CA2808152C - Hppd variants and methods of use - Google Patents

Abstract

The present invention relates to an isolated nucleic acid comprising a nucleotide sequence encoding a mutated HPPD protein, wherein said mutated HPPD protein has HPPD activity, wherein in said mutated HPPD protein at least one amino acid has been replaced so that the resulting amino acid sequence comprises at least one amino acid selected from certain amino acids at specific positions important for conferring an increased HPPD inhibitor tolerance. The present invention also relates to proteins encoded by the nucleic acid of the invention, to chimeric genes, plant cells comprising the nucleic acid of the invention operably linked to a plant- expressible promoter and optionally a transcription termination and polyadenylation region, plants essentially consisting of the plant cells of the invention and methods of obtaining transgenic plants.

Description

HPPD variants and methods of use Description The present invention relates to an isolated nucleic acid comprising a nucleotide sequence encoding a mutated HPPD protein, wherein said mutated HPPD protein has HPPD activity, wherein in said mutated HPPD protein at least one amino acid has been replaced so that the resulting amino acid sequence comprises at least one amino acid selected from certain amino acids at specific positions important for conferring an increased HPPD inhibitor tolerance. The present invention also relates to proteins encoded by the nucleic acid of the invention, to chimeric genes, plant cells comprising the nucleic acid of the invention operably linked to a plant-expressible promoter and optionally a transcription termination and polyadenylation region, plants essentially consisting of the plant cells of the invention and methods of obtaining transgenic plants.
In this specification, a number of documents including patent applications and manufacturer's manuals are cited.
HPPD (hydroxyphenylpyruvate dioxygenase) proteins are enzymes which catalyse the reaction in which para-hydroxyphenylpyruvate (abbreviated herein as HPP), a tyrosine degradation product, is transformed into homogentisate (abbreviated herein as HG), the precursor in plants of tocopherol and plastoquinone (Crouch N.P.
et al.
(1997) Tetrahedron, 53, 20, 6993-7010, Fritze et al., (2004), Plant Physiology 134:1388-1400). Tocopherol acts as a membrane-associated antioxidant.
Plastoquinone, firstly acts as an electron carrier between PSII and the cytochrome , .

2 b6/f complex and secondly, is a redox cofactor for phytoene desaturase, which is involved in the biosynthesis of carotenoids.
Up to now, more than 700 nucleic acid sequences from various organisms present in NCB! database were annotated as coding for a putative protein having an HPPD
domain. But for most of these sequences, it has not been proven that the protein would have an HPPD enzymatic activity either in an in vitro assay or an in in planta approach, nor that such HPPD protein can confer herbicide tolerance to HPPD
inhibitor herbicides when expressed in a plant. Several HPPD proteins and their primary sequences have been described in the state of the art, in particular the HPPDs of bacteria such as Pseudomonas (Ruetschi et al, Eur. J. Biochem., 205, 459-466, 1992, WO 96/38567), of plants such as Arabidopsis (WO 96/38567, Genebank AF047834), carrot (WO 96/38567, Genebank 87257), Avena sativa (WO
02/046387), wheat (WO 02/046387), Brachiaria platyphylla (WO 02/046387), Cenchrus echinatus (WO 02/046387), Lolium rigidum (WO 02/046387), Festuca arundinacea (WO 02/046387), Setaria faberi (WO 02/046387), Eleusine indica (WO

02/046387), Sorghum (WO 02/046387), Coccicoides (Genebank COlTRP),of Coptis japonica ONO 06/132270), Chlamydomonas reinhardtii (ES 2275365), or of mammals such as mouse or pig.
Most plants synthesize tyrosine via arrogenate (Abou-Zeid et al. (1995), Applied Env Microb 41: 1298-1302; Bonner et al., (1995), Plant Cells Physiol. 36, 1013-1022;
Byng et al., (1981), Phytochemistry 6:1289-1292; Connely and Conn (1986), Z.
Naturforsch 41c: 69-78; Gaines et al., (1982), Plants 156: 233-240). In these plants, the HPP is derived only from the degradation of tyrosine. On the other hand, in organisms such as the yeast Sacharomyces cerevisiae or the bacterium Escherichia coil, HPP is a tyrosine precursor, and it is synthesized by the action of an enzyme, prephenate dehydrogenase (hereinafter referred to as PDH), which converts prephenate to HPP (Lingens et al., (1967) European J. Biochem 1:363-374;
Sampathkumar and Morrisson (1982), Bloch Biophys Acta 701: 204-211). In these organisms, the production of HPP is therefore directly connected to the aromatic

3 amino acid biosynthetic pathway (shikimate pathway), and not to the tyrosine degradation pathway.
Inhibition of HPPD leads to uncoupling of photosynthesis, deficiency in accessory light-harvesting pigments and, most importantly, to destruction of chlorophyll by UV-radiation and reactive oxygen species (bleaching) due to the lack of photo protection normally provided by carotenoids (Norris et al. (1995), Plant Cell 7: 2139-2149).
Bleaching of photosynthetically active tissues leads to growth inhibition and plant death.
At present, most commercially available HPPD inhibitor herbicides belong to one of these four chemical families:
1) the triketones, e.g. sulcotrione [i.e. 242-chloro-4-(methylsulfonyl)benzoy1]-1,3-cyclohexanedione], mesotrione [i.e.244-(methylsulfony1)-2-nitrobenzoy1]-1,3-cyclohexanedione]; tembotrione [i.e.242-chloro-4-(methylsulfony1)-3-[(2,2,2,-tri-fluoroethoxy)methyl] benzoyI]-1,3-cyclo-hexanedione]; tefuryltrione [i.e. 242-chloro-

4-(methylsulfony1)-3-[[(tetrahydro-2-furanyl)methoxy]methyl]benzoy1]-1,3 cyclohexaned ione]]; bicyclopyrone [i.e. 4-hydroxy-34[2-[(2-methoxyethoxy)methy1]-6-(trifluoromethyl)-3-pyridinyl]carbonyl]bicyclo[3.2.1]oct-3-en-2-one] ;
Benzobicyclon [i.e. 3-(2-chloro-4-mesylbenzoy1)-2-phenylthiobicyclo[3.2.1]oct-2-en-4-one]
2) The diketonitriles, e.g. 2-cyano-3-cyclopropy1-1-(2-methylsulphony1-4-trifluoromethylpheny1)-propane-1,3-dione and 2-cyano-1-[4-(methylsulphonyI)-2-trifluoromethylpheny1]-3-(1-methylcyclopropyl)propane-1,3-dione;
3) the isoxazoles, e.g. isoxaflutole [i.e.(5-cyclopropy1-4-isoxazoly0[2-(methylsulfony1)-4-(trifluoromethyl)phenyllmethanone]. In plants, the isoxaflutole is rapidly metabolized in DKN, a diketonitrile compound which exhibits the HPPD inhibitor property; and 4) the pyrazolinates, e.g. topramezone [i.e.[3-(4,5-dihydro-3-isoxazoly1)-2-methyl-4-(methylsulfonyl) phenylli5-hydroxy-1-methy1-1H-pyrazol-4-yl)methanone], and . v pyrasulfotole [(5-hydroxy-1,3-dimethylpyrazol-4-y1(2-mesy1-4-trifluaromethylphenyOmethanone]; pyrazofen [2-[4-(2,4-dichlorobenzoy1)-1,3-dimethylpyrazol-5-yloxy]acetophenone].
These HPPD-inhibiting herbicides can be used against grass and/or broad leaf weeds in crop plants that display metabolic tolerance, such as maize (Zea mays) in which they are rapidly degraded (Schulz et al., (1993). FEBS letters, 318, 162-166;
Mitchell et al., (2001) Pest Management Science, Vol 57, 120-128; Garcia et al., (2000) Biochem., 39, 7501-7507; Pa!lett et al., (2001) Pest Management Science, Vol 57, 133-142). In order to extend the scope of these HPPD-inhibiting herbicides, several efforts have been developed in order to confer to plants, particularly plants without or with an underperforming metabolic tolerance, a tolerance level acceptable under agronomic field conditions.
Besides the attempt of by-passing HPPD-mediated production of homogentisate (US
6,812,010), overexpressing the sensitive enzyme so as to produce quantities of the target enzyme in the plant which are sufficient in relation to the herbicide has been performed (W096/38567). Overexpression of HPPD resulted in better pre-emergence tolerance to the diketonitrile derivative (DKN) of isoxaflutole (IFT), but tolerance was not sufficient for tolerance to post-emergence treatment (Matringe et al., (2005), Pest Management Science 61: 269-276).
In WO 04/024928, the inventors have sought to increase the prenylquinone biosynthesis (e.g., synthesis of plastoquinones, tocopherols) in the cells of plants by increasing the flux of the HPP precursor into the cells of these plants. This has been done by connecting the synthesis of said precursor to the "shikimate" pathway by overexpression of a PDH enzyme. They have also noted that the transformation of plants with a gene encoding a PDH enzyme makes it possible to increase the tolerance of said plants to HPPD inhibitors.

Another strategy was to mutate the HPPD in order to obtain a target enzyme which, while retaining its properties of catalysing the transformation of HPP into homogentisate, is less sensitive to HPPD inhibitors than is the native HPPD
before mutation.

5 This strategy has been successfully applied for the production of plants tolerant to 2-cyano-3-cyclopropy1-1-(2-rnethylsulphony1-4-trifluoromethylpheny1)-propane-1,3-dione and to 2-cyano-144-(methylsulphony1)-2-trifluoromethylphenyl]-3-(1-methylcyclopropyl)propane-1,3-dione (EP496630), two HPPD-inhibiting herbicides belonging to the diketonitriles family (WO 99/24585). Pro215Leu, Gly336G1u, Gly33611e, and more particularly Gly336Trp (positions of the mutated amino acid are indicated with reference to the Pseudomonas HPPD) were identified as mutations which are responsible for an increased tolerance to pre-emergence treatment with these diketonitrile herbicides without causing an alteration of the activity of the enzyme.
More recently, introduction of a Pseudomonas HPPD gene into the plastid genome of tobacco and soybean has shown to be more effective than nuclear transformation, conferring even tolerance to post-emergence application of isoxaflutole (Dufourmantel et al., 2007, Plant Biotechnol J.5(1):118-33).
In the patent application WO 2009/144079, a nucleic acid sequence encoding a mutated hydroxyphenylpyruvate dioxygenase (HPPD) at position 336 of the Pseudomonas fluorescens HPPD protein and its use for obtaining plants which are tolerant to HPPD inhibitor herbicides is disclosed.
In WO 2002/046387, several domains of HPPD proteins originated from plants have been identified that may be relevant to confer tolerance to various HPPD
inhibitor herbicides but no in planta nor biochemical data have been shown to confirm the impact of the as described domain functions.
In WO 2008/150473, the combination of two distinct tolerance mechanisms ¨ a modified Avena sativa gene coding for a mutant HPPD enzyme and a CYP450

6 Maize monooxygenase (nsf1 gene) ¨ was exemplified in order to obtain an improved tolerance to HPPD inhibitor herbicides, but no data have been disclosed demonstrating the synergistic effects based on the combination of both proteins.
US 2010/0197503 suggests a number of mutations at different positions within or close to the active site of the HPPD taken from Avena sativa and examined some of them for their inhibition by certain HPPD inhibitors such as sulcotrione in vitro and in planta.
.. Despite these successes obtained for the development of plants showing tolerance to several HPPD inhibitors herbicides described above, it is still necessary to develop and/or improve the tolerance of plants to newer or to several different HPPD
inhibitors, particularly HPPD inhibitors belonging to the classes of the triketones (e.g.sulcotrione, mesotrione, tembotrione, benzobicyclon and bicyclopyrone) and the pyrazolinates (e.g., topramezone and pyrasulfotole).
Accordingly, the present invention relates to an isolated nucleic acid comprising a nucleotide sequence encoding a mutated HPPD protein, wherein said mutated HPPD protein has HPPD activity, wherein in said mutated HPPD protein at least one amino acid has been replaced so that the resulting amino acid sequence comprises at least one amino acid selected from:
a) Ala, Asp, Glu, Phe, Gly, Lys, Gin, Arg, Ser, Thr, Val, Tyr, Ile, Leu or Met at a position in an HPPD protein, said position corresponding to position 250 of the amino acid sequence of SEQ ID No. 2;
b) Ala, Asp, Glu, Ile, Lys, Leu, Asn, Pro, Arg,Ser Gly, His or Tyrõ at a position in an HPPD protein, said position corresponding to position 251 of the amino acid sequence of SEQ ID No. 2;
c) Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Gin, Arg, Ser, Val, Thr or Tyr at a position in an HPPD protein, said position corresponding to position 252 of the amino acid sequence of SEQ ID No.;

7 d) Ala, Phe, His, Gin, Val, Trp, lie, Leu or Met at a position in an HPPD
protein, said position corresponding to position 253 of the amino acid sequence of SEQ ID
No. 2;
e) Leu, Val, or Met at a position in an HPPD protein, said position corresponding to position 265 of the amino acid sequence of SEQ ID No. 2;
f) Leu, Gin, Arg, Val, Tyr, Ala, Ile, Lys or Met at a position in an HPPD
protein, said position corresponding to position 268 of the amino acid sequence of SEQ ID
No. 2;
g) Ala, Thr or Val at a position in an HPPD protein, said position corresponding to position 269 of the amino acid sequence of SEQ ID No. 2;
h) Ala, Ile, Asn, Pro, Thr or Val at a position in an HPPD protein, said position corresponding to position 280 of the amino acid sequence of SEQ ID No. 2;
i) Leu, Gln, Val, Ala, Phe, Gly, Met, Arg or Ser at a position in an HPPD
protein, said position corresponding to position 293 of the amino acid sequence of SEQ ID
No. 2;
j) Ile, Met, Ala, Pro, Ser, Thr or Val at a position in an HPPD protein, said position corresponding to position 294 of the amino acid sequence of SEQ ID No. 2;
k) Gin, His or Asn at a position in an HPPD protein, said position corresponding to position 307 of the amino acid sequence of SEQ ID No. 2;
I) lie, Met, Asn or Leu at a position in an HPPD protein, said position corresponding to position 335 of the amino acid sequence of SEQ ID No. 2;
m) Leu or Met at a position in an HPPD protein, said position corresponding to position 368 of the amino acid sequence of SEQ ID No. 2;
n) Phe or Tyr at a position in an HPPD protein, said position corresponding to position 381 of the amino acid sequence of SEQ ID No. 2;
o) Phe or Ser at a position in an HPPD protein, said position corresponding to position 392 of the amino acid sequence of SEQ ID No. 2;
p) Phe or Tyr at a position in an HPPD protein, said position corresponding to position 419 of the amino acid sequence of SEQ ID No. 2;
q) Asp, Lys, Asn, Ala, Cys, Glu, Gly, His, Ile, Leu, Met, Gin, Ser, Thr or Val at a position in an HPPD protein, said position corresponding to position 421 of the amino acid sequence of SEQ ID No. 2;
r) Ala, Gly, Met, Pro or Thr at a position in an HPPD protein, said position corresponding to position 422 of the amino acid sequence of SEQ ID No. 2;

8 s) Ala, Phe, Ile or Val at a position in an HPPD protein, said position corresponding to position 424 of the amino acid sequence of SEQ ID No. 2;
t) Ile, Pro, Arg, Ser Ala, Gly, Lys, Asn or Gin at a position in an HPPD
protein, said position corresponding to position 425 of the amino acid sequence of SEQ ID
No. 2;
u) Glu, Phe, Thr, Val Ala, Gin or Ser at a position in an HPPD protein, said position corresponding to position 426 of the amino acid sequence of SEQ ID No. 2;
v) Ile, Met, Gln, Val Phe or Leu at a position in an HPPD protein, said position corresponding to position 431 of the amino acid sequence of SEQ ID No. 2; and w) at least one amino acid deletion or replacement at any one of positions 228, 248, 270, 271, 379 and/or 427.
Unless indicated otherwise, the specific definitions or specific features of certain embodiments can be introduced into any other embodiment of the present invention.
According to the present invention, a "nucleic acid" is understood as being a nucleotide sequence which can be of the DNA or RNA type, preferably of the DNA

type, and in particular double-stranded, whether it be of natural or synthetic origin, in particular a DNA sequence in which the codons which encode the HPPD according to the invention have been optimized in accordance with the host organism in which it is to be expressed (e.g., by replacing codons with those codons more preferred or most preferred in codon usage tables of such host organism or the group to which such host organism belongs, compared to the original or source organism).
An "isolated nucleic acid/DNA/protein", as used in the present application, refers to a nucleic acid/DNA/protein which is not naturally-occurring (such as an artificial or synthetic DNA with a different nucleotide sequence than the naturally-occurring DNA, or a modified protein) or which is no longer in the natural environment wherein it was originally present, e.g., a DNA coding sequence associated with a heterologous regulatory element (such as a bacterial coding sequence operably-linked to a plant-expressible promoter) in a chimeric gene, a DNA transferred into another host cell, such as a transgenic plant cell.

9 The terminology relating to nucleic acid or protein "comprising" a certain nucleotide sequence or amino acid sequence, as used throughout the text, refers to a nucleic acid or protein including or containing at least the described sequence, so that other nucleotide or amino acid sequences can be included at the 5' (or N-terminal) and/or 3' (or C-terminal) end, e.g. (the nucleotide sequence of) a selectable marker protein, (the nucleotide sequence of) a transit peptide, and/or a 5' leader sequence or a 3' trailer sequence. Similarly, use of the term "comprise", "comprising" or "comprises"
throughout the text and the claims of this application should be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. The term "comprising" also includes the term "consisting of".
In accordance with the present invention, the term "mutated HPPD protein"
which is interchangeably used with the term "mutant HPPD protein" denotes an HPPD
protein having an amino acid sequence which does not occur in nature. As opposed to the term "isolated" referred to above, the term "mutated" cannot refer to the environment of the (amino acid or protein) sequence in question, such as it being isolated from its natural environment or being coupled to a heterologous (amino acid or protein) sequence, but only refers to the amino acid sequence defining said mutated HPPD
protein which cannot be found anywhere in nature but arose from a unmutated or wild-type starting amino acid sequence. In other words, in arriving at the nucleic acid of the present invention encoding a mutated HPPD protein, a starting amino acid sequence of a naturally existing protein has to be taken and to be modified by man by replacing at least one amino acid as defined in the present application.
The sequence which encodes an original unmutated HPPD which will be mutated according to the invention can be of any origin. In particular, it can be of bacterial, plant or animal origin. Advantageous examples which may be cited are bacteria of the Pseudomonas sp. type, for example Pseudomonas fluorescens, or otherwise cyanobacteria of the Synechocystis genus. The sequence can also be of plant origin, in particular derived from dicotyledonous plants, umbelliferous plants, or otherwise monocotyledonous plants. Advantageous examples which may be cited are plants . .
such as tobacco, Arabidopsis, Daucus carotta, Zea mays (corn), wheat, barley, Avena sativa, wheat, Brachiaria platyphylla, Cenchrus echinatus, Lolium rigidum, Festuca arundinacea, Setaria faberi, Eleusine indica, and Sorghum. The coding sequences, and the way of isolating and cloning them, are described in the 5 previously cited references. In a particular embodiment of the invention, the HPPD is from a bacterial origin, particularly from Pseudomonas sp., more particularly from Pseudomonas fluorescens, Rhodococcus sp., Blepharisma japonicum, Synechococcus sp., Picrophilus torrid us, Kordia algicida or from a plant origin, particularly from Arabidopsis thaliana or Avena sativa. The HPPD to make the

10 mutation (s) in for the purpose of the invention, can be any naturally-occurring HPPD, or any active fragment thereof or any variant thereof wherein some amino acids (1 to 10 amino acids) have been replaced, added or deleted for cloning purposes, to make a transit peptide fusion, and the like, which retains HPPD activity, i.e. the property of catalysing the conversion of para-hydroxyphenylpyruvate to homogentisate.
The mutated HPPD protein according to the present invention has HPPD activity, i.
e., as described above, catalyses the reaction in which para-hydroxyphenylpyruvate is transformed into homogentisate. Preferentially, the catalytic activity of the isolated mutated HPPD of the present invention, when tested in vitro, does not differ from that of the unmutated reference HPPD protein by more than 70%, preferably more than 50%, more preferably more than 30%, even more preferably more than 20%
when assayed under identical conditions and in the absence of the HPPD
inhibitor herbicides described above. The catalytic activity of an HPPD enzyme may be defined by various methods well-known in the art. WO 2009/144079 describes various suitable screening methods.
Initial screens may be performed with the nucleic acid encoding the mutated HPPD
protein of the invention being expressed in bacteria.
Colorimetric Screening Test for Active HPPD Enzymes:
A YT-broth-type culture medium with 1% agarose, 5mM L-Tyrosine and 42mM
Succinate, which contains the selection agent for the vector pSE420 (Invitrogen,

11 Karlsruhe, Germany) is poured into deep well plates. E.coli culture in the exponentional growth phase which contains the vector pSE420-HPPDx (HPPDx means any gene coding for a putative HPPD enzyme/protein) is applied to each well.
After 16 hours at 37 C, the wells which do not contain the culture medium, those which have been seeded with an E. coil culture containing the empty vector pSE420 are transparent, or those which have been seeded with an E. coli culture containing a vector pSE420-HPPDx containing a gene coding for an inactive HPPD are transparent, while the weels seeded with an E. coli culture containing the vector pSE420-HPPDx coding for an active HPPD are brown. It has been previously demonstrated that this test refelects the HPPD activity, whatever the orgin of this activity is, and allows the identification of HPPD activities (US 6,768,044), i.e. at a qualitative level.
Further and more elaborate screens may be carried out in plant cells or plants expressing the mutated HPPD protein of the invention.
The same screenings may also be used when examining of whether a mutated HPPD protein is capable of modulating, such as decreasing or increasing, the tolerance of a plant to at least one HPPD herbicide inhibitor which will be referred to further below, with the difference that at least one of such an HPPD inhibitor is added. Examples of HPPD inhibitors to be used in those screenings include tembotrione, mesotrione, pyrasulfotole, bicyclopyrone, topramezone and sulcotrione.
A screening method which is simple to implement is to determine the dose of HPPD
inhibitor which fully inhibits the original unmutated HPPD, and which is lethal for the cells which express this unmutated HPPD, and to subject the mutated cells to this predetermined dose, and thereafter to isolate the mutated cells which have withstood this lethal dose, and then to isolate and to clone the gene which encodes the mutated HPPD.
Alternatively, at the quantitative level data like p150 (1)150-value means the log value of the concentration of inhibitor necessary to inhibit 50% of the enzyme activity in molar concentration) can be obtained by employing the isolated and purified HPPD

12 polypeptide, i.e. the mutated vs. the unmutated HPPD polypeptide and in presence or absence of the any respective HPPD inhibitor herbicide.
The terms "tolerance", "tolerant" or "less sensitive" denotes the lack of susceptibility of a plant expressing the mutated HPPD protein of the present invention to substances, particularly herbicides, which inhibit HPPD proteins, optionally in comparison with the plant's own HPPD protein or with any known HPPD protein.
More specifically, said terms mean the relative levels of inherent tolerance of the HPPD screened according to a visible indicator phenotype of the strain or plant transformed with a nucleic acid comprising the gene coding for the respective HPPD
protein in the presence of different concentrations of the various HPPD
inhibitors.
Dose responses and relative shifts in dose responses associated with these indicator phenotypes (formation of brown colour, growth inhibition, bleaching, herbicidal effect etc) are conveniently expressed in terms, for example, of GR50 (concentration for 50% reduction of growth) or MIC (minimum inhibitory concentration) values where increases in values correspond to increases in inherent tolerance of the expressed HPPD, 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 values correspond to increased levels of inherent tolerance of the expressed HPPD.
Herbicides can suitably be applied pre-emergence or post emergence.
Likewise, tolerance level of the nucleic acid or gene encoding an HPPD protein according to the invention, or the mutated HPPD protein of the invention is screened via transgenesis, regeneration, breeding and spray testing of a test plant such as tobacco, or a crop plant such as soybean or cotton. In line with the results obtained by such screening, such plants are at least 2-4 times more tolerant to HPPD
inhibitors like tembotrione, mesotrione, diketonitrile, and/or bicyclopyrone, pyrasulfotole, than plants that do not contain any exogenous gene encoding an HPPD protein, or than plants that contain a gene comprising an Arabidopsis thaliana

13 HPPD-encoding DNA, under control of the same promoter as the nucleic acid encoding the mutated HPPD protein of the invention. Accordingly, the term "capable of increasing the tolerance of a plant to at least one herbicide acting on HPPD"
denotes a tolerance increased in a plant by at least the factor of 2, alternatively at least the factor of 3 or 4 or even 5 or 6 as compared to a plant only expressing it's endogenous HPPD or a plant expressing an Arabidopsis thaliana HPPD. In this regard, the term "herbicide acting on HPPD" is not limited to substances which are known and/or used as herbicides but to any substances which inhibits the catalytic activity of HPPD proteins.
In an alternative embodiment of the nucleic acid encoding a mutated HPPD
polypeptide comprising at least one of the mutations as defined above, the HPPD
protein comprises a) a His at a position in an HPPD protein, said position corresponding to position 226 of the amino acid sequence of SEQ ID No. 2;
b) a Ser at a position in an HPPD protein, said position corresponding to position 267 of the amino acid sequence of SEQ ID No. 2;
c) an Asn at a position in an HPPD protein, said position corresponding to position 282 of the amino acid sequence of SEQ ID No. 2;
d) a His at a position in an HPPD protein, said position corresponding to position 308 of the amino acid sequence of SEQ ID No. 2;
e) a Tyr at a position in an HPPD protein, said position corresponding to position 342 of the amino acid sequence of SEQ ID No. 2;
f) a Glu at a position in an HPPD protein, said position corresponding to position 394 of the amino acid sequence of SEQ ID No. 2;
g) a Gly at a position in an HPPD protein, said position corresponding to position 420 of the amino acid sequence of SEQ ID No. 2; and h) an Asn at a position in an HPPD protein, said position corresponding to position 423 of the amino acid sequence of SEQ ID No. 2 In the mutated HPPD protein encoded by the nucleic acid of the invention at least one amino acid has been deleted or replaced as defined above.

14 The replacement or deletion can be effected in the nucleic acid sequence which encodes the original unmutated, i. e. naturally occurring HPPD as defined above by any means which is appropriate for replacing, in the said sequence, the codon which encodes the amino acid to be replaced with the codon which corresponds to the amino acid which is to replace it, or by deleting a codon, with the said codons being widely described in the literature and well known to the skilled person.
Several molecular biological methods can be used to achieve this replacement or deletion. A preferred method for preparing a mutated nucleic acid sequence according to the invention and the corresponding protein comprises carrying out site-directed mutagenesis on codons encoding one or more amino acids which are selected in advance. The methods for obtaining these site-directed mutations are well known to the skilled person and widely described in the literature (in particular:
Directed Mutagenesis: A Practical Approach, 1991, Edited by M.J. McPHERSON, IRL PRESS), or are methods for which it is possible to employ commercial kits (for example the U. S. E. mutagenesis kit from PHARMACIA). After the site-directed mutagenesis, it is useful to select the cells which contain a mutated HPPD
which is less sensitive to an HPPD inhibitor by using an appropriate screening aid.
Appropriate screening methods to achieve this have been described above.
In accordance with the present invention, the term "said position corresponding to position X", X being any number to be found in the respective context in the present application, does not only include the respective position in the SEQ ID No.
referred to afterwards but also includes any sequence encoding an HPPD protein, where, after alignment with the reference SEQ ID No., the respective position might have a different number but corresponds to that indicated for the reference SEQ ID
No.
Whereas HPPD sequences may be very diverse and may only show a low sequence identity of about 30%, HPPD proteins are characterized by a common three dimensional consensus structure which is achieved despite a low sequence identity.
Due to specific positions being conserved within HPPD proteins, alignment of HPPD
proteins can be effected by by applying various alignment tools in a senseful manner.

. .
Methods of aligning nucleic acid or amino acid sequences and, accordingly, determining the sequence identity of two or more sequences, are well-known in the art. They include performing mathematical algorithms such as the algorithm of Myers and Miller (1988) CABIOS 4:11-17 or the local alignment algorithm of Smith and 5 Waterman (1981) Adv. Appl. Math. 2:482-489; the global alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci USA 872264 and that of Brutlag et al.
(Comp.
App. Biosci. 6:237-245 (1990 )).
Such algorithms can be implemented in computer programs including but not limited 10 to CLUSTALX, ALIGN, GAP, BESTFIT, BLAST, FASTDB and FASTA.
For example, when using BESTFIT (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711)or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a

15 reference sequence, the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.
The identity between a first sequence and a second sequence, also referred to as a global sequence alignment, is determined using the FASTDB computer program based on the algorithm of Brutlag and colleagues (Comp. App. Biosci. 6:237-245 (1990)). In a sequence alignment the query and subject sequences are both DNA
sequences. The result of said global sequence alignment is in percent identity.
Preferred parameters used in a FASTDB alignment of DNA sequences to calculate percent identity are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30, Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or the length of the subject nucleotide sequence, whichever is shorter.
The present invention is based on the results of a combination of a comparison of the amino acid sequences of HPPD proteins from various organisms and the analysis of the substrate binding and inhibitor binding site of selected HPPD
proteins

16 using X-ray crystallography. Using this combined approach, it was possible to determine key positions in HPPD proteins, where an amino acid can be replaced with one of a defined set of other amino acids in order to modulate HPPD
catalytic activity and the affinity to at least one HPPD herbicide inhibitor to a plant expressing the mutated HPPD protein.
Superposition of the 3D structure of HPPD from Arabidopsis thaliana (1TFZ) (Yang et al., 2004, Biochemistry 43, 10414-10423) with the 3D structures of HPPD
from other species such as Pseudomonas fluorescens (1CJX) (Serre et al., 1999, Structure Fold Des. 7, 977-988), Streptomyces avermitilis (1147) (Brownlee et at., 2004, Biochemistry 43, 6370-6377), Homo sapiens (3ISQ) (PDB ID: 31sq Pilka et al, Structural Genomics Consortium (SGC). Crystal structure of human 4-Hydroxyphenylpyruvate dioxygenase), Rattus norvegicus (1SQ1) (Yang et al., 2004, Biochemistry 43, 10414-10423) shows that they have the same folding and corresponding amino acids are at equivalent position in the 3D structure of the protein. Since the species with known 3D structures are very diverse in their amino acid sequence, it can be assumed that all HPPD sequences have the same basic folding even though the overall sequence identity is low. The sequence and the structure of Arabidopsis thaliana has been used as reference structure in the present invention. Figure 1 shows the superposition of the structure of A.thaliana HPPD with the structure of (a) Pseudomonas fluorescens, (b) Streptomyces avermitilis, (c) Homo sapiens and (d) Rattus norvegicus. In order to define the binding site of the substrate and/or inhibitors, amino acids were selected which play a role in catalysis or inhibitor binding. This includes amino acids in the active site and amino acids from the C-terminal helix. The 3D arrangement is demonstrated in Figure 2 which displays the amino acids defined as binding site in case of (a) Arabidopsis thaliana, (b) Pseudomonas fluorescens, (c) Streptomyces avermitilis, (d) Homo sapiens and (e) Rattus norvegicus. The amino acid numbering of the Pseudomonas Fluorescens structure (1cjx) was changed into the numbering according to SEQ ID No. 10.
The 36 amino acids defining the binding site including their position are listed in Table 1 for (a) Arabidopsis thaliana, (b) Pseudomonas fluorescens, (c) Streptomyces avermitilis, (d) Homo sapiens and (e) Rattus norvegicus.

, .

17 Table 1: Amino acids forming the binding site in A.thaliana, P.fluorescens, S.avermitilis, H.sapiens, R. norvegicus Arabidopsis Pseudomonas Streptomyces Homo Rattus thaliana fluorescens avermitilis sapiens norvegicus Amino Amino Amino Amino Amino Position Acid Position Acid Position Acid Position Acid Position Acid

18 An exemplary alignment of HPPD proteins is given in Table 2a for the HPPD
proteins with known 3D structures. Table 2a gives the numbering of the amino acids of the Arabidopsis sequence and also the amino acids which are common within these HPPD sequences, with these amino acids being designated by an asterisk.
On the basis of such an alignment and from the definition of the Arabidopsis amino acid by its position and its nature, it is easy to identify the position of the corresponding amino acid in another HPPD sequence. Figure 2 shows that this can be done with the alignment of sequences of different plant, mammalian and bacterial origin, demonstrating that this method of alignment, which is well known to a skilled person, can be generalized to any other sequence. An alignment of different HPPD
sequences is also described in Patent Application WO 97/49816.
Table 2a: Alignment of HPPD sequences with known crystal structures i.e.
A.thaliana, P.fluorescens, S.avermitilis, H.sapiens, R. norvegicus Pos. 1TFZ Pos. 1= Pos. 1T47 Pos. 3ISQ Pos. 1SQI Common A. thaliana P. fluorescens S. avermitilis H.
Sapiens R. norvegicus amino acid

19 Pos. 1TFZ Pos. 1C,1X Pos. 1T47 Pos. 31SQ Pos. 1SQ1 Common A. thaliana P. fluorescens S. avermitilis H. Sapiens R. norvegicus amino acid , , 19 A - 6.1-1 8 G 8 G

20 S - 7 H 9 A 9 P

21 S - 8 T 10 K 10 K

22 P - 9 P 11 P 11 P

23 G - - -_

24 F - - -

25 K - - -

26 L - - -

27 V - - -_

28 G - _ ...
, 29 F - - -_ _ , _ _ , 39 S6 E'15 A 17 L 17 L

_ Pos. 1TFZ Pos. 1 CJX Pos. 1T47 Pos. 3ISQ
Pos. 1SQI Common A. thaliana P. fluorescens S. avermitilis H. Sapiens R. norvegicus amino acid 52 F 19 F 28 F 24 F 24 F x Pos, 1TFZ Pos. 1CJK Pos. 1T47 Pos. 3ISQ Pos. 1SQI Common A. thaliana P. fluorescens S. avermitilis H. Sapiens R. norvegicus amino acid . , Pos. 1TFZ Pos. 1CJX Pos. 1T47 Pos. 3ISQ Pos. 1SQI Common A. thaliana P. fluorescens S. avermitilis H. Sapiens R. norvegicus amino acid 103 ' Y 63 P 79 I 75 L ' 75 L

108 S 68 S 84 P 77 P 77 ' P
109 A - ' 85 W 78 W 78 W

111 ' E - 87 ' H ' 80 K 80 K ' 112 I - ' 88 F 81 E 81 E

114 P - 90 A 83 G 83 ' G ' 116 T - . . , I
-, 120 I - - - , , , , 130 S ' 69 Y 91 D 84 D 84 D
131 F 70 F 92 H 85 H ' 85 H

. .

Pos. 1TFZ Pos. 1 C,IX Pos. 1T47 Pos. 31S0 Pos. 1SQ1 Common A. thaliana P. fluorescens S. avermitilis H. Sapiens R. norvegicus amino acid 136 G 7= 5 G 97 G 90 G 90 G X

140 R 7= 9 C 101 V 94 K 94 K

145 E 8= 4 R 106 E 99 E 99 E

. .

Pos. 1TFZ, Pos. 1CJX Pos. 1147 Pos. 3ISQ Pos. 1SQ1 Common A. thaliana P. fluorescens S. avermitilis H. Sapiens R. norvegicus amino acid - 1= 61 - A 100 A 1= 22 A 115 A 115 A

164 S - 1= 25 - V 118 M 118 V

- 1= 66 - P - 1= 27 E 120 E 120 E

169 V 104 H 130 E 123 ¨ V 123 V

180 K 115 " P 141 A 134 A 134 A

1= 82 Y 117 I 143 I 136 L 136 L

- 1= 84 D 119 G 145 T 138 T 138 T

= , Pos. 1TFZ Pos. 1CJX Pos. 1T47 Pos. 31SQ Pos. 1SQ1 Common A. thaliana P. fluorescens S. avermitilis H. Sapiens R. norvegicus amino acid 202 F 138 Y 163 Y 1= 56 F 156 F

207 E 143 V 168 V 1= 61 E 161 E

209 V 145 L 170 A 1= 63 P 163 P
210 E 146 E 171 A 1= 64 A 164 T

Y

. , Pos. 1TFZ Pos. 1CJX Pos. 1T47 Pos. 3ISQ Pos. 1SQ1 Common A. thaliana P. fluorescens S. avermitilis H. Sapiens R. norvegicus amino acid 225 D 161 D r 186 D 182 D 182 D x 226 H 162 H 187 H 183 H 183 H x 228 V 164 T 189 V 1= 85 V 185 V

230 N 166 N 191 N 187 N 187 N x - 169 R 194 L " 190 D 190 D

233 E 171 R 196 R 1= 92 E 192 E

236 P 174 Y 199 E 1= 95 S 195 S

241 V 179 Y 204 Y 2= 00 Y 200 Y

=

Pos, 1TFZ Pos. 1CJX Pos. 1T47 Pos. 3ISQ Pos. 1SQI Common A. thaliana P. fluorescens S. avermitilis H. Sapiens R. norvegicus amino acid 247 F 185 F 210 F 206 F 206 F x 257 D 195 G 220 D 216 Q 2= 16 Q

262 E 197 Y 225 Y 221 Y 2= 21 Y
263 S 198 T 226 S 222 S 2= 22 S

265 L 200 L 228 L 224 L 224 L x 267 S 202 S 230 S 226 S 226 S x , Pos. 1TFZ Pos. 1CJX Pos. 1T47 Pos. 3ISQ Pos. 1SQ1 Common A. thaliana P. fluorescens S. avermitilis H.
Sapiens R. norvegicus amino acid 271 A 206 S 234 A 230 A 230 A x 280 P 215 P 243 P 239 P 239 P x 282 N 217 N 245 N 241 N 241 N x 283 E 218 E 246 E 242 E 242 E x 289 K 222 K 252 K 248 K 248 K x 293 Q 226 Q 255 Q 251 Q 251 Q x 294 I 227 I 256 I 252 I 252 I x . ,

29 Pos. 1TFZ Pos. 1C.JX Pos. 1T47 Pos. 31SQ Pos. 1SQ1 Common A. thaliana P. fluorescens S. avermitilis H. Sapiens R. norvegicus amino acid 303 3 236 G 265 G 261 G 261 G x 305 G 238 G 267 G 263 G 263 G x 307 Q 240 Q 269 Q 265 Q 265 Q x 308 H 241 H 270 H 2= 66 H 266 H x 310 A 243 A 272 A 268 A 268 A x 315 D 248 D 277 D 273 D 273 D x 318 R 251 K 280 E 2= 76 T 276 T
319 T 252 T 281 T 2= 77 A 277 T

Pos. 1TFZ Pos. 1 C.IX Pos. 1T47 Pos. 3ISQ Pos. 1SQI
Common A. thaliana P. fluorescens S. avermitilis H. Sapiens R. norvegicus amino acid 331 G 260 G 289 G 285 G 285 G x 332 F 261 M 290 V 286 L 2= 86 M

334 F 263 F 292 F 288 F 288 F x 338 P 267 P 296 P 292 P 292 P x 342 Y 271 Y 299 Y 295 Y 2= 95 Y x 343 Y 272 V 300 Y 296 Y 296 Y x 346 L 275 L 303 L 299 L 299 L x 355 S 284 E 312 V 308 I 3= 08 I
356 D 285 P 313 P 309 K 3= 09 Q

Pos. 1TFZ Pos. 1 CJX Pos. 1T47 Pos. 3ISQ Pos.
1SQI Common A. thaliana P. fluorescens S. avermitilis H. Sapiens R. norvegicus amino acid 359 I 290 Q - 3= 14 I 314 M

365 L 296 L 320 L 320 L 320 L x 377 L 308 L 332 L 32 L 332 L x 378 L 309 L 333 L 333 L 333 L x 379 Q 310 Q 334 Q 3= 34 Q 334 Q x 380 I 311 I 335 I 335 I 335 I x 381 F 312 F 336 F 336 F 336 F x 382 T 313 S 337 T 3= 37 T 337 T

Pos. I TFZ Pos. 1= Pos. 1147 Pos. 3ISQ Pos. 1 SQI Common A. thaliana P. fluorescens S. avermitilis H. Sapiens R. norvegicus amino acid 388 R - 343 R 343 R 3= 43 R

392 F 321 F 347 F 347 F 347 F x 393 I 322 F 348 F 348 L 3= 48 L
394 E 323 E 349 E 349 E 349 E x 396 I 325 I 351 I 351 I 351 I x 398 R 327 R 353 R 353 R 3= 53 R x = =

Pos. 1TFZ Pos. 1= Pos. 1T47 Pos. 3ISQ Pos. 1SQ I Common A thaliana P. fluorescens S. avermitilis H. Sapiens R. norvegicus amino acid 418 G 332 G 358 G 358 G 358 G x 419 F 333 F 359 F 359 F 359 F x 420 G 334 G - 360 G 360 G 360 G x 422 G 336 G 362 G 362 G 362 G x 423 N 337 N 363 N 363 N 363 N x 424 F 338 F 364 F 364 F 364 F x 426 E 340 .7- A - 366 A 366 S 366 S
427 - L 341 L 367 L 367 L 367 L x 428 F 342 F 368 F 368 F 368 F x 432 E 346 E 372 E 372 E 372 E x Pos. 1TFZ
Pos. 1CJX Pos. 1T47 Pos. 3ISQ Pos. 1SQI Common A. thaliana P. fluorescens S. avermitilis H.
Sapiens R. norvegicus amino acid A sequence analysis of more than 700 HPPD sequences from public data bases including sequences of HPPD proteins and predicted HPPD proteins such as from plants, mammals, fungi and bacteria was performed using ClustalX. The alignment was corrected using the information of the available 3D structures. Identical amino acid sequences with different identifiers were included only once and some sequences with obvious sequence errors were excluded. The alignment also includes incomplete sequences. Table 2b shows the sequence alignment for a representative set of HPPD proteins and includes sequences from plants, bacteria, mammals.

Table 2b: Alignment of a representative set of HPPD sequences CLUSTAL X (1.81) multiple sequence alignment RATTUS NORVEGICUS ---MTT ----------------------------------YSNKGPKPE
HOMO-SAPIENS ---------------------------------------------------------- GAKPE
XENOPUS-LAEVIS -MTS ----------------------------------YTDKGPKPD
BLEPHARISMA-JAPONICUS-----NT? ------------------------------------------YDKQETRPD

RTSPLYDSDGYVPAPAALVVGG
ASPERGILLUS-FUMIGATUS ---MAPSAISTS ----------------------------PPPTDRVSSS
MAGNAPORTHE-GRISEA ---MSPSAITESPRN -------------------------SVVDHTSGLQVDSLAVQGP
CANDIDA-ALBICANS
PICHIA-STIPITIS ----LLKELPFLPTSS ------------------------DPITEPDIDELLSDGHVNSKYP
15 RIODOCOCCUS-SP. ---MTIEQTLTD ----------------------------KERLAGLDLGQLEQLVGLVEYDGTRD
RHODOCOCCUS-ERYTHROPOLIS ---MTVEQTLTD ----------------------------DEQLAGLDLEQLRQLVGLVEYDSDHD
JANIBACTER-SP.-HTCC2649 MTDTTITNPTTDTASQPTPLDLTPUREANLNLEOLKOLVGLVEYDESKD
STREPTOMYCES-AVERMITILIS ---MTQTTHHTP ----------------------------DTARQAD

LQNTEYGLKKLFDEAED

SVDYGLEKIFDGAQD
GEMMATIMONAS-AURANTIACA ---MATLTT -------------------------------PEIGTEQD
PICROPHILUS-TORRIDUS --------------------------------------------------MYGKNLISELRE
ARABIDOPSIS-THALIANA --------------------------------------------------MGHQNAAvSENQNHDDGAAS--SPGFKLvGFSKFVRKNPKSD
BRASSICA-RAPA-SUBSP.-PEKINENSI ----------------------------------------MGHENAAVSENQHHDDAATTSASPGFKLVGFSKFVRKNPKSD
25 COPTIS-JAPONICA-VAR.-DISSECTA -------- MVPST -----------------------ASNLKLVGHTNFVHNNPKSD
HEVEA-BRASILIENSIS ------------------ MGKENDSVPSS ---------------------APGFKLLGFSNFVRTNPRSD
MEDICAGO-TRUNCATULA ----------------- MAIETETQTQT ---------------------QTGFKLVGEKNEVRANPKSD
GLYCINE-MAX ------------------------- MCNEIQAQAQAQ --------------------AQPGFKLVGFKNFVRTNPKSD
SOLANUM-LYCOPERSICUM ----MC--MGKETLS--TTDTTG -----------------ATFKLVGFNNFIRANPRSD

30 SOLANUM-TUBEROSUM ----MG--MGKETLSTTTTDETG -----------------ATFKLG-FNNFIRANPRSD
NICOTIANA-BENTHAMIANA ----MGKLETVTTTSATAADDSSEL ---------------TTNFKLVGFKNFIRTNPRSD
DAUCUS-CAROTA ----MGKKQSEAEILSSNSSNTSP ----------------ATFKLVGENNFVRANPKSD
SOLENOSTEMON-SCUTELLARIOIDES -MG --- QESTAAAAVVP ------------------AEFKLVGHKNFVRSNPMSD
HORDEUM-VULGARE --------------------- MPPTPTTPAAT- GAAAAVTPEHARP ------HRMVRFNPRSD

RRMVRFNPRSD
AVENA-SATIVA ----------------------------------------------------------MPPTPATATGA---AAAAVTPEHAARS---EPRVVRVNPRSD
ORYZA-SATIVA ----------------------------------------------------------MPPTPTPTATTGAVSAAAAAGENAGFRLVGHRRFVRANPRSD
SORGHUM-BICOLOR -------------------------------------------------------MPPTPTTAAAT-GAAVAAASAEQAAFRLVGHRNFVRVNPRSD
ZEA-MAYS --------------------------------------------------------------MPPTPTAAAAG-AAVAAASAAEQAAFRLVGHRNFVRFNPRSD

KRKLVGCANFVRSNPLSD
OTAURI ------------------------------ MTTSAS --------------------------GRKLVGHANFVRCNPLSD

AKRHKLVGCKNFVFENNPMD
SYNECHOCOCCUS-SP. -----------------------------------------------------MNPSIR
VIERIO-SP.-MED222 -----------------------------------------------------MVDTYN
MARINOMONAS-SP.-MED121 ---------------------------------------------MNTALKIDYSIN
PSEUDOMONAS-FLUORESCENS -----------------------------------------------ADLYEN
SULFITOBACTER-SP.-NAS-14.1 --------------------------------------------MGPFPHDAEKSKITDEN

MGPFPHDAPKSEITDEN
BDELLOVISRIO-BACTERIOVORUS --------------------------------------------MAQVTEKN
RATTUS NORVEGICUS
RGRELHFHSVTFWVGNAK--QAASFYCNKMGFEPLAYKGLETGSREVVSH
HOMO-SAPIENS
RGRELHFHSVTFWVGNAK--QAASFYCSKMGFEPLAYRGLETGSREVVSH
XENOPUS-LAEVIS
VGRYLAFDHITFYVGNAK--QAAAYYATRFGENPIAYRGLETGHRDVCTH
BLEPHARISMA-JAPONICUS
LGEFYGFHHVRFYVSNSE--QAASFYTSRFGFSPVAYEGLETGNQKFCTN
MYCOSPHAERELLA-GRAMINICOLA
EVNYRGYHHAEWWVGNAK--QVAQFYITRMGFEPVAHKGLETGSRFFASH
ASPERGILLUS-FUMIGATUS
LASYKGYDHVHWYVGNAK--QAASYYITRMGFKRIAYRGLETGCRSVCSH
MAGNAPORTHE-GRI SEA

CANDIDA-ALBICANS ------------------------------------------------------MSKYLDLAMGEKEVAYKGLETGSKLIGAH
PICHIA-STIPITIS
TDGFIKFFSLKICSSNAK--QMSKYLQLAMDFKEIAYKGLENDSRLVGAH
RHODOCOCCUS-SP.
PFPVSGWDAVVWVVGNAT--QTAHYFQSAFGMTLVAYSGETTGNRDHHSF
RHODOCOCCUS-ERYTHROPOLIS
PFPVSGWDGLEWIVCNAT--QTSHFFQSAFGMELVAYSGPSTGNRDHHAF
JANIBACTER-SP.-HTCC2649 PFPVTGWDAIVETVGNAT--QAAAYYQGTWGMELVGYSGPENGNRDHKAF
STREPTOMYCES-AVERMITILIS
PFPVKGMDAVVFAVGNAK--QAAHYYSTAFGMQLVAYSGPENGSRETASY

KORDIA-ALGICIDA
FLPLLGTDYVELYVGNAK- -QSAHFYKTAFGFQSEAYAGLETGLTDRVSY
LEEUWENHOEKIELLA-BLANDENSIS -ME FLPLLGTDYVEFYVGNAK--QAAHEYKTAFGFQSEAYSGLEIGKKDRVSY
GEMMATIMONAS-AURANTIACA
AFPINGTDYVEFYVGNAK--QASHYYRAAFGYSLVAYRGPETGVRDRASY
PICROPHILUS-TCRRIDUS
KEIFKRLHHVEFYVSSAK--TWSYFMNRGLGFKTVAYAGPETGIRDKISY
ARABIDOPSIS-THALIANA
KEKVKRFHHIEFWCGDAT--NVARRFSWGLGMRFSAKSDLSTGNMVHASY
BRASSICA-RAPA-SUBSP.-PEKINENSI KEKVKREHHIEFWCGDAT--NVARRFSWGLGMRFSAKSDLSTGNMVHASY
COPTIS-JAPONICA-VAR.-DISSECTA
KFHVKKEHHIEFWSTDAT--NTARRFSWGLGMPMVAKSDLSIGNMVHASY
HEVEA-BRASILIENSIS
LEKVKREHHVEFWCTDAT--NTACRFSWGLGMPFVAKSDLSTGNVTHASY
MEDICAGO-TRUNCATULA
RENVKREHHVEFWCIDAT--NTARRFSHGLGMPIVAKSDLSIGNLTHASY
GLYCINE-MAX
RFQVNRFHHIEFWCIDAT--NASRRFSWGLGMPIVAKSDLSTGNQIHASY
SOLANUM-LYCOPERSICUM
FFSVKREHHIEFWCGDAT--NTSRRFSWSLGMPITAKSDLSTGNSVHASY
SOLANUM-TUBEROSUM
FFSVKREHHIEFWCGDAT--NTSRRFSWSLGMPITAKSDLSIGNSVHASY

FFSVKREHHIEFWCGDAT- -NTSRRFSWSLGMPIAAKSDLSTGNSVHASY
DAUCUS-CAROTA
HFAVKREHKIEFWCGDAT--NTSRRFSWGLGMPLVAKSDLSIGNSVHASY
SOLENOSTEMON-SCUTELLARIOIDES HERVHRFEHVEFWCGDAT--NTSRRFSWGLGMPLVAKSOLSIGNSAHASY
HORDEUM-VULGARE
RFKILSEHHVEFWCADAA--SAAGRFAFALGAPLAARSDLSTGNSAHASQ
TRITICUM-AESTIVUM
REHTLSEHHVEFWCADAA--SAAGRFAFALGAPLAARSDLSTGNSVHASQ
AVENA-SATIVA
RFPVLSEHHVELWCADAA--SAAGRFSFALGAPLAARSDLSTGNSAHASL
ORYZA-SATIVA
RFQALAFHHVELWCADAA--SAAGRFAFALGAPLAARSDLSIGNSAHASL
SORGHUM-BICOLOR
RFHTLAFHHVELWCADAA--SAAGRFSEGLGAPLAARSDLSIGNTAHASL
ZEA-MAYS
RFHTLAFHHVELWCADAA--SAAGRFSEGLGAPLAARSDLSTGNSAHASL
AB095005_OLUCIMARINUS
AFECEKEDHIEFWCGDAT--NAAAREGVGLGMGLRCKSDATIGNGTYASY
OTAURI
AFECVGFDHVEFWCGDAT--NAASRFGVGLGMSLRAKSDASIGNGIYASY

LETMRKEHHVEFWCROAT--TTAARFAVGLGMQLVAKSDLTTGNARYASY
SYNECHOCOCCUS-SP. --IVQGIHHLHFYLWDLP--RWREHFCRVWGFRVASDAGN -- TL
VIBRIO-SP.-MED222 PLGTDGFEFVEYTAVDHKGIEQLKALLVSLGFAEIAKHRSKE --AW
MARINOMONAS-SP.-MED121 PLGTDGFEFVEYTAADEKGIADLKALFVSLGFTEVAKHRSKE --VW
PSEUDOMONAS-FLUORESCENS PMGLMGFEFIEFASPTPG-- -TLEPIFEIMGFTKVATHRSKN
VH
SULFITOBACTER-SP.-NAS-14.1 PAGTDGFEFVEFASADPQ---ELRDLETRMGYAHVANHKTQK --IE
OCEANICOLA-RATsENSIS-HTCC2597 PAGTDC,FERVEPARPDPQ---ELROLFSKMC,YELVGRHKSKD
VF.
BDELLOVIBRIO-BACTERIOVORUS PVGINGVDFIEYSGPDAH---FFEQVEKRYAFKEVGQVHGKN --IK
RATTUS NORVEGICUS VIKQGK --IVFVLCSALNPWN ------------------HOMO-SAPIENS VIKQGK--IVEVLSSALNPWN -------------------XENOPUS-LAEVIS VVRQNN - -ATFVFQSPLNPGN -----------------BLEPHARISMA-JAPONICUS VVRSNH--VVIAFTSALTPED -------------------MYCOSPHAERELLA-GRAMINICOLA VVQNNG--VRFVFTSPVRSSA -------------------ASPERGILLUS-FUMIGATUS VVRNGD --ITFILTSPLRS --------------------MAGNAPORTHE-GRTSEA LVGKED--VRFVFTSPIRSHVH ------------------CANDIDA-ALBICANS
VMQNGS--ITLEIINTLETIDDDNVLKFPFFQNDLNKFRNINHEYFLENF
PICHIA-STIPITIS
VIRNGD--VTLEIVNTLETVEDDNVLKEPYFEKDLKQFPQLNESKYLRDF
RHOD000CCUS-SP. VLESGA--VRFVIKGAVNPDSP ------------------RHODOCOCCUS-ERYTHROPOLIS VLKSGA--VREVVKGAVDPASP ------------------JANIBACTER-SP.-HTCC2649 VLKSGS --IRFVLKGAVSPDSP
STREPTOMYCES-AVERMITILIS VLTNGS --ARFVLTSVIKPATPW ----------------KORDIA-ALGICIDA VLKQDK--IRLVLTTPLGKGGE ------------------LEEUWENHOEKIELLA -BLANDENSIS -ME VLRQDK --IRLVLTSPLGSASP ----------------GEMMATIMONAS-AURANTIACA LMQQGK--IRLVLTTSITADTP - ----------------PICROPHILUS-TORRIDUS VMSQGT --ARISFTSSMNDDSY -----------------ARABIDOPSIS-THALIANA LLTSGD--LRFLFTAPYSP---SLSAGEI -----------BRASSICA-RAPA-SUBSP.-PEKINENSI LLTSGO--LRFLETAPYSP---SLSAGEN ---------COPTIS -JAPONICA -VAR. -DISSECTA LLRSGE --LNELFTAPYSP - - -SIAGNT ----HEVEA-BRASILIENSIS LLRSGD --LSELFTAPYSP---TIASMENF ---------MEDICAGO-TRUNCATULA LLRSGD- -LNFLFSAAYSP - --SISLSS ---------GLYCINE-MAX LLRSGD--LSFLFSAPYSP---SLSAGSS -----------SOLANUM-LYCOPERSICUM LLRSVSGELQFVFTAPYSP---SISVPS ------------SOLANUM-TUBEROSUM LLRSVSGELQFVFTAPYSP---SISVPS ------------NICOTIANA-BENTHAMIANA LLRPVSGSLQELFTAPYSP---SISTPS ------------DAUCUS-CAROTA LVRSAN- -LSFVFTAPYSP ---STITSSG ---------SOLENOSTEMON-SCUTELLARIOIDES LLRSGE--LSFVFTAPYSP---SLAEPS ------------HORDEUM-VULGARE LLRSGS- -LAFLETAPYAN -- GCDAA -----------TRITICUM-AESTIVUM LLRSGN--LAFLETAPYAN -- GCDAA ------------AVENA-SATIVA LLRSGA - -LAFLFTAPYAPPPQEAATAAA ---------ORYZA-SATIVA LLRSAS--VAELFTAPYGGDHGVGADAAT -----------=

SORGHUM-BICOLOR LLRSGA--LAFLETAPYAB -- GADAA ------------ZEA-MAYS LIRSGS--ESFLETAPYAH-----GADAA -----------A3095005_OLUCIMARINUS AMKSND--LTFVFTAPYG ----- VESGGSR --------OTAURI AMKSHD--LTFVFTAPYGDDERAVGCGGSS ----------5 mICROMONAS-PUSILLA-CcmP1545 vLQSND--LRFvFSAPYDVPEGEENDDARS
SYNECHOCOCCUS-SP. ELEQGS--LRLRLSQPARAG --------------------VIBRIO-SP.-MED222 LYRQGD--INFIVNEQPHS ---------------------MARINOMONAS-SP.-MED121 LYROND--INFIVNSEPNS ---------------------PSEUDOMONAS-FLUORESCENS LYRQGE--INLILNNEPNS ---------------------SULFITOBACTER-SP.-NAS-14.1 LWQQGD--ITYVLNADPDS --------------------BDELLOVIBRIO-BACTERIOVORUS LFRQGD--INFILNCEPHT ---------------------RATTUS NORVEGICUS
HOMO-SAPIENS
XENOPUS-LAEVIS
BLEPHARISMA-JAPONICUS
MYCOSPHAERELLA-GRAMINICOLA
ASPERGILLUS-FUMIGATUS
MAGNAPORTHE-GRISEA
CANDIDA-ALBICANS
KLTTDDLIFDFVNSRIESLSFKSDYLKFGKQFYNNIIRSDDYQESMAKVS
PICHIA-STIPITIS
KITTNDLVEDEVNSRIESFSVSPNABYFRRKLYNKIVSSRAFRNNMFDYN
RIODOCOcCUS-SP.
RHODOCOCCUS-ERYTHROPOLIS
JANIBACTER-SP.-HTCC2649 STREPTOMYCES-AVERMITILIS
KORDIA-ALGICIaA
LEEUWENBOEKIELLA-BLANDENSIS-ME -----------------------------------------GEMmATIMONAS-AURANTIAcA
PICROPHILUS-TORRIDUS
ARABIDOPSIS-THALIANA
BRASSICA-RAPA-SUBSP.-PEKINENSI -----------------------------------------COPTIS-JAPONICA-VAR.-DISSECTA
HEVEA-BRASILIENSIS
MEDICAGO-TRUNCATULA
GLYCINE-MAX
SOLANUM-LYCOPERSICUM
SOLANUM-TUBEROSUM
NICOTIANA-BENTHAMIANA
DAUCUS-CAROTA
SOLENOSTEMON-SCUTELLARIOIDES
HORDEUM-VULGARE
TRITICUM-AESTIVUM
AVENA-SATIVA
ORYZA-SATIVA
SORGHUM-BICOLOR
MA-MAYS
AB095005_OLUCIMARINUS
OTAURI

SYNECHOCOCCUS-SP.
VIBRIO-SP.-MED222 MARINOMONAS-SP.-MED121 PSEUDOMONAS-FLUORESCENS
SULFITOBACTER-SP.-NAS-14.1 BDELLOVIBRIO-BACTERIOVORUS
RATTUS NORVEGICUS ------------------------------------------------------KEMGDHLVKHGDGVKDIAFEVEDCEHIVQK
HOMO- SAPIENS ----------------------------------------------------------KEMGDHLVKHGDGVKDIAFEVEDCDYIVQK
KENOPUS-LAEVIS ---------------------------------------------------------HPISDHVAmHGDGVKDVAFSVEDCRGIYKR
BLEPHARISMA-JAPONICUS --------------------------------------------------NEVNRHVGKHSDGVODIAFSVSDARGMYEK
MYCOSPHAERELLA-GRAMINICOLA ------------------------------------------RQTLKAAPLADQARLDEMYDHLDKHGDGVKDVAFEVDDVLAVYEN

=

ASPERGILLUS-FUMIGATUS --------------------------------------------------LDQVDREPPEEQELLKEIHAHLEKHGDGVKDVAFEVDSVDSVEYA
MAGNAPORTHE-GRISEA -----------------------------------------------------LPEDEPISDEDRALLKEMHAHLEKHGDAVKDVCFEVDNVQGVYER
CANDIDA-ALBICANS
NEIVQTINNSEEIYNDMMECTLIQKFLKLHAEGVMDIAENVNDVDTIENR
PICHIA-STIPITIS
NLILNVINNSEVIYNDIMECTLIQKFLKTHGEGVMDISFLVEDVITIFDK
RHODOCOCCuS-5F. ------------------------------------------------------LIDHHRThGDGVVDIALAVPDVDKCIAE
RHODOCOCCUS-ERYTHROPOLIS -----------------------------------------------LIEHHSRHGDGIRDIALSVPDVDKCIAH
JANIBACTER-SP.-HTCC2649 ------------------------------------------------LIAHHTKHGDGVVDISLEVPDVDKCIAQ
STREPTOMYCES-AVERMITILIS -----------------------------------------------GHFLADHVAEHGDGVVDLAIEVPDARAAHAY
KORDIA-ALGICIDA --------------------------------------------------------INEHIDLHGDGVKVVALWVEDATKAFEE
LEEUWENHOEKIELLA -BLANDENSIS -ME ---------------------------------INEHIVKHGDGVKVVALWVEDARSAFEE
GEMMATIMONAS-AURANTIACA ------------------------------------------------IAEHVHRHGDOvRDYALWVDDARLAYET
PICROPHILUS-TORRIDUS ---------------------------------------------------ISNHVKKHGDGVKDIALEVDDLDEAKSL
ARABIDOPSIS-THALIANA ---------------------------------------------------KPTTTASIPSFDHGSCRSFFSSHGLGVRAVAIEVEDAESAFSI
BRASSICA-RAPA-SUBSP.-PEKINENSI -----------------------------------------PPITTASIPSFDHVTYRSFESSHGLGVRAVAVEVEDAEAAFSI
COPTIS-JAPONICA-VAR.-DISSECTA ---------------------------------------LTHTASIPTYSHNLARLFASTHGLAVRAIAIEVQDAELAYNI
BEVEA-BRASILIENSIS -----------------------------------------------------SHTATASIPTESHEACRNFSAKHGLGVRAIAlEVEDAEIAYNT
MEDICAGO-TRUNCATULA ----------------------------------------------------PSSTAAIPTESASTCFSFSASHGLAVRAVAVEVEDAEVAFTT
GLYCINE-MAX ------------------------------------------------------------AASSASIPSFDAATCLAFAAKHGEGVRAIALEVADAEAAFSA
SOLANUM-LYCOPERSICOM ---------------------------------------------------TAGIPSFSTPTYRDFTAKHGLGVRAVALEVENAYLAFSA
SOLANUM-TUBEROSUM ---------------------------------------------------TAGIPSFSTSTHRDFTAKHGLGVRAVALEVENAYLAFSA
NICOTIANA-BENTHAMIANA --------------------------------------------------SAAIPSFSTSTHRSFAATHGLGVRAVALEVENAYTAFSA
DAUCUS-CAROTA ----------------------------------------------------------SAAIPSFSASGFHSFAAKHGLAVRAIALEVADVAAAFEA
SOLENOSTEMON-SCUTELLARIOIDES -------------------------------------------SASIPTFSFSDBRAFTSSHGLAVRAVAIQVDSASSAYSA
HORDEUM-VULGARE --------------------------------------------------------TASLPSFSADAARRFSADHGIAVRSVALRVADAAEAFRA
TRITICUM-AESTIVUM ---------------------------------------------------TASLPSFSADAARRFSADHGLAVRSIALRVADAAEAFRA
AVENA-SATIVA -----------------------------------------------------------TASIPSFSADAARTFAAAHGLAVRSVGVRVADAAEAFRV
ORYZA-SATIVA -----------------------------------------------------------TASIPSFSPGAARRFAADHGLAVHAVALRVADAADAFRA
SORGHUM-BICOLOR --------------------------------------------------------TASLPSFSAAEARRFAADHGLAVRAVALRVADAEDAFRA
ZEA -MAYS --------------------------------------------------------------TAALPSFSAAAARRFAADHGLAVRAVALRVADAEDAFR---HEGRAMMRFFEKHGLAARAVGVRVKDARAAYEE
OTAURI -----------------------------------------------------------------VNVPHPG---NERGAMMRFFERHGLAARAVGLRVGDARAAYEE

SMFEKSGVLSHDPSFMRTFCERHGLAVRAVCLLVDDAAVAFYT
SYNECHOCOCCUS-SP. ------------------------------------------------------DEVDRHLQRDGPGVVDVALAVGEQELPALA
VIBRIO-SP.-MED222 ------------------------------------------------------QAEAFAKVHGPSVCGMAFRVNEATAAMEQ
MARINOMONAS-SP.-MED121 ----------------------------------------------QSEAFARIHGPSVCGMAERVKDASLAMQH
PSEUDOMONAS-FLUORESCENS ------------------------------------------------IASYFAAEHGPSVCGMAFRVKDSQKAYNR
SULFITOBACTER-SP.-NAS-14.1 ---------------------------------------------FAAGFVAEHGPCAPSMGWRVVDAQKALDH

HAATFIEEHGPCAPSMGWRVVDAQHAFDH
BDELLOVIBRIO-BACTERIOVORUS ---------------------------------------------FATDFAKLHGPCVNATGFRVIDADQAFKT
RATTUS NORVEGICUS ARERGAKIVREPWVEED -----------------------KFGKVKFAVLQTYG-DITHILVEK-- -HOMO -SAPIENS ARERGAKIMREPWVEQD -- KFGKVKFAVLQTYG-DTTHTLVEK-XENOPUS-LAEVIS AIERGAKSVREPWEESD -----------------------EFGTVVMATIQTYG-DTTHTFVER---BLEPHARISMA-JAPONICUS AIAKGCKSFREPQVLQD -----------------------QFGSVIIASLQTYG-DTVHTLVQN---MYCOSPHAERELLA-GRAMINICOLA AVANGAESVSSPHTDSC ----------------------DEGDVISAAIKTYG-DTTHTFIQR---ASPERGILLUS-FUMIGATUS ATNNGAKIVSQPRTLED -----------------------DNGQVRVATIQTYG-ETTHILVER---MAGNAPORTHE-GRISEA AVQQGAVSIAPPKTLSDK ----------------------EHGSVTMAVIQTYG-DTTHTLLSR---CANDIDA-ALBICANS AIKAGSGIIRLPKIISD -----------------------ENGVVKLATISIPNSDIQHTLIEN---PICHIA-STTPITIS AVAAGAGIIRLPKIISD -----------------------CNGSVRLGTISIPKTDIQHTLIEN---RHODOCOCCUS-SP. ARAQGATVLDEPHDVTD -----------------------DHGTVRLAAIATYG-DTRHILVDR---RHODOCOCCUS-ERYTHROPOLIS AITQGATVLSEPHDITD -----------------------EHGTVRLASIATYG-ETRHTLVDR---JANIBACTER-SP.-HTCC2649 AKAAGARVVQEAETVSD -----------------------EFGSVRIGAIATYG-ETRHTLVQRTVD
STREPTOMYCES-AVERMITILIS AIEHGARSVAEPYELKD -----------------------EHGTVVLAAIATYG-KTRHTLVDR---KORDIA-ALGICIDA TTKRGAKPYMEPTKEED -----------------------ENGYVIRSGIYTYG-ETVHVFVER-- -LEEUWENHOEKIELLA-BLANDENSIS-ME TTKRGAKPFMEPEVEKD ------------------EHGEVVRSGIYTYG-ETVHMFVER- - -GEMMATIMONAS-AURANTIACA AIARGAIPIQEPQVYSD -----------------------EHGEVVIAAIGTYG-DTIHSLVER---PICROPHILUS-TORRIDDS IEKYGTKVS-KINEIKD -----------------------GNGKIRTAEIKTYG-ETVHTLIET---ARABIDOPSIS-THALIANA SVANGAIPSSPPIVLNE -----------------------AVTIAEVKLYG-DVVLRYVSYKAE
BRASSICA-RAPA-SUBSP.-PEKINENSI SVSNGAVPSSPPIVIND ------------------------AVTIAEVKLYG-DVVLRYVSYKVA
COPTIS-JAPONICA-VAR.-DISSECTA SVANGAKPSSSPIKLDE ----------------------GVVLSEIQLYG-DVVLRYLSFKNT
HEVEA-BRASILIENSIS SVARGALPMGGPITLDN -----------------------RAVVAEVHLYG-DVVLRYISYKNS
MEDICAGO-TRUNCATULA SVNLGAIPSSPPVILEN -----------------------NVKLAEVHLYG-DVVLRYVSYNDL
GLYCINE-MAX SVAKGAEPASPPITLVDD ----------------------RTGFAEVRLYG-DVVLRYVSYKDA
SOLANUM-LYCOPERSICUM SVARGAKPREEPVTIDE -----------------------HVAVAEVHLYG-DVVLREVSLVKD
SOLANUM-TUBEROSUM SVSRGAKPRFEPVTIDE -----------------------HVAVAEVHLYG-DVVLRFVSFVKD

. .

N/COTIANA-BENTHAMIANA SVSRGAKPMFEPVTIDG -----------------------OVAMAEVHLYG-DVVLRFMS-LKD
DAUCUS-CAROTA SVARGARPASAPVELDD -----------------------OAWLAEVELYG-DVVLRFVSFGRE
SOLENOSTEMON-SCUTELLARIOIDES AVSRaAKPVSPPVVLADC ----------------------ETAIAEVHLYG-DTVLRFVSCGSG
HORDEUM-VULGARE SRARGARPAFAPVDLCR -----------------------GFAFAEVELYG-DVVLRFVSHPDG
TRITICUM-AESTIVUM SVDGGARPAFSPVDLGR -----------------------GEGFAEVELYG-DVVLRFVSHPDD
AVENA-SATIVA SVAGGARPAFAPADLGH -----------------------GFGLAEVELYG-DVVLRFVSYPDE
ORYZA-SATIVA SVAAGARPAFQPADLGG -----------------------GFGLAEVELYG-DVVLRFVSHPDG
SORGHUM-BICOLOR SVAAGARPAPEPVELGL -----------------------GFRLAEVELYG-DVVLRYVSYPDD
ZEA-MAYS -------------------------------------------------------------------GFRLAEVELYG-DVVLRYVSYPDG
AB095005_OLUCIMARINUS
AVKAGARGVLAPTVLTHTVDDGCAKGGQVIAEIELYG-DVVLREVNATDG
OTAURI
AMKRGARGVLEPTEMRHEKHDGCVMGTQIISEvELYG-DVVLRFVSRADG

SPAFSSACDGFAR----VSEVELYG-DVVLRYYSFHAG
SYNECHOCOCCUS-SP. ELLRGRG ---------------------------------AOLAWIP-AAAALCLHIPYG
VIBRIO-SP.-MED222 AFKGGGEEYK-TEIGP ------------------------MELSIPAIYGIG-ESLLYFVDRYGK
MARINOMONAS-SP.-MED121 ALANGAKEFS-GNLGA ------------------------MELKLPAVYGIG-ESTLYFIDRYGD
PSEUDOMONAS-FLUORESCENS ALELGAQPIH-IDTGP ------------------------MELNLPAIKGIG-GAPLYLIDRFGE
SULFITOBACTER-SP.-NAS-14.1 AVSKGAEEYT-GAG --------------------------KVLDVPAIKGIG-GSLIYFVDQYYD

KVMDVPAILGIG-GSLIYFIEDYYE
BDELLOVIBRIO-BACTERIOVORUS AVARGARPYEGNEHOK ------------------------GATPFPAIYGIG-DSLIYFMDQKNQ
RATIOS NORVEGICUS -INYTG -- RFLPGFEAPTYKDT ----------------LLPKLPSCNLEIIDHIVGNQ
HOMO-SAPIENS -MNYIG -- QFLPGYEAPAFMDP ----------------LLPKLPKCSLEMIDHIVGNQ
XENOPUS-LAEVIS -TNYKAP--AHVFLPNYRA-CEVDP ---------------INNVLPTVKLLNVDHVVGNQ
BLEPHARISmA-JAPONICUS -VDYTG -- PFLPGFRAITKDDP ----------------LNSAFPOVNYD1I0HVvGNQ
MYCOSPRAERELLA-GRAMINICOLA -TTYTG -- PFLPGYRSCTIVDS ----------------ANKFLPPVNLEAIDHCVGNQ
ASPERGILLUS-FUMIGATUS -GSYHG ----------------------------------AFLPGYRMETGVEDP----ISQLLPGVHLNRIDHCVGNQ
MAGNAPORTHE-GRISEA -DNFRG ----------------------------------TFLPGFRDVNRQPAA----YSALAP-VPLQRIDHCVGNQ
CANDIDA-ALBICANS -INYTG ----------------------------------PFLPGESQPIYPLADYYQVQLNMMPPVNLTVLDHCVENY
PICHIA-STIPITIS -IDYTG ----------------------------------PFLPNYSESVIQYNSKYYDOMQNIPTVSFQCIDHCVENY
RHODOCOCCUS-SP. -SHYTG -- PYLPGYTARTSGHT ----------------FRDGAPERLFQALDHVVONV
RHODOCOCCUS-ERYTHROPOLIS -SRYTG -- PYLPGYVERTSSYR ----------------KRDGAPKRIFQALDHVVGNV
JANIBACTER-SP.-HICC2649 GQTYSG -- PYLPGYVARSSSFV ----------------KROGAPKRLFOALDHIVGNV
STREPTOMYCES-AVERMITILIS -TGYDG-----PYLPGYVAAAP ------------------IVEPPAHRTFQAIDHCVGNV
KORDIA-ALGICIDA -KNYNG -- VFLPGYQRWES -------------------HYNPEPVGLKFIDHMVGNV
LEEUWENHOEKIELLA-BLANDENSIS-ME -KNYNG --- QFLPGYRKWES --------------------DYNPEPTGLKYIDHMVGNV
GEMMATIMONAS-AURANTIACA -RNYNG -- VFLPGFKAVTP -------------------HYQPSDVGLKYIDHCVGNV
PICROPHILUS-TORRIDUS -GDYNG -- VFMPGYEESEI -------------------NSKNTGIKKIDHIVGNV
ARABIDOPSIS-THALIANA DTE -- KSEFLPGFERVEDASSF ----------------P-LDYGIRRLDHAVGNV
BRASSICA-RAPA-SUBSP.-PEKINENSI TV ----------- FLPRFETVDDTSSF ----------------P-LDYGIRRLDHAVGNV
COPTIS-JAPONICA-VAR.-DISSECTA N----QSCP---FLPGFEEVGEVSSS -----------------RGLDEGIRRLDHAVGNV
HEVEA-BRASILIENSIS NPNLNDSSPDSWFLPKFESVDEASSF --------------P-LDYGIRRLDHAVGNV
MEDICAGO-TRUNCATULA NP---NQNPNLFFLPGFERVSDESSN --------------SSLDFGIRRLDHAVGNV
GLYCINE-MAX APOAPHADPSRWFLPGFEAAASSSSF --------------PELDYGIRRLDHAVGNV
SOLANUM-LYCOPERSICUM ADTL-------IFLPGFEAMDETSSF - ------------KELDYGIHRLDHAVGNV
SOLANUM-TUBEROSUM EDSL --- IFLPGFEAMDETSSF ----------------KELDYGIRRLDHAVGNV
NICOTIANA-BENTHAMIANA ADSL --- VFLPGFNAMDETASY - --------------KELDYGIRRLDHAVGNV
DAUCUS-CAROTA KG ----- LFLPGFEAVEGTASF ----------------PDLDYGIRRLDHAvGNV
SOLENOSTEMON-SCUTELLARIOIDES ADO -------------------------------------WFLPGFEVVGDGVSC-------QELDYGIRRLDHAVGNV
HORDEUM-VULGARE TDVP ---- FLPGFEGVTNP -------------------DAVDYGLTRFDHVVGNV
TRITICUM-AESTIVUM TDVP ---- FLPGFEGVSNP -------------------DAVDYGLTRFDHVVGNV
AVENA-SATIVA TDLP ---- FLPGFERVSSP -------------------GAVDYGLTRFDHVVGNV
ORYZA-SATIVA ADAP ---- FLPGFEGVSNP -------------------GAVDYGLRRFDHVVGNV
SORGHUM-BICOLOR ADAS ---- FLPGFVGVISP -------------------GAADYGLRREDHIvGNV
ZEA-MAYS AAGEP --- FLPGFEGVASP -------------------GAADYGLSRFDHIVGNV
AB095005_OLUCIMARINUS ---------------- FDGDFLCNYSATRDAP -----------------DVSYGLQRLDHAVGNV
OTAURI ---------------------------------- FDGDFLCNYEATRDVP -----------------SVSYGLRRLDHAVGNV

EKNA----KPKTFLPGYEDVPLEPPHT------TPLTYGLQRLDHAVGNV
SYNECHOCOCCUS-SP. IRHS ---- LIPGPLDAAPAEAG ----------------LFSHWDHVVLNV
VIBRIO-SP.-MED222 -QSIYD----VDFRFYDDAEQRMAEA --------------NVGLYEIDHLTHNV
MAR1NOMONAS-SP.-MED121 -KSIYD----VDFNEYENYQEKMLSH --------------QAGLYEVDHLTHNV
PSEUDOMONAS-FLUORESCENS GSSIYD----IDFVYLEGVERNPVG ---------------AGLKVIDHLTHNV
SULFITOBACTER-SP.-NAS-14.1 -TSPYN----EEYDWIAQS--KPAG ---------------VGFYYLDHLTHNV

VGFYYLDHLTHNV
BDELLOVIBRIO-BACTERIOVORUS -DKLYN----EIFQVKPED-KAPVG ---------------VGFTVVDHFTNNV
, =
RATTUS NORVEGICUS
PDQEMESASEWYLKNLQFHREWSVDDTQVHTEYSSLRSIVVANYEESIKM
HOMO-SAPIENS
PDQEMVSASEWYLKNLQFHRFWSVDDTQVHTEYSSLRSIVVANYEESIKM
XENOPUS-LAEVIS
PDDMMVPVAEWYEKMLMEHRFWSVDDTQMHTDYSAIRSIVVIDYDEVIKM
BLEPHARISMA-JAPONICUS
PCGDMTPTVEWYEKYLEFHRYWSADESVIHTDYSALRSVVVADWDEVIKM

DWDEMSDACIDEYERCLGFHRFWSVDDEDICTEFSALKSIVMSSPNQVVICM
ASPERGILLUS-FUMIGATUS
DWDEMDKVCEYYEKALGEHRFWSVDDKQICTEYSALKSIVMASPNEVVKM
MAGNAPORTHE-GRI SEA
DWDDMRAACDFYERCLSEHRFWSVDDNQISTDFSALNSIVMASPNNVVKM
CANDIDA-ALBICANS
SWNQMMEQAKLYADMFGEHKYWSVDEDDISTGETALRSIVMSSSNGOIKM
PICHIA-STIPITIS
SWNQMMAQAKLYASLFGEHKYWSADDHDIATDNTALRSIVMASGNGKIKM
10 RHODOCOCCUS-SP.
ELGKMDHWVDFYNRVMGFTNMAEFVGEDIATDYSALMSKVVSNGNHRVKF
RHODOCOCCUS-ERYTHROPOLIS
ELCKMDQWVDFYNRVMGETNMAEFVGGDIATDYSALMSKVVSSONHRVKF
JANIBACTER-SP.-HTCC2649 ELGKMDEWVSFYNRVMGFVNMAEFVGDDIATDYSALMSKVVANGNHRVKF
STREPTOMYCES-AVERMITILIS
ELGRMNEWVGFYNKVMGETNMKEFVCDDIATEYSALMSKVVADGILKVKF
KORDIA-ALGICIDA
GWGEMKEWCEFYAKVMGFAQIISFTDDDISTDFTALMSKVMSNGNGRIKF

GWGEMNTWVKWYEDVMGEVNFLTEDDKQITTEYSALMSKVMSNGNGRIKF
GEMMATIMONAS-AURANTIACA
ELGKMNQWVGYYADVLGERNLITEDDTDINTEYSSLMSKVMANGNDRIKF
PICROPHILUS-TORRIDUS YEGEMDSWVNFYIEKLGFEHLITEDDKDIRTDYSALRSKVVKY-NDDIVF
ARABIDOPSIS-THALIANA P--ELGPALTYVAGFTGEHQFAEFTADDVGTAESGLNSAVLASNDEMVLL
BRASSICA-RAPA-SUBSP.-PEKINENSI P--ELGPALTYLSRLTGEHOFAEFTADDVGTAESGLNSAVLANNDETVLL
20 COPTIS-JAPONICA-VAR.-DISSECTA P--NLAEAIGYLKEFTGEHEFAEFTAEDVGTTESGLNSIVLASNDEMVLL
HEVEA-BRASILIENSIS P--ELAPAVSYVKEFTGFHEFAEFTAEDVGTSESGLNSLVLANNEDTVLL
MEDICAGO-TRUNCATULA P--ELSSAVKYVKQFTGFHEFAEFTAEDVGTSESGLNSVVLANNEETVLL
GLYCINE -MAX P--ELAPAVRYLKGFSGFHEFAEFTAEDVGTSESGLNSVVLANNSETVLL
SOLANUM-LYCOPERSICUM P--ELGPVVDYIKAFTGEHEFAEFTAEDVGTAESGLNSVVLANNDETVLL

ELGPVVDYIKEFTGEHEFAEFTAEDVGTAESGLNSVVLANNDETVLL
NICOTIANA-BENTHAMIANA P--ELGPAVDYIKRFTGFHEFAEFTSEDVGTAESGINSMVVANNDETVLL
DAUCUS-CAROTA T--ELGPVVEYIKGFTGEHEFAEFTAEDVGTLESGLNSVVLANNEEMVLL
SOLENOSTEMON-SCUTELLARIOI DES P--KLEPVVDYLKKFTGEHEFAEFTAEDVGTAESGLNSVVLANNNENVLF
HORDEUM-VULGARE P--ELAPAAAYIAGFTGEHEFAEFTAEDVGTTESGLNSVVLANNSEGVLL

ELAPAAAYVACFAGFREFAEFTTEDVCTAESCLNsmVLANNSECVLL
AVENA-SATIVA P--EMAPVIDYMKGFLGEHEFAEFTAEDVGTTESGLNSVVLANNSEAVLL
ORYZA-SATIVA P--ELAPVAAYISGFTGEHEFAEFTAEDVCTAESGLNSVVLANNAETVLL
SORGHUM-BICOLOR P--ELAPAAAYFAGFTGEHEFAEFTAEDVGTTESGLNSMVLANNAENVLL
ZEA-MAYS P--ELAPAAAYFAGFTGFHEFAEFTTEDVGTAESGLNSMVLANNSENVLL

DLIETVDYITKVTGEHEFAEFTAEDIGTIDSGLNSMVLANNNEYVLL
OTAURI H--NLLETVDYIMKITGEHEFAEFTAEDIGTIDSGLNSMVLANNNEYVLL

NLLETVDYITAMTGMHEFAEFTAEDVGTVDSGLNSMVLANDDEMILL
SYNECHOCOCCUS-SP. EQGSLQAAADWYGRVLGWRRLYRYS -IGTATSGLESVVVGDPEAGIQW
VIBRIO-SP. -MED222 KOGNMDVWSGEYERLGNFREIRYFDIEGKLIG---LVSRAMTSPCGKIRI
40 MARINOMONAS-SP.-MED121 MRGNMDHWAGEYENIGNFREIRYFDIEGKLTG---LVSRAMTSPCGKIRI
PSEUDOMONAS-FLUORESCENS YRGRMVYWANFYEKLENFREARYFDIKGEYTG---LTSKAMSAPDGMIRI
SULFITOBACTER-SP.-NAS-14.1 FKGNMDVWFKFYGDLFNFREIRFFDIEGKFTG- - -LTSRALTSPCGRIRI

LFSRALTSPCGRIRI
BDELLOVIBRIO-BACTERIOVORUS PKGEMDKWQHFYEDIFGEYEAKYFDIRGSKTG---LLSRAMRSPCGKFSV
RATTUS NORVEGICUS PINEPAPGRK -KSQIQEYVDYNGGAGVQHIALRTEDIITTIRHLRER -HOMO-S-APIENS PINEPAPGKK-KSQIQEYVDYNGGAGVQHIALKTEDIITAIRHLRER---xENOPus-LAEvIs PINEPAPOKK-KSQIQEFVEYYCGAOVQHIALRTDDILRDVSAMRAR - -BLEPRARISMA-JAPONICUS PINEPADGLR-KSQIQEYVEYYGGAGVQHIALKVNDIISVISTLRAR- - -MYCOSPHAERELLA-GRAMINICOLA PINEPAHGKK-KSQIEEYVDEYNGPGVQHIALRTPNIIEAVSNLRSR---ASPERGILLUS-FUMIGATUS PINEPAKGKK-QSQIEEYVDEYNGAGVQHIALLTDDIIRDITNLKAR---MAGNAPORTHE-GRISEA PINEPAKGKK -RSQIEEYVTFNSGAGVQHIALLTSDIITTVEAMRSR -CANDIDA-ALBICANS PINEPVKSIM-KGQIEEENDENGGPGIQHIAFRTNNIIETVMALMOR---PICHIA-STIPITIS PINEPVKSKM-RGQIEEFHDENGGPGVQHIALRTNDIIDTVCALLAR---RHODOCOCCUS-SP. PLNEPALAKK-RSOIDEYLDFYRGPGAQHLALATNDILTAVDOLTAE---RHODOCOCCUS-ERYTHROPOLIS PLNEPAIAKK-RSQIDEYLEFYQGPGAQHLALATNDILGAVDALVDE---JANIBACTER-SP.-HTCC2649 PLNEPAIAKK -RSQIDEYLDFYQGPGAQHLAVATNDILRSVDELRKE-- -STREPTOMYCES-AVERMITILIS PINEPALAKK-KSQIDEYLEFYGGAGVQHIALNTGDIVETVRTMRAA---KORDIA-ALGICIDA PINEPAEGKK-KSQIEEYLDFYNGSGVQHIAVATDNIIDTVSQMRER---LEEUWENHOEKIELLA-BLANDENSIS-ME PINEPAEGIK-KSQIEEYLDFYEGPGVQHLAVATDDIVKIVAALKAR---GEMMATIMONAS-AURANTIACA PINEPASOKK-KSQIEEYLDFYGGPGAQHLALATDDILATVTALRDR-- -PTCROPHILUS-TORRIDUS PINEPAKGLR-KSQIEEYLDYYRSEGVOHIALLTDDIIKTVSMMEEN---ARABIDOPSIS-THALIANA
PINEPVHGTKRKSQIQTYLEHNEGAGLQHLALMSEDIERTLREMRERSS-BRASSICA-RAPA-SUBSP.-PEKINENSI
PVNEPVHGTKRKSQIQTYLEHNEGAGVQHLALMSEDIFRTLREMRKRSG-. .
= 41 COPTIS-JAPONICA-VAR.-DISSECTA
PMNERVYGTKRKSQIQTYLEHNEGAGVQHLALVSEDIFTTLREMRRRSG-HEVEA-BRASILIENSIS
PLNEPVEGTKRKSQIQTYLEHNEGAGLQHLALVSEDIFKTLREMRRRSG-MEDICAGO-TRUNCATULA
PMNEPVYGTKRKSQIETYLEHNEGAGLQHLALMSADIFRTLREMRKRSG-GLYCINE-MAX
PLNEPVYGTKRKSQIETYLEHNEGAGVQHLALVTHDIFTTLREMRKRSF-SOLANUM-LYCOPERSICUM
PLNEPVYGTKRKSQIQTYLEHNEGAGVQHLALVTEDIETTLREMWKRSG-SOLANUM-TUBEROSUM
PMNEPVYGTKRKSQIQTYLEHNEGAGVQHLALVTEDIERTLREMRKRSG-NICOTIANA-BENTHAMIANA
PLNEPVYGTKRKSQIQTYLEHNEGAGVQHLALVTEDIFFTLKEMRKRSG-DAUCUS-CAROTA
PLNEPVYGTKRKSQIQTYLEHNEGAGVQHLALVSEDIFRTLREMRKRSC-SOLENOSTEMON-SCUTELLARIOIDES
PLNEPVYGTKRKSQIQTYLDHNEGAGVQHLALITEDIFRTLREMRKRSE-HORDEUM-VULGARE
PLNEPVHGTKRRSQIQTFLEHHGGPGVQHIAVASSDVLATLRKMRARSA-TRITICUM-AESTIVUM
PLNEPVHGTKRRSQIQTFLEHHGGSGVOHIAVASSDVLRILREMRARSA-AVENA-SATIVA
PLNEPVHGTKRRSQIQTYLEYRGGPGVQHIALASNOVERTLREMRARTP-ORYZA-SATIVA
PLNEPVHGTKRRSQIQTYLDHHGGPGVQHIALASDDVLGTEREMRARSA-SORGHUM-BICOLOR
PLNEPVHGTKRRSQIQTYLDHHGGPGVQHMALASDDVERTLREMQARSA-ZEA-MAYS
PLNEPVHGTKRRSQIQTFLDHHGGPGVQHMALASDDVLRTLREMQARSA-AB095005_OLUCIMARINUS
PVNEPTEGTKRKSQIQTYLEQNNGPGLQHLALKTDDIFATVREMRKYSHL
OTAURI

PVNEPTEGTKRKSQIQTYLECNNGAGLQHLALKSDDVFATVREMRKHGGG
SYNECHOCOCCUS-SP. AINEPTCAAS---QIQEFLHAHGGPGIQHAALHSSDIVASLRRER
-- VIBRIO-SP.-MED222 PINE-SSDDK--SQIEEFIREYNGEGIQHIALATDDIYKTVKTLRDR MARINOMONAS-SP.-MED121 PINE-SSDDK--SQIEEFLNQYNGEGIQHIAMSSSDIYETVRQLKAG PSEUDOMONAS-FLUORESCENS PLNEESSKGA--GQIEEFLMQFNGEGIQHVAFLTDDLVKTWDALKKI SULFITOBACTER-SP.-NAS-14.1 PINE-BACTERIOVORUS PINE-PTEEK--SQIQEYLDEYKGSGIQHIALLTHDINYSLESLKNS RATTUS
NORVEGICUS --GMEFLAVP-SSYYRLL RENLKTSK -IQVKEN MDV
HOMO-SAPIENS --GLEFLSVP-STYYKQL ---------------REKLKTAK -IKVKEN IDA
XENOPUS-LAEVIS --GLEFLTIP-RTYYKNL ---------------RARLSMSK -VQVEED LAE
BLEPHARISMA-JAPONICUS --GVEFLEVP-PKYYDSL --------------- RKRLAHSA -VQIEED LKR
MYCOSPHAERELLA-GRAMINICOLA --GVEFISVP-UTYYENM ---------------RLRLKAAG -MKLEES FDI
ASPERGILLUS-FUMIGATUS --GVEFIKVP-DTYYEDI ---------------KVRLKKAG -LTLHED YET
MAGNAPORTHE-GRI SEA --GVEFIEVP-HTYYDTM ---------------RRRLKTEKRDWELQED FDR
CANDIDA-ALBICANS --GVEFNHTS-ENYYNNL ---------------KQRLNNDG -IKLYED FDT
PICHIA-STIPITIS --GIEFNTAS-DKYYTNL --------------- ERLLREDD -VALFED FDT
RHODOCOCCUS-SP. --GVEFLATP-DSYYEDP --------------- ELRARIG-NVRAP IAE
RHODOCOCCUS-ERYTHROPOLIS --GIEFLSTP-ASYYEDP --------------- ELRARIG-EVRVP IEE
JANIBACTER-SP.-HICC2649 --GVEFLDTP-DAYYDDP --------------- EMRARIG-EVRVP IEE
STREPTOMYCES-AVERMITILIS --GVQFLDTP-DSYYDT ---------------- LGEWVG-DTRVP VDT
KORDIA-ALGICIDA --GVEFLYVP-DTYYDD ---------------- LLERVG-DIDED VEE
LEEUWENHOEKIELLA-BLANDENSIS-ME --GVEFLPPPPQAYYDD ---------------------IPRRLGAMMDTMKED LNK
GEMMATIMONAS-AURANTIACA --GVEFLSVP-TSYYED ---------------- LQERVG-KIDEK LEE
PICROPHILUS-TORRIDUS --GIEFLKTP-GSYYES ---------------- LSSRIG-SIDED LNE
ARABIDOPSIS-THALIANA IGGFDEMPSPPPTYYQN -----------------------LKKRVG---DVLSDD----QIKE
BRASSICA-RAPA-SUBSP.-PEKINENSI VGGFDFMPSPPPTYYKN -------------------------LKNRVG---DVLSEE----QIEE
COPTIS-JAPONICA-VAR.-DISSEOTA VGGFEFMPSPPPTYYKN -----------------------HEVEA-BRASILIENSIS VGGFDEMPSPPPTYYRN -----------------------LKNRVG---DVLIDE----QIKE
MEDICAGO-TRUNCATULA VGGFEFMPSPPVTYYRN -----------------------LKNRVG---DVLSDE----QIKE
GLYCINE-MAX LGGFEEMPSFPFTYYAN -----------------------LHNRAA---DVLIVD----QIKQ
SOLANUM-LYCOPERSICUM VGGFEEMPAPPPTYYKN ----------------------- -LRSRAG---DVLSDE----QIQA
SOLANUM-TUBEROSUM VGGFEEMPUPPTYYKN ------------------------LKSRAG---DVLSDE----QIQA
NICOTIANA-BENTHAMIANA VGGFEFMPSPPPTYYKN -----------------------LKNRAG---DVLIDE----QIQA
DAUCUS-CAROTA LGGFEEMPSPPPTYYKN -----------------------LKNRVG---DVLSDE----QIKE
SOLENOSTEMON-SCUTELLARIOIDES VGGFEFMPSPPPTYYRN -----------------------LKSRAG---DVLSDE----QIEE
HORDEUM-VULGARE MGGFDFLPPPLPKYYEG -----------------------VRRLAG---DVLSEA----QIKE
TRITICUM-AESTIVUM MGGFDFLPPRCRKYYEG -----------------------VRRIAG---DVLSEA----QIKE
AVENA-SATIVA MGGFEFMAPPQAKYYEG -----------------------VRRIAG---OVLSEE----QIKE
ORYZA-SATIVA MGGFEFLAPPPPNYYDG -----------------------VRRRAG---DVLSEE----QINE
SORGHUM-BICOLOR MGGFEFMAPPAPEYYDG -----------------------VRRRAG---DVLTEA----QIKE
ZEA-MAYS MGGFEFMAPPTSDYYDG -----------------------VRRRAG---DVLTEA----QIKE
AB095005_OLUCIMARINUS RGGFDFQAPASDDYYKQ -----------------------LKAKIG---DALNDE----QYAL
OTAURI HGGFDFQAPASDDYYKH -----------------------LKEKIG---DALTDE----QYAL

LKARVGE--DALTER----QFKE
SYNECHOCOCCUS-SP. QGGVDFLQVAP-QYYTS -----------------------LERELGL--ALRSALGQAISWQD
VIBRIO-SP.-MED222 --GMDFMPTP-DTYYEKVDDRVKGHG- ------ ED --- TDL

. =

MARINOMONAS-SP.-MED121 --GLKFMSTP-DTYYAKVNDRVVGHG ----- ED -----LEK
PSEUDOMONAS-FLUORESCENS --GMRFMTAPPDTYYEMIEGRLPDHG ----- EP -----VDQ
SULFITOBACTER-SP.-NAS-14.1 --GLKEMPGPPETYYKMSKURVTGHQ ----- EP -----LDR

IDR
BDELLOVIbRIO-BACTEKIOVORUS --EIQFLTPFPHSYYEMIPERVPGVT ----- ED -----RATTUS NORVEGICUS LEELKILVDYD -----------------------------EKGYLLQIFTKPMQDRPTLFLEVI
HOMO-SAPIENS LEELKILVDYD -----------------------------EKGYLLOIFTKPVQDRPTLFLEVI
XENOPUS-LAEVIS IEKLSILVDFD -----------------------------EEGYLLQIFTKPLEDRPTLFIEII
BLEPHARISMA-JAPONICUS IEDLHILVDFD -----------------------------DRGYLLQIFTKPVEDRPTLFYEII
MYCOSPHAERELLA-GRAMINICOLA IQKLNILIDFD -----------------------------EGGYLLQLFTKPLMDRPTVFIEII
ASPERGILLUS-FUMIGATUS IRSLDILIDFD -----------------------------EGGYLLQLFTKHLMDRPTVFIEII
MAGNAPORTHE-GRISEA LVRNNILIDYD -----------------------------EGGYLLQIFTRPUMDRPTVFIEII
CANDIDA-ALBICANS
LRSLNILIDYDPSTKPKPKSKRKRNNKCNYLLQIFSKPLHDRPTLFIEII
PICHIA-STIPITIS LRKLNILVDYDISTR -------------------------NKKTGICNYLLQIFTKPLHDRPTLFIEII
RHODOCOCCUS-SP. LQKRGILVDRD -----------------------------EDGYLLQIFTKPLVDRPTVFFELI
RHODOCOCCUS-ERYTHROPOLIS LQKRGILVDRD -----------------------------EDGYLLQIFTKPIGDRPTVFFEII
JANIBACTER-SP.-HTCC2549 LKSRKILVDRD -----------------------------EDGYLLQIFTKPLGDRPTVFFEII
STREPTOMYCES-AVERMITILIS LRELKILADRD -----------------------------EDGYLLQIFTKPVQDRPTVFFEII
KORDIA-ALGICIDA LKKHGILIDRD -----------------------------EEGYLLQLFTKTIVDRPTMFFEVI
LEEUWENHOEKIELLA-BLANDENSIS-ME LQELSILVDAD ----------------------------------EEGYLLQIFTKPLQDRPTLFFEII
GEMMATIMONAS-AURANTIACA LAALGILVDRD -----------------------------PDGYLLQIFTKPVEDRPTLFFEII
PICROPHILUS-TORRIDUS IEKHNILVDRD -----------------------------ENGYLLQIFTKPVTDRPTFFFEVI
ARABIDOPSIS-THALIANA CEELGILVDR ------------------------------DDQGTLLQIFTKPLGDRPTIFIEII
BRASSICA-RAPA-SUBSP.-PEKINENSI CEELGILVDR --------------------------------DDQGTLLQIFTKPLGDRPTIFIEII
COPTIS-JAPONICA-VAR.-DISSECTA CEELGILVDR ------------------------------DAQGILLQIFTKPVGDRPTIFVEII
HEVEA-BRASILIENSIS CEELGILVDR ------------------------------DDQGILLQIFTKPVGDRPTIFIEII
MEDICAGO-TRUNCATULA CEELGILVDR ------------------------------DDQGILLQIFTKPIGDRPTIFIEII
GLYCINE-MAX CEELGILVDR ------------------------------DDQGTLLQIFTKPVGDRPTIFIXII
SOLANUM-LYCOPERSICUM
CEELGILVDR --- --- --- DDQGTLLQIFTKPVGDRPTIFIEII
SOLANUM-TUBEROSUM CEDLGILVDR ------------------------------DDQGTLLQIFTKPVGDRPTIFIEII
NICOTIANA-BENTHAMIANA CEDLGILVDR ------------------------------DACCUS-CAROTA CEDLGILVDR ------------------------------DDQGILLQIFTKPVGDRPTLFIEII
SOLENOSTEMON-SCUTELLARIOIDES CEKLGILIDR ------------------------------DDQGILLQIFTKPVGDRPTIFIEII
HORDEUM-VULGARE CQELGVLVDR ------------------------------DDQGVILOIFTKPVGDRPTLFLEMI
TRITICUM-AESTIVUM CQELGVLVDR ------------------------------DDOGVILOIFTKPVGDRPTLFLEMI
AVENA-SATIVA CQELGVLVDR ------------------------------DDQGVILQIFTKPVGDRPIFFLEMI
ORYZA-SATIVA CQELGVLVDR ------------------------------DDQGVILQIFTKPVGDRPTFFLEMI
SORGHUM-BICOLOR CQELGVLVDR ------------------------------DDOGVILOIFTKPVGDRPTLFLEII
ZEA-MAYS CQELGVLVDR ------------------------------DDQGVLLQIFTKPVGDRPTLFLEII
AB095005_OLUCIMARINUS VEELGLLVDK ------------------------------DDQGVLIQVFTKPVGDRPTLFLEII
OTAURI VEELGLLVDK ------------------------------DDQGVLIQVFTKPVGDRPTLFLEII

DDQGVLVQIFTKPLGDRPTVFIEII
SYNECHOCOCCUS-SP. LVEWILLDATLPASDG ------------------------QDRPLLLQTFTQPLFGRPTFFFEVI
VIBRIO-SP.-MED222 LRDLRVLIDGAPTKDG ------------------------ILLQIFTQTVIG--PVFFEII
MARINOMONAS-SP.-MED121 LQDLNILIDGAPLKDG ------------------------ILLQIFTDTVIG--PVFFEII
PSEUDOMONAS-FLUORESCENS LQARGILLDGSSVEGDK -----------------------RLLLQIFSETLMG--PVFFEFI
SULFITOBACTER-SP.-NAS-14.1 MKKHGILIDGEGVVDGGE ----------------------TRILLQIFSKTVIG--PIFFEFI
ocEANIcoLA-BATsENsis-HTcC2597 mKKHGILIDGEGvvDGGE ----------------------TRILLQIFSKTVIG--PIFFEFI
BDELLOVIBRIO-BACTERIOVORUS LEKNAILVDGD -KTG ----------------------KYLLQIFTKNTFG--PIFYELI
RATTUS NORVEGICUS QRHN ------------------------------------HOGFGAGNENSLFKAFEEEQAIRGN
HOMO-SAPIENS QRHN ------------------------------------HQGFGAGNFNSLFKAFEEEQNLRGN
XENOPUS-LAEVIS QRHN ------------------------------------HQGFGAGNFKALFESIEMEQAIRGN
BLEPHARISMA-JAPONICUS QRHN ------------------------------------NNGFGIGNFKALFESLEQEQERRGN
MYCOSPHAERELLA-GRAMINICOLA QRNN ------------------------------------FDGFGAGNFKSIFEAIEREQDLRGN
ASPERGILLUS-FUMIGATUS QRHN ------------------------------------FSGFGAGNEKSIFEAIEREQAIRGN
MAGNAPORTHE-GRI SEA QRNE ------------------------------------FDGFGAGNFKSLFEAIEREQAERGN
CANDIDA-ALBICANS ORHE ------------------------------------HNGFGKGTFKGLFESIEEOQKLRGT
PICHIA-STIPITIS QRHH ------------------------------------HNGFGKGTFKGLFETIEEQQRIRGT
RHODOCOCCUS-SP. ERHG ------------------------------------SIGFGIGNFKALFEAIEREQAARGN
RHODOCOCCUS-ERYTHROPOLIS ERHG ------------------------------------SLGEGIGNFKALFEAIEREQAARGN
JANTBACTER-SP.-HTCC2649 ERHG ------------------------------------SLGFGKGNFKALFESIEREQDARGN
STREPTOMYCES-AVERMITILIS ERHG ------------------------------------SMGEGKGNFKALFEAIEREQEKRGN
KORDIA-ALGICIDA QRKG ------------------------------------AQSFGVGNFKALFEAIEREQAARGT

LEEUWENHOEKIELLA-BLANDENSIS-ME QRMG -------------------------------------AKGFGAGNFKALFESIEREQAQRGT
GEMMATIMONAS-AURANTIACA QRKG ------------------------------------ATSFGKGNFRALFEAIEREQELRGN
PICROPHILUS-TORRIDCS QRKG ------------------------------------ARSEGNGNEKALFEATEREQAKRGN
ARABIDOPSIS-THALIANA QRVGCMMKDEEGKA --YQSGGCGGFGKGNFSELFKSIEEYEKTLEA
bRA561CA-RAPA-SUBSP.-FEKINENSI QKIGCMKKDEEGRV --YQSGGGGGFGKGNFSELKKSIEEYEKTLEA
COPTIS-JAPONICA-VAR.-DISSECTA QRLGCMLKDEEGKT --YOKAGCGGEGKGNESELFKSIEEYEKTLEA
HEVEA-BRASILIENSIS QRVGCMIKDETGKE --YQKGGCGGEGKGNESELFKSIEEYEKTLEA
MEDICAGO-TRUNCATULA QRVGCMLKDEEGKE --YOKGGCGGEGKGNESELFKSIEEYEKTLET
GLYCINE-MAX QRIGCMVEDEEGKV --YQKGACGGEGKGNESELFKSIEEYEKTLEA
SOLANUM-LYCOPERSICUM QRIGCMLKDEKGQV -- YOKGGCGGEGKGNESELFRSIEEYEKMLEA
SOLANUM-TUBEROSUM QRIGCMLKDENGQV --YQKGGCGGFGKGNFSELFRSIEEYEKMLEA
NICOTIANA-BENTHAMIANA QRIGCMLKDEKGQV -- YQKGGCGGEGKGNEWELFRSIEEYEKTL--DAUCUS-CAROTA QRVGCMLKDDAGQM --YQKGGCGGEGKGNESELFKSIEEYEKTLEA
SOLENOSTEMON-SCUTELLARIOIDES QRVGCMMKDEEGKM --YQKGGCGGFGKGNFSELFKSIEEYEKMLES
HORDEUM-VULGARE QRIGCMEKDERGEE -- YQKGGCGGEGKGNESELFKSIEDYEKSLEA
TRITICUM-AESTIVUM QRIGCMEKDERGEE --YQKGGCGGEGKGNESELEKSIEDYEKSLEA
AVENA-SATIVA QRIGCMEKDEVGQE --YQKGGCGGEGKGNESELFKSIEDYEKSLEV
ORYZA-SATIVA QRIGCMEKDESGQE --YQKGGCGGFGKGNFSELFKSIEEYEKSLEA
SORGHUM-BICOLOR QRIGCMEKDEKGQE --YQKGGCGGEGKGNESQLEKSIEDYEKSLEA
ZEA-MAYS QRIGCMEKDEKGQE -- YQKGGCGGFGKGNFSQLFKSIEDYEKSLEA
AB095005_OLUCIMARINUS QRIGCMRRKADSES --------------------------FEQAAGCGGEGKGNESELFKSIEAYEATLQI
OTAURI QRVGCMRKKADTDE --------------------------LEQVAGCGGFGKGNFSELFKSIEAYEKTLNI

QRIGCLREVKSADANAPPRIEQAGGCGGEGKGNESELFKSIENYERTLKM
SYNEOHOCOOCUS-SP. QRLG--------------------GATGEGEANFQALFEALERQQRQRHQ
VlBRIO-SP.-MED222 QRKG ------------------------------------NEGFGEGNFKALFESIEEDQIRRGV
MARINOMONAS-SP.-MED121 QRKG ------------------------------------NEGFGEGNFKALFESIEEDQIRRGV
PSEUDOMONAS-FLUORESCENS QRKG ------------------------------------DDGFGEGNFKALFESIERDQVRRGV
SULFITOBACTER-SP.-NAS-14.1 ERKG ------------------------------------DDGFGEGNFKALFESIEQEQIDSGE

DDGFGEGNFKALFESIEQEQIDNGE
BDELLOVIDRIO-BACTERIOVORUS QRKG -----------------------------HDGFGDGNFQALFRAIERDQRERGY
RATTUS NORVEGICUS LTDLETNGVRSGM --HOMO-SAPIENS LTNMETNGVVPGMAENLYFQ
XENOPUS-LAEVIS
BLEPHARISMA-JAPONICUS LI -------------MYCOSPHAERELLA-GRAMINICOLA
ASPERGILLUS-FUMIGATUS LV -------------MAGNAPORTHE-GRI SEA
CANDIDA-ALBICANS FVKSQNN --------PICHIA-STIPITIS LVQVDEDDDSQQST - --RHODOCOCCUS-SP.
RHODOCOCCUS-ERYTHROPOLIS
JANIBACTER-SP.-HTCC2649 STREPTOMYCES-AVERMITILIS
KORDIA-ALGICIDA
LEEUWENHOEKIELLA-BLANDENSIS-ME L --------GEMMATIMONAS-AURANTIACA
PICROPHILuS-ToRRIDus I. -------------ARABIDOPSIS-THALIANA KQLVG ----------BRASSICA-RAPA-SUBSP.-PEKINENSI KQLVG -----------COPTIS-JAPONICA-VAR.-DISSECTA KANVVAA -------HEVEA-BRASILIENSIS KRNAEAR --------MEDICAGO-TRUNCATULA RRTA -----------GLYCINE-MAX KRTA -----------SOLANUM-LYCOPERSICUM KHVNQVAAVE -----SOLANUM-TUBEROSUM KBVNQVAAA ------NICOTIANA-BENTHAMIANA
DAUCUS-CAROTA KQITGSAAA ------SOLENOSTEMON-SCUTELLARIOIDES KLVTKTAMA ------HORDEUM-VULGARE KQSAAVQGS ------TRITICUM-AESTIVUM KQSAAVQGS ------AvENA-SATIVA KQSVvAQKS ------ORYZA-SATIVA KQAPTVQGS ------SORGHUM-BICOLOR KQAAAAQGS ------ZEA-MAYS KQAAAAAAAQGS --AB095005_OLUCIMARINUS
OTAURI

SYNECHOCOCCUS-SP. ALT? ----------VIBRIO-SP.-MED222 LDDA ----------MARINOMONAS-SP.-MED121 LNDA ----------PSEUDOMONAS-FLUORESCENS LATD ----------SULFITOBACTER-SP.-NAS-14.1 LSEA ----------BDELLOVIBRIO-BACTERTOVORUS LT ------------The overall sequence identity between individual full length HPPD sequences is in general quite low and is shown for the representative HHPD proteins in Table 3.
Table 4a shows the sequence alignment of the binding pocket. In contrast, the sequence identity of the 36 amino acids forming the binding site is significantly higher which is shown for the representative HPPD proteins in Table 4b. In particular, the amino acids at 8 positions are strictly conserved in all species and illustrate that these amino acids have a key role (e.g. His226, His308, Glu394 binding the iron required for catalysis). These positions, with reference to the HPPD from Arabidopsis (SEQ ID No. 2) are His226, Ser267, Asn282, His308, Tyr342, Glu394, Gly420, Asn423 (Table 5a). A mutation of any of the amino acids at either of these positions will most likely lead to an inactive protein. The variability at other positions within the binding site is higher. Table 5b shows the 28 variable positions in the binding site and the amino acids which were identified at these positions using the sequence alignment. Some positions have only limited variability which reflects their role in the 3D environment. An example for this represents the position 269. All HPPD
proteins have at this position either a Val, Ala or Thr. Looking at the 3D structures it seems that at this position a small apolar amino acid is required and a mutation to a polar amino acid such as Arg, His or Lys will disturb the protein structure locally.
Another example represents position 379. Most sequences have a glutamine at position 379.
However, there are also some bacterial sequences which have a histidine at this position. Looking at the 30 structure, it seems that only few amino acids are tolerated at this position. GIn379 in A.thaliana stabilizes via its H-bond donor the side chain conformation of the strictly conserved Glu394 which in turn interacts with the catalytic iron. In addition, with its H-bond acceptors GIn379 stabilizes the side chain conformation of the strictly conserved Asn423 which in turn interacts with the strictly conserved Tyr342. Only glutamine, asparagine and histidine have an H-bond donor . .
=
and acceptor required for the stabilization of this particular 3D arrangement which very likely play a key role in the interaction of the C-terminal helix with the core of the HPPD protein. Limited variability is also seen at position 381 with either a phenylalanine or a tyrosine in all HPPD sequences. The aromatic ring stabilizes the 5 binding of HPPD inhibitors and very likely also the binding of the substrate to the HPPD binding. However, the presence of the additional hydroxyl group in tyrosine compared to phenylalanine does not disturb the catalytic activity. The third category of positions includes those positions which display a very high natural variability.
These positions may not be crucial for substrate binding and catalysis but influence 10 inhibitor binding. These positions include adjacent positions in strand 248 to 255 and positions in the C-terminal helix 419-427. It can be assumed that the interaction of this particular strand and the C-terminal helix with the core of the protein plays a crucial role in inhibitor binding. Table 5b includes for each variable position those amino acids which have been identified in the sequence alignments using all known 15 HPPD sequences.

Table 3: Pairwise sequence identity of HPPD sequences from the representative set of HPPD proteins RATTUS_NORYEOICUS 103 90 62 55 52 63 50 44 41 42 39 41 45 45 40 48 45 37 37 XENOBJS-LAE\40 62 63 100 60 54 57 52 44 44 43 42 43 49 48 61 49 45 37 37 36 BLEPH8RI6MA-JAP0I4CUS 55 55 130 103 49 49 47 41 40 42 41 41 45 42 4e 48 45 ASPEROILLUS-FU410ATUS 53 55 57 49 es 100 63 45 43 43 42 44 46 49 51 49 46 RH00000CCUS-SP. 42 42 43 42 44 43 41 37 38 100 135 77 62 48 49 55 96 34 34 STREPTOMYCES-AVERM775.16 45 48 49 45 46 45 46 30 37 62 60 63 100 50 53 58 K091215-ALO1C1DA 45 46 48 42 48 49 45 41 37 48 47 47 50 1120 73 60 s3 35 35 GEMMATIMONAS=AURANTIACA

P1CROPHLUS.TORRIDUS

ARA86)OPSIS-Tli5LIAMA

8RASSICA3RAPA=SUBSP -PEHINENSI 37 37 37 35 34 33 34 28 26 34 35 35 38 35 36 HEVEA-BRASILIENSIS

27 27 27 30 N, 30 27 27 27 29 .. 0 S0LANUM-LYc04'ERSicu4 38 37 X 35 34 33 33 28 26 36 36 36 37 34 39 39 35 72 Lo SOLANUM=TUBEROSUM 35 37 36 35 33 33 33 28 25 35 36 35 37 35 35 39 36 72 74 oI
HCOTIANA.BENTHAXANA X 38 301 35 33 33 34 29 27 35 35 35 37 34 38 38 36 70 73 72 74 74 74 07 88 133 76 75 El 60 61 133 84 64 55 56 56 31 31 30 27 27 26 63 63 63 85 64 83 65 64 63 82 81 100 84 83 54 se 55 32 29 29 27 26 28 31 $809500530LUCIMARINUS 34 34 36 34 33 32 34 26 25 36 35 36 38 34 313 40 32 NECHOCOCCU653. 31 31 30 31 30 29 30 27 26 34 32 32 34 31 34 36 33 31 31 29 28 29 29 30 30 31 29 30 33 32 30 32 30 32 ze 2e 29 100 25 27 26 23 24 ze VBR6)-SP A1ED222 29 29 3i 30 33 35 33 33 32 30 33 31 34 37 37 37 33 28 2B

MARNOM0NAS-SP:5ED121 28 29 32 31 34 34 30 32 29 31 X 30 33 35 38 35 33 28 28 31 29 3o 3o 29 29 30 30 31 30 30 29 29 30 31 27 23 28 27 74 1M 56 51 49 44 11:0 =

Table 4a: Sequence alignment of amino acids forming the binding pocket in the representative set of HPPD sequences HOMO-SAPIENS

RATTUS NORVEGICUS
HVHFWSVLSIVVAPNQIQHLYLQFFEFGAGNFNSLF
XENOPU-LAEVIS
HVHFWSVLSIVVTPNOIOHLYLQFFEFGAGNFKALI
ASPERGILLUS-FUMIGATUS
HVHFWSVLSIVMAPNQIQHIYLQFFEFGAGNEKSLI
MAGNAPORTHE-GRISEA
HVHFWSVLSIVMAPNQIQHIYLQFFEFGAGNFKSLI
MYCOSPHAERELLA-GRAMINIOOLA
HVHFWSVLSIVMSPNQIQHIYLQFFEFGAGNFKSLI
CANDIDA-ALHICANS
HVHYWSVLSIVMSPNQIQHNYLQFFEFGKGTFKGLI
PICHIA-STIPITIS
HVHYWSALSIVMAPNQIQHNYLQFFEFGKGTFKGLI
ARABIDOPSIS-THALIANA
HVHFAEFLSAVLAPNQIQHMYLQFFEFGKGNFSELI
BRASSICA-RAPA-SUBSP.-PEKINENSI
HVHFAEFLSAVLAPNQIQHMYLQFFEFGKGNFSELI
HEVEA-BRASILIENSIS
HVHFAEFLSLVLAPNQIQHMYLQFFEFGKGNFSELI
COPTIS-JAPONICA-VAR.-DISSECTA
HVHFAEFLSIVLAPNQIQHMYLQFFEFGKGNFSELI
SOLENOSTEMON-SCUTELLARIOIDES
HVHFAEFLSVVLAPNQIQHMYLQFFEFGKGNFSELI
AVENA-SATIVA
HVHFAEFLSVVLAPNQIQHMYLQFFEFGKGNFSELI
DAUCUS-CAROTA
HVHFAEFLSVVLAPNOIQHMYLQFFEFGKGNFSELI
SOLANUM-TUBEROSUM
HVHFAEFLSVVLAPNQIQHMYLQFFEFGKGNFSELI
SOLANUM-LYCOPERSICUM
HVHFAEFLSVVLAPNQIQHMYLQFFEFGKGNFSELI
MEDICAGO-TRUNCATULA
HVHFAEFLSVVLAPNQIQHMYLQFFEFGKGNFSELI
GLYCINE-MAX
HVHFAEFLSVVLAPNQIQHMYLQFFXFGKGNFSELI
HORDEUM-VULGARE
HVBFAEFLSVVLAPNOIOHLYLQFFEFGKGNESELI
ORYZA-SATIVA
HVHFAEFLSVVLAPNQIQHLYLQFFEFGKGNFSELI
SORGHUM-BICOLOR
HVHFAEFLSMVLAPNQIQHMYLQFFEFGKGNFSQLI
ZEA-MAYS
HVHFAEFLSMVLAPNQIQHMYLQFFEFGKGNFSQLI

HVHFAEFLSMVLAPNQIQHQYLOFFEFGKGNESELI
OTAURI
HVHFAEFLSMVLAPNQIQHQYLQFFEFGKGNFSELI

HVHFAEFLSMVLAPNQIQHQYLQFFEFGKGNFSELI
TRITICUM-AESTIVUM
HVHFAEFLSMVLAPNQIQHLYLQFFEFGKGNFSELI
NICOTIANA-BENTHAMIANA
HVHFAEFLSMVVAPNQIQHMYLQFFEFGKGNFWELI
RHODOCOCCUS-SP.
HVTMAEFLSKVVSPNQIQHLYLQFFEFGIGNFKALI
RHODOCOCCUS-ERYTHROPOLIS

STREPTOMYCES-AVERMITILIS
HVTMKEFLSKVVAPNQIQHLYLOFFEFGKGNFKALI
JANIBACTER-SP.-HTCC2649 HVVMAEFLSKVVAPNQIQHLYLQFFEFGKGNFKALI
KORDIA-ALGICIDA
HVAIISFLSKVMSPNQIQHLYLQFFEFGVGNFKALI
LEEUWENHOEKIELLA-BLANDENSIS-ME
HVVFLTFLSKVMSPNOIQHLYLQFFEFGAGNFKALI
PICROPHILUS-TORRIDUS
HVELITFLSKVVKPNQIQHLYLQFFEFGNGNFKALI
GEMMATIMONAS-AURANTIACA
HVRLITFLSKVMAPNQIQHLYLQFFEFGKGNFRALI
VIBRIO-SP.-MED222 HTRIRYFLSRAMTPNQIQHMYLQFFEFGEGNFKALI
MARINOMONAS-SP.-MED121 HTRIRYFLSRAMTPNQIQHMYLQFFEFGEGNFKALI
SULFITOBACTER-SP.-NAS-14.1 HTRIRFFLSRALTPNQIQHMYLQFFEFGEGNFKALI

HTRIRFFLSRALTPNQIQHMYLQFFEFGEGNFKALI
PSEUDOMONAS-FLUORESCENS
HTRARYFLSKAMSPNQIQHMYLQFFEFGEGNFKALI
BDELLOVIBRIO-BACTERIOVORUS
HTYAKYFLSRAMRPNQIQHLYLQFFEFGDGNFQALI
BLEPHARISMA-JAPONICUS
HVHYWSALSVVVAPNOIQHLYLQFFEFGIGNFKALL
SYNECHOCOCCUS-SP.
HVRLYRYLSVVVGANQIQHLYLQFFEFGEANFQALL

Table 4b: Pairwise sequence identity of amino acids forming the binding site HPPD in the representative set RAT1US_N0RVE-4369US 103 100 89 69 89 86 75 75 72 72 72 75 72 72 72 72 72 72 ASPER0ILLUS-91.1910A7US 69 89 89 100 100 97 83 63 75 75 75 76 75 75 75 75 CANDIDA-ALEICA601 75 75 81 B3 ea 86 1C0 94 69 93 69 72 69 69 69 59 69 69 67 4RAB6)0PSIS-TH4LIANA 72 72 72 75 75 72 69 72 100 100 97 97 97 97 97 97 97 CCP9S-JAP0NICA-VAR.-0ISSECTA 75 75 75 78 78 76 72 75 97 97 97 100 87 97 97 87 69 69 67 64 72 81 C) 49) 61 n) 67 69 69 67 64 72 61 co co Ln 94 92 92 92 92 92 92 ag 72 72 75 78 67 69 67 72 64 64 67 67 64 61 69 56 7.,) 67 64 67 75 54 N) SOR0MUM-aCOLCR 72 72 72 75 75 72 69 72 94 94 94 94 94 94 94 94 94 94 92 92 (9 .413095005_OLUC4ARIMB 72 72 72 75 75 72 69 72 94 94 94 94 94 94 94 94 94 84 97 97 94 99 97 97 97 160 92 78 78 at 83 72 75 72 78 64 54 67 67 64 67 72 81 1CO11ANA-BENT'9A96ANA 75 75 75 75 75 72 69 72 92 92 92 92 92 92 92 92 92 92 75 81 81 78 78 78 78 78 81 el 92 92 100 94 81 81 83 83 69 69 69 69 72 72 78 67 JA7.BACTER-SP.91TOC2649 72 72 78 72 72 69 63 72 81 01 e1 81 91 81 &I 131 81 81 78 63 83 61 81 91 81 81 63 83 92 92 94 100 91 83 e3 83 69 69 69 69 72 89 78 72 72 89 75 75 72 72 72 72 72 75 72 83 83 61 83 136 100 83 83 72 72 69 es 78 1313NATIVA4AS=ADRANTIACA 8697272 7269 69727575757575757575757572 TB

4ARINOM0NAS-SP At79121 58 58 69 67 67 67 64 64 67 67 67 67 67 67 67 67 67 SULM08ACTER,99.-64A9-141 58 58 69 64 54 64 61 61 69 69 69 69 69 69 69 69 69 69 69 69 67 67 67 69 69 67 67 67 67 67 69 69 69 69 72 69 es 69 94 94 100 100 991,ECH000CCUS-59. 64 64 67 58 S3 56 56 56 58 58 58 58 61 61 61 61 61 61 58 . .

Table 5a: Amino acid positions which are strictly conserved shown for the crystal structures Arabidopsis Pseudomonas Streptomyces Homos Rattus thaliana fluorescens avermitilis apiens norvegicus Amino Amino Amino Amino Amino Position Acid Position Acid Position Acid Position Acid Position Acid Table 5b: Amino acid position with the amino acids at this positions in the known crystal structures and the amino acids a these position within all HPPD
sequences Arabidopsis Pseudomonas Streptomyces Homo Rattus Natural thaliana tluorescens ayennitilis sapiens nowegicus Variabaility Amino Amino Amino Amino Amino Amino Pos. Acid Pos. Acid Pos. Acid Pos. Acid Pos, Acid Acid 431 I 345 i 371 I 371 F 371 F IFVMLQR

, Table 5c: Amino acid position with the amino acids at this positions in the known crystal structures and most common amino acids a these position within all HPPD
sequences Arabidopsis Pseudomonas Streptomyces Homo Rattus Most thaliana fluorescens avermitilis sapiens norvegicus common Amino Amino Amino Amino Amino Amino Pos. Acid Pos. Acid Pos. Acid Pos. Acid Pos. Acid Acid . , Arabidopsis Pseudomonas Streptomyces Homo Raftus Most thaliana fluorescens avermitilis sapiens norvegicus common Amino Amino Amino Amino Amino Amino Pos. Acid Pos. Acid Pos. Acid Pos. Acid Pos, Acid Acid Not all amino acids occurring at the variable positions have the same likelihood to be present in an active protein. In some cases most sequences have the same amino acids at a particular position while other amino acids are present at that position in only few HPPD sequences. An example is position 392. Most sequences have at a corresponding position a phenylalanine while few i.e. the burkholdeda sequences have a serine. Whereas in some rare cases, the rare amino acids may be the result of a sequencing error, in most other cases the resulting protein is active.
Table 5c shows a list with the most common amino acids at the variable positions.
The influence of the amino acids at the variable positions is different. Some of these positions are crucial for catalysis and/or the interaction of HPPD with an inhibitor while others may have less impact. For instances changes at positions 269 and which are in direct contact with the inhibitor and the substrate will very likely have a great impact upon catalysis and inhibitor binding. Also modifications at positions involved in the helix movement induced by inhibitor or substrate binding such as positions 252, 421 and 422 are likely to have high impact on inhibitor binding. In contrast modifications at positions such as 293 are less likely to influence inhibitor binding because this position is quite distant from the active site. Table 6a shows the position with very high impact and Table 6b with high impact on activity and inhibitor binding.
From these observations, the present inventors drew the conclusion that amino acids are preferred in the binding sites which naturally occur at the corresponding positions.
This means, that those modifications which exchange a naturally occurring amino acid into another are likely providing a catalytically active HPPD protein which may exert a modified or even increased tolerance to HPPD herbicide inhibitors.
Even . .
õ

more promising are those mutant proteins which have the amino acid most often found in nature at the variable position chosen from Table 5c.
Table 6a: Amino acid positions with high priority shown for the x-ray structures Arabidopsis Pseudomonas Streptomyces Homo Rattus thaliana tluorescens avermitilis sapiens norvegicus Amino Amino Amino Amino Amino Pos. Acid Pos. Acid Pos. Acid Pos. Acid Pos. Acid 426 E 340 A 366 A 366 S 366 S .

Table 6b: Amino acid positions with very high priority shown for the x-ray structures Arabidopsis Pseudomonas Streptomyces Homo Rattus Ma'am fluorescens avermitilis sapiens norvegicus Amino Amino Amino Amino Amino Pos. Acid Pos. Acid Pos. Acid Pos. Acid Pos. Acid In another embodiment, in the isolated nucleic acid of the invention as defined above, said at least on amino acid is selected from a) Ala, Asp, Glu, Phe, Thr, Val or Tyr at a position in an HPPD protein, said position corresponding to position 250 of the amino acid sequence of SEQ ID
No. 2;
b) Ala, Asp, Glu, Ile, Lys, Leu, Pro or Arg at a position in an HPPD
protein, said position corresponding to position 251 of the amino acid sequence of SEQ ID
No. 2;
c) Glu, Thr, Arg, Ser or Tyr at a position in an HPPD protein, said position corresponding to position 252 of the amino acid sequence of SEQ ID No. 2;
d) Ala, Phe, Val or Trp at a position in an HPPD protein, said position corresponding to position 253 of the amino acid sequence of SEQ ID No. 2;
e) Leu or Val at a position in an HPPD protein, said position corresponding to position 265 of the amino acid sequence of SEQ ID No. 2;
f) Leu, Gin, Arg, Val or Tyr at a position in an HPPD protein, said position corresponding to position 268 of the amino acid sequence of SEQ ID NO: 2;
g) Ala or Val at a position in an HPPD protein, said position corresponding to position 269 of the amino acid sequence of SEQ ID No. 2;

h) Ala, Pro, Thr or Val at a position in an HPPD protein, said position corresponding to position 280 of the amino acid sequence of SEQ ID No. 2;
i) Gin at a position in an HPPD protein, said position corresponding to position 307 of the amino acid sequence of SEQ ID No. 2;
5 j) Ile or Met at a position in an HPPD protein, said position corresponding to position 335 of the amino acid sequence of SEQ ID No. 2;
k) Leu at a position in an HPPD protein, said position corresponding to position 368 of the amino acid sequence of SEQ ID No. 2;
I) Phe at a position in an HPPD protein, said position corresponding to position 10 392 of the amino acid sequence of SEQ ID No. 2;
m) Asp, Lys, or Asn at a position in an HPPD protein, said position corresponding to position 421 of the amino acid sequence of SEQ ID No. 2;
n) Ala or Gly at a position in an HPPD protein, said position corresponding to position 422 of the amino acid sequence of SEQ ID No. 2;
15 o) Ile, Arg or Ser at a position in an HPPD protein, said position corresponding to position 425 of the amino acid sequence of SEQ ID No. 2 p) Glu, Phe or Val at a position in an HPPD protein, said position corresponding to position 426 of the amino acid sequence of SEQ ID No. 2;
q) Ile, Met, Gin or Val or Phe at a position in an HPPD protein, said position 20 corresponding to position 431 of the amino acid sequence of SEQ ID No.
2;
r) Ala, Cys, Thr or Val at a position in an HPPD protein, said position corresponding to position 228 of the amino acid sequence of SEQ ID No. 2;
s) Ala, Glu, His, Lys, Gln, Arg, Thr or Tyr at a position in an HPPD
protein, said position corresponding to position 248 of the amino acid sequence of SEQ ID
25 No. 2;
t) Ile, Leu, Met or Val at a position in an HPPD protein, said position corresponding to position 270 of the amino acid sequence of SEQ ID No. 2;
u) Ala, Glu, Lys, Arg, Ser, Thr or Val at a position in an HPPD protein, said position corresponding to position 271 of the amino acid sequence of SEQ ID
30 No. 2;
v) His or Gin at a position in an HPPD protein, said position corresponding to position 379 of the amino acid sequence of SEQ ID No. 2;

=

w) Leu or Arg at a position in an HPPD protein, said position corresponding to position 427 of the amino acid sequence of SEQ ID No. 2.
In an alternative embodiment of the nucleic acid of the invention, said at least one .. amino acid is selected from a) Glu, Thr, Arg, Ser or Tyr at a position in an HPPD protein, said position corresponding to position 252 of the amino acid sequence of SEQ ID No. 2;
b) Ala or Val at a position in an HPPD protein, said position corresponding to position 269 of the amino acid sequence of SEQ ID No. 2;
c) Ala, Pro, Thr or Val at a position in an HPPD protein, said position corresponding to position 280 of the amino acid sequence of SEQ ID No. 2;
d) Ile or Met at a position in an HPPD protein, said position corresponding to position 335 of the amino acid sequence of SEQ ID No. 2;
e) Leu at a position in an HPPD protein, said position corresponding to position 368 of the amino acid sequence of SEQ ID No. 2;
f) Aspõ Asn or Lys at a position in an HPPD protein, said position corresponding to position 421 of the amino acid sequence of SEQ ID No. 2;
g) Ala or Gly at a position in an HPPD protein, said position corresponding to position 422 of the amino acid sequence of SEQ ID No. 2;
h) Ala, Cys, Thr or Val at a position in an HPPD protein, said position corresponding to position 228 of the amino acid sequence of SEQ ID No. 2;
i) Ala, Glu, His, Lys, Gin, Arg, Thr or Tyr at a position in an HPPD
protein, said position corresponding to position 248 of the amino acid sequence of SEQ ID
No. 2;
j) Ile, Leu, Met or Val at a position in an HPPD protein, said position corresponding to position 270 of the amino acid sequence of SEQ ID No. 2;
k) Ala, Glu, Lys, Arg, Ser, Thr or Val at a position in an HPPD protein, said position corresponding to position 271 of the amino acid sequence of SEQ ID
No. 2;
I) His or Gin at a position in an HPPD protein, said position corresponding to position 379 of the amino acid sequence of SEQ ID No. 2;

m) Leu or Arg at a position in an HPPD protein, said position corresponding to position 427 of the amino acid sequence of SEQ ID No. 2.
Various sequences of HPPD proteins or predicted HPPD proteins are known in the art. These include the HPPD sequences of Streptomyces avermitilis (Genebank SAV11864), Daucus carota (Genebank DCU 87257), Arabidopsis thaliana (Genebank AF047834), Mycosphaerella graminicola (Genebank AF038152), oryza sativa / rice [8A026248], Zea mays / corn [ACN36372], avena sativa [ABZ23427], Pseudomonas fluorescens [ABF50055], Synechococcus sp.
[YP_473959], Blepharisma japonicum [BAF91881], Rhodococcus RHA1 sp. ro0240 FP_7020051, Rhodococcus RHA1 sp. ro0341 [Y13_703002], Picrophilus torridus [YP_0241471, Kordia algicida [ZP_02161490], Sorghum bicolor [X13_0024533591, Triticum aestivum / wheat [AAZ67144], or Hordeum vulgare / barley [048604].
The sequence of the HPPD protein taken as a starting point may be any amino acid sequence encoding a catalytically active HPPD protein. In one embodiment of the isolated nucleic acid of the invention, said HPPD protein comprises the amino acid sequence of SEQ ID No. 4 [Oryza sativa], wherein the resulting amino acid sequence comprises at least one amino acid selected from a) Ala, Asp, Glu, Gly, Lys, Gln, Arg, Ser, Thr, Val, Tyr, Ile, Leu or Met at position 247 of the amino acid sequence of SEQ ID No. 4 (corresponding to position 250 of SEQ ID No. 2);
b) Asp, Glu, Ile, Lys, Leu, Asn, Pro, Arg, Ser, Gly, His or Tyr at position 248 of the amino acid sequence of SEQ ID No. 4 (corresponding to position 251 of SEQ ID
No. 2);
Phe, Gly, His, Ile, Leu, Met, Asn, Gin, Arg, Ser, Val, Tyr or Thr at position 249 of the amino acid sequence of SEQ ID No. 4 (corresponding to position 252 of SEQ
ID No. 2) ;
d) Ala, His, Gin, Val, Trp, lie, Leu or Met at position 250 of the amino acid sequence of SEQ ID No. 4 (corresponding to position 253 of SEQ ID No. 2);

e) Val or Met at position 262 of the amino acid sequence of SEQ ID No. 4 (corresponding to position 265 of SEQ ID No. 2);
f) Leu, Gin, Arg, Tyr, Ala, Ile, Lys or Met at position 265 of the amino acid sequence of SEQ ID No. 4 (corresponding to position 268 of SEQ ID No. 2);
g) Ala or Thr at position 266 of the amino acid sequence of SEQ ID No. 4 (corresponding to position 269 of SEQ ID No. 2);
h) Ala, Ile, Asn, Thr or Val at position 277 of the amino acid sequence of SEQ
ID No.
4 (corresponding to position 280 of SEQ ID No. 2);
i) Leu, Val, Ala, Phe, Gly, Met, Arg or Ser at position 290 of the amino acid sequence of SEQ ID No. 4 (corresponding to position 293 of SEQ ID No. 2);
j) Met, Ala, Pro, Ser, Thr or Val at position 291 of the amino acid sequence of SEQ
ID No. 4 (corresponding to position 294 of SEQ ID No. 2);
k) Ile, Met or Asn at position 332 of the amino acid sequence of SEQ ID No. 4 (corresponding to position 335 of SEQ ID No. 2);
l) Met at position 365 of the amino acid sequence of SEQ ID No. 4 (corresponding to position 368 of SEQ ID No. 2);
m) Tyr at position 378 of the amino acid sequence of SEQ ID No. 4 (corresponding to position 381 of SEQ ID No. 2);
n) Ser at position 389 of the amino acid sequence of SEQ ID No. 4 (corresponding to position 392 of SEQ ID No. 2);
o) Tyr at position 416 of the amino acid sequence of SEQ ID No. 4 (corresponding to position 419 of SEQ ID No. 2);
p) Asp, Phe, Asn, Ala, Cys, Glu, Gly, His, Ile, Leu, Met, Gin, Ser, Thr or Val at position 418 of the amino acid sequence of SEQ ID No. 4 (corresponding to position 421 of SEQ ID No. 2);
q) Ala, Met, Pro or Thr at position 419 of the amino acid sequence of SEQ ID
No. 4 (corresponding to position 422 of SEQ ID No. 2);
r) Ala, Ile or Val at position 421 of the amino acid sequence of SEQ ID No. 4 (corresponding to position 424 of SEQ ID No. 2);
s) Ile, Pro, Arg, Ala, Gly, Lys, Asn or Gin at position 422 of the amino acid sequence of SEQ ID No. 4 (corresponding to position 425 of SEQ ID No. 2);

. , . .

t) Phe, Thr, Val, Ala, Gin or Ser at position 423 of the amino acid sequence of SEQ
ID No. 4 (corresponding to position 426 of SEQ ID No. 2);
u) Met, Gin, Val, Phe or Leu at position 428 of the amino acid sequence of SEQ
ID
No. 4 (corresponding to position 431 of SEQ ID No. 2);
v) Phe at position 431 of the amino acid sequence of SEQ ID No. 4 (corresponding to position 434 of SEQ ID No. 2);
w) Ala, Cys, Gly or Thr at position 225 of the amino acid sequence of SEQ ID
No. 4 (corresponding to position 228 of SEQ ID No. 2);
x) Ala, Glu, Gly, Lys, Leu, Asn, Gin, Arg, Ser, Thr or Tyr at position 245 of the amino acid sequence of SEQ ID No. 4 (corresponding to position 248 of SEQ ID No. 2);
y) Ala, Ile, Met or Val at position 267 of the amino acid sequence of SEQ ID
No. 4 (corresponding to position 270 of SEQ ID No. 2);
z) Glu, His, Ile, Lys, Leu, Met, Arg, Ser, Thr or Val at position 268 of the amino acid sequence of SEQ ID No. 4 (corresponding to position 271 of SEQ ID No. 2);
aa)His at position 376 of the amino acid sequence of SEQ ID No. 4 (corresponding to position 379 of SEQ ID No. 2);
bb)Arg at a position in an HPPD protein, said position corresponding to position 424 of the amino acid sequence of SEQ ID No. 4 (corresponding to position 427 of SEQ ID No. 2).
In another embodiment of the isolated nucleic acid of the invention, said HPPD

protein comprises the amino acid sequence of SEQ ID No. 6 [Zea mays], wherein the resulting amino acid sequence comprises at least one amino acid selected from a) Ala, Asp, Glu, Gly, Lys, Gin, Arg, Ser, Thr, Val, Tyr, Ile, Leu or Met at position 224 of the amino acid sequence of SEQ ID No. 6 (corresponding to position 250 of SEQ ID No. 2);
b) Asp, Glu, lie, Lys, Leu, Asn, Pro, Arg, Ser, Gly, His or Tyr at position 225 of the amino acid sequence of SEQ ID No. 6 (corresponding to position 251 of SEQ
ID No. 2);
c) Phe, Gly, His, Ile, Leu, Met, Asn, Gin, Arg, Ser, Val, Tyr or Thr at position 226 of the amino acid sequence of SEQ ID No. 6 (corresponding to position 252 of SEQ ID No. 2);

, d) Ala, His, Gin, Val, Trp, Ile, Leu or Met at position 227 of the amino acid sequence of SEQ ID No. 6 (corresponding to position 253 of SEQ ID No. 2);
e) Val or Met at position 239 of the amino acid sequence of SEQ ID No. 6 (corresponding to position 265 of SEQ ID No. 2);
5 f) Leu, Gin, Arg, Val, Tyr, Ala, Ile or Lys at position 242 of the amino acid sequence of SEQ ID No. 6 (corresponding to position 268 of SEQ ID No. 2);
g) Ala or Thr at position 243 of the amino acid sequence of SEQ ID No. 6 (corresponding to position 269 of SEQ ID No. 2);
h) Ala, Ile, Asn, Thr or Val at position 254 of the amino acid sequence of SEQ ID
10 No. 6 (corresponding to position 280 of SEQ ID No. 2);
i) Leu, Val, Ala, Phe, Gly, Met, Arg or Ser at position 267 of the amino acid sequence of SEQ ID No. 6 (corresponding to position 293 of SEQ ID No. 2);
j) Met at position 268 of the amino acid sequence of SEQ ID No. 6 (corresponding to position 294 of SEQ ID No. 2);
15 k) His or Asn at position 281 of the amino acid sequence of SEQ ID No. 6 (corresponding to position 307 of SEQ ID No. 2);
I) Ile, Asn or Leu at position 309 of the amino acid sequence of SEQ ID
No. 6 (corresponding to position 335 of SEQ ID No. 2);
m) Met at position 342 of the amino acid sequence of SEQ ID No. 6 (corresponding 20 to position 368 of SEQ ID No. 2);
n) Tyr at position 355 of the amino acid sequence of SEQ ID No. 6 (corresponding to position 381 of SEQ ID No. 2);
o) Ser at position 366 of the amino acid sequence of SEQ ID No. 6 (corresponding to position 392 of SEQ ID No. 2);
25 p) Tyr at position 393 of the amino acid sequence of SEQ ID No. 6 (corresponding to position 419 of SEQ ID No. 2);
q) Asp, Phe, Asn, Ala, Cys, Glu, Gly, His, Ile, Leu, Met, Gin, Ser, Thr or Val at position 395 of the amino acid sequence of SEQ ID No. 6 (corresponding to position 421 of SEQ ID No. 2);
30 r) Ala, Met, Pro or Thr at position 396 of the amino acid sequence of SEQ ID No.
6 (corresponding to position 422 of SEQ ID No. 2);

s) Ala, Ile or Val at position 398 of the amino acid sequence of SEQ ID No.

(corresponding to position 424 of SEQ ID No. 2);
t) Ile, Pro, Arg, Ala, Gly, Lys, Asn or Gin at position 399 of the amino acid sequence of SEQ ID No. 6 (corresponding to position 425 of SEQ ID No. 2);
u) Glu, Phe, Thr, Val, Ala or Ser at position 400 of the amino acid sequence of SEQ ID No. 6 (corresponding to position 426 of SEQ ID No. 2);
v) Met, Gin, Val, Phe or Leu at position 405 of the amino acid sequence of SEQ ID
No. 6 (corresponding to position 431 of SEQ ID No. 2);
w) Ala, Cys, Gly or Thr at a position in an HPPD protein, said position corresponding to position 202 of the amino acid sequence of SEQ ID No. 6 (corresponding to position 228 of SEQ ID No. 2);
x) Ala, Glu, Gly, Lys, Leu, Asn, Gin, Arg, Ser, Thr or Tyr at position 222 of the amino acid sequence of SEQ ID No. 6 (corresponding to position 248 of SEQ
ID No. 2);
y) Ala, Ile, Met or Val at position 244 of the amino acid sequence of SEQ ID
No. 6 (corresponding to position 270 of SEQ ID No. 2);
z) Glu, His, Ile, Lys, Leu, Met, Arg, Ser, Thr or Val at position 245 of the amino acid sequence of SEQ ID No. 6 (corresponding to position 271 of SEQ ID No.
2);
aa) Gin at position 353 of the amino acid sequence of SEQ ID No. 6 (corresponding to position 379 of SEQ ID No. 2);
ab) Arg at position 401 of the amino acid sequence of SEQ ID No. 6 (corresponding to position 427 of SEQ ID No. 2).
In another embodiment of the isolated nucleic acid of the invention, said HPPD
protein comprises the amino acid sequence of SEQ ID No. 8 [Avena sativa], wherein the resulting amino acid sequence comprises at least one amino acid selected from a) Ala, Asp, Glu, Gly, Lys, Gin, Arg, Ser, Thr, Val, Tyr, Ile, Leu or Met at position 241 of the amino acid sequence of SEQ ID No. 8 (corresponding to position 250 of SEQ ID No. 2), preferably Ala;

. .

b) Asp, Glu, Ile, Lys, Leu, Asn, Pro, Arg, Ser, Gly, His or Tyr at position 242 of the amino acid sequence of SEQ ID No. 8 (corresponding to position 251 of SEQ
ID No. 2), preferably Arg or Lys;
c) Phe, Gly, His, Ile, Leu, Met, Asn, Gin, Arg, Ser, Val, Tyr or Thr at position 243 of the amino acid sequence of SEQ ID No. 8 (corresponding to position 252 of SEQ ID No. 2), preferably Tyr;
d) Ala, His, Gin, Val, Trp, Ile, Leu or Met at position 244 of the amino acid sequence of SEQ ID No. 8 (corresponding to position 253 of SEQ ID No. 2), preferably Val;
e) Val or Met at position 256 of the amino acid sequence of SEQ ID No. 8 (corresponding to position 265 of SEQ ID No. 2);
f) Leu, Gin, Arg, Tyr, Ala, Ile, Lys or Met at position 259 of the amino acid sequence of SEQ ID No. 8 (corresponding to position 268 of SEQ ID No. 2);
g) Ala or Thr at position 260 of the amino acid sequence of SEQ ID No. 8 (corresponding to position 269 of SEQ ID No. 2), preferably Ala;
h) Ala, Ile, Asn, Thr or Val at position 271 of the amino acid sequence of SEQ ID
No. 8 (corresponding to position 280 of SEQ ID No. 2);
i) Leu, Val, Ala, Phe, Gly, Met, Arg or Ser at position 284 of the amino acid sequence of SEQ ID No. 8 (corresponding to position 293 of SEQ ID No. 2);
j) Met, Ala, Pro, Ser, Thr or Val at position 285 of the amino acid sequence of SEQ ID No. 8 (corresponding to position 294 of SEQ ID No. 2);
k) His or Asn at position 265 of the amino acid sequence of SEQ ID
No. 8 (corresponding to position 307 of SEQ ID No. 2);
I) Ile or Asn or Leu at position 326 of the amino acid sequence of SEQ ID No. 8 (corresponding to position 335 of SEQ ID No. 2);
m) Met at position 359 of the amino acid sequence of SEQ ID No. 8 (corresponding to position 368 of SEQ ID No. 2);
n) Tyr at position 372 of the amino acid sequence of SEQ ID No. 8 (corresponding to position 381 of SEQ ID No. 2);
o) Ser at position 383 of the amino acid sequence of SEQ ID No. 8 (corresponding to position 392 of SEQ ID No. 2);

. .
' p) Tyr at position 410 of the amino acid sequence of SEQ ID No. 8 (corresponding to position 419 of SEQ ID No. 2);
q) Asp, Phe, Asn, Ala, Cys, Glu, Gly, His, Ile, Leu, Met, Gin, Ser, Thr or Val at position 412 of the amino acid sequence of SEC) ID No. 8 (corresponding to position 421 of SEQ ID No. 2), preferably Lys;
r) Ala, Met, Pro or Thr at position 413 of the amino acid sequence of SEQ
ID No.
8 (corresponding to position 422 of SEQ ID No. 2);
s) Ala, Ile or Val at position 415 of the amino acid sequence of SEQ ID No.

(corresponding to position 424 of SEQ ID No. 2);
t) Ile, Pro, Arg, Ala, Gly, Lys, Asn or Gin at position 416 of the amino acid sequence of SEQ ID No. 8 (corresponding to position 425 of SEQ ID No. 2);
u) Phe, Thr, Val, Ala, Gin or Ser at position 417 of the amino acid sequence of SEQ ID No. 8 (corresponding to position 426 of SEQ ID No. 2);
v) Met, Gin, Val, Phe or Leu at position 422 of the amino acid sequence of SEQ ID
No. 8 (corresponding to position 431 of SEQ ID No. 2);
w) Ala, Cys, Gly or Thr at position 219 of the amino acid sequence of SEQ
ID No.
8 (corresponding to position 228 of SEQ ID No. 2);
x) Ala, Glu, Gly, Lys, Leta, Asn, Gin, Arg, Ser, Thr or Tyr at position 239 of the amino acid sequence of SEQ ID No. 8 (corresponding to position 248 of SEQ
ID No. 2);
y) Ala, Ile, Met or Val at position 261 of the amino acid sequence of SEQ
ID No. 8 (corresponding to position 270 of SEQ ID No, 2);
z) Glu, His, Ile, Lys, Leu, Met, Arg, Ser, Thr or Vat at position 262 of the amino acid sequence of SEQ ID No. 8 (corresponding to position 271 of SEQ ID No.
2);
aa) His at position 370 of the amino acid sequence of SEQ ID No. 8 (corresponding to position 379 of SEQ ID No. 2);
bb) Arg at position 418 of the amino acid sequence of SEQ ID No. 8 (corresponding to position 427 of SEQ ID No. 2).
In another embodiment, of the isolated nucleic acid of the invention, said HPPD
protein comprises the amino acid sequence of SEQ ID No. 10 [Pseudomonas . , ' , fluorescens], wherein the resulting amino acid sequence comprises at least one amino acid selected from a) Phe, Asp, Glu, Gly, Lys, Gin, Arg, Ser, Thr, Val, Tyr, Ile, Leu or Met at position 188 of the amino acid sequence of SEQ ID No. 10 (corresponding to position 250 of SEQ ID No. 2), preferably Ala;
b) Ala, Asp, Glu, Ile, Lys, Leu, Asn, Pro, Ser, Gly, His or Tyr at position 189 of the amino acid sequence of SEQ ID No. 10 (corresponding to position 251 of SEQ
ID No. 2), preferably Ala or Lys;
c) Phe, Gly, His, Ile, Leu, Met, Asn, Gin, Arg, Ser, Val, Glu or Thr at position 190 of the amino acid sequence of SEQ ID No. 10 (corresponding to position 252 of SEQ ID No. 2), preferably Ser or Tyr;
d) Ala, His, Gin, Val, Trp, Ile, Leu or Met at position 191 of the amino acid sequence of SEQ ID No. 10 (corresponding to position 253 of SEQ ID No. 2), preferably Phe or Val;
e) Val or Met at position 200 of the amino acid sequence of SEQ ID No. 10 (corresponding to position 265 of SEQ ID No. 2);
f) Leu, Gin, Arg, Val Tyr, Ala, Ile or Met at position 203 of the amino acid sequence of SEQ ID No. 10 (corresponding to position 268 of SEQ ID No. 2);
g) Thr or Val at position 204 of the amino acid sequence of SEQ ID No. 10 (corresponding to position 269 of SEQ ID No. 2), preferably Val;
h) Ala, Ile, Asn, Thr or Val at position 215 of the amino acid sequence of SEQ ID
No. 10 (corresponding to position 280 of SEQ ID No. 2);
i) Leu, Val, Ala, Phe, Gly, Met, Arg or Ser at position 226 of the amino acid sequence of SEQ ID NO:SEQ ID No. 10 (corresponding to position 293 of SEQ
ID No. 2);
j) Met, Thr, Ala, Pro, Ser or Val at position 227 of the amino acid sequence of SEQ ID No. 10 (corresponding to position 294 of SEQ ID No. 2);
k) His or Asn at position 240 of the amino acid sequence of SEQ ID No. 10 (corresponding to position 307 of SEQ ID No. 2);
I) Ile, Asn or Leu at position 264 of the amino acid sequence of SEQ ID No.

(corresponding to position 335 of SEQ ID No. 2);

. .
, .
m) Met at position 295 of the amino acid sequence of SEQ ID No. 10 (corresponding to position 368 of SEQ ID No. 2);
n) Tyr at position 312 of the amino acid sequence of SEQ ID No. 10 (corresponding to position 381 of SEQ ID No. 2);
5 o) Ser at position 321 of the amino acid sequence of SEQ ID No. 10 (corresponding to position 392 of SEQ ID No. 2);
p) Tyr at position 333 of the amino acid sequence of SEQ ID No. 10 (corresponding to position 419 of SEQ ID No. 2);
q) Asp, Lys, Asn, Ala, Cys, Gly, His, Ile, Leu, Met, Gin, Ser, Thr or Val at position 10 335 of the amino acid sequence of SEQ ID No. 10 (corresponding to position 421 of SEQ ID No. 2), preferably Lys;
r) Ala, Met, Pro or Thr at position 336 of the amino acid sequence of SEQ
ID No.
10 (corresponding to position 422 of SEQ ID No. 2);
s) Ala, Ile or Val at position 338 of the amino acid sequence of SEQ ID No.

15 (corresponding to position 424 of SEQ ID No. 2);
t) Ile, Pro, Ala, Asn, Gly or Gin at position 339 of the amino acid sequence of SEQ
ID No. 10 (corresponding to position 425 of SEQ ID No. 2);
u) Phe, Thr or Val at position 340 of the amino acid sequence of SEQ ID No.

(corresponding to position 426 of SEQ ID No. 2);
20 v) Met, Phe, Leu, Gin or Val at position 345 of the amino acid sequence of SEQ ID
No. 10 (corresponding to position 431 of SEQ ID No. 2);
w) Ala, Cys, Gly or Val at position 164 of the amino acid sequence of SEQ
ID No.
10 (corresponding to position 228 of SEQ ID No. 2);
x) Ala, Glu, Gly, His, Lys, Leu, Asn, Gin, Ser, Thr or Tyr at position 186 of the 25 amino acid sequence of SEQ ID No. 10 (corresponding to position 248 of SEQ
ID No. 2);
y) Ala, Ile, Leu or Val at position 205 of the amino acid sequence of SEQ
ID No.
10 (corresponding to position 270 of SEQ ID No. 2);
z) Ala, Glu, His, Ile, Lys, Leu, Met, Arg, Thr or Val at position 206 of the amino 30 acid sequence of SEQ ID No. 10 (corresponding to position 271 of SEQ ID
No.
2);

. .
. .

aa) His at position 310 of the amino acid sequence of SEQ ID No. 10 (corresponding to position 379 of SEQ ID No. 2);
bb) Arg at position 341 of the amino acid sequence of SEQ ID No. 10 (corresponding to position 427 of SEQ ID No. 2).
In another embodiment of the isolated nucleic acid of the invention, said HPPD

protein comprises the amino acid sequence of SEQ ID No. 14 [Synechococcus sp.], wherein the resulting amino acid sequence comprises at least one amino acid selected from a) Ala, Asp, Glu, Phe, Gly, Lys, Gin, Arg, Ser, Thr, Val, Tyr, Ile or Met at position 169 of the amino acid sequence of SEQ ID No. 14 (corresponding to position 250 of SEQ ID No. 2), preferably Ala or Phe;
b) Ala, Asp, Glu, Ile, Lys, Leu, Asn, Pro, Arg or Ser, Gly or His at position 170 of the amino acid sequence of SEQ ID No. 14 (corresponding to position 251 of SEQ ID No. 2), preferably Ala, Arg or Lys;
c) Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Gln, Ser, Val, Tyr or Thr at position 171 of the amino acid sequence of SEQ ID No. 14 (corresponding to position 252 of SEQ ID No. 2), preferably Glu, Ser or Tyr;
d) Ala, Phe, His, Gin, Val or Ile, Leu or Met at position 172 of the amino acid sequence of SEQ ID No, 14 (corresponding to position 253 of SEQ ID No. 2), preferably Phe or Val;
e) Val or Met at position 181 of the amino acid sequence of SEQ ID No. 14 (corresponding to position 265 of SEQ ID No. 2);
f) Leu, Gin, Arg, Tyr, Ala, Ile, Lys or Met at position 184 of the amino acid sequence of SEQ ID No. 14 (corresponding to position 268 of SEQ ID No. 2);
g) Ala or Thr at position 185 of the amino acid sequence of SEQ ID No. 14 (corresponding to position 269 of SEQ ID No. 2), preferably Ala;
h) Ile, Asn, Pro, Thr or Val at position 196 of the amino acid sequence of SEQ ID
No. 14 (corresponding to position 280 of SEQ ID No. 2), preferably Pro;
i) Leu, Val, Ala, Phe, Gly, Met, Arg or Ser at position 206 of the amino acid sequence of SEQ ID No. 14 (corresponding to position 293 of SEQ ID No. 2);

j) Met , Ala, Pro, Ser, Thr or Val at position 207 of the amino acid sequence of SEQ ID No. 14 (corresponding to position 294 of SEQ ID No. 2);
k) His or Asn at position 220 of the amino acid sequence of SEQ ID No. 14 (corresponding to position 307 of SEQ ID No. 2);
I) Ile, Met or Asn at position 244 of the amino acid sequence of SEQ ID No.

(corresponding to position 335 of SEQ ID No. 2), preferably Met;
m) Met at position 286 of the amino acid sequence of SEQ ID No. 14 (corresponding to position 368 of SEQ ID No. 2);
n) Phe or Tyr at position 301 of the amino acid sequence of SEQ ID No. 14 (corresponding to position 381 of SEQ ID No. 2);
o) Ser at position 312 of the amino acid sequence of SEQ ID No. 14 (corresponding to position 392 of SEQ ID No. 2);
p) Tyr at position 325 of the amino acid sequence of SEQ ID No. 14 (corresponding to position 419 of SEQ ID No. 2);
q) Asp, Phe, Lys, Asn, Ala, Cys, Gly, His, Ile, Leu, Met, Gin, Ser, Thr or Val at position 327 of the amino acid sequence of SEQ ID No. 14 (corresponding to position 421 of SEQ ID No. 2), preferably Lys;
r) Gly, Met, Pro or Thr at position 328 of the amino acid sequence of SEQ ID No.
14 (corresponding to position 422 of SEQ ID No. 2);
s) Ala, Ile or Val at position 330 of the amino acid sequence of SEQ ID No. 14 (corresponding to position 424 of SEQ ID No. 2);
t) Ile, Pro, Arg, Ser, Ala, Gly, Lys or Asn at position 331 of the amino acid sequence of SEQ ID No. 14 (corresponding to position 425 of SEQ ID No. 2);
u) Glu, Phe, Thr, Val, Gln or Ser at position 332 of the amino acid sequence of SEQ ID No. 14 (corresponding to position 426 of SEQ ID No. 2), preferably Glu;
v) Ile, Met, Gln, Val or Phe at position 337 of the amino acid sequence of SEQ ID
No. 14 (corresponding to position 431 of SEQ ID No. 2);
w) Ala, Cys, Gly or Thr at position 145 of the amino acid sequence of SEQ
ID No.
14 (corresponding to position 228 of SEQ ID No. 2);
x) Ala, Glu, Gly, His, Lys, Leu, Asn, Gln, Ser, Thr or Tyr at position 167 of the amino acid sequence of SEQ ID No. 14 (corresponding to position 248 of SEQ
ID No. 2);

. .

y) Ala, Ile, Leu or Met at position 186 of the amino acid sequence of SEQ
ID No.
14 (corresponding to position 270 of SEQ ID No. 2);
z) Ala, Glu, His, Ile, Lys, Leu, Met, Arg, Ser, Thr or Val at position 187 of the amino acid sequence of SEQ ID No. 14 (corresponding to position 271 of SEQ
ID No. 2);
aa) His at a position at position 299 of the amino acid sequence of SEQ ID No.

(corresponding to position 379 of SEQ ID No. 2);
bb) Arg at a position at position 333 of the amino acid sequence of SEQ ID No.

(corresponding to position 427 of SEQ ID No. 2).
In another embodiment of the isolated nucleic acid of the invention, said HPPD

protein comprises the amino acid sequence of SEQ ID No. 16 [Blepharisma japonicum], wherein the resulting amino acid sequence comprises at least one amino acid selected from a) Ala, Asp, Glu, Phe, Gly, Lys, Gin, Arg, Ser, Thr, Val, Ile, Leu or Met at position 209 of the amino acid sequence of SEQ ID No. 16 (corresponding to position 250 of SEQ ID No. 2), preferably Ala or Phe;
b) Ala, Asp, Glu, Ile, Lys, Leu, Asn, Pro, Arg, Ser, Gly, His or Tyr at position 210 of the amino acid sequence of SEQ ID No. 16 (corresponding to position 251 of SEQ ID No. 2), preferably Ala, Arg, Lys;
c) Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Gin, Arg, Val, Tyr or Thr at position 211 of the amino acid sequence of SEQ ID No. 16 (corresponding to position 252 of SEQ ID No. 2), preferably Glu or Tyr;
d) Phe, His, Gin, Val, Trp, Ile, Leu or Met at position 212 of the amino acid sequence of SEQ ID No. 16 (corresponding to position 253 of SEQ ID No. 2), preferably Phe or Val;
e) Val or Met at position 224 of the amino acid sequence of SEQ ID No. 16 (corresponding to position 265 of SEQ ID No. 2);
f) Leu, Gin, Arg, Tyr, Ala, Ile, Lys or Met at position 227 of the amino acid sequence of SEQ ID No. 16 (corresponding to position 2268 of SEQ ID No. 2);
g) Ala or Thr at position 228 of the amino acid sequence of SEQ ID No. 16 (corresponding to position 269 of SEQ ID No. 2), preferably Ala;

h) Ala, Ile, Asn, Thr or Val at position 239 of the amino acid sequence of SEQ ID
No. 16 (corresponding to position 280 of SEQ ID No. 2);
i) Leu, Val, Ala, Phe, Gly, Met, Arg or Ser at position 251 of the amino acid sequence of SEQ ID No. 16 (corresponding to position 293 of SEQ ID No. 2);
j) Met, Ala, Pro, Ser, Thr or Val at position 252 of the amino acid sequence of SEQ ID No. 16 (corresponding to position 294 of SEQ ID No. 2);
k) His or Asn at position 265 of the amino acid sequence of SEQ ID No.

(corresponding to position 307 of SEQ ID No. 2);
I) Ile, Met or Asn at position 289 of the amino acid sequence of SEQ ID
No. 16 (corresponding to position 335 of SEQ ID No. 2), preferably Met;
m) Met at position 323 of the amino acid sequence of SEQ ID No. 16 (corresponding to position 368 of SEQ ID No. 2);
n) Tyr at position 336 of the amino acid sequence of SEQ ID No. 16 (corresponding to position 381 of SEQ ID No. 2);
o) Ser at position 347 of the amino acid sequence of SEQ ID No. 16 (corresponding to position 292 of SEQ ID No. 2);
p) Tyr at position 359 of the amino acid sequence of SEQ ID No. 16 (corresponding to position 419 of SEQ ID No. 2);
q) Asp, Phe, Lys, Asn, Ala, Cys, Glu, Gly, His, Leu, Met, Gin, Ser, Thr or Val at position 361 of the amino acid sequence of SEQ ID No. 16 (corresponding to position 421 of SEQ ID No. 2), preferably Lys;
r) Ala, Met, Pro or Thr at position 362 of the amino acid sequence of SEQ
ID No.
16 (corresponding to position 422 of SEQ ID No. 2);
s) Ala, Ile or Val at position 364 of the amino acid sequence of SEQ ID No.

(corresponding to position 424 of SEQ ID No. 2);
t) Ile, Pro, Arg, Ser, Ala, Gly, Asn or Gin at position 365 of the amino acid sequence of SEQ ID No. 16 (corresponding to position 425 of SEQ ID No. 2);
u) Glu, Phe, Thr, Val, Gln or Ser at position 366 of the amino acid sequence of SEQ ID No. 16 (corresponding to position 426 of SEQ ID No. 2), preferably Glu;
v) Ile, Met, Gln, Val or Phe at position 371 of the amino acid sequence of SEQ
ID
No. 16 (corresponding to position 431 of SEQ ID No. 2);

. , ' , w) Ala, Cys, Gly or Thr at a position in an HPPD protein, said position corresponding to position 185 of the amino acid sequence of SEQ ID No. 16 (corresponding to position 228 of SEQ ID No. 2);
x) Ala, Glu, Gly, Lys, Leu, Asn, Gin, Arg, Ser, Thr or Tyr at a position in an HPPD
5 protein, said position corresponding to position 207 of the amino acid sequence of SEQ ID No. 16 (corresponding to position 248 of SEQ ID No. 2);
y) Ala, Ile, Leu or Met at a position in an HPPD protein, said position corresponding to position 229 of the amino acid sequence of SEQ ID No. 16 (corresponding to position 270 of SEQ ID No. 2);
10 z) Glu, His, Ile, Lys, Leu, Met, Arg, Ser, Thr or Val at a position in an HPPD
protein, said position corresponding to position 230 of the amino acid sequence of SEQ ID No. 16 (corresponding to position 271 of SEQ ID No. 2);
aa) His at a position in an HPPD protein, said position corresponding to position 334 of the amino acid sequence of SEQ ID No, 16 (corresponding to position 15 379 of SEQ ID No. 2);
bb) Arg at a position in an HPPD protein, said position corresponding to position 367 of the amino acid sequence of SEQ ID No, 16 (corresponding to position 427 of SEQ ID No. 2).
20 In another embodiment, of the isolated nucleic acid of the invention, said HPPD
protein comprises the amino acid sequence of SEQ ID No. 18 [Rhodococcus RHA1 sp. ro0240], wherein the resulting amino acid sequence comprises at least one amino acid selected from a) Ala, Asp, Glu, Phe, Gly, Lys, Gin, Arg, Ser, Thr, Val, Tyr, Ile or Leu at position 25 231 of the amino acid sequence of SEQ ID No, 18 (corresponding to position 250 of SEQ ID No. 2), preferably Phe or Ala;
b) Asp, Glu, Ile, Lys, Leu, Asn, Pro, Arg, Ser, Gly, His or Tyr at position 232 of the amino acid sequence of SEQ ID No. 18 (corresponding to position 251 of SEQ
ID No. 2), preferably Arg or Lys;
30 c) Phe, Gly, His, Ile, Leu, Met, Asn, Gin, Arg, Ser, Val, Tyr or Thr at position 233 of the amino acid sequence of SEQ ID No. 18 (corresponding to position 252 of SEQ ID No. 2), preferably Ser or Tyr;

d) Ala, His, Gin, Val, Trp, Ile, Leu or Met at position 234 of the amino acid sequence of SEQ ID No. 18 (corresponding to position 253 of SEQ ID No. 2), preferably Val;
e) Val or Met at position 246 of the amino acid sequence of SEQ ID No. 18 (corresponding to position 265 of SEQ ID No. 2);
f) Leu, Gin, Arg, Val, Tyr, Ala, Ile or Met at position 249 of the amino acid sequence of SEQ ID No. 18 (corresponding to position 268 of SEQ ID No. 2);
g) Ala or Thr at position 250 of the amino acid sequence of SEQ ID No. 18 (corresponding to position 269 of SEQ ID No. 2), preferably Ala;
h) Ala, Ile, Asn, Thr or Val at position 261 of the amino acid sequence of SEQ
ID
No. 18 (corresponding to position 280 of SEQ ID No. 2);
i) Leu, Val, Ala, Phe, Gly, Met, Arg or Ser at position 273 of the amino acid sequence of SEQ ID No. 18 (corresponding to position 293 of SEQ ID No. 2);
j) Met, Ala, Pro, Ser, Thr or Val at position 274 of the amino acid sequence of SEQ ID No. 18 (corresponding to position 294 of SEQ ID No. 2);
k) His or Asn at position 287 of the amino acid sequence of SEQ ID No. 18 (corresponding to position 307 of SEQ ID No. 2);
I) Ile, Met or Asn at position 311 of the amino acid sequence of SEQ ID
No. 18 (corresponding to position 335 of SEQ ID No. 2), preferably Met;
m) Met at position 343 of the amino acid sequence of SEQ ID No. 18 (corresponding to position 368 of SEQ ID No. 2);
n) Tyr at position 356 of the amino acid sequence of SEQ ID No. 18 (corresponding to position 381 of SEQ ID No. 2);
a) Ser at position 367 of the amino acid sequence of SEQ ID No. 18 (corresponding to position 392 of SEQ ID No. 2);
p) Tyr at position 379 of the amino acid sequence of SEQ ID No. 18 (corresponding to position 419 of SEQ ID No. 2);
q) Asp, Lys, Asn, Ala, Cys, Glu, Gly, His, Leu, Met, Gin, Ser, Thr or Val at position 381 of the amino acid sequence of SEQ ID No. 18 (corresponding to position 421 of SEQ ID No. 2), preferably Lys;
r) Ala, Met, Pro or Thr at position 382 of the amino acid sequence of SEQ
ID No.
18 (corresponding to position 422 of SEQ ID No. 2);

s) Ala, Ile or Val at position 384 of the amino acid sequence of SEQ ID No, (corresponding to position 424 of SEQ ID No. 2);
t) Ile, Pro, Arg, Ser, Ala, Gly, Asn or Gin at position 385 of the amino acid sequence of SEQ ID No. 18 (corresponding to position 425 of SEQ ID No. 2);
u) Glu, Phe, Thr, Val, Gin or Ser at position 386 of the amino acid sequence of SEQ ID No. 18 (corresponding to position 426 of SEQ ID No. 2), preferably Glu;
v) Met, Gin, Phe, Val, Phe or Leu at position 391 of the amino acid sequence of SEQ ID No. 18 (corresponding to position 431 of SEQ ID No. 2);
w) Ala, Cys, Gly or Thr at a position in an HPPD protein, said position corresponding to position 207 of the amino acid sequence of SEQ ID No. 1 (corresponding to position 228 of SEQ ID No. 2)8;
x) Ala, Glu, Gly, His, Lys, Leu, Asn, Gin, Arg, Ser or Tyr at a position in an HPPD
protein, said position corresponding to position 229 of the amino acid sequence of SEQ ID No. 18 (corresponding to position 248 of SEQ ID No. 2);
y) Ala, Ile, Leu or Met at a position in an HPPD protein, said position corresponding to position 251 of the amino acid sequence of SEQ ID No. 18 (corresponding to position 270 of SEQ ID No. 2);
z) Ala, Glu, His, Ile, Lys, Leu, Met, Arg, Thr or Val at a position in an HPPD protein, said position corresponding to position 252 of the amino acid sequence of SEQ
ID No. 18 (corresponding to position 271 of SEQ ID No. 2);
aa) His at a position in an HPPD protein, said position corresponding to position 354 of the amino acid sequence of SEQ ID No. 18 (corresponding to position 379 of SEQ ID No. 2);
bb) Arg at a position in an HPPD protein, said position corresponding to position 387 of the amino acid sequence of SEQ ID No. 18 (corresponding to position 427 of SEQ ID No. 2).
In another embodiment, of the isolated nucleic acid of the invention, said HPPD
protein comprises the amino acid sequence of SEQ ID No. 30 [Rhodococcus RHA1 sp. 0341], wherein the resulting amino acid sequence comprises at least one amino acid selected from a) Ala, Asp, Glu, Phe, Gly, Lys, Gin, Arg, Ser, Thr, Val, Tyr, Ile or Leu at position 232 of the amino acid sequence of SEQ ID No. 30 (corresponding to position 250 of SEQ ID No. 2), preferably Phe or Ala;
b) Asp, Glu, Ile, Lys, Leu, Asn, Pro, Arg, Ser, Gly, His or Tyr at position 233 of the amino acid sequence of SEQ ID No. 30 (corresponding to position 251 of SEQ ID
No. 2), preferably Arg or Lys;
c) Phe, Gly, His, Ile, Leu, Met, Asn, Gin, Arg, Ser, Val, Tyr or Thr at position 234 of the amino acid sequence of SEQ ID No. 30 (corresponding to position 252 of SEC) ID No. 2), preferably Ser or Tyr;
d) Ala, His, Gin, Val, Trp, Ile, Leu or Met at position 235 of the amino acid sequence of SEQ ID No. 30 (corresponding to position 253 of SEQ ID No. 2), preferably Val;
e) Val or Met at position 247 of the amino acid sequence of SEQ ID No. 30 (corresponding to position 265 of SEQ ID No. 2);
f) Leu, Gin, Arg, Val, Tyr, Ala, Ile or Met at position 250 of the amino acid sequence of SEQ ID No. 30 (corresponding to position 268 of SEQ ID No. 2);
g) Ala or Thr at position 251 of the amino acid sequence of SEQ ID No. 30 (corresponding to position 269 of SEQ ID No. 2), preferably Ala;
h) Ala, Ile, Asn, Thr or Val at position 262 of the amino acid sequence of SEQ
ID No.
30 (corresponding to position 280 of SEQ ID No. 2);
i) Leu, Val, Ala, Phe, Gly, Met, Arg or Ser at position 274 of the amino acid sequence of SEQ ID No. 30 (corresponding to position 293 of SEQ ID No. 2);
j) Met, Ala, Pro, Ser, Thr or Val at position 275 of the amino acid sequence of SEQ
ID No. 30 (corresponding to position 294 of SEQ ID No. 2);
k) His or Asn at position 288 of the amino acid sequence of SEQ ID No. 30 (corresponding to position 307 of SEQ ID No. 2);
I) Ile, Met or Asn at position 312 of the amino acid sequence of SEQ ID
No. 30 (corresponding to position 335 of SEQ ID No. 2), preferably Met;
m) Met at position 344 of the amino acid sequence of SEQ ID No. 30 (corresponding to position 368 of SEQ ID No. 2);
n) Tyr at position 357 of the amino acid sequence of SEQ ID No. 30 (corresponding to position 381 of SEQ ID No. 2);

. , ' 0) Ser at position 368 of the amino acid sequence of SEQ ID No. 30 (corresponding to position 392 of SEQ ID No. 2);
p) Tyr at position 380 of the amino acid sequence of SEQ ID No. 30 (corresponding to position 419 of SEQ ID No. 2);
q) Asp, Phe, Lys, Asn, Ala, Cys, Glu, Gly, His, Leu, Met, Gin, Ser, Thr or Val at position 382 of the amino acid sequence of SEQ ID No. 30 (corresponding to position 421 of SEQ ID No. 2), preferably Lys;
r) Ala, Met, Pro or Thr at position 383 of the amino acid sequence of SEQ ID
No. 30 (corresponding to position 422 of SEQ ID No. 2);
s) Ala, Ile or Val at position 385 of the amino acid sequence of SEQ ID No. 30 (corresponding to position 424 of SEQ ID No. 2);
t) Ile, Pro, Arg, Ser, Ala, Gly, Asn or Gin at position 386 of the amino acid sequence of SEQ ID No. 30 (corresponding to position 425 of SEQ ID No. 2);
u) Glu, Phe, Thr, Val, Gin or Ser at position 387 of the amino acid sequence of SEQ
ID No. 30 (corresponding to position 426 of SEQ ID No. 2), preferably Glu;
v) Met, Gin, Phe, Val, Phe or Leu at position 392 of the amino acid sequence of SEQ ID No. 30 (corresponding to position 431 of SEQ ID No. 2);
w) Ala, Cys, Gly or Thr at a position in an HPPD protein, said position corresponding to position 208 of the amino acid sequence of SEQ ID No. 30 (corresponding to position 228 of SEQ ID No. 2);
x) Ala, Glu, Gly, His, Lys, Leu, Asn, Gin, Arg, Ser or Tyr at a position in an HPPD
protein, said position corresponding to position 230 of the amino acid sequence of SEQ ID No. 30 (corresponding to position 248 of SEQ ID No. 2);
y) Ala, Ile, Leu or Met at a position in an HPPD protein, said position corresponding to position 252 of the amino acid sequence of SEQ ID No. 30 (corresponding to position 270 of SEQ ID No. 2);
z) Glu, His, Ile, Lys, Leu, Met, Arg, Thr or Val at a position in an HPPD
protein, said position corresponding to position 253 of the amino acid sequence of SEQ ID
No.
(corresponding to position 271 of SEQ ID No. 2);
30 aa)His at a position in an HPPD protein, said position corresponding to position 355 of the amino acid sequence of SEQ ID No. 30 (corresponding to position 379 of SEQ ID No. 2);

. .
' , bb)Arg at a position in an HPPD protein, said position corresponding to position 388 of the amino acid sequence of SEQ ID No. 30 (corresponding to position 427 of SEQ ID No. 2).
5 In another embodiment, of the isolated nucleic acid of the invention, said HPPD
protein comprises the amino acid sequence of SEQ ID No. 20 [Picrophilus torridus], wherein the resulting amino acid sequence comprises at least one amino acid selected from a) Ala, Asp, Glu, Phe, Gly, Lys, Gin, Arg, Ser, Thr, Val, Tyr, Ile or Met at position 10 201 of the amino acid sequence of SEQ ID No. 20 (corresponding to position 250 of SEQ ID No. 2), preferably Phe or Ala;
b) Ala, Asp, Glu, Lys, Leu, Asn, Pro, Arg or Ser, Gly, His or Trp at position 202 of the amino acid sequence of SEQ ID No. 20 (corresponding to position 251 of SEQ ID No. 2), preferably Ala, Arg or Lys;
15 c) Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Gin, Arg, Ser, Val or Tyr at position 203 of the amino acid sequence of SEQ ID No. 20 (corresponding to position 252 of SEQ ID No. 2), preferably Glu, Ser or Tyr;
d) Ala, His, Gin, Val, Trp, Ile, Leu or Met at position 204 of the amino acid sequence of SEQ ID No. 20 (corresponding to position 253 of SEQ ID No. 2), 20 preferably Val;
e) Val or Met at position 216 of the amino acid sequence of SEQ ID No. 20 (corresponding to position 265 of SEQ ID No. 2);
f) Leu, Gin, Arg, Val, Tyr, Ala, Ile or Met at position 219 of the amino acid sequence of SEQ ID No. 20 (corresponding to position 268 of SEQ ID No. 2);
25 g) Ala or Thr at position 220 of the amino acid sequence of SEQ ID
No. 20 (corresponding to position 269 of SEQ ID No. 2), preferably Ala;
h) Ala, Ile, Asn, Thr or Val at position 230 of the amino acid sequence of SEQ ID
No. 20 (corresponding to position 280 of SEQ ID No. 2);
i) Leu, Val, Ala, Phe, Gly, Met, Arg or Ser at position 242 of the amino acid 30 sequence of SEQ ID No. 20 (corresponding to position 293 of SEQ
ID No. 2);
j) Met, Ala, Pro, Ser, Thr or Val at position 243 of the amino acid sequence of SEQ ID No. 20 (corresponding to position 294 of SEQ ID No. 2);

. .
' k) His or Asn at position 256 of the amino acid sequence of SEQ ID
No. 20 (corresponding to position 307 of SEQ ID No. 2);
I) lie, Met or Asn at position 280 of the amino acid sequence of SEQ
ID No. 20 (corresponding to position 335 of SEQ ID No. 2), preferably Met;
m) Met at position 310 of the amino acid sequence of SEQ ID No. 20 (corresponding to position 368 of SEQ ID No. 2);
n) Phe or Tyr at position 323 of the amino acid sequence of SEQ ID No. 20 (corresponding to position 381 of SEQ ID No. 2);
o) Ser at position 334 of the amino acid sequence of SEQ ID No. 20 (corresponding to position 392 of SEQ ID No. 2);
p) Tyr at position 346 of the amino acid sequence of SEQ ID No. 20 (corresponding to position 419 of SEQ ID No. 2);
q) Asp, Phe, Lys, Ala, Cys, Glu, Gly, His, Ile, Leu, Met, Gin, Ser, Thr or Val at position 348 of the amino acid sequence of SEQ ID No. 20 (corresponding to position 421 of SEQ ID No. 2), preferably Lys;
r) Ala, Met, Pro or Thr at position 349 of the amino acid sequence of SEQ
ID No.
(corresponding to position 422 of SEQ ID No. 2);
s) Ala, Ile or Val at position 351 of the amino acid sequence of SEQ ID No.

(corresponding to position 424 of SEQ ID No. 2);
20 t) Ile, Pro, Arg, Ser, Ala, Gly, Lys or Gin at position 352 of the amino acid sequence of SEQ ID No. 20 (corresponding to position 425 of SEQ ID No. 2);
u) Glu, Phe, Thr, Val, Ala or Gin at position 353 of the amino acid sequence of SEQ ID No. 20 (corresponding to position 426 of SEQ ID No. 2), preferably Glu;
v) Met, Gin, Phe, Val, Phe or Leu at position 358 of the amino acid sequence of SEQ ID No. 20 (corresponding to position 431 of SEQ ID No. 2);
w) Ala, Cys, Gly or Thr at position 177 of the amino acid sequence of SEQ
ID No.
20 (corresponding to position 228 of SEQ ID No. 2);
x) Ala, Glu, Gly, His, Lys, Leu, Asn, Gin, Arg, Ser, Thr or Tyr at position 199 of the amino acid sequence of SEQ ID No. 20 (corresponding to position 248 of SEQ
ID No. 2);
y) Ala, Ile, Leu or Met at a position at position 221 of the amino acid sequence of SEQ ID No. 20 (corresponding to position 270 of SEQ ID No. 2);

, .

z) Ala, Glu, His, Ile, Leu, Met, Arg, Ser, Thr or Val at position 222 of the amino acid sequence of SEQ ID No. 20 (corresponding to position 271 of SEQ ID No.
2);
aa) His at position 321 of the amino acid sequence of SEQ ID No. 20 (corresponding to position 379 of SEQ ID No. 2);
bb) Arg at position 354 of the amino acid sequence of SEQ ID No. 20 (corresponding to position 427 of SEQ ID No. 2).
In another embodiment, of the isolated nucleic acid of the invention, said HPPD
protein comprises the amino acid sequence of SEQ ID No. 22 [Kordia algicidal wherein the resulting amino acid sequence comprises at least one amino acid selected from a) Ala, Asp, Glu, Phe, Gly, Lys, Gin, Arg, Ser, Thr, Val, Tyr, Leu or Met at position 219 of the amino acid sequence of SEQ ID No. 22 (corresponding to position 250 of SEQ ID No. 2), preferably Phe or Ala;
b) Ala, Asp, Glu, Lys, Leu, Asn, Pro, Arg or Ser, Gly, His or Trp at position 220 of the amino acid sequence of SEQ ID No. 22 (corresponding to position 251 of SEQ ID No. 2), preferably Ala, Arg or Lys;
C) Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Gin, Arg, Val, Tyr or Thr at position 221 of the amino acid sequence of SEQ ID No, 22 (corresponding to position 252 of SEQ ID No. 2), preferably Glu or Tyr;
d) Ala, His, Gin, Val Trp, Ile, Leu or Met at position 222 of the amino acid sequence of SEQ ID No. 22 (corresponding to position 253 of SEQ ID No. 2), preferably Val;
e) Val or Met at position 234 of the amino acid sequence of SEQ ID No. 22 (corresponding to position 265 of SEQ ID No. 2);
f) Leu, Gin, Arg, Val, Tyr, Ala, Ile or Met at position 237 of the amino acid sequence of SEQ ID No. 22 (corresponding to position 268 of SEQ ID No. 2);
g) Ala or Thr at position 238 of the amino acid sequence of SEQ ID No. 22 (corresponding to position 269 of SEQ ID No. 2), preferably Ala;
h) Ala, Ile, Asn, Thr or Val at position 249 of the amino acid sequence of SEQ ID
No. 22 (corresponding to position 280 of SEQ ID No. 2);

. .
, .

i) Leu, Val, Ala, Phe, Gly, Met, Arg or Ser at position 261 of the amino acid sequence of SEQ ID No. 22 (corresponding to position 293 of SEQ ID Na. 2);
j) Met, Ala, Pro, Ser, Thr or Val at position 262 of the amino acid sequence of SEQ ID No. 22 (corresponding to position 294 of SEQ ID No. 2);
k) His or Asn at position 275 of the amino acid sequence of SEQ ID No. 22 (corresponding to position 307 of SEQ ID No. 2);
I) Ile, Met or Asn at position 299 of the amino acid sequence of SEQ
ID No. 22 (corresponding to position 335 of SEQ ID No. 2), preferably Met;
m) Met at position 329 of the amino acid sequence of SEQ ID No. 22 (corresponding to position 368 of SEQ ID No. 2);
n) Tyr at position 342 of the amino acid sequence of SEQ ID No. 22 (corresponding to position 381 of SEQ ID No. 2);
o) Ser at position 353 of the amino acid sequence of SEQ ID No. 22 (corresponding to position 392 of SEQ ID No. 2);
p) Tyr at position 365 of the amino acid sequence of SEQ ID No. 22 (corresponding to position 419 of SEQ ID No. 2);
q) Asp, Phe, Lys, Asn, Ala, Cys, Glu, Gly, His, Ile, Leu, Met, Gin, Ser or Thr at position 367 of the amino acid sequence of SEQ ID No. 22 (corresponding to position 421 of SEQ ID No. 2), preferably Lys;
r) Ala, Met, Pro or Thr at position 368 of the amino acid sequence of SEQ ID
No.
22 (corresponding to position 422 of SEQ ID No. 2);
s) Ala, Ile or Val at position 370 of the amino acid sequence of SEQ ID No.

(corresponding to position 424 of SEQ ID No. 2);
t) Ile, Pro, Arg, Ser, Ala, Gly, Asn or Gln at position 371 of the amino acid sequence of SEQ ID No. 22 (corresponding to position 425 of SEQ ID No. 2);
u) Glu, Phe, Thr, Val, Gin or Ser at position 372 of the amino acid sequence of SEQ ID No. 22 (corresponding to position 426 of SEQ ID No. 2), preferably Glu;
v) Met, Gin, Phe, Val, Phe or Leu at position 377 of the amino acid sequence of SEQ ID No. 22 (corresponding to position 431 of SEQ ID No. 2);
w) Ala, Cys, Gly or Thr at position 195 of the amino acid sequence of SEQ ID
No.
22 (corresponding to position 228 of SEQ ID No. 2);

x) Glu, Gly, His, Lys, Leu, Asn, Gin, Arg, Ser, Thr or Tyr at position 217 of the amino acid sequence of SEQ ID No. 22 (corresponding to position 248 of SEQ
ID No. 2);
y) Ala, Ile, Leu or Val at position 239 of the amino acid sequence of SEQ
ID No.
22 (corresponding to position 270 of SEQ ID No. 2);
z) Ala, Glu, His, Ile, Lys, Leu, Met, Arg, Thr or Val at position 240 of the amino acid sequence of SEQ ID No. 22 (corresponding to position 271 of SEQ ID No.
2);
aa) His at a position at position 340 of the amino acid sequence of SEQ ID No.

(corresponding to position 379 of SEQ ID No. 2);
bb) Arg at a position at position 373 of the amino acid sequence of SEQ ID No.

(corresponding to position 427 of SEQ ID No. 2).
In another embodiment, of the isolated nucleic acid of the invention, said HPPD
protein comprises the amino acid sequence of SEQ ID No. 24 [Sorghum bicolor], wherein the resulting amino acid sequence comprises at least one amino acid selected from a) Ala, Asp, Glu, Gly, Lys,G1n, Arg, Ser, Thr, Val Tyr, Ile, Leu or Met at position 241 of the amino acid sequence of SEQ ID No. 24 (corresponding to position 250 of SEQ ID No. 2), preferably Ala;
b) Asp, Glu, Lys, Leu, Asn, Pro, Arg, Ser, Gly, His or Trp at position 242 of the amino acid sequence of SEQ ID No. 24 (corresponding to position 251 of SEQ
ID No. 2), preferably Arg or Lys;
C) Phe, Gly, His, Ile, Leu, Met, Asn, Gin, Arg, Val, Tyr or Thr at position 243 of the amino acid sequence of SEQ ID No. 24 (corresponding to position 252 of SEQ
ID No. 2), preferably Tyr;
d) Ala, His, Gin, Val, Trp, Ile, Leu or Met at position 244 of the amino acid sequence of SEQ ID No. 24 (corresponding to position 253 of SEQ ID No. 2), preferably Val;
e) Val or Met at position 256 of the amino acid sequence of SEQ ID No. 24 (corresponding to position 265 of SEQ ID No. 2);

. .
4 µ
f) Leu, Gin, Arg, Val, Tyr, Ala, Ile or Lys at position 259 of the amino acid sequence of SEQ ID No. 24 (corresponding to position 268 of SEQ ID No. 2);
g) Ala or Thr at position 260 of the amino acid sequence of SEQ ID No. 24 (corresponding to position 269 of SEQ ID No. 2), preferably Ala;
5 h) Ala, Ile, Asn, Thr or Val at position 271 of the amino acid sequence of SEQ ID
No. 24 (corresponding to position 280 of SEQ ID No. 2);
i) Leu or Val, Ala, Phe, Gly, Met, Arg or Ser at position 284 of the amino acid sequence of SEQ ID No. 24 (corresponding to position 293 of SEQ ID No. 2);
j) Met, Ala, Pro, Ser, Thr or Val at position 285 of the amino acid sequence of 10 SEQ ID No. 24 (corresponding to position 294 of SEQ ID No. 2);
k) His or Asn at position 298 of the amino acid sequence of SEQ ID No. 24 (corresponding to position 307 of SEQ ID No. 2);
I) Ile, Asn or Leu at position 326 of the amino acid sequence of SEQ
ID No. 24 (corresponding to position 335 of SEQ ID No, 2);
15 m) Met at position 359 of the amino acid sequence of SEQ ID No. 24 (corresponding to position 368 of SEQ ID No. 2);
n) Tyr at position 372 of the amino acid sequence of SEQ ID No. 24 (corresponding to position 381 of SEQ ID No. 2);
o) Ser at position 383 of the amino acid sequence of SEQ ID No. 24 20 (corresponding to position 392 of SEQ ID No. 2);
p) Tyr at position 410 of the amino acid sequence of SEQ ID No. 24 (corresponding to position 419 of SEQ ID No. 2);
q) Asp, Phe, Asn, Ala, Cys, Glu, Gly, His, Ile, Leu, Met, Gin, Ser, Thr or Val at position 412 of the amino acid sequence of SEQ ID No. 24 (corresponding to 25 position 421 of SEQ ID No. 2);
r) Ala, Met, Pro or Thr at position 413 of the amino acid sequence of SEQ
ID No.
24 (corresponding to position 422 of SEQ ID No. 2);
s) Ala, Ile or Val at position 415 of the amino acid sequence of SEQ ID No.

(corresponding to position 424 of SEQ ID No. 2);
30 t) Ile, Pro, Arg, Als, Gly, Lys, Asn or Gin at position 416 of the amino acid sequence of SEQ ID No. 24 (corresponding to position 425 of SEQ ID No. 2);

U) Phe, Thr, Val, Ala, Gin or Ser at position 417 of the amino acid sequence of SEQ ID No. 24 (corresponding to position 426 of SEQ ID No. 2);
v) Met, Gin, Phe, Val, Phe or Leu at position 422 of the amino acid sequence of SEQ ID No. 24 (corresponding to position 431 of SEQ ID No, 2);
w) Ala, Cys, Gly or Thr at position 219 of the amino acid sequence of SEQ ID
No.
24 (corresponding to position 228 of SEQ ID No. 2);
x) Ala, Glu, Gly, Lys, Leu, Asn, Gin, Arg, Ser, Thr or Tyr at position 239 of the amino acid sequence of SEQ ID No. 24 (corresponding to position 248 of SEQ
ID No. 2);
y) Ala, Ile, Met or Val at position 261 of the amino acid sequence of SEQ ID
No.
24 (corresponding to position 270 of SEQ ID No. 2);
z) Giu, His, Ile, Lys, Leu, Met, Arg, Ser, Thr or Val at position 262 of the amino acid sequence of SEQ ID No. 24 (corresponding to position 271 of SEQ ID No.
2);
aa) His at position 370 of the amino acid sequence of SEQ ID No. 24 (corresponding to position 379 of SEQ ID No. 2);
bb) Arg at position 418 of the amino acid sequence of SEQ ID No. 24 (corresponding to position 427 of SEQ ID No. 2).
In another embodiment, of the isolated nucleic acid of the invention, said I-IPPD
protein comprises the amino acid sequence of SEQ ID No. 26 [Triticum aestivum /
wheat], wherein the resulting amino acid sequence comprises at least one amino acid selected from a) Ala, Asp, Glu, Gly, Lys,G1n, Arg, Ser, Thr, Val, Tyr, lie, Leu or Met at position 237 of the amino acid sequence of SEQ ID No. 26 (corresponding to position 250 of SEQ ID No. 2), preferably Ala;
b) Asp, Glu, Lys, Leu, Asn, Pro, Arg or Ser, Gly, His or Trp at position 238 of the amino acid sequence of SEQ ID No. 26 (corresponding to position 251 of SEQ
ID No. 2), preferably Arg or Lys;
c) Phe, Gly, His, Ile, Leu, Met, Asn, Gin, Arg, Val or Tyr or Thr at position 239 of the amino acid sequence of SEQ ID No. 26 (corresponding to position 252 of SEQ ID No. 2), preferably Tyr;

d) Ala, His, Gin, Val Trp, Ile, Leu or Met at position 240 of the amino acid sequence of SEQ ID No. 26 (corresponding to position 253 of SEQ ID No. 2), preferably Val;
e) Val or Met at position 252 of the amino acid sequence of SEQ ID No. 26 (corresponding to position 265 of SEQ ID No. 2);
f) Leu, Gin, Arg, Val, Tyr, Ala, Ile or Lys at position 255 of the amino acid sequence of SEQ ID No. 26 (corresponding to position 268 of SEQ ID No. 2);
g) Ala or Thr at position 256 of the amino acid sequence of SEQ ID No. 26 (corresponding to position 269 of SEQ ID No. 2), preferably Ala;
h) Ala, Ile, Asn, Thr or Val at position 267 of the amino acid sequence of SEQ
ID
No. 26 (corresponding to position 280 of SEQ ID No. 2);
i) Leu, Val, Ala, Phe, Gly, Met, Arg or Ser at position 280 of the amino acid sequence of SEQ ID No. 26 (corresponding to position 293 of SEQ ID No. 2);
j) Met, Ala, Pro, Ser, Thr or Val at position 281 of the amino acid sequence of SEQ ID No. 26 (corresponding to position 294 of SEQ ID No. 2);
k) His or Asn at position 294 of the amino acid sequence of SEQ ID No. 26 (corresponding to position 307 of SEQ ID No. 2);
I) Ile, Met or Asn at position 322 of the amino acid sequence of SEQ ID
No. 26 (corresponding to position 335 of SEQ ID No. 2), preferably Met;
m) Met at position 355 of the amino acid sequence of SEQ ID No. 26 (corresponding to position 368 of SEQ ID No. 2);
n) Tyr at position 368 of the amino acid sequence of SEQ ID No. 26 (corresponding to position 381 of SEQ ID No. 2);
o) Ser at position 379 of the amino acid sequence of SEQ ID No. 26 (corresponding to position 392 of SEQ ID No. 2);
p) Tyr at position 406 of the amino acid sequence of SEQ ID No. 26 (corresponding to position 419 of SEQ ID No. 2);
q) Asp, Phe, Asn, Ala, Cys, Glu, Gly, His, Ile, Leu, Met, Gin, Ser, Thr or Val at position 408 of the amino acid sequence of SEQ ID No. 26 (corresponding to position 421 of SEQ ID No. 2);
r) Ala, Met, Pro or Thr at position 409 of the amino acid sequence of SEQ
ID No.
26 (corresponding to position 422 of SEQ ID No. 2);

. .

s) Ala, Ile or Val at position 411 of the amino acid sequence of SEQ ID No.

(corresponding to position 424 of SEQ ID No. 2);
t) Ile, Pro, Arg, Ala, Gly, Lys, Asn or Gin at position 412 of the amino acid sequence of SEQ ID No. 26 (corresponding to position 425 of SEQ ID No. 2);
u) Phe, Thr or Val, Ala, Gin or Ser at position 413 of the amino acid sequence of SEQ ID No. 26 (corresponding to position 426 of SEQ ID No. 2);
v) Met, Gin, Phe, Leu or Val at position 418 of the amino acid sequence of SEQ ID
No. 26 (corresponding to position 431 of SEQ ID No. 2);
w) Ala, Cys, Gly or Thr at a position in an HPPD protein, said position corresponding to position 215 of the amino acid sequence of SEQ ID No. 26 (corresponding to position 228 of SEQ ID No. 2);
x) Ala, Glu, Gly, Lys, Leu, Asn, Gin, Arg, Ser, Thr or Tyr at position 235 of the amino acid sequence of SEQ ID No. 26 (corresponding to position 248 of SEQ
ID No. 2);
y) Ala, Ile, Met or Val at position 257 of the amino acid sequence of SEQ ID
No.
26 (corresponding to position 270 of SEQ ID No. 2);
z) Glu, His, Ile, Lys, Leu, Met, Arg, Ser, Thr or Val at position 258 of the amino acid sequence of SEQ ID No. 26 (corresponding to position 271 of SEQ ID No.
2);
aa) His at a position at position 366 of the amino acid sequence of SEQ ID No.

(corresponding to position 379 of SEQ ID No. 2);
bb) Arg at position 414 of the amino acid sequence of SEQ ID No. 26 (corresponding to position 427 of SEQ ID No. 2).
In another embodiment, of the isolated nucleic acid of the invention, said HPPD
protein comprises the amino acid sequence of SEQ ID No. 2 [Arabidopsis thaliana], wherein the resulting amino acid sequence comprises at least one amino acid selected from (a) Ala, Asp, Glu, Gly, Lys, Gin, Arg, Ser, Thr, Val, Tyr, Ile, Leu or Met at position 250 of the amino acid sequence of SEQ ID No. 2;
(b) Asp, Glu, Ile, Lys, Leu, Asn, Pro, Arg, Ser, Gly, His or Trp at position 251 of the amino acid sequence of SEQ ID No. 2;

(c) Phe, Gly, His, Ile, Leu, Met, Asn, Gin, Arg, Ser, Val, Tyr or Thr at position 252 of the amino acid sequence of SEQ ID No. 2;
(d) Ala, His, Gin, Val, Trp, Ile, Leu or Met at position 253 of the amino acid sequence of SEQ ID No. 2;
(e) Val or Met at a position at position 265 of the amino acid sequence of SEQ
ID
No. 2;
(f) Leu, Gin, Arg, Val, Tyr, Ile, Lys or Met at position 268 of the amino acid sequence of SEQ ID No. 2;
(g) Ala or Thr at position 269 of the amino acid sequence of SEQ ID No. 2;
(h) Ala, Ile, Asn, Thr or Val at position 280 of the amino acid sequence of SEQ ID
No. 2;
(i) Gin, Val, Ala, Phe, Gly, Met, Arg or Ser at a in an HPPD protein, said position corresponding to position 293 of the amino acid sequence of SEQ ID No. 2;
(j) Met, Ala, Pro, Ser, Thr or Val at position 294 of the amino acid sequence of SEQ ID No. 2;
(k) His or Asn at position 307 of the amino acid sequence of SEQ ID No. 2;
(I) Ile, Asn or Leu at position 335 of the amino acid sequence of SEQ ID
No. 2;
(m) Met at position 368 of the amino acid sequence of SEQ ID No. 2;
(n) Tyr at position 381 of the amino acid sequence of SEQ ID No. 2;
(o) Ser at position 392 of the amino acid sequence of SEQ ID No. 2;
(p) Tyr at position 419 of the amino acid sequence of SEQ ID No. 2;
(q) Asp, Phe, Asn, Ala, Cys, Glu, Gly, His, Ile, Leu, Met, Gin, Ser, Thr or Val at position 421 of the amino acid sequence of SEQ ID No. 2;
(r) Ala, Met, Pro or Thr at position 422 of the amino acid sequence of SEQ
ID No.
2;
(s) Ala, Ile or Val at position 424 of the amino acid sequence of SEQ ID
No. 2;
(t) Ile, Pro, Arg, Ala, Gly, Lys, Asn or Gin at position 425 of the amino acid sequence of SEQ ID No. 2;
(u) Phe, Thr, Val, Ala, Gin or Ser at position 426 of the amino acid sequence of SEQ ID No. 2;
(v) Met, Gin, Val, Phe or Leu position 431 of the amino acid sequence of SEQ ID
No. 2;

. .
(w) Ala, Cys, Gly or Thr at position 228 of the amino acid sequence of SEQ ID
No.
2;
(x) Ala, Glu, Gly, Lys, Leu, Asn, Gin, Arg, Ser, Thr or Tyr at position 248 of the amino acid sequence of SEQ ID No. 2;
5 (y) Ala, Ile, Met or Val at position 270 of the amino acid sequence of SEQ ID No. 2;
(z) Glu, His, Ile, Lys, Leu, Met, Arg, Ser, Thr or Val at position 271 of the amino acid sequence of SEQ ID No. 2;
(aa) His at position 379 of the amino acid sequence of SEQ ID No. 2; or (bb) Arg at position 427 of the amino acid sequence of SEQ ID No. 2.
In a further embodiment of the nucleic acid of the invention, in said mutated HPPD
protein at least one amino acid has been replaced so that the resulting amino acid sequence comprises (a) Glu, Ser or Tyr at position 252; (b) Val or Ala at position 269;
(c) Pro, Val or Ala at position 280; (d) Asp, Lys or Asn at position 421; and (e) Gly or Ala at postion 42Z These positions correspond to amino acid sequence of SEQ ID
No 2.
In a further embodiment of the nucleic acid of the invention, in said mutated HPPD
protein at least two amino acids have been replaced.
An isolated nucleic acid comprising a nucleotide sequence encoding a mutated HPPD protein, wherein said mutated HPPD protein has HPPD activity, wherein in said mutated HPPD protein at least one amino acid at position 228, 248, 270, 271, 379 and/or 427 has been replaced by another amino acid.
In an alternative embodiment of the nucleic acid of the invention having at least one amino acid at position 228, 248, 270, 271, 379 and/or 427 deleted or replaced by another amino acid as defined above, said mutated HPPD protein comprises a) a His at a position in an HPPD protein, said position corresponding to position 226 of the amino acid sequence of SEQ ID No. 2;

b) a Ser at a position in an HPPD protein, said position corresponding to position 267 comprising the amino acid sequence of SEQ ID No. 2 or at a position corresponding thereto in a different HPPD enzyme;
c) an Asn at a position in an HPPD protein, said position corresponding to position 282 of the amino acid sequence of SEQ ID No. 2;
d) a His at a position in an HPPD protein, said position corresponding to position 308 of the amino acid sequence of SEQ ID No. 2;
e) a Tyr at a position in an HPPD protein, said position corresponding to position 342 of the amino acid sequence of SEQ ID No. 2;
f) a Glu at a position in an HPPD protein, said position corresponding to position 394 of the amino acid sequence of SEQ ID No. 2;
g) a Gly at a position in an HPPD protein, said position corresponding to position 420 of the amino acid sequence of SEQ ID No. 2; and h) an Asn at a position in an HPPD protein, said position corresponding to position 423 of the amino acid sequence of SEQ ID No. 2_ In another embodiment of the isolated nucleic acid as defined above, in said mutated HPPD protein at least one amino acid has been replaced so that the resulting amino acid sequence has at least one selected from a. Ala, Cys, Gly, Thr or Val at a position in an HPPD protein, said position corresponding to position 228 of the amino acid sequence of SEQ ID No. 2;
b. Ala, Glu, Gly, His, Lys, Leu, Asn, Gln, Arg, Ser, Thr or Tyr at a position in an HPPD protein, said position corresponding to position 248 of the amino acid sequence of SEQ ID No. 2;
c. Ala, Ile, Leu, Met or Val at a position in an HPPD protein, said position corresponding to position 270 of the amino acid sequence of SEQ ID No. 2;
d. Ala, Glu, Hits, Ile, Lys, Leu, Met, Arg, Ser, Thr or Val at a position in an HPPD
protein, said position corresponding to position 271 of the amino acid sequence of SEQ ID No. 2;
e. His or Gin at a position in an HPPD protein, said position corresponding to position 379 of the amino acid sequence of SEQ ID No. 2; and f. Leu or Arg at a position in an HPPD protein, said position corresponding to position 427 of the amino acid sequence of SEQ ID No. 2.
In another embodiment of the isolated nucleic acid as defined above, in said mutated HPPD protein at least one amino acid has been replaced so that the resulting amino acid sequence has at least one selected from a. Val or Thr at a position in an HPPD protein, said position corresponding to position 228 of the amino acid sequence of SEQ ID No. 2;
b. Leu, Met or Val at a position in an HPPD protein, said position corresponding to position 270 of the amino acid sequence of SEQ ID No. 2;
c. Ala or Ser at a position in an HPPD protein, said position corresponding to position 271 of the amino acid sequence of SEQ ID No. 2, d. Gin at a position in an HPPD protein, said position corresponding to position 379 of the amino acid sequence of SEQ ID No. 2; and e. Leu at a position in an HPPD protein, said position corresponding to position 427 of the amino acid sequence of SEQ ID No. 2.
In another embodiment of the nucleic acid as defined above, said mutated HPPD
protein is capable of increasing the tolerance of a plant to at least one herbicide acting on HPPD (also called HPPD inhibitor herbicide).
In another embodiment, the present invention relates to a protein encoded by the isolated nucleic acid of the invention.
In a further embodiment, the present invention relates to a chimeric gene comprising a coding sequence comprising the nucleic acid of the invention operably linked to a plant-expressible promoter and optionally a transcription termination and polyadenylation region.
As a regulatory sequence which functions as a promoter in plant cells and plants, use may be made of any promoter sequence of a gene which is naturally expressed in plants, in particular a promoter which is expressed especially in the leaves of plants, such as for example "constitutive" promoters of bacterial, viral or plant origin, or "light-dependent" promoters, such as that of a plant ribulose-biscarboxylase/oxygenase (RuBisCO) small subunit gene, or any suitable known promoter-expressible which may be used. Among the promoters of plant origin, mention will be made of the histone promoters as described in EP 0 507 698 Al, the rice actin promoter (US 5,641,876), or a plant ubiquitin promoter (US
5,510,474).
Among the promoters of a plant virus gene, mention will be made of that of the cauliflower mosaic virus (CaMV 19S or 35S, Sanders et al. (1987), Nucleic Acids Res. 15(4)1543-58.), the circovirus (AU 689 311) or the Cassava vein mosaic virus (CsVMV, US 7,053,205).
In one embodiment of this invention, a promoter sequence specific for particular regions or tissues of plants can be used to express the HPPD proteins of the invention, such as promoters specific for seeds (Datla, R. et al., 1997, Biotechnology Ann. Rev. 3, 269-296), especially the napin promoter (EP 255 378 Al), the phaseolin promoter, the glutenin promoter, the helianthinin promoter (WO 92/17580), the albumin promoter (WO 98/45460), the oleosin promoter (WO 98/45461), the SAT1 promoter or the SAT3 promoter (PCT/US98/06978).
Use may also be made of an inducible promoter advantageously chosen from the phenylalanine ammonia lyase (PAL), HMG-CoA reductase (HMG), chitinase, glucanase, proteinase inhibitor (PI), PR1 family gene, nopaline synthase (nos) and vspB promoters (US 5 670 349, Table 3), the HMG2 promoter (US 5 670 349), the apple beta-galactosidase (ABG1) promoter and the apple aminocyclopropane carboxylate synthase (ACC synthase) promoter (WO 98/45445).
According to the invention, use may also be made, in combination with the promoter, of other regulatory sequences, which are located between the promoter and the coding sequence, such as transcription activators ("enhancers"), for instance the translation activator of the tobacco mosaic virus (TMV) described in Application WO
87/07644, or of the tobacco etch virus (TEV) described by Carrington & Freed 1990, . .
, J. Virol. 64: 1590-1597, for example, or introns such as the adh1 intron of maize or intron 1 of rice actin.
As a regulatory terminator or polyadenylation sequence, use may be made of any corresponding sequence of bacterial origin, such as for example the nos terminator of Agrobacterium tumefaciens, of viral origin, such as for example the CaMV

terminator, or of plant origin, such as for example a histone terminator as described in published Patent Application EP 0 633 317 Al.
A method of obtaining a mutated HPPD protein capable of modulating the tolerance of a plant to at least one herbicide acting on HPPD, wherein said mutated HPPD

protein has HPPD activity, the method comprising i. providing an HPPD protein, said HPPD optionally comprising an amino acid sequence, wherein a) a His is present at a position in an HPPD protein, said position corresponding to position 226 of the amino acid sequence of SEQ ID No. 2;
b) a Ser is present at a position in an HPPD protein, said position corresponding to position 267 of the amino acid sequence of SEQ ID No. 2;
c) an Asn is present at a position in an HPPD protein, said position corresponding to position 282 of the amino acid sequence of SEQ ID No. 2;
d) a His is present at a position in an HPPD protein, said position corresponding to position 308 of the amino acid sequence of SEQ ID No. 2;
e) a Tyr is present at a position in an HPPD protein, said position corresponding to position 342 of the amino acid sequence of SEQ ID No. 2;
f) a Glu is present at a position in an HPPD protein, said position corresponding to position 394 of the amino acid sequence of SEQ ID No. 2;
g) a Gly is present at a position in an HPPD protein, said position corresponding to position 420 of the amino acid sequence of SEQ ID No. 2;
and h) an Asn is present at a position in an HPPD protein, said position corresponding to position 423 of the amino acid sequence of SEQ ID No. 2 . .
, ii) replacing at least one amino acid in said HPPD enzyme so that the resulting amino acid sequence has at least one selected from a. Ala, Asp, Glu, Phe, Gly, Lys,G1n, Arg, Ser, Thr, Val, Ile, Leu, Met or Tyr at a position in an HPPD protein, said position corresponding to position 250 of 5 the amino acid sequence of SEQ ID No. 2;
b. Ala, Asp, Glu, Ile, Lys, Leu, Asn, Pro, Arg, Gly, His, Tyr or Ser at a position in an HPPD protein, said position corresponding to position 251 of the amino acid sequence of SEQ ID No. 2;
c, Giu, Phe, Gly, His, Ile, Leu, Met, Asn, Gin, Arg, Ser, Val, Thr or Tyr at a 10 position in an HPPD protein, said position corresponding to position 252 of the amino acid sequence of SEQ ID No. 2;
d, Ala, Phe, His, Gin, Val, Ile, Leu, Met or Trp at a position in an HPPD
protein, said position corresponding to position 253 of the amino acid sequence of SEQ ID No. 2;
15 e. Leu, Met or Val at a position in an HPPD protein, said position corresponding to position 265 of the amino acid sequence of SEQ ID No. 2;
f. Leu, Gin, Arg, Val, Ala, lie, Lys, Met or Tyr at a position in an HPPD protein, said position corresponding to position 268 of the amino acid sequence of SEQ ID No. 2;
20 g. Ala, Thr or Val at a position in an HPPD protein, said position corresponding to position 269 of the amino acid sequence of SEQ ID No. 2;
h. Ala, Ile, Asn, Pro, Thr or Val at a position in an HPPD protein, said position corresponding to position 280 of the amino acid sequence of SEQ ID No. 2;
i. Leu, Gin, Ala, Phe, Gly, Met, Arg, Ser or Val at a position in an HPPD
25 protein, said position corresponding to position 293 of the amino acid sequence of SEQ ID No. 2;
j. Ile, ala, Pro, Ser, Thr, Val or Met at a position in an HPPD protein, said position corresponding to position 294 of the amino acid sequence of SEQ
ID No. 2;
30 k. Gin, His or Asn at a position in an HPPD protein, said position corresponding to position 307 of the amino acid sequence of SEQ ID No. 2;

I. Ile, Met, Leu or Asn at a position in an HPPD protein, said position corresponding to position 335 of the amino acid sequence of SEQ ID No. 2;
m. Leu or Met at a position in an HPPD protein, said position corresponding to position 368 of the amino acid sequence of SEQ ID No. 2;
n. Phe or Tyr at a position in an HPPD protein, said position corresponding to position 381 of the amino acid sequence of SEQ ID No. 2;
o. Phe or Ser at a position in an HPPD protein, said position corresponding to position 392 of the amino acid sequence of SEQ ID No. 2;
p. Phe or Tyr at a position in an HPPD protein, said position corresponding to position 419 of the amino acid sequence of SEQ ID No. 2;
q. Asp, Phe, Lys, Asn, Ala, Cys, Glu, Gly, His, Ile, Leu, Met, Gin, Ser, Thr or Val at a position in an HPPD protein, said position corresponding to position 421 of the amino acid sequence of SEQ ID No. 2;
r. Ala, Gly, Met, Pro or Thr at a position in an HPPD protein, said position corresponding to position 422 of the amino acid sequence of SEQ ID No. 2;
s. Ala, Phe, Ile or Val at a position in an HPPD protein, said position corresponding to position 424 of the amino acid sequence of SEQ ID No. 2 or;
t. Ile, Pro, Arg, Ala, Gly, Lys, Asn, Gin or Ser at a position in an HPPD
protein, said position corresponding to position 425 of the amino acid sequence of SEQ ID No. 2;
u. Glu, Phe, Thr, Ala, Gin, Ser or Val at a position in an HPPD protein, said position corresponding to position 426 of the amino acid sequence of SEQ
ID No. 2;
v. Ile, Met, Gin, Phe, Leu or Val at a position in an HPPD protein, said position corresponding to position 431 of the amino acid sequence of SEQ ID No. 2;
w. an amino acid deletion or replacement at least one position in an HPPD
protein, said position corresponding to at least one of positions 228, 248, 270, 271, 379 and 427 of the amino acid sequence of SEQ ID No. 2;
iii) determining the inhibition of the resulting HPPD protein by at least one herbicide acting on HPPD;

wherein an inhibition of the resulting protein of less or more than that observed with a reference HPPD protein is indicative that the resulting protein is capable of modulating the tolerance of a plant to said herbicide.
It is to be understood that also the (more specific) amino acids and positions listed above for other embodiments, such as the nucleic acid of the invention, may be applied to the method of obtaining a mutated HPPD protein as described above.
In an alternative embodiment of the method of obtaining a mutated HPPD protein as described above, said mutated HPPD protein is capable of increasing the tolerance of a plant to at least one herbicide acting on HPPD.
Within the above method of obtaining in mutated HPPD protein, different herbicides acting on HPPD may be chosen. Accordingly, in another embodiment of the method of obtaining a mutated HPPD protein as described above, wherein said mutated HPPD protein is capable of increasing the tolerance of a plant to at least one herbicide acting on HPPD, the herbicide acting on HPPD is selected from triketones, or pyrazolinates, preferably tembotrione, mesotrione, topramezone or sulcotrione, bicyclopyrone, pyrasulfotole, pyrazolate, benzofenap and tefuryitrione, particularly tembotrione and such plants containing the HPPD of the invention have an agronomically acceptable tolerance to an HPPD inhibitor herbicide particularly to triketones, or pyrazolinates, preferably tembotrione, mesotrione, topramezone or sulcotrione, bicyclopyrone, pyrasulfotole, pyrazolate, benzofenap and tefuryltrione, particularly tembotrione.
In another embodiment, the present invention relates to a method of producing a transgenic plant comprising introducing into a said plant genome the nucleic acid of the present invention operably linked to a plant expressible promoter, the chimeric gene of the invention or a nucleic acid encoding the HPPD enzyme identified by the method of of claim 27 or 28.

In an alternative embodiment of the method of producing a transgenic plant as described above, the nucleic acid of the invention, wherein said mutated HPPD
protein is capable of increasing the tolerance of a plant to at least one herbicide acting on HPPD, or a nucleic acid identified by the method of obtaining a mutated HPPD protein, wherein said mutated HPPD protein is capable of increasing the tolerance of a plant to at least one herbicie acting on HPPD, both operably linked to a plant expressible promoter, or the chimeric gene of the invention comprising a nucleic acid, wherein said mutated HPPD protein is capable of increasing the tolerance of a plant to at least one herbicie acting on HPPD, is introduced into said plant.
In another embodiment, the present invention relates to a plant cell comprising the isolated nucleic acid of the invention or the chimeric gene of the invention in its genetic information.
The present invention also relates to a plant, a part of a plant or plant tissue consisting essentially of the plant cells of the invention.
Furthermore, the present invention relates to a plant obtainable from the method of obtaining a mutated HPPD protein capable of modulating or increasing the tolerance of a plant to at least one herbicide acting on HPPD in all alternative aspects described above.
The plant of the present invention can be any plant. Non-limiting examples of plants of the invention include wheat, cotton, canola, rice, corn, soy bean, sorghum,canola, sunflower, tobacco, sugarbeet, cotton, maize, wheat, barley, rice, sorghum, tomato, mango, peach, apple, pear, strawberry, banana, melon, potato, carrot, lettuce, cabbage, onion, 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.
.. The present invention also relates to a seed of the plant of the invention.

Furthermore, the invention relates to progeny of the plant of the invention or the seed of the invention.
In a further embodiment, the present invention relates to a method of modulating a plant's tolerance to at least one herbicide acting on HPPD comprising introducing the isolated nucleic acid of the invention operably linked to a plant expressible promoter or the chimeric gene of the invention into a plant's genome In an alternative embodiment, the present invention relates to a method of increasing a plant's tolerance to at least one herbicide acting on HPPD or of obtaining a plant tolerant to an HPPD inhibitor herbicide comprising introducing the isolated nucleic acid of the invention, wherein said nucleic acid encodes a mutated HPPD
protein which is capable of increasing the tolerance of a plant to at least one herbicide acting on HPPD, operably linked to a plant expressible promoter or the chimeric gene of the invention comprising a nucleic acid of the invention, wherein said nucleic acid encodes a mutated HPPD protein which is capable of increasing the tolerance of a plant to at least one herbicide acting on HPPD, into a plant's genome.
Furthermore, the present invention relates to a method for controlling weeds comprising spraying at least one herbicide acting on HPPD on or around a crop plant, wherein said crop plant comprises the nucleic acid of the present invention, wherein said mutated HPPD protein is capable of increasing the tolerance of a plant to at least one herbicide acting on HPPD, operably linked to a plant expressible promoter or the chimeric gene of the invention comprising the nucleic acid of the invention, wherein said mutated HPPD protein is capable of increasing the tolerance of a plant to at least one herbicide acting on HPPD. In alternative embodiment of the method of controlling weeds, the tolerance of said plant to at least one herbicie acting on HPPD
is increased.
In addition, the present invention relates to the use of a chimeric gene of the invention or the nucleic acid of the invention operably linked to a plant expressible promoter for modulating the tolerance of a plant to at least one herbicide acting on HPPD.
In an alternative embodiment, the present invention relates to the use of a chimeric 5 gene of the invention or the nucleic acid of the invention operably linked to a plant expressible promoter for increasing the tolerance of a plant to at least one herbicide acting on HPPD. In this embodiment of the invention, the chimeric gene used comprises the nucleic acid of the invention, wherein the mutated HPPD protein encoded thereby is capable of increasing the tolerance of a plant to at least one 10 herbicide acting on HPPD. Alternatively, if a nucleic acid operably linked to a plant expressible promoter is used, said nucleic is chose that the mutated HPPD
protein encoded thereby is capable of increasing the tolerance of a plant to at least one herbicide acting on HPPD.
15 The present invention also relates to the plant cell of the invention and the plant of the invention which may comprise a further useful trait as described further below.
While a number of herbicide-tolerant crop plants are presently commercially available, one issue that has arisen for many commercial herbicides and 20 herbicide/crop combinations is that individual herbicides typically have incomplete spectrum of activity against common weed species. For most individual herbicides which have been in use for some time, populations of herbicide resistant weed species and biotypes have become more prevalent (see, e.g., Tranel and Wright (2002) Weed Science 50: 700-712; Owen and Zelaya (2005) Pest Manag. Sci. 61:
25 301-311). Transgenic plants which are resistant to more than one herbicide have been described (see, e.g., W02005/012515). However, improvements in every aspect of crop production, weed control options, extension of residual weed control, and improvement in crop yield are continuously in demand.
30 The HPPD protein or gene of the invention is advantageously combined in plants with other genes which encode proteins or RNAs that confer useful agronomic properties to such plants. Among the genes which encode proteins or RNAs that confer useful agronomic properties on the transformed plants, mention can be made of the DNA sequences encoding proteins which confer tolerance to one or more herbicides that, according to their chemical structure, differ from HPPD
inhibitor herbicides, and others which confer tolerance to certain insects, those which confer tolerance to certain diseases, DNAs that encodes RNAs that provide nematode or insect control, etc...
Such genes are in particular described in published PCT Patent Applications WO
91/02071 and W095/06128.
Among the DNA sequences encoding proteins which confer tolerance to certain herbicides on the transformed plant cells and plants, mention can be made of a bar or PAT gene or the Streptomyces coelicolor gene described in W02009/152359 which confers tolerance to glufosinate herbicides, a gene encoding a suitable EPSPS which confers tolerance to herbicides having EPSPS as a target, such as glyphosate and its salts (US 4,535,060, US 4,769,061, US 5,094,945, US
4,940,835, US 5,188,642, US 4,971,908, US 5,145,783, US 5,310,667, US 5,312,910, US
5,627,061, US 5,633,435), or a gene encoding glyphosate oxydoreductase (US
5,463,175).
Among the DNA sequences encoding a suitable EPSPS which confer tolerance to the herbicides which have EPSPS as a target, mention will more particularly be made of the gene which encodes a plant EPSPS, in particular maize EPSPS, particularly a maize EPSPS which comprises two mutations, particularly a mutation at amino acid position 102 and a mutation at amino acid position 106 (WO 2004/074443), and which is described in Patent Application US 6566587, hereinafter named double mutant maize EPSPS or 2mEPSPS, or the gene which encodes an EPSPS isolated from Agrobacterium and which is described by sequence ID No. 2 and sequence ID No. 3 of US Patent 5,633,435, also named CP4.
Among the DNA sequences encoding a suitable EPSPS which confer tolerance to the herbicides which have EPSPS as a target, mention will more particularly be made of the gene which encodes an EPSPS GRG23 from Arthrobacter globiformis, but also the mutants GRG23 ACEI, GRG23 ACE2, or GRG23 ACE3, particularly the mutants or variants of GRG23 as described in W02008/100353, such as GRG23(ace3)R173K of SEQ ID No. 29 in W02008/100353.

In the case of the DNA sequences encoding EPSPS, and more particularly encoding the above genes, the sequence encoding these enzymes is advantageously preceded by a sequence encoding a transit peptide, in particular the "optimized transit peptide" described in US Patent 5,510,471 or 5,633,448.
In WO 2007/024782, plants being tolerant to glyphosate and at least one ALS
(acetolactate synthase) inhibitor are disclosed. More specifically plants containing genes encoding a GAT (Glyphosate-N-Acetyltransferase) polypeptide and a polypeptide conferring resistance to ALS inhibitors are disclosed.
In US 6855533, transgenic tobacco plants containing mutated Arabidopsis ALS/AHAS genes were disclosed.
In US 6,153,401, plants containing genes encoding 2,4-D-monooxygenases conferring tolerance to 2,4-D (2,4-dichlorophenoxyacetic acid) by metabolisation are disclosed.
In US 2008/0119361 and US 2008/0120739, plants containing genes encoding Dicamba monooxygenases conferring tolerance to dicamba (3,6-dichloro-2-methoxybenzoic acid) by metabolisation are disclosed.
All the above mentioned herbicide tolerance traits can be combined with those performing HPPD tolerance which are subject matter of this invention.
Among the DNA sequences encoding proteins concerning properties of tolerance to insects, mention will more particularly be made of the Bt proteins widely described in the literature and well known to those skilled in the art. Mention will also be made of proteins extracted from bacteria such as Photorhabdus (WO 97/17432 & WO
98/08932).
Among such DNA sequences encoding proteins of interest which confer novel properties of tolerance to insects, mention will more particularly be made of the Bt Cry or VIP proteins widely described in the literature and well known to those skilled in the art. These include the Cry1F protein or hybrids derived from a Cryl F
protein (e.g., the hybrid Cry1A-Cry1F proteins described in US 6,326,169; US
6,281,016; US

6,218,188, or toxic fragments thereof), the Cry1A-type proteins or toxic fragments thereof, preferably the Cry1Ac protein or hybrids derived from the Cry1Ac protein (e.g., the hybrid Cryl Ab-Cry1Ac protein described in US 5,880,275) or the Cry1Ab or Bt2 protein or insecticidal fragments thereof as described in EP451878, the Cry2Ae, Cry2Af or Cry2Ag proteins as described in W002/057664 or toxic fragments thereof, the Cry1A.105 protein described in WO 2007/140256 (SEQ ID No. 7) or a toxic fragment thereof, the VIP3Aa19 protein of NCBI accession ABG20428, the VIP3Aa20 protein of NCB! accession ABG20429 (SEQ ID No. 2 in WO
2007/142840), the VIP3A proteins produced in the C0T202 or C0T203 cotton events (WO 2005/054479 and WO 2005/054480, respectively), the Cry proteins as described in W001/47952, the VIP3Aa protein or a toxic fragment thereof as described in Estruch et al. (1996), Proc Natl Acad Sci U S A. 28;93(11):5389-94 and US 6,291,156, the insecticidal proteins from Xenonhabdus (as described in W098/50427), Serratia (particularly from S. entomophila) or Photorhabdus species strains, such as Tc-proteins from Photorhabdus as described in W098/08932 (e.g., Waterfield et al., 2001, Appl Environ Microbiol. 67(11):5017-24; Ffrench-Constant and Bowen, 2000, Cell Mol Life Sci.; 57(5):828-33). Also any variants or mutants of any one of these proteins differing in some (1-10, preferably 1-5) amino acids from any of the above sequences, particularly the sequence of their toxic fragment, or .. which are fused to a transit peptide, such as a plastid transit peptide, or another protein or peptide, is included herein.

The FIGURES show:
FIGURE1: Superpositon of the x-ray structure of HPPD from Arabidopsis thaliana (dark grey) and (a) Pseudomonas fluorescens (light grey), (b) Streptomyces avermitilis, (c) Homo sapiens, (d) Rattus norvegicus. The structures are shown as ribbon plot.
F1GURE2: Amino acids forming the binding site of (a) Arabidopsis thaliana, (b) Pseudomonas fluorescens (c) Streptomyces avermitilis (d) Homo sapiens, and (e) Rattus norvegicus (represented by bold lines). The core of the HPPD protein is shown as Caoha trace and the iron is marked.
F1GURE3: Brown test color in Escherichia coil SEQUENCES LISTING
SEQ ID No. 1: Nucleic acid sequence encoding Arabidopsis thaliana HPPD
SEQ ID No. 2: Protein encoded by SEQ ID No. 1 SEQ ID No. 3: Nucleic acid sequence encoding Oryza sativa HPPD
SEQ ID No. 4: Protein encoded by SEQ ID No. 3 SEQ ID No. 5: Nucleic acid sequence encoding Zea mays HPPD
SEQ ID No. 6: Protein encoded by SEQ ID No. 5 SEQ ID No. 7: Nucleic acid sequence encoding Avena sativa HPPD
SEQ ID No. 8: Protein encoded by SEQ ID No. 7 SEQ ID No. 9: Nucleic acid sequence encoding Pseudomonas fluorescens HPPD
SEQ ID No. 10: Protein encoded by SEQ ID No. 9 SEQ ID No. 13: Nucleic acid sequence encoding Synechococcus sp. HPPD
SEQ ID No. 14: Protein encoded by SEQ ID No. 13 SEQ ID No. 15: Nucleic acid sequence encoding Blepharsima japonicum HPPD
SEQ ID No. 16: Protein encoded by SEQ ID No 15 . .

SEQ ID No. 17: Nucleic acid sequence encoding Rhodococcus RHA1 isolate ro0341 HPPD
SEQ ID No. 18: Protein encoded by SEQ ID No. 17 SEQ ID No. 19: Nucleic acid sequence encoding Picrophilus torridus HPPD
SEQ ID No. 20: Protein encoded by SEQ ID No. 19 SEQ ID No. 21: Nucleic acid sequence encoding Kordia algicida HPPD
SEQ ID No. 22: Protein encoded by SEQ ID No. 21 SEQ ID No. 23: Nucleic acid sequence encoding Sorghum bicolor HPPD
SEQ ID No. 24: Protein encoded by SEQ ID No. 23 SEQ ID No. 25: Nucleic acid sequence encoding Triticum aestivum HPPD
SEQ ID No. 26: Protein encoded by SEQ ID No. 25 SEQ ID No. 27: Nucleic acid sequence encoding Hordeum vulgare HPPD
SEQ ID No. 28: Protein encoded by SEQ ID No. 27 SEQ ID No. 29: Nucleic acid sequence encoding Rhodococcus RHA1 isolate ro0240 HPPD
SEQ ID No. 30: Protein encoded by SEQ ID No. 29 SEQ ID No. 31: Nucleic acid sequence encoding Arabidopsis thaliana wild type HPPD polypeptide, plus containing at the 5' end a nucleic acid encoding an alanine and 6 histidine amino acids SEQ ID No. 32: Protein encoded by SEQ ID No. 31 SEQ ID No. 33: Nucleic acid sequence encoding mutant HPPD
polypeptide, plus containing at the 5' end a nucleic acid encoding an alanine and 6 histidine amino acids SEQ ID No. 34: Protein encoded by SEQ ID No. 33, SEQ ID No. 35: Nucleic acid sequence encoding mutant HPPD
polypeptide, plus containing at the 5' end a nucleic acid encoding an alanine and 6 histidine amino acids SEQ ID No. 36: Protein encoded by SEQ ID No. 35, SEQ ID No. 37: Nucleic acid sequence encoding mutant HPPD polypeptide, plus containing at the 5' end a nucleic acid encoding an alanine and 6 histidine amino acids SEQ ID No. 38: Protein encoded by SEQ ID No. 37, SEQ ID No. 39: Nucleic acid sequence encoding mutant HPPD polypeptide, plus containing at the 5' end a nucleic acid encoding art alanine and 6 histidine amino acids SEQ ID No. 40: Protein encoded by SEQ ID No. 39, SEQ ID No. 41: Nucleic acid sequence encoding mutant HPPD polypeptide, plus containing at the 5' end a nucleic acid encoding an alanine and 6 histidine amino acids SEQ ID No. 42: Protein encoded by SEQ ID No. 41, SEQ ID No. 43: Nucleic acid sequence encoding mutant HPPD polypeptide, plus containing at the 5' end a nucleic acid encoding an alanine and 6 histidine amino acids SEQ ID No. 44: Protein encoded by SEQ ID No. 43, SEQ ID No. 45: Nucleic acid sequence encoding mutant HPPD polypeptide, plus containing at the 5' end a nucleic acid encoding an alanine and 6 histidine amino acids SEQ ID No. 46: Protein encoded by SEQ ID No. 45 SEQ ID No. 47: Nucleic acid sequence encoding mutant HPPD polypeptide, plus containing at the 5' end a nucleic acid encoding an alanine and 6 histidine amino acids SEQ ID No. 48: Protein encoded by SEQ ID No. 47 SEQ ID No. 49: Nucleic acid sequence encoding mutant HPPD polypeptide, plus containing at the 5' end a nucleic acid encoding an alanine and 6 histidine amino acids SEQ ID No. 50: Protein encoded by SEQ ID No. 49, =

The Examples illustrate the invention Example1:
A mutation of strictly conserved amino acids leads to an inactive protein.
The point mutants S267A (SEQ ID No. 34) in A.thaliana and N282A (SEQ ID No.
36) in A.thaliana are inactive. The amino acid position given are referring to the position of SEQ ID No. 2.
Mutant HPPD polypeptides of the presenting invention have amino acids changes at one or more positions relative to the starting wild type sequence from which they are derived.
The DNA sequence SEQ ID No. 2 encoding the wild HPPD protein from Arabidopsis thaliana (1335 bp; Genebank AF047834; WO 96/38567) was cloned into the vector pSE420(RI)NX (modified from the the cloning and expression vector pSE420(RI)NX

(5261 bp) is based on the plasmid pSE420 by Invitrogen (Karlsruhe, Germany)).
At the 5' end, directly downstream to the ATG was inserted a nucleic acid sequence coding for an alanine amino acid and a nucleic acid sequence encoding a N-terminal HIS6-Tag (6x HIS, encoded by: cat cat cat cac cat cat). The resulting sequence is presented as SEQ ID No. 31. Upstream to the ATG, two additional cysteine base pairs were added in order to obtain a sequence corresponding to the recognition site of the restriction enzyme Ncol and downstream to the stop codon the sequences corresponding to the recognition site of the restriction enzyme Xbal were added. The resulting plasmid was then used to transform E. Coll cells B1_21 (DE3) with 50pg/m1 kanamycin or 100pg/mIcarbenicillin selection as described in European patent applications "EP09015984.9"; EP09015985.6"; EP 09015986.4" EP 09015987.2";
and EP09015988.0" all filed on 23. December 2009.

Quick test of the activity of HPPD protein: Production of brown color Colorimetric Screening Test for Active HPPD Enzymes:
A YT-broth-type culture medium with 1% agarose, 5mM L-Tyrosine and 42mM
Succinate, which contains the selection agent for the vector pSE420 is poured into deep well plates. E.coli culture in the exponentional growth phase which contains the vector pSE420-HPPDx (any gene coding for a putative HPPD enzyme/protein) is applied to each well. After 16 hours at 37 C, the wells which do not contain the culture medium, those which have been seeded with an E. coil culture containing the empty vector pSE420 are transparent, or those which have been seeded with an E.
coli culture containing a vector pSE420 containing a gene coding for an inactive HPPD are transparent, while the wells seeded with an E. coil culture containing the vector pSE420-HPPD coding for active HPPD are brown. It has been previously demonstrated that this test refelects the HPPD activity, whatever the orgin of this activity, and allows the identification of HPPD activities (US 6,768,044).
As it can be seen on Figure 3, the culture from bacteria containing the gene coding for the mutant HPPD did not develop a brown color while the one containing the gene coding of the wild type HPPD developed a strong brown color reflecting the activity of the HPPD enzymes. It can be concluded that the two HPPD mutants are not able to convert HPP into homogentisate. The two HPPD mutants are inactive.
One can conclude that the amino acid positions 267 and 282 (referring to position in SEQ ID No. 2) are essential for HPPD activity.
The expression of the HPPD protein was then done as following.
Overnight cultures grown at 37 C were used to inoculate LB media at a ratio 1:100.
Cells were allowed to grow until OD reached 0.5, then expression was initiated from the trp-lac (trc) promoter by induction with 1 mM IPTG which binds to the lac .. repressor and causes its dissociation from the lac operon. Expression was carried out over 15h at 28 C.

. =

To prepare the pre-starter culture, 2 mL of TB medium (100 pg*mL-1 carbenicillin) were inoculated with 50 pL of an E coil K-12 BL21 glycerol stock. The pre-starter culture was incubated at 37 C with shaking at 140 rpm for 15 h. 200p1 of the pre-starter culture was used to initiate the starter culture (5mL TB supplement with 100 pg*L-1), which was incubated 3 h at 37 C.
To prepare the main culture, 400 mL of TB medium (100 pg*ml.:1 carbenicillin) were inoculated with 4 mL of the starter culture. This starter culture was incubated at 37 C with shaking at 140 rpm until 0D600 0.5 was reached. Then recombinant protein expression was induced with 400 pl of 1M IPTG solution. The cells were allowed to grow for an additional hour under these conditions, then the temperature was lowered to 28 C and the culture was shaken at 140 rpm for 15 h. Cells were harvested by centrifugation at 6000 x g for 15 min at 4 C. Then cell pellets were stored at -80 C.
Isolation and purification of His6-AtHPPD in native form Lysis of cells Cells were lysed using Lysozyme, an enzyme that cleaves the 1,4-0-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in peptidoglycan which forms the bacterial cell wall. Cell membranes were then disrupted by the internal pressure of the bacterial cell. In addition, the lysis buffer contained Benzonase Nuclease, an endonuclease that hydrolyzes all forms of DNA
and RNA without damaging proteins and thereby largely reduces viscosity of the cell lysate. Lysis under native conditions was carried out on ice.
For purification of His6-tagged proteins the QIAexprese Ni-NTA Fast Start Kit was used following the user manual instruction.
Purification of His6-tagged proteins by immobilized metal ion affinity chromatography (IMAC) =

The cleared cell lysate (10 mL) obtained after centrifugation of the lysis reaction was loaded onto a Ni-NTA Fast Start Column from the QlAexpress Ni-NTA Fast Start Kit (Qiagen, Hi!den, Germany) and purification was carried out according to the instruction manual. The Hiss-tagged protein was eluted with 2.5 mL of elution buffer.
Desalting of HPPD solutions by gel filtration I-IPPD solutions eluted from a Ni-NTA Fast Start Column with 2.5 mL of elution buffer were applied to a Sephadex G-25 PD-10 column (GE Healthcare, Freiburg, Germany) following the user manual instruction. After the whole sample had entered the gel bed, elution was performed with 3.5 mL of storage buffer.
The HPPD solutions eluted from the desalting column were frozen at -80 C in 1 mL
aliquots.
Determination of HPPD protein concentration using the Bradford protein assay Protein concentration was determined using the standard Bradford assay (Bradford, (1976), Anal Biochem 72: 248-254).
Determination of purity of HPPD solutions using SDS-PAGE
The integrity of the eluted protein was checked by SDS-PAGE protein gel electrophoresis using the gel NuPAGE Novex 4-12 % Bis-Tris Gels (Invitrogen, Karlsruhe, Germany), approximately 10 pg of protein were loaded. 10 pL of Laemmli Sample Buffer was added to 1-10 pL of protein solution and the mixture was incubated at 90 C for 10 min. After short centrifugation step, the whole mixture was loaded into a slot of an SDS gel previously fixed in a XCell SureLockTM Novex Mini-Cell gel chamber filled with NuPAGE MOPS SDS Running Buffer (diluted from the 20 x-solution with ddH20). A voltage of 150 was then applied to the gel chamber for 1 h. For staining of protein bands, the gel was immersed in Coomassie Brilliant Blue R-250 Staining Solution. For destaining of the polyacrylamide gel, it was immersed in Coomassie Brilliant Blue R-250 Destaining Solution until protein bands appear blue on a white gel.

=

The HPPD activity was checked by the standard spectrophotmetric assay (method extensively described in WO 2009/144079) In this content, p150-value means the log value of the concentration of inhibitor __ necessary to inhibit 50% of the enzyme activity in molar concentration.
p150-values for HPPD inhibitors were determined from dose-response plots of HPPD
activity versus inhibitor concentration using the assay extensively described in WO 2009/144079 at 2 mM fixed HPP concentration and 3 minutes fixed incubation time using the ID Business Solutions Ltd. XLfit software suite.
Table 7: Determination of pI50 HPPD enzymes (Arabidopsis thaliana wild type HPPD "SEQ ID No. 32", the mutants S267A (SEQ ID No. 34) and N282A
__ (SEQ ID No. 36)" and the tolerance to the several listed below HPPD
inhibitors tembotrione, diketonitrile, mesotrione, bicyclopyrone, pyrasulfotole, sulcotrione, pyrazolate, tefuryltrione, and benzofenap. The symbol ">" means that the value was far higher than the one indicated but could not be precisely calculated within in the range of concentration of inhibitor tested (2.5x10-6, 5.0x10-6, 1.0x10-5, 2.5x10-5, __ 6.3x10-5, and 2.5x10-4M).
Bicyclopyrone Benzofenap Diketonitrile Mesotrione VVT
5.2 >5.6 >5.6 >5.6 (SEQ ID No. 32) nd - la (P) nd - la (P) nd la (P) nd - la (P) (SEQ ID No. 34) nd - la (P) nd - la (P) nd - la (P) nd la (P) (SEQ ID No. 36) Pyrasulfotole Pyrazolate Sulcotrione Tefuryltrione Tembotrione WT
5.4 5.4 >5.6 >5.6 >5.6 (SEQ ID No. 32) nd - la (P) nd - la (P) nd - la (P) nd - la (P) nd - la (P) (SEQ ID No. 34) nd la (P) nd - la (P) nd - la (P) nd - la (P) nd - la (P) (SEQ ID No. 36) As previously demonstrated, the 2 protein mutants are not able to convert HPP
into Homogentisate, confirming that the 2 mutant proteins are inactive. It confirms the hypothesis that the position 267 and 282 (referring to position in SEQ ID No.
2) are absolutely essential for obtaining an active HPPD
Example 2: Single point mutants displayed increased tolerance to HPPD
inhibitor herbicides:
Kinetic characterization and evaluation of tolerance to HPPD inhibitors of HPPD
enzyme "SEQ ID No. 32".
.. The HPPD activity was checked by the standard spectrophotmetric assay (method extensively described in WO 2009/144079) =
Determination of HPPD in vitro kinetic properties Km, Vmax, and kcat values for different HPPD enzyme preparations and K1, K1=k0n, and Ki=Koff for different HPPD inhibitors were determined using a HPLC assay for measurements of HPPD activity. The assay mixtures contained in a volume of 1 ml 150 mM Tris-HCI buffer at pH 7.8, 10 mM sodium ascorbate, 650 units of bovine catalase (Sigma C30 (Sigma-Aldrich, Munich,Germany), 34 mg protein/ml, 23,000 .. units/mg), and appropriate amounts of HPP, purified HPPD enzyme and HPPD
inhibitors. For Km, Vmax, and kcat value determination HPP concentrations in the assay mixture were varied between 10 and 400 pM. For K., Ki=Kon, and KA=Koff value determination 2 mM HPP was used. All assays were started by the addition of HPPD enzyme to the assay mixture and stopped at a series of times between 0 and 240 s by addition of 200 pl of the reaction mixture to reaction assay tubes containing __ 20 p110% perchloric acid. Precipitated protein was pelleted by a 5 minute centrifugation at 10,000 g. 100 pl of the supernatant were loaded onto a 250 x 4mm Knauer (Berlin, Germany) Eurospher 100-5 C18-column equilibrated with 10%
methanol, 0.1% trifluoroacetic acid (buffer A). The column was eluted, also at 1.5 ml/min, using a 4 minute wash with buffer A, followed by a 3 min wash with 95%
__ methanol and by a further 2 minute wash with buffer A. The elution of HGA
(homogentisic acid) and HPP (hydroxyphenylpyruvate) was monitored at 292 nm.
HGA elutes at around 5 minutes and HPP elutes later. A standard set of concentrations of HGA were used to provide a standard curve in order to calibrate the 292 nm absorbance of the HGA peak versus HGA concentration.
__ For Km and Võax value determinations the initial rates of the HPPD reaction at different substrate concentrations were determined from plots of HGA formed versus time and fitted to the Michaelis-Menten equation for unireactant enzymes using the ID Business Solutions Ltd. (www.idbs.com) XLfit software suite. For the determination of Ki, Ki=Kon, and K.1=K0ff values the time-courses of the HPPD
__ reaction at different inhibitor concentrations were fitted to the equations for Mechanism A, competitive inhibition, for tight-binding inhibitors (Cha, S.
(1975) Tight-binding inhibitors ¨ I. Kinetic behaviour. Biochemical Pharmacology 24, 2177-2185) using the ID Business Solutions Ltd. XLfit software suite __ Table 8 Protein SEQ ID No. 32 consists of protein SEQ ID No. 2 with an insertion of one amino acid Ala and six amino acids His directly behind the first methionine.
Kinetic characterization of HPPD enzymes (Arabidopsis thaliana "SEQ ID No. 32"
__ and the respective tolerance to the HPPD inhibitors tembotrione and diketonitrile.
In below given table1, "Km" (Michaelis-Menten constant) means the kinetic parameter that is used to characterise an enzyme, and ills defined as the concentration of =

substrate that permits half maximal rate of the reaction. Km is further defined as the substrate concentration at which the reaction rate reaches half of its maximum value (Vmax/2) where Vmax has the meaning of being the maximum velocity of the reaction.
'<0n-A(1 equals the association rate constant of the enzyme-substrate binding and Kaff=KA equals the rate constant of the enzyme-inhibitor complex dissociation.
Ki defines the inhibition constant.
HPP Tembotrione Diketonitrile Km Vmax k1 k1 Kik1 k1K1 (PM) (IiM) (M-1 s-1) (s-1) (pM) (M-1 s-1) (s-1) (1A11) SEQ ID 6.3 1.2 2.3E+05 3.5E-03 0.015 6.1E+05 1.1E-02 0.018 No. 32 The kinetic parameters Km and Vmax of the plant HPPD "SEQ ID No. 32"
summarized in Table 8 are well in the range of the specific activities measured from purified plant HPPD as for example for the carrot HPPD (Garcia et al. 2000, Biochemistry, 39, 7501-7507). The Arabidopsis thaliana HPPD is also sensitive in the smimilar range as the carrot HPPD to diketonitrile. It is the first time, that the kinetic parameter of Arabidopsis thaliana HPPD in presence of tembotrione are measured and being in a comparable range as the one measured in presence of the HPPD inhibitor diketonitrile.
Determination of HPPD activity in presence of several HPPD inhibitors In this content, plarvalue means the log value of the concentration of inhibitor necessary to inhibit 50% of the enzyme activity in molar concentration.
p150-values for HPPD inhibitors were determined from dose-response plots of HPPD
activity versus inhibitor concentration using the assay extensively described in WO 2009/144079 at 2 mM fixed HPP concentration and 3 minutes fixed incubation time using the ID Business Solutions Ltd. XLfit software suite.

. , Table 9: Determination of pI50 HPPD enzymes (Arabidopsis (haliana "SEQ ID
No. 32" and some Arabidopsis HPPD single point mutants "SEQ ID No. 35, SEQ ID No. 40, SEQ ID No. 42, SEQ ID No. 44, SEQ ID No, 46, SEQ ID No. 48, and SEQ ID No. 50") and their respective tolerance to the several listed below HPPD
inhibitors tembotrione, diketonitrile, mesotrione, bicyclopyrone, pyrasulfotole, sulcotrione, pyrazolate, tefuryltrione, and benzofenap. The symbol ">" means that the value was far higher than the one indicated but could not be precisely calculated within in the range of concentration of inhibitor tested (2.5x10-6, 5.0x10-6, 1.0x10'5, 2.5x10-5, 6.3x10-5, and 2.5x10-4M).
Bicyclopyrone Benzofenap Diketonitrile Mesotrione Pyrasulfotole SEQ ID No. 32 5.2 >5.6 >5.6 >5.6 5.4 E252S 4.3 5.2 4.9 5.3 4.3 (SEQ ID No. 36) V269A 4.3 5.2 5.2 5.2 4.4 (SEQ ID No. 40) P280A 4.3 5.2 5.2 5.3 4.4 SEQ ID No. 42 L368M 4.5 >5.6 5.6 5.4 5.0 SEQ ID No. 44 K4211 4.2 5.1 5.0 5.2 4.3 SEQ ID No. 46 K421Q 4.7 >5.6 >5.6 >5.6 4.8 SEQ ID No. 48 K421V 4.2 5.2 5.1 5.3 4.4 SEQ ID No. 50 . .

Pyrazolat Sulcotrione Tefuryltrione Ternbotrione SEQ ID No. 32 5.4 >5.6 >5.6 >5.6 E2525 4.7 5.1 5.2 5.5 (SEQ ID No. 35) V269A 4.4 5.0 5.4 >5.6 (SEQ ID No. 40) P280A 4.7 5.0 ' 5.5 >5.6 SEQ ID No. 42 L368M 4.9 5.2 >5.6 >5,6 SEQ ID No. 44 - K421I 4.5 5.0 5.2 5.6 SEQ ID No. 46 K421Q 5.0 >5.6 >5.6 >5.6 SEQ ID No. 48 K421V 4.5 5.1 5.4 >5.6 SEQ ID No, 50 Measurements show that the pI50 in case of the point mutants is reduced compared to the wild-type protein_ In Table 9, it can be clearly seen that the HPPD mutants are all affected in their tolerance to at least one HPPD inhibitors and some to several HPPD inhibitors.
All the tested mutants are more active (do show an increased tolerance concerning the various applyied HPPD inhibitors) in the presence of at least one of the selected HPPD inhibitors, therefore the mutant enzyme-inhibitor complex should be less stable the wild-type enzyme-inhibitor complex. It can be concluded that the positions 252, 269, 280, 368 and 421 (reference taken in SEQ ID No. 2) are all important in the modulation of tolerance of HPPD enzyme to HPPD inhibitors.

111a SEQUENCE LISTING IN ELECTRONIC FORM
In accordance with Section 111(1) of the Patent Rules, this description contains a sequence listing in electronic form in ASCII
text format (file: 30725-1494 Seq 13-MAR-13 vl.txt).
A copy of the sequence listing in electronic form is available from the Canadian Intellectual Property Office.
The sequences in the sequence listing in electronic form are reproduced in the following table.
SEQUENCE TABLE.
<110> Bayer CropScience AG
<120> HPPD variants and methods of use <130> 30725-1494 <140> To be assigned <141> 2013-03-05 <160> 50 <170> PatentIn version 3.3 <210> 1 <211> 1335 <212> DNA
<213> Arabidopsis thaliana <400> 1 atgggccacc aaaacgccgc cgtttcagag aatcaaaacc atgatgacgg cgctgcgtcg 60 tcgccgggat tcaagctcgt cggattttcc aagttcgtaa gaaagaatcc aaagtatgat 120 aaattcaagg ttaagcgctt ccatcacatc gagttctggt gcggcgacgc aaccaacgtc 180 gctcgtcgct tctcctgggg tctggggatg agattctccg ccaaatccga tctttccacc 240 ggaaacatgg ttcacgcctc ttacctactc acctccggtg acctccgatt ccttttcact 300 gctccttact ctccgtctct ctccgccgga gagattaaac cgacaaccac agcttctatc 360 ccaaqtttcq atcacggctc ttgtcgttcc ttcttctctt cacatggtct cggtgttaga 420 gccgttgcga ttgaagtaga agacgcagag tcagctttct ccatcagtgt agctaatggc 480 gctattcctt cgtcgcctcc tatcgtcctc aatgaagcag ttacgatcgc tgaggttaaa 540 ctatacggcq atgttgttct ccgatatgtt agttacaaag cagaagatac cgaaaaatcc 600 gaattcttgc cagggttcga gcgtgtagag gatqcgtcgt cgttcccatt ggattatggt 660 atccggcggc ttgaccacgc cgtgggaaac gttcctgagc ttggtccggc tttaacttat 720 gtagcggggt tcactggttt tcaccaattc gdagagttca cagcagacga cgttggaacc 780 gccgagagcg gtttaaattc agcggtcctg gctagcaatg atgaaatggt tcttctaccg 840 attaacgagc cagtgcacgg aacaaagagg aagagtcaga ttcagacgta tttggaacat 900 aacgaaggcg cagggctaca acatctggct ctgatgagtg aagacatatt caggaccctg 960 agagagatga ggaagaggag cagtattgga ggattcgact tcatgccttc tcctccgcct 1020 acttactacc agaatctcaa gaaacgggtc ggcgacgtgc tcagcgatga tcagatcaag 1080 gagtgtgagg aattagggat tcttgtagac agagatgatc aagggacgtt gcttcaaatc 1140 111b ttcacaaaae cactaggtga caggccgacg atatttatag agataatcca gagagtagga 1200 tgcatgatga aagatgagga agggaaggct taccagagtg gaggatgtgg tggttttggc 1260 aaaggcaatt tctctgagct cttcaagtec attgaagaat acgaaaagac tcttgaagcc 1320 aaacagttag tggga 1335 <210> 2 <211> 445 <212> PRT
<213> Arabidopsis thalisns <400> 2 Met Gly His Gin Asn Ala Ala Val Ser Glu Asn Gin Asn His Asp Asp Gly Ala Ala Ser Ser Pro Gly Phe Lys Leu Val Gly Phe Ser Lys Phe Val Arg Lys Asn Pro Lys Ser Asp Lys Phe Lys Val Lys Arg Phe His His Ile Glu Phe Trp Cys Gly Asp Ala Thr Asn Val Ala Arg Arg Phe Ser Trp Gly Leu Gly Met Arg Phe Ser Ala Lys Ser Asp Leu Set Thr Gly Asn Met Val His Ala Ser Tyr Leu Lou Thr Ser Gly Asp Leu Arg Phe Leu Phe Thr Ala Pro Tyr Ser Pro Ser Leu Ser Ala Gly Glu Ile Lys Pro Thr Thr Thr Ala Scr Ile Pro Ser Phe Asp His Gly Ser Cys Arg Ser Phe Phe Ser Ser His Gly Leu Gly Val Arg Ala Val Ala Ile Glu Val Glu Asp Ala Glu Ser Ala Phe Ser Ile Ser Val Ala Asn Gly Ala Ile Pro Ser Ser Pro Pro Ile Val Leu Asn Glu Ala Val Thr Ile Ala Glu Val Lys Leu Tyr Gly Asp Val Val Leu Arg Tyr Val Ser Tyr Lys Ala Glu Asp Thr Glu Lys Ser Glu Phe Leu Pro Gly Phe Glu Arg Val Glu Asp Ala Ser Ser Phe Pro Leu Asp Tyr Gly Ile Arg Arg Lou Asp His Ala Val Gly Asn Val Pro Glu Leu Gly Pro Ala Leu Thr Tyr Val Ala Gly Phe Thr Gly Phe His Gin Phe Ala Glu Phe Thr Ala Asp Asp Val Gly Thr Ala Glu Ser Gly Leu Asn Ser Ala Val Leu Ala Ser Asn Asp Glu Met Val Leu Leu Pro Ile Asn Glu Pro Val His Gly Thr Lys Arg Lys Ser Gin Ile Gin Thr Tyr Leu Glu His Asn Glu Gly Ala Gly Leu Gin His Leu Ala Leu Met Ser Glu Asp Ile Phe Arg Thr Leu Arg Glu Met Arg Lys Arg Ser Ser Ile Gly Gly Phe Asp Phe Met Pro Ser Pro Pro Pro Thr Tyr Tyr Gin Asn Leu Lys Lys Arg Val Gly Asp 111c Val Leu Ser Asp Asp Gin Ile Lys Glu Cys Glu Glu Leu Gly Ile Leu Val Asp Arg Asp Asp Gin Gly Thr Leu Leu Gin Ile Phe Thr Lys Pro Leu Gly Asp Arg Pro Thr Ile Phe Ile Glu Ile Ile Gin Arg Val Gly Cys Met Met Lys Asp Glu Glu Gly Lys Ala Tyr Gin Ser Gly Gly Cys Gly Gly Phe Gly Lys Gly Asn Phe Ser Glu Leu Phe Lys Ser Ile Glu Glu Tyr Glu Lys Thr Leu Glu Ala Lys Gin Leu Val Gly <210> 3 <211> 1341 <212> DNA
<213> Oryza sativa <400> 3 atgcctccca ctcccacccc caccgccacc accggcgccg tctcggccgc tgcggcggcg 60 ggggagaacg cggggttccg cctcgtcggg caccgccgct tcgtccgcgc caacccgcgg 120 agcgaccggt tccaggcgct cgcgttccac cacgtogagc tctqgtgcgc cgacgccgcg 180 tccgccgcgg gccggttcgc cttcgccctg ggcgcgccgc tcgccgccag gtccgacctc 240 tccacgggga actccgcgca cgcctccctc ctcctccgct ccgcctccgt cgcgttcctc 300 ttcaccgccc cctacggcgg cgaccacggc gtcggcgcgg acgcggccac caccgcctcc 360 atcccttcct tctccccagg cgccgcgcgg aggttcgccg cggaccacgg cctcgcggtg 420 cacgccgtgg cgctgcgcgt cgccgacgcg gccgacgcct tccgcgccag cgtcgcggcc 480 ggtgcgcgcc cggcgttcca gcccgccgac ctcggcqgtg gcttcggcct cgcggaggtg 540 gagctctacg gcgacgtcgt gctccgattc gtcagccacc cggacggcgc cgacgcgccc 600 ttcctcccgg gtttcgaggg cgtcagcaac ccgggcgccg tggactacgg cctccgccgg 660 ttcgaccacg tcgtcggcaa cgtgccggag ctcgctccgg tagccgcgta catctccggg 720 ttcaccgggt tccacgagtt cgccgagttc accgccgagg acgtgggcac cgccgagagc 780 ggcctcaact cggtggtgct cgccaacaac geggagaccg tgctgctgcc gctcaacgag 840 ccggtgcacg gcaccaagcg gcggagccag atacagacgt acctggacca ccacggcggc 900 ccgggggtgc agcacatcgc gctggccagc gacgacgtgc tcgggacgct gagggagatg 960 cgggcgcgct ccgccatggg cggcttcgag ttcttggcgc cgccgccgcc caactactac 1020 gacggcgtgc ggcggcgcgc cggggacgtg ctctcggagg agcagatcaa cgagtgccag 1080 gagctcgggg tgctcgtqga cagggatgac cagggqgtgt tgctccagat cttcaccaag 1140 ccagtaggag acaggccaac ctttttcttg gagatgatac aaaggattgg gtgcatggag 1200 aaggatgaga gtgggcagga gtaccagaag ggcggctgcg gcgggtttgg gaagggcaac 1260 ttctcggagc tgttcaagtc cattgaggag tatgagaaat cccttgaagc caagcaagcc 1320 cctacagttc aaggatccta g 1341 <210> 4 <211> 446 <212> PRT
<213> Oryza sativa <400> 4 Met Pro Pro Thr Pro Thr Pro Thr Ala Thr Thr Gly Ala Val Ser Ala Ala Ala Ala Ala Gly Glu Asn Ala Gly Phe Arg Lou Val Gly His Arg 111d Arg Phe Val Arg Ala Asn Pro Arg Ser Asp Arg Phe Gln Ala Leu Ala Phe His His Val Glu Leu Trp Cys Ala Asp Ala Ala Ser Ala Ala Gly Arg Phe Ala Phe Ala Leu Gly Ala Pro Leu Ala Ala Arg Ser Asp Leu Ser Thr Gly Asn Ser Ala His Ala Ser Leu Leu Leu Arg Ser Ala Ser Val Ala Phe Leu Phe Thr Ala Pro Tyr Gly Gly Asp His Gly Val Gly Ala Asp Ala Ala Thr Thr Ala Ser Ile Pro Ser Phe Ser Pro Gly Ala Ala Arg Arg Phe Ala Ala Asp His Gly Leu Ala Val His Ala Val Ala Leu Arg Val Ala Asp Ala Ala Asp Ala Phe Arg Ala Ser Val Ala Ala Gly Ala Arg Pro Ala Phe Gln Pro Ala Asp Leu Gly Gly Gly Phe Gly Leu Ala Glu Val Glu Leu Tyr Gly Asp Val Val Leu Arg Phe Val Ser His Pro Asp Gly Ala Asp Ala Pro Phe Leu Pro Gly Phe Glu Gly Val Ser Asn Pro Gly Ala Val Asp Tyr Gly Leu Arg Arg Phe Asp His Val Val Gly Asn Val Pro Glu Leu Ala Pro Val Ala Ala Tyr Ile Ser Gly Phe Thr Gly Phe His Glu Phe Ala Glu Phe Thr Ala Glu Asp Val Gly Thr Ala Glu Ser Gly Leu Asn Ser Val Val Leu Ala Asn Asn Ala Glu Thr Val Leu Leu Pro Leu Asn Glu Pro Val His Gly Thr Lys Arg Arg Ser Gln Ile Gln Thr Tyr Leu Asp His His Gly Gly Pro Gly Val Gln His Ile Ala Leu Ala Ser Asp Asp Val Lau Gly Thr Leu Arg Glu Met Arg Ala Arg Ser Ala Met Gly Gly Phe Glu Phe Leu Ala Pro Pro Pro Pro Asn Tyr Tyr Asp Gly Val Arg Arg Arg Ala Gly Asp Val Leu Ser Glu Glu Gln Ile Asn Glu Cys Gln Glu Leu Gly Val Leu Val Asp Arg Asp Asp Gln Gly Val Leu Leu Gln Ile Phe Thr Lys Pro Val Gly Asp Arg Pro Thr Phe Phe Leu Glu Met Ile Gln Arg Ile Gly Cys Met Glu Lys Asp Glu Ser Gly Gln Glu Tyr Gln Lys Gly Gly Cys Gly Gly Phe Gly Lys Gly Asn Phe Ser Glu Leu Phe Lys Ser Ile Glu Glu Tyr Glu Lys Ser Leu Glu Ala Lys Gln Ala Pro Thr Val Gln Gly Ser <210> 5 <211> 1281 b fle nTD ATO ETV PTV ATO dsy oaa aAy JeS TEA aAL ay a TPA TEA dsV
091 881 OgT G6I
ATO JAI nai nTO TPA nTD eIV na7 6IV aqd AID blci aqd PTV dgV nTD
OT GET OEI
PTV dsy 2TV TEA ETV naq PTV TPA PTV 6IV TPA PIV ne7 ATE) sTH dsy SZI OZT GTT
ETV ETV 214d bali 6JV eTV PTV eTV eTV leS aqd ass oad neq PTV PTV

J1-11, erd EDI ciGV uTV AID sTH eTV JAI 0.1d eTV qL qd naq aqd asS

naq las ATE) aes bay naq narl neri aes eTV sTH ETV IBS nsV AID agI

las nari dsv las 61V PTV PTV nal Old PTV AID nP7 ATD a-Td 19S GU

SIV ATE) PTV PTV JaS ETV ETV dsv sTy sA3 dai neq GTE) TEA STH Sill cf7 017 ge e-Td PTV nal 6LGTH Pqd 6.71/ ciski Jas 6.1v ()ad usv aqa 611/ WA sqd OE SZ
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18ZI b eqoo4ebbbe o4obeo54ob 09ZT eoftobaoba obeeobeepo buebqq000q bre5e54e4-4 ebbeb24epo 4b8e244b4o 00ZI bP05040143 882556eecb boT4656365 0640bbqbb6 82E83O2428 6e2056b6ee OPTI 6854Ebbee6 2bbqp0b466 bo4ebbeee0 oqe04ee266 .440446q0b0 2e33bbe026 2200E,0440; ppe33q0640 64636bbv30 26qebbbe3e bbq56-40646 00-[ 6b6u405ef.6 epobqbebbe eqlebeopob eebbneoqob qboebbbboo 606366056e 096 b4bo5bDe5l 240e4De5o0 4e0e0o3qo0 bobbqeo4ab ebo;4obbob bbleoobbaq 006 obababOeob qpbe666rE; oboebbeoao 6450260860 6e00664obo 66qe0e06eo 08 6460E6000o 563560epou 03ebb40344 608280E286 P006206306 0622302356 QSL 0e364bb336 ebouu34060 3343540645 3e8beb5340 2v38203534 061564u334 OZL peepqDpbbo 5268560600 eobbb45oe5 fiebboeboeo 1.q.be5Do5ol abeftepoqg 099 585502oqq.0 EtooLoqwe 4op5o5oo5 popbobbwb e563364602 236634E104e 009 oeopeboqab 5e36264356 50e-4086005 606E1563000 60554605 bbrboqqbbb 06G 63364034.10 30bpbobb60 6036056025 b3o0u43be6 qbop466004 3646346086 08V 356peq3436 eb3qb6ebo3 63qopelpoq4 0663633443 3613-ebbu600 53'96336346 017 oboowbobo qboobibobq bbobowobb ovoceboobe oboai.obobb oboboDboob 09E o0b03q3443 0-4.0005463 5306=2=6 l0boph0060 65oe0En50e qopobobboe ou 34.4p4004qo oloapoolob boowbopao baobabooqb obbeaboboo aoveDbbboe 0D,z. 334oqopeb0 owb0e0b00 6340633606 3666433660 41.paioqqob 3366636306 081 opqopbbpbo 8600606466 434.36266.46 3e30e33446 3bo4o6383e 3044353386 ee344015334 boagoeepbo peoobbbgbo qoobooggbo bbobbeobeb 09 bobeobeoge 0653653564 6 06=8356 3062350363 obeoeoppop eboopoobae G <006>
sAem 882 <-2>
VNO <ZTZ>
ITT

111f Pro Phe Leu Pro Gly She Glu Gly Val Ala Ser Pro Gly Ala Ala Asp Tyr Gly Leu Ser Arg Phe Asp His Ile Val Gly Asn Val Pro Glu Leu Ala Pro Ala Ala Ala Tyr Phe Ala Gly She Thr Gly Phe His Glu Phe Ala Glu Phe Thr Thr Glu Asp Val Gly Thr Ala Glu Ser Gly Leu Asn Ser Met Val Leu Ala Asn Asn Ser Glu Asn Val Leu Leu Pro Leu Asn Glu Pro Val His Gly Thr Lys Arg Arg Ser Gin Ile Gin Thr Phe Leu Asp His His Gly Gly Pro Gly Val Gin His Met Ala Leu Ala Ser Asp Asp Val Leu Arg Thr Leu Arg Glu Met Gin Ala Arg Ser Ala Met Gly Gly She Glu She Met Ala Pro Pro Thr Ser Asp Tyr Tyr Asp Gly Val Arg Arg Arg Ala Gly Asp Val Leu Thr Glu Ala Gin Ile Lys Glu Cys Gin Glu Leu Gly Val Leu Val Asp Arg Asp Asp Gln Gly Val Leu Leu Gin Ile Phe Thr Lys Pro Val Gly Asp Arg Pro Thr Lou Phe Leu Glu Ile Ile Gin Arg Ile Gly Cys Met Glu Lys Asp Glu Lys Gly Gin Glu Tyr Gin Lys Gly Gly Cys Gly Gly She Gly Lys Gly Asn Phe Ser Gin Leu She Lys Ser Ile Glu Asp Tyr Glu Lys Ser Leu Glu Ala Lys Gin Ala Ala Ala Ala Ala Ala Ala Gin Gly Ser <210> 7 <211> 1323 <212> DNA
<213> Avena sativa <400> 7 atgccgccca cccccgccac cgccaccggc gccgccgcgg ccgccgtgac tccagagcac 60 gcggcccgga gctttccccg agtggtccgc gtcaacccgc gcagcgaccg attcbccgtg 120 ctctccttcc accacgtcga gctctggtgc gccgacgccg cctcagcggc cggacgcttc 180 tccttcgcgc tcggcgcgcc gctcgccgcc cggtccgacc tctccacggg gaactccgcg 240 cacgcctccc tcctgctccg ctcgggcgcc ctcgccttcc tcttcacggc gccctacgcg 300 ccgccgccgc aggaggccgc cacggccgca gccaccgcct ccatcccctc cttctccgcc 360 gacgccgcgc ggacgttcgc cgccgcccac ggcctcgcgg tgcgctccgt cggggtbcgc 420 gtcgctgacg ccgccgaggc cttccgcgtc agcgtagccg gcggcgctcg cccggccttc 480 gccccagccg acctcggcca tggcttcggc ctcgccgagg tcgagctcta cggcgacgtc 540 gtgctacgct tcgtcagcta cccggacgag acagacctgc cattcctgcc agggttcgag 600 cgcgtgagca gccccggcgc cgtggactac ggcctcacgc ggttcgacca cgtcgtgggc 660 aacgtcccgg agatggcccc ggtcatagac tacatgaaag gcttcttggg gttccacgag 720 ttcgccgagt tcaccgccga ggacgtgggc acgaccgaga gcgggctcaa ctcggtggtg 780 ctcgccaaca actccgaggc cgtgctgctg ccgctcaacg agcccgtgca cggcacaaag 840 cgacggagcc agatacagac gtacctggag tatcacggcg ggcccggcgt gcagcacatc 900 gcgctcgcca gcaacgacgt gctcaggacg ctcagggaga tgcgggcgcg cacgcccatg 960 111g ggcggcttcg agttcatggc gccaccgcag gcgaaatact atgaaggcgt gcggcgcatc 1020 gcaggtgacg tgctctcgga agagcagatc aaggaatgcc aggagctggg ggtgctagtc 1080 gacagggatg atcaaggggt gttgctccaa atcttcacca agccagtagg ggacaggcca 1140 acgtttttcc tggagatgat ccaaagaatc gggtgcatgg agaaggacga ggtcgggcaa 1200 gagtaccaga agggtggctg cggcgggttt ggcaagggca atttctccga gctgttcaag 1260 tccattgagg actatgagaa atcccttgag gtcaagcaat ctgttgtagc tcagaaatcc 1320 tag 1323 <210> 8 <211> 440 <212> PRT
<213> Avena sativa <400> 8 Met Pro Pro Thr Pro Ala Thr Ala Thr Gly Ala Ala Ala Ala Ala Vol Thr Pro Glu His Ala Ala Arg Ser Phe Pro Arg Vol Val Arg Val Asn Pro Arg Ser Asp Arg Phe Pro Val Leu Ser Phe His His Val Glu Leu Trp Cys Ala Asp Ala Ala Ser Ala Ala Gly Arg Phe Ser Phe Ala Leu Gly Ala Pro Leu Ala Ala Arg Ser Asp Leu Ser Thr Gly Asn Ser Ala His Ala Ser Leu Leu Leu Arg Ser Gly Ala Leu Ala Phe Leu Phe Thr Ala Pro Tyr Ala Pro Pro Pro Gin Glu Ala Ala Thr Ala Ala Ala Thr Ala Ser Ile Pro Ser Phe Ser Ala Asp Ala Ala Arg Thr Phe Ala Ala Ala His Gly Leu Ala Val Arg Ser Val Gly Val Arg Val Ala Asp Ala Ala Glu Ala Phe Arg Val Ser Val Ala Gly Gly Ala Arg Pro Ala Phe Ala Pro Ala Asp Leu Gly His Gly Phe Gly Leu Ala Glu Vol Glu Leu Tyr Gly Asp Vol Val Leu Arg Phe Vol Ser Tyr Pro Asp Glu Thr Asp Leu Pro Phe Leu Pro Gly Phe Glu Arg Val Ser Ser Pro Gly Ala Val Asp Tyr Gly Leu Thr Arg Phe Asp His Val Val Gly Asn Vol Pro Glu Met Ala Pro Vol Ile Asp Tyr Met Lys Gly Phe Leu Gly Phe His Gin Phe Ala Glu Phe Thr Ala Glu Asp Val Gly Thr Thr Glu Ser Gly Leu Asn Ser Val Vol Leu Ala Asn Asn Ser Glu Ala Val Leu Leu Pro Leu Asn Glu Pro Val His Gly Thr Lys Arg Arg Ser Gin Ile Gin Thr Tyr Leu Glu Tyr His Gly Gly Pro Gly Val Gln His Ile Ala Leu Ala Ser Asn Asp Vol Leu Arg Thr Leu Arg Glu Met Arg Ala Arg Thr Pro Met 111h Gly Gly Phe Glu Phe Met Ala Pro Pro Gin Ala Lys Tyr Tyr Glu Gly Val Arg Arg Ile Ala Gly Asp Val Leu Ser Glu Glu Gin Ile Lys Glu Cys Gin Glu Leu Gly Val Lou Val Asp Arg Asp Asp Gin Gly Val Leu Leu Gin Ile Phe Thr Lys Pro Val Gly Asp Arg Pro Thr Phe Phe Lou Glu Met Ile Gin Arg Ile Gly Cys Net Giu Lys Asp Glu Val Gly Gin Glu Tyr Gin Lys Gly Gly Cys Gly Gly Phe Gly Lys Gly Asn Phe Ser Clu Lou Phe Lys Ser Ile Glu Asp Tyr Glu Lys Ser Lou Glu Val Lys Gin Ser Val Val Ala Gin Lys Ser <210> 9 <211> 1077 <212> DNA
<213> Pseudomonas fluorescens <400> 9 atggcagatc tatacgaaaa cccaatgggc ctgatgggct ttgaattcat cgaattcgcg 60 tcgccgacgc cgggtaccct ggagccgatc ttcgagatca tgggcttcac caaagtcgcg 120 acccaccgtt ccaagaacgt gcacctgtac cgccagggcg agatcaacct gatcctcaac 180 aacgagccca acagcatcgc ctcctacttt gcggccgaac acggcccgtc ggtgtgcggc 240 atggcgttcc gcgtgaagga ctcgcaaaag gcctacaacc gcgccctgga actcggcgcc 300 cagccgatcc atattgacac cgggccgatg gaattgaacc tgccggcgat caagggcatc 360 ggcggcgcgc cgttgtacct gatcgaccgt ttcggcgaag gcagctcgat ctacgacatc 420 gacttcgtgt acctcgaagg tgtggagcgc aatccggtcg gtgcaggtct caaagtcatc 480 gaccacctga cccacaacgt cLatcgcgyc cycaLggtct actgggccaa cttctacgag 540 aaattgttca acttccgtga agcgcgttac ttcgatatca agggcgagta caccggcctg 600 acttccaagg ccatgagtgc gccggacggc atgatccgca tcccgctgaa cgaagagtcg 660 tccaagggcg cggggcagat cgaagagttc ctgatgcagt tcaacggcga aggcatccag 720 cacgtggcgt tcctcaccga cgacctggtc aagacctggg acgcgttgaa gaaaatcggc 780 .atgcgcttca tgaccgcgcc gccagacact tattacgaaa tgctcgaagg ccgcctgcct 840 gaccacggcg agccggtgga tcaactgcag gcacgcggta tcctgctgga cggatcttcc 900 gtggaaggcg acaaacgcct gctgcLgcag atcttctcgg aaaccctgat gggcccggtg 960 ttcttcgaat tcatccagcg caagggcgac gatgggtttg gcgagggcaa cttcaaggcg 1020 ctgttcgagt ccatcgaacg tgaccaggtg cgtcgtggtg tattgaccgc cgattaa 1077 <210> 10 <211> 358 <212> PRT
<213> Pseudomonas fluorescens <400> 10 Met Ala Asp Leu Tyr Glu Asn Pro Met Gly Lou Met Cly Phe Glu Phe Ile Glu Phe Ala Ser Pro Thr Pro Gly Thr Lou Glu Pro Ile Phe Glu Ile Met Gly Phe Thr Lys Val Ala Thr His Arg Ser Lys Asn Val His 111i Leu Tyr Arg Gin Gly Glu Ile Asn Leu Ile Leu Asn Asn Glu Pro Asn 50 55 '60 Ser Ile Ala Ser Tyr Phe Ala Ala Glu His Gly Pro Ser Val Cys Gly Met Ala Phe Arg Val Lys Asp Ser Gin Lys Ala Tyr Asn Arg Ala Leu Glu Leu Gly Ala Gin Pro Ile His Ile Asp Thr Gly Pro Met Glu Leu Asn Leu Pro Ala Ile Lys Gly Ile Gly Gly Ala Pro Leu Tyr Leu Ile Asp Arg Phe Gly Glu Gly Ser Ser Ile Tyr Asp Ile Asp Phe Val Tyr Leu Glu Gly Val Glu Arg Asn Pro Val Gly Ala Gly Leu Lys Val Ile Asp His Leu Thr His Asn Val Tyr Arg Gly Arg Met Val Tyr Trp Ala Asn Phe Tyr Glu Lys Leu Phe Asn Phe Arg Glu Ala Arg Tyr Phe Asp Ile Lys Gly Glu Tyr Thr Gly Leu Thr Ser Lys Ala Met Ser Ala Pro Asp Gly Met Ile Arg Ile Pro Leu Asn Glu Glu Ser Ser Lys Gly Ala Gly Gin Ile Glu Glu Phe Leu Met Gin Phe Asn Gly Glu Gly Ile Gin His Val Ala Phe Leu Thr Asp Asp Leu Val Lys Thr Trp Asp Ala Leu 245 250 255 =
Lys Lys Ile Gly Met Arg Phe Met Thr Ala Pro Pro Asp Thr Tyr Tyr Glu Met Leu Glu Gly Arg Leu Pro Asp His Gly Glu Pro Vol Asp Gin Leu Gin Ala Arg Gly Ile Leu Leu Asp Gly Ser Ser Val Glu Gly Asp Lys Arg Leu Leu Leu Gin Ile Phe Ser Glu Thr Leu Met Gly Pro Val Phe Phe Glu Phe Ile Gin Arg Lys Gly Asp Asp Gly Phe Gly Glu Gly Asn Phe Lys Ala Leu Phe Glu Ser Ile Glu Arg Asp Gin Val Arg Arg Gly Val Leu Thr Ala Asp <210> 11 <400> 11 <210> 12 <400> 12 111j <210> 13 <211> 1053 <212> DNA
<213> Synechococcus sp.
<400> 13 atgaacccgt ccattcgaat tgtccaaggg atccaccacc tgcacttcta cctttgggat 60 ctgccccgtt ggcgggaaca cttttgtcgg qtttqqggct tccgggtggc aagcgacgcc 120 ggcaacaccc tggagctgga gcagggatcc ctgcgcttgc gcctgtctca gccggcacgg 180 gcgggggacg aggtggaccg ccatttgcag cggcaLgggc cgggggtggt ggatgtggcc 240 ttggcggtgg gagagcagga gctaccggcc ttggcggagc tgttgcgggg ccgaggcgcc 300 caactggcgt ggatcccggc agcagcggcg ctctgcctcc acacccccta cgggatCcgg 360 cattctctga tocctggccc cttggatgcc gcccctgccg aagcgggcct gttttcccac 420 tgggatcacg tggtgttgaa cgtggagcag ggatccctgc aggcggcagc cgactggtat 480 gggcgggtgc tgggctggcg gcggctgtac cgctacagca tcggcaccgc cacctccggc 540 ctggaaagcg tggtggtggg ggatccggaa gcggggatcc aatgggccat caacgagccc 600 acctgtgccg ettcccagat tcaggagttt ttgcatgccc atggcggccc gggcattcag 660 cacgcggcgc tgcacagctc agacattgtt gccagcctgc gccggttgcg gcagggggga 720 gtggactttt tgcaagtggc gccgcagtac tacaccagcc tggaaaggga gctggggttg 780 gcgctccgtt ctgcccttgg gcaggccatc tcctggcaag acctggtgga gcagcagatc 840 cttctggatg ctaccctgcc cgcttctgat ggccaggatc gcccccttct gctgcagacc 900 tttacccagc ccctctttgg tcggcccacc tttttctttg aagtcattca acggctaggc 960 ggggccacgg gctttggcga ggccaatttt caggctttgt tcgaggccct ggaacggcaa 1020 cagcgacagc gacaccaqgc gctgacccct tag 1053 <210> 14 <211> 350 <212> PRT
<213> Synechococcus sp.
<400> 14 Net Asn Pro Ser Ile Arg Ile Vol Gin Gly Ile His His Leu His Phe 1 5 10 15 =
Tyr Leu Trp Asp Leu Pro Arg Trp Arg Glu His Phe Cys Arg Val Trp Gly Phe Arg Val Ala Ser Asp Ala Gly Asn Thr Leu Glu Leu Glu Gin Gly Ser Leu Arg Leu Arg Leu Ser Gin Pro Ala Arg Ala Gly Asp Glu Vol Asp Arg His Leu Gin Arg His Gly Pro Gly Val Val Asp Val Ala Leu Ala Val Gly Glu Gin Glu Leu Pro Ala Leu Ala Glu Leu Leu Arg Gly Arg Gly Ala Gin Leu Ala Trp Ile Pro Ala Ala Ala Ala Leu Cys 1.00 105 110 Leu His Thr Pro Tyr Gly Ile Arg His Ser Leu Ile Pro Gly Pro Leu Asp Ala Ala Pro Ala Glu Ala Gly Leu Phe Ser His Trp Asp His Val Val Leu Asn Vol Glu Gin Gly Ser Leu Gin Ala Ala Ala Asp Trp Tyr Gly Arg Val Leu Gly Trp Arg Arg Leu Tyr Arg Tyr Ser Ile Gly Thr Ala Thr Ser Gly Leu Glu Ser Val Val Vol Gly Asp Pro Glu Ala Gly 111k Ile Gin Trp Ala Ile Asn Glu Pro Thr Cys Ala Ala Ser Gin Ile Gin Glu Phe Leu His Ala His Gly Gly Pro Gly Ile Gin His Ala Ala Leu His Ser Ser Asp Ile Val Ala Ser Leu Arg Arg Leu Arg Gin Gly Gly Val Asp Phe Leu Gin Val Ala Pro Gin Tyr Tyr Thr Ser Leu Glu Arg Glu Leu Gly Leu Ala Leu Arg Ser Ala Leu Gly Gin Ala Ile Ser Trp Gin Asp Leu Val Glu Gin Gin Ile Leu Leu Asp Ala Thr Leu Pro Ala Ser Asp Gly Gin Asp Arg Pro Lou Leu Leu Gin Thr Phe Thr Gin Pro Leu Phe Gly Arg Pro Thr Phe Phe Phe Glu Val Ile Gin Arg Leu Gly Gly Ala Thr Gly Phe Gly Glu Ala Asn Phe Gin Ala Leu She Glu Ala Leu Glu Arg Gin Gin Arg Gin Arg His Gin Ala Leu Thr Pro <210> 15 <211> 1149 <212> DNA
<213> Biepharisma japonicum <400> 15 atgacttatt acgacaagca agaaacgcgt ccagatcttg gcgaattcta tggtttccat 60 cacgttcgtt tttacgtctc caactcagag caagccgctt cgttctacac atctcgcttt 120 gggttttctc cggttgccta tgaaggattg gaaacaggaa accaaaaatt ctgtaccaat 180 gtcgtccgaa gcaaccatgt agtcatcgct tttacctcag ctctcactcc tgaagacaat 240 yaagLydauc yLuauyLtyy uaaycaLayL
ydLygdyLLu auyauaLLyu uLLLugLyLa 300 agtgacgcaa gagggatgta tgagaaagcg atagctaaag gctgtaaaag cttccgtgag 360 ccacaggttt tacaagatca atttggatct gttataatag cgtctctcca gacttatgga 420 gacactgttc acacattagt ccaaaatgtc gactatacag gacccttttt gcctggcttc 480 agagcaatca caaaagatga tccattaaac tctgcctttc ctcaggtaaa ttatgacatt 540 attgatcatg ttgtaggaaa tcagcctggt ggcgatatga ctcctacagt agaatggtat 600 gagaaatatc tagaatttca tcgatattgg tctgctgatg agtctgtaat ccataccgat 660 tattcagcat taaggtctgt tgtggttgct gattgggatg aagtgatcaa aatgcctatt 720 aatgagcctg ctgatggact tagaaaaagt caaatccaag aatatgtcga atattatggt 780 ggagcaggcg tacaacatat tgccttaaaa gtcaatgata ttatttcagt aataagcacc 840 ttaagggcta gaggtgtgga attcttagaa gttcctccta aatattatga tagcttaaga 900 aaaagacttg cgcattctgc ggtacaaatt gaagaagact taaaaagaat tgaagacctt 960 catattttgg ttgactttga cgaccgtggg tatttacttc agattttcac aaaaccagta 1020 gaagacagac ctactctgtt ttatgaaatt attcaaagac ataataacaa tggattcgga 1080 attggaaatt ttaaagccct atttgaatca ttggaacaag agcaagaaag aagaggtaat 1140 ttgatctaa 1149 <210> 16 <211> 382 <212> PRT
<213> Blepharisma japonicum <400> 16 Met Thr Tyr Tyr Asp Lys Gin Glu Thr Arg Pro Asp Lou Gly Glu Phe Tyr Gly Phe His His Val Arg Phe Tyr Val Ser Asn Ser Glu Gin Ala Ala Ser Phe Tyr Thr Ser Arg Phe Gly Phe Ser Pro Val Ala Tyr Glu Gly Leu Glu Thr Gly Asn Gin Lys Phe Cys Thr Asn Val Val Arg Ser Asn His Val Val Ile Ala Phe Thr Ser Ala Leu Thr Pro Glu Asp An Glu Val Asn Arg His Val Gly Lys His Ser Asp Gly Val Gin Asp Ile Ala Phe Ser Val Ser Asp Ala Arg Gly Met Tyr Glu Lys Ala Ile Ala Lys Gly Cys Lys Ser Phe Arg Glu Pro Gin Val Leu Gin Asp Gin Phe Gly Ser Val Ile Ile Ala Ser Leu Gin Thr Tyr Gly Asp Thr Val His Thr Leu Val Gin Asn Val Asp Tyr Thr Gly Pro Phe Leu Pro Gly Phe Arg Ala Ile Thr Lys Asp Asp Pro Leu Asn Ser Ala Phe Pro Gin Val Asn Tyr Asp Ile Ile Asp His Val Val Gly Asn Gin Pro Gly Gly Asp Net Thr Pro Thr Val Glu Trp Tyr Glu Lys Tyr Leu Glu Phe His Arg Tyr Trp Ser Ala Asp Glu Ser Val Ile His Thr Asp Tyr Ser Ala Leu Arg Ser Val Val Val Ala Asp Trp Asp Glu Val Ile Lys Net Pro Ile Asn Glu Pro Ala Asp Gly Lou Arg Lys Ser Gin Ile Gln Glu Tyr Val Glu Tyr Tyr Gly Gly Ala Gly Val Gin His Ile Ala Lou Lys Val Asn Asp Ile Ile Ser Val Ile Ser Thr Leu Arg Ala Arg Gly Val Glu Phe . Leu Glu Val Pro Pro Lys Tyr Tyr Asp Ser Leu Arg Lys Arg Leu Ala His Ser Ala Val Gin Ile Glu Glu Asp Leu Lys Arg Ile Glu Asp Leu His Ile Leu Val Asp Phe Asp Asp Arg Gly Tyr Leu Leu Gin Ile Phe Thr Lys Pro Val Glu Asp Arg Pro Thr Leu Phe Tyr Glu Ile Ile Gin Arg His Asn Asn Asn Gly Phe Gly Ile Gly Asn Phe Lys Ala Lou Phe Glu Ser Leu Glu Gin Glu Gin Glu Arg Arg Gly Asn Leu Ile <210> 17 <211> 1206 <212> DNA
<213> Rhodococcus sp.

11 lm <400> 17 atgacgatcg agcagactct caccgacaag gaacgcctgg caggtctcga cctcggccag 60 ctcgagcagt tggtcgggct cgtcgagtac gacggcaccc gcgacccgtt cccggtcagc 120 ggctgggatg ccgtcgtctg ggtggtcggc aacgccaccc agaccgccca ctacttccag 180 tccgcgttcg ggatgaccct cgtcgcctac tccggaccca ccaccggcaa ccgcgaccac 240 cacagcttcg tcctcgaatc cggggccgtc cgcttcgtca tcaaaggcgc cgtgaacccg 300 gacagccccc tgatcgacca ccaccgcacc cacggcgacg gcgtcgtcga catcgccctc 360 gccgtccccg acgtcgacaa gtgcatcgcc cacgcccgcg cccagggcgc caccgtcctc 420 gacgaacccc acgacgtgac cgacgaccac ggcaccgtcc gcctcgccgc gatcgccacc 480 tacggcgaca cccgccacac cctcgtcgac cgcagccact acaccggccc ctacctgccc 540 ggctacaccg cccgcacctc cggccacacc aaacgggacg gggcacccaa gcgcctgttc 600 caggccctcg accacgtcgt cggcaacgtc gaactcggca agatggacca ctgggtcgac 660 ttctacaacc gggtcatggg ctttacgaac atggccgagt tcgtcggcga ggacatcgcc 720 accgactact ccgcgctgat gagcaaggtc gtctccaacg gcaaccaccg ggtcaagttc 780 cccctcaacg aacccgccct cgccaagaaa cgctcgcaga tcgacgaata cctcgacttc 840 taccgcggcc ccggcgccca gcacctggcc ctggccacca atgacatcct caccgccgtc 900 gaccagctga ccgccgaggg cgtcgagttc ctggccaccc ccgactccta ctacgaggac 960 cccgaactgc gggcccggat cggcaacgtc cgcgccccca tcgccgaact gcagaaacgc 1020 ggcatcctcg tcgaccgcga cgaagacggc tacctgctgc agatcttcac caaacccctc 1080 gtcgaccggc ccaccgtgtt cttcgaactc atcgaacgcc acggctccct cggcttcggc 1140 atcggcaact tcaaagccct cttcgaggcc atcgaacgcg aacaagccgc ccgcggaaac 1200 ttctga 1206 <210> 18 <211> 401 <212> PRT
<213> Rhodococcus sp.
<400> 18 Met Thr Ile Glu Gin Thr Leu Thr Asp Lys Glu Arg Leu Ala Gly Leu Asp Leu Gly Gin Leu Glu Gin Leu Val Gly Leu Val Glu Tyr Asp Gly Thr Arg Asp Pro Phe Pro Val Ser Gly Trp Asp Ala Val Val Trp Val Val Gly Asn Ala Thr Gin Thr Ala His Tyr Phe Gin Ser Ala Phe Gly Met Thr Leu Val Ala Tyr Ser Gly Pro Thr Thr Gly Asn Arg Asp His His Ser Phe Val Leu Glu Ser Gly Ala Val Arg Phe Val Ile Lys Gly Ala Val Asn Pro Asp Ser Pro Leu Ile Asp His His Arg Thr His Gly Asp Gly Val Val Asp Ile Ala Leu Ala Val Pro Asp Val Asp Lys Cys Ile Ala His Ala Arg Ala Gin Gly Ala Thr Val Leu Asp Glu Pro His Asp Val. Thr Asp Asp His Gly Thr Val Arg Leu Ala Ala Ile Ala Thr Tyr Gly Asp Thr Arg His Thr Leu Val Asp Arg Ser His Tyr Thr Gly Pro Tyr Leu Pro Gly Tyr Thr Ala Arg Thr Ser Gly His Thr Lys Arg Asp Gly Ala Pro Lys Arg Leu Phe Gin Ala Leu Asp His Val Val Gly 111n Asn Val Glu Leu Gly Lys Met Asp His Trp Val Asp Phe Tyr Asn Arg Val Met Gly Phe Thr Asn Met Ala Glu Phe Val Gly Glu Asp Ile Ala Thr Asp Tyr Ser Ala Leu Met Ser Lys Val Val Ser Asn Gly Asn His Arg Val Lys Phe Pro Lou Asn Glu Pro Ala Lou Ala Lys Lys Arg Ser Gln Ile Asp Glu Tyr Leu Asp Phe Tyr Arg Gly Pro Gly Ala Gln His Leu Ala Leu Ala Thr Asn Asp Ile Leu Thr Ala Val Asp Gln Leu Thr Ala Glu Gly Val Glu Phe Leu Ala Thr Pro Asp Ser Tyr Tyr Glu Asp Pro Glu Leu Arg Ala Arg Ile Gly Asn Val Arg Ala Pro Ile Ala Glu Leu Gln Lys Arg Gly Ile Leu Val Asp Arg Asp Glu Asp Gly Tyr Leu Lou Gln Ile Phe Thr Lys Pro Leu Val Asp Arg Pro Thr Val Phe Phe Glu Leu Ile Glu Arg His Gly Ser Leu Gly Phe Gly Ile Gly Asn Phe Lys Ala Leu Phe Glu Ala Ile Glu Arg Glu Gln Ala Ala Arg Gly Asn Phe <210> 19 <211> 1107 <212> DNA
<213> Picrophilus torridus <400> 19 atgtatggca aaaatttaat ctcagaacta agggaaaagg agatctttaa acgattacat 60 cacgtggaat tttacgttag cagtgccaaa acatggtcat atttcatgaa caggggtctt 120 ggatttaaaa cagtggcata tgccggtcca gaaaccggga taagggacaa gatatcctat 180 gttatgtccc agggcactgc aaggatatct tttacatcat caatgaatga tgatagctat 240 atatcgaatc atgttaaaaa acacggggat ggcgtaaagg atatagcact tgaggtcgat 300 gatctggacg aggcaaaaag cctgatagaa aagtatggaa caaaggtttc aaaaataaat 360 gaaataaagg atggaaatgg aaagataaga actgcagaga taaaaacgta cggtgaaacc 420 gttcatacat taatagaaac cggggattac aatggcgtat tcatgcccgg ttatgaggaa 480 tctgaaataa attcaaaaaa cactgggata aaaaagatcg atcatatagt tggaaatgtc 540 tatgagggcg agatggatag ctqggttaat ttttacatag aaaaacttgg ctttgagcat 600 ttaataacct ttgatgataa agatataaga actgattaca gcgcattaag atcaaaggtt 660 gtaaaataca atgacgatat cgtatttcca ataaatgagc ctgcaaaggg cttaagaaaa 720 tcacagatag aggaatatct tgactattac aggtctgagg gcgttcagca catagcactg 780 ttaactgatg atataataaa aactgtatcc atgatggagg aaaacggcat agaattttta 840 aaaacaccag gatcatacta tgaatcccta tcatcaagga taggctcaat agacgaggat 900 ttaaatgaaa tagagaaaca taacatactt gtggatcgtg atgagaacgg atacctatta 960 cagatcttca caaagcctgt tactgacagg ccaacgttct tctttgaggt catacagaga 1020 aagggtgcaa ggtcattcgg caacggtaac tttaaggcac tttttgaggc gatagaaagg 1080 gagcaggcaa agagaggaaa cctatga 1107 <210> 20 <211> 368 <212> PRT
<213> Picrophilus torridus <400> 20 Met Tyr Gly Lys Asn Leu Ile Ser Glu Leu Arg Glu Lys Glu Ile Phe Lys Arg Leu His His Val Glu Phe Tyr Val Ser Ser Ala Lys Thr Trp Ser Tyr Phe Met Asn Arg Gly Leu Gly Phe Lys Thr Val Ala Tyr Ala Gly Pro Glu Thr Gly Ile Arg Asp Lys Ile Ser Tyr Val Met Ser Gin Gly Thr Ala Arg Ile Ser Phe Thr Ser Ser Met Asn Asp Asp Ser Tyr Ile Ser Asn His Val Lys Lys His Gly Asp Gly Val Lys Asp Ile Ala Leu Glu Val Asp Asp Leu Asp Glu Ala Lys Ser Leu Ile Glu Lys Tyr Gly Thr Lys Val Ser Lys Ile Asn Glu Ile Lys Asp Gly Asn Gly Lys Ile Arg Thr Ala Glu Ile Lys Thr Tyr Gly Glu Thr Val His Thr Leu Ile Glu Thr Gly Asp Tyr Asn Gly Val Phe Met Pro Gly Tyr Glu Clu Ser Glu Ile Asn Ser Lys Asn Thr Gly Ile Lys Lys Ile Asp His Ile Val Gly Asn Val Tyr Glu Gly Glu Met Asp Ser Trp Val Asn Phe Tyr Ile Glu Lys Leu Gly Phe Glu His Leu Ile Thr Phe Asp Asp Lys Asp Ile Arg Thr Asp Tyr Ser Ala Leu Arg Ser Lys Val Val Lys Tyr Asn Asp Asp Ile Val Phe Pro Ile Asn Glu Pro Ala Lys Gly Leu Arg Lys Ser Gin Ile Glu Glu Tyr Leu Asp Tyr Tyr Arg Ser Glu Gly Val Gin His Ile Ala Leu Leu Thr Asp Asp Ile Ile Lys Thr Val Ser Met Met Glu Glu Asn Gly Ile Glu Phe Leu Lys Thr Pro Gly Ser Tyr Tyr Glu Ser Leu Ser Ser Arg Ile Gly Ser Ile Asp Glu Asp Leu Asn Glu Ile Glu Lys His Asn Ile Leu Val Asp Arg Asp Glu Asn Gly Tyr Leu Leu Gin Ile Phe Thr Lys Pro Val Thr Asp Arg Pro Thr Phe Phe Phe Glu Val Ile Gin Arg Lys Gly Ala Arg Ser Phe Gly Asn Gly Asn Phe Lys Ala Leu Phe Glu Ala Ile Glu Arg Glu Gin Ala Lys Arg Gly Asn Leu <210> 21 <211> 1164 <212> DNA
<213> Kordia algicida 111p <400> 21 atggcagcag aaataaaaaa cttaaaagat ttacaaaata cagaatacgg actcaaaaaa 60 ttatttgacg aagcagaaga ctttettcca cttttaggaa cagactacgt agaattatac 120 gtcgggaacg ccaaacaatc ggcacatttc tacaaaacgg cttttggttt tcaatcagaa 180 gcttacgcag gattggaaac aggattaacc gacagagttt catacgtatt aaaacaagat 240 aaaattcgct tggtcttaac aacaccatta ggaaaaggtg gcgaaatcaa tgagcatatc 300 gatttacacg gcgatggcgt aaaagtagta gcactttggg tagaagatgc tadaaaagcc 360 tttgaagaaa cgaccaaaag aggcgcaaaa ccgtacatgg aaccaacaaa agaagaagat 420 gaaaacggat atgtaattcg ctcaggaatc tatacgtacg gagaaacggt tcatgttttt 480 gtagaacgta aaaactataa cggagtottt ttaccaggat atcaaagatg ggaatctcac 540 tacaatccgg agccagttgg cttaaaattc atcgatcaca tggtaggaaa tgtaggttgg 600 ggagaaatga aagaatggtg tgaattctac gcgaaagtaa tgggatttgc gcaaattatc 660 tcctttacag atgatgatat ttctaccgat tttactgcgt tgatgagtaa agtaatgagt 720 aatggaaatg gtagaatcaa atttccaatc aatgaacccg cagaaggaaa aaagaaatcg 780 caaattgaag aatatctaga cttttacaat ggttcaggag tacaacatat tgcggttgct 840 acagacaata ttattgatac ggtttcgcaa atgcgcgaac gtggagtaga attcttatac 900 gttccagata catattatga tgacttgtta gaacgtgttg gcgacatcga tgaagatgta 960 gaagaactca aaaaacacgg aatcttaatt gatcgtgatg aagaaggata cttattgcag 1020 ttatttacca aaaccattgt agacagacca acaatgttct ttgaagtcat tcagcgtaaa 1080 ggcgcacaat catttggagt aggaaacttt aaagctttat ttgaagcgat agaaagagaa 1140 caagctgctc gcggaacatt gtaa 1164 <210> 22 <211> 387 <212> PRT
<213> Kordia algicida <400> 22 Met Ala Ala Glu Ile Lys Asn Leu Lys Asp Lou Gin Asn Thr Glu Tyr Gly Leu Lys Lys Leu Phe Asp Glu Ala Glu Asp Phe Leu Pro Leu Leu Gly Thr Asp Tyr Val Glu Leu Tyr Val Gly Asn Ala Lys Gin Ser Ala His Phe Tyr Lys Thr Ala Phe Gly Phe Gin Ser Glu Ala Tyr Ala Gly Leu Glu Thr Gly Leu Thr Asp Arg Val Ser Tyr Val Leu Lys Gin Asp Lys Ile Arg Leu Val Leu Thr Thr Pro Leu Gly Lys Gly Gly Glu Ile Asn Glu His Ile Asp Leu His Gly Asp Gly Val Lys Val Val Ala Leu Trp Val Glu Asp Ala Thr Lys Ala Phe Glu Glu Thr Thr Lys Arg Gly Ala Lys Pro Tyr Met Glu Pro Thr Lys Glu Glu Asp Glu Asn Gly Tyr Val Ile Arg Ser Gly Ile Tyr Thr Tyr Gly Glu Thr Val His Val Phe Val Glu Arg Lys Asn Tyr Asn Gly Val Phe Leu Pro Gly Tyr Gin Arg Trp Glu Her His Tyr Asn Pro Glu Pro Val Gly Leu Lys Phe Ile Asp His Met Val Gly Asn Val Gly Trp Gly Glu Met Lys Glu Trp Cys Glu EZET fm4 0zEi obbbepq a6vofmob4o oeeobeeiob ee.644oso46 esbeb4sq4s .6.6r5q4gool ogn epeoprqbiob epop43qqop pebbbepobb 44455.5affq. bqobbibboe p8poopqer6 003I 2vnbbbbee5 ebqebbevele E64e0b4abb 04effeeeo4 4eoDrebebb4 4044b44bDp orn epoo5epeb5 f6b4beoabe eooeDq4oqv emoD436405 qbobbbeooe b4?be69o96 0801 b4U4ob465 EtieqoePbele oq6q5e6brE 44r5vaeobb ebboeoqobq barbbbboob OZOI o6o65066.6 qboaboebqe ququeboyo bobb33qoab obbi,e344bu boT4o6bobb 06 bquoDbboqD
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OZZ STZ OIZ
dEV atid, eqd 19S 91I eTI eTD eTV e4d AID 4aW TPA sArl eTV 1A1 04d bITT

111r <210> 24 <211> 440 <212> PRT
<213> Sorghum bicolor <400> 24 Met Pro Pro Thr Pro Thr Thr Ala Ala Ala Thr Gly Ala Ala Val Ala Ala Ala Ser Ala Glu Gln Ala Ala Phe Arg Leu Val Gly His Arg Asn Phe Val Arg Val Asn Pro Arg Ser Asp Arg Phe His Thr Leu Ala Phe His His Val Glu Leu Trp Cys Ala Asp Ala Ala Ser Ala Ala Gly Arg Phe Ser Phe Gly Leu Gly Ala Pro Leu Ala Ala Arg Ser Asp Leu Ser Thr Gly Asn Thr Ala His Ala Ser Leu Leu Leu Arg Ser Cly Ala Leu Ala Phe Leu Phe Thr Ala Pro Tyr Ala His Gly Ala Asp Ala Ala Thr Ala Her Leu Pro Ser Phe Ser Ala Ala Glu Ala Arg Arg Phe Ala Ala Asp His Gly Leu Ala Val Arg Ala Val Ala Leu Arg Val Ala Asp Ala Glu Asp Ala Phe Arg Ala Ser Val Ala Ala Gly Ala Arg Pro Ala Phe Glu Pro Val Glu Leu Gly Leu Gly She Arg Leu Ala Glu Val Glu Leu Tyr Gly Asp Val Val Leu Arg Tyr Val Ser Tyr Pro Asp Asp Ala Asp Ala Her Phe Leu Pro Gly Phe Val Gly Val Thr Ser Pro Gly Ala Ala Asp Tyr Gly Leu Arg Arg Phe Asp His Ile Vol Gly An Vol Pro Glu Leu Ala Pro Ala Ala Ala Tyr Phe Ala Gly Phe Thr Gly Phe His Glu Phe Ala Glu She Thr Ala Glu Asp Val Gly Thr Thr Glu Ser Gly Leu Asn Ser Met Val Leu Ala Asn Asn Ala Glu Asn Val Leu Leu Pro Leu Asn Glu Pro Val His Gly Thr Lys Arg Arg Ser Gln Ile Gln Thr Tyr Leu Asp His His Gly Gly Pro Gly Val Gln His Met Ala Leu Ala Ser Asp Asp Val Leu Arg Thr Leu Arg Glu Met Gln Ala Arg Ser Ala Met Gly Gly Phe Glu Phe Met Ala Pro Pro Ala Pro Glu Tyr Tyr Asp Gly Val Arg Arg Arg Ala Gly Asp Val Leu Thr Glu Ala Gln Ile Lys Glu Cys Gln G1u Leu Gly Val Leu Val Asp Arg Asp Asp Gln Gly Val Leu Leu Gln Ile Phe Thr Lys Pro Val Gly Asp Arg Pro Thr Leu Phe Leu Glu Ile Ile Gln Arg Ile Gly Cys Met Glu Lys Asp Glu Lys Gly Gln ills Glu Tyr Gin Lys Gly Gly Cys Gly Gly Phe Gly Lys Cly Asn Phe Ser Gin Leu Phe Lys Ser Ile Glu Asp Tyr Glu Lys Ser Leu Glu Ala Lys Gin Ala Ala Ala Ala Gin Gly Ser <210> 25 <211> 1311 <212> DNA
<213> Triticum aestivum <400> 25 atgccgccca cccccaccac ccccgcagcc accggcgccg gcgctgccgc cgcggtgacg 60 ccggagcacg cgcggccgcg ccgaatggtc cgcttcaacc cgcgcagcga ccgcttccac 120 acgctatcct tccaccacgt cgagttctgg tgcgcggacg ccgcctccgc cgccggccgc 180 ttcgccttcg cgctcggcgc gccgctcgcc gccaggtccg acctctccac ggggaactcc 240 gtgcacgcct cccagctgct ccgctcgggc aacctcgcct tcctcttcac cgcgccctac 300 gcgaacggct gcgacgccgc caccgcctcc ctgccctcct tctccgccga cgccgcgcgc 360 cggttctccg cggaccacgg gctcgcagtg cgctccatag cactgcgcgt cgcagacgcc 420 gcagaggcct tccgcgccag cgtcgacgga ggcgcgcgcc cggccttcag ccccgtggac 480 ctcggccgcg gotteggctt cgcggaggtc gagctctacg gcgacgtcgt gctccgcttc 540 gtcagtcacc cggatgacac ggacgtgccc ttcttgccgg ggttcgaggg cgtgagcaac 600 ccggatgccg tggactacgg cctgacgcgg ttcgaccacg tcgtcggcaa cgteccggag 660 cttgcccccg ccgccgcata cgtcgccggg ttcgcggggt tccacgagtt cgccgagttc 720 acgacggagg acgtgggcac ggccgagagc gggctcaant cgatggtgct cgccaacaan 780 tcggagggcg tgctgctgcc gctcaacgag ccggtgcacg gcaccaagcg ccggagccag 840 atacagacgt tcctggaaca ccacggcggc tcgggcgtgc agcacatcgc ggtggccagc 900 agcgacgtgc tcaggacgct cagggagatg cgtgcgcgct ccgccatggg cggcttcgac 960 ttcctgccac cccgctgccg aaagtactac gaaggcgtgc ggcgcatcgc cggggatgtg 1020 ctctcggagg cgcagatcaa ggaatgccag gagctggggg tgctcgtcga cagggacgac 1080 caaggggtgt tgctacaaat cttcacaaag ccagtggggg acaggccaac gctgttcctg 1140 gagatgatcc aaaggatcgg gtgcatggag aaggacgaga gaggggaaga gtaccagaag 1200 ggtggctgcg gcgggttcgg caaaggcaac ttctccgagc tgttcaagtc cattgaagat 1260 tacgagaagt cccttgaagc caagcaatct gctgcagttc agggatcata g 1311 <210> 26 <211> 436 <212> PRT
<213> Triticum aestivum <400> 26 Met Pro Pro Thr Pro Thr Thr Pro Ala Ala Thr Gly Ala Gly Ala Ala Ala Ala Val Thr Pro Glu His Ala Arg Pro Arg Arg Met Val Arg Phe Asn Pro Arg Ser Asp Arg Phe His Thr Leu Ser Phe His His Val Glu Phe Trp Cys Ala Asp Ala Ala Ser Ala Ala Gly Arg Phe Ala Phe Ala Lela Gly Ala Pro Leu Ala Ala Arg Ser Asp Leu Ser Thr Gly Asn Ser Val His Ala Ser Gin Leu Leu Arg Ser Gly Asn Leu Ala Phe Leu Phe lilt Thr Ala Pro Tyr Ala Asn Gly Cys Asp Ala Ala Thr Ala Ser Lela Pro Ser Phe Ser Ala Asp Ala Ala Arg Arg Phe Ser Ala Asp His Gly Leu Ala Vol Arg Ser Ile Ala Leu Arg Vol Ala Asp Ala Ala Glu Ala Phe Arg Ala Ser Val Asp Gly Gly Ala Arg Pro Ala Phe Ser Pro Vol Asp Leu Gly Arg Gly Phe Gly Phe Ala Glu Val Glu Leu Tyr Gly Asp Val Vol Leu Arg Phe Val Ser His Pro Asp Asp Thr Asp Val Pro Elle Leu Pro Gly Phe Glu Gly Val Ser Asn Pro Asp Ala Val Asp Tyr Gly Leu Thr Arg Phe Asp His Val Val Gly Asn Val Pro Glu Leu Ala Pro Ala Ala Ala Tyr Val Ala Gly Phe Ala Gly Phe His Glu Phe Ala Glu Phe Thr Thr Glu Asp Vol Gly Thr Ala Glu Ser Gly Leu Asn Ser Met Vol Leu Ala Asn Asn Ser Glu Gly Vol Leu Leu Pro Leu Asn Glu Pro Val His Gly Thr Lys Arg Arg Ser Gin Ile Gin Thr Phe Leu Glu His His Gly Gly Ser Gly Val Gin His Ile Ala Val Ala Ser Ser Asp Vol Leu Arg Thr Len Arg Glu Met Arg Ala Arg Ser Ala Met Gly Gly Phe Asp Phe Leu Pro Pro Arg Cys Arg Lys Tyr Tyr Glu Gly Val Arg Arg Ile Ala Gly Asp Val Leu Ser Glu Ala Gin Ile Lys Glu Cys Gin Glu Leu Gly Vol Leu Val Asp Arg Asp Asp Gin Gly Val Leu Leu Gin Ile Phe Thr Lys Pro Vol Gly Asp Arg Pro Thr Leu Phe Leu Glu Met Ile Gin Arg Ile Gly Cys Met Glu Lys Asp Glu Arg Gly Glu Glu Tyr Gin Lys Gly Gly Cys Gly Gly Phe Gly Lys Gly Asn Phe Ser Glu Leu Phe Lys Ser Ile Glu Asp Tyr Glu Lys Ser Leu Glu Ala Lys Gin Ser Ala Ala Val Gin Gly Ser 435 =
<210> 27 <211> 1305 <212> DNA
<213> Hordeum vulgare <400> 27 atgccgccca cccccaccac ccccgcggct accggcgccg ccgccgcggt gacgccggag 60 cacgcgcgac cgcaccgaat ggtccgcttc aacccgcgca gcgaccgctt ccacacgctc 120 tccttccacc acgtcgagtt ctggtgcgcg gacgccgcct ccgccgccgg ccgcttcgcg 180 ttcgcgctcg gcgcgccgct cgccgccagg tccgacctct ccacggggaa ctccgcgcac 240 111u gcctcccagc tgctccgctc gggctccctc gccttcctct tcaccgcgcc ctacgccaac 300 ggctgcgacg ccgccaccgc ctccctgccc tccttctccg ccgacgccgc gcgccggttc 360 tccgccgacc acgggatcgc ggtgcgctcc gtagcgctgc gcgtcgcaga cgccgccgag 420 gccttccgcg ccagtcgtcg acggggcgcg cgcccggcct tcgcccccgt ggacctcggc 480 cgcggcttcg cgttcgcgga ggtcgagctc tacggcgacg tcgtgctccg cttcgtcagc 540 cacccggacg gcacggacgt gcccttcttg ccggggttcg agggcgtaac caacccggac 600 gccgtggact acggcctgac gcggttcgac cacgtcgtcg gcaacgtccc ggagcttgcc 660 cccgccgcag cctacatcgc cgggttcacg gggttccacg agttcgccga gttcacggcg 720 gaggacgtgg gcacgaccga gagcgggctc aactcggtgg tgctcgccaa caactcggag 780 ggcgtgctgc tgccgctcaa cgagccggtg cacggcacca agcgccggag ccagatacag 840 acgttcctgg aacaccacgg cggcccgggc gtgcagcaca tcgcggtggc cagcagtgac 900 gtgctcagga cgctcaggaa gatgcgtgcg cgctccgcca tgggcggctt cgacttcctg 960 ccacccccgc tgccgaagta ctacgaaggc gtgcgacgcc ttgccgggga tgtcctctcg 1020 gaggcgcaga tcaaggaatg ccaggagctg ggtgtgctcg tcgataggga cgaccaaggg 1080 gtgttgctcc aaatcttcac caagccagta ggggacaggc cgaccttgtt cctggagatg 1140 atccagagga tcgggtgcat ggagaaggac gagagagggg aagagtacca gaagggtggc 1200 tgcggcgggt tcggcaaagg caacttctcc gagctgttca agtccattga agattacgag 1260 aagtcccttg aagccaagca atctgctgca gttcagggat catag 1305 <210> 28 <211> 434 <212> PRT
<213> Hordeum vulgare <400> 28 Met Pro Pro Thr Pro Thr Thr Pro Ala Ala Thr Gly Ala Ala Ala Ala Val Thr Pro Glu His Ala Arg Pro His Arg Met Val Arg Phe Asn Pro Arg Ser Asp Arg Phe His Thr Leu Ser Phe His His Val Glu Phe Tip Cys Ala Asp Ala Ala Ser Ala Ala Gly Arg Phe Ala Phe Ala Leu Gly Ala Pro Leu Ala Ala Arg Ser Asp Leu Ser Thr Gly Asn Ser Ala His Ala Ser Gin Leu Leu Arg Ser Gly Ser Lou Ala Phe Lou Phe Thr Ala Pro Tyr Ala Asn Gly Cys Asp Ala Ala Thr Ala Ser Lau Pro Ser Phe Ser Ala Asp Ala Ala Arg Arg Phe Ser Ala Asp His Gly Ile Ala Val Arg Set Val Ala Leu Arg Val Ala Asp Ala Ala Glu Ala Phe Arg Ala Ser Arg Arg Arg Gly Ala Arg Pro Ala Phe Ala Pro Val Asp Leu Gly Arg Gly Phe Ala Phe Ala Glu Val Glu Leu Tyr Gly Asp Val Val Leu Arg Phe Val Ser His Pro Asp Gly Thr Asp Val Pro Phe Leu Pro Gly Phe Glu Gly Val Thr Asn Pro Asp Ala Val Asp Tyr Gly Lou Thr Arg Phe Asp His Val Val Gly Asn Val Pro Glu Lou Ala Pro Ala Ala Ala Tyr Ile Ala Gly Phe Thr Gly Phe His Glu Phe Ala Glu Phe Thr Ala 111v Glu Asp Val Gly Thr Thr Glu Ser Gly Leu Asn Ser Val Val Leu Ala Asn Asn Ser Glu Gly Val Leu Leu Pro Leu Asn Glu Pro Val His Gly Thr Lys Arg Arg Ser Gin Ile Gin Thr Phe Leu Glu His His Gly Gly Pro Gly Val Gin His Ile Ala Val Ala Ser Ser Asp Val Leu Arg Thr Leu Arg Lys Met Arg Ala Arg Ser Ala Met Gly Gly Phe Asp Phe Leu Pro Pro Pro Leu Pro Lys Tyr Tyr Glu Gly Val Arg Arg Leu Ala Gly Asp Val Leu Ser Glu Ala Gin Ile Lys Glu Cys Gin Glu Leu Gly Val Leu Val Asp Arg Asp Asp Gin Gly Val Leu Leu Gin Ile Phe Thr Lys Pro Val Gly Asp Arg Pro Thr Leu Phe Leu Glu Met Ile Gin Arg Ile Gly Cys Met Glu Lys Asp Glu Arg Gly Glu Glu Tyr Gin Lys Gly Gly Cys Gly Gly Phe Gly Lys Gly Asn Phe Ser Glu Leu Phe Lys Ser Ile Glu Asp Tyr Glu Lys Ser Leu Glu Ala Lys Gin Ser Ala Ala Val Gin Gly Ser <210> 29 <211> 1209 <212> DNA
<213> Rhodococcus sp.
<400> 29 atgactaccg ccgacattcg cctgacgccc cgcgaggtgg ccgcacatct ggagaccgac 60 gagctccggc agttggtcgg gctcgtcgaa cacgacgacg cgtcggatcc gtttcccgtg 120 gtcgcgatgg atgccgtggt gttcgtgtgc ggcaacgcga cgcagagcac gcagtacttc 180 gtctccacgt ggggcatgac cctcgtcgcc tacgccgggc cggagaccgg tcagcgctcg 240 cacaagtcct tcgtcctcga gtcggggtcg gcacggttcg tgctgcacgg cgccgtcgat 300 ccgaagagcc cgctcgcgga ccatcaccgg gcgcacggcg acggcgtggt ggacctggcg 360 atggaagttc tcgacgtcga ccgctgcatc gcgcatgcac gctcgcaggg ggccaccatt 420 ctcgaggagc cgcgcgacgt cacggatcag ttcggcaccg tgcggctcgc ggcgatcgcc 480 acgtacggca gcacccggca caccatcgtc qaccgaagcc gatacgacgg cccctacctc 540 cccggattcg tcgcgcgctc cagcggtttc gcggcgcgac cgggtaaacc cccgcgattg 600 ttccaggcgc tcgaccacgc cgtcggcaac gtcgagatgg gccggatgga tcactgggtc 660 cggttctaca accgcgtcat gggcttcacg aacatggccg aattcgtcgg cgacgacatc 720 gccacggagt actcggcgct gatgtcgaag gtcgtggcga acggcaatca ccgggtgaag 780 ttcccgctca acgaacccgc ggtgggaaag aagaagtcgc agatcgacga atatctcgag 840 ttctacggtg agccgggctg ccagcatctg gccctcgcga ccggagacat cctcgcgacg 900 gtggacgcgt tgcgggccga gggtgLcgaa ttcctgaaca cacccgacgc gtactacgag 960 gacccacagc tgcgcgcccg gatcggcagg gtgcgggtgc cggtggagga actgcagaag 1020 cgcggaatcc tcgtcgaccg cgacgaggac ggatacctcc tgcagatctt caccaaaccg 1080 ctcggcgacc ggccgaccgt gttcttcgag gtgatcgaac ggcacggttc gctcgggttc 1140 ggggcgggta acttccaggc cctgttcgaa tccatcgagc gtgaggaggc ggcgcgcggc 1200 aatctgtga 1209 111w <210> 30 <211> 402 <212> PRT
<213> Rhodococcus sp.
<400> 30 Met Thr Thr Ala Asp Ile Arg Lou Thr Pro Arg Glu Val Ala Ala His Leu Glu Thr Asp Glu Leu Arg Gin Leu Val Gly Leu Val Glu His Asp Asp Ala Ser Asp Pro Phe Pro Vol Val Ala Met Asp Ala Vol Val Phe Val Cys Gly Asn Ala Thr Gin Ser Thr Gin Tyr Phe Val Ser Thr Trp Gly Met Thr Leu Val Ala Tyr Ala Gly Pro Glu Thr Gly Gin Arg Ser His Lys Ser Phe Val Leu Glu Ser Gly Her Ala Arg Phe Val Leu His Gly Ala Val Asp Pro Lys Ser Pro Lou Ala Asp His His Arg Ala His Gly Asp Gly Val Vol Asp Leu Ala Met Glu Vol Leu Asp Val Asp Arg Cys Ile Ala His Ala Arg Ser Gin Gly Ala Thr Ile Leu Glu Glu Pro Arg Asp Val Thr Asp Gin Phe Gly Thr Val Arg Leu Ala Ala Ile Ala Thr Tyr Gly Ser Thr Arg His Thr Ile Val Asp Arg Ser Arg Tyr Asp Gly Pro Tyr Leu Pro Gly Phe Val Ala Arg Ser Ser Gly Phe Ala Ala Arg Pro Gly Lys Pro Pro Arg Leu Phe Gin Ala Leu Asp His Ala Vol Gly Asn Val Glu Met Gly Arg Met Asp His Trp Val Arg Phe Tyr Asn Arg Val Met Gly Phe Thr Asn Met Ala Glu Phe Vol Gly Asp Asp Ile Ala Thr Glu Tyr Ser Ala Leu Met Her Lys Vol Val Ala Asn Gly Asn His Arg Val Lys Phe Pro Leu Asn Glu Pro Ada Val Gly Lys Lys Lys Ser Gin Ile Asp Glu Tyr Leu Glu Phe Tyr Gly Glu Pro Gly Cys Gin His Leu Ala Leu Ala Thr Gly Asp Ile Lou Ala Thr Val Asp Ala Leu Arg Ala Glu Gly Val Glu Phe Leu Asn Thr Pro Asp Ala Tyr Tyr Glu Asp Pro Gin Lou Arg Ala Arg Ile Gly Arg Val Arg Val Pro Val Glu Glu Leu Gin Lys Arg Gly Ile Leu Val Asp Arg Asp Glu Asp Gly Tyr Leu Lou Gin Ile Phe Thr Lys Pro Leu Gly Asp Arg Pro Thr Val Phe Phe Glu Val Ile Glu Arg His Gly Ser Leu Gly Phe Gly Ala Gly Asn 111x Phe Gin Ala Leu Phe Glu Ser Ile Glu Arg Glu Gin Ala Ala Arg Gly Aon Leu <210> 31 <211> 1353 <212> DNA
<213> Artificial sequence <220>
<223> Nucleic acid sequence encoding Arabidopsis HPPD plus containing at the 5 end a nucleic acid encoding an Ala and 6 His amino acids <220>
<221> misc_feature <222> (4)..(6) <223> sequence coding for an Ala <220>
<221> misc_feature <222> (7)..(24) <223> sequence coding for an His Tag containing 6 His <400> 31 atggctcatc accatcacca tcaccaaaac gccgccgttt cagagaatca aaaccatgat 60 gacggcgctg cgtcgtcgcc gggattcaag ctcgtcggat tttccaagtt cgtaagaaag 120 aatccaaagt ctgataaatt caaggttaag cgcttccatc acatcgagtt ctggtgoggc 180 gacgcaacca acgtcgctcg tcgcttctcc tggggtctgg ggatgagatt ctccgccaaa 240 tccgatcttt ccaccggaaa catggttcac gcctcttacc tactcacctc cggtgabctc 300 cgattccttt tcactgctcc ttactctccg tctctctccg ccggagagat taaaccgaca 360 accacagctt ctatcccaag tttcgatcac ggctcttgtc yUculAutt ctcgtcacat 420 ggtctcggtg ttagagccgt tgcgattgaa gtagaagacg cagagtcagc tttctccatc 480 agtgtagcta atggcgctat tccttcgtcg cctcctatcg tcctcaatga agcagttacg 540 atcgctgagg ttaaactata cggcgatgtt gttctccgat atgttagtta caaagcagaa 600 gataccgaaa aatccgaatt cttgccaggg ttcgagcgtg tagaggatgc gtcgtcgttc 660 ccattggatt atggtatccg gcggcttgac cacgccgtgg gaaacgttcc tgagcttggt 720 ccggctttaa cttatgtage ggggttcact ggttttcacc aattcgcaga gttcacagca 780 gacgacgttg gaaccgccga gagcggttta aattcagcgg tcctggctag caatgatgaa 840 atggttcttc taccgattaa cgagccagtg cacggaacaa agaggaagag tcagattcag 900 acgtatttgg aacataacga aggcgcaggg ctacaacatc tggctctgat gagtgaagac 960 atattcagga ccctgagaga gatgaggaag aggagcagta ttggaggatt cgacttcatg 1020 ccttctcctc cgcctactta ctaccagaat ctcaagaaac gggtcggcga cgtgctcagc 1080 gatgatcaga tcaaggagtg tgaggaatta gggattcttg tagacagaga tgatcaaggg 1140 acgttgcttc aaatcttcac aaaaccacta ggtgacaggc cgacgatatt tatagagata 1200 atccagagag taggatgcat gatgaaagat gaggaaggga aggcttacca gaqtggagga 1260 tgtggtggtt ttggcaaagg caatttctct gagctcttca agtccattga agaatacgaa 1320 aagactcttg aagccaaaca gttagtggga tga 1353 <210> 32 <211> 450 <212> PRT
<213> Artificial sequence lily <220>
<223> encoded by SEQ ID No. 31 <220>
<221> MISC_FEATURE
<222> (2)..(2) <223> Ala <220>
<221> MISC_FEATURE
<222> (3)..(8) <223> His Tag made of 6 His <400> 32 Met Ala His His His His His His Gln Asn Ala Ala Val Ser Glu Asn Gln Asn His Asp Asp Gly Ala Ala Ser Ser Pro Gly Phe Lys Leu Val Gly Phe Ser Lys Pile Val Arg Lys Asn Pro Lys Ser Asp Lys Phe Lys Val Lys Arg Phe His His Ile Glu Phe Trp Cys Gly Asp Ala Thr Asn Val Ala Arg Arg Phe Ser Trp Gly Leu Gly Met Arg Phe Ser Ala Lys Ser Asp Leu Ser Thr Gly Asn Met Val His Ala Ser Tyr Leu Leu Thr Ser Gly Asp Leu Arg Phe Leu Phe Thr Ala Pro Tyr Her Pro Her Leu Ser Ala Gly Glu Ile Lys Pro Thr Thr Thr Ala Ser Ile Pro Ser Phe Asp His Gly Ser Cys Arg Ser Phe Phe Ser Ser His Gly Leu Gly Val Arg Ala Val Ala Ile Glu Val Glu Asp Ala Glu Ser Ala Phe Ser Ile Ser Val Ala Asn Gly Ala Ile Pro Ser Ser Pro Pro Ile Val Leu Asn Glu Ala Val Thr Ile Ala Glu Val Lys Leu Tyr Gly Asp Val Val Leu Arg Tyr Val Ser Tyr Lys Ala Glu Asp Thr Glu Lys Ser Glu Phe Leu Pro Gly Phe Glu Arg Val Glu Asp Ala Ser Ser Phe Pro Leu Asp Tyr Gly Ile Arg Arg Leu Asp His Ala Val Gly Asn Val Pro Glu Leu Gly Pro Ala Leu Thr Tyr Val Ala Gly Phe Thr Gly Phe His Gln Phe Ala Glu Phe Thr Ala Asp Asp Val Gly Thr Ala Glu Ser Gly Leu Asn Ser Ala Val Leu Ala Ser Asn Asp Glu Met Val Leu Leu Pro Ile Asn Glu Pro Val His Gly Thr Lys Arg Lys Ser Gln Ile Gln Thr Tyr Leu Glu His Asn Glu Gly Ala Gly Leu Gin His Leu Ala Leu Met Ser Glu Asp Ile Phe Arg Thr Leu Arg Glu Met Arg Lys Arg Ser Ser Ile Gly Gly 111z Phe Asp Phe Met Pro Ser Pro Pro Pro Thr Tyr Tyr Gin Asn Leu Lys Lys Arg Val Gly Asp Val Leu Ser Asp Asp Gin Ile Lys Glu Cys Glu Glu Leu Gly Ile Leu Val Asp Arg Asp Asp Gin Gly Thr Leu Leu Gin Ile Phe Thr Lys Pro Leu Gly Asp Arg Pro Thr Ile Phe Ile Glu Ile Ile Gin Arg Val Gly Cys Met Met Lys Asp Glu Glu Gly Lys Ala Tyr Gin Ser Gly Gly Cys Gly Gly Phe Gly Lys Gly Asn Phe Ser Glu Leu Phe Lys Ser Ile Glu Glu Tyr Glu Lys Thr Leu Glu Ala Lys Gin Leu Val Gly <210> 33 <211> 1353 <212> DNA
<213> Artificial sequence <220>
<223> nucleotide sequence encoding mutant HPPD polypeptide <220>
<221> misc_feature <222> (4)..(6) <223> sequence coding for Ala <220>
<221> miscjeature <222> (7)..(24) <223> sequence coding an His Tag containing 6 His <400> 33 atggctcatc accatcacca tcaccaaaac gccgccgttt cagagaatca aaaccatgat 60 gacggcgctg cgtcgtcgcc gggattcaag ctcgtcggat tttccaagtt cgtaagaaag 120 aatccaaagt ctgataaatt caaggttaag cgcttccatc acatcgagtt ctggtgcggc 180 gacgcaacca acgtcgctcg tcgcttctcc tggggtctgg ggatgagatt ctccgccaaa 240 tccgatcttt ccaccggaaa catggttcac gcctcttacc tactcacctc cggtgacctc 300 cgattccttt tcactgctcc ttactctccg tctctctccg ccggagagat taaaccgaca 360 accacagctt ciateccaag tttcgatcac ggctcttgtc gttccttctt ctcgtcacat 420 ggtctcggtg ttagagccgt tgcgattgaa gtagaagagg cagagtcagc tttctccatc 480 agtgtagcta atggcgctat tccttcgtcg cctcctatcg tcctcaatga aggagttacg 540 atcgctgagg ttaaactata cggcgatgtt gttctccgat atgttagtta caaagcagaa 600 gataccgaaa aatccgaatt cttgccaggg ttcgagcgtg tagaggatgc gtcgtcgttc 660 ccattggatt atggtatccg gcggcttgac cacgccgtgg gaaacgttcc tgagcttggt 720 ccggctttaa cttatgtagc ggggttcact ggttttcacc aattcgcaga gttcacagca 780 gacgacgttg gaaccgccga gagcggttta aatgcggcgg tcctggctag caatgatgaa 840 atggttcttc taccgattaa cgagccagtg cacggaacaa agaggaagag tcagattcag 900 acgtatttgg aacataacga aggcgcaggg ctacaacatc tggctctgat gagtgaagac 960 atattcagga ccctgagaga gatgaggaag aggagcagta ttggaggatt cgacttcatg 1020 ccttctcctc cgcctactta ctaccagaat ctcaagaaac gggtcggcga cgtgctcagc 1080 gatgatcaga tcaaggagtg tgaggaatta gggattcttg tagacagaga tgatcaaggg 1140 111aa acgttgcttc aaatcttcac aaaaccacta ggtgacaggc cgacgatatt tatagagata 1200 atccagagag taggatgcat gatgaaagat gaggaaggga aggcttacca gagtggagga 1260 tgtggtggtt ttggcaaagg caatttctct gagctcttca agtccattga agaatacgaa 1320 aagactcttg aagccaaaca gttagtggga tga 1353 <210> 34 <211> 450 <212> PET
<213> Artificial sequence <220>
<223> encoded by SEQ ID No. 33 <220>
<221> MISC_FEATURE
<222> (2)..(2) <223> Ala <220>
<221> MISCJEATURE
<222> (3)..(8) <223> His Tag made of 6 His <400> 34 Met Ala His His His His His His Gin Asn Ala Ala Val Ser Glu Asn Gin Asn His Asp Asp Gly Ala Ala Ser Ser Pro Gly Phe Lys Leu Val Gly Phe Ser Lys Phe Val Arg Lys Asn Pro Lys Ser Asp Lys Phe Lys Val Lys Arg Phe His His Ile Glu the Trp Cys Gly Asp Ala Thr Asn Val Ala Arg Arg Phe Se/ Trp Gly Leu Gly Met Arg Phe Ser Ala Lys Ser Asp Leu Ser Thr Gly Asn Met Val His Ala Ser Tyr Leu Leu Thr Ser Gly Asp Leu Arg the Leu Phe Thr Ala Pro Tyr Ser Pro Ser Leu Ser Ala Gly Glu Ile Lys Pro Thr Thr Thr Ala Ser Ile Pro Ser Phe Asp His Gly Ser Cys Arg Ser Phe the Ser Ser His Gly Leu Gly Val Arg Ala Val Ala Ile Glu Val Glu Asp Ala Glu Ser Ala Phe Ser Ile Ser Val Ala Asn Gly Ala Ile Pro Ser Ser Pro Pro Ile Val Leu Asn Glu Ala Val Thr Ile Ala Glu Val Lys Leu Tyr Gly Asp Val Val Leu Arg Tyr Val Ser Tyr Lys Ala Glu Asp Thr Glu Lys Ser Glu Phe Leu Pro Gly Phe Glu Arg Val Glu Asp Ala Ser Ser the Pro Leu Asp Tyr Gly Ile Arg Arg Leu Asp His Ala Val Gil, Asn Val Pro Glu Leu Gly 111bb Pro Ala Leu Thr Tyr Val Ala Gly Phe Thr Gly Phe His Gin Phe Ala Glu Phe Thr Ala Asp Asp Val Gly Thr Ala Glu Ser Gly Leu Asn Ala Ala Vol Leu Ala Ser Asn Asp Glu Met Val Leu Leu Pro Ile Asn Glu Pro Val His Gly Thr Lys Arg Lys Ser Gin Ile Gin Thr Tyr Leu Glu His Asn Glu Gly Ala Gly Leu Gin His Leu Ala Leu Met Ser Glu Asp Ile Phe Arg Thr Leu Arg Glu Met Arg Lys Arg Ser Ser Ile Gly Gly Phe Asp Phe Met Pro Ser Pro Pro Pro Thr Tyr Tyr Gin Asn Leu Lys Lys Arg Val Gly Asp Val Leu Ser Asp Asp Gin Ile Lys Glu Cys Glu Glu Leu Gly Ile Leu Val Asp Arg Asp Asp Gin Gly Thr Leu Leu Gin Ile Phe Thr Lys Pro Leu Gly Asp Arg Pro Thr Ile Phe Ile Glu Ile Ile Gin Arg Val Gly Cys Met Met Lys Asp Glu Glu Gly Lys Ala Tyr Gin Ser Gly Gly Cys Gly Gly Phe Gly Lys Giy Asn Phe Ser Glu Leu Phe Lys Ser Ile Glu Glu Tyr Glu Lys Thr Leu Glu Ala Lys Gin Leu Val Gly <210> 35 <211> 1353 <212> DNA
<213> Artificial sequence <220>
<223> nucleotide sequence encoding mutant IIPPD polypeptide <220>
<221> misc_feature <222> (4)¨(6) <223> sequence coding for Ala <220>
<221> misc_feature <222> (7)..(24) <223> sequence coding for an His Tag containing 6 His <400> 35 atggctcatc accatcacca tcaccaaaac gccgccgttt cagagaatca aaaccatgat 60 gacggcgctg cgtcgtcgcc gggattcaag ctcgtcggat tttccaagtt cgtaagaaag 120 aatccaaagt ctgataaatt caaggttaag cgcttccatc acatcgagtt ctggtgcggc 180 gacgcaacca acgtcgctcg tcgcttctcc tggggtctgg ggatgagatt ctccgccaaa 240 tccgatcttt ccaccggaaa catggttcac gcctcttacc tactcacctc cggtgacctc 300 cgattccttt tcactgctcc ttactctccg tctctctccg ccggagagat taaaccgaca 360 accacagctt ctatcccaag tttcgatcac ggctcttgtc gttccttctt ctcgtcacat 420 Mcc ggtctcggtg ttagagccgt tgcgattgaa gtagaagacg cagagtcagc tttctccatc 480 agtgtagcta atggcgctat tccttcgtcg cctcctatcg tcctcaatga agcagttacg 540 atcgctgagg ttaaactata cggcgatgtt gttctccgat atgttagtta caaagcagaa 600 gataccgaaa aatccgaatt cttgccaggg ttcgagcgtg tagaggatgc gtcgtcgttc 660 ccattggatt atggtatccg gcggettgac cacgccgtgg gaaacgttcc tgagcttggt 720 ccggctttaa cttatgtagc ggggttcact ggttttcacc aattcgcaga gttcacagca 780 gacgacgttg gaaccgccga gagcggttta aattcagcgg tcctggctag caatgatgaa 840 atggttcttc taccgattgc cgagccagtg cacggaacaa agaggaagag tcagattcag 900 acgtatttgg aacataacga aggcgcaggg ctacaacatc tggctctgat gagtgaagac 960 atattcagga ccctgagaga gatgaggaag aggagcagta ttggaggatt cgacttcatg 1020 ccttctcctc cgcctactta ctaccagaat ctcaagaaac gggtcggcga cgtgctcagc 1080 gatgatcaga tcaaggagtg tgaggaatta gggattcttg tagacagaga tgatcaaggg 1140 acgttgcttc aaatcttcac aaaaccacta ggtgacaggc cgacgatatt tatagagata 1200 atccagagag taggatgcat gatgaaagat gaggaaggga aggcttacca gagtggagga 1260 tgtggtggtt ttggcaaagg caatttctct gagctcttca agtccattga agaatacgaa 1320 aagactcttg aagccaaaca gttagtggga tga 1353 <210> 36 <211> 450 <212> PRT
<213> Artificial sequence <220>
<223> encoded by SEQ ID No. 35 <220>
<221> MISCJEATURE
<222> (2)..(2) <223> Ala <220>
<221> MISC_FEATURE
<222> (3)..(8) <223> His Tag made of 6 His <400> 36 Met Ala His His His His His His Gln Asn Ala Ala Val Ser Glu Asn Gin Asn His Asp Asp Gly Ala Ala Ser Ser Pro Gly Phe Lys Leu Val Gly Phe Ser Lys Phe Val Arg Lys Asn Pro Lys Ser Asp Lys Phe Lys Val Lys Arg Phe His His Ile Glu Phe Trp Cys Gly Asp Ala Thr Asn Val Ala Arg Arg Phe Ser Trp Gly Leu Gly Met Arg Phe Ser Ala Lys Ser Asp Lau Ser Thr Gly Asn Met Val His Ala Ser Tyr Leu Leu Thr Ser Gly Asp Leu Arg Phe Leu Phe Thr Ala Pro Tyr Ser Pro Ser Leu Ser Ala Gly Glu Ile Lys Pro Thr Thr Thr Ala Ser Ile Pro Ser Phe Asp His Gly Ser Cys Arg Ser Phe Phe Ser Ser His Gly Leu Gly Val ill dd Arg Ala Val Ala Ile Glu Val Glu Asp Ala Glu Ser Ala Phe Ser Ile Ser Vol Ala Asn Gly Ala Ile Pro Ser Ser Pro Pro Ile Val Leu Asn Glu Ala Val Thr Ile Ala Glu Val Lys Leu Tyr Gly Asp Val Vol Leu Arg Tyr Val Ser Tyr Lys Ala Glu Asp Thr Glu Lys Ser Glu Phe Leu Pro Gly Phe Glu Arg Val Glu Asp Ala Ser Ser ?he Pro Leu Asp Tyr Gly Ile Arg Arg Leu Asp His Ala Val Gly Asn Val Pro Glu Lou Gly Pro Ala Leu Thr Tyr Val Ala Gly Phe Thr Gly Phe His Gin Phe Ala Glu Phe Thr Ala Asp Asp Val Gly Thr Ala Glu Ser Gly Leu Asn Ser Ala Val Leu Ala Ser Asn Asp Glu Met Vol Leu Leu Pro Ile Ala Glu Pro Val His Gly Thr Lys Arg Lys Ser Gin Ile Gin Thr Tyr Leu Glu His Asn Glu Gly Ala Gly Leu Gin His Leu Ala Lou Met Ser Glu Asp Ile Phe Arg Thr Leu Arg Glu Met Arg Lys Arq Ser Ser Ile Gly Gly Phe Asp Phe Met Pro Ser Pro Pro Pro Thr Tyr Tyr Gin Asn Leu Lys Lys Arg Val Gly Asp Vol Leu Ser Asp Asp Gin Ile Lys Glu Cys Glu Glu Leu Gly lie Leu Val Asp Arg Asp Asp Gin Gly Thr Leu Leu Gin Ile Phe Thr Lys Pro Lou Gly Asp Arg Pro Thr Ile Phe Ile Glu Ile Ile Gin Ary Val Gly Cys Met Met Lys Asp Glu Glu Gly Lys Ala Tyr Gin Ser Gly Gly Cys Gly Gly Phe Gly Lys Gly Asn Phe Ser Glu Leu Phe Lys Ser Ile Glu Glu Tyr Glu Lys Thr Leu Glu Ala Lys Gin Leu Vol Gly <210> 37 <211> 1353 <212> DNA
<213> Artificial sequence <220>
<223> nucleotide sequence encoding mutant HPPD polypeptide <220>
<221> misc_feature <222> (4)..(6) <223> sequence coding for Ala Mee <220>
<221> misc_feature <222> (7)..(24) <223> sequence coding for an His Tag containing 6 His <400> 37 atggctcatc accatcacca tcaccaaaac gccgccgttt cagagaatca aaaccatgat 60 gacggcgctg cgtcgtcgcc gggattcaag ctcgtcggat tttccaagtt cgtaagaaag 120 aatccaaagt ctgataaatt caaggttaag cgcttccatc acatcgagtt ctggtgcggc 180 gacgcaacca acgtcgctcg tcgcttctoc tggggtctgg ggatgagatt ctccgccaaa 240 tccgatcttt ccaccggaaa catggttcac gcctcttacc tactcacctc cggtgacctc 300 cgattccttt tcactgctcc ttactctccg tctctctccg ccggagagat taaaccgaca 360 accacagctt ctatcccaag tttcgatcac ggctcttgtc gttccttctt ctcgtcacat 420 ggtctcggtg ttagagccgt tgcgattgaa gtagaagacg cagagtcagc tttctccatc 480 agtgtagcta atggcgctat tccttcgtcg cctcctatcg tcctcaatga aggagttacg 540 atcgctgagg ttaaactata cggcgatgtt gttctccgat atgttagtta caaagcagaa 600 gataccgaaa aatccgaatt cttgccaggg ttcgagcgtg tagaggatgc gtcgtcgttc 660 ccattggatt atggtatccg gcggcttgac cacgccgtgg gaaacgttcc tgagcttggt 720 ccggctttaa cttatgtagc ggggttcact ggttttcacc aattcgcatc tttcacagca 780 gacgacgttg gaaccgccga gagcggttta aattcagcgg tcctggctag caatgatgaa 840 atggttcttc taccgattaa cgagccagtg cacggaacaa agaggaagag tcagattcag 900 acgtatttgg aacataacga aggcgcaggg ctacaacatc tggctctgat gagtgaagac 960 atattcagga ccctgagaga gatgaggaag aggagcagta ttggaggatt cgacttcatg 1020 ccttctcctc cgcctactta ctaccagaat ctcaagaaac gggtcggcga cgtgctcagc 1080 gatgatcaga tcaaggagtg tgaggaatta gggattcttg tagacagaga tgatcaaggg 1140 acgttgettc aaatcttcac aaaaccacta ggtgacaggc cgacgatatt tatagagata 1200 atccagagag taggatgcat gatgaaagat gaggaaggga aggcttacca gagtggagga 1260 tgtggtggtt ttggcaaagg caatttctct gagctcttca agtccattga agaatacgaa 1320 aagactcttg aagccaaaca gttagtggga tga 1353 <210> 38 <211> 450 <212> PRT
<213> Artificial sequence <220>
<223> encoded by SEQ ID No. 37 <220>
<221> MISC_FEATURE
<222> (2)..(2) <223> Ala <220>
<221> MISC_FEATURE
<222> (3)..(8) <223> His Tag made of 6 His <400> 38 Met Ala His His His His His His Gin Asn Ala Ala Val Ser Glu Asn Gln Asn His Asp Asp Gly Ala Ala Ser Ser Pro Gly Phe Lys Leu Val Gly Phe Sex Lys Phe Val Arg Lys Asn Pro Lys Ser Asp Lys Phe Lys 111ff Val Lys Arg Phe His His Ile Glu Phe Trp Cys Gly Asp Ala Thr Asn Val Ala Arg Arg Phe Ser Trp Gly Leu Gly Met Arg Phe Ser Ala Lys Ser Asp Lou Ser Thr Gly Asn Met Val His Ala Ser Tyr Leu Leu Thr Ser Gly Asp Leu Arg Phe Leu Phe Thr Ala Pro Tyr Ser Pro Ser Leu Ser Ala Gly Glu Ile Lys Pro Thr Thr Thr Ala Ser Ile Pro Ser Phe Asp His Gly Ser Cys Arg Ser Phe Phe Ser Ser His Gly Leu Gly Val Arc Ala Val Ala Ile Glu Val Glu Asp Ala Glu Ser Ala Phe Ser Ile Ser Val Ala Asn Gly Ala He Pro Ser Ser Pro Pro Ile Val Leu Asn Glu Ala Val Thr Ile Ala Glu Val Lys Leu Tyr Gly Asp Val Val Leu Arg Tyr Val Ser Tyr Lys Ala Glu Asp Thr Glu Lys Ser Glu Phe Leu Pro Gly Phe Glu Arg Val Glu Asp Ala Ser Ser Phe Pro Leu Asp Tyr Gly Ile Arg Arq Leu Asp His Ala Val Gly Asn Val Pro Glu Leu Gly Pro Ala Leu Thr Tyr Val Ala Gly Phe Thr Gly Phe His Gin Phe Ala Ser Phe Thr Ala Asp Asp Val Gly Thr Ala Glu Ser Gly Leu Asn Ser Ala Val Leu Ala Ser Asn Asp Glu Met Val Leu Leu Pro Ile Asn Glu Pro Val His Gly Thr Lys Arg Lys Ser Gin Ile Gin Thr Tyr Leu Glu His Asn Glu Gly Ala Gly Leu Gin His Leu Ala Leu Met Ser Glu Asp Ile Phe Arg Thr Leu Arg Glu Met Arg Lys Arg Ser Ser Ile Gly Gly Phe Asp Phe Met Pro Ser Pro Pro Pro Thr Tyr Tyr Gin Asn Leu Lys Lys Arg Val Gly Asp Val Leu Ser Asp Asp Gin Ile Lys Glu Cys Glu Glu Leu Gly Ile Leu Val Asp Arg Asp Asp Gin Gly Thr Leu Leu Gin Ile Phe Thr Lys Pro Leu Gly Asp Arg Pro Thr Ile Phe Ile Glu Ile Ile Gin Arg Val Gly Cys Met Met Lys Asp Glu Glu Gly Lys Ala Tyr Gin Ser Gly Gly Cys Gly Gly Phe Gly Lys Gly Asn Phe Ser Glu Leu Phe Lys Ser Ile Glu Glu Tyr Glu Lys Thr Leu Glu Ala Lys Gin Leu Val Gly =

<210> 39 <211> 1353 Ca 02808152 2013-03-27 111gg <212> DNA
<213> Artificial sequence <220>
<223> nucleotide sequence encoding mutant HPPD polypeptide <220>
<221> misc_feature <222> (4)..(6) <223> sequence coding for Ala <220>
<221> misc_feature <222> (7)..(24) <223> sequence coding for an His Tag containing 6 His <400> 39 atggctcatc accatcacca tcaccaaaac gccgccgttt cagagaatca aaaccatgat 60 gacggcgctg cgtcgtcgcc gggattcaag ctcgtcggat tttccaagtt cgtaagaaag 120 aatccaaagt ctgataaatt caaggttaag cgcttccatc acatcgagtt ctggtgcggc 180 gacgcaacca acgtcgctcg tcgcttctcc tggggtctgg ggatgagatt ctccgccaaa 240 tccgatcttt ccaccggaaa catggttcac gcctcttacc tactcacctc cggtgacctc 300 cgattccttt tcactgctcc ttactctccg tctctctccg ccggagagat taaaccgaca 360 accacagctt ctatcccaag tttcgatcac ggctcttgtc gttccttctt ctcgtcacat 420 ggtctcggtg ttagagccgt tgcgattgaa gtagaagacg cagagtcagc tttctccatc 480 agtgtagcta atggcgctat tccttcgtcg cctcctatcg tcctcaatga agcagttacg 540 atcgctgagg ttaaactata cggcgatgtt gttctccgat atgttagtta caaagcagaa 600 gataccgaaa aatccgaatt cttgccaggg ttcgagcgtg tagaggatgc gtcgtcgttc 660 ccattggatt atggtatccg goggcttgac cacgccgtgg gaaacgttcc tgagcttggt 720 ccggctttaa cttatgtagc ggggttcact ggttttcacc aattcgcaga gttcacagca 780 gacgacgttg gaaccgccga gagcggttta aattcagcgg cgctggctag caatgatgaa 840 atggttcttc taccgattaa cgagccagtg cacggaacaa agaggaagag tcagattcag 900 acgtatttgg aacataacga aggcgcaggg ctacaacatc tggctctgat gagtgaagac 960 atattcagga ccctgagaga gatgaggaag aggagcagta ttggaggatt cgacttcatg 1020 ccttctcctc cgcctactta ctaccagaat ctcaagaaac gggtcggcga cgtgctcagc 1080 gatgatcaga tcaaggagtg tgaggaatta gggattcttg tagacagaga tgatcaaggg 1140 acgttgcttc aaatcttcac aaaaccacta ggtgacaggc cgacgatatt tatagagata 1200 atccagagag taggatgcat gatgaaagat gaggaaggga aggcttacca gagtggagga 1260 tgtggtggtt ttggcaaagg caatttctct gagctcttca agtccattga agaatacgaa 1320 aagactcttg aagccaaaca gttagtggga tga 1353 <210> 40 <211> 450 <212> PRT
<213> Artificial sequence <220>
<223> encoded by SEQ ID No. 39 <220>
<221> MISC_FEATURE
<222> (2)..(2) <223> Ala 111 hh <220>
<221> MISC_FEATURE
<222> (3)..(8) <223> His Tag made of 6 His <400> 40 Met Ala His His His His His His Gin Asn Ala Ala Val Ser Glu Asn Gin Asn His Asp Asp Gly Ala Ala Ser Ser Pro Gly Phe Lys Leu Val Gly Phe Ser Lys Phe Val Arg Lys Asn Pro Lys Ser Asp Lys Phe Lys Val Lys Arg Phe His His Ile Glu Phe Trp Cys Gly Asp Ala Thr Asn Val Ala Arg Arg Phe Ser Trp Gly Leu Gly Met Arg Phe Ser Ala Lys Ser Asp Leu Ser Thr Gly Asn Met Val His Ala Ser Tyr Leu Leu Thr Ser Gly Asp Leu Arg Phe Leu Phe Thr Ala Pro Tyr Ser Pro Ser Leu Ser Ala Gly Glu Ile Lys Pro Thr Thr Thr Ala Ser Ile Pro Ser Phe Asp His Gly Ser Cys Arg Ser Phe Phe Ser Ser His Gly Leu Gly Val Arg Ala Val Ala Ile Glu Val Glu Asp Ala Glu Ser Ala Phe Ser Ile Ser Val Ala Asn Gly Ala Ile Pro Ser Ser Pro Pro Ile Val Leu Asn Glu Ala Val Thr Ile Ala Glu Val Lys Leu Tyr Gly Asp Val Val Leu Arg Tyr Val Ser Tyr Lys Ala Glu Asp Thr Glu Lys Ser Glu Phe Leu Pio Gly Phe Glu A/A- Val Glu Asp Ala Su/ Her Phe Pro Leu Asp Tyr Gly Ile Arg Arg Leu Asp His Ala Val Gly Asn Val Pro Glu Leu Gly Pro Ala Leu Thr Tyr Val Ala Gly Phe Thr Gly Phe His Gin Phe Ala Glu Phe Thr Ala Asp Asp Val Gly Thr Ala Glu Ser Gly Leu Asn Ser Ala Ala Leu Ala Ser Asn Asp Glu Met Val Leu Leu Pro Ile Asn Glu Pro Val His Gly Thr Lys Arg Lys Ser Gin Ile Gin Thr Tyr Leu Glu His Asn Glu Gly Ala Gly Leu Gin His Leu Ala Leu Met Ser Glu Asp lie Phe Arg Thr Lou Arg Glu Met Arg Lys Arg Ser Ser Ile Gly Gly Phe Asp Phe Met Pro Ser Pro Pro Pro Thr Tyr Tyr Gin Asn Leu Lys Lys Arg Val Gly Asp Val Leu Ser Asp Asp Gin Ile Lys Glu Cys Glu Glu Leu Gly Ile Leu Val Asp Arg Asp Asp Gin Gly Thr Leu Leu Gin Ile Phe Thr Lys Pro Leu Gly Asp Arg Pro Thr Ile Phe Ile Glu Ile 111ii Ile Gin Arg Val Gly Cys Met Met Lys Asp Glu Glu Gly Lys Ala Tyr Gin Ser Gly Gly Cys Gly Gly Phe Gly Lys Gly Asn Phe Ser Glu Lou Phe Lys Ser Ile Glu Glu Tyr Glu Lys Thr Leu Glu Ala Lys Gin Leu Val Gly <210> 41 <211> 1353 <212> DNA
<213> Artificial sequence <220>
<223> nucleotide sequebc encoding mutant HOOD polypeptide <220>
<221> misc_feature <222> (4)..(6) <223> sequence coding for Ala <220>
<221> misc_feature <222> (7)..(24) <223> sequence coding for an His Tag containing 6 His <400> 41 atggctcatc accatcacca tcaccaaaac gccgccgttt cagagaatca aaaccatgat 60 gacggcgctg cgtcgtcgcc gggattcaag ctcgtcggat tttccaagtt cgtaagaaag 120 aatccaaagt ctgataaatt caaggttaag cgcttccatc acatcgagtt ctggtgcggc 180 gacgcaacca acgtogctog tcgcttctcc tggggtctgg ggatgagatt ctccgccaaa 240 tccgatcttt ccaccggaaa catggttcac gcctcttacc tactcacctc cggtgacctc 300 cgattccttt tcactgctcc ttactctccg tctctctccg ccggagagat taaaccgaca 360 accacagctt ctatcccaag tttcgatcac ggctcttgtc gttccttctt ctcgtcacat 420 ggtctcggtg ttagagccgt tgcgattgaa gtagaagacg cagagtcagc tttctccatc 480 agtgtagcta atggcgctat tccttcgtcg cctcctatcg tcctcaatga agcagttacg 540 atcgctgagg ttaaactata cggcgatgtt gttctccgat atgttagtta caaagcagaa 600 gataccgaaa aatccgaatt cttgccaggg ttcgagcgtg tagaggatgc gtcgtcgttc 660 ccattggatt atggtatccg gcggcttgac cacgccgtgg gaaacgttcc tgagcttggt 720 ccggctttaa cttatgtagc ggggttcact ggttttcacc aattcgcaga gttcacagca 780 gacgacgttg qaaccgccga qaqcqqttta aattcagogg tcctqqctaq caatqatgaa 840 atggttcttc tagcgattaa cgagccagtg cacggaacaa agaggaagag tcagattcag 900 acgtatttgg aacataacga aggcgcaggg ctacaacatc tggctctgat gagtgaagac 960 atattcagga ccctgagaga gatgaggaag aggagcagta ttggaggatt cgacttcatg 1020 ccttctcctc cgcctactta ctaccagaat ctcaagaaac gggtcggcga cgtgctcagc 1080 gatgatcaga tcaaggagtg tgaggaatta gggattcttg tagacagaga tgatcaaggg 1140 acgttgcttc aaatcttcac aaaaccacta ggtgacaggc cgacgatatt tatagagata 1200 atccagagag taggatgcat gatgaaagat gaggaaggga aggcttacca gagtggagga 1260 tgtggtggtt ttggcaaagg caatttctct gagctcttca agtccattga agaatacgaa 1320 aagactcttg aagccaaaca gttagtggga tga 1353 <210> 42 <211> 450 . CA 02808152 2013-03-27 111jj <212> PRT
<213> Artificial sequence <220>
<223> encoded by SEQ ID No. 41 <220>
<221> MISC_FEATURE
<222> (2)..(2) <223> Ala <220>
<221> MISC_FEATURE
<222> (3)..(8) <223> His Tag made of 6 His <400> 42 Met Ala His His His His His His Gin Asn Ala Ala Val Ser Glu Asn Gin Asn His Asp Asp Gly Ala Ala Ser Ser Pro Gly Phe Lys Leu Val Gly Phe Ser Lys Phe Val Arg Lys Asn Pro Lys Ser Asp Lys Phe Lys Val Lys Arg Phe His His Ile Glu She Trp Cys Gly Asp Ala Thr Asn Val Ala Arg Arg Phe Ser Trp Gly Leu Gly Met Arg Phe Ser Ala Lys Ser Asp Leu Ser Thr Gly Asn Met Val His Ala Ser Tyr Leu Leu Thr Ser Gly Asp Lou Arg Phe Leu Phe Thr Ala Pro Tyr Ser Pro Ser Leu Ser Ala Gly Glu Ile Lys Pro Thr Thr Thr Ala Ser Ile Pro Ser Phe Asp His Gly Ser Cys Arg Ser Phe Phe Ser Ser His Gly Leu Gly Val Arg Ala Val Ala Ile Glu Val Glu Asp Ala Glu Ser Ala Phe Ser Ile Ser Val Ala Asn Gly Ala Ile Pro Ser Ser Pro Pro Ile Val Leu Asn Glu Ala Val Thr Ile Ala Glu Val Lys Leu Tyr Gly Asp Val val Leu Arg Tyr Val Ser Tyr Lys Ala Glu Asp Thr Glu Lys Ser Glu Phe Leu Pro Gly Phe Glu Arg Val Glu Asp Ala Ser Ser Phe Pro Leu Asp Tyr Gly Ile Arg Arg Leu Asp His Ala Val Gly Asn Val Pro Glu Leu Gly Pro Ala Leu Thr Tyr Val Ala Gly She Thr Gly Phe His Gin Phe Ala Glu Phe Thr Ala Asp Asp Val Gly Thr Ala Glu Ser Gly Leu Asn Ser Ala Val Lou Ala Ser Asn Asp Glu Met Val Leu Leu Ala Ile Asn Glu Pro Val His Gly Thr Lys Arg Lys Ser Gin Ile Gin Thr Tyr Leu Glu = CA 02808152 2013-03-27 111 kk His Asn Glu Gly Ala Gly Leu Gin His Leu Ala Leu Met Ser Glu Asp Ile Phe Arg Thr Leu Arg Glu Met Arg Lys Arg Ser Ser Ile Gly Gly Phe Asp Phe Met Pro Ser Pro Pro Pro Thr Tyr Tyr Gin Asn Leu Lys Lys Arg Val Gly Asp Val Leu Ser Asp Asp Gin Ile Lys Glu Cys Glu Glu Leu Gly Ile Leu Val Asp Arg Asp Asp Gln Gly Thr Leu Lou Gin Ile Phe Thr Lys Pro Leu Gly Asp Arg Pro Thr Ile Phe Ile Glu Ile Ile Gin Arg Val Gly Cys Met Met Lys Asp Glu Glu Gly Lys Ala Tyr Gin Ser Gly Gly Cys Gly Gly Phe Gly Lys Gly Asn Phe Ser Glu Leu Phe Lys Ser Ile Glu Glu Tyr Glu Lys Thr Leu Glu Ala Lys Gin Lou Val Gly <210> 43 <211> 1353 <212> DNA
<213> Artificial sequence <220>
<223> nucleotide sequence encoding mutant HPPD polypeptide <220>
<221> misc_feature <222> (4)..(6) <223> sequence coding for Ala <220>
<221> misc_feature <222> (7)..(24) <223> sequence coding for an His Tag containing 6 His <400> 43 atggctcatc accatcacca tcaccaaaac gccgccgttt cagagaatca aaaccatgat 60 gacggcgctg cgtcgtcgcc gggattcaag ctcgtcggat tttccaagtt cgtaagaaag 120 aatccaaagt ctgataaatt caaggttaag cgcttccatc acatcgagtt ctggtgcggc 180 gacgcaacca acgtcgctcg tcgcttctcc tggggtctgg ggatgagatt ctccgccaaa 240 tccgatcttt ccaccggaaa catggttcac gcctcttacc tactcacctc cggtgacctc 300 cgattccttt tcactgctcc ttactctccg tctatatccg ccggagagat taaaccgaca 360 accacagctt ctatcccaag tttcgatcac ggctcttgtc gttcattatt ctcgtcacat 420 ggtctcggtg ttagagccgt tgcgattgaa gtagaagacg cagagtcagc tttctccatc 480 agtgtagcta atggcgctat tccttcgtcg cctcctatcg tcctcaatga agcagttacg 540 atcgctgagg ttaaactata cggcgatgtt gttctccgat atgttagtta caaagcagaa 600 gataccgaaa aatccgaatt cttgccaggg ttcgagcgtg tagaggatgc gtcgtcgttc 660 ccattggatt atggtatccg gcggcttgac cacgccgtgg gaaacgttcc tgagattggt 720 ccggctttaa cttatgtagc ggggttcact ggttttcacc aattcgcaga gttcacagca 780 gacgacgttg gaaccgccga gagcggttta aattcagcgg tcctggctag caatgatgaa 840 atggttcttc taccgattaa cgagccagtg cacggaacaa agaggaagag tcagattcag 900 , = . CA 02808152 2013-03-27 acgtatttgg aacataacga aggcgcaggg ctacaacatc tggctctgat gagtgaagac 960 atattcagga ccctgagaga gatgaggaag aggagcagta ttggaggatt cgacttcatg 1020 ccttctcctc cgcctactta ctaccagaat ctcaagaaac gggtcggcga cgtgctcagc 1080 gatgatcaga tcaaggagtg tgaggaatta gggattatgg tagacagaga tgatcaaggg 1140 acgttgcttc aaatcttcac aaaaccacta ggtgacaggc cgacgatatt tatagagata 1200 atccagagag taggatgcat gatgaaagat gaggaaggga aggcttacca gagtggagga 1260 tgtggtggtt ttggcaaagg caatttctct gagotottca agtccattga agaatacgaa 1320 aagactattg aagccaaaca gttagtggga tga 1353 <210> 44 <211> 450 <212> PRT
<213> Artificial sequence <220>
<223> encoded by SEQ ID No. 43 <220>
<221> MISC_FEATURE
<222> (2)..(2) <223> Ala <220>
<221> MISC_FEATURE
<222> (3)..(8) <223> His Tag made of 6 His <400> 44 Met Ala His His His His His His Gin Asn Ala Ala Val Ser Glu Asn Gln Asn His Asp Asp Gly Ala Ala Ser Ser Pro Gly Phe Lys Leu Val Gly Phe Ser Lys Phe Val Arg Lys Asn Pro Lys Ser Asp Lys Phe Lys Val Lys Arg Phe His His Ile Glu Phe Trp Cys Gly Asp Ala Thr Asn Val Ala Arg Arg Phe Ser Trp Gly Leu Gly Met A/9- Phe Ser Ala Lys Ser Asp Leu Ser Thr Gly Asn Met Val His Ala Ser Tyr Leu Leu Thr Ser Gly Asp Leu Arg Phe Leu Phe Thr Ala Pro Tyr Ser Pro Ser Leu Ser Ala Gly Glu Ile Lys Pro Thr Thr Thr Ala Ser Ile Pro Ser Phe Asp His Gly Ser Cys Arg Ser Phe Phe Ser Ser His Gly Leu Gly Val Arg Ala Val Ala Ile Clu Val Glu Asp Ala Glu Ser Ala Phe Ser Ile Ser Val Ala Asn Gly Ala Ile Pro Ser Ser Pro Pro Ile Val Leu Asn Glu Ala Val Thr Ile Ala Glu Val Lys Leu Tyr Gly Asp Val Val Leu Arg Tyr Val Ser Tyr Lys Ala Glu Asp Thr Glu Lys Ser Glu Phe Leu , CA 02808152 2013-03-27 111mm Pro Gly Phe Glu Arg Val Glu Asp Ala Ser Ser Phe Pro Leu Asp Tyr Gly Ile Arg Arg Leu Asp His Ala Val Gly Asn Val Pro Glu Leu Gly Pro Ala Leu Thr Tyr Val Ala Gly Phe Thr Gly Phe His Gin Phe Ala Glu Phe Thr Ala Asp Asp Val Gly Thr Ala Glu Ser Gly Leu Asn Ser Ala Val Leu Ala Ser Asn Asp Glu Met Val Leu Leu Pro Ile Asn Glu Pro Val His Gly Thr Lys Arg Lys Ser Gin Ile Gin Thr Tyr Leu Glu His Asn Glu Gly Ala Gly Leu Gin His Leu Ala Leu Met Ser Glu Asp Ile Phe Arg Thr Leu Arg Glu Met Arg Lys Arg Ser Ser Ile Gly Gly Phe Asp Phe Met Pro Ser Pro Pro Pro Thr Tyr Tyr Gin Asn Leu Lys Lys Arg Val Gly Asp Val Leu Ser Asp Asp Gin Ile Lys Giu Cys Glu Glu Leu Gly Ile Met Val Asp Arg Asp Asp Gin Gly Thr Leu Leu Gin Ile Phe Thr Lys Pro Leu Gly Asp Arg Pro Thr Ile Phe Ile Glu Ile Ile Gin Arg Val Gly Cys Met Met Lys Asp Glu Glu Gly Lys Ala Tyr Gin Ser Gly Gly Cys Gly Cly Phe Cly Lys Cly Asia Phe Ser Glu Lou Phe Lys Ser Ile Glu Glu Tyr Glu Lys Thr Leu Glu Ala Lys Gin Leu Val Gly <210> 45 <211> 1353 <212> DNA
<213> Artificial sequence <220>
<223> nucleotide sequence encoding mutant HPPD polypeptide <220>
<221> misc_feature <222> (4)¨(6) <223> sequence coding for Ala <220>
<221> misc_feature <222> (7)..(24) <223> sequence coding for an His Tag containing 6 His <400> 45 atggctcatc accatcacca tcaccaaaac gccgccgttt cagagaatca aaaccatgat 60 gacggegotg cgtcgtcgcc gggattcaag ctcgtcggat tttccaagtt cgtaagaaag 120 aatccaaagt ctgataaatt caaggttaag cgcttccatc acatcgagtt ctggtgcggc 180 =. = CA 02808152 2013-03-27 111nn gacgcaacca acgtcgctcg tcgcttctcc tggggtctgg ggatgagatt ctccgccaaa 240 tccgatcttt ccaccggaaa catggttcac gcctottacc tactcacctc cggtgacctc 300 cgattccttt tcactgctcc ttactctccg tctctctccg ccggagagat taaaccgaca 360 accacagctt ctatcccaag tttcgatcac ggctettgtc gttcottatt ctcgtcacat 420 ggtctcggtg ttagagccgt tgcgattgaa gtagaagacg cagagtcagc tttctccatc 480 agtgtagcta atggcgctat tccttcgtcg cctcctatcg tcctcaatga agcagttacg 540 atcgctgagg ttaaactata cggcgatgtt gttctccgat atgttagtta caaagcagaa 600 gataccgaaa aatccgaatt cttgccaggg ttcgagcgtg tagaggatgc gtcgtcgttc 660 ccattggatt atggtatccg gcggcttgac cacgccgtgg gaaacgttcc tgagcttggt 720 ccggctttaa cttatgtagc ggygttcact ggttttcacc aattcgcaga gttcacagca 780 gacgacgttg gaaccgccga gagcggttta aattcagcgg tcctggctag caatgatgaa 840 atggttcttc taccgattaa cgagccagtg cacggaacaa agaggaagag tcagattcag 900 acgtatttqg aacataacqa aggcgcaggg ctacaacatc tggctctgat gagtgaagac 960 atattcagga ccctgagaga gatgaggaag aggagcagta ttggaggatt cgacttcatg 1020 ccttctcctc cgcctactta ctaccagaat ctcaagaaac gggtcggcga cgtgctcagc 1080 gatgatcaga tcaaggagtg tgaggaatta gggattcttg tagacagaga tgatcaaggg 1140 acgttgcttc aaatcttcac aaaaccacta ggtgacaggc cgacgatatt tatagagata 1200 atccagagag taggatgcat gatgaaagat gaggaaggga aggcttacca gagtggagga 1260 tgtggtggtt ttggcattgg caatttctct gagctcttca agtccattga agaatacgaa 1320 aagactcttg aagccaaaca gttagtggga tga 1353 <210> 46 <211> 450 <212> PRT
<213> Artificial sequence <220>
<223> encoded by SEQ ID No. 45 <220>
<221> MISC_FEATURE
<222> (2)..(2) <223> Ala <220>
<221> MISC_FEATORE
<222> (3)..(8) <223> His Tag made of 6 His <400> 46 Net Ala His His His His His His Gln Asn Ala Ala Val Ser Glu Asn Gin Asn His Asp Asp Gly Ala Ala Ser Ser Pro Gly Phe Lys Leu Val Gly Phe Ser Lys Phe Val Arg Lys Asn Pro Lys Ser Asp Lys Phe Lys Val Lys Arg Phe His His Ile (flu Phe Trp Cys Gly Asp Ala Thr Asn Val Ala Arg Arg Phe Ser Trp Gly Leta Gly Met Arg Phe Ser Ala Lys Ser Asp Leu Ser Thr Gly Asn Met Val His Ala Ser Tyr Leu Leu Thr Ser Gly Asp Leu Arg Phe Leu Phe Thr Ala Pro Tyr Ser Pro Ser Leu , . CA 02808152 2013-03-27 Ser Ala Gly Glu Ile Lys Pro Thr Thr Thr Ala Ser Ile Pro Ser Phe Asp His Gly Ser Cys Arg Ser Phe Phe Ser Ser His Gly Leu Gly Val Arg Ala Val Ala Ile Glu Val Glu Asp Ala Glu Ser Ala Phe Ser Ile Ser Val Ala Asn Gly Ala Ile Pro Ser Ser Pro Pro Ile Val Leu Asn Glu Ala Val Thr Ile Ala Glu Val Lys Leu Tyr Gly Asp Val Val Leu Arg Tyr Val Ser Tyr Lys Ala Glu Asp Thr Glu Lys Ser Glu Phe Leu Pro Gly Phe Glu Arg Val Glu Asp Ala Ser Ser Phe Pro Leu Asp Tyr Gly Ile Arg Arg Leu Asp His Ala Val Gly Asn Val Pro Glu Leu Gly Pro Ala Leu Thr Tyr Val Ala Gly Phe Thr Gly Phe His Gin Phe Ala Glu Phe Thr Ala Asp Asp Val Gly Thr Ala Glu Ser Gly Leu Asn Ser Ala Val Leu Ala Ser Asn Asp Glu Met Val Leu Leu Pro Ile Asn Glu Pro Val His Gly Thr Lys Arg Lys Ser Gin Ile Gin Thr Tyr Leu Glu His Asn Glu Gly Ala Gly Leu Gin His Leu Ala Leu Met Ser Glu Asp Ile Phe Arg Thr Lou Arg Glu Met Arg Lys Arg Ser Ser Ile Gly Gly Phe Asp Phe Met Pro Ser Pro Pro Pro Thr Tyr Tyr Gin Asn Leu Lys Lys Arg Val Gly Asp Val Leu Ser Asp Asp Gin Ile Lys Glu Cys Glu Clu Leu Gly Ile Lou Val Asp Arg Asp Asp Gin Gly Thr Leu Leu Gin Ile Phe Thr Lys Pro Leu Gly Asp Arg Pro Thr Ile Phe Ile Glu Ile Ile Gin Arg Val Gly Cys Met Met Lys Asp Glu Glu Gly Lys Ala Tyr Gin Ser Gly Gly Cys Gly Gly Phe Gly Ile Gly Asn Phe Ser Glu Leu Phe Lys Ser Ile Glu Glu Tyr Glu Lys Thr Leu Glu Ala Lys Gin Leu Val Gly <210> 47 <211> 1353 <212> DNA
<213> Artificial sequence <220>
<223> nucleotide sequence encoding mutant HPPD polypeptide <220>
<221> misc_feature , CA 02808152 2013-03-27 "pp <222> (4)..(6) <223> sequence coding for Ala <220>
<221> raise feature <222> (7)..(24) <223> sequence coding for an His Tag containing 6 His <400> 47 atggctcatc accatcacca tcaccaaaac gccgccgttt cagagaatca aaaccatgat 60 gacggcgctg cgtcgtcgcc gggattcaag ctcgtcggat tttccaagtt cgtaagaaag 120 aatccaaagt ctgataaatt caaggttaag cgcttccatc acatcgagtt ctggtgcggc 180 gacgcaacca acgtcgctcg tcgcttctcc tggggtctgg ggatgagatt ctccgccaaa 240 tccgatcttt ccaccggaaa catggttcac gcctcttacc tactcacctc cggtgacctc 300 cgattccttt tcactgctcc ttactctccg tctctctccg ccggagagat taaaccgaca 360 accacagctt ctatcccaag tttcgatcac ggctcttgtc gttccttctt ctcgtcacat 420 ggtctcggtg ttagagccgt tgcgattgaa gtagaagacg cagagtcagc tttctccatc 480 agtgtagcta atggcgctat tccttcgtcg cctcctatcg tcctcaatga agcagttacg 540 atcgctgagg ttaaactata cggcgatgtt gttctccgat atgttagtta caaagcagaa 600 gataccgaaa aatccgaatt cttgccaggg ttcgagcgtg tagaggatgc gtcgtcgttc 660 ccattggatt atggtatccg gcggcttgac cacgccgtgg gaaacgttcc tgagcttggt 720 ccggctttaa cttatgtagc ggggttcact ggttttcacc aattcgcaga gttcacagca 780 gacgacgttg gaaccgccga gagcggttta aattcagcgg tcctggctag caatgatgaa 840 atggttcttc taccgattaa cgagccagtg cacggaacaa agaggaagag tcagattcag 900 acgtatttgg aacataacga aggcgcaggg ctacaacatc tggctctgat gagtgaagac 960 atattcagga ccctgagaga gatgaggaag aggagcagta ttggaggatt cgacttcatg 1020 ccttctcctc cgcctactta ctaccagaat ctcaagaaac gggtcggcga cgtgctcagc 1080 gatgatcaga tcaaggagtg tgaggaatta gggattcttg tagacagaga tgatcaaggg 1140 acgttgcttc aaatcttcac aaaaccacta ggtgacaggc cgacgatatt tatagagata 1200 atccagagag taggatgcat gatgaaagat gaggaaggga aggcttacca gagtggagga 1260 tgtggtggtt ttggccaggg caatttctct gagctcttca agtccattga agaatacgaa 1320 aagactcttg aagccaaaca gttagtggga tga 1353 <210> 48 <211> 450 <212> PRT
<213> Artificial sequence <220>
<223> encoded by SEQ ID No. 47 <220>
<221> MISC_FEATURE
<222> (2)..(2) <223> Ala <220>
<221> MISC FEATURE
<222> (3).7(8) <223> His Tag made of 6 His <400> 48 Met Ala His His His His His His Gin Asn Ala Ala Val Ser Glu Asn . CA 02808152 2013-03-27 4 =
=
111qg Gin Asn His Asp Asp Gly Ala Ala Ser Ser Pro Gly Phe Lys Leu Val Gly Phe Ser Lys Phe Val Arg Lys Asn Pro Lys Her Asp Lys Phe Lys Val Lys Arg Phe His His Ile Glu Phe Trp Cys Gly Asp Ala Thr Asn Val Ala Arg Arg Phe Ser Trp Gly Leu Gly Met Arg Phe Ser Ala Lys Ser Asp Leu Ser Thr Gly Asn Met Val His Ala Ser Tyr Leu Lou Thr Ser Gly Asp Leu Arg Phe Leu Phe Thr Ala Pro Tyr Ser Pro Ser Leu Ser Ala Gly Glu Ile Lys Pro Thr Thr Thr Ala Ser Ile Pro Ser Phe Asp His Gly Ser Cys Arg Ser Phe Phe Ser Ser His Gly Leu Gly Val Arg Ala Val Ala Ile Glu Val Glu Asp Ala Glu Ser Ala Phe Ser lie Ser Val Ala Asn Gly Ala Ile Pro Ser Ser Pro Pro Ile Val Leu Asn Glu Ala Val Thr Ile Ala Glu Val Lys Leu Tyr Gly Asp Val Val Leu Arg Tyr Val Ser Tyr Lys Ala Glu Asp Thr Glu Lys Ser Glu Phe Leu Pro Gly Phe Glu Arg Val Glu Asp Ala Ser Ser Phe Pro Leu Asp Tyr Gly Ile Arg Arg Leu Asp His Ala Val Cly Asn Val Pro Glu Leu Gly Pro Ala Leu Thr Tyr Val Ala Gly Phe Thr Gly Phe His Gin Phe Ala Glu Phe Thr Ala Asp Asp Val Gly Thr Ala Glu Ser Gly Leu Asn Ser Ala Val Leu Ala Ser Asn Asp Glu Met val Leu Leu Pro Ile Asn Glu Pro Val His Gly Thr Lys Arg Lys Ser Gin Ile Gin Thr Tyr Leu Glu His Asn Glu Gly Ala Gly Leu Gin His Leu Ala Leu Met Ser Glu Asp Ile Phe Arg Thr Leu Arg Glu Met Arg Lys Arg Ser Ser Ile Gly Gly Phe Asp Phe Met Pro Ser Pro Pro Pro Thr Tyr Tyr Gin Asn Leu Lys Lys Arg Val Gly Asp Val Leu Ser Asp Asp Gin Ile Lys Glu Cys Glu Glu Leu Gly Ile Leu Val Asp Arg Asp Asp Gin Gly Thr Leu Leu Gin Ile Phe Thr Lys Pro Leu Gly Asp Arg Pro Thr Ile Phe Ile Glu Ile Ile Gin Arg Val Gly Cys Met Met Lys Asp Glu Glu Gly Lys Ala Tyr Gin Ser Gly Gly Cys Gly Gly Phe Gly Gin Gly Asn Phe Ser Glu Leu Phe Lys Ser Ile Glu Glu Tyr Glu Lys Thr Leu Glu Ala Lys Gin Lou Val Gly 111rr <210> 49 <211> 1353 <212> DNA
<213> Artificial sequence <220>
<223> nucleotide sequence encoding mutant HPPD polypeptide <220>
<221> misc_feature <222> (4)..(6) <223> sequence coding for Ala <220>
<221> misc_feature <222> (7)..(24) <223> sequence coding for an His Tag containing 6 His <400> 49 atggctcatc accatcacca tcaccaaaac gccgccgttt cagagaatca aaaccatgat 60 gacggcgctg cgtcgtcgcc gggattcaag ctcgtcggat tttccaagtt cgtaagaaag 120 aatccaaagt ctgataaatt caaggttaag cgcttccatc acatcgagtt ctggtgcggc 180 gacgcaacca acgtcgctcg tcacttctcc tggggtctgg ggatgagatt ctccgccaaa 240 tccgatcttt ccaccggaaa catggttcac gcctcttacc tactcacctc cggtgacctc 300 cgattccttt tcactgctcc ttactctccg tctctctccg ccggagagat taaaccgaca 360 accacagctt ctatcccaag tttcgatcac ggctcttgtc gttccttctt ctcgtcacat 420 ggtctcggtg ttagagccgt tgcgattgaa gtagaagacg cagagtcagc tttctccatc 480 agtgtagcta atggcgctat tcattcgtcg cctcctatcg tcctcaatga aggagttacg 540 atcgctgagg ttaaactata cggcgatgtt gttctccgat atgttagtta caaagcagaa 600 gataccgaaa aatccgaatt cttgccaggg ttcgagcgtg tagaggatgc gtcgtcgttc 660 ccattggatt atggtatccg gcggcttgac cacgccgtgg gaaacgttcc tgagcttggt 720 ccggctttaa cttatgtagc gaggttcact ggttttcacc aattcgcaga gttcacagca 780 gacgacgttg gaaccgccga gagcggttta aattcagagg tcctggctag caatgatgaa 840 atggttottc taccgattaa cgagccagtg cacggaacaa agaggaagag tcagattcag 900 acgtatttgg aacataacga aggcgcaggg ctacaacatc tggctctgat gagtgaagac 960 atattcagga ccctgagaga gatgaggaag aggagcagta ttggaggatt cgacttcatg 1020 cottctcctc cgcctactta ctaccagaat ctcaagaaac gggtcggcga cgtgctcagc 1080 gatgatcaga tcaaggagtg tgaggaatta gggattcttg tagacagaga tgatcaaggg 1140 acgttgcttc aaatcttcac aaaaccacta ggtgacaggc cgacgatatt tatagagata 1200 atccagagag taggatgcat gatgaaagat gaggaaggga aggcttacca gagtggagga 1260 tgtggtggtt ttggcgtggg caatttctct gagctcttca agtccattga agaatacgaa 1320 aagactcttg aagccaaaca gttagtggga tga 1353 <210> 50 <211> 450 <212> PRT
<213> Artificial sequence <220>
<223> encoded by SEQ ID No. 49 <220>
<221> MISC_FEATURE
<222> (2)..(2) <223> Ala * CA 02808152 2013-03-27 hiss <220>
<221> MISC_FEATURE
<222> (3)..(8) <223> His Tag made of 6 His <400> 50 Met Ala His His His His His His Gin Asn Ala Ala Val Ser Glu Asn Gin Asn His Asp Asp Gly Ala Ala Ser Ser Pro Gly She Lys Leu Val Gly She Ser Lys She Val Arg Lys Asn Pro Lys Ser Asp Lys Phe Lys Val Lys Arg Phe His His Ile Glu She Trp Cys Gly Asp Ala Thr Asn Val Ala Arg Arg Phe Ser Trp Gly Leu Gly Met Arg She Ser Ala Lys Ser Asp Leu Ser Thr Gly Asn Met Val His Ala Ser Tyr Leu Leu Thr Ser Gly Asp Leu Arg Phe Leu She Thr Ala Pro Tyr Ser Pro Ser Leu Ser Ala Gly Glu Ile Lys Pro Thr Thr Thr Ala Ser Ile Pro Ser Phe Asp His Gly Ser Cys Arg Ser Phe Phe Ser Ser His Gly Leu Gly Val Arg Ala Val Ala Ile Glu Val Glu Asp Ala Glu Ser Ala She Ser Ile Ser Val Ala Asn Gly Ala Ile Pro Ser Ser Pro Pro Tie Val Leu Asn Glu Ala Val Thr Ile Ala Glu Val Lys Leu Tyr Gly Asp Val Val Leu Arg Tyr Val Ser Tyr Lys Ala Glu Asp Thr Glu Lys Ser Glu Phe Leu Pro Gly Phe Glu Ay Vul Glu Asp Ala Ser Ser She Pro Leu Asp Tyr Gly Ile Arg Arg Leu Asp His Ala Val Gly Asn Val Pro Glu Leu Gly Pro Ala Leu Thr Tyr Val Ala Gly She Thr Gly Phe His Gin She Ala Giu Phe Thr Ala Asp Asp Val Gly Thr Ala Glu Ser Gly Leu Asn Ser Ala Val Leu Ala Ser Asn Asp Glu Met Val Leu Leu Pro Ile Asn Glu Pro Val His Gly Thr Lys Arg Lys Ser Gin Ile Gin Thr Tyr Leu Glu His Ann Glu Gly Ala Gly Leu Gin His Leu Ala Leu Met Ser Glu Asp Ile Phe Arg Thr Leu Arg Glu Met Arg Lys Arg Ser Ser Ile Gly Gly She Asp Phe Met Pro Ser Pro Pro Pro Thr Tyr Tyr Gin Asn Leu Lys Lys Arg Val Gly Asp Val Leu Ser Asp Asp Gin Ile Lys Glu Cys Glu Glu Lou Gly Ile Leu Val Asp Arg Asp Asp Gin Gly Thr Leu Leu Gin Ile Phe Thr Lys Pro Leu Gly Asp Arg Pro Thr Ile Phe Ile Glu Ile = CA 02808152 2013-03-27 Hitt Ile Gin Arg Val Gly Cys Met Met Lys Asp Glu Glu Gly Lys Ala Tyr Gin Ser Gly Gly Cys Gly Gly Phe Gly Val Gly Asn Phe Ser Glu Leu Phe Lys Ser Ile Glu Glu Tyr Glu Lys Thr Leu Glu Ala Lys Gin Leu Val Gly The following embodiments are provided:
Embodiment 1. An isolated nucleic acid comprising a nucleotide sequence encoding a mutated HPPD protein, wherein said mutated HPPD protein has HPPD activity, and wherein said mutated HPPD protein comprises a replacement of the amino acid at the position corresponding to position 421 of SEQ ID No. 2 with Asp or Asn.
Embodiment 2. The isolated nucleic acid of embodiment 1, wherein said HPPD
protein is derived from Streptomyces avermitilis (Genebank SAV11864), Daucus carota (Genebank DCU 87257), Arabidopsis thaliana (Genebank AF047834), Mycosphaerella graminicola (Genebank AF038152), oryza sativa / rice [BAD26248], Zea mays /
corn [ACN36372], Avena sativa [ABZ23427], Pseudomonas fluorescens [ABF50055], Synechococcus sp. [YP 473959], Blepharisma japonicum [BAF91881], Rhodococcus RHA1 sp. ro0240 [YP_702005], Rhodococcus RHA1 sp. ro0341 [YP_703002], Picrophilus torridus [YP_024147], Kordia algicida [ZP_02161490], Sorghum bicolor [XP_002453359], Triticum aestivum / wheat [AAZ67144], or Hordeum vulgare /
barley [048604].
Embodiment 3. The isolated nucleic acid of embodiment 1, wherein the amino acid sequence of said mutated HPPD protein is a variant of the amino acid sequence of SEQ
ID No. 4 that comprises Asp or Asn, at position 418 of the amino acid sequence of SEQ
ID No. 4 (corresponding to position 421 of SEQ ID No. 2).
Embodiment 4. The isolated nucleic acid of embodiment 1, wherein the amino acid sequence of said mutated HPPD protein is a variant of the amino acid sequence of SEQ
ID No. 6 that comprises Asp or Asn at position 395 of the amino acid sequence of SEQ
ID No. 6 (corresponding to position 421 of SEQ ID No. 2).
Embodiment 5. The isolated nucleic acid of embodiment 1, wherein the amino acid sequence of said mutated HPPD protein is a variant of the amino acid sequence of SEQ
Date Recue/Date Received 2022-12-02 ID No. 8 that comprises Asp or Asn at position 412 of the amino acid sequence of SEQ
ID No. 8 (corresponding to position 421 of SEQ ID No. 2).
Embodiment 6. The isolated nucleic acid of embodiment 1, wherein the amino acid sequence of said mutated HPPD protein is a variant of the amino acid sequence of SEQ
ID No. 10 that comprises Asp or Asn at position 335 of the amino acid sequence of SEQ
ID No. 10 (corresponding to position 421 of SEQ ID No. 2).
Embodiment 7. The isolated nucleic acid of embodiment 1, wherein the amino acid sequence of said mutated HPPD protein is a variant of the amino acid sequence of SEQ
ID No. 14 that comprises Asp or Asn at position 327 of the amino acid sequence of SEQ
ID No. 14 (corresponding to position 421 of SEQ ID No. 2).
Embodiment 8. The isolated nucleic acid of embodiment 1, wherein the amino acid sequence of said mutated HPPD protein is a variant of the amino acid sequence of SEQ
ID No. 16 that comprises Asp or Asn at position 361 of the amino acid sequence of SEQ
ID No. 16 (corresponding to position 421 of SEQ ID No. 2).
Embodiment 9. The isolated nucleic acid of embodiment 1, wherein the amino acid sequence of said mutated HPPD protein is a variant of the amino acid sequence of SEQ
ID No. 18 that comprises Asp or Asn at position 381 of the amino acid sequence of SEQ
ID No. 18 (corresponding to position 421 of SEQ ID No. 2).
Embodiment 10. The isolated nucleic acid of embodiment 1, wherein the amino acid sequence of said mutated HPPD protein is a variant of the amino acid sequence of SEQ
ID No. 30 that comprises Asp or Asn at position 382 of the amino acid sequence of SEQ
ID No. 30 (corresponding to position 421 of SEQ ID No. 2).
Embodiment 11. The isolated nucleic acid of embodiment 1, wherein the amino acid sequence of said mutated HPPD protein is a variant of the amino acid sequence of SEQ
Date Recue/Date Received 2022-12-02 ID No. 20 that comprises Asp at position 348 of the amino acid sequence of SEQ
ID No.
20 (corresponding to position 421 of SEQ ID No. 2).
Embodiment 12. The isolated nucleic acid of embodiment 1, wherein said the amino acid sequence of mutated HPPD protein is a variant of the amino acid sequence of SEQ
ID No. 22 that comprises Asp or Asn at position 367 of the amino acid sequence of SEQ
ID No. 22 (corresponding to position 421 of SEQ ID No. 2).
Embodiment 13. The isolated nucleic acid of embodiment 1, wherein said the amino acid sequence of mutated HPPD protein is a variant of the amino acid sequence of SEQ
ID No. 24 that comprises Asp or Asn at position 412 of the amino acid sequence of SEQ
ID No. 24 (corresponding to position 421 of SEQ ID No. 2).
Embodiment 14. The isolated nucleic acid of embodiment 1, wherein said the amino acid sequence of mutated HPPD protein is a variant of the amino acid sequence of SEQ
ID No. 26 that comprises Asp or Asn at position 408 of the amino acid sequence of SEQ
ID No. 26 (corresponding to position 421 of SEQ ID No. 2).
Embodiment 15. The nucleic acid of any one of embodiments Ito 14, wherein said mutated HPPD protein increases the tolerance of a plant to at least one herbicide acting on HPPD.
Embodiment 16. A protein encoded by the isolated nucleic acid of any one of embodiments 1 to 15.
Embodiment 17. A gene or a nucleic acid comprising a coding sequence comprising the nucleic acid of any one of embodiments 1 to 15 operably linked to a plant-expressible promoter and optionally a transcription termination and polyadenylation region.
Date Recue/Date Received 2022-12-02 Embodiment 18. A method of obtaining a mutated HPPD protein modulating the tolerance of a plant to at least one herbicide acting on HPPD, wherein said mutated HPPD protein has HPPD activity, the method comprising i) providing an HPPD protein;
ii) replacing the amino acid at position 421 of SEQ ID NO. 2 in said HPPD
protein so that the resulting amino acid sequence comprises Asp, Phe, or Asn at the position corresponding to position 421 of the amino acid sequence of SEQ ID No. 2;
iii) determining the inhibition of the resulting HPPD protein by at least one herbicide acting on HPPD, wherein an inhibition of the resulting protein of less or more than that observed with a reference HPPD protein is indicative that the resulting protein modulates the tolerance of a plant to said herbicide.
Embodiment 19. The method of embodiment 18, wherein said mutated HPPD protein increases the tolerance of a plant to at least one HPPD inhibitor herbicide.
Embodiment 20. The nucleic acid of embodiment 15 or the method of embodiment 19, wherein the herbicide acting on HPPD is tembotrione.
Embodiment 21. A method of producing a transgenic plant comprising introducing into said plant the nucleic acid of any one of embodiments 1 to 15 and 20, operably linked to a plant expressible promoter or the chimeric gene of embodiment 17.
Embodiment 22. The method of embodiment 21, wherein the nucleic acid of embodiment 16 or a nucleic acid identified by the method of embodiment 18 or 19, both operably linked to a plant expressible promoter, or the chimeric gene of embodiment 17 comprising the nucleic acid of embodiment 15 is introduced into said plant.
Embodiment 23. A plant cell comprising the isolated nucleic acid of any one of embodiments 1 to 15 or the chimeric gene of embodiment 17 in its genetic information.
Date Recue/Date Received 2022-12-02 Embodiment 24. A cell of a plant, a cell of a part thereof or a cell of plant tissue comprising the isolated nucleic acid of any one of embodiments 1 to 15 or the chimeric gene of embodiment 17 in its genetic information.
Embodiment 25. A cell of a plant comprising the isolated nucleic acid of any one of embodiments 1 to 15 or the chimeric gene of embodiment 17 in its genetic information obtained from the method of embodiment 21 or 22.
Embodiment 26. The cell of embodiment 24 or 25 which is a cell comprising the isolated nucleic acid of any one of embodiments 1 to 15 or the chimeric gene of embodiment 17 in its genetic information of a plant selected from wheat, cotton, canola, rice, corn, soybean, and sorghum.
Embodiment 27. A cell of a seed of a plant comprising the isolated nucleic acid of any one of embodiments 1 to 15 or the chimeric gene of embodiment 17 in its genetic information as defined in any one of embodiments 24 to 26.
Embodiment 28. A cell of progeny of a plant cell comprising the isolated nucleic acid of any one of embodiments Ito 15 or the chimeric gene of embodiment 17 in its genetic information as defined in any one of embodiments 23 to 26 or a cell of progeny of a seed cell as defined in embodiment 27.
Embodiment 29. A method of modulating a plant's tolerance to at least one herbicide acting on HPPD or of obtaining a plant tolerant to an HPPD inhibitor herbicide comprising introducing the isolated nucleic acid of any one of embodiments 1 to 15 operably linked to a plant expressible promoter or the chimeric gene of embodiment 17 into a plant's genome.
Embodiment 30. A method for controlling weeds comprising spraying at least one HPPD
inhibitor herbicide on or around a crop plant, wherein said crop plant contains the nucleic acid according to embodiment 15 operably linked to a plant expressible Date Recue/Date Received 2022-12-02 promoter or the chimeric gene of embodiment 17 comprising the nucleic acid of embodiment 15.
Embodiment 31. Use of a chimeric gene of embodiment 17 or the nucleic acid of any .. one of embodiments 1 to 15 operably linked to a plant expressible promoter for modulating the tolerance of a plant to at least one HPPD inhibitor herbicide applied for controlling weeds.
Embodiment 32. The method of any one of embodiments 22, 29 or 30 or the use of .. embodiment 31, wherein the tolerance of a plant to at least one HPPD
inhibitor herbicide is increased.
Date Recue/Date Received 2022-12-02

Claims (32)

CLAIMS:

1. An isolated nucleic acid comprising a nucleotide sequence encoding a mutated HPPD protein, wherein said mutated HPPD protein has HPPD activity, and wherein said mutated HPPD protein comprises a replacement of the amino acid at the position corresponding to position 421 of SEQ ID No. 2 with Asp or Asn.

2. The isolated nucleic acid of claim 1, wherein said HPPD protein is derived from Streptomyces avermitilis (Genebank SAV11864), Daucus carota (Genebank DCU 87257), Arabidopsis thaliana (Genebank AF047834), Mycosphaerella graminicola (Genebank AF038152), oryza sativa / rice [BAD26248], Zea mays / corn [ACN36372], Avena sativa [ABZ23427], Pseudomonas fluorescens [ABF50055], Synechococcus sp. [YP_473959], Blepharisma japonicum [BAF91881], Rhodococcus RHA1 sp. r00240 [YP_702005], Rhodococcus RHA1 sp. ro0341 [YP_703002], Picrophilus torridus [YP_024147], Kordia algicida [ZP_02161490], Sorghum bicolor [XP_002453359], Triticum aestivum / wheat [AAZ67144], or Hordeum vulgare / barley [048604].

3. The isolated nucleic acid of claim 1, wherein the amino acid sequence of said mutated HPPD protein is a variant of the amino acid sequence of SEQ ID No.
4 that comprises Asp or Asn, at position 418 of the amino acid sequence of SEQ ID No. 4 (corresponding to position 421 of SEQ ID No. 2).

4. The isolated nucleic acid of claim 1, wherein the amino acid sequence of said mutated HPPD protein is a variant of the amino acid sequence of SEQ ID No.
6 that comprises Asp or Asn at position 395 of the amino acid sequence of SEQ ID No. 6 (corresponding to position 421 of SEQ ID No. 2).

5. The isolated nucleic acid of claim 1, wherein the amino acid sequence of said mutated HPPD protein is a variant of the amino acid sequence of SEQ ID No.
8 that comprises Asp or Asn at position 412 of the amino acid sequence of SEQ ID No. 8 (corresponding to position 421 of SEQ ID No. 2).

6. The isolated nucleic acid of claim 1, wherein the amino acid sequence of said mutated HPPD protein is a variant of the amino acid sequence of SEQ ID No.
that comprises Asp or Asn at position 335 of the amino acid sequence of SEQ ID No. 10 (corresponding to position 421 of SEQ ID No. 2).

7. The isolated nucleic acid of claim 1, wherein the amino acid sequence of said mutated HPPD protein is a variant of the amino acid sequence of SEQ ID No.
14 that comprises Asp or Asn at position 327 of the amino acid sequence of SEQ ID No. 14 (corresponding to position 421 of SEQ ID No. 2).

8. The isolated nucleic acid of claim 1, wherein the amino acid sequence of said mutated HPPD protein is a variant of the amino acid sequence of SEQ ID No.
16 that comprises Asp or Asn at position 361 of the amino acid sequence of SEQ ID No. 16 (corresponding to position 421 of SEQ ID No. 2).

9. The isolated nucleic acid of claim 1, wherein the amino acid sequence of said mutated HPPD protein is a variant of the amino acid sequence of SEQ ID No.
18 that comprises Asp or Asn at position 381 of the amino acid sequence of SEQ ID No. 18 (corresponding to position 421 of SEQ ID No. 2).

10. The isolated nucleic acid of claim 1, wherein the amino acid sequence of said mutated HPPD protein is a variant of the amino acid sequence of SEQ ID No.
30 that comprises Asp or Asn at position 382 of the amino acid sequence of SEQ ID No. 30 (corresponding to position 421 of SEQ ID No. 2).

11. The isolated nucleic acid of claim 1, wherein the amino acid sequence of said mutated HPPD protein is a variant of the amino acid sequence of SEQ ID No.
20 that comprises Asp at position 348 of the amino acid sequence of SEQ ID
No. 20 (corresponding to position 421 of SEQ ID No. 2).

12. The isolated nucleic acid of claim 1, wherein said the amino acid sequence of mutated HPPD protein is a variant of the amino acid sequence of SEQ ID No.
22 that comprises Asp or Asn at position 367 of the amino acid sequence of SEQ ID No. 22 (corresponding to position 421 of SEQ ID No. 2).

13. The isolated nucleic acid of claim 1, wherein said the amino acid sequence of mutated HPPD protein is a variant of the amino acid sequence of SEQ ID No.
24 that comprises Asp or Asn at position 412 of the amino acid sequence of SEQ ID No. 24 (corresponding to position 421 of SEQ ID No. 2).

14. The isolated nucleic acid of claim 1, wherein said the amino acid sequence of mutated HPPD protein is a variant of the amino acid sequence of SEQ ID No.
26 that comprises Asp or Asn at position 408 of the amino acid sequence of SEQ ID No. 26 (corresponding to position 421 of SEQ ID No. 2).

15. The nucleic acid of any one of claims 1 to 14, wherein said mutated HPPD
protein increases the tolerance of a plant to at least one herbicide acting on HPPD.

16. A protein encoded by the isolated nucleic acid of any one of claims 1 to 15.

17. A gene or a nucleic acid comprising a coding sequence comprising the nucleic acid of any one of claims 1 to 15 operably linked to a plant-expressible promoter and optionally a transcription termination and polyadenylation region.

18. A method of obtaining a mutated HPPD protein modulating the tolerance of a plant to at least one herbicide acting on HPPD, wherein said mutated HPPD
protein has HPPD activity, the method comprising i) providing an HPPD protein;
ii) replacing the amino acid at position 421 of SEQ ID NO. 2 in said HPPD
protein so that the resulting amino acid sequence comprises Asp, Phe, or Asn at the position corresponding to position 421 of the amino acid sequence of SEQ ID No. 2;
iii) determining the inhibition of the resulting HPPD protein by at least one herbicide acting on HPPD, wherein an inhibition of the resulting protein of less or more than that observed with a reference HPPD protein is indicative that the resulting protein modulates the tolerance of a plant to said herbicide.

19. The method of claim 18, wherein said mutated HPPD protein increases the tolerance of a plant to at least one HPPD inhibitor herbicide.

20. The nucleic acid of claim 15 or the method of claim 19, wherein the herbicide acting on HPPD is tembotrione.

21. A method of producing a transgenic plant comprising introducing into said plant the nucleic acid of any one of claims 1 to 15 and 20, operably linked to a plant expressible promoter or the chimeric gene of claim 17.

22. The method of claim 21, wherein the nucleic acid of claim 16 or a nucleic acid identified by the method of claim 18 or 19, both operably linked to a plant expressible promoter, or the chimeric gene of claim 17 comprising the nucleic acid of claim 15 is introduced into said plant.

23. A plant cell comprising the isolated nucleic acid of any one of claims 1 to 15 or the chimeric gene of claim 17 in its genetic information.

24. A cell of a plant, a cell of a part thereof or a cell of plant tissue comprising the isolated nucleic acid of any one of claims 1 to 15 or the chimeric gene of claim 17 in its genetic information.

25. A cell of a plant comprising the isolated nucleic acid of any one of claims 1 to 15 or the chimeric gene of claim 17 in its genetic information obtained from the method of claim 21 or 22.

26. The cell of claim 24 or 25 which is a cell comprising the isolated nucleic acid of any one of claims 1 to 15 or the chimeric gene of claim 17 in its genetic information of a plant selected from wheat, cotton, canola, rice, corn, soybean, and sorghum.

27. A cell of a seed of a plant comprising the isolated nucleic acid of any one of claims 1 to 15 or the chimeric gene of claim 17 in its genetic information as defined in any one of claims 24 to 26.

28. A cell of progeny of a plant cell comprising the isolated nucleic acid of any one of claims 1 to 15 or the chimeric gene of claim 17 in its genetic information as defined in any one of claims 23 to 26 or a cell of progeny of a seed cell as defined in claim 27.

29. A method of modulating a plant's tolerance to at least one herbicide acting on HPPD or of obtaining a plant tolerant to an HPPD inhibitor herbicide comprising introducing the isolated nucleic acid of any one of claims 1 to 15 operably linked to a plant expressible promoter or the chimeric gene of claim 17 into a plant's genome.

30. A method for controlling weeds comprising spraying at least one HPPD
inhibitor herbicide on or around a crop plant, wherein said crop plant contains the nucleic acid according to claim 15 operably linked to a plant expressible promoter or the chimeric gene of claim 17 comprising the nucleic acid of claim 15.

31. Use of a chimeric gene of claim 17 or the nucleic acid of any one of claims 1 to 15 operably linked to a plant expressible promoter for modulating the tolerance of a plant to at least one HPPD inhibitor herbicide applied for controlling weeds.

32. The method of any one of claims 22, 29 or 30 or the use of claim 31, wherein the tolerance of a plant to at least one HPPD inhibitor herbicide is increased.