U.S. patent application number 11/623624 was filed with the patent office on 2007-06-07 for use of ketol-acid reductoisomerase inhibitors to prevent or treat fungal infection of plants.
Invention is credited to Renaud Dumas, Geraldine Effantin, Marc-Henri Lebrun, Valerie Morin, Jean-Luc Zundel.
Application Number | 20070129337 11/623624 |
Document ID | / |
Family ID | 8867134 |
Filed Date | 2007-06-07 |
United States Patent
Application |
20070129337 |
Kind Code |
A1 |
Dumas; Renaud ; et
al. |
June 7, 2007 |
Use Of Ketol-Acid Reductoisomerase Inhibitors To Prevent Or Treat
Fungal Infection Of Plants
Abstract
The invention concerns the use of ketol-acid reductoisomerase
inhibitors for treating fungal diseases affecting crops. The
invention concerns methods for treating crops against fungal
diseases comprising applying a ketol-acid reductoisomerase
inhibitor. The invention also concerns methods for identifying
novel fungicidal compounds comprising a step which consists in
identifying ketol-acid reductoisomerase inhibitors.
Inventors: |
Dumas; Renaud;
(Bourgoin-Jallieu, FR) ; Lebrun; Marc-Henri;
(Lyon, FR) ; Zundel; Jean-Luc; (Lyon, FR) ;
Effantin; Geraldine; (Saint Colombe, FR) ; Morin;
Valerie; (Lyon, FR) |
Correspondence
Address: |
BAKER & BOTTS L.L.P.
30 ROCKEFELLER PLAZA
44TH FLOOR
NEW YORK
NY
10112-4498
US
|
Family ID: |
8867134 |
Appl. No.: |
11/623624 |
Filed: |
January 16, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10797248 |
Mar 10, 2004 |
7166706 |
|
|
11623624 |
Jan 16, 2007 |
|
|
|
PCT/FR02/03073 |
Sep 10, 2002 |
|
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10797248 |
Mar 10, 2004 |
|
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Current U.S.
Class: |
514/124 ;
514/640 |
Current CPC
Class: |
C12Q 1/32 20130101; G01N
2500/04 20130101; A01N 37/28 20130101; A01N 61/00 20130101; A01N
57/20 20130101 |
Class at
Publication: |
514/124 ;
514/640 |
International
Class: |
A61K 31/66 20060101
A61K031/66; A61K 31/15 20060101 A61K031/15; A01N 57/00 20060101
A01N057/00; A01N 33/24 20060101 A01N033/24 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 10, 2001 |
FR |
01/11,689 |
Claims
1. A method for treating crops against fungal diseases, comprising
applying a fungicidal composition comprising an effective amount of
a ketol-acid reductoisomerase inhibitor, wherein the ketol-acid
reductoisomerase is selected from the group consisting of: SEQ ID
NO:1, SEQ ID NO:2 and SEQ ID NO:3.
2. The method of claim 1, wherein the ketol-acid reductoisomerase
inhibitor is dimethylphosphinoyl-2-hydroxyacetate.
3. The method of claim 1, wherein the ketol-acid reductoisomerase
inhibitor is N-hydroxy-N-isopropyloxamate.
Description
[0001] This application is a divisional of U.S. application Ser.
No. 10/797,248, now U.S. Pat. No. 7,166,706, filed on Mar. 10,
2004, which is a continuation of International Patent application
No. PCT/FR02/03073 filed Sep. 10, 2002 and published in French as
WO 03/022056 on Mar. 20, 2003, which claims priority to French
Patent Application No. FR 01/11,689 filed Sep. 10, 2001.
FIELD OF THE INVENTION
[0002] The present invention relates to the use of ketol-acid
reductoisomerase inhibitors for treating fungal diseases affecting
crops.
BACKGROUND OF THE INVENTION
[0003] Fungi are responsible for devastating epidemics which can
lead to considerable losses of crops of various plant species. The
principle of employing inhibitors of enzymes from pathogenic fungi,
and of using these enzymes in tests for identifying novel molecules
which are active against these fungi is known per se. However,
simple characterization of a fungal enzyme is not sufficient to
achieve this aim, the enzyme chosen as a target for potential
fungicidal molecules also has to be essential to the life of the
fungus, its inhibition by the fungicidal molecule resulting in
death of the fungus, or essential to the pathogenesis of the
fungus, its inhibition not being lethal for the fungus, but simply
inhibiting its pathogenic potency. The identification of metabolic
pathways and of enzymes essential to the pathogenesis and to the
survival of the fungus is therefore necessary for the development
of novel fungicidal products.
[0004] Ketol-acid reductoisomerase is an enzyme which has been well
characterized in plants and microorganisms such as bacteria and
yeast. This enzyme is the second enzyme of the biosynthetic pathway
for branched-chain amino acids; it catalyzes conversion of the
substrate 2S-2-acetolactate (AL) or 2S-2-aceto-2-hydroxybutyrate
(AHB) to 2,3-dihydroxy-3-isovalerate (DHIV) or to
2,3-dihydroxy-3-methylvalerate (DHIM), respectively. This reaction
requires the presence of magnesium ions (Mg.sup.2+) and occurs in
two steps: isomerization of a methyl or ethyl group, followed by
reduction by NADPH. A great deal of knowledge has been acquired
regarding plant reductoisomerase as a target for herbicides
(Wittenbach et al., Plant Physiol. 96, No. 1, Suppl., 94, 1991;
Schulz et al., FEBS Lett., 238:375-378, 1988) and ketol-acid
reductoisomerase inhibitors have been described as herbicides
(EP106114; U.S. Pat. No. 4,594,098, EP196026, EP481407, WO
94/23063, CA2002021). However, these compounds have not shown
effective herbicidal activity on plants.
[0005] A subject of the present invention is methods for treating
crops against fungal diseases, comprising applying a ketol-acid
reductoisomerase inhibitor. It has been found that inactivation of
the ILV5 gene encoding ketol-acid reductoisomerase in Magnaporthe
grisea results in inhibition of fungal growth. This inhibition of
fungal growth is also observed in vivo in the presence of
inhibitors specific for ketol-acid reductoisomerase. M. grisea is
pathogenic for many species of crop plants such as rice.
SUMMARY OF THE INVENTION
[0006] The invention relates to the use of ketol-acid
reductoisomerase inhibitors for treating fungal diseases affecting
crops. The invention provides antifungal compositions comprising a
ketol-acid reductoisomerase inhibitor. The invention also provides
methods for treating crops against fungal diseases comprising
applying a ketol-acid reductoisomerase inhibitor, e.g. to seeds,
roots, and/or shoots. The invention further provides methods for
identifying novel fungicidal compounds comprising identifying
ketol-acid reductoisomerase inhibitors.
DESCRIPTION OF THE FIGURES
[0007] FIG. 1: Comparison of the protein sequences of the
reductoisomerases of M. grisea and N. crassa and of the yeast S.
cerevisiae. Sequence alignment using the CLUSTAL W(1.4) software.
Symbols: ":" marks similar amino acids; "*" marks identical amino
acids.
[0008] FIG. 2: Growth assay for M. grisea in the presence of the
inhibitor N-hydroxy-N-isopropyloxamate (IpOHA) and under various
culture conditions.
[0009] The effect of the inhibitor IpOHA is tested on the
pathogenic fungus M. grisea by following the changing growth of
this fungus in the presence of various concentrations of inhibitor,
in various culture media and over a period of 7 days.
[0010] 200 .mu.l of culture medium, minimum medium (MM) or minimum
medium+leucine, valine and isoleucine at 0.3 mM (MM+ILV), is
inoculated with a suspension of spores of the M. grisea strain P1.2
at a final concentration of 10.sup.5 spores/ml. The microplate is
incubated at ambient temperature and the optical density at 630 nm
(OD.sub.630) is measured on days 0, 3, 4, 5, 6 and 7 (D0, D3, D4,
D5, D6 and D7). The optical density provides a measurement of the
growth of the mycelium.
[0011] FIG. 3: Influence of the concentration of NADPH on the
enzyme activity of the yeast reductoisomerase. The enzyme activity
measurements are carried out at 25.degree. C. in 1 ml of the
following reaction medium: 10 mM MgCl.sub.2, from 0 to 175 .mu.M
NADPH, 0.48 mM AHB, and in 50 mM sodium Hepes buffer, pH 7.5. The
curve is Michaelis Menten model-adjusted. The K.sub.M for NADPH is
1.6 .mu.M.
[0012] FIGS. 4A and 4B: Influence of the concentration of substrate
AHB [A] and AL [B] on the enzyme activity of the yeast
reductoisomerase. The enzyme activity measurements are carried out
at 25.degree. C. in the following reaction medium: 10 mM
MgCl.sub.2, 250 .mu.M NADPH and in 1 ml of 50 mM sodium Hepes
buffer, pH 7.5 and in the presence of AHB [A] and of AL [B]. The
curves are Michaelis Menten model-adjusted. A The K.sub.M for AHB
is 104 .mu.M. B The K.sub.M for AL is 266 .mu.M.
[0013] FIG. 5: Influence of the concentration of magnesium on the
enzyme activity of the yeast reductoisomerase. The enzyme activity
measurements are carried out at 25.degree. C. in 1 ml of the
following reaction medium: from 0 to 40 mM of MgCl.sub.2, 250 .mu.M
NADPH, 0.48 mM AHB, and 50 mM sodium Hepes buffer, pH 7.5. The
curve is Michaelis Menten model-adjusted. The K.sub.M for the
Mg.sup.2+ is 968 .mu.M.
[0014] FIGS. 6A and 6B: Stoichiometry for binding of the inhibitors
dimethylphosphinoyl-2-hydroxyacetate and
N-hydroxy-N-isopropyloxamate to the yeast reductoisomerase. The
inhibitors Hoe 704 [A] and IpOHA [B] (0.1 nmol) are incubated with
varying amounts of enzyme (0 to 0.4 nmol) for 20 minutes at
25.degree. C. with 25 nmol of NADPH and 0.25 .mu.mol of Mg.sup.2+
in a volume of 10 .mu.l. The enzyme activity measurements are
carried out at 25.degree. C. in 1 ml of the following reaction
medium: 10 mM MgCl.sub.2, 250 .mu.M NADPH, 0.48 mM AHB, and in 50
mM sodium Hepes buffer, pH 7.5.
[0015] FIGS. 7A and 7B: Kinetics of inhibition of the yeast
reductoisomerase in the presence of the inhibitors
dimethylphosphinoyl-2-hydroxyacetate [A] and
N-hydroxy-N-isopropyloxamate [B]. The enzyme activity measurements
are carried out at 25.degree. C. in 1 ml of the following reaction
medium: 10 mM MgCl.sub.2, 250 .mu.M NADPH, 0.48 mM AHB in 50 mM
sodium Hepes buffer, pH 7.5, and in the presence of enzyme 110
nM.
[0016] The reactions are initiated by simultaneously adding varying
amounts of inhibitors dimethylphosphinoyl-2-hydroxyacetate [A] and
N-hydroxy-N-isopropyloxamate [B] (from 2 .mu.M to 30 .mu.M) to the
reaction medium. The curves are plotted according to the equation
(1), which makes it possible to determine the values for K.sub.obs,
the apparent rate of formation of the enzyme-inhibitor complex.
[0017] FIGS. 8A and 8B: Kinetics of inhibition of the yeast
reductoisomerase in the presence of the inhibitors
dimethylphosphinoyl-2-hydroxyacetate [A] and
N-hydroxy-N-isopropyloxamate [B]. The enzyme activity measurements
are carried out at 25.degree. C. in 1 ml of the following reaction
medium: 10 mM MgCl.sub.2, 250 .mu.M NADPH, 0.48 mM AHB in 50 mM
sodium Hepes buffer, pH 7.5, and in the presence of enzyme at 110
nM.
[0018] The reactions are initiated by simultaneously adding varying
amounts of inhibitors [A] and [B] (from 2 .mu.M to 50 .mu.M) to the
reaction medium.
[0019] FIGS. 9A and 9B: Determination of the association constant
k.sub.0 for the dimethylphosphinoyl-2-hydroxyacetate [A] and for
the N-hydroxy-N-isopropyloxamate [B] on the yeast reductoisomerase
using the representation 1/Kobs as a function of the concentration
of substrate AHB. The enzyme activity measurements are carried out
at 25.degree. C. in 1 ml of the following reaction medium: 10 mM
MgCl.sub.2, 250 .mu.M NADPH, from 125 .mu.M to 2.375 mM AHB, and in
50 mM sodium Hepes buffer, pH 7.5, and in the presence of enzyme at
110 nM. The reactions are initiated by simultaneously adding
varying amounts of substrate AHB (from 125 .mu.M to 2.375 mM) and
the inhibitors [A] (10 .mu.M) and [B] (15 .mu.M) to the reaction
medium.
[0020] The curves are plotted according to the equation (3), which
makes it possible to determine the values for k.sub.0, the rate of
association of the inhibitor with the enzyme.
DESCRIPTION OF THE SEQUENCE LISTING
[0021] SEQ ID NO:1: Magnaporthe grisea ketol-acid
reductoisomerase.
[0022] SEQ ID NO:2: Saccharomyces cerevisiae ketol-acid
reductoisomerase.
[0023] SEQ ID NO:3: Neurospora crassa ketol-acid
reductoisomerase.
[0024] SEQ ID NO:4: Magnaporthe grisea ketol-acid reductoisomerase
gene cDNA.
[0025] SEQ ID NO:5: Magnaporthe grisea ketol-acid
reductoisomerase.
[0026] SEQ ID NO:6: Magnaporthe grisea ketol-acid reductoisomerase
gene.
[0027] SEQ ID NOS:7-18: Primers for PCR.
DESCRIPTION OF THE INVENTION
[0028] A subject of the present invention is methods for treating
crops against fungal diseases by applying an effective amount of a
ketol-acid reductoisomerase inhibitor.
[0029] A subject of the invention is a method for combating, in a
curative or preventive capacity, phytopathogenic fungi affecting
crops, characterized in that an effective (agronomically effective)
and nonphytotoxic amount of a ketol-acid reductoisomerase inhibitor
is applied to the soil where the plants are growing or where they
are likely to grow, to the leaves and/or the fruits of the plants
or to the seeds of the plants. The expression "effective and
nonphytotoxic amount" is intended to mean an amount of inhibitor
which is sufficient to control or destroy the fungi present or
likely to appear on the crops, and which does not result in any
appreciable symptom of phytotoxicity for said crops. Such an amount
can vary within a wide range depending on the fungus to be
combated, the type of crop, the climatic conditions and the
compounds included in the fungicidal composition according to the
invention. This amount can be determined by systematic field
trials, which are within the scope of those skilled in the art.
[0030] The methods according to the invention are useful for
treating the seeds of cereals (wheat, rye, triticale and barley in
particular), of potato, of cotton, of pea, of rapeseed, of maize or
of flax, or else the seeds of forest trees, or else genetically
modified seeds of these plants. The present invention also relates
to foliar application to the plant crops, i.e. to the foliage, the
flowers, the fruits and/or the trunks of the plants concerned.
Among the plants targeted by the methods according to the
invention, mention may be made of rice, maize, cotton, cereals,
such as wheat, barley or triticale, fruit trees, in particular
apple trees, pear trees, peach trees, grapevine, banana trees,
orange trees, lemon trees, etc., oil-producing crops, for example
rapeseed or sunflower, market-garden and vegetable crops, tomatoes,
salads, protein-producing crops, pea, Solanaceae, for example
potato, beetroot, flax, and forest trees, and also genetically
modified homologues of these crops.
[0031] Among the plants targeted by the method according to the
invention, mention may be made of: [0032] wheat, as regards
combating the following seed diseases: fusaria (Microdochium nivale
and Fusarium roseum), stinking smut (Tilletia caries, Tilletia
controversa or Tilletia indica), septoria disease (Septoria
nodorum); loose smut (Ustilago tritici); [0033] wheat, as regards
combating the following diseases of the parts of the plant above
ground: cereal eyespot (Tapesia yallundae, Tapesia acuiformis),
take-all (Gaeumannomyces graminis), foot blight (F. culmorum, F.
graminearum), head blight (F. culmorum, F. graminearum,
Microdochium nivale), black speck (Rhizoctonia cerealis), powdery
mildew (Erysiphe graminisforma specie tritici), rusts (Puccinia
striiformis and Puccinia recondita) and septoria diseases (Septoria
tritici and Septoria nodorum), net blotch (Drechslera
tritici-repentis); [0034] barley, as regards combating the
following seed diseases: net blotch diseases (Pyrenophora graminea,
Pyrenophora teres and Cochliobolus sativus), loose smut (Ustilago
nuda) and fusaria (Microdochium nivale and Fusarium roseum); [0035]
barley, as regards combating the following diseases of the parts of
the plant above ground: cereal eyespot (Tapesia yallundae), net
blotch diseases (Pyrenophora teres and Cochliobolus sativus),
powdery mildew (Erysiphe graminisforma specie hordei), dwarfleaf
rust (Puccinia hordei) and leaf blotch (Rhynchosporium secalis);
[0036] potato, as regards combating tuber diseases (in particular
Helminthosporium solani, Phoma tuberosa, Rhizoctonia solani,
Fusarium solani), and mildew (Phytopthora infestans); [0037]
potato, as regards combating the following foliage diseases: early
blight (Alternaria solani), mildew (Phytophthora infestans); [0038]
cotton, as regards combating the following diseases of young plants
grown from seeds: damping-off and collar rot (Rhizoctonia solani,
Fusarium oxysporum), black root rot (Thielaviopsis basicola);
[0039] protein-producing crops, for example pea, as regards
combating the following seed diseases: anthracnose (Ascochyta pisi,
Mycosphaerella pinodes), fusaria (Fusarium oxysporum), gray mold
(Botrytis cinerea), mildew (Peronospora pisi); [0040] oil-producing
crops, for example rapeseed, as regards combating the following
seed diseases: Phoma lingam, Alternaria brassicae and Sclerotinia
sclerotiorum; [0041] maize, as regards combating seed diseases:
(Rhizopus sp., Penicillium sp., Trichoderma sp., Aspergillus sp.
and Gibberella fujikuroi); [0042] flax, as regards combating seed
diseases: Alternaria linicola; [0043] forest trees, as regards
combating damping-off (Fusarium oxysporum, Rhizoctonia solani);
[0044] rice, as regards combating the following diseases of the
parts above ground: blast disease (Magnaporthe grisea), black speck
(Rhizoctonia solani); [0045] vegetable crops, as regards combating
the following diseases of seedlings or of young plants grown from
seeds: damping-off and collar rot (Fusarium oxysporum, Fusarium
roseum, Rhizoctonia solani, Pythium sp.); [0046] vegetable crops,
as regards combating the following diseases of the parts above
ground: gray mold (Botrytis sp.), powdery mildews (in particular
Erysiphe cichoracearum, Sphaerothecafuliginea, Leveillula taurica),
fusaria (Fusarium oxysporum, Fusarium roseum), leaf spot
(Cladosporium sp.), alternaria leaf spot (Alternaria sp.),
anthracnose (Colletotrichum sp.), septoria leaf spot (Septoria
sp.), black speck (Rhizoctonia solani), mildews (for example,
Bremia lactucae, Peronospora sp., Pseudoperonospora sp,
Phytophthora sp); [0047] fruit trees, as regard diseases of the
parts above ground: monilia disease (Monilia fructigenae, M. laxa),
scab (Venturia inaequalis), powdery mildew (Podosphaera
leucotricha); [0048] grapevine, as regards foliage diseases: in
particular gray mold (Botrytis cinerea), powdery mildew (Uncinula
necator), black rot (Guignardia biwelli), mildew (Plasmopara
viticola); [0049] beetroot, as regards the following diseases of
the parts above ground: cercosporia blight (Cercospora beticola),
powdery mildew (Erysiphe beticola), leaf spot (Ramularia
beticola).
[0050] Ketol-acid reductoisomerase is a well-characterized enzyme
which is found in plants and microorganisms (bacteria, yeast,
fungi). The methods of the present invention use ketol-acid
reductoisomerase inhibitors. In a present embodiment, the invention
relates to the use of inhibitors of fungal ketol-acid
reductoisomerase, more preferably of inhibitors of the ketol-acid
reductoisomerase of a phytopathogenic fungus, for treating fungal
diseases affecting crops. In a particular embodiment of the
invention, the ketol-acid reductoisomerase inhibitors inhibit the
ketol-acid reductoisomerase of Magnaporthe grisea and/or of
Saccharomyces cerevisiae and/or of Neurospora crassa. In another
particular embodiment, the ketol-acid reductoisomerase inhibitor is
an inhibitor of the enzyme activity of the ketol-acid
reductoisomerase of SEQ ID NO:1, of SEQ ID NO:2, of SEQ ID NO:3
and/or of SEQ ID NO:5.
[0051] Any ketol-acid reductoisomerase inhibitor can be used in the
methods according to the invention. Ketol-acid reductoisomerase
inhibitors are well known to those skilled in the art, and these
inhibitors have in particular been described in EP106114; U.S. Pat.
No. 4,594,098, EP196026, EP481407, WO 94/23063, CA2002021 and WO
97/37660.
[0052] In a particular embodiment of the invention, the ketol-acid
reductoisomerase inhibitor is a reaction intermediate analog which
binds to the active site of the ketol-acid reductoisomerase.
[0053] Preferably, the ketol-acid reductoisomerase inhibitor is
dimethylphosphinoyl-2-hydroxyacetate.
[0054] More preferably, the ketol-acid reductoisomerase inhibitor
is N-hydroxy-N-isopropyloxamate.
[0055] In a preferred embodiment of the invention, the ketol-acid
reductoisomerase inhibitor is in the form of a fungicidal
composition. The invention also relates to fungicidal compositions
comprising an effective amount of at least one ketol-acid
reductoisomerase inhibitor. The fungicidal compositions according
to the invention comprise, besides the inhibitor, agriculturally
acceptable solid or liquid carriers and/or surfactants which are
also agriculturally acceptable. The usual inert carriers and the
usual surfactants can in particular be used. These fungicidal
compositions according to the invention can also contain any type
of other ingredients, such as, for example, protective colloids,
adhesives, thickeners, thixotropic agents, penetrating agents,
stabilizers, sequestering agents, etc. More generally, the
ketol-acid reductoisomerase inhibitors can be combined with all the
solid or liquid additives corresponding to the conventional
techniques of formulation.
[0056] A subject of the present invention is also fungicidal
compositions comprising a ketol-acid reductoisomerase inhibitor and
another fungicidal compound. Mixtures with other fungicides are
particularly advantageous, in particular mixtures with
acibenzolar-S-methyl, azoxystrobin, benalaxyl, benomyl,
blasticidin-S, bromuconazole, captafol, captan, carbendazim,
carboxin, carpropamide, chlorothalonil, fungicidal compositions
based on copper, or on copper derivatives such as copper hydroxide
or copper oxychloride, cyazofamide, cymoxanil, cyproconazole,
cyprodinil, dichloran, diclocymet, dichloran, diethofencarb,
difenoconazole, diflumetorim, dimethomorph, diniconazole,
discostrobin, dodemorph, dodine, edifenphos, epoxyconazole,
ethaboxam, ethirimol, famoxadone, fenamidone, fenarimol,
fenbuconazole, fenhexamid, fenpiclonil, fenpropidine,
fenpropimorph, ferimzone, fluazinam, fludioxonil, flumetover,
fluquinconazole, flusilazole, flusulfamide, flutolanil, flutriafol,
folpet, furalaxyl, furametpyr, guazatine, hexaconazole, hymexazol,
imazalil, iprobenphos, iprodione, isoprothiolane, kasugamycin,
kresoxim-methyl, mancozeb, maneb, mefenoxam, mepanipyrim, metalaxyl
and its entiomeric forms such as metalaxyl-M, metconazole,
metiram-zinc, metominostrobin, oxadixyl, pefurazoate, penconazole,
pencycuron, phosphoric acid and its derivatives such as fosetyl-AI,
phthalide, picoxystrobin, probenazole, prochloraz, procymidone,
propamocarb, propiconazole, pyraclostrobin, pyrimethanil,
pyroquilon, quinoxyfen, silthiofam, simeconazole, spiroxamine,
tebuconazole, tetraconazole, thiabendazole, thifluzamide,
thiophanate, e.g. thiophanate-methyl, thiram, tridimefon,
triadimenol, tricyclazole, tridemorph, trifloxystrobin,
triticonazole, derivatives of valinamide such as, for example,
iprovalicarb, vinclozolin, zineb and zoxamide. The mixtures thus
obtained have a wider spectrum of activity. The compositions
according to the invention may also comprise one or more
insecticides, bactericides or acaricides or pheromones or other
compounds having a biological activity.
[0057] The subject of the present invention is also methods for
producing a fungicidal composition using a ketol-acid
reductoisomerase inhibitor.
[0058] The subject of the present invention is also methods for
preparing fungicidal compounds, comprising identifying compounds
which inhibit the enzyme activity of ketol-acid
reductoisomerase.
[0059] The enzyme reaction is carried out in the presence of the
test compound in order to measure the inhibition of the enzyme
activity of the ketol-acid reductoisomerase. All biochemical assays
for measuring the enzyme activity of ketol-acid reductoisomerase
and therefore for identifying compounds which inhibit this enzyme
activity can be used in the methods according to the invention. The
biochemical assays are well known to those skilled in the art
(Dumas et al., Biochem. J. 288:865-874, 1992; Dumas et al.,
Biochem. J. 301:813-820, 1994; Dumas et al., Febs Letters
408:156-160, 1997, Halgand et al., Biochemistry 37:4773-4781, 1998,
Wessel et al., Biochemistry 37:12753-12760, 1998; Halgand et al.,
Biochemistry 38:6025-6034, 1999).
[0060] The enzyme reactions are advantageously carried out in
solution in a suitable buffer. The use of this type of reaction
medium makes it possible to perform a large number of reactions in
parallel and therefore to test a large number of compounds in a
microplate format, for example.
[0061] Preferably, the methods for identifying compounds which
inhibit the enzyme activity of ketol-acid reductoisomerase comprise
bringing these compounds into contact with the ketol-acid
reductoisomerase in the presence of magnesium, of NADPH in the
substrate, and measuring this enzyme activity.
[0062] Advantageously, in the methods according to the invention,
measurement of the enzyme activity comprises measuring the decrease
in absorption of NADPH at 340 nm, and the substrate used for the
enzyme reaction is 2-acetolactate (AL) or 2-aceto-2-hydroxybutyrate
(AHB). It is understood that any other method for measuring enzyme
activity known to those skilled in the art may be used in the
methods according to the invention.
[0063] Any ketol-acid reductoisomerase can be used in the methods
according to the invention. Ketol-acid reductoisomerases have been
characterized in several organisms, such as plants bacteria, yeast
and fungi. The corresponding genes have been cloned, making it
possible to determine the protein sequence of this enzyme (Dumas et
al., Biochem. J. 277:69-475, 1991; Curien et al., Plant Mol. Biol.
21:717-722, 1993; Dumas et al., Biochem. J. 294:821-828, 1993; Biou
et al., EMBO J. 16:3405-3415, 1997; Dumas et al., Biochemistry
34:6026-6036, 1995; Dumas et al., Accounts of Chemical Research
34:399-408, 2001; Sista et al., Gene, 120:115-118, 1992;
Zelenaya-Troitskaya et al., EMBO J. 14:3268-3276, 1995).
[0064] In a preferred embodiment of the invention, the ketol-acid
reductoisomerase used in the methods according to the invention is
represented in SEQ ID NO:1, in SEQ ID NO:2, in SEQ ID NO:3 and/or
in SEQ ID NO:5.
[0065] Preferably, the ketol-acid reductoisomerase is isolated,
purified or partially purified from its natural environment. The
ketol-acid reductoisomerase can be prepared using various methods.
These methods are in particular purification from natural sources
such as cells naturally expressing these polypeptides, production
of recombinant polypeptides by appropriate host cells and
subsequent purification thereof, production by chemical synthesis
or, finally, a combination of these various approaches. These
various methods of production are well known to those skilled in
the art.
[0066] In a first embodiment of the invention, the ketol-acid
reductoisomerase is purified from an organism which naturally
produces this enzyme, such as, for example, bacteria such as E.
coli, yeasts such as S. cerevisiae, or fungi such as N. crassa or
M. grisea.
[0067] In a preferred embodiment of the invention, the ketol-acid
reductoisomerase is overexpressed in a recombinant host organism.
The methods for engineering DNA fragments and the expression of
polypeptides in host cells are well known to those skilled in the
art and have, for example, been described in "Current Protocols in
Molecular Biology" Volumes 1 and 2, F. M. Ausubel et al., published
by Greene Publishing Associates and Wiley-Interscience (1989) or in
Molecular Cloning, T. Maniatis, E. F. Fritsch, J. Sambrook
(1982).
[0068] Preferably, the methods for identifying compounds which
inhibit the enzyme activity of ketol-acid reductoisomerase comprise
expressing the ketol-acid reductoisomerase in the host organism,
purifying the ketol-acid reductoisomerase produced by the host
organism, bringing these compounds into contact with the purified
ketol-acid reductoisomerase in the presence of magnesium, of NADPH
and of substrate, and measuring the enzyme activity.
[0069] In a preferred embodiment, all these methods comprise an
additional step in which it is determined whether said compounds
which inhibit the enzyme activity of the ketol-acid
reductoisomerase inhibit fungal growth and/or pathogenesis.
[0070] The present invention therefore relates to methods for
identifying compounds which inhibit fungal growth and/or
pathogenesis by inhibiting the enzyme activity of ketol-acid
reductoisomerase. These methods consist in subjecting a compound,
or a mixture of compounds, to an appropriate assay for identifying
the ketol-acid reductoisomerase-inhibiting compounds and in
selecting the compounds which react positively to said assay, where
appropriate in isolating them, and then in identifying them.
[0071] Preferably, the appropriate assay is an assay for the enzyme
activity of the ketol-acid reductoisomerase as defined above.
[0072] Preferably, a compound identified according to these methods
is then tested for its antifungal properties and for its ability to
inhibit the pathogenesis and/or the growth of the fungus for
plants, according to methods known to those skilled in the art.
Preferably, the compound is evaluated using phenotypic tests such
as pathogenesis assays on leaves or on whole plants.
[0073] According to the invention, the term "compound" is intended
to mean any chemical compound or mixture of chemical compounds,
including peptides and proteins.
[0074] According to the invention, the term "mixture of compounds"
is understood to mean at least two different compounds, such as,
for example, the (dia)stereoisomers of a molecule, mixtures of
natural origin derived from the extraction of biological material
(plants, plant tissues, bacterial cultures, yeast cultures or
fungal cultures, insect, animal tissues, etc.) or unpurified or
totally or partially purified reaction mixtures, or else mixtures
of products derived from combinatorial chemistry techniques.
[0075] Finally, the present invention relates to novel fungal
pathogenesis-inhibiting compounds which inhibit the enzyme activity
of ketol-acid reductoisomerase, in particular the compounds
identified by the methods according to the invention and/or the
compounds derived from the compounds identified by the methods
according to the invention.
[0076] Preferably, the fungal pathogenesis-inhibiting compounds
which inhibit the enzyme activity of ketol-acid reductoisomerase
are not general enzyme inhibitors. Also preferably, the compounds
according to the invention are not compounds already known to have
fungicidal activity and/or activity on fungal pathogenesis.
[0077] A subject of the invention is also a method for treating
plants against a phytopathogenic fungus, characterized in that it
comprises treating said plants with a compound identified by a
method according to the invention.
[0078] The present invention also relates to a method for preparing
a fungal pathogenesis-inhibiting compound, said method comprising
the steps consisting in identifying a fungal
pathogenesis-inhibiting compound which inhibits the enzyme activity
of ketol-acid reductoisomerase by the method of identification
according to the invention, and then in preparing said identified
compound by the usual methods of chemical synthesis, of enzymatic
synthesis and/or of extraction of biological material. The step for
preparing the compound can be preceded, where appropriate, by an
"optimization" step by which a compound derived from the compound
identified by the method of identification according to the
invention is identified, said derived compound then being prepared
by the usual methods.
EXAMPLES
Example 1
Cloning of the Magnaporthe grisea ILV5 Gene
[0079] An internal fragment of the M. grisea ILV5 gene was
amplified by PCR from the genomic DNA of this fungus using pairs of
degenerate primers corresponding to protein domains which are
conserved between fungal reductoisomerases. The PCR product
obtained was then cloned into the plasmid pGEM-T-Easy (Promega),
sequenced, and amplified by PCR with a new pair of primers. The
latter PCR product was used as a homologous probe for screening an
M. grisea cosmid DNA library. The sequence of the M. grisea ILV5
gene was then produced using one of the positive clones and
oligonucleotides derived from the sequence of the PCR product
already obtained.
1.1. Isolation of an Internal Fragment of the M. grisea ILV5 Gene
by Amplification Using Degenerate Oligonucleotides
1.1.1 Choice of Degenerate Oligonucleotides
[0080] Amplification of an internal fragment of the N. grisea ILV5
gene was carried out by PCR using degenerate oligonucleotides.
These degenerate oligonucleotides were chosen based on comparison
of the protein sequences of the reductoisomerases of N. Crassa and
of S. cerevisiae. This comparison made it possible to demonstrate 4
domains which are conserved between these two fungal
reductoisomerase sequences, which should be present in the
reductoisomerase of M. grisea. These 4 conserved domains consist of
a succession of at least 7 conserved amino acids. The sequence of
the degenerate oligonucleotides was detennined from that of the
amino acids of the conserved domains translated according to the
genetic code. In order for the degree of degeneracy (number of
codons for a given amino acid) to be as low as possible, amino
acids such as arginine, leucine or serine are to be avoided since
six codons correspond thereto. The amino acids methionine and
tryptophan are, for their part, desired, since a single codon
corresponds thereto. The degree of degeneracy should also be low at
the 3' end of the oligonucleotide, in order to increase the
specificity of amplification. We were able to define four
oligonucleotides: oligonucleotides 1(+) and 3(+) on the (+) strand;
and oligonucleotides 2(-) and 4(-) on the (-) strand of the M.
grisea DNA. Thus, the PCR amplification can be carried out with
four different pairs of degenerate oligonucleotides: 1(+) and 2(-);
1(+) and 4(-); 3(+) and 2(-); 3(+) and 4(-).
1.1.2. Amplification of an Internal Fragment of the M. grisea ILV5
Gene Using Degenerate Oligonucleotides
[0081] The optimum conditions for amplification of the M. grisea
ILV5 gene were determined by varying the pair of primers and their
hybridization temperature. The first three amplification cycles
were carried out at a variable hybridization temperature
(42.degree. C., 50.degree. C. or 55.degree. C.), whereas the
hybridization temperature for the other cycles is 55.degree. C.
Positive controls were performed with the genomic DNA of N. crassa
and of S. cerevisiae under the same conditions as for the genomic
DNA of M. grisea. At a hybridization temperature of 42.degree. C.,
the S. cerevisiae DNA fragments were amplified at the expected
size, i.e. 590 bp, 610 bp, 440 bp and 470 bp, with the pairs of
primers (1-4), (1-2), (3-4) and (3-2), respectively. The
amplification profiles for the S. cerevisiae genomic DNA with the
pairs of primers (1-4) and (3-2) are, however, complex. For a
hybridization temperature of 50.degree. C., N. crassa DNA fragments
were amplified at the expected size, i.e. 660 bp, 685 bp, 523 bp
and 544 bp, with the pairs of primers (1-4), (1-2), (3-4) and (3-2)
respectively, although the amplification profiles with the pairs of
primers (1-4) and (3-4) are complex. The various pairs of
degenerate oligonucleotides made it possible to amplify a fragment
of expected size from the yeast genomic DNA and from the N. crassa
genomic DNA. These degenerate oligonucleotides could therefore be
used to amplify the M. grisea ILV5 gene. The amount of
oligonucleotides used, tested at a hybridization temperature of
42.degree. C., does not appear to have any influence on the
amplification of the M. grisea genomic DNA. At a hybridization
temperature in the first PCR cycles of 42.degree. C., the
amplification profiles for the M. grisea genomic DNA, obtained with
the various pairs of primers, are quite complex. At a hybridization
temperature of 50.degree. C., a simplification of the amplification
profiles for the M. grisea genomic DNA was observed for most of the
pairs of primers, in particular for the pair of primers (1-2) which
makes it possible to amplify a single DNA fragment at the expected
size (685 bp). A hybridization temperature in the first PCR cycles
of 55.degree. C. does not make it possible to increase the
specificity of the amplification with the pairs of primers (1-2)
and (3-2) and reduces its yield. The best conditions for
amplification from the M. grisea genomic DNA were therefore
obtained at a primer hybridization temperature in the first PCR
cycles of 50.degree. C., with the pair of oligonucleotides
(1-2).
1.1.3. Cloning of a PCR-Amplified Internal Fragment of the M.
grisea ILV5 Gene, in the Plasmid pGEM-T-Easy
[0082] The internal fragment of the M. grisea ILV5 gene was
amplified by PCR from the M. grisea genomic DNA, with the pair of
primers (1-2), at a primer hybridization temperature of 50.degree.
C. for 3 cycles, and then 55.degree. C. for the other amplification
cycles. This PCR product, of approximately 680 bp, is purified
after separation by agarose gel electrophoresis and then cloned
into the plasmid pGEM-T-easy. The bacterial colonies obtained after
transformation, and using a white/blue selection system (X-Gal),
showed three different phenotypes: white, blue, and white with the
center of the colony being blue (called white/blue colonies). 30
colonies of various phenotypes were analyzed by PCR using the
universal primers Sp6 and T7, which hybridize on either side of the
cloning site of the plasmid pGEM-T-easy. The 20 white colonies and
the 10 white/blue colonies are positive. In fact, from these
colonies, a DNA fragment was amplified at the expected size (810
bp), which corresponds to the size of the insert (680 bp) plus the
distance separating the insert from each of the primers Sp6 and T7
(129 bp). Two clones of different phenotypes, white and white/blue,
were then chosen in order to be sequenced. These are clones no. 4
(white) and no. 20 (white/blue).
1.1.4. Analysis of the Sequence of the Cloned Internal Fragment of
the M. grisea ILV5 Gene
[0083] Comparison of the nucleotide sequences of the two clones no.
4 and no. 20 showed that they correspond to the same DNA fragment
cloned in different orientations into the plasmid pGEM-T-easy,
which might explain their phenotypic difference (white and
white/blue). The double-stranded nucleotide sequence of this cloned
fragment was thus obtained. The homology of this nucleotide
sequence with those encoding known proteins was sought using the
Blastx program from NCBI (National Center for Biotechnology
Information). This program compares the translated sequences of the
six reading frames of a nucleotide sequence with all the protein
sequences contained in the databases. Significant homology between
the nucleotide sequence of the cloned fragment and the protein
sequence of the N. crassa reductoisomerase (error of e-102) was
identified. The percentage amino acid identity within the region
defined by the software for comparing these two sequences is 94%.
The fragment cloned into the plasmid pGEM-T-easy therefore
corresponds to the internal fragment of the M. grisea ILV5 gene.
Although the sequence of the internal fragment of the M. grisea
ILV5 gene and that of the N. crassa ILV5 gene exhibits strong
homology, a difference exists at the center of the sequence of the
M. grisea ILV5 gene. This difference might correspond to the
presence of an intron within the M. grisea sequence, since a 77 bp
intron exists in N. crassa at this position. The position in the
sequence of this intron was sought. A 5' splicing consensus motif
was identified in the nucleotide sequence of the internal fragment
of the M. grisea ILV5 gene, along with a 3' splicing consensus
motif and a lariat sequence. The putative intron (86 bp) of the
internal fragment of the M. grisea ILV5 gene was therefore
identified. Splicing of the intron of the sequence of the internal
fragment of the M. grisea ILV5 gene made it possible to obtain a
"theoretical" cDNA fragment. The fragment of the protein sequence
of the M. grisea reductoisomerase then deduced from this
"theoretical" cDNA was compared with that of the N. crassa
reductoisomerase, showing very strong identity between the primary
sequences of these two enzymes (FIG. 1).
1.2 Screening of an M. grisea Cosmid Library with a Probe for the
M. grisea ILV5 Gene
1.2.1. Construction of the Homologous Probe for the M. grisea ILV5
Gene
[0084] An internal fragment of the M. grisea ILV5 gene was
amplified by PCR, from clone no. 4 using the primers 13U and 549L
defined on the basis of the sequence of the ILV5 gene cloned into
the plasmid pGEM-T-easy. This fragment, after purification on
agarose gel, was used as a matrix to prepare a labeled probe for
the ILV5 gene.
1.2.2. Screening of the M. grisea Guy11 Cosmid Library by PCR Using
Primers Specific for M. grisea ILV5 Gene
[0085] The Guy11 cosmid library is represented in the form of 28
pools of 96 DNA mini preparations corresponding to the 96 different
cosmids present in a 96-well plate (2688 clones). The M. grisea
ILV5 gene was sought in this cosmid library by performing a PCR
amplification on these pools of DNA mini preparations using the
primers 300U and 549L defined on the basis of the known sequence of
the ILV5 gene. A fragment of expected size (249 bp) was amplified
from the pools no. 17; 19; 20; 21; 27; 28 and 29. The search for
the ILV5 gene was continued by hybridizing, with the probe for the
ILV5 gene, the cosmids from plates no. 17; 19; 20; 21; 27; 28 and
29.
1.2.3. Screening of the M. grisea Guy11 Cosmid Library by
Hybridization with the Homologous Probe for the M. grisea ILV5
Gene
[0086] The bacterial colonies derived from plates no. 17; 19; 20;
21; 27; 28 and 29 were replicated on nylon membranes and hybridized
with the probe for the M. grisea ILV5 gene. This hybridization made
it possible to select the cosmids G6 and A7 from plates no. 20 and
27, respectively, and the cosmids B5 and B6 from plate no. 29.
1.2.4. Characterization of the M. grisea ILV5 Gene Using the Cosmid
20/G6
(a) Sequencing of the M. grisea ILV5 Gene Using the Cosmid
20/G6
[0087] The sequencing of the ILV5 gene was carried out in steps.
The first sequencing reaction was carried out using divergent
primers chosen from the known sequence of the internal fragment of
the M. grisea ILV5 gene obtained by PCR. Based on this new ILV5
gene sequence, further primers were defined in order to carry out
other sequencing reactions, until the M. grisea ILV5 gene had been
entirely sequenced. The sequences translated from the entire
nucleotide sequence of the ILV5 gene according to the six reading
frames were compared with the protein sequence of the N. crassa
reductoisomerase; in order to locate the position of the
translation-initiating ATG, of the translation-terminating stop
codon, and of the various possible introns. Thus, the
translation-initiating ATG was identified on the nucleotide
sequence of the ILV5 gene and serves as reference (+1) from which
the other elements of the sequence are positioned. Three putative
introns were located in the nucleotide sequence of the M. grisea
ILV5 gene. The first intron is thought to be located between
positions (in bp) 199 and 280, the second intron at position
314-390 and the third intron is thought to be located between
positions 670 and 755 of the M. grisea ILV5 gene. The
translation-terminating stop codon is thought to be at position
1449 of the sequence of the M. grisea ILV5 gene. Isolation of the
cDNA of the M. grisea ILV5 gene was undertaken in order to verify
the position of the suspected introns by comparison of the protein
sequences of M. grisea and of N. crassa.
(b) Isolation of the cDNA of the M. grisea ILV5 Gene and Search for
the Introns of the M. grisea Gene
[0088] The cDNA of the M. grisea ILV5 gene was isolated by carrying
out a PCR amplification from a cDNA library of the isolate P1.2
(RNA of mycelium cultured in complete medium) using the
oligonucleotides defined on the basis of the ILV5 gene sequence:
oligonucleotides 22U and 1603L. Oligonucleotide 22U is located
before the translation-initiating ATG and oligonucleotide 1603L is
located 93 bp after the translation-terninating STOP codon. Two
fragments are amplified with this pair of primers: a fragment less
than 500 bp in size and a fragment amplified at the expected size,
i.e. 1.6 kb. The fragment amplified at the expected size is
purified after separation by agarose gel electrophoresis and cloned
into the plasmid pGEM-T-easy. The 24 bacterial clones obtained
after transformation are analyzed by PCR using the pair of primers
22U and 1603 L. These 24 clones possess the cDNA of the M. grisea
ILV5 gene, since a DNA fragment was in fact amplified at the
expected size (1.6 kb). Clone no. 18 was chosen in order to be
sequenced using the universal primers Sp6 and T7. Comparison of the
nucleotide sequence of the cDNA of the ILV5 gene allowed us to
determine the exact position of the introns. The three introns are
located at the positions predicted by the comparison of the protein
sequences of the N. crassa reductoisomerases and of the
translations of the M. grisea ILV5 gene. In N. crassa, the ILV5
gene has 4 introns which are positioned differently and are
different in length compared to the M. grisea ILV5 gene.
(c) Protein Sequence of the M. grisea Reductoisomerase, Deduced
from All the Data Acquired on the M. grisea ILV5 Gene
[0089] The protein sequence of the M. grisea ILV5 gene was deduced
from the cDNA sequence of this gene. Comparison of the protein
sequences of the reductoisomerases of M. grisea, of N. crassa and
of S. cerevisiae shows very strong identity between the
reductoisomerases of these three species. In fact, the percentage
identity between the sequences of the M. grisea and N. crassa
reductoisomerases is 86%. The percentage identity between the M.
grisea and yeast reductoisomerases is 70%, and that between the N.
crassa and yeast reductoisomerases is 72% (FIG. 1). N. crassa and
M. grisea are very similar fungal species (pyrenomycetes), which
might explain the high percentage identity between the
reductoisomerases of these two species.
(d) Study of the Expression of the M. grisea Reductoisomerase in
this Fungus Subjected to Various Conditions of Stress
[0090] M. grisea total RNA originating from a mycelium subjected to
various conditions of stress was extracted and then transferred
onto a membrane before being hybridized with the homologous probe
for the M. grisea ILV5 gene. The ILV5 gene is expressed
constitutively. It is thus expressed at the same level during a
hyperosmotic stress or a nitrogen-based nutritional deficiency for
an induction by cAMP, a thermal shock or an oxidative stress. It is
not, however, expressed during a carbon-based nutritional
deficiency.
Example 2
Disruption of the Magnaporthe grisea ILV5 Gene
[0091] After isolation and characterization of the ILV5 gene, the
aim was to obtain mutants of the M. grisea ILV5 gene in order to
test their pathogenic potency. The technique used to disrupt the
ILV5 gene is insertional mutagenesis by transposition in vitro.
2.1. Subcloning of the M. grisea ILV5 Gene in the Plasmid pBC
SK+
[0092] The subcloning of the ILV5 gene is carried out in the
plasmid pBC SK+ before transposon-based insertional mutagenesis.
The cosmid 20/G6 containing the ILV5 gene carries a gene for
resistance to ampicillin; it cannot therefore be used directly as a
target for the insertional mutagenesis. In fact, a double selection
with kanamycin and ampicillin would not be selective enough for the
target plasmids which have integrated the transposon, since the
transposon-donating plasmid (pGPS.sub.3 Hygro.sup.R) is also
resistant to kanamycin and to ampicillin. The ILV5 gene was
therefore subcloned into a plasmid carrying a gene for resistance
to chloramphenicol, the plasmid pBC SK+. The clones which have
integrated the transposon into the target plasmid (pBC SK+ carrying
the ILV5 gene) may be selected for their double resistance to
kanamycin and to chloramphenicol. In addition, the size of the
cosmid insert is too large (40 kb). A fragment of approximately 15
kb containing the ILV5 gene, subcloned into the plasmid pBC SK+,
was chosen. In fact, the probability that the transposon will
integrate into the ILV5 gene is greater when the size of the
fragment carrying the ILV5 gene is decreased. The ILV5 gene is
approximately 3 kb long, the probability that the transposon will
integrate into the gene present in the cosmid (46 kb) is 6.5%,
whereas the probability of integration of the transposon into the
ILV5 gene subcloned into the plasmid pBC SK+ (18 kb) is
approximately 3 times higher, i.e. approximately 17%. Subcloning of
the genomic DNA fragment carrying the ILV5 gene was carried out by
positioning this gene at the center of the insert. This type of
construct facilitates integration of the mutated ILV5 gene into the
M. grisea genome by homologous recombination. Mapping of the region
of the cosmid 20/G6 carrying ILV5 made it possible to choose a 15
kb ClaI-ClaI fragment in which the ILV5 gene is relatively well
centered. In fact, the ILV5 gene is ordered in the 5' position by 5
kb of the genomic sequence and in the 3' position by 5.5 kb. After
digestion of the cosmid 20/G6 with ClaI, the 15 kb fragment
containing the ILV5 gene was purified after separation on agarose
gel and subcloned into the plasmid pBC SK+. Twenty-four colonies
obtained after transformation were analyzed by PCR using the pair
of primers 22U and 1603L specific for the ILV5 gene. The 5 clones
amplify a fragment at the expected size (1.6 kb) possessing the
ILV5 gene. Clone no. 19 was chosen to perform the in vitro
transposition-based insertional mutagenesis of the ILV5 gene.
2.2. In Vitro Transposition-Based Insertional Mutagenesis of the M.
grisea ILV5 Gene
[0093] The in vitro transposition-based insertional mutagenesis of
the M. grisea ILV5 gene was carried out with the GPS.TM. (New
England Biolabs) using clone no. 19. The plasmid pGPS.sub.3
Hygro.sup.R, which carries a gene for resistance to kanamycin and
to hygromycin in the transprimer, is used as transposon donor. The
plasmid pBC SK+, carrying the ILV5 gene and the gene for resistance
to chloramphenicol, corresponds to the target plasmid. Once the
insertional mutagenesis has been carried out, thermocompetent
DH5.alpha. bacteria or electrocompetent DH10B bacteria are
transformed with the "mutagenesis mixture". After transformation,
the bacterial clones possessing an integration of the transposon in
the target plasmid pBC SK+ are selected by virtue of their
resistance both to kanamycin and chloramphenicol. This double
resistance could also be conferred on the bacterial clones having
both the target plasmid and the donor plasmid intact. Destruction
of the donor plasmid by digestion with the P1-SceI enzyme makes it
possible to overcome this problem. In order to determine whether
the transposon has integrated into the M. grisea ILV5 gene, the
selected bacterial clones are analyzed by PCR using the pair of
primers 22U and 1603L, located on either side of the coding region
of the ILV5 gene. Specifically, when the transposon Tn7 inserts
into the ILV5 gene, there is no amplification with the pair of
primers 22U and 1603L since the size of the DNA fragment located
between these two primers is too great to be amplified (4.3 kb).
This size corresponds to the sum of the size of the coding region
of the M. grisea ILV5 gene (1.6 kb) and of the Tn7 transposon of
2.7 kb. After transformation of the thermocompetent DH5 cells, an
absence of amplification was observed for 4 colonies (clones no. 3;
4; 8; 18) out of 32 colonies tested (12.5%). These bacterial clones
therefore possess an insertion of the transprimer into the ILV5
gene. After transformation of the electrocompetent DH10B cells, out
of 38 colonies tested by PCR, just one (2.6%) exhibits an absence
of amplification (clone no. 29). Bacterial clones no. 3; 8; 18 and
29 were sequenced using divergent primers Tn7L and Tn7R located at
each end of the transposon, in order to locate the exact position
of the site of insertion of the Tn7 transposon within the sequence
of the ILV5 gene. The transposon became integrated 21 bp before the
ATG for clone no. 18, 9 bp after the ATG for clone no. 8, 809 bp
after the ATG for clone no. 3 and 1176 bp after the ATG for clone
no. 29. We chose clone no. 8 for the transformation of M. grisea.
In fact, in this clone, the transposon was integrated at the
beginning of the coding region of the M. grisea ILV5 gene (+9 bp
after the ATG), resulting in inactivation of the ILV5 gene.
2.3. Transformation of the M. grisea Strain P1.2 with the M. grisea
ILV5 Gene Disrupted in its Coding Region
[0094] The insert of clone no. 8 containing the M. grisea ILV5 gene
disrupted in its coding region (9 bp after the ATG) is re-excised
from the plasmid by digestion with the ClaI enzyme, and purified on
agarose gel. It corresponds to the linearized construct. The
plasmid pBC SK+ originating from the undigested clone no. 8
corresponds to the "circular" construct. Transformation of the M.
grisea strain P1.2. protoplasts is carried out either with 5 .mu.g
of the linearized construct or with 4 .mu.g of the "circular"
construct. The positive transformation control is performed using 3
.mu.g of plasmid pCB1003 carrying a gene for resistance to
hygromycin, and the negative control is carried out without DNA. 62
transformants are obtained for the linearized construct and 24 for
the "circular" construct. These 86 transformants were subcultured
on complete medium supplemented with hygromycin at 120 g/l and on
the minimum medium supplemented with hygromycin at 120 mg/l or at
60 mg/l. This type of subculturing makes it possible to identify
the ilv5.sup.- transformants which are auxotrophic for leucine,
valine and isoleucine, and which, consequently, do not grow on
minimum medium. 8 transformants were thought to be auxotrophic out
of the 62 (13%) obtained with the linearized construct, whereas 2
transformants out of 24 (8.3%) were thought to be so with the
"circular" construct. The better efficiency in obtaining ilv5.sup.-
mutants with the linearized construct compared to the "circular"
construct might be explained by the fact that homologous
recombination is facilitated with a linearized construct. These
transformants are subcultured on "rice flour" medium in order to
make them sporulate, the spores are then plated out on complete
TNKYE glucose medium and left to germinate in order to effect a
single-spore isolation. The single spores are subcultured on TNKYE
glucose medium supplemented with hygromycin at 120 mg/l in order to
purify the transformants. Identification of the auxotrophic
transformants is carried out by subculturing these colonies derived
from these single spores on minimum medium, or minimum medium
supplemented with leucine, with valine and with isoleucine at 0.3
mM and on complete TNKYE glucose medium. Thus, genetically purified
and stable transformants were obtained. Out of 10 potential
transformants auxotrophic for leucine, valine and isoleucine, 8
(80%) were found to be effectively auxotrophic. The two
nonauxotrophic transformants must have corresponded to a mixture of
genetically different populations (ilv5.sup.+ and ilv5.sup.-) which
evolved toward a majority of ilv5.sup.+ during growth of the
transformant on nonselective medium before single-spore
purification.
Example 3
Phenotypic Characterization of the Magnaporthe grisea ilv5
Transformants Auxotrophic for Leucine, Valine and Isoleucine and
Study of their Pathogenic Potency
3.1. Effect of Disruption of the ILV5 Gene on the Growth and
Development of the M. grisea Transformants
[0095] The development of the ilv5.sup.- transformants was tested
on various culture media. Thus, on nitrate minimum medium, the
ilv5.sup.- transformants are incapable of growing whereas their
growth is possible on minimum medium supplemented with valine,
leucine and isoleucine at 0.3 mM. The development of the ilv5.sup.-
transformants on minimum medium+valine, leucine and isoleucine at
0.3 mM is, however, different than that of the wild-type M. grisea
strain P1.2. Their growth is in fact slowed down and their mycelium
is gray/green, low, flat and sporulating, and not aerial like the
wild-type strain. The presence of leucine is not necessary for
growth of the ilv5.sup.- transformants, since the results obtained
on minimum medium supplemented with isoleucine and valine at 0.3 mM
are identical to those obtained on minimum medium supplemented with
leucine, valine and isoleucine at 0.3 mM. The development of the
ilv5.sup.- transformants or minimum medium supplemented with valine
and isoleucine at 0.3 mM can be improved by supplementing the
minimum medium with a final concentration of valine and of
isoleucine of 1 mM. On complete medium, the ilv5 transformants
exhibit a phenotype which is relatively similar to the wild-type
strain: their mycelium is gray/white, and more or less aerial (less
aerial than the wild-type strain). The addition of pantotheine, the
oxidized form of pantothenate which is involved in leucine
biosynthesis, at 1 mg/1 to the minimum medium+valine and isoleucine
at 0.3 mM does not improve the development of the ilv5.sup.-
transformants. The sporulation of the ilv5.sup.- transformants is
slower and ten times less on "rice flour" agar medium compared to
the wild-type strain. The sporulation of the ilv5.sup.-
transformants is almost identical to the wild-type strain when
valine and isoleucine are added to the "rice flour" agar medium at
a final concentration of 1 mM.
3.2. Tests for the Pathogenic Potency of the ilv5.sup.- Auxotrophic
Mutants on Chopped up Barley Leaves under Artificial Survival
Conditions
[0096] The tests for the pathogenic potency of the M. grisea
ilv5.sup.- transformnants were carried out by swabbing or by
depositing blocks of test transformant spore suspension onto
chopped up barley leaves. One inoculation is carried out using a
wet Q-tip soaked in a suspension of spores (3.times.10.sup.4
spores/ml in general) and used to swab the fragments of barley
leaves under artificial survival conditions (on 1% agar-in-water
medium, 2 mg/1 kinetin). The other type of inoculation consists in
depositing drops of 30 .mu.l at three different places on the
surface of the barley leaves. The symptoms are observed after
incubating for 5 to 9 days at 26.degree. C.
[0097] The lesions caused by these mutants are smaller in size than
for the wild-type strain and they are 75% fewer in number (see
Table 1 below). TABLE-US-00001 TABLE 1 Test for the pathogenic
potency of the ilv5.sup.-transformants on barley leaves under
artificial survival conditions. Transformants Average number of
lesions per leaf* A L57 (ilv5.sup.+) 4 L64 (ilv5.sup.+) 4 L71
(ilv5.sup.-) 1 (-80%) L85 (ilv5.sup.-) 1 (-80%) B L41 (ilv5.sup.+)
10 L21 (ilv5.sup.-) 2 (-80%) A. Suspension of spores of the
transformants prepared at 3 .times. 10.sup.4 spores ml.sup.-1 B.
Suspension of spores of the transformants prepared at 10.sup.5
spores ml.sup.-1 *Number of lesions caused by the M. grisea
transformants on barley leaves or in a swab test, 5 days after
inoculation (L41 and L21) and 7 days after inoculation (L57, L64,
L71, L85).
[0098] The lesions caused by the ilv5.sup.- mutants are also
atypical and they appear later. They appear after incubation for 6
to 9 days at 26.degree. C., against 4 to 9 days for the wild-type
strain (see Table 2 below).
[0099] Some lesions caused by the ilv5.sup.- transformants appear
at the ends of the barley leaves (Table 2). Injuries at the ends of
the leaves might explain these lesions, since they might facilitate
penetration of the fungus. Tests for the pathogenic potency of
whole plants were consequently carried out in order, firstly, to
confirm the existence of a decrease in the pathogenic potency of
the ilv5.sup.- transformants and, secondly, to estimate the
decrease in pathogenesis of the ilv5.sup.- mutants and the
importance of injuries for penetration of the ilv5.sup.- fungus.
TABLE-US-00002 TABLE 2 Evolution of the symptoms caused by the M.
grisea transformants on barley leaves under artificial survival
conditions Trans- Drop test Swab test formants 4.sup.th day
9.sup.th day 4.sup.th day 9.sup.th day L71 Absence of Atypical
Absence of Absence of lesion (ilv5.sup.-) lesion lesions lesion
(except at the ends of the leaf) L73 Absence of Some rare Absence
of Absence of (ilv5.sup.-) lesion lesions lesion lesion L85 One
lesion Atypical Rare Rare atypical (ilv5.sup.-) lesions lesions
lesions L63 Absence of Atypical Absence of Absence of (ilv5.sup.-)
lesion lesions lesion lesion L64 Sporulating Gray Some Gray
(ilv5.sup.+) lesions sporulating sporulating sporulating lesions
lesions lesions
3.3. Tests for the Pathogenic Potency of the ilv5.sup.- Auxotrophic
Mutants on Whole Plants
[0100] The tests for the pathogenic potency of the ilv5.sup.-
transformants were carried out by spraying a suspension of spores
(10.sup.4 and 3.times.10.sup.4 spores.ml.sup.-1) of these
transformants onto barley plants. Gelatin at a final concentration
of 0.5% (w/v) is added to this spore suspension to enable better
adhesion of the spores to the surface of the leaves. The plants are
placed in a humid chamber overnight after the inoculation. The
symptoms are generally observed after incubation for 5 to 10 days
at ambient temperature for barley.
[0101] A notable decrease in the symptoms of blast disease on
barley is observed in the ilv5.sup.- transformants compared to the
ilv5.sup.+ transformant. After incubation for 10 days at ambient
temperature, the number of lesions per leaf (approximately 12 cm)
varies from 6 to 9 for the ilv5.sup.- transformants (L87, L74 and
C24) against 37 on average for the ilv5.sup.+ transformant L57 (see
table 3). The number of lesions caused by the ilv5.sup.- mutants is
therefore reduced by 80% compared to that obtained with the
wild-type strain. In addition, the lesions are twice as small for
the ilv5.sup.- mutants than for the ilv5.sup.+ transformant (6
mm.sup.2 against 13 mm.sup.2), and these lesions appear twice as
slowly. Specifically, the number of lesions doubled for the
ilv5.sup.- mutants between the 5.sup.th and the 10.sup.th day of
the experiment, whereas it remained identical for the wild type.
The appearance of certain lesions caused by the ilv5.sup.-
transformants at the ends of the barley leaves under artificial
survival conditions suggested that the penetration of the fungus
could be facilitated by the injuries inflicted on the barley leaves
during the pathogenesis test. However, the results of the
pathogenesis tests carried out on the ilv5.sup.- transformants on
whole plants clearly show a decrease in the pathogenic potency of
these mutants of the same order (80%) as on barley leaves under
artificial survival conditions. TABLE-US-00003 TABLE 3 Test for the
pathogenic potency of the ilv5.sup.- transformants on whole plants
(barley) Average number of lesions Average lesion size
Transformants per barley leaf of 12 cm (in mm.sup.2) L57
(ilv5.sup.+) 37 .+-. 9 13 L87 (ilv5.sup.-) 9 .+-. 2 (-75%) 6 (-50%)
C24 (ilv5.sup.-) 6 .+-. 2 (-83%) 4 (-70%) L74 (ilv5.sup.-) 8 .+-. 3
(-80%) 6 (-50%)
[0102] The inoculation of the barley plants was carried out by
spraying a suspension of spores (3.times.104 spores. ml.sup.-1) of
the transformants the pathogenic potency of which it was desired to
test. The plants were placed in a humid chamber overnight after the
inoculation. The symptoms were observed after incubation for 10
days at ambient temperature. The percentage decreases in the number
and the size of the lesions per leaf, calculated with respect to
the values obtained for the transformant L57 (ilv5.sup.+), are
indicated in brackets.
Example 4
Fungicidal Effect of the Ketol-Acid Reductoisomerase Inhibitors
4.1. Fungicidal Effect of the Ketol-Acid Reductoisomerase
Inhibitors on the Phytopathogenic Fungus Magnaporthe grisea
[0103] The fungicidal effect of the inhibitors
dimethylphosphinoyl-2-hydroxyacetate and
N-hydroxy-N-isopropyloxamate was tested on the pathogenic fungus M.
grisea by following the evolution of the growth of this fungus in
the presence of various concentrations of inhibitor, over a period
of 7 days. Thus, a given culture medium (nitrate minimum medium
(MM); MM+leucine, valine and isoleucine at 0.3 mM; complete TNKYE
glucose medium (MC); MC+leucine, valine and isoleucine at 0.3 mM)
is inoculated with a suspension of spores of the M. grisea strain
P1.2 at a final concentration of 10.sup.5 spores/ml. A range of
dilutions of the test product, N-hydroxy-N-isopropyloxamate (at pH
5), is prepared in water such that the product is 100 times
concentrated. 200 .mu.l of inoculated medium are distributed into a
96-well microplate, to which wells are added 2 .mu.l of
100.times.-concentrated product. The N-hydroxy-N-isopropyloxamate
is tested at final doses of 3; 1; 0.3; 0.1 and 0.03 .mu.M in
minimum media and 3 and 0.3 .mu.M in complete media. The microplate
is incubated at ambient temperature and reading of the optical
density at 630 nm of this microplate makes it possible to follow
the growth of the fungus under these various culture conditions at
times 0 (beginning of the test) and on days 3, 4, 5, 6 and 7.
[0104] This test was carried out by inoculating various culture
media with a suspension of spores of the wild-type M. grisea strain
P1.2. The first experiments were carried out in nitrate minimum
medium (MM) and various concentrations of inhibitor were tested. It
was the 6.sup.th and 7.sup.th day of growth before the appearance
of significant inhibition of growth in MM medium was observed in
the presence of dimethylphosphinoyl-2-hydroxyacetate at 2 mM. The
growth is in fact decreased by 50% compared to that of the control
without inhibitor. In the presence of the inhibitor
N-hydroxy-N-isopropyloxamate (0.3 to 1 .mu.M), from the 3.sup.rd
day, approximately 50% inhibition of fungal growth is obse-rved in
MM medium. The greater inhibition of the M. grisea growth by
N-hydroxy-N-isopropyloxamate compared to
dimethylphosphinoyl-2-hydroxyacetate led us to carry out a more
thorough study of this inhibitor.
[0105] Growth assays for the M. grisea fungus were carried out over
a period of 7 days, in the presence of the inhibitor
N-hydroxy-N-isopropyloxamate (at concentrations ranging from 0.03
.mu.M to 3 .mu.M) in various media (MM and MM supplemented with
leucine, valine and isoleucine at 0.3 mM). In minimum medium,
N-hydroxy-N-isopropyloxamate strongly inhibits the growth of M.
grisea. This inhibition of growth by N-hydroxy-N-isopropyloxamate
(from 0.3 .mu.M) is observed from the 3.sup.rd day of growth, and
remains similar on the following days (FIG. 2). We therefore chose
to calculate the ID.sub.80 (concentration of inhibitor such that
the inhibition in fungal growth is 80%) and the ID.sub.50
(concentration of inhibitor such that the inhibition of growth is
50%) on the 5.sup.th day of growth. Thus, the growth of the M.
grisea fungus is decreased by 80% compared to the nontreated
control, at an N-hydroxy-N-isopropyloxamate concentration of 1
.mu.M (ID.sub.80). An N-hydroxy-N-isopropyloxamate concentration of
0.3 .mu.M (ID.sub.50) inhibits fungal growth by 50% (Table 4).
TABLE-US-00004 TABLE 4 Study of the effect of the inhibitor
N-hydroxy-N-isopropyloxamate on the growth of the M. grisea fungus
N-hydroxy-N-isopropyloxamate in .mu.M 0 (control) 0.03 0.1 0.3 1 3
Minimum 100 92 89 72 56 13 medium (MM) MM + ILV 100 77.5 83.5 81
72.5 71.5
[0106] The effect of the inhibitor N-hydroxy-N-isopropyloxamate was
tested on the pathogenic fungus M. grisea by following the
evolution of the growth of this fungus in the presence of various
concentrations of inhibitor, in various culture media and over a
period of 7 days. The values given in the table correspond to the
mean percentages of growth of the M. grisea fungus, obtained on the
5.sup.th day of growth from two experiments. The control (wild-type
strain of M. grisea) corresponds to a 100% growth rate. 200 .mu.l
of culture medium, minimum medium (MM) or minimum medium +leucine,
valine and isoleucine at 0.3 mM (MM+ILV), were inoculated with a
suspension of spores of the M. grisea strain P1.2 at a final
concentration of 10.sup.5 spores.ml.sup.-1. The microplate was
incubated at ambient temperature and the optical density at 630 nm
(OD.sub.630) was measured on days 0, 3, 4, 5, 6 and 7 (D0, D3, D4,
D5, D6 and D7).
[0107] The toxicity of the N-hydroxy-N-isopropyloxamate on the
growth of the M. grisea fungus is lifted by supplementing the
minimum medium with valine, leucine and isoleucine at 0.3 mM,
whatever the concentration of N-hydroxy-N-isopropyloxamate used.
This lifting of N-hydroxy-N-isopropyloxamate toxicity by adding
valine, leucine and isoleucine to the minimum medium shows that
this toxicity comes from specific inhibition of the biosynthetic
pathway for these amino acids, by inhibiting the
reductoisomerase.
[0108] In fact, the N-hydroxy-N-isopropyloxamate acting
specifically on the M. grisea reductoisomerase strongly inhibits
the growth of M. grisea at very low concentrations. The
reductoisomerase and the inhibitor N-hydroxy-N-isopropyloxamate
proved to be a good target/fungicide couple.
[0109] We also sought to determine whether the inhibitor
N-hydroxy-N-isopropyloxamate had an effect on germination of the M.
grisea spores. A microscopic observation of the M. grisea spores
during the experiments carried out previously in minimum medium, in
the presence of N-hydroxy-N-isopropyloxamate at 1 and 3 .mu.M on
days 0 and 2 showed that spore germination was not blocked.
Additional tests were carried out in order to determine whether, at
high concentrations, N-hydroxy-N-isopropyloxamate could block spore
germination. Thus at 10 mM, N-hydroxy-N-isopropyloxamate does not
inhibit M. grisea spore germination either in water or in minimum
medium, at 24 and at 72 hours. The inhibition of M. grisea growth
by N-hydroxy-N-isopropyloxamate only manifests itself after
germination, during growth of the hyphae. It may therefore be
supposed that use of the amino acid (valine and isoleucine) stores
present in the spore could, initially, allow germination.
Limitation of the amino acid stores and more or less rapid
exhaustion thereof could act as a factor limiting M. grisea
growth.
4.2 Fungicidal Effect of the Ketol-Acid Reductoisomerase Inhibitors
on Other Fungi
[0110] The toxicity of IpOHA with respect to other fungal species
such as Pythium ultimum, Botrytis cinerea, Ustilago nuda and
Mycosphaerella graminicola was measured under the same conditions
as for M. grisea (culture in liquid minimum medium in 96-well
microplates). The growth of Botrytis cinerea is not affected by the
highest concentration of IpOHA used (30 .mu.M), which shows that
this species is resistant to IpOHA. The growth of Ustilago nuda and
of Pythium ultimum is inhibited by IpOHA starting from 10 .mu.M.
The growth of Mycosphaerella graminicola is inhibited from 0.3
.mu.M (Table 5). When the minimum medium (MM-liq) is supplemented
with isoleucine, leucine and valine (1 mM), the IpOHA toxicity is
lifted for all the sensitive fungal species. On the other hand,
with a concentration of 0.3 mM, the inhibition is lifted in
Ustilago nuda only for IpOHA concentrations of less than 30 .mu.M.
IpOHA has a strong action on the growth of Mycosphaerella
graminicola. TABLE-US-00005 TABLE 5 Toxicity of IpOHA for various
fungal species Level of sensitivity ID.sub.80 ID.sub.50 to IpOHA
Botrytis cinerea / / Resistant Pythium ultimum 30 10 Moderately
sensitive Ustilago nuda 30 3 Moderately sensitive Mycosphaerella
graminicola 3 1 Sensitive Magnaporthe grisea 3 1 Sensitive
ID.sub.50: concentration which inhibits fungal growth by 50%.
ID.sub.80: concentration which inhibits fungal growth by 80%.
Example 5
Biochemical Studies of the Ketol-Acid Reductoisomerase of S.
cerevisiae
[0111] The coding sequence of the yeast ILV5 gene without the
transit peptide was overexpressed in E. coli in order to obtain
large amounts of enzyme so as to facilitate the biochemical study
thereof and in particular the structural study thereof.
[0112] The strategy employed for studying the yeast
reductoisomerase was as follows: the yeast reductoisomerase ILV5
gene was initially amplified by PCR without the portion encoding
the transit peptide. The PCR reaction product was then cloned into
an IPTG-inducible expression vector pET. The reductoisomerase was
then overproduced in E. coli and purified, and then its biochemical
properties were studied.
5.1 PCR Amplification and Cloning of the Portion of the Yeast
Reductoisomerase ILV5 Gene Encoding the Mature Protein
[0113] The signal peptide, which allows adjusting of the yeast
reductoisomerase into its cellular compartment, the mitochondriun,
is cleaved when the protein has penetrated into the mitochondrium.
We therefore chose to clone the ILV5 gene without the region
encoding the transit peptide in order to overproduce the yeast
reductoisomerase corresponding to the mature protein in E.
coli.
[0114] The region of the yeast ILV5 gene located between the end of
the transit peptide and the translation-terminating STOP codon was
amplified by PCR from the genomic DNA of S. cerevisiae with the
pairs of primers (1'-3') and (2'-3'). The size of the DNA fragments
amplified with the pairs of primers (1'-3') and (2'-3') is 1079 bp
and 1124 bp, respectively. These DNA fragments were purified after
separation by electrophoresis, and then digested and cloned into
the vector PET-23d at Sall/NcoI. A double digestion of the cloning
vector and of the PCR reaction product with the NcoI and SalI
enzymes in fact enables the yeast ILV5 gene to be cloned into the
vector PET-23d, in the correct orientation. The plasmid pET-23d
(Tebu), carrying the gene for resistance to ampicillin, is used as
an inducible expression vector to produce a large amount of the
yeast reductoisomerase in E. coli. This type of vector has a T7
phage promoter, which is recognized by the T7 RNA polymerase but
not by the RNA polymerase of E. coli. Production of the cloned
protein takes place after IPTG induction of the strain BL21 pLysS
(resistant to chloramphenicol); in this bacterial strain, the T7
RNA polymerase gene is under the control of the IPTG-inducible lac
promoter. The strain BL21 pLysS transformed with the plasmid
pET-23d, carrying the S. cerevisiae ILV5 gene, is called BL21
pLysS-pET-23d-reductoisomerase. Construct no. 1 corresponds to the
cloning of the fragment amplified with the primers (1'-3') and
construct no. 2 corresponds to the cloning of the DNA fragment
amplified with the primers (2'-3'). A SalI/NcoI double digestion of
the 12 clones obtained after transformation of the DH5 cells with
construct no. 1 and of the 6 clones transformed with construct no.
2 makes it possible to re-excise the fragment cloned into the
vector pET and to thus verify the presence of one of the two
constructs in the various clones. Analysis of the digestion
profiles for these bacterial clones showed that they all possess
the corresponding construct.
[0115] Two clones were selected: clone PET 1-4 (construct no. 1)
and clone PET 2-1 (construct no. 2); they were used to transform
BL21 pLysS cells in order to overproduce the yeast reductoisomerase
in E. coli.
5.2 Purification of the Yeast Reductoisomerase Overproduced in E.
coli
[0116] Overexpression of the "short" form (construct no. 1) and of
the "long" form (construct no. 2) of the yeast reductoisomerase was
induced in E. coli with IPTG. The bacterial strain BL21 pLysS,
transformed with the plasmid pET 23-d containing the yeast ILV5
gene without the region encoding the transit peptide, is cultured
at 28.degree. C. with shaking in LB medium supplemented with
carbenicillin (100 mg/l) and with chloramphenicol (30 mg/l) until a
density equivalent to an OD.sub.600 of approximately 0.6 is
obtained. The IPTG is then added at a final concentration of 0.4 mM
and the bacteria are left in culture at 28.degree. C. with shaking
for approximately 15 hours. This bacterial culture is then
centrifuged (30 minutes, 4500 rpm); the bacterial pellet is
resuspended in 15 ml of buffer (10 mM KH.sub.2--K.sub.2PO.sub.4 (pH
7.5), 1 mM EDTA, 1 mM DTT and protease inhibitors: 1 mM benzamidine
HCl, 5 mM aminocaproic acid) and sonicated using a Vibra-cell
disruptor (Sonics and Materials, Danbury, Conn., U.S.A) for 15
minutes, at power 4, 40% of the total lysis time. The cell extract
is centrifuged (20 minutes, 15 000 rpm); the supernatant,
containing the soluble proteins, is then conserved at -80.degree.
C.
[0117] Analysis of the total protein fractions and of the soluble
protein fractions on acrylamide gel showed that the "short" and
"long" forms of the yeast reductoisomerase are present in the
soluble protein fraction and that they represent approximately 25
to 30% of these proteins. Since the most probable position for the
yeast reductoisomerase transit peptide cleavage site is located
between amino acids 47 and 48 of the protein sequence of this
enzyme (Petersen, G. L. et al., NAR 14, 24:9631-9650, 1986), we
chose to continue working with the short form of the yeast
reductoisomerase.
[0118] Purification of the short form of the reductoisomerase was
therefore carried out in two steps using the soluble protein
fraction; first, on an anion exchange column (Q-Sepharose), and
then on a permeation column (Superdex 75). The soluble protein
extract (15.5 ml; 227.8 mg of proteins), which contains the yeast
reductoisomerase (crude extract), is applied to an anion exchange
column, HiLoad 16/10 Q-Sepharose (Pharmacia), connected to a
Pharmacia FPLC system, pre-equilibrated with 10 mM
KH.sub.2--K.sub.2PO.sub.4 buffer/1 mM EDTA/1 mM DTT. The enzyme is
eluted with 78 ml of this same buffer (flow rate=1 ml/min; fraction
size=3 ml). The chromatographic fractions containing the yeast
reductoisomerase are concentrated to 1.6 ml by centrifugation at
5500 rpm in a macrosep--10 unit (filtron). This extract (27.7 mg)
is then applied to a HiLoad 16/60 Superdex 75 column (Pharmacia)
connected to a Pharmacia FPLC system, pre-equilibrated with 25 mM
Hepes-KOH buffer. The enzyme is eluted with 58 ml of this same
buffer (flow rate=1 ml/min; fraction size=1 ml). The
chromatographic fractions containing the yeast reductoisomerase
(18.99 mg) are concentrated to 9.7 mg/ml by centrifugation at 5500
rpm in a 10K microsep (filtron) and conserved at -80.degree. C.
[0119] After injection of the soluble protein fraction
(approximately 230 mg) onto the Q-Sepharose column, the yeast
reductoisomerase is diluted with 10 mM KH.sub.2--K.sub.2PO.sub.4
buffer/1 mM EDTA/1 mM DTT. There is in fact no need to elute this
enzyme through the action of an increasing concentration gradient
of phosphate buffer since preliminary experiments have shown that
this enzyme is not retained by the column. After this first
purification step, approximately 30 mg of protein were recovered
and the yield from the purification in terms of activity is 55%
(Table 6). TABLE-US-00006 TABLE 6 Steps for purifying the yeast
reductoisomerase overexpressed in E. coli Specific Total activity
Amount of activity (total activity/ proteins (.mu.mol of mg.sup.-1
of (mg) NADPH proteins) Purification Brad- oxidized Brad- Yield
steps ford 205 min.sup.-1) ford 205 in % Crude extract 227.82 n.d.
134.16 0.59 n.d. 100 of soluble proteins Q-Sepharose 27.72 14.34
73.22 2.64 5.11 54.58 fraction pool Superdex 75 18.99 8.4 45.85 2.4
5.46 34.18 fraction pool The activities were determined in the
following reaction medium: 50 mM sodium Hepes, pH 7.5; 10 mM
MgCl.sub.2; 250 .mu.M NADPH and 0.48 mM AHB. The proteins were
assayed according to the Bradford method (Bradford) or by measuring
the absorbence at 205 nm (205). n.d. = not determined.
[0120] Analysis of the Q-Sepharose fraction pool on acrylamide gel
shows that, after this first purification step, the enzyme is
virtually pure. The Q-Sepharose fraction pool is injected onto the
gel filtration column and the yeast reductoisomerase is eluted with
25 mM Hepes-KOH buffer. After this 2.sup.nd purification step,
approximately 20 mg of pure protein are recovered; the final yield
from the two steps for purifying the yeast reductoisomerase is
approximately 34%.
5.3 Studies of the Kinetic Properties of the Yeast Reductoisomerase
Ovelproduced in E. coli
[0121] The kinetic parameters for the yeast reductoisomerase were
determined by following the evolution of the enzyme reaction in the
spectrophotometer under saturating conditions with respect to
magnesium, to NADPH and to AHB or AL substrate. All the enzyme
activity measurements were carried out in a quartz cuvette with an
optical path of 1 cm, containing sodium Hepes buffer (50 mM, pH
7.5), 10 mM MgCl.sub.2, 250 .mu.M NADPH, in a final volume of 1 ml,
at 25.degree. C. The enzyme reaction was initiated by adding 0.48
mM of AHB or of AL. The evolution of this reaction was followed by
virtue of the decrease in absorption of NADPH at 340 nm.
5.3.1. Determination of the Optimum pH for Activity of the Purified
Recombinant Yeast Reductoisomerase
[0122] In order to determine the optimum conditions for carrying
out the kinetic measurements on the yeast reductoisomerase, the
optimum pH for activity of this enzyme was determined, by measuring
the activity of the purified enzyme in buffers of varying pH. The
optimum pH for activity of the yeast reductoisomerase is 7.5,
although studies had shown that that of plant reductoisomerase is
8.2. This difference in optimum pH for reductoisomerase activity
between plants and yeast is explained by the cellular location of
these two enzymes. The plant reductoisomerase is located in the
chloroplast, the pH of which is 8.2 in light, whereas the yeast
reductoisomerase is located in the mitochondrium, the pH of which
is 7.5. The optimum pH for activity of the reductoisomerase is
therefore well suited to the cellular environment in which they are
found.
5.3.2. Determination of the Kinetic Parameters for the Purified
Recombinant Yeast Reductoisomerase
[0123] The affinity of the yeast reductoisomerase for its various
ligands was studied.
[0124] (a) Specific Activities
[0125] The specific activities of the yeast reductoisomerase for
the AHB and AL substrates are 6 and 1 .mu.mol of NADPH
oxidized.min.sup.-1.mg.sup.-1 of protein, respectively. The ratio
of the maximum rate (V.sub.m) of enzyme reaction in the presence of
the AHB substrate to the maximum rate (V.sub.m) of enzyme reaction
in the presence of the acetolactate substrate is therefore
unchanged for the yeast enzyme compared to the plant enzyme
(V.sub.m AHBN.sub.m AL=6). The specific activities of the plant
reductoisomerase for the AHB and AL substrates are, moreover, 6 and
1 .mu.mol of NADPH oxidized.min.sup.-1.mg.sup.-1 of protein,
respectively.
[0126] (b) Affinities for the Cofactors NADPH and NADH
[0127] The affinity of the yeast reductoisomerase for NADPH is very
high (FIG. 3). In fact, a Km for this cofactor of 1.6 .mu.M was
measured. This K.sub.M is relatively similar to that measured for
the plant enzyme (K.sub.M=5 .mu.M). The K.sub.M NADPH value
obtained for the purified yeast reductoisomerase is coherent with
the value obtained for the partially purified yeast enzyme
(K.sub.M<2.5 .mu.M; Hawkes et al., 1989). However, unlike the
plant enzyme, which is capable of using NADH, with a very low
affinity (K.sub.M NADH=645 .mu.M in the presence of AHB and of
Mg.sup.2+; Dumas et al., Biochem. J., 288:865-874, 1992), the yeast
enzyme appears to be incapable of using NADH. No enzyme activity
was detected in the presence of NADH (at 300 .mu.M) under
saturating conditions with respect to AHB substrate and to
magnesium, perhaps due to the affinity for NADH being even lower
than that of the plant reductoisomerase. The yeast reductoisomerase
is thought to use NADPH as a hydrogen donor with a specificity
which is even more marked than the plant enzyme.
(c) Affinities for the AHB and AL Substrates
[0128] The affinity of the yeast reductoisomerase for the AHB
substrate (K.sub.M=104 .mu.M for the racemic form) is approximately
5 times lower than that of the plant reductoisomerase; whereas the
affinity of the yeast reductoisomerase for the AL substrate
(K.sub.M=266 .mu.M for the racemic form) is 10 times lower than the
plant reductoisomerase (FIG. 4). These K.sub.M values are quite
close to those obtained for the partially purified enzyme of N.
crassa. Specifically, the K.sub.M AHB and AL values for the N.
crassa reductoisomerase are 160 .mu.M and 320 .mu.M,
respectively.
[0129] The most noteworthy difference in biochemical properties
between the yeast reductoisomerase and plant reductoisomerase is
the affinity of this enzyme for magnesium.
(d) Affinities for Magnesium
[0130] The affinity of the yeast reductoisomerase (K.sub.M=968
.mu.M) is in effect 200 times lower than that of the plant enzyme
(K.sub.M=approximately 5 .mu.M). See FIG. 5. This low affinity of
the yeast reductoisomerase for magnesium could be a characteristic
of the fungal reductoisomerase. In fact, studies carried out on the
N. crassa reductoisomerase have shown that this enzyme has a
K.sub.M magnesium of 580 .mu.M in the presence of NADPH and of the
AHB substrate (Kritani et al., J. Biological Chemistry,
241:2047-2051, 1965). Comparison of the primary sequences of the
yeast, N. crassa and plant reductoisomerases showed that the main
difference between these three enzymes was the absence within the
flingal protein of a 140 amino acid sequence involved in the
interaction between the two monomers of the plant enzyme. The two
domains known to bind Mg.sup.2+ ions in the plant enzyme are,
however, present in the yeast enzyme. The study of a monomeric
mutant of the plant reductoisomerase obtained by deleting 7 amino
acids in the 140 amino acid dimerization region showed that the
enzyme exhibits a much lower affinity for magnesium (KM=640 .mu.M)
than the wild-type form of the enzyme (Wessel et al., Biochemistry,
37:12753-12760, 1998). The quaternary structure of the plant
reductoisomerase is therefore thought to play a role in stabilizing
the active site of the plant enzyme and the high affinity sites for
magnesium. Thus, although the yeast reductoisomerase clearly
possesses the two Mg.sup.2+ ion-binding domains, the absence of the
sequence of this 140 amino acid region within the yeast enzyme
could explain the lower affinity of the yeast reductoisomerase for
magnesium by virtue of the spatial organization of the active site
of this enzyme, which is probably different from that of the plant
enzyme. Although the magnesium-binding sites of the yeast
reductoisomerase have a much lower affinity for this cation, the
affinity of the yeast enzyme for NADPH is similar to that of the
plant enzyme. It may therefore be supposed that the conformation of
the yeast reductoisomerase could be such that the amino-terminal
domain which is involved in the binding of NADPH is relatively
similar to that of the plant enzyme and that the carboxy-terminal
domain which is responsible for the binding of the two magnesium
atoms and of the substrate is different than that of the plant
enzyme. A knowledge of the quaternary structure of the yeast
reductoisomerase through crystallization of the enzyme would make
it possible to explain this low affinity for Mg.sup.2+ ions.
5.4 Study of the Effect of the Inhibitors
N-hydroxy-N-isoipropyloxamate and
Dimethylphosphinoyl-2-Hydroxyacetate on the Yeast
Reductoisomerase
5.4.1 Study of the Stoichiometry of Binding of the Inhibitors
N-hydroxy-N-Isopropyloxamate and
Dimethylphosphinoyl-2-Hydroxyacetate to the Yeast
Reductoisomerase
[0131] The study of the stoichiometry of binding of the inhibitors
N-hydroxy-N-isopropyl-oxamate and
dimethylphosphinoyl-2-hydroxyacetate was carried out by incubating
varying amounts of enzyme (from 0.1 to 0.4 nmol) with a constant
amount of inhibitor for 20 minutes, with 25 nmol of NADPH and 0.25
.mu.mol of MgCl.sub.2, in a final volume of 10 .mu.l. The reactions
are initiated by adding 0.48 mM AHB in 50 mM sodium Hepes buffer,
pH 7.5, containing 10 mM MgCl.sub.2.
[0132] In the presence of 0.1 nmol of
dimethylphosphinoyl-2-hydroxyacetate, an enzyme activity is
detected only with amounts of enzyme greater than 0.09 nmol.
Similarly, in the presence of 0.1 nmol of
N-hydroxy-N-isopropyloxamate, an enzyme activity is detected only
for amounts of enzyme greater than 0.1 nmol (FIG. 6). In addition,
for these two inhibitors, when the enzyme is in excess relative to
the inhibitor in the reaction medium, the enzyme activities
increase in parallel with those of the control without inhibitor.
N-hydroxy-N-isopropyloxamate and
dimethylphosphinoyl-2-hydroxyacetate are therefore thought to act
on the yeast reductoisomerase as irreversible inhibitors. In fact,
in the case of irreversible inhibition, in the presence of a low
amount of enzyme, all the enzyme complexes with the inhibitor. The
enzyme activity is then zero, since there is no longer any free
enzyme in the reaction medium. When the amount of enzyme present in
the reaction medium is greater than the amount of inhibitor, the
free enzyme in excess in the medium then behaves like the control
without inhibitor (straight line parallel to the control).
Moreover, since 0.1 umol of inhibitor (N-hydroxy-N-isopropyloxamate
or dimethylphosphinoyl-2-hydroxyacetate) is necessary to completely
inhibit 0.1 nmol of enzyme, the stoichiometry of binding of the
inhibitors to the yeast reductoisomerase is, consequently, 1 mol of
inhibitor per mole of enzyme.
5.4.2 Study of the Rate of Binding of the Inhibitors
N-hydroxy-N-isopropyloxamate and
Dimethylphosphinoyl-2-Hydroxyacetate to the Yeast
Reductoisomerase
[0133] The effect of the inhibitors N-hydroxy-N-isopropyloxamate
and dimethylphosphinoyl-2-hydroxyacetate on the yeast
reductoisomerase was followed over time by measuring the decrease
in absorbence of NADPH at 340 nm, in a spectrophotometer. Each
measurement is carried out under saturating conditions with respect
to MgCl.sub.2 (10 mM) and with respect to NADPH (0.25 mM) for a
period of 6 minutes in the presence of a given concentration of
inhibitor (N-hydroxy-N-isopropyloxamate=15 gM or
dimethylphosphinoyl-2-hydroxyacetate=10 .mu.M) and of enzyme (110
nM); the concentration of AHB substrate used ranges from 250 .mu.M
to 2375 .mu.M. The study of the stoichiometry of binding of the
inhibitors N-hydroxy-N-isopropyloxamate and
dimethylphosphinoyl-2-hydroxyacetate to the yeast reductoisomerase
showed that these inhibitors behave like irreversible inhibitors.
Equation (1) therefore makes it possible to describe the kinetics
of formation of the reaction product, this equation being
applicable to irreversible inhibitors. The parameters m1, m2 and
m3, defined on the basis of equation (1), are obtained directly
from the adjustment of the experimental curves using the
KaleidaGraph program, along with the errors associated with the
determination of these parameters.
[0134] Equation (1):
OD.sub.340=m.sub.1+(m.sub.2-m.sub.1)e.sup.(-m3t) where the
parameters are as follows: [0135] OD.sub.340 is the optical density
measured on a spectrophotometer at 340 nm at time "t" [0136]
m.sub.1 the optical density when "t" tends toward infinity [0137]
m.sub.2 the initial optical density [0138] m.sub.3 the product of
the concentration of inhibitor multiplied by the apparent
disappearance constant for NADPH.
[0139] For a reversible inhibition, two cases may occur: either the
inhibitor binds directly to the enzyme in a single step, or it
binds to the enzyme in two steps, forming a reversible
enzyme/inhibitor intermediate complex. The graphic representation
m.sub.3 as a function of the concentration inhibitor makes it
possible to define the type of irreversible inhibition. Thus, to
determine whether the inhibitors N-hydroxy-N-isopropyloxamate and
dimethylphosphinoyl-2-hydroxyacetate bind to the yeast
reductoisomerase in one or two steps, the effect of these
inhibitors on the enzyme activity of this enzyme is followed over
time (6 min) by varying the concentration of inhibitor, for given
concentrations of enzyme (110 nM) and AHB (0.48 mM) (FIG. 7). For a
simple irreversible inhibition, without formation of the reversible
enzyme/inhibitor intermediate complex, the apparent rate of
formation of the enzyme/inhibitor complex (K.sub.obs or m.sub.3) is
a linear function of the concentration of inhibitor. If the
irreversible inhibition involves the existence of a reversible
intermediate complex, the graphic representation m.sub.3 as a
function of the concentration of inhibitor is a hyperbola. For the
inhibitors N-hydroxy-N-isopropyloxamate and
dimethylphosphinoyl-2-hydroxyacetate, the graphic representation
m.sub.3 as a function of the concentration of inhibitor is linear,
suggesting that the inhibition of the yeast reductoisomerase by
these products occurs in a single step (FIG. 8), as is the case for
inhibition of the plant reductoisomerase. However, even taking into
account experimental errors, the graphic representations m.sub.3 as
a finction of the concentration of inhibitor do not pass through
the origin for the two inhibitors. These two inhibitors,
N-hydroxy-N-isopropyloxamate and
dimethylphosphinoyl-2-hydroxyacetate, might not be entirely
irreversible. In this case, the dissociation constant for the
enzyme/inhibitor complex, k.sub.0, could have a non-negligible
value. The mechanism of inhibition (competitive or noncompetitive)
can be determined by studying the effect of the concentration of
substrate on the apparent rate of formation of the enzyme/inhibitor
complex. This determination is carried out using the graphic
representation 1/m.sub.3 as a function of the concentration of
substrate. For competitive inhibitors, the graphic representation
1/m.sub.3 as a function of the concentration of substrate is
linear. For noncompetitive inhibitors, the parameter m.sub.3 is
independent of the concentration of substrate. For the inhibitors
N-hydroxy-N-isopropyloxamate and
dimethylphosphinoyl-2-hydroxyacetate, the inverse of m.sub.3 varies
in a linear fashion as a function of the concentration of AHB
substrate. N-hydroxy-N-isopropyloxamate and
dimethylphosphinoyl-2-hydroxyacetate therefore behave like
inhibitors of the yeast reductoisomerase which are competitive with
respect to the AHB substrate (FIG. 9). The inhibitor/yeast enzyme
association constant (k.sub.0) is then calculated by virtue of
equation (2). Equation .times. .times. ( 2 ) .times. : ##EQU1## k
obs = k 0 [ I ] 1 + [ S ] K M S + k - 0 ##EQU1.2## [0140]
k.sub.obs=m.sub.3=apparent rate of formation of the
enzyme/inhibitor complex [0141] k.sub.0=inhibitor/enzyme
association constant [0142] k.sub.0=inhibitor/enzyme dissociation
constant [0143] K.sup.S.sub.M=Michaelis-Menten constant for the AHB
substrate [0144] [I]=concentration of inhibitor [0145]
[S]=concentration of substrate
[0146] For irreversible inhibitors, the k.sub.0 can be considered
to be negligible, the k.sub.0 is then calculated by virtue of
equation (3) using the KaleidaGraph program. Equation .times.
.times. ( 3 ) .times. : ##EQU2## 1 m 3 = 1 k 0 [ I ] + 1 K M S [ I
] k 0 [ S ] ##EQU2.2##
[0147] This equation can be used for the inhibitor
N-hydroxy-N-isopropyloxamate, but not for
dimethylphosphinoyl-2-hydroxyacetate. For
dimethylphosphinoyl-2-hydroxyacetate, the graphic representation
1/m.sub.3 as a function of the concentration of substrate is
linear, but does not pass through the origin. For
N-hydroxy-N-isopropyloxamate, a graphic representation 1/m.sub.3 as
a function of the concentration of substrate is linear and the
value on the y-axis at the origin is negligible. A hypothesis which
may explain this result is: the inhibitor
dimethylphosphinoyl-2-hydroxyacetate is perhaps not completely
irreversible. A linear regression line then makes it possible to
obtain the value of the k.sub.0 for
dimethylphosphinoyl-2-hydroxyacetate. The values of k.sub.0
corresponding to the inhibitors N-hydroxy-N-isopropyloxamate and
dimethylphosphinoyl-2-hydroxyacetate are 12 433 M.sup.-1.s.sup.-1
and 7721 M.sup.-1.s.sup.-1, respectively. Thus, unlike the plant
reductoisomerase (ko for N-hydroxy-N-isopropyloxamate=1900
M.sup.-1.s.sup.-1 and k.sub.0 for
dimethylphosphinoyl-2-hydroxyacetate=22 000 M.sup.-1.s.sup.-1),
N-hydroxy-N-isopropyloxamate is a better inhibitor of the yeast
enzyme than dimethylphosphinoyl-2-hydroxyacetate.
5.5. Structural Study of the Yeast Reductoisomerase
[0148] The quaternary structure of the yeast reductoisomerase was
studied according to two different approaches: mass spectrometry
and gel filtration.
[0149] The existence of two different states of oligomerization was
demonstrated by means of mass spectrometry under nondenaturing
conditions. This technique showed that the yeast reductoisomerase
is present mainly in dimeric form, but a minor monomeric form is
also present. The dimer is represented by a charge state
distribution [D+18H].sup.18+ to [D+21H].sup.21+. A small presence
of monomer of the yeast reductoisomerase, represented by the charge
states [M+12H].sup.12+ to [M+14H].sup.14+, is demonstrated on this
same mass spectrum. The yeast reductoisomerase could therefore be
in equilibrium between a monomeric form and a dimeric form.
[0150] Application, to gel filtration (Superdex 75), of the pool of
yeast reductoisomerase fractions obtained after the 1.sup.st
purification step shows that the reductoisomerase is eluted in a
single peak and that its molecular mass is estimated at 67 kDa.
Now, the expected molecular mass of the monomeric form of this
enzyme is approximately 40 kDa and 80 kDa for the dimeric form
under nondenaturing conditions. The molecular mass which is
intermediate between the monomeric and dimeric forms of the yeast
reductoisomerase confirms the existence of an equilibrium between
these two forms of the enzyme. Only a rapid dynamic equilibrium
between these two forms could explain a single elution peak being
obtained in gel filtration. Specifically, if this equilibrium was
slow, two elution peaks would have been observed on exiting gel
filtration; one would correspond to the monomeric form, and the
other to the dimeric form of the enzyme.
DOCUMENTS CITED
[0151] All sequences, patents, patent applications or other
published documents cited anywhere in this specification are herein
incorporated in their entirety by reference to the same extent as
if each individual sequence, publication, patent, patent
application or other published document was specifically and
individually indicated to be incorporated by reference.
Sequence CWU 1
1
18 1 402 PRT Magnaporthe grisea TRANSIT (1)..(51) Putative
mitochondrial transit peptide 1 Met Ser Ala Arg Gly Phe Ser Lys Ala
Leu Arg Pro Met Ala Arg Gln 1 5 10 15 Leu Ala Thr Pro Ala Val Gln
Arg Arg Ser Phe Val Ala Ala Ser Ser 20 25 30 Met Val Arg Ala Thr
Arg Lys Ala Ala Val Ala Pro Thr Gln Gln Gln 35 40 45 Ile Arg Gly
Val Lys Thr Met Asp Phe Ala Gly His Lys Glu Gln Val 50 55 60 Trp
Glu Arg Ala Asp Trp Pro Lys Glu Lys Leu Leu Glu Tyr Phe Lys 65 70
75 80 Asp Asp Thr Leu Ala Leu Ile Gly Tyr Gly Ser Gln Gly His Gly
Gln 85 90 95 Gly Leu Asn Leu Arg Asp Asn Gly Leu Asn Val Ile Ile
Gly Val Arg 100 105 110 Lys Asp Gly Lys Ser Trp Lys Asp Ala Val Gln
Asp Gly Trp Val Pro 115 120 125 Gly Lys Asn Leu Phe Glu Val Asp Glu
Ala Ile Ser Arg Gly Thr Val 130 135 140 Ile Met Asn Leu Leu Ser Asp
Ala Ala Gln Ser Glu Thr Trp Pro Ala 145 150 155 160 Leu Lys Pro Gln
Ile Thr Lys Gly Lys Thr Leu Tyr Phe Ser His Gly 165 170 175 Phe Ser
Pro Val Phe Lys Asp Leu Thr Lys Val Glu Val Pro Thr Asp 180 185 190
Val Asp Val Ile Leu Cys Ala Pro Lys Gly Ser Gly Arg Thr Val Arg 195
200 205 Ser Leu Phe Arg Glu Gly Arg Gly Ile Asn Ser Ser Phe Ala Val
Tyr 210 215 220 Gln Asp Val Thr Gly Glu Ala Glu Glu Lys Ala Ile Ala
Leu Gly Val 225 230 235 240 Ala Ile Gly Ser Gly Tyr Leu Tyr Lys Thr
Thr Phe Glu Lys Glu Val 245 250 255 Tyr Ser Asp Leu Tyr Gly Glu Arg
Gly Cys Leu Met Gly Gly Ile His 260 265 270 Gly Met Phe Leu Ala Gln
Tyr Glu Val Leu Arg Glu Arg Gly His Ser 275 280 285 Pro Ser Glu Ala
Phe Asn Glu Thr Val Glu Glu Ala Thr Gln Ser Leu 290 295 300 Tyr Pro
Leu Ile Gly Ala Asn Gly Met Asp Trp Met Tyr Glu Ala Cys 305 310 315
320 Ser Thr Thr Ala Arg Arg Gly Ala Ile Asp Trp Ser Pro Arg Phe Lys
325 330 335 Asp Ala Leu Lys Pro Val Phe Asn Gln Leu Tyr Asp Ser Val
Lys Asp 340 345 350 Gly Ser Glu Thr Gln Arg Ser Leu Asp Tyr Asn Ser
Gln Pro Asp Tyr 355 360 365 Arg Glu Lys Tyr Glu Ala Glu Met Glu Glu
Ile Arg Asn Leu Glu Ile 370 375 380 Trp Arg Ala Gly Lys Ala Val Arg
Ser Leu Arg Pro Glu Asn Gln Lys 385 390 395 400 Gln Lys 2 395 PRT
Saccharomyces cerevisiae TRANSIT (1)..(47) mitochondrial transit
peptide gbX04969 1993-09-12 2 Met Leu Arg Thr Gln Ala Ala Arg Leu
Ile Cys Asn Ser Arg Val Ile 1 5 10 15 Thr Ala Lys Arg Thr Phe Ala
Leu Ala Thr Arg Ala Ala Ala Tyr Ser 20 25 30 Arg Pro Ala Ala Arg
Phe Val Lys Pro Met Ile Thr Thr Arg Gly Leu 35 40 45 Lys Gln Ile
Asn Phe Gly Gly Thr Val Glu Thr Val Tyr Glu Arg Ala 50 55 60 Asp
Trp Pro Arg Glu Lys Leu Leu Asp Tyr Phe Lys Asn Asp Thr Phe 65 70
75 80 Ala Leu Ile Gly Tyr Gly Ser Gln Gly Tyr Gly Gln Gly Leu Asn
Leu 85 90 95 Arg Asp Asn Gly Leu Asn Val Ile Ile Gly Val Arg Lys
Asp Gly Ala 100 105 110 Ser Trp Lys Ala Ala Ile Glu Asp Gly Trp Val
Pro Gly Lys Asn Leu 115 120 125 Phe Thr Val Glu Asp Ala Ile Lys Arg
Gly Ser Tyr Val Met Asn Leu 130 135 140 Leu Ser Asp Ala Ala Gln Ser
Glu Thr Trp Pro Ala Ile Lys Pro Leu 145 150 155 160 Leu Thr Lys Gly
Lys Thr Leu Tyr Phe Ser His Gly Phe Ser Pro Val 165 170 175 Phe Lys
Asp Leu Thr His Val Glu Pro Pro Lys Asp Leu Asp Val Ile 180 185 190
Leu Val Ala Pro Lys Gly Ser Gly Arg Thr Val Arg Ser Leu Phe Lys 195
200 205 Glu Gly Arg Gly Ile Asn Ser Ser Tyr Ala Val Trp Asn Asp Val
Thr 210 215 220 Gly Lys Ala His Glu Lys Ala Gln Ala Leu Ala Val Ala
Ile Gly Ser 225 230 235 240 Gly Tyr Val Tyr Gln Thr Thr Phe Glu Arg
Glu Val Asn Ser Asp Leu 245 250 255 Tyr Gly Glu Arg Gly Cys Leu Met
Gly Gly Ile His Gly Met Phe Leu 260 265 270 Ala Gln Tyr Asp Val Leu
Arg Glu Asn Gly His Ser Pro Ser Glu Ala 275 280 285 Phe Asn Glu Thr
Val Glu Glu Ala Thr Gln Ser Leu Tyr Pro Leu Ile 290 295 300 Gly Lys
Tyr Gly Met Asp Tyr Met Tyr Asp Ala Cys Ser Thr Thr Ala 305 310 315
320 Arg Arg Gly Ala Leu Asp Trp Tyr Pro Ile Phe Lys Asn Ala Leu Lys
325 330 335 Pro Val Phe Gln Asp Leu Tyr Glu Ser Thr Lys Asn Gly Thr
Glu Thr 340 345 350 Lys Arg Ser Leu Glu Phe Asn Ser Gln Pro Asp Tyr
Arg Glu Lys Leu 355 360 365 Glu Lys Glu Leu Asp Thr Ile Arg Asn Met
Glu Ile Trp Lys Val Gly 370 375 380 Lys Glu Val Arg Lys Leu Arg Pro
Glu Asn Gln 385 390 395 3 400 PRT Neurospora crassa TRANSIT
(1)..(53) putative mitochondrial transit peptide gbM84189.1
1996-05-23 3 Met Ala Ala Arg Asn Cys Thr Lys Ala Leu Arg Pro Leu
Ala Arg Gln 1 5 10 15 Leu Ala Thr Pro Ala Val Gln Arg Arg Thr Phe
Val Ala Ala Ala Ser 20 25 30 Ala Val Arg Ala Ser Val Ala Val Lys
Ala Val Ala Ala Pro Ala Arg 35 40 45 Gln Gln Val Arg Gly Val Lys
Thr Met Asp Phe Ala Gly His Lys Glu 50 55 60 Glu Val His Glu Arg
Ala Asp Trp Pro Ala Glu Lys Leu Leu Asp Tyr 65 70 75 80 Phe Lys Asn
Asp Thr Leu Ala Leu Ile Gly Tyr Gly Ser Gln Gly His 85 90 95 Gly
Gln Gly Leu Asn Leu Arg Asp Asn Gly Leu Asn Val Ile Val Gly 100 105
110 Val Arg Lys Asn Gly Lys Ser Trp Glu Asp Ala Ile Gln Asp Gly Trp
115 120 125 Val Pro Gly Lys Asn Leu Phe Asp Val Asp Glu Ala Ile Ser
Arg Gly 130 135 140 Thr Ile Val Met Asn Leu Leu Ser Asp Ala Ala Gln
Ser Glu Thr Trp 145 150 155 160 Pro His Ile Lys Pro Gln Ile Thr Lys
Gly Lys Thr Leu Tyr Phe Ser 165 170 175 His Gly Phe Ser Pro Val Phe
Lys Asp Leu Thr Lys Val Glu Val Pro 180 185 190 Thr Asp Val Asp Val
Ile Leu Val Ala Pro Lys Gly Ser Gly Arg Thr 195 200 205 Val Arg Ser
Leu Phe Arg Glu Gly Arg Gly Ile Asn Ser Ser Phe Ala 210 215 220 Val
Tyr Gln Asp Val Thr Gly Lys Ala Lys Glu Lys Ala Val Ala Leu 225 230
235 240 Gly Val Ala Val Gly Ser Gly Tyr Leu Tyr Glu Thr Thr Phe Glu
Lys 245 250 255 Glu Val Tyr Ser Asp Leu Tyr Gly Glu Arg Gly Cys Leu
Met Gly Gly 260 265 270 Ile His Gly Met Phe Leu Ala Gln Tyr Glu Val
Leu Arg Glu Arg Gly 275 280 285 His Ser Pro Ser Glu Ala Phe Asn Glu
Thr Val Glu Glu Ala Thr Gln 290 295 300 Ser Leu Tyr Pro Leu Ile Gly
Ala His Gly Met Asp Trp Met Phe Asp 305 310 315 320 Ala Cys Ser Thr
Thr Ala Arg Arg Gly Ala Ile Asp Trp Thr Pro Lys 325 330 335 Phe Lys
Asp Ala Leu Lys Pro Val Phe Asn Asn Leu Tyr Asp Ser Val 340 345 350
Lys Asn Gly Asp Glu Arg Lys Arg Ser Leu Glu Tyr Asn Ser Gln Pro 355
360 365 Asp Tyr Arg Glu Arg Tyr Glu Ala Glu Leu Asp Glu Ile Arg Asn
Leu 370 375 380 Glu Ile Trp Arg Ala Gly Lys Arg Ser Leu Arg Pro Glu
Asn Gln Lys 385 390 395 400 4 1356 DNA Magnaporthe grisea 5'UTR
(1)..(43) CDS (44)..(1246) 3'UTR (1247)..(1356) polyA_site
(1322)..(1330) 4 ttgtttttct tggttcctta ttctaccttg tcacacaaca aac
atg tct gct cgc 55 Met Ser Ala Arg 1 ggt ttc tca aag gct ttg agg
cca atg gcc cgc caa ttg gcc act ccc 103 Gly Phe Ser Lys Ala Leu Arg
Pro Met Ala Arg Gln Leu Ala Thr Pro 5 10 15 20 gcc gtt cag agg cgt
acc ttc gtg gct gct tct agc atg gtg cgg gcc 151 Ala Val Gln Arg Arg
Thr Phe Val Ala Ala Ser Ser Met Val Arg Ala 25 30 35 acc agg aaa
gcc gcc gtc gct ccc act cag cag cag atc cgt ggt gtc 199 Thr Arg Lys
Ala Ala Val Ala Pro Thr Gln Gln Gln Ile Arg Gly Val 40 45 50 aag
acc atg gat ttt gct ggc cac aag gag cag gtc tgg gag cgt gcc 247 Lys
Thr Met Asp Phe Ala Gly His Lys Glu Gln Val Trp Glu Arg Ala 55 60
65 gac tgg ccc aag gag aag ctg ctg gag tac ttc aag gac gac acc ctt
295 Asp Trp Pro Lys Glu Lys Leu Leu Glu Tyr Phe Lys Asp Asp Thr Leu
70 75 80 gcc ctc atc ggc tat ggt tcg cag ggc cac ggc cag ggt ctt
aac ctc 343 Ala Leu Ile Gly Tyr Gly Ser Gln Gly His Gly Gln Gly Leu
Asn Leu 85 90 95 100 cgc gac aac ggc ctc aac gtc atc atc ggt gtg
cgc aag gac gga aag 391 Arg Asp Asn Gly Leu Asn Val Ile Ile Gly Val
Arg Lys Asp Gly Lys 105 110 115 tcg tgg aag gac gcc gtc cag gac ggc
tgg gtt ccc ggc aag aac ctc 439 Ser Trp Lys Asp Ala Val Gln Asp Gly
Trp Val Pro Gly Lys Asn Leu 120 125 130 ttc gag gtc gac gag gcc atc
tcg cgc ggt acc gtc atc atg aac ctt 487 Phe Glu Val Asp Glu Ala Ile
Ser Arg Gly Thr Val Ile Met Asn Leu 135 140 145 ctg agc gac gct gcc
cag agc gag acg tgg cct gct ctg aag ccc cag 535 Leu Ser Asp Ala Ala
Gln Ser Glu Thr Trp Pro Ala Leu Lys Pro Gln 150 155 160 atc act aag
ggc aag act ctc tac ttc tcg cac ggt ttc tct ccc gtc 583 Ile Thr Lys
Gly Lys Thr Leu Tyr Phe Ser His Gly Phe Ser Pro Val 165 170 175 180
ttc aag gac ctc acc aag gtc gag gtc ccc acc gac gtc gac gtc atc 631
Phe Lys Asp Leu Thr Lys Val Glu Val Pro Thr Asp Val Asp Val Ile 185
190 195 ctc tgc gcc ccc aag ggc tcc ggc cgc act gtc cgc tcg ctc ttc
cgc 679 Leu Cys Ala Pro Lys Gly Ser Gly Arg Thr Val Arg Ser Leu Phe
Arg 200 205 210 gag ggt cgt ggc atc aac tcc tcc ttc gcc gtc tac cag
gac gtg act 727 Glu Gly Arg Gly Ile Asn Ser Ser Phe Ala Val Tyr Gln
Asp Val Thr 215 220 225 ggc gag gct gaa gag aag gct atc gct ctc ggt
gtt gcc att ggc agt 775 Gly Glu Ala Glu Glu Lys Ala Ile Ala Leu Gly
Val Ala Ile Gly Ser 230 235 240 ggt tac ctc tac aag acc acc ttc gag
aag gag gtc tac tct gac ctg 823 Gly Tyr Leu Tyr Lys Thr Thr Phe Glu
Lys Glu Val Tyr Ser Asp Leu 245 250 255 260 tac ggt gag cgt ggc tgc
ctg atg ggt ggt atc cac ggt atg ttc ctt 871 Tyr Gly Glu Arg Gly Cys
Leu Met Gly Gly Ile His Gly Met Phe Leu 265 270 275 gcc cag tac gag
gtt ctc cgc gag cgt ggc cac agc ccc tcg gag gct 919 Ala Gln Tyr Glu
Val Leu Arg Glu Arg Gly His Ser Pro Ser Glu Ala 280 285 290 ttc aac
gag act gtc gag gag gcc acc cag tct ctc tac ccc ctg atc 967 Phe Asn
Glu Thr Val Glu Glu Ala Thr Gln Ser Leu Tyr Pro Leu Ile 295 300 305
ggt gcc aac ggc atg gac tgg atg tac gag gcc tgc tct acc act gct
1015 Gly Ala Asn Gly Met Asp Trp Met Tyr Glu Ala Cys Ser Thr Thr
Ala 310 315 320 cgt cgt ggt gcc att gac tgg agc ccc cgc ttc aag gac
gcc ctc aag 1063 Arg Arg Gly Ala Ile Asp Trp Ser Pro Arg Phe Lys
Asp Ala Leu Lys 325 330 335 340 ccc gtc ttc aac cag ctc tac gac tcg
gtc aag gac ggc tct gag act 1111 Pro Val Phe Asn Gln Leu Tyr Asp
Ser Val Lys Asp Gly Ser Glu Thr 345 350 355 cag cgc tcg ctc gac tac
aac agc cag ccc gac tac cgc gag aag tac 1159 Gln Arg Ser Leu Asp
Tyr Asn Ser Gln Pro Asp Tyr Arg Glu Lys Tyr 360 365 370 gag gcc gag
atg gag gag atc cgc aac ctg gag atc tgg agg gcg ggt 1207 Glu Ala
Glu Met Glu Glu Ile Arg Asn Leu Glu Ile Trp Arg Ala Gly 375 380 385
aag gct gtg cgc agc ctc cgt cct gag aac cag aag taa actgtatatt 1256
Lys Ala Val Arg Ser Leu Arg Pro Glu Asn Gln Lys 390 395 400
tgcttccaag ttccggttaa atgccagtgg atctctacag agtgctggtg ggtggtcaat
1316 ttgttgaaaa ataatctgga gtcatcgcta catttcttgg 1356 5 400 PRT
Magnaporthe grisea 5 Met Ser Ala Arg Gly Phe Ser Lys Ala Leu Arg
Pro Met Ala Arg Gln 1 5 10 15 Leu Ala Thr Pro Ala Val Gln Arg Arg
Thr Phe Val Ala Ala Ser Ser 20 25 30 Met Val Arg Ala Thr Arg Lys
Ala Ala Val Ala Pro Thr Gln Gln Gln 35 40 45 Ile Arg Gly Val Lys
Thr Met Asp Phe Ala Gly His Lys Glu Gln Val 50 55 60 Trp Glu Arg
Ala Asp Trp Pro Lys Glu Lys Leu Leu Glu Tyr Phe Lys 65 70 75 80 Asp
Asp Thr Leu Ala Leu Ile Gly Tyr Gly Ser Gln Gly His Gly Gln 85 90
95 Gly Leu Asn Leu Arg Asp Asn Gly Leu Asn Val Ile Ile Gly Val Arg
100 105 110 Lys Asp Gly Lys Ser Trp Lys Asp Ala Val Gln Asp Gly Trp
Val Pro 115 120 125 Gly Lys Asn Leu Phe Glu Val Asp Glu Ala Ile Ser
Arg Gly Thr Val 130 135 140 Ile Met Asn Leu Leu Ser Asp Ala Ala Gln
Ser Glu Thr Trp Pro Ala 145 150 155 160 Leu Lys Pro Gln Ile Thr Lys
Gly Lys Thr Leu Tyr Phe Ser His Gly 165 170 175 Phe Ser Pro Val Phe
Lys Asp Leu Thr Lys Val Glu Val Pro Thr Asp 180 185 190 Val Asp Val
Ile Leu Cys Ala Pro Lys Gly Ser Gly Arg Thr Val Arg 195 200 205 Ser
Leu Phe Arg Glu Gly Arg Gly Ile Asn Ser Ser Phe Ala Val Tyr 210 215
220 Gln Asp Val Thr Gly Glu Ala Glu Glu Lys Ala Ile Ala Leu Gly Val
225 230 235 240 Ala Ile Gly Ser Gly Tyr Leu Tyr Lys Thr Thr Phe Glu
Lys Glu Val 245 250 255 Tyr Ser Asp Leu Tyr Gly Glu Arg Gly Cys Leu
Met Gly Gly Ile His 260 265 270 Gly Met Phe Leu Ala Gln Tyr Glu Val
Leu Arg Glu Arg Gly His Ser 275 280 285 Pro Ser Glu Ala Phe Asn Glu
Thr Val Glu Glu Ala Thr Gln Ser Leu 290 295 300 Tyr Pro Leu Ile Gly
Ala Asn Gly Met Asp Trp Met Tyr Glu Ala Cys 305 310 315 320 Ser Thr
Thr Ala Arg Arg Gly Ala Ile Asp Trp Ser Pro Arg Phe Lys 325 330 335
Asp Ala Leu Lys Pro Val Phe Asn Gln Leu Tyr Asp Ser Val Lys Asp 340
345 350 Gly Ser Glu Thr Gln Arg Ser Leu Asp Tyr Asn Ser Gln Pro Asp
Tyr 355 360 365 Arg Glu Lys Tyr Glu Ala Glu Met Glu Glu Ile Arg Asn
Leu Glu Ile 370 375 380 Trp Arg Ala Gly Lys Ala Val Arg Ser Leu Arg
Pro Glu Asn Gln Lys 385 390 395 400 6 3827 DNA Magnaporthe grisea
promoter (1)..(1460) 5'UTR (1461)..(1503) intron (1700)..(1781)
intron (1814)..(1890) intron (2170)..(2255) 3'UTR (2952)..(3061)
terminator (3062)..(3827) 6 ccgagtttcc cgaccccgac gacatcctca
actttaggct tatcatcgag cccggcgagg 60 gcatgtaccg cggcgggcga
ttcactttcg acttcaagat taaccaaaac ttcccgcatg 120 agccaccaaa
ggtcttgtgt gaacagaaga tctaccatcc caatatcgat ctagagggca 180
aggtttgcct gaacattctg cgtgaggatt ggaagccggt cctgaacctc aacgccgtca
240 ttgttggtct tcaggtacgt ccaaatatag ctttgatgcc cgacctgtta
catcctatat 300 gtgacacaac atactcaaaa gactcgtagc tctatacgtg
agcagtggca caatgtaaaa 360 caactggaat cagaaaacgc taaccagacc
acttcgtgtc gtcttagttc cttttcctcg 420 agcccaacgc ctcggacccg
ctgaacaagg acgcggctga agacctcaga
agcaaccggg 480 agggcttcaa acgtaacgtc cggacctcca tgggcggagg
ggcagtcaag gggatttctt 540 tcgatcgcgt tctgaaataa agataggcgg
aaggaaggca aactctgctg caaagagaac 600 atgataccct cctactaaga
ctcaagagtc gcaacctgtg ccatgggaac gatcatttct 660 atcccgaccg
cgaaacctac gcgtcggcgt cccagcggtg gctatgagtg agtcatctgg 720
atgcctggaa cattcaggac aatagacccc agggagtcat ctcagtgccc agaaggcaaa
780 catggttgaa tgcgagaata agccggggag gtgaatggat ggcgacgagg
gttttctcgc 840 tggccattgg acttcctaga cctagttcta aatacggagt
atcggaacat aagatgggtg 900 gggcaagcgg tgtcgatttt tagcgatttc
ggcggtctgt attttcaaca ttggcggttt 960 ctgctttctt atttcaccct
ctattaagat ctacctcatc tccccaataa atacataaat 1020 ctagatcgtt
ttcatgtctt taagaacata gattgaggct ttttgtacgt gaatggctgg 1080
tttcaaaaga atgactcgat tggctgagta atgaatttta tctcgtggtt gttattatgg
1140 aattaggcga aagcctatcc ggtcgtcgtc gataacgctg acccaagagg
cttctccggt 1200 ttggagagct gctggttctt ggtagatcaa agcggggcat
ctgggtctgg ggctccgtta 1260 tgcgggcggt ctccaccgga gctaccctgc
ttttcaaaac ggtccgatat cgctggccta 1320 ggaaaatttt gtacccacaa
tgcctaagtc ggagctgctc cttagtgttg agttttgttg 1380 aagctcgctc
ctaaattcat cttcattcga ctttccattg tcggtgaata cggattccct 1440
ttctctccat ctccactcaa ttgtttttct tggttcctta ttctaccttg tcacacaaca
1500 aacatgtctg ctcgcggttt ctcaaaggct ttgaggccaa tggcccgcca
attggccact 1560 cccgccgttc agaggcgtac cttcgtggct gcttctagca
tggtgcgggc caccaggaaa 1620 gccgccgtcg ctcccactca gcagcagatc
cgtggtgtca agaccatgga ttttgctggc 1680 cacaaggagc aggtctgggg
tgagttggac agctcaattg ctcaattcgc ggctcaatga 1740 attcgtaaac
tgacaggctt tttttcggtc taccaatcta gagcgtgccg actggcccaa 1800
ggagaagctg ctggtgagtc atgtcatttt tttttgcctg acaattccac caacttcaag
1860 cagtcaaaat actaatcact tgaactacag gagtacttca aggacgacac
ccttgccctc 1920 atcggctatg gttcgcaggg ccacggccag ggtcttaacc
tccgcgacaa cggcctcaac 1980 gtcatcatcg gtgtgcgcaa ggacggaaag
tcgtggaagg acgccgtcca ggacggctgg 2040 gttcccggca agaacctctt
cgaggtcgac gaggccatct cgcgcggtac cgtcatcatg 2100 aaccttctga
gcgacgctgc ccagagcgag acgtggcctg ctctgaagcc ccagatcact 2160
aagggcaagg tatgtggcgc ttaagactga ccgtttcttt ttacttaccc cgctgcttta
2220 taagaataaa aaagagctaa caagtcttta tgtagactct ctacttctcg
cacggtttct 2280 ctcccgtctt caaggacctc accaaggtcg aggtccccac
cgacgtcgac gtcatcctct 2340 gcgcccccaa gggctccggc cgcactgtcc
gctcgctctt ccgcgagggt cgtggcatca 2400 actcctcctt cgccgtctac
caggacgtga ctggcgaggc tgaagagaag gctatcgctc 2460 tcggtgttgc
cattggcagt ggttacctct acaagaccac cttcgagaag gaggtctact 2520
ctgacctgta cggtgagcgt ggctgcctga tgggtggtat ccacggtatg ttccttgccc
2580 agtacgaggt tctccgcgag cgtggccaca gcccctcgga ggctttcaac
gagactgtcg 2640 aggaggccac ccagtctctc taccccctga tcggtgccaa
cggcatggac tggatgtacg 2700 aggcctgctc taccactgct cgtcgtggtg
ccattgactg gagcccccgc ttcaaggacg 2760 ccctcaagcc cgtcttcaac
cagctctacg actcggtcaa ggacggctct gagactcagc 2820 gctcgctcga
ctacaacagc cagcccgact accgcgagaa gtacgaggcc gagatggagg 2880
agatccgcaa cctggagatc tggagggcgg gtaaggctgt gcgcagcctc cgtcctgaga
2940 accagaagta aactgtatat ttgcttccaa gttccggtca aatgccagtg
gatctctaca 3000 gagtgctggt gggtggtcaa tttgttgaaa aataatctgg
agtcatcgct acatttcttg 3060 gaatatcgcg gggttctgtg tccaaaaagc
ttgcggtatt tcgatcgggt ttgcttttaa 3120 tgtttatcag atttcatctt
tgtctgggat tacatcagtc tactatacct cgccatttta 3180 aattggtatc
tttttttcgt ctttacctgc ttattcaact gtccccatgc tactgtgccg 3240
cttttgattc atcttctcca tggcgccttg gcagttgttt tgcgagttcc acaaaacctc
3300 cttcttggtc accaccaagg gtgatccaaa tgtcaaccca tcgccgggca
tcctcgtagc 3360 gctcttcgga cctaatccat tcttcctcac ccacgcgctc
aaggtatacg gtacgcaagc 3420 cgcaggcttt tgcgcccttg aggtcgccta
agtgggcagc aaccatggca acctcatccg 3480 ggcgcaggcc caagccctca
acagctccca agtacgtcct tgggtttggc ttgtatgcac 3540 caaagtcctc
agccgagaag agacgatcga accccaggcc atcgtggtag gtgttgaggt 3600
cctgcagcag agactggttg ccgttagaca aggcggccgt gaccagaccc cggctcgcca
3660 gccgcgcgag accttgacgg gtatcgggcc agggctccag cctatgccac
gcccgactca 3720 actccagcac ctcatcgtcc gtgaacaggc cggctagtcc
tcgctcctcg agcagacgct 3780 cgaggctctc gcggtggtgc gtgtcaatgt
ccttccaggg cgtgatg 3827 7 20 DNA Artificial sequence Description of
the artificial sequence primer 1 7 gaytayttya araaygayac 20 8 20
DNA Artificial sequence Description of the artificial sequence
primer 2 VARIANT (9)...(12) n = a, c, g, or t 8 atgttyytng
cncartayga 20 9 20 DNA Artificial sequence Description of the
artificial sequence primer 3 VARIANT (6)...(18) n = a, c, g, or t 9
gayggntggg tnccnggnaa 20 10 21 DNA Artificial sequence Description
of the artificial sequence primer 4 VARIANT (6)...(18) n = a, c, g,
or t 10 atgggnggna tacayggnat g 21 11 29 DNA Artificial sequence
Description of the artificial sequence primer 2' 11 ctgcttccat
ggcaccagct gcccgtttc 29 12 29 DNA Artificial sequence Description
of the artificial sequence primer 1' 12 ctacccccat ggtgaagcaa
atcaacttc 29 13 36 DNA Artificial sequence Description of the
artificial sequence primer 3' 13 gcacttgata ttattgtcga ctttattggt
tttctg 36 14 20 DNA Artificial sequence Description of the
artificial sequence primer 13U 14 aacgacaccc ttgccctcat 20 15 20
DNA Artificial sequence Description of the artificial sequence
primer 300U 15 accgtttctt tttacttacc 20 16 20 DNA Artificial
sequence Description of the artificial sequence primer 549L 16
gcgatagcct tctcttcagc 20 17 20 DNA Artificial sequence Description
of the artificial sequence primer 22U 17 ttgtttttct tggttcctta 20
18 20 DNA Artificial sequence Description of the artificial
sequence Primer 1603L 18 ccaagaaatg tagcgatgac 20
* * * * *