8
5
G5
1



C9
0
G9
9



D2
10
H8
8



D4
2
W/T_SD
0



D5
0
W/T_SD
0



D6
2.5
W/T_SD
0



D8
3
W/T_TC
0



E1
2
W/T_TC
0



E10
0
W/T_TC
0







Compared to untreated controls all the plants are affected by treatment at 500 g/ha and are smaller and growth is set back. However, unlike controls that show white meristems and that are essentially dead many of the HST transgenics show green meristems and are recovering and some show essentially no bleaching. Plants are scored on a scale from 0 to 10 with 0 meaning plant substantially bleached/burnt and meristem dead/white and 10 meaning that the entire plant looks green and undamaged.






In addition to the above experiments two lines of Arabidopsis HST expressing transgenic plants, E9 and F6 were treated with haloxydine at 250 g/ha in a similar manner to the methods described above but at a slightly later growth stage (4-5 leaf). After assessment 8 DAT the w/t plants were 55 and 65% damaged, line F6 plants were 50 and 75% damaged whereas line E9 plants were only 20 and 40% damaged.


At 25 DAT the w/t and F6 plants remained stunted, bleached and small (50-70% damage) whereas the E9 plants now appeared as healthy and similar sized to untreated control plants. Thus expression of the Arabidopsis HST confers resistance to haloxydine.


The plants are assessed at various times after treatment up to 28 DAT. Those events (e.g C8, G9, E9) showing the least damage from HST herbicides are grown on to flowering, bagged and allowed to self. The seed from selected events are collected sown on again in pots and tested again for herbicide resistance in a spray test for herbicide resistance. Single copy events amongst the T1 plant lines are identified by their 3:1 segregation ratio (for example, dependent on the construct, by both kanamycin selection and wrt herbicide resistance phenotype) and by quantitative RT-PCR.


Example 5
Production and Further Testing of T1 and T2 Transgenic Plants Transformed to Express Arabidopsis HST, Avena HPPD or Pseudomonas HPPD

T0 transgenic tobacco plant lines B8 and G9 described above in the foregoing examples are selfed. About 50 of the resultant seed from each selfing each line are planted out into a soil/peat mixture in 3 inch pots, grown in the glass house for 7-10 d and sprayed with 500 g/ha of compound 2.3 (all as described in the foregoing examples). For each line about three quarters of the plants display visible resistance to the herbicide and of these a few plants (possible homozygotes at a single insertion event) appear the most resistant. A few of these more highly tolerant T1 plants are selfed again to produce batches of T2 seed.


6 Seed from w/t Samsun tobacco and 8 T1 seed from events B8 and G9 are also planted out in 3 inch pots, grown on for 7 to 12 d and then, as described in the foregoing examples, the plantlets spray tested for resistance to various chemicals and assessed at 14 DAT. Chemicals are formulated in 0.2% X77 and sprayed at a spray volume of 200 l/ha. Results are depicted in Table 4 below. The results clearly demonstrate the heritability of the herbicide resistance phenotype. The results also show that, aside from obvious non-transgenic segregants, the transgenic Arabidopsis HST Tobacco T1 plants display resistance to HST herbicides as exemplified using compounds 2.15 and 2.30 but that the phenotype is specific and the plants are not significantly tolerant to the other two herbicides tested, norflurazon and atrazine.









TABLE 4







Assessment of herbicide % damage to T1 progeny plants of Arabidopsis


HST lines B8 and G9 at 14 DAT with various herbicides.














Compound
Compound
Norflurazon
Atrazine


Line
Rep
2.15 1 kg/ha
2.30 150 g/ha
150 g/ha
500 g/ha















B8
1
5
60
35
100



2
10
0
15
100



3
15
0
15
100



4
0
0
15
100



5
0
0
15
100



6
0
0
10
100



7
0
0
15
100



8
0
60
35
100


G9-2
1
0
0
15
100



2
0
65
20
100



3
0
65
15
100



4
0
0
15
100



5
0
0
15
100



6
0
0
20
100



7
40
0
35
100



8
0
0
40
100


WT
1
30
60
20
100


control
2
30
60
15
100



3
20
70
10
100



4
50
75
15
100



5
50
75
15
100



6
50
70
15
100









Tobacco plants expressing the wild-type HPPD gene of Pseudomonas fluorescens strain 87-79 under operable control of the double enhanced 35S CMV promoter region, Nos3′ terminator and TMV translational enhancer were provided as detailed in Example 4 of WO0246387. A T0 event exhibiting tolerance to mesotrione was selfed to produce a single insertion T1 line (exhibiting 3:1 segregation of the herbicide tolerance and kanamycin selection phenotypes) which was again further selfed to provide the T2 line designated C2.


Seed of wild-type tobacco plants, C2 tobacco plants and of the T1 progeny of a further Arabidopsis-HST expressing tobacco line, D2 were planted out in 3 inch pots, grown on, sprayed with compounds 1.1, 2.30 and 3.6 at the rates shown in table 5. Percent damage scores were assessed at 7 DAT. Aside from the presumptive non-transgenic segregants the D2 line containing the Arabidopis HST expression construct offered the highest level of tolerance to the two HST herbicides with the Psuedomonas HPPD, C2 line, offering only marginal tolerance under the conditions of this test.









TABLE 5







Testing of wild-type (WT) Samsun, C2 and D2 tobacco lines versus various HST inhibitors.


Results depict % damage at 7 DAT
























Rate

















Compound
gai/ha
WT
WT
WT
C2
C2
C2
C2
D2
D2
D2
D2
D2
D2
D2
D2


























1.1
25
75
75
75
75
75
75
75
30
55
35
65
65
100
65
55


2.30
150
75
80
85
90
70
80
80
40
65
40
50
55
60
60
90



300
75
70
80
85
85
85
85
90
60
80
60
95
98
70
90



450
70
70
75
65
70
85
85
70
35
20
35
55
55
60
65


3.6
150
80
80
80
60
60
60
60
70
10
70
10
30
20
65
25



300
80
80
75
65
65
65
70
40
65
35
7
85
80
85
75



450
85
85
85
65
65
65
65
25
55
25
65
70
65
80
75
















TABLE 6







Testing of T0 lines of Arabidopsis HST tobacco treated with


mesotrione.










Damage %










Line
5 DAT
10 DAT












367
25
10


332
30
10


404
45
20


426
45
20


351
50
25


wt
55
25


wt
55
60


wt
55
60


wt
50
35


wt
60
60


wt
60
30





5 lines of tobacco transformed with Arabidopsis HST were less damaged 10 DAT with 10 g/ha mesotrione than like-treated wild-type lines. Expression of Arabidopsis HST confers a degree of tolerance to the HPPD herbicide, mesotrione.






Example 6
Resistance of Plants Expressing Avena HPPD to HST Herbicides

Seed of segregating T1 lines of tobacco expressing the wild-type HPPD gene of Avena sativa under operable control of the double enhanced 35S CMV promoter region, Nos3′ terminator and TMV translational enhancer were provided as described in WO0246387. About 30-40 T1 seed derived from selling a mesotrione-tolerant T0 event were grown up to 7-10 d old plantlets sprayed and assessed as described above and the results (at 6 DAT) are depicted in the Table 7 below. Under the conditions of the experiment the Avena HPPD appears to offer a degree tolerance to both HST inhibitors 2.30 and 3.6.









TABLE 7







Testing of wild-type (WT) Samsun tobacco and an Avena HPPD


expressing tobacco lines versus mesotrione, compound 2.30 and


compound 3.6. Results depict percent damage at 6 DAT.













Mesotrione
Compound 2.30
Compound 3.6


Line
Rep.
100 g/ha
150 g/ha
300 g/ha














WT
1
70
70
65



2
80
70
65



3
80
70
65


Avena HPPD
1
5
55
10



2
10
55
10



3
20
55
5



4
0
55
5



5
0
55
3



6
0
55
10



7
0
40
10



8
5
50
15



9
10
45
5



10
5
40
10



11
2
45
10



12
5
50
10



13
0
50
5



14
0
60
5



15
8
30
15



16
5
40
15



17
3
60
5



18
15
60
10



19
3
60
5



20
0
70
5



21
15
65
5



22
10
70
10



23
5
55
8



24
0
55
5



25
5
55
5



26
0
45
10



27
80
55
15



28
5
55
10



29
3
45
5



30
3
60
10









Example 7
Tobacco, Transformation and Selection of HST-Expressing Transformants on HST Herbicides

A master plate of Agrobacterium tumefaciens containing the binary vector pBinAT HST (described in example 6) is used to inoculate 10 ml LB containing 100 mg/1 Rifampicin plus 50 mg/1 Kanamycin using a single bacterial colony. This is incubated overnight at 28° C. shaking at 200 rpm.


This entire overnight culture is used to inoculate a 50 ml volume of LA (plus antibiotics). Again this is cultured overnight at 28° C. shaking at 200 rpm. The Agrobacterium cells are pelleted by centrifuging at 3000 rpm for 15 minutes then resuspended in MS medium with 30 g/1 sucrose, pH 5.9 to an OD (600 nM)=0.6. This suspension is dispensed in 25 ml aliquots into petri dishes.


Clonal micro-propagated tobacco shoot cultures are used to excise young (not yet fully expanded) leaves. The mid rib and outer leaf margins are removed and discarded, the remaining lamina is cut into 1 cm squares. These are transferred to the Agrobacterium suspension for 20 minutes. Explants are then removed, dabbed on sterile filter paper to remove excess suspension then transferred to NBM medium (MS medium, 30 g/1 sucrose, 1 mg/1 BAP, 0.1 mg/1 NAA, pH 5.9, solidified with 8 g/1 Plantagar), with the abaxial surface of each explant in contact with the medium. Approximately 7 explants are transferred per plate, which are then sealed then maintained in a lit incubator at 25° C., 16 hour photoperiod for 3 days.


Explants are then transferred to NBM medium containing 0.5 mg/1 Haloxydine plus antibiotics to prevent further growth of Agrobacterium (200 mg/1 timentin with 250 mg/1 carbenicillin). Further subculture on to this same medium is then performed every 2 weeks.


As shoots start to regenerate from the callusing leaf explants these are removed to Shoot elongation medium (MS medium, 30 g/1 sucrose, 8 g/1 Plantagar, 1 mg/1 Haloxydine (or similar or concentration of any other HST herbicide at an appropriate discriminating concentration), 200 mg/1 timentin, 250 mg/1 carbenicillin, pH 5.9). Shoots that root and continue to proliferate are analysed for stable integration of the HST transgene by PCR. Ultimately these rooted shoots are transferred to soil and progressed under glasshouse conditions. T1 seed is produced from selected T0 lines, Thus it is found that the use of a HST gene in combination with a HST-inhibitor herbicide provides a means for the selection of transgenic plant tissue


Example 8
Preparation and Testing of Stable Transgenic Plants Lines Expressing a Heterologous HPPD Enzyme

Transgenic lines of tobacco, soyabean and corn etc. can be engineered to express various heterologous HPPDs derived from, for example Avena (SEQ ID #26), Wheat (SEQ ID #27), Pseudomonas fluorescens (SEQ ID # 25) and Shewanella colwelliana (SEQ ID #28) as, for example, described in WO 02/46387.


The seed from selected events are collected sown on again in pots and tested again for herbicide resistance in a spray test for resistance to HPPD herbicide (for example mesotrione). Single copy events amongst the T1 plant lines are identified by their 3:1 segregation ratio (wrt kanamycin and or herbicide) and by quantitative RT-PCR. Seed from the thus selected T1 tobacco (var Samsun) lines are sown in 3 inch diameter pots containing 50% peat, 50% John Innes soil no 3. After growth to the 3 leaf stage, plants are sprayed, as described above, in order to test for herbicide tolerance relative to like-treated non-transgenic tobacco plants.


Control tobacco plants and transgenic T1 plants expressing either the Pseudomonas or the wheat HPPD gene are sprayed at 37, 111, 333 and 1000 g/ha rates of HST inhibitors and, for example, compound 2.3.


Plants are assessed and scored for % damage at 16 DAT.









TABLE 8







Comparing % damage observed 16 DAT of w/t tobacco plants with


transgenic plants expressing either Pseudomonas or Wheat HPPD














Pseudomonas

Wheat
w/t


Compound
RATE g/ha
HPPD
HPPD
tobacco














Mesotrione
37
3
0
66



111
3
2
57



333
43
0
57



1000
67
13
54


Compound 2.3
37
0
0
17



111
3
0
40



333
6
3
47



1000
10
8
57









It is thus observed that expression of either HPPD gene provides tobacco with a high level of resistance to treatment with the HST inhibitor, 2.3 as well as to mesotrione. Treated w/t tobacco plants show white bleached meristems whereas transgenic HPPD expressing plants have green healthy meristems and new leaves and look almost undamaged. In this test plants were relatively large at the time of spraying and thus controls were not completely controlled.


Example 9
Preparation of Transgenic Plants Lines Expressing Different Heterologous HST and HPPD Enzymes and Stacked Combinations Thereof
Glasshouse Testing for Herbicide Tolerance

The full length Arabidopsis (plus start codon) HST seq ID 11 is cloned behind a double 35s CMV promoter sequence and a TMV translational enhancer sequence and in front of the 3′ terminator from the nos gene as described previously. As described above this expression construct is cloned into a binary vector (pBIN 35S Arabidopsis HST) that is transformed into tobacco to produce populations of 30-50 transgenic events which are subdivided at the callus stage to produce 2-4 clonal plants from each transgenic ‘event’ which are then regenerated and transferred into soil before transfer to the glass house and testing.


In just the same way the Chlamydomonas HST gene sequence (AM285678) is codon-optimised for tobacco and cloned behind the double Cauliflower mosaic virus 35S promoter and Tobacco mosaic virus enhancer sequences and in front of a nos gene terminator, cloned into a binary vector and transformed into tobacco to produce a population of T0 plants.


Exactly as described in example 5 of WO 02/46387 the wheat HPPD gene sequence (Embl DD064495) is cloned behind an Arabidopsis Rubisco small subunit (SSU) promoter and in front of a nos gene terminator to produce an SSU Wheat HPPD nos expression cassette' which is cloned into a binary vector and transformed into tobacco to produce a population of 30-50 transgenic events.


A “pBin Arabidopsis HST/Wheat HPPD” vector is also built in order to provide a population of plants that co-express the HST and HPPD enzymes. The SSU Wheat HPPD nos cassette (described above) is cloned into the EcoRI site of the pBin 35S Arabidopsis HST vector (described above) to generate the HST/HPPD expression construct and binary vector. Again this is transformed into tobacco to produce a population of primary transformants.


Alternatively, transgenic plants expressing both HPPD and HST are produced by first transforming to express either a heterologous HST or HPPD and then the progeny tissue are subsequently transformed with a construct designed to express the other enzyme. For example, as described in WO 02/46387 tobacco plants are transformed to express wheat, Avena or Pseudomonas HPPD under expression control of the Arabidopsis small subunit of rubisco promoter, TMV translational enhancer and nos gene 3′ terminator. Examples of T0 events highly tolerant to mesotrione are selfed on to make T1 seed. Approximately 100 of these T1 seeds from a single event are surface sterilised using 1% Virkon for 15 minutes then following washing in sterile water plated onto MS medium with 20 g/1 sucrose, 100 mg/1 Kanamycin, pH 5.8 solidified with 8 g/1 plantagar. Individual plants are picked from the mixed population of hemizygous and homozygous plants that germinate, grown on in vitro and micropropagated to provide a clonal recombinant shoot culture. Leaves from these shoot cultures are subject to transformation using constructs and selection methods described previously. To initially evaluate whether co-expression of HPPD and HST results in elevated levels of plant resistance to mesotrione compared to expression of HPPD alone, shoot culture derived leaf explants from HPPD only and HPPD plus HST transformants are plated onto NBM medium containing a range of mesotrione concentrations between 0.1 to 5 mg/1. Explants from transgenics combining HPPD and HST may exhibit green callus and more limited bleaching of regenerating shoots at higher mesotrione concentrations than the HPPD only ‘background’ explants from the clonal single plant, HPPD event derived material. ‘Control’ plantlets are regenerated from the untransformed (with HST) HPPD-expressing clonal background material derived from a single plant of a single event. T0 HST transgenic plantlets that are additionally transformed with HST are selected against this background as described in the previous example on haloxydine, and are also regenerated. Plantlets are micropropagated into further clones, rooted and grown on in pots in the glass house as described in the previous examples.









TABLE 9







T0 populations of tobacco events containing, alternatively, the


expression cassettes described above having 1) the 35 S Arabidopsis


HST gene, 2) the SSU wheat HPPD gene or 3) the 35 S Chlamydomonas


HST gene. Assessments of herbicidal damage at various times after


spray with 100 g of compound 2.30. The parameter “ht” refers


to the % in height reduction relative to control plants, whilst the


parameter “blch” refers to the % bleaching observed at the


meristem relative to control plants. It is clear that transformation


with all three constructs confers tolerance to compound 2.30. The


highest number of plants showing the least damage (about 50% of the


events being <30% stunted) were observed in the populations


transformed with either of the two HST genes.
















ht

blch

ht

blch












ARABIDOPSIS HST T0 TOBACCO LINES











7 DAT
26 DAT















WT

80

85

95

100


WT

80

70

90

85


WT

85

75

75

35


WT

80

75

90

65


WT

75

70

80

50


WT

80

80

95

85


 807

30

30

75

25


 811

0

5

5

0


 812

0

10

15

0


 813

30

15

30

0


 814

50

15

50

0


 816
nr

nr

nr

nr


 817

0

5

5

0


 819
nr

nr

nr

nr


 823

70

50

30

100


 826

0

70

60

85


 828
nr

nr

nr

nr


 836

0

5

5

0


 838

80

10

80

25


 841

80

20

75

0


 844

0

0

5

0


 845

40

40

90

90


 846

70

70

90

90


 848

30

100

50

90


 852

80

80

90

90


 853

30

25

90

85


 855

5

0

15

0


 857

10

20

35

0


 858

10

0

10

0


 859
nr

nr

nr

nr


 860

70

0

30

0


 861

15

30

30

0


 865

20

10

5

0


 867
nr

nr

nr

nr


 874

0

5

5

0


 881

40

0

10

0







WHEAT HPPD T0 TOBACCO LINES










7 DAT
21 DAT















WT

80

85

95

100


WT

80

70

90

85


WT

85

75

75

35


WT

80

75

90

65


WT

75

70

80

50


WT

80

80

95

85


1173

50

35

45

0


1176

50

40

85

90


1181

60

50

80

70


1190

90

60

85

70


1193

70

35

85

70


1202

60

30

30

0


1203

70

35

55

50


1207

40

60

50

0


1210

50

35

50

0


1219

70

30

55

0


1224

60

70

80

70


1228

30

30

40

0


1230

70

40

40

0


1231

70

40

50

0


1234

50

50

60

0


1250

50

35

60

0


1255

70

70

75

70


1257





70

65


1259

20

30

50

0


1260

90

10

95

45


1261





45

0


1265

20

40

40

0


1267

70

30

70

50


1273

30

40

55

0


1275

40

30

50

0


1182

90

10

90

90







CHLAMYDOMONAS HST T0 TOBACCO LINES










17 DAT
26 DAT















WT

80

85

95

100


WT

80

70

90

85


WT

85

75

75

35


WT

80

75

90

65


WT

75

70

80

50


WT

80

80

95

85


 510

85

75

90

90


 511

40

15

60

0


 513

20

0

20

0


 516
nr

nr

nr

nr


 518

15

0

30

0


 519
nr

nr

nr

nr


 521

10

0

20

0


 524
nr

nr

nr

nr


 527

55

15

70

0


 528

25

0

30

0


 529

10

0

20

0


 530

30

0

30

0


 534

0

0

10

0


 535
nr

nr

nr

nr


 536

5

0

30

0


 539

0

0

20

0


 543

15

0

20

0


 545

55

20

80

25


 546

8

0

20

0


 551
nr

nr

nr

nr


 552

20

0

35

0


 553
nr

nr

nr

nr


 557
nr

nr

nr

nr


 558
nr

nr


90

85


 559

40

15

50

0


 563

20

0

25

0


 566

50

0

75

0


 568

55

65

75

85


 572

30

10

35

0


 574

35

10

30

0


 577

90

90

95

90


 578
nr

nr

nr

nr
















TABLE 10







T0 populations of tobacco events containing, alternatively, the expression cassettes described above


having 1) the Arabidopsis HST gene, 2) the wheat HPPD gene or 3) the Chlamydomonas HST gene. Assessments


of herbicidal damage at various times after spray with 40 g of compound 1.1. The parameter “ht” refers to the % in


height reduction relative to control plants, whilst the parameter “blch” refers to the % bleaching observed at the


meristem relative to control plants. All three constructs provide tolerance to compound 1.1. The highest number


of least damaged plants (more than 50% of the events <35% stunted) were observed in plants transformed with


either of the two HST genes.










ARABIDOPSIS HST

WHEAT HPPD

CHLAMYDOMONAS HST
















7 DAT
21 DAT

7 DAT
21 DAT

14 DAT






























ht

blch

ht

blch


ht

blch

ht

blch


ht

blch


































WT

60

70

70

80
WT

60

70

70

80
WT

60

40


WT

65

65

80

70
WT

65

65

80

70
WT

70

70


WT

60

60

85

80
WT

60

60

85

80
WT

60

60


WT

75

75

60

45
WT

75

75

60

45
WT

70

75


WT

75

70

55

30
WT

75

70

55

30
WT

75

70


WT

70

80

65

45
WT

70

80

65

45
WT

65

65


807

60

70

90

90
1173

10

30

40

0
510

30

15


811

20

20

25

0
1176

50

50

65

25
511

30

15


812

0

15

5

0
1181

10

40

40

0
513

20

5


813

0

30

35

0
1190

70

70

80

25
516

15

5


814

80

20

40

0
1193

50

50

45

0
518

30

15


816
nr

nr


20

0
1202

40

40

40

0
519
nr

nr


817

0

15

15

0
1203

70

70

100

100
521

10

0


819
nr

nr

nr

nr

1207

70

50

80

80
524
nr

nr


823

90

20

90

90
1210

70

60

80

80
527

40

35


826

70

70

80

80
1219

40

40

45

0
528

35

15


828
nr

nr

nr

nr

1224

40

45

40

0
529

15

0


836

30

10

35

0
1228

10

60

50

0
530

10

5


838

40

15

40

0
1230

70

50

40

0
534

30

5


841

70

25

35

0
1231

50

50

80

25
535

20

10


844

40

25

35

0
1234

50

60

90

90
536

25

0


845

80

40

85

90
1250

90

50

90

20
539

5

0


846

80

40

70

60
1255

90

30

90

20
543

20

0


848
nr

nr


30

0
1257

70

70

80

75
545

25

20


852

50

50

50

0
1259

70

70

80

10
546

15

0


853

90

20
nr

nr

1260

30

40

65

0
551

30

15


855

0

10

20

0
1261

70

60

90

90
552

40

15


857

40

30

35

0
1265
nr

nr


50

0
553

10

10


858

50

20

40

0
1267

70

70

85

80
557

25

15


859
nr

nr

nr

nr

1273

10

50

40

0
558

15

5


860

40

25

40

0
1275

60

60

80

20
559

25

15


861

40

20

40

0
1182

40

50

55

0
563

55

10


865

0

30

25

0









566

50

25


867

70

60

40

0









568

35

15


874

0

25

15

0









572

30

15


881
nr

nr


35

0









574
NR

NR




















577

80

80




















547

30

15




















578

50
















TABLE 11







T0 populations of tobacco events containing, alternatively, the


expression cassettes described above having 1) the Arabidopsis HST


gene, 2) the wheat HPPD gene stacked with the Arabidopsis HST gene or


3) the Chlamydomonas HST gene. Assessments of herbicidal damage


at various times after spray with 500 g of compound 2.30. The parameter


“ht” refers to the % in height reduction relative to control


plants, whilst the parameter “blch” refers to the % bleaching


observed at the meristem relative to control plants. All three constructs


provide tolerance to compound 2.30. The highest number of least damaged


plants (more than 50% of the events <30% stunted) were observed in


plants transformed with the stacked combination of the HPPD and


HST genes.
















ht

blch

ht

blch












ARABIDOPSIS HST T0 TOBACCO LINES











7 DAT
26 DAT















WT

85

70

85

90


WT

85

70

85

90


WT

85

75

100

100


WT

90

75

90

80


WT

90

80

80

70


WT

90

80

70

40


807

50

80

95

95


811

60

60

60

100


812

30

30

30

0


813

20

25

60

0


814

60

40

60

0


816

20

0

35

0


817

30

25

30

0


819
nr

nr

nr

nr


823

60

70

45

100


826

50

100

90

95


828
nr

nr

nr

nr


836

20

10

50

0


838

60

50

90

95


841

30

25

30

0


844

50

25

50

0


845

85

85

95

95


846

80

100

95

95


848

40

40

50

0


852

50

100

90

95


853

60

80

95

95


855

0

20

30

0


857

30

25

35

0


858

50

20

55

0


859
nr

nr

nr

nr


860

50

25

20

0


861

10

5

25

0


865

30

25

30

0


867
nr

nr


95

95


874

30

20

20

0


881
nr

nr


35

0








ARABIDOPSIS HST + WHEAT HPPD T0 TOBACCO LINES











7 DAT
26 DAT















WT

85

70

85

90


WT

85

70

85

90


WT

85

75

100

100


WT

90

75

90

80


WT

90

80

80

70


WT

90

80

70

40


694

30

90

90

90


695

50

25

25

0


704

30

20

30

0


705
nr

nr


85

50


706

0

20

50

0


714
nr

nr

nr

nr


716

50

15

55

0


718

15

15

12

0


722

60

15

60

0


724

80

30

90

90


727

0

20

12

0


728

75

20

30

0


730

60

60

95

95


739

0

25

35

0


740

45

30

10

0


742

20

15

30

0


745
nr

nr

nr

nr


747

60

60

90

90


749

35

30

30

0


751

30

15

10

0


752

30

20

15

0


757

45

55

80

100


758

20

20

10

0


759
nr

nr


80

20


762

40

20

30

0


768

30

30

30

0


769

50

25

35

0


773

50

20

35

0


776
nr

nr

nr

nr


778

0

20

12

0


786

40

25

85

30








CHLAMYDOMONAS HST T0 TOBACCO LINES











17 DAT
26 DAT















WT

85

70

85

90


WT

85

70

85

90


WT

85

75

100

100


WT

90

75

90

80


WT

90

80

80

70


WT

90

80

70

40


510

60

10

50

0


511

45

0

40

0


513

35

0

30

0


516

60

10

70

0


518

50

0

75

0


519
nr

nr

nr

nr


521

40

0

45

0


524






nr


527

80

35

85

20


528

50

10

65

30


529

70

70

80

70


530

50

0

45

0


534

50

0

35

0


535
nr

nr


60

65


536

50

20

55

25


539

45

0

35

0


543

80

70

80

80


545

90

85

85

70


546

40

0

45

0


551

75

65

90

80


552

45

0

55

0


553

60

0

40

0


557

35

0

40

0


558

70

65

75

70


559

70

70

75

0


563

45

0

30

0


566

50

10

65

0


568

90

60

90

75


572

80

50

70

65


574

45

15

60

0


577

80

80

90

90


578

90

85

90

80
















TABLE 12







T0 populations of tobacco events containing, alternatively, the expression


cassettes described above having 1) the Arabidopsis HST gene or 2)


the Chlamydomonas HST gene. Assessments of herbicidal damage at


various times after spray with 15 g/ha of mesotrione. A number of


plant lines containing the Chlamydomonas HST gene showed


some tolerance to mesotrione with 3 lines in particular greening up


and recovering to a significantly greater extent than like-treated


control plants.


TREATMENT WITH 15 g/ha of mesotrione











ARABIDOPSIS


CHLAMYDOMONAS




HST TOBACCO
HST TOBAC












21 DAT

21 DAT











ht
ht
















WT

90
WT

90


WT

85
WT

85


WT

85
WT

85


WT

75
WT

75


WT

75
WT

75


WT
nr

WT
nr


807
NR

510

70


811

80
511

55


812

80
513
nr


813

90
516

85


814

85
518

65


816

80
519
nr


817

90
521

65


819
nr

524
nr


823

80
527

80


826

90
528

85


828
nr

529

65


836

70
530

65


838

80
534

70


841

75
535

55


844

90
536

65


845

85
539

55


846

80
543

80


848

85
545

65


852

90
546

75


853

90
551

60


855
nr

552

65


857

85
553
nr


858

90
557

80


859
nr

558

70


860

85
559

65


861

85
563

70


865

90
566

80


867

90
568

70


874

75
572

70


881

90
574

70





577

100





578

70









Example 10
Construction of Soybean Transformation Vectors

A binary vector (17107) for dicot (soybean) transformation is, for example, constructed, with the Arabidopsis UBQ3 promoter driving expression of the Chlamydomonas HST coding sequence (SEQ ID # 15), followed by Nos gene 3′ terminator. The gene is codon optimized for soybean expression based upon the predicted amino acid sequence of the HST gene coding region. The amino acid sequence of the protein encoded by Chlamydomonas HST gene is provided in SEQ ID # 5. Optionally the transformation vector also contains two PAT gene cassettes (one with the 35S promoter and one with the CMP promoter, and both PAT genes are followed by the nos terminator) for glufosinate based selection during the transformation process.


A similar binary vector (17108) is similarly constructed but also comprising an expression cassette expressing the soyabean codon-optimized Avena HPPD gene. In this case there is no PAT gene and selection is carried out using a HPPD herbicide or, as described herein, a HST herbicide.


Example 11
Soybean to Plant Establishment and Selection

Soybean transformation is achieved using methods well known in the art. T0 plants were taken from tissue culture to the greenhouse where they are transplanted into saturated soil (Redi-Earth® Plug and Seedling Mix, Sun Gro Horticulture, Bellevue, Wash.) mixed with 1% granular Marathon® (Olympic Horticultural Products, Co., Mainland, Pa.) at 5-10 g/gal Redi-Earth® Mix in 2″ square pots. The plants are covered with humidity domes and placed in a Conviron chamber (Pembina, N. Dak.) with the following environmental conditions: 24° C. day; 18° C. night; 23 hr photoperiod; 80% relative humidity.


After plants became established in the soil and new growth appeared (˜1-2 weeks), plants are sampled and tested for the presence of desired transgene by Taqman™ analysis using appropriate probes for the HST and/or HPPD genes, or promoters (for example prCMP and prUBq3). All positive plants and several negative plants are transplanted into 4″ square pots containing MetroMix® 380 soil (Sun Gro Horticulture, Bellevue, Wash.). Sierra 17-6-12 slow release fertilizer is incorporated into the soil at the recommended rate. The negative plants serve as controls for the spray experiment. The plants are then relocated into a standard greenhouse to acclimatize (˜1 week). The environmental conditions are: 27° C. day; 21° C. night; 12 hr photoperiod (with ambient light); ambient humidity. After acclimatizing (−1 week), the plants are ready to be sprayed with the desired herbicides.


Example 12
HPPD/HST Herbicide Mixtures. Effect of Adding Small Amounts of HPPD Inhibitor on the Herbicidal Activity of HST Herbicides

Tobacco (var Samsun) plantlets germinated aseptically in agar made up in 1/3 strength Murashige and Skoog salts medium along with various doses of herbicide. Bleaching damage to emerging plantlets is assessed 7 DAT. The plantlets are kept covered under clear perspex and grown at 18° C. (night) and 24° C. (day) under a 16 h day (˜500-900 umol/m2), 8 h darkness regime. Herbicide affected plantlets are bleached white and grow less. Synergistic/antagonistic responses are calculated using the Colby formula (Colby, S. R. (Calculating synergistic and antagonistic responses of herbicide Combinations”, Weeds, 15, p. 20-22, 1967).









TABLE 13







HST + HPPD herbicide effects on tobacco seedlings in agar.


% Injury













Plus
plus





Mesotrione
Mesotrione



[Haloxydine]/
Haloxydine
(0.004 ppm)
(0.004 ppm)


ppm
only
(OBSERVED)
(EXPECTED)
(O − E)














37.5
100
100
100
0


18.8
100
100
100
0


9.4
90
100
94
6


4.7
70
100
80
20


2.4
50
90
67.5
22.5


1.2
35
70
58
12


0.6
20
50
48
2


0.3
10
50
41.5
8.5


0.75%
0
35
35
0


v/v DMSO





The % bleaching observed 7 DAT of germinating tobacco seeds in agar is assessed with various doses of haloxydine and 0.75% v/v DMSO in the presence or absence of 0.004 ppm mesotrione. At this dose the mesotrione by itself consistently gives 35% bleaching damage and the expected values for the damage in mixture with the various doses of haloxydine are therefore calculated accordingly as described by Colby (1967).






In an alternative herbicide test procedure tobacco (var Samsun) plantlets germinated aseptically in agar made up in 1/3 strength Murashige and Skoog salts medium are transferred after 4 d to float on top of 2.9 ml of sterile liquid culture medium (half strength Murashige and Skoog medium containing 30 mM sucrose) in wells of 12 well plates. Test compounds are added at various doses and bleaching damage is assessed after 14-20 DAT. The plantlets are kept covered under clear perspex and grown at 18° C. (night) and 24° C. (day) under a 1611 day (˜500-900 umol/m2), 8 h darkness regime Plantlets continue to grow and produce new tissue over the 14-20 DAT period but are bleached and grow less in the presence of controlling concentrations of herbicide.









TABLE 14







HST + HPPD herbicide effects on tobacco seedlings growing on liquid.


% Injury












Plus



Compound 2.13/

Mesotrione
plus Mesotrione


ppm
compound 2.13 only
(0.001 ppm)
(0.0005 ppm)













23
100
100
100


7.67
50
100
100


2.56
0
90
90


0.85
0
90
40


0.28
0
80
0


0.09
0
80
0


0.03
0
60
0


0.01
0
40
0


0.75% v/v DMSO
0
20
0





The % bleaching observed 20 DAT of tobacco seedlings on liquid culture medium is assessed versus the presence of various concentrations of the HST herbicide, compound 2.13 with 0.75% v/v DMSO in the presence or absence of 0.001 or 0.0005 ppm of mesotrione. At these doses the mesotrione by itself produced either minimal, 20%, or zero bleaching damage.






From the data provided it is apparent that addition of a low dose of mesotrione synergises the herbicidal effect of the HST inhibitor, haloxydine on agar grown tobacco plantlets.









TABLES 15a AND 15b





HST + HPPD herbicide injury on tobacco seedlings growing on liquid


culture medium.







TABLE 15a









Haloxydine ppm
Haloxydine alone
+mesotrione 0.001 ppm





47
100
100


16
100
100


5.2
100
100


1.7
100
100


0.58
90
100


0.19
50
100


0.75% DMSO
0
5










TABLE 15b









Compound 2.15 ppm
Compound 2.15 alone
+mesotrione 0.001 ppm





47
100
100


16
100
100


5.2
40
70


1.7
5
80


0.58
0
5


0.19
5
0


0.75% DMSO
0
5





The % bleaching observed 14 DAT of tobacco seedlings on liquid culture medium is assessed versus the presence of various concentrations of the HST herbicides, haloxydine (Table 15a), and compound 2.15 (Table 15b), with 0.75% v/v DMSO in the presence or absence of 0.001 ppm mesotrione. At this dose mesotrione produced minimal (0-5% v) damage.






In liquid culture the synergising effect of mesotrione on the activity of HST inhibitor, 2.13 is even more apparent than that on haloxydine. Even addition of a dose of mesotrione that itself produces no visible damage at all results in levels of 40, 90 and 100% bleaching injury at doses of compound 2.13 where the expected level of control (according to Colby) is only 0, 0 and 50%. Similarly, at the higher dose of mesotrione, 80 or 90% bleaching is observed across a range of rates of compound 2.13 where only 20% is expected. Similarly, under similar conditions and in repeat experiments, there are clear synergistic effects of low amounts of mesotrione on the injury observed down haloxydine and compound 2.15 dose responses.


Example 13
Glass House Weed Control by Mixtures of HST and HPPD Herbicides

Weed seeds are planted out in trays containing suitable soil (for example 50% peat, 50% John Innes soil no 3) and grown in the glass house conditions under 24-27° C. day; 18-21° C. night and approximately a 14 h (or longer in UK summer) photoperiod. Humidity is ˜65% and light levels up to 2000 μmol/m2 at bench level. Trays are sprayed with test chemicals dissolved in water with 0.2-0.25% X-77 surfactant and sprayed from a boom on a suitable track sprayer moving at about 2 mph in a suitable track sprayer (for example a DeVries spray chamber with the nozzle about 2 inches from the plant tops). Spray volume is suitably 500-1000 l/ha. Sprays are carried out both pre-emergence and over small plants at about 7-12 d post-emergence


Plants are assessed 14 DAT and herbicidal damage is scored on a scale from 0 to 100%. The HST inhibitor compound 2.30 (designated compound A in table 16 and 17), haloxydine (compound 1.1) and compound 2.13 (designated compound AE in tables 16 and 17) are sprayed at rates between 0 and 500 g/ha. Mesotrione is applied at a very low rate of 1 g/ha at which it causes essentially no (<10% damage).









TABLE 16







Postemergence Control of Weeds by HST/HPPD Herbicide Mixtures. Numbers


reported correspond to the % damage observed.


POST-EMERGENCE APPLICATION











SETFA
ECHCG
ALOMY


















Rate

1 g ai ha−1


1 g ai ha−1


1 g ai ha−1



a.I.
g/ha
no-mesotrione
mesotrione
colby
no-mesotrione
mesotrione
colby
no-mesotrione
mesotrione
colby




















Mesotrione
1

0


8


0



Compound A
500
70
75
5
67
75
6
23
35
12



250
65
75
10
65
68
0
15
22
7



125
57
62
5
57
53
−7
13
20
7



63
33
42
8
42
42
−5
5
10
5



31
32
42
10
18
38
13
3
3
0



16
23
18
−5
12
17
−2
2
2
0


Compound AE
500
0
0
0
0
20
12
7
5
−2



250
0
0
0
0
22
13
2
3
2



125
0
0
0
0
15
7
0
0
0



63
0
0
0
0
8
0
0
3
3



31
0
0
0
0
5
−3
0
2
2



16
0
0
0
0
5
−3
0
0
0


Haloxidine
500
100
100
0
98
100
2
95
95
0



250
100
100
0
83
97
12
90
92
2



125
93
97
3
70
78
6
78
83
5



63
80
92
12
57
75
15
53
68
15



31
68
82
13
52
67
11
27
33
7



16
42
27
−15
33
57
18
13
23
10
















TABLE 17





Pre-Emergence Weed Control by HPPD/HST Herbicide Mixtures


PRE-EMERGENCE APPLICATION



















AMARE
SOLNI
IPOHE




















1 g ai ha−1


1 g ai ha−1


1 g ai ha−1




Rate
no-mesotrione
mesotrione
colby
no-mesotrione
mesotrione
colby
no-mesotrione
mesotrione
colby









a.I.
g/ha
% Damage at 14 DAA (N = 3)




















Mesotrione
1

2


17


0



Compound A
500
73
90
16
45
75
21
3
17
13



250
37
47
9
3
30
11
2
0
−2



125
10
40
29
2
8
−11
0
0
0



63
0
10
8
0
3
−13
0
0
0



31
0
0
−2
0
7
−10
0
0
0



16
0
0
−2
0
0
−17
0
0
0


Compound AE
500
60
70
9
85
87
−1
17
17
0



250
53
57
3
68
78
5
0
5
5



125
32
45
12
33
53
9
0
0
0



63
28
33
4
22
38
4
0
7
7



31
8
23
13
12
23
−3
0
0
0



16
0
7
5
0
5
−12
0
0
0


Haloxidine
500
100
100
0
100
100
0
83
87
3



250
100
100
0
95
100
4
80
67
−13



125
100
100
0
87
92
3
62
52
−10



63
88
93
5
48
75
18
15
8
−7



31
73
75
1
27
60
21
0
0
0



16
38
42
2
0
47
30
0
0
0














SETFA
ECHCG
ALOMY




















1 g ai ha−1


1 g ai ha−1


1 g ai ha−1




Rate
no-mesotrione
mesotrione
colby
no-mesotrione
mesotrione
colby
no-mesotrione
mesotrione
colby









a.I.
g/ha
% Damage at 14 DAA (N = 3)




















Mesotrione
1

0


0


0



Compound A
500
22
32
10
18
27
8
10
20
10



250
3
7
3
5
5
0
2
0
−2



125
2
0
−2
2
0
−2
0
0
0



63
0
0
0
0
0
0
0
0
0



31
0
0
0
0
0
0
0
0
0



16
0
0
0
0
0
0
0
0
0


Compound AE
500
0
0
0
3
2
−2
2
0
−1



250
0
0
0
0
2
2
0
0
0



125
0
0
0
0
3
3
0
0
0



63
0
0
0
0
3
3
0
0
0



31
0
0
0
0
0
0
0
0
0



16
0
0
0
0
0
0
0
0
0


Haloxidine
500
97
98
2
88
95
7
85
83
−2



250
95
95
0
92
83
−8
82
78
−3



125
88
90
2
73
78
5
67
77
10



63
50
58
8
40
53
13
42
50
8



31
25
32
7
25
23
−2
13
25
12



16
0
10
10
13
20
7
3
8
5









Example 14
Further Studies Showing Synergy Between HPPD and HST Herbicides

In a further test, the results of which are depicted in tables 18 and 19, weed seeds are planted out in trays containing 50% peat/50% John Innes no. 3 soil and grown in the glass house at 24-27 C day; 18-21 C night and approximately a 15 h photoperiod. Humidity is ˜65% and light levels at bench level are up to 2 mmol/m2. Again all spray chemicals are dissolved in 0.2% X77 surfactant and sprayed from a boom on a track sprayer moving at 2 mph with the nozzle set about 2 inches above the plant tops. The spray volume is 5001/ha. Sprays are carried out both pre-emergence and post-emergence over small plants at about 7-12 d post-emergence. Plants are assessed at 14 DAT with herbicidal damage scored on a scale from 0 to 100%. The HPPD inhibiting herbicide is compound A22 (4-hydroxy-3-[[2-(2-methoxyethoxy)methyl]-6-(trifluoromethyl)-3-pyridinylicarbonyl]-bicyclo[3.2.1]oct-3-en-2-one) which is sprayed at 2 g/ha both alone and in mixture with various HST herbicides. Results and spray rates are depicted in Tables 18 and 19. Again the Colby formula has been used to calculate synergy scores observed following treatment with the mixture based on the results obtained with the single components alone. Positive synergy is observed between the HPPD herbicide and a wide variety of HST inhibitor herbicides applied both pre and postemergence across a variety of weeds.









TABLE 18







Postemergence weed control of a range of weeds. % control scores following sprays with a variety


of HST inhibitors alone and in mixture with A22 (single replicate tests only)









SCORES 14 DATPOST_EMERGENCE TREATMENT





















Rate
SIDSP
Colby
DIGSA
Colby
PANMI
Colby
BRAPL
Colby
SETFA
Colby
ECHCG
Colby


Compound
(g/ha)
Obs
(O-E)
Obs
(O-E)
Obs
(O-E)
Obs
(O-E)
Obs
(O-E)
Obs
(O-E)























Compound A22 alone
2
20

5

50

2

10

30




2
15

5

55

2

2

30


Compound 2.15 alone
250
15

0

0

5

2

2



62.5
10

0

0

0

0

0



15.625
0

0

0

0

0

0


Compound 2.15 + 2 g/ha
250
60
30.125
10
5
50
−2.5
30
23.1
10
4.08
60
28.6


compound A22
62.5
50
24.25
10
5
50
−2.5
20
18
0
−4
50
20



15.625
30
12.5
0
−5
40
−12.5
10
8
0
−4
40
10


Compound 3.5 alone
250
10

5

70

65

40

30



62.5
10

0

15

30

10

15



15.625
2

0

5

2

2

5


Compound 3.5 + 2 g/ha
250
30
4.25
10
0.25
75
−10.75
50
−15.7
55
12.6
60
9


compound A22
62.5
25
−0.75
2
−3
75
15.375
40
8.6
30
16.4
50
9.5



15.625
25
5.85
0
−5
70
15.125
2
−1.96
5
−0.92
50
16.5


Compound 2.21 alone
250
40

20

70

80

60

40



62.5
20

10

30

30

20

10



15.625
5

0

10

5

2

0


Compound 2.21 + 2 g/ha
250
50
−0.5
60
36
80
−5.75
80
−0.4
60
−1.6
65
7


compound A22
62.5
50
16
50
35.5
70
3.25
75
43.6
50
26.8
65
28



15.625
30
8.375
20
15
75
17.75
30
23.1
50
44.08
50
20


Compound 2.14 alone
250
50

20

90

60

70

65



62.5
30

20

60

60

60

50



15.625
15

10

40

30

25

30


Compound 2.14 + 2 g/ha
250
60
1.25
60
36
80
−15.25
80
19.2
75
3.8
80
4.5


compound A22
62.5
50
7.75
40
16
80
−1
60
−0.8
70
8.4
70
5



15.625
50
20.125
30
15.5
75
3.5
50
18.6
55
27
60
9


Compound 2.19 alone
250
50

70

85

80

80

75



62.5
30

50

70

70

60

45



15.625
15

30

40

20

30

30


Compound 2.19 + 2 g/ha
250
70
11.25
80
8.5
90
−2.875
80
−0.4
80
−0.8
85
2.5


compound A22
62.5
65
22.75
75
22.5
80
−5.75
70
−06
80
18.4
80
18.5



15.625
50
20.125
60
26.5
80
8.5
60
38.4
80
47.2
80
29


Compound 2.18 alone
250
70

50

70

80

85

70



62.5
60

30

65

60

60

40



15.625
15

1

10

5

10

2


Compound 2.18 + 2 g/ha
250
70
−5.25
75
22.5
85
−0.75
80
−0.4
85
−0.6
85
6


compound A22
62.5
50
−17
60
26.5
80
−3.375
70
9.2
80
18.4
80
22



15.625
50
20.125
40
34.05
70
12.75
50
43.1
65
51.4
60
28.6


Compound 2.9 alone
250
20

15

70

70

65

70



62.5
20

10

50

55

50

40



15.625
10

2

30

15

20

20


Compound 2.9 + 2 g/ha
250
70
36
50
30.75
85
−0.75
75
4.4
80
13.6
85
6


compound A22
62.5
70
36
40
25.5
80
3.75
70
14.1
80
28
75
17



15.625
50
24.25
40
33.1
75
8.25
50
33.3
60
36.8
70
26


Compound 2.3 alone
250
55

45

65

70

60

40



62.5
40

35

55

50

40

30



15.625
10

2

2

20

5

5


Compound 2.3 + 2 g/ha
250
50
−12.875
60
12.25
85
1.625
70
−0.6
60
−1.6
65
7


compound A22
62.5
50
−0.5
55
16.75
75
−3.625
60
9
60
17.6
60
9



15.625
40
14.25
50
43.1
75
21.55
50
28.4
60
51.2
60
26.5


Compound 2.8 alone
250
50

60

80

85

60

65



62.5
45

20

50

55

65

40



15.625
30

5

40

20

5

10


Compound 2.8 + 2 g/ha
250
70
11.25
65
3
90
−0.5
90
4.7
65
3.4
70
−5.5


compound A22
62.5
60
5.375
60
36
75
−1.25
80
24.1
60
−6.4
70
12



15.625
50
7.75
50
40.25
70
−1.5
60
38.4
50
41.2
55
18


Compound 2.17 alone
250
50

50

55

50

40

40



62.5
15

5

10

15

10

5



15.625
5

0

0

1

0

0


Compound 2.17 + 2 g/ha
250
55
−3.75
60
7.5
70
−8.625
40
−11
55
12.6
50
−8


compound A22
62.5
50
20.125
50
40.25
70
12.75
40
23.3
50
36.4
50
16.5



15.625
40
18.375
40
35
60
7.5
35
32.02
35
31
30
0


Compound 2.16 alone
250
50

40

70

70

65

50



62.5
40

2

40

30

30

15



15.625
5

0

5

10

1

2


Compound 2.16 + 2 g/ha
250
75
16.25
70
27
75
−10.75
70
−0.6
75
9.3
70
5


compound A22
62.5
70
19.5
40
33.1
60
−11.5
40
8.6
50
18.6
60
19.5



15.625
40
18.375
40
35
60
5.125
20
8.2
40
37.02
50
18.6


Compound 2.28 alone
250
50

70

70

75

80

70



62.5
40

50

35

50

50

40



15.625
15

10

1

5

2

2


Compound 2.28 + 2 g/ha
250
75
16.25
80
8.5
85
−0.75
80
4.5
75
−5.4
80
1


compound A22
62.5
75
24.5
70
17.5
80
10.875
70
19
75
24
80
22



15.625
70
40.125
60
45.5
75
22.025
50
43.1
65
61.04
60
28.6


Compound 2.25 alone
250
40

10

55

60

40

30



62.5
30

2

10

30

5

2



15.625
10

0

2

15

1

1


Compound 2.25 + 2 g/ha
250
60
9.5
60
45.5
75
−3.625
80
19.2
60
18.8
70
19


compound A22
62.5
40
−2.25
30
23.1
70
12.75
50
18.6
30
23.1
50
18.6



15.625
30
4.25
15
10
60
6.55
30
13.3
25
22.02
40
9.3


Compound 2.20 alone
250
55

80

85

95

80

90



62.5
50

60

45

50

65

60



15.625
10

20

15

30

25

20


Compound 2.20 + 2 g/ha
250
80
17.125
85
4
90
−2.875
90
−5.1
85
4.6
85
−8


compound A22
62.5
70
11.25
80
18
85
11.125
70
19
80
14.3
80
8



15.625
65
39.25
60
36
80
20.375
70
38.6
80
53.5
80
36


Compound 6.1 alone
250
20

30

80

75

70

50



62.5
15

10

50

40

20

5



15.625
5

0

2

10

0

0


Compound 6.1 + 2 g/ha
250
60
26
70
36.5
97
6.5
80
4.5
70
−0.6
75
10


compound A22
62.5
50
20.125
50
35.5
75
−1.25
30
−11.2
60
38.4
75
41.5



15.625
45
23.375
40
35
65
11.55
20
8.2
30
28
50
20


















TABLE 19









SCORES 14 DATPRE_EMERGENCE TREATMENT





















Rate
SIDSP
Colby
DIGSA
Colby
PANMI
Colby
BRAPL
Colby
SETFA
Colby
ECHCG
Colby


Compound
(g/ha)
Obs
(O-E)
Obs
(O-E)
Obs
(O-E)
Obs
(O-E)
Obs
(O-E)
Obs
(O-E)










Pre-emergence weed control of a range of weeds. % control scores at 14 DAT following sprays with


a variety of HST inhibitors alone and in mixture with A22.




















Compound A22 alone
2
2

2

0

0

5

5




2
0

0

0

0

0

0


Compound 2.15 alone
250
0

0

0

0

0

0



62.5
0

0

0

0

0

0



15.625
0

0

0

0

0

0


Compound 2.15 + 2 g/ha
250
2
1
0
−1
0
0
0
0
0
−2.5
30
27.5


compound A22
62.5
2
1
0
−1
0
0
0
0
0
−2.5
20
17.5



15.625
0
−1
0
−1
0
0
0
0
0
−2.5
0
−2.5


Compound 3.5 alone
250
2

0

0

0

0

0



62.5
0

0

0

0

0

0



15.625
0

0

0

0

0

0


Compound 3.5 + 2 g/ha
250
5
2.02
0
−1
0
0
0
0
0
−2.5
2
−0.5


compound A22
62.5
1
0
0
−1
0
0
0
0
0
−2.5
2
−0.5



15.625
0
−1
0
−1
0
0
0
0
0
−2.5
0
−2.5


Compound 2.21 alone
250
1

0

0

5

2

5



62.5
0

0

0

2

0

0



15.625
0

0

0

0

0

0


Compound 2.21 + 2 g/ha
250
10
8.01
85
84
15
15
10
5
10
5.55
30
22.625


compound A22
62.5
0
−1
0
−1
0
0
0
−2
1
−1.5
0
−2.5



15.625
0
−1
0
−1
0
0
0
0
0
−2.5
0
−2.5


Compound 2.14 alone
250
5

5

2

20

20

50



62.5
0

0

0

2

2

5



15.625
0

0

0

0

0

0


Compound 2.14 + 2 g/ha
250
50
44.05
30
24.05
50
48
30
10
40
18
97
45.75


compound A22
62.5
0
−1
0
−1
0
0
10
8
20
15.55
60
52.625



15.625
0
−1
0
−1
0
0
0
0
0
−2.5
20
17.5


Compound 2.19 alone
250
10

20

60

40

60

90



62.5
2

0

0

10

5

50



15.625
0

0

0

0

0

0


Compound 2.19 + 2 g/ha
250
65
54.1
80
59.2
65
5
70
30
70
9
80
−10.25


compound A22
62.5
65
62.02
30
29
NC
NC
20
10
5
−2.375
60
8.75



15.625
30
29
2
1
2
2
0
0
2
−0.5
10
7.5


Compound 2.18 alone
250
30

0

50

40

25

20



62.5
30

0

0

10

20

20



15.625
0

0

0

0

0

0


Compound 2.18 + 2 g/ha
250
60
29.3
30
29
65
15
60
20
60
33.125
80
58


compound A22
62.5
20
−10.7
10
9
0
0
5
−5
2
−20
2
−20



15.625
0
−1
0
−1
0
0
0
0
1
−1.5
0
−2.5


Compound 2.9 alone
250
40

65

60

50

30

60



62.5
0

0

0

2

0

0



15.625
0

0

0

0

0

0


Compound 2.9 + 2 g/ha
250
40
−0.6
50
−15.35
35
−25
50
0
60
28.25
90
29


compound A22
62.5
2
1
1
0
0
0
2
0
20
17.5
75
72.5



15.625
1
0
0
−1
0
0
0
0
0
−2.5
60
57.5







Pre-emergence weed control of a range of weeds. % control scores following sprays


with a variety of HST inhibitors alone and in mixture with A22.




















Compound 2.3 alone
250
20

40

50

40

30

50




62.5
0

0

0

2

5

10



15.625
0

0

0

0

0

0


Compound 2.3 + 2 g/ha
250
5
−15.8
10
−30.6
20
−30
20
−20
40
8.25
80
28.75


compound A22
62.5
0
−1
5
4
0
0
10
8
30
22.625
70
57.75



15.625
NC
NC
0
−1
0
0
0
0
20
17.5
30
27.5


Compound 2.8 alone
250
2

30

60

15

10

50



62.5
0

0

0

1

0

2



15.625
0

0

0

0

0

0


Compound 2.8 + 2 g/ha
250
2
−0.98
2
−28.7
5
−55
20
5
30
17.75
60
8.75


compound A22
62.5
2
1
1
0
0
0
20
19
30
27.5
55
50.55



15.625
0
−1
0
−1
0
0
0
0
10
7.5
40
37.5


Compound 2.17 alone
250
0

0

0

2

10

10



62.5
0

0

0

0

0

0



15.625
0

0

0

0

0

0


Compound 2.17 + 2 g/ha
250
2
1
1
0
0
0
10
8
40
27.75
60
47.75


compound A22
62.5
0
−1
0
−1
0
0
0
0
2
−0.5
2
−0.5



15.625
0
−1
0
−1
0
0
0
0
1
−1.5
1
−1.5


Compound 2.16 alone
250
0

0

40

50

45

60



62.5
0

0

0

5

10

15



15.625
0

0

0

0

0

0


Compound 2.16 + 2 g/ha
250
2
1
10
9
40
0
35
−15
30
−16.375
50
−11


compound A22
62.5
0
4
15
14
0
0
2
−3
10
−2.25
20
2.875



15.625
5
−1
0
−1
1
1
0
0
5
2.5
25
22.5


Compound 2.28 alone
250
10

30

60

50

60

80



62.5
0

0

0

2

25

50



15.625
0

0

0

0

0

0


Compound 2.28 + 2 g/ha
250
20
9.1
30
−0.7
55
−5
60
10
70
9
97
16.5


compound A22
62.5
1
0
15
14
5
5
10
8
15
−11.875
20
−31.25



15.625
0
−1
0
−1
0
0
2
2
10
7.5
20
17.5


Compound 2.25 alone
250
2

5

10

20

15

30



62.5
1

0

0

2

1

0



15.625
0

0

0

0

0

0


Compound 2.25 + 2 g/ha
250
10
7.02
0
−5.95
10
0
40
20
15
−2.125
30
−1.75


compound A22
62.5
0
−1.99
0
−1
0
0
5
3
15
11.525
40
37.5



15.625
0
−1
0
−1
0
0
0
0
0
−2.5
0
−2.5


Compound 2.20 alone
250
5

20

30

60

65

90



62.5
0

0

2

10

20

15



15.625
0

0

0

0

0

1


Compound 2.20 + 2 g/ha
250
5
−0.95
10
−10.8
50
20
60
0
45
−20.875
85
−5.25


compound A22
62.5
5
4
2
1
0
−2
20
10
50
28
85
67.875



15.625
0
−1
10
9
0
0
2
2
10
7.5
20
16.525


Compound 6.1 alone
250
0

2

40

10

20

60



62.5
0

0

0

0

2

1



15.625
0

0

0

0

0

0


Compound 6.1 + 2 g/ha
250
80
79
80
77.02
80
40
97
87
98
76
85
24


compound A22
62.5
10
9
40
39
20
20
10
10
15
10.55
30
26.525



15.625
0
−1
0
−1
0
0
20
20
30
27.5
50
47.5









The data provided in the above tables indicate that, in many cases, addition of even low (sub-lethal) levels of mesotrione improves weed control by HST inhibiting herbicides both pre and post emergence.

Claims
  • 1. A method of selectively controlling weeds at a locus comprising crop plants and weeds, wherein the method comprises application to the locus of a weed controlling amount of a pesticide composition comprising an homogentisate solanesyltransferase (HST) inhibiting herbicide and/or hydroxyphenyl pyruvate dioxygenase (HPPD) inhibiting herbicide, wherein the crop plants comprise at least one recombinant polynucleotide which comprises a region which encodes an HST.
  • 2. A method according to 1, wherein the crop plants contain an additional recombinant polynucleotide which comprises a region which encodes a hydroxyphenyl pyruvate dioxygenase (HPPD).
  • 3. A method of selectively controlling weeds at a locus comprising crop plants and weeds, wherein the method comprises application to the locus of a weed controlling amount of a pesticide composition comprising an homogentisate solanesyltransferase (HST) inhibiting herbicide, wherein the crop plants comprise at least one recombinant polynucleotide which comprises a region which encodes a HPPD enzyme.
  • 4. A method according to claim 1, wherein the pesticide composition comprises an HST-inhibiting herbicide and a hydroxyphenyl pyruvate dioxygenase (HPPD) inhibiting herbicide
  • 5. A method according to claim 1, wherein the HST inhibiting herbicide is selected from the group consisting of a compound of Formula (IIa),
  • 6. A method according to claim 1, wherein the HPPD-inhibiting herbicide is selected from the group consisting of mesotrione, sulcotrione, isoxaflutole, tembotrione, topramezone, benzofenap, pyrazolate, pyrazoxyfen, pyrasulfotole, ketospiradox or the free acid thereof, 4-hydroxy-3-[[2-(2-methoxyethoxy)methyl]-6-(trifluoromethyl)-3-pyridinyl]carbonyl]-bicyclo[3.2.1]oct-3-en-2-one, [2-chloro-3-(2-methoxyethoxy)-4-(methylsulfonyl)phenyl](1-ethyl-5-hydroxy-1H-pyrazol-4-yl)-methanone, α-(cyclopropylcarbonyl)-2-(methylsulfonyl)-β-oxo-4-(trifluoromethyl)-benzenepropanenitrile, and (2,3-dihydro-3,3,4-trimethyl-1,1-dioxidobenzo[b]thien-5-yl)(5-hydroxy-1-methyl-1H-pyrazol-4-yl)-methanone.
  • 7. A method according to claim 1, wherein the HST enzyme is derived from Arabidopsis thaliana, Glycine max, Oryza sativa or Chlamydomonas reinhardtii.
  • 8. A method according to claim 1, wherein the HST enzyme is selected from the group consisting of the HST enzymes depicted as SEQ ID NO. 1 to 10.
  • 9. A method according to claim 1 wherein the crop plant comprises a further recombinant polynucleotide encoding a further herbicide tolerance enzyme.
  • 10. A method according to claim 9, wherein the further herbicide tolerance enzyme is selected from the group consisting of, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), Glyphosate acetyl transferase (GAT), Cytochrome P450, phosphinothricin acetyltransferase (PAT), Acetolactate synthase (ALS), Protoporphyrinogen oxidase (PPGO), Phytoene desaturase (PD) and dicamba degrading enzymes.
  • 11. A method according to claim 1, wherein the pesticide composition comprises one or more additional herbicides.
  • 12. A method according to claim 11, wherein the one or more additional herbicides is selected from the group consisting of glyphosate (including agrochemically acceptable salts thereof); glufosinate (including agrochemically acceptable salts thereof); chloroacetanilides e.g alachlor, acetochlor, metolachlor, S-metholachlor; photo system II inhibitors e.g triazines such as ametryn, atrazine, cyanazine and terbuthylazine, triazinones such as hexazinone and metribuzin, and ureas such as chlorotoluron, diuron, isoproturon, linuron and terbuthiuron; ALS-inhibitors e.g sulfonyl ureas such as amidosulfuron, chlorsulfuron, flupyrsulfuron, halosulfuron, nicosulfuron, primisulfuron, prosulfuron, rimsulfuron, triasulfuron, trifloxysulfuron and tritosulfuron; diphenyl ethers e.g acifluorofen and fomesafen.
  • 13. A method according to claim 12, wherein the one or more additional herbicides is glyphosate.
  • 14. A method according to claim 1, further comprising application to the locus of an insecticide and/or a fungicide.
  • 15. A recombinant polynucleotide which comprises a region which encodes an HST-enzyme operably linked to a plant operable promoter, wherein the region which encodes an HST-enzyme does not include the polynucleotide sequence depicted in SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 14 or SEQ ID NO. 15.
  • 16. A recombinant polynucleotide according to claim 15, wherein the HST is selected from the group consisting of SEQ ID NO. 3, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO.10.
  • 17. A recombinant polynucleotide comprising (i) a region which encodes a HST enzyme operably linked to a plant operable promoter and (ii) at least one additional region, which encodes a herbicide tolerance enzyme selected from the group consisting of hydroxyphenyl pyruvate dioxygenase (HPPD), 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), Glyphosate acetyl transferase (GAT), Cytochrome P450, phosphinothricin acetyltransferase (PAT), Acetolactate synthase (ALS), Protoporphyrinogen oxidase (PPGO), Phytoene desaturase (PD) and dicamba degrading enzymes, operably linked to a plant operable promoter.
  • 18. A recombinant polynucleotide according to claim 17, comprising (i) a region which encodes a HST enzyme and (ii) at least one additional region which encodes an HPPD.
  • 19. A recombinant polynucleotide according to claim 17, comprising at least two additional regions encoding a herbicide tolerance enzyme.
  • 20. A recombinant polynucleotide according to claim 19, wherein the polynucleotide comprises (i) a region which encodes a HST enzyme, (ii) a region which encodes a HPPD enzyme and (iii) a region which encodes a glyphosate tolerance enzyme.
  • 21. A vector comprising a recombinant polynucleotide according to claim 15.
  • 22. A plant cell which is tolerant to an HST-inhibiting herbicide and/or an HPPD-inhibiting herbicide—said plant cell comprising a recombinant polynucleotide according to claim 15.
  • 23. A plant cell according to claim 22, wherein the plant is selected from corn, soybean, wheat, barley, sugar beet, rice and sugarcane.
  • 24. A HST-inhibitor and/or HPPD-inhibitor tolerant plant which comprises a plant cell according to claim 22.
  • 25. Seed, or material derived therefrom, comprising a plant cell according to claim 22.
  • 26. A method of providing a transgenic plant which is tolerant to HST-inhibiting and/or HPPD-inhibiting herbicides which comprises transformation of plant material with a recombinant polynucleotide which comprises a region which encodes an HST and, optionally, a region encoding a HPPD, selection of the transformed plant material using an HST-inhibitor and/or an HPPD inhibitor, and regeneration of that material into a morphological normal fertile plant.
  • 27. A method according to 26, wherein the recombinant polynucleotide further comprises a region encoding the target for a non-HST inhibitor herbicide and/or a region encoding a protein capable of conferring on plant material transformed with the region resistance to insects, fungi and/or nematodes.
  • 28. A morphologically normal fertile whole plant obtained by the method of claim 26.
  • 29. (canceled)
  • 30. (canceled)
  • 31. (canceled)
  • 32. (canceled)
  • 33. A synergistic herbicidal composition comprising a HPPD-inhibiting herbicide and a HST-inhibiting herbicide.
  • 34. A herbicidal composition according to claim 33, wherein the HPPD-inhibiting herbicide is selected from the group consisting of mesotrione, sulcotrione, isoxaflutole, tembotrione, tobramezone, benzofenap, pyrazolate, pyrazoxyfen, pyrasulfotole, ketosbiradox or the free acid thereof, 4-hydroxy-3-[[2-(2-methoxyethoxy)methyl]-6-(trifluoromethyl)-3-pyridinyl]carbonyl]-bicyclo[3.2.1]oct-3-en-2-one, [2-chloro-3-(2-methoxyethoxy)-4-(methylsulfonyl)phenyl](1-ethyl-5-hydroxy-1H-pyrazol-4-yl)-methanone, α-(cyclopropylcarbonyl)-2-(methylsulfonyl)-β-oxo-4-(trifluoromethyl)-benzenepropanenitrile, and (2,3-dihydro-3,3,4-trimethyl-1,1-dioxidobenzo[b]thien-5-yl)(5-hydroxy-1-methyl-1H-pyrazol-4-yl)-methanone.
  • 35. A synergistic herbicidal composition comprising a HPPD-inhibiting herbicide and a HST-inhibiting herbicide selected from the group consisting of a compound of formula (IIa), a compound of formula (IIb), a compound of formula (IIc), a compound of formula (IId), a compound of formula (IIe) and a compound of formula (IIf) each as defined in claim 5.
  • 36. A herbicide composition according to claim 33, wherein the molar ratio of the HPPD-inhibiting herbicide to the HST-inhibiting herbicide in the composition is from 100:1 to 1:100.
  • 37. A herbicide composition according to claim 36, wherein the molar ratio of the HPPD-inhibiting herbicide to the HST-inhibiting herbicide in the composition is from 1:1 to 1:20.
  • 38. A herbicidal composition according to claim 33, further comprising one or more additional pesticidal ingredient(s).
  • 39. A herbicidal composition according to claim 38, wherein the one or more additional pesticidal ingredient(s) comprises a herbicide.
  • 40. A herbicidal composition according to claim 39, wherein the additional herbicide is selected from the group consisting of glyphosate (including agrochemically acceptable salts thereof); glufosinate (including agrochemically acceptable salts thereof); chloroacetanilides e.g alachlor, acetochlor, metolachlor, S-metholachlor; photo system II inhibitors e.g triazines such as ametryn, atrazine, cyanazine and terbuthylazine, triazinones such as hexazinone and metribuzin, and ureas such as chlorotoluron, diuron, isoproturon, linuron and terbuthiuron; ALS-inhibitors e.g sulfonyl ureas such as amidosulfuron, chlorsulfuron, flupyrsulfuron, halosulfuron, nicosulfuron, primisulfuron, prosulfuron, rimsulfuron, triasulfuron, trifloxysulfuron and tritosulfuron; diphenyl ethers e.g acifluorofen and fomesafen.
  • 41. A herbicidal composition according to claim 40, wherein the additional is herbicide selected from the group consisting of glyphosate, glufosinate, atrazine and S-metolachlor.
  • 42. A method of selectively controlling weeds at a locus comprising crop plants and weeds comprising applying to the locus a weed controlling amount of a herbicidal composition according to claim 33.
  • 43. A method according to claim 42, wherein the HPPD-inhibiting herbicide present in the composition is applied to the locus at a rate which is normally sub-lethal to the weeds when the HPPD-inhibiting herbicide is applied alone.
  • 44. A method according to claim 43, wherein the HST-inhibiting herbicide is applied from 10 to 2000 g/ha and the HPPD-inhibiting herbicide is applied from 5 to 1000 g ai/ha.
  • 45. A method according to claim 42, wherein the crop plants comprise at least one recombinant polynucleotide which comprises a region which encodes a herbicide tolerance enzyme.
  • 46. A method according to claim 45, wherein the herbicide tolerance enzyme is selected from the group consisting of HST, HPPD, EPSPS, GAT, Cytochrome P450, PAT, ALS, PPGO, Phytoene desaturase (PD) and dicamba degrading enzymes.
  • 47. A method according to claim 42, wherein the crop plants are selected from the group consisting of corn, wheat, barley, rice, soybean, sugar beet and sugar cane.
  • 48. (canceled)
  • 49. A vector comprising a recombinant polynucleotide according to claim 17.
Priority Claims (1)
Number Date Country Kind
0816880.9 Sep 2008 GB national
PCT Information
Filing Document Filing Date Country Kind 371c Date
PCT/GB09/02188 9/14/2009 WO 00 3/15/2011