Note: Descriptions are shown in the official language in which they were submitted.
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Description
Utilization of an antifungal protein from Streptomyces
tendae 'ag<~inst plant pathogenic fungi
The present invention relates to the use of an
antifunga=_ protein (AFP) having a molecular weight of
approx. 10 kDa from Streptomyces tendae against plant
pathogenic: fungi fr.~om the Ascomycetes family.
Antifungal. proteins have been described for some time,
some of these proteins also being termed PR proteins
(plant pathogenesis--related proteins) in the case of
plants (Bol, J.F. & Linthorst, H.J.M. (1990), Annu.
Rev. Phytopathol., 28, 113). These proteins are formed
by plants under strc=ss conditions, for example in the
case of viral or fungal infection, inducing an active
defense mechanism of the plant which is termed "induced
resistance" (Lindhorst, H.J.M., (1991) Cri. Rev. Plant
Sci., 10(2), 123). :3ome of these proteins have, for
example, a chitin<~se activity or (3-1,3-glucanase
activity.
Proteins with an antifungal activity have already been
detected from microorganisms too, some of these
proteins also showing chitinase activity or (3-1,3-
glucanase activity. Other proteins, in contrast, act
via an interaction ofthe fungal cell wall.
For example, an antifungal protein (AFP) having a size
of approx. 10 kDa which is active against the fungal
species Paecilomyce;s varriotii, Byssoclamis nivea,
Pencillium puberulum and Eupenicillium terrum has been
isolated from Streptomyces tendae. However, the action
was restricted to these species. Other species such as,
for example, Paec.ilomyces carneus, Paecilomyces
lilacenus, PeniciLlium chrysogenum or Penicillium
claviforme are not inhibited. In addition, the use of
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AFP is not justified commercially either since the
fungal species mentioned are not plant pathogenic.
The object of the present invention was therefore to
find an antifungal protein which is active against
plant pathogenic fungi.
Surprisin~~ly, it has now been found that AFP from
Streptomy~~es tendae is active against plant pathogenic
fungi from the Ascomycetes family.
The subject-matter of present the invention is
therefore the use of- an antifungal protein (AFP) with a
molecular weight of approx. 10 kDa from Streptomyces
tendae against plant pathogenic fungi from the
AscomycetE~s family, in particular against Botrytis
cinera.
The antif:ungal prot=ein mentioned contains a typical
amino-terminal. signal sequence, i.e. a hydrophilic
N-terminu;~ is followed by a hydrophobic transmembrane
region, which cause extracellular secretion. Moreover,
two form, are known, namely a shorter form with a
molecular weight o:~ approx. 9 860 Da and a longer form
with a moJ_ecular weight of approx. 10 300 Da.
Since AFP is secreted by Streptomyces tendae into the
culture medium, it can be isolated for example directly
from the' Streptomyces tendae culture filtrate,
following methods k:gown to the skilled worker. As an
alternati~re, AFP can be produced by genetic
engineering, the genre isolated preferably being cloned
into a so-called multicopy vector, for example p1J702
(Hopwood, D.A. et a,_. (1985) Genetic Manipulation of
Streptomyc:es. A Laboratory Manual. Norwich, U.K.) and
using this construc=tion to transform a suitable strain,
for example thF~ non-nikk;~mycin-producing strain
Streptomyc:es tendac: NP9. An example of an AFP-encoding
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nucleic acid is shown in SEQ ID No. 1. When this
preferred transform<~nt was grown, for example, for 7
days in :?00 ml of nutrient solution, approx. 6 mg of
AFP were obtained, whi~~h corresponds to 30 mg of AFP/1.
The production of AFP by genetic engineering via a
preferably controlled fermentation, is therefore
especially preferred.
For the use in accordance with the invention, AFP can
be applied directly for example in the form of a
formulation together with preferably at least one
additiona7_ auxiliary. To this end, for example, plants
attacked by fungi or endangered by fungal attack are
sprayed with the formulation described. The AFP-
concentration of the formula i.s generally from approx.
10 ~g/ml up to approx. 500 mg/ml. Suitable auxiliaries
are, for example, protease inhibitors, stabilizers such
as glycerol, sucro:~e, salts, solvents or generally
known substances from the field of crop protection.
So-called transgenic plants which are capable of
producing AFP themselves are another possibility of
providing protection against fungal infection.
Another embodiment :is therefore the use according to
the invention of AFP, the AFP being formed in a
transgenic plant. 'rhe transgenic plant is, preferably,
genetically engineered maize, cotton, potato, banana,
Arabidopsis, casava, tobacco, oilseed rape (canola),
potato, sugar beet, cereals such as, for example,
wheat, barley or oats, strawberries, vegetables such
as, for example, cabbage, legumes such as, for example,
peas or beans, tomato, lettuce or melon.
To generate a transgenic plant, a nucleic acid encoding
AFP, for example ir_ 'she form of naked DNA, viral DNA or
RNA, or in the form of plasmid DNA, is introduced into
a plant cell. An e:Kample of an AFP-encoding nucleic
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acid is ~;hown in SEQ ID No. 1. However, the present
invention also covers those nucleic acids which differ
from the nucleic acid sequence of SEQ ID No. 1 owing to
the degeneracy of the genetic code, but which encodes
the same AFP amino acid sequence. Moreover, the
invention covers thaw mutants or variants of the
nucleic acid of SE;Q ID No. 1 which encode an AFP
protein which is active against plant pathogenic fungi
of the Ascomycetes family, in particular against
Botrytis cinera. 'Ih.ese include, for example, fusion
proteins of the AFP ;pratein with other foreign proteins
or an AFP protein with an N-terminally deleted
methionine. Other examples of variants are nucleic
acids which hybridize an the stringent conditions with
the nucleic acid of SEQ ID No. 1. The stringent
hybridization conditior~s can be determined for example
by Sambrook, J. et al. in Molecular Cloning, A
Laboratory Manual, 2nd Edition, Cold Spring Habour
Laboratory Press, 1989. Transgenic plants are
subsequently regenerated from the transformed plant
cells. Preferably, an AFP-encoding nucleic acid can be
introduced into the plant cell by means of recombinant
agrobacteria, by e:lE>ctroporation, by bombardment with
microparticles and/or by means of polyethylene glycol.
The infection with recombinant Agrobacterium
tumefaciens bacteria in plant cells of dicots is
described, for examp7_e~, by Klee, H. et al.. (1997) Annu.
Rev. Plant Physiol. 38, 467 or EP-B2-0122791). The
infection with re:ombinant Agrobacterium tumefaciens
bacteria in plant cells of monocots is described, for
example, by Ishida, 'Y. et al. (1996) Nature Biotech.
14, 745 with reference to maize (Zea mays L.). The
agrobacteria used fcr this purpose have a T-DNA into
which the AFP-expressing gene and, if appropriate, a
suitable promoter, for example a plant phaseolin
promoter (see, for example, EP-B2-0122791) or a viral
35S promoter (see, fcr example, Ishida, Y. et al.
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(1996), above) had been inserted. The T-DNA can be
transferred into plant cells for example by coculturing
the recornbinant agrobacteria together with immature
embryos of the plant in question (see, for example,
Ishida, '~. et a1. (1996), above). To select the
transformed plant cc=_lls, it is preferred to use T-DNA
construct; which are capable of expressing, in the
plant cell, a resistance gene from the AFP gene to be
expressed.. A suitable resistance gene is, for example,
the gene encoding phosphinothricin acetyltransferase
(see, for example, Ishida, Y. et al. (1996}, above).
Thus, successfully transformed plant cells can be
selected for example by means of the corresponding
antibiotic phosphinothricin. The regeneration of a
plant from transformed plant cells is generally known
(see, for example, ~~agi et al. (1995), Nature Biotech,
13, 481-485, Ishida, Y. et al. (1996), above, or
Schopke et al. (1996;x, Nature Biotech, 14, 731-735.
In addition to the use of Agrobacterium tumefaciens as
transformation means, other transformation methods are
known and suitable, such as, for example, electro-
poration, the bombardment with microparticles or the
use of polyethylene glycol (see, for example, Potrykos,
(1991) Annu. Rev. Plant Physiol. Mol. Biol., 42, 205;
Estruch, J.J. et al. (1997), Nature Biotech., 15, 137-
141 or Dingermann, 'T. (1995) BIOforum, 18, 252). For
example, casava (Schopke et al. (1996), above) or
banana (Sagi, et a:l. (1995), above) have already been
transformed by bombarding plant cells with DNA-coated
particles. In this method too, the AFP gene to be
expressed is cloned into a suitable vector which
preferably has a resistance gene allowing the
subsequent selection of the transformed plant cells.
The recombinant erector is preferably applied to
tungsten particles with which, for example, suspensions
of embryogenic cel~.s are bombarded (see, for example,
Sagi, et al. (195), above). The cell suspensions
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treated thus are sL.bsequently selected for transformed
plant ce7.ls using a suitable antibiotic, for example
phosphinothricin, and a transgenic plant is regenerated
therefrom, for example as already described above.
A considerable advantage of the present invention is
that AFP is active against plant pathogenic fungi from
the Ascomycetes family, in particular against Botrytis
Cinera, which have .already developed a resistance in
particular to one or more crop protection agents.
The examples which follow and the figure are intended
to describe the invention in greater detail without
imposing any limitation:
Sequence description
SEQ ID No. 1 shows a nucleic acid sequence encoding
an ar~tifungal (223) protein (AFP) from
Streptomyces tendae.
Examples
Example 1:
To prepare test plates, 50 ~1 of a spore suspension of
Botrytis cinerae were added to 100 ml of test agar
(20 g/1 malt extra~~t, 10 g/1. glucose, 2 g/1 yeast
extract, 0.5 ammor~:ium sulfate, i5 g/1 agar, pH 6.0).
25 ml of this were poured into Petri dishes. The final
spore concentratiorv was 105 spores/ml. Sterile anti-
biotic test disks (Sc:~leicher and Schuell, Dassel, Frg)
0.5 cm in diameter were subsequently applied to a test
plate. Then, 5, 10, 15 and 20 ail of the AFP solution
(100 mg/ml lyophili.z~~d culture filtrate of S. tendae-
transformants) were subsequently pipetted onto the test
disks and the test plate was incubated for 4 days at
30°C. The diameter of the inhibitory zone was 0.7 cm
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for 5 ~1 of AFP solution, 1 cm diameter for 10 ~1,
1.3 cm for 15 ul ofsolution and 1.9 cm for 20 ~1.
Example 2:
To prepare test plates, 50 ~l of a spore suspension of
Botrytis cinerae ZF 3629 (resistant to BCM (Benlat,
BenomylTM, Hoechst Sc:~ering AgrEvo GmbH, Frankfurt,
carbendazem) were added to 100 ml of test agar (20 g/1
malt extract, 10 g,Jl glucose, 2 g/1 yeast extract, 0.5
ammonium :sulfate, 15 g/1 agar, pH 6.0). 25 ml of this
were pou:=ed into Petri dishes. The final spore
concentration was :LOS spores/ml. Sterile antibiotic
test disks. (Schleic:h~=r and Schuell, Dassel, Frg) 0.5 cm
in diameter were swbsequently applied to a test plate.
Then, 5, 10, 15 and 20 ~1 of the AFP solution
(100 mg/ml lyophilized culture filtrate of S. tendae-
transformants) were subsequently pipetted onto the test
disks and the test plate was incubated for 4 days at
30°C. The diameter of the inhibitory zone was 0.9 cm
for 5 ~tl of AFP solution, 1.3 cm diameter for 10 ul,
1.9 cm for 15 ~1 of ~~olution and 2.2 cm for 20 ul.
Example 3 - Improved production by fermentation:
In a flask equipped with a hose, 100 ml of the
preculture medium (103 g/1 sucrose, 20 g Tryptic Soy
Broth (Oxoid, Wesel), 10 g/1 MgCl~ and 10 g/1 yeast
extract) were inoculated with an agar section of strain
S. tendae. The culture was grown for 5 days at 28°C at
200 rpm on an orbit:~l shaker.
10 1 of the production medium (103 g/1 sucrose, 20 g
Tryptic Soy Broth (Uxoid, Wesel), 10 g/1 MgCl2, 10 g/1
yeast extract, 100 ~Cl desmophen, pH 7.5) were
inoculated with 7_00 ml of the above-described
preculture.
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The fermenter employed was a Biostat E (Braun,
Melsungenl . Fermentation was carried out for 4 days at
28°C, 700 rpm and 0.5 vvm of air. Thereupon,
fermentation was stopped, and the culture supernatant
was harvested by cE=_ntrifugation at 3000 rpm and 4°C.
The batch was subsequently dried by lyophilization. It
was possible to employ the powder directly. If 15 ~l of
the lyophilizate (1.00 mg/ml) were employed against
Botrytis cinerae in an inhibitory-zone test, an
inhibitory zone of 1.7 cm and, against Botrytis cinerae
ZF 3629, an inhibitory zone of 2.3 cm were detected.
