Note: Descriptions are shown in the official language in which they were submitted.
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PePtide with Inhibitory Activity Towards Plant Pathogenic Fungi
This invention reiates to a method of controlling plant pathogens by a peptide which is
provided by genetically modifying the plant to produce the peptide, and to genes and plants
useful in that method. In particular, this invention relates to a lactofe!ricin and its use in
controlling plant pathogens.
Numerous fungi and bacteria are serious pests of economically-important agricultural
crops. Various methods of controlling dise~ses in plants have ~een used with varying
degress of sucess. One method has been to apply an antimicrobial chemical to crops. This
method has numerous, art-recognized problems. Alternatively, a more recent method
involves the use of biological control organisms ("biocontrol") which are natural competitors
or inhibitors of the pest organism. However, it is difficult to apply biocontrol to large areas,
and even more difficult to cause those living organisms to remain in the treated area for an
extended period of time. More recently, techniques in recombinant DNA have provided the
opportunity to insert into plant cells cloned genes which express antimicrobial compounds.
However, this technology has given rise to concerns about eventual microbial resistance to
well-known, naturally occurring antimicrobials. Thus, a continuing need exists to identify
naturally occurring antimicrobial compounds which can be formed by plant cells directly by
translation of a single gene.
It is particularly interesting to identify novel peptides and DNA sequences encoding
said peptides since peptides having antipathogenic and antimicrobial activity are already
known in the art. For example, there are many so-called Iytic peptides from animal, plant,
insect, and microbial sources including the mammalian defensins, cecropins, thionins,
mellitins, insect defensins, and the like.
Another class of peptide with antipathogenic activity is represented by hydrolytic
enzymes such as chitinase and ~-1,3-glucanase which are known to inhibit fungal growth
(Schlumbaum, et al., 1986; Mauch, et al., 19B8).
Enzymatic hydrolysates of the milk protein lactoferrin are also shown to have
antimicrobial properties. Lactoferrin is an iron binding glycoprotein present in most
biological fluids of mammals, including milk, tears, and mucous secretions (Brock, J., Arch.
Dis. Child. 55: 417 (1980); Bullen, J. Rev. Infect. Dis. 3: 1127 (1981)). Lactoferrin has
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been ~ssoci~ted with the promotion of cell growth, regulation of hematopoiesis, and
protection against microbial infection and immune modulating properties in vitro. Little is
known, however, about the function of la.;tofer,i" in vivo. Extracellular lactoferrin has been
reported to have antibacterial and antiviral activities. Work with bovine lactoferrin has found
that the anlil,liclobial activity of an enzymatic hydrolysate generated by digestion with
porcine pepsin is stronger than that of the whole protein against an Escherichia coli Iysate
(Tomita, et al., J. Dairy Sci. 74:4137-4142 (1991).
The antifungal activity of lactoferrin has also been demonstrated in humans. Thesusceptibility of Candida al~fcans to inhibition and inactivation by la~toferl icin B has been
examined by Bellamy et al., Med. Microbiol. lmmunolo4. 182: 97-105 (1993). Its effect is
lethal, causing a rapid Ioss of colony-forming capability, suggesting that the active peptides
of lactoferrin could potentially contribute to the host defense against C. albicans.
Hurnan laclo~e, ril, cDNA was recently expressed in tob~cco suspension cells to test the
antibacterial activity of la~arl in in plants. Transgenic calli produced a single peptide of 48 kD
which is significantly smaller than the full length la~;t l~r,i" protein. Total protein extracts made
from transgenic tobAcco callus exhibited much higher antibacterial activity than commercially
available purified lactoferrin as determined by the decrease of colony forming units when tested
with four phytopathogenic species of bacteria. The full length lactoferrin was never detected in
transgenic calli implying that the lac~oferli" gene product had undergone posttranslational
processing. Since a human lactoferrin gene was expressed in Aspergfllus nidulans with the
production of large amounts of full-length lactoferrin, it was theorized that full length plant-
produced lactoferrin does notundergo proper folding and the unfolded part is degraded. The
truncated protein exhibiting antibacterial activity in calli was identified as the amino- or carboxy-
terminal end of the protein.
The present invention provides an enzymatic hydrolysate of lactoferrin that can be
safely expressed in plants to provide dise~se resistance. Genetic constructs colllprisi"g a
synthetic gene for lactoferricin are prepared and used to provide transgenic disease-
resistant plants. A method of transforming plants to express la~toferll..in which is
expressed in plants to provide disease resistance to those plants is also described.
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Figure 1: Plasmid pClB7703, containing the lactoferricin B gene (from position 199~-2079)
under control of the ubiquitin promoter (15-1995) and the nos terminator (207~-
2339).
Figure 2: Plasmid pClB7704, simi~ar to pClB7703 with the ubi-SynPat-nos selection
cassette (from position 15-2845).
Figure 3: Plasmid 7705, containing the ubi-SynPat-nos -- ubi-lactoferricin B-nos fragment in
an Nptll containing plasmid.
Figure 4: Plasmid 7706, containing the ubi-lactoferricin B-nos fragment in an Nptll
containing plasmid.
The present invention utilizes lactoferricins, which are enzymatic hydrolysates of
lactoferrins having antipathogenic activity and corresponding to the N-terminal region of a
lactoferrin. Lactoferrins are proteins produced in mammalian milk which have structural and
functional homologies among the different species, although there is some variation in the
precise amino acid sequence from species to species. For use in the present invention, the
preferred lactoferricins include lactoferricin B (bovine lactoferricin) or lactoferricin H (human
lactoferricin). Lactoferricins are now found to inhibit the growth of a number of
agronomically important pathogens and to be capable of expression in plants thereby
conferring disease resistance.
Lactoferricin B (or bovine lactoferricin) is a peptide of 2~ amino acids long:
Phe-Lys-Cys-Arg-Arg-Trp-Gln-Trp--Arg-Met-Lys-Lys-Leu-Gly-Ala-Prc-
Ser-Ile-Thr-Cys-Val-Arg-Arg-Ala-Phe (SEQ ID NO:1)
having exact homology with the N-terminus of the whole bovine lactoferrin sequence.
Lactoferricin H (or human lactoferricin) corresponds to amino acids 1-33 of human
lactoferrin, and is thus a 33 amino acid peptide of sequence as follows:
G R R R R S V Q W C A V S Q P E A T K C E Q W Q R N M R K V R G P
(SEQ ID NO: 2).
When expressed, these peptides may include an N-terminal methionine residue
corresponding to a start codon.
Based on the protein sequence, a lactoferricin gene is designed for expression in
planta. The codon usage of this synthetic gene is optimized for expression in monocot
plants, by using codons for each of the amino acids that are used most frequently in maize
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expressed genes. The maize codon usage table has been described by Murray, Lotzer and
Eberle, Nucl. Acids Res. 17,477-498,1989. Altematively, the codon usage of the synthetic
gene can be optimized following different strategies such as the one described in
EP-A-359 472.
The designed sequence for the plant optimized lac~uler.ic;, I B gene comprises the
following coding sequence:
5'-TTC-AA~-TGC-CGC-CGC-TGG-CAG-TGG-CGC-ATG-AAG-AAG-CTG-GGC-GCC-CCC-
AGC-ATC-ACC-TGC-GTG-CGC-AG&-GCC-TTC-3'(SF~ ID NO:3)
When a stop codon (TAA)iS added to the 3' end, the sequence is as follows:
5'-TTC-AAG-TGC-CGC-CGC-TGG-CAG-TGG-CGC-ATG-AAG-AAG-CTG-GGC-GCC-CCC-
AGC-ATC-ACC-TGC-GTG-CGC-AGG-GCC-TTC-TAA-3' (SEQ ID NO:4)
The plant optimized sequence for the lactoferricin H gene is as follows:
5'-GGC-CGC-CGC-C~C-CGC-AGC-GTG-CAG-TGG-TGC-GCC-GTG-AGC-CAG-CCC-GAG-
GCC-ACC-AAG-TGC-TTC-CAG-TGG-CAG-CGC-AAC-ATG-CGC-AAG-GTG-CGC-GGC-
ccc-3 ~ (SE~ ID NO:5)
Preferably, a methionine start codon and a stop codon (TAG) are added. In addition, the
ACC sequence from the Kozak consensus sequence is added to improve the liklihood of
efficient translation. This results in the Met-Lactoferricin H gene sequence shown below,
with start and stop codons underlined:
5'-ACC-ATG-GGC-CGC-CGC-CGC-CGC-AGC-GTG-CAG-TGG-TGC-GCC-GTG-AGC-CAG-
CCC-GAG-GCC-ACC-AAG-TGC-TTC-CAG-TGG-CAG-CGC-AAC-ATG-CGC-AAG-GTG-
CGC-GGC-CCC-TAG-3~(SEQ ID NO:6)
A structural coding sequence encoding lactoferricin is expressed in a plant cell under
control of a promoter capable of functioning in a plant cell to cause the production of RNA
sequences and a 3' non-translated region causing polyadenylation of the transcribed RNA.
Examples of promoters capable of functioning in plants or plant cells, i.e., those capable of
driving expression of the associated structural genes such as lactoferricin in plant cells,
include the cauliflower mosaic virus (CaMV) 1 9S or 35S promoters and CaMV double
promoters; nopaline synthase promoters; pathogenesis-related (PR) protein promoters;
small subunit of ribulose bisphosphate carboxylase (ssuRUBlSCO) promoters, and the like.
Preferred are the rice actin promoter (McElroy et at., Mol. Gen. Genet. 231: 150 (1991)), a
ubiquitin promoter such as the maize ubiquitin promoter (EP 0 342 926; Taylor et al., Plant
CelJ Rep. 12: 491 (1993)), and the Pr-1 promoter from tol~eco, ArAhidoFsis, or maize. Also
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preferred are the 35S promoter and an enhanced or double 35S promoter such as that
described in Kay et al., Science 236: 1299-1302 (1987) and the double 35S promoter
cloned into pCGN2113, deposited as ATCC 40587. The promoters themselves may be
modified to manipulate promoter ~l-er,gll, to increase la.:lofer,i~il, ex~,ression, in accordance
with art-recognized procedures.
Signal or transit peptides may be fused to the lactoferricin coding sequence in the
chimeric DNA constructs of the invention to direct transport of the expressed lactoferricin
peptide to the desired site of action. Examples of signal peptides inctude those natively
linked to the plant pathogenesis-related proteins, e.g. PR-1, PR-2, and the like. See, e.g.,
Payne et al., Plant Mol. Biol. 11:89-94 (1988). Examples of transit peptides include the
chloroplast transit peptides such as those described in Von Heijne et al., Plant Mol. Biol.
f~ep. 9:104-126 (1991 ); Mazur et al., Plant Physiol. 85: 11 10 (1987); Vorst et a/., Gene 65:
59 (1988), and mitochondrial transit peptides such as those described in Boutry et al.,
Nature 328.~40-342 (1987). The relevant r~isclosures of these publications are
incorporated herein by reference in their entirety. For cytoplasmic expression, a methionine
start codon is added.
The chimenc DNA construct(s) of the invention may contain multiple copies of a
promoter or multiple copies of the lactoferricin structural genes. In addition, the construct(s)
may include coding sequences for markers and coding sequences for other peptides such
as signal or transit peptides, each in proper reading frame with the other functional
elements in the DNA molecule. The preparation of such constructs are within the ordinary
level of skill in the art.
Useful markers include peptides providing herbicide, antibiotic or drug resistance,
such as, for example, resistance to hygromycin, kanamycin, G418, gentamycin, lincomycin,
methotrexate, glyphosate, phosphinothricin, or the like. These markers can be used to
select cells transformed with the chimeric DNA constructs of the invention from
untransformed cells. Other useful markers are peptidic enzymes which can be easily
detected by a visible reaction, for example a color reaction, for example luciferase,
B-glucuronidase, or B-galactosid~se.
Transgenic plants are obtained by
(a) inserting recombinant DNA molecules according to the invention into the genome of a
plant cell;
(b) obtaining transformed plant cells; and
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(c) regenerating therefrom the transgenic plants which express lactoferricin in an amount
effective to reduce damage from ~ise~ce.
The recolllbinant DNA molecu'es can be introduced into the plant cell in a number of
art-recognized ways. Those skilled in the art will appreciate that the choice of method might
depend on the type of plant, i.e. monocot or dicot, targeted for transformation. Suitable
methods of transforming plant cells include microinjection lCrossway et al., BioTechniques
4.320-334 (1986)), electroporation (Riggs et a/, Proc. Natl. Acad. Sci. USA 83:5602-~606
(1986), Agrobacterium mediated transformation (Hinchee et al., Biotechnology ~:915-921
(1988)), direct gene transfer (Paszl~owski etal., EMBOJ. 3.~717-2722 (1984)), and ballistic
particle acceleration using devices available from Agracetus, Inc., Madison, Wisconsin and
Dupont, Inc., Wilmington, Delaware (see, for example, Sanford et aL, U.S. Patent4,945,050; and McCabe et al., Biotechnology 6.~23-926 (1988)). Also see, Weissinger et
al., Annual ~ev. Genet. 22:42~-477 (1988); Sanford et al., Particulate Science and
Technology ~.~7-37 (1987)(onion); Christou et al., Plant Physiol. 8~671 -674
(1988)(soybean); McCabe etal., Bio/Technology6:923-926 (1988)(soybean); Datta etal.,
Bio~Technology 8:736-740 (1990)(rice); Klein et al., Proc. Natl. Acad. Sci. USA,854305-4309 (1988)(maize); Klein etal., Bio/Technology6:5~9-563 (1988)(maize); Klein et
al., Plant Physiol. 91:440-444 (1988)(maize); Fromm etal., Bio/Tect~nology 8.~33-839
(1990); and Gordon-Kamm etal., PlantCell2:603-618 (lg90)(maize).
The selection of a promoter used in expression cassettes will determine the spatial
and temporal expression pattern of the transgene in the transgenic plant. Selected
promoters will express transgenes in specific cell types (such as leaf epidermal cells,
mesophyll cells, root cortex cells) or in specific tissues or organs (roots, leaves or flowers,
for example) and this selection will reflect the desired location of expression of the
transgene. Alternatively, the selected promoter may drive expression of the gene under a
light-induced or other temporally regulated promoter. A further alternative is that the
selected promoter be chemically regulated. This would provide the possibility of inducing
expression of the transgene only when desired and caused by treatment with a chemical
inducer.
A variety of transcriptional terminators are available for use in expression c~ssettes.
These are responsible for the termination of transcription beyond the transgene and its
correct polyadenylation. Appropriate transcriptional terrrlinators and those which are known
to function in plants and include the CaMV 35S terminator, the tml terminator, the nopaline
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synthase terminator, the pea rbcS E9 terminator. These can be used in both
monocotyledons and dicotyledons.
Numerous sequences have been found to enhance gene expression from within the
transcriptional unit and these sequences can be used in conjunction with the genes of this
invention to increase their expression in transgenic plants.
Various intron sequences have been shown to enhance e~ression, particularly in
monocotyledonous cells. For example, the introns of the maize Adh 1 gene have been
found to significantly enhance the expression of the wild-type gene under its cognate
promoter when introduced into maize cells. Intron 1 was found to be particularly effective
and enhanced expression in fusion constructs with the chloramphenicol acetyltransferase
gene (Callis etal., Genes Develop. 1: 1183-1200 (1987)). In the same experimental
system, the intron from the maize bronze1 gene had a similar effect in enhancingexpression (Callis et al., supra). Intron sequences have been routinely incorporated into
plant transformation vectors, typically within the non-translated leader.
A number of non-translated leader sequences derived from viruses are also known to
enhance expression, and these are particularly effective in dicotyledonous cells.
Specifically, leader sequences from Tobacco Mosaic Virus (TMV, the '~-sequence"), Maize
Chlorotic Mottle Virus (MCMV), and Alfalfa Mosaic Virus (AMV) have been shown to be
effective in enhancing expression (e.g. Gallie etal. NL~CI. Acids Res. 1~: 8693-8711 (1987);
Skuzeski et al. Plant Molec. Biol. 15: 65-79 (1990))
Various mechanisms for targeting gene products are known to exist in plants and the
sequences controlling the functioning of these mechanisms have been characterized in
some detail. For example, the targeting of gene products to the chloroplast is controlled by
a signal sequence found at the amino terminal end of various proteins and which is cleaved
during chloroplast import yielding the mature protein (e.g. Comai et a/. J. Biol. Chem. 263:
15104-15109 (1988)). These signal sequences can be fused to heterologous gene
products to effect the import of heterologous products into the chloroplast (van den Broeck
et al. Nature 373: 358-363 (1985)). DNA encoding for appropriate signal sequences can be
isolated from the 5' end of the cDNAs encoding the RUBISCO protein, the CAB protein, the
EPSP synthase enzyme, the GS2 protein and many other proteins which are known to be
chloroplast localized.
Other gene products are localized to other organelles such as the mitochondrion and
the peroxisome (e.g. Unger etal. PlantMolec. Biol. 13: 411-418 (1989)). The cDNAs
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encoding these products can also be manipulated to effect the targeting of heterologous
gene producSs to these organelles. Examples of such sequences are the nuclear-encoded
ATPases and specific aspartate amino transferase isoforms for mitochondria. Targeting to
cellular protein bodies has been described by Rogers et at., Proc. Nat/. Acad. Sci. USA 8~.
6512-6516 (1985)).
In addition sequences have been characterized which cause the targeting of gene
products to other cell compartments. Amino terminal sequences are responsible for
targeting to the ER, the apoplast, and extracellular secretion from aleurone cells (Koehler &
Ho, Plant Cell 2: 769-783 (1990)). Additionally, amino terminal sequences in conjunction
with carboxy terminal sequences are responsible for vacuolar targeting of gene products
(Shinshi et al., Plan~ Molec. Biol. 14: 357-368 (1990)).
By the fusion of the appropriate targeting sequences described above to transgene
sequences of interest it is possible to direct the transgene product to any organelle or cell
compartment. For chloroplast targeting, for example, the chloroplast signal sequence from
the RUBISCO gene, the CAB gene, the EPSP synthase gene, or the GS2 gene is fused in
frame to the amino terminal ATG of the transgene. The signal sequence selected should
include the known cleavage site and the fusion constructed should take into account any
amino acids after the cleavage site which are required for cleavage. In some cases this
requirement may be fulfilled by the addition of a small number ot amino acids between the
cleavage site and the transgene ATG or alternatively replacement of some amino acids
within the transgene sequence. Fusions constructed for chloroplast import can be tested
for efficacy of chloroplast uptake by in vitro translation of m vitro transcribed constructions
followed by in vitro chloroplast uptake using techniques described by (Bartlett et al. In:
Edelmann etal. (Eds.) Methods in Chloroplast Molecular Bioloqy, ElseYier. pp 1081-1091
(1982); Wasmann etal. Mol. Gen. Genet. 205: 446-453 (1986)). These construction
techniques are weli known in the art and are equally applicable to mitochondria and
peroxisomes. The choice of targeting which may be required for ex,uression of the
transgenes will depend on the cellular localization of the precursor required as the starting
point for a given pathway. This will usually be cytosolic or chloroplastic, although it may in
some cases be mitochondrial or peroxisomal. The products of transgene expression will not
normally require targeting to the ER, the apoplast or the vacuole.
The above described mechanisms for cellular targeting can be utilized not only in
conjunction with their cognate promoters, but also in conjunction with heterologous
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g
promoters so as to effect a specific cell targeting goal under the transcriptional regulation of
a promoter which has an expression pattern different to that of the promoter from which the
targeting signal deriYes.
Transformation techniques for dicotyledons are well known in the art and includeAgrobacterium-based techniques and techniques which do not require Agrobacterium.
Non-Agrobacterium techniques involve the uptake of exogenous genetic material directly by
protoplasts or cells. This can be accomplished by PEG or electroporation mediated uptake,
particle bombardment-mediated delivery, or microinjection. Examples of these techniques
are described by Paszkowski et al., EMBO J 3: 2717-2722 (1984), Potrykus et al., Mol.
Gen. Genet. 199: 169-177 ~1985), Reich et al., Biotecl~nology 4: 1001-1004 (1986), and
Klein et al., Nature 327: 70-73 (1987). In each case the transformed cells are regenerated
to whole plants using standard techniques known in the art.
Agrobacteriur~mediated transformation is a preferred technique for transformation of
dicotyledons because of its high efficiency of transformation and its broad utility with many
different species. The many crop species which are routinely transformable by
Agrobacterium include tobacco, tomato, sunflower, cotton, oilseed rape, potato, soybean,
alfalfa and poplar (EP 0 317 511 (cotton); EP 0 249 432 (tomato, to Calgene); WO87107299 (Brassica, to Calgene); US 4,795,855 (poplar);). Agrobacterium transformation
typically involves the transfer of the binary vector carrying the foreign DNA of interest (e.g.
pClB200 or pClB2001) to an appropriate AgrobacteriL~m strain which may depend of the
complement of vir genes carried by the host Agrobacter~um strain either on a co~resident Ti
plasmid or chromosomally (e.g. strain CIB542 for pClB200 and pClB2001 (Uknes et al.
P~ant CelJ ~: 159-169 (1993)). The transfer of the recombinant binary vector to
Agrobacterium is accomplished by a triparental mating procedure using E. coii carrying the
recombinant binary vector, a helper E. coli strain which carries a plasmid such as pRK2013
and which is able to mobilize the recombinant binary vector to the target Agrobacterium
strain. Alternatively, the recombinant binary vector can be trans~er,ed to Agrobacterium by
DNA transformation (Hofgen & Willmitzer, Nucl. Acids Res. 16: 9877(1988)).
Transformation of the target plant species by recombinant Agrobacterium usually
involves co-cultivation of the Agrobacterium with explants from the plant and follows
protocols well known in the art. Transformed tissue is regenerated on selectable medium
carrying the antibiotic or herbicide resistance marker present between the binary plasmid T-
DNA borders.
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Transformation of most monocotyledon species has now also become routine.
Preferred techniques include direct gene transfer into protoplasts using PEG or
electroporation techniques, and particle bombardment into callus tissue. Transformations
can be undertaken with a single DNA species or multiple DNA species (i.e. co-
transformation) and both these techniques are s~liPhle for use with this invention. Co-
transformation may have the advantage of avoiding complex vector construction and of
generating transgenic plants with unlinked loci for the gene of interest and the selectable
marker, enabling the removal of the selectable marker in subsequent generations, should
this be regarded desirable. However, a disadvantage of the use of co-transformation is the
less than 100% frequency with which separate DNA species are integrated into the genome
(Schocher eta/. Biotechnology4: 1093-1096 (1986)).
Patent Applications EP O 292 435, EP O 392 225 and WO 93/07278 describe
techniques for the preparation of callus and protoplasts from an élite inbred line of maize,
transformation of protoplasts using PEG or electloporation, and the regeneration of maize
piants from transformed protoplasts. Gordon-Kamm et al., Plant Cell 2: 603-618 ~t990))
and Fromm et al., Biotechnology 8: 833-839 (19gO)) have published techniques fortransformation of A188-derived maize line using particle bombardment. Furthermore,
application WO 93/07278 and Koziel et al., Biotechnology 11: 194-200 (1993)) describe
techr~iques for the transformation of élite inbred lines of maize by particle bombardment.
This technique utilizes immature maize embryos of 1.5-2.5 mm length excised from a maize
ear 14-15 days after pollination and a PDS-lOOOHe Biolistics device for bombardment.
Transformation of rice can also be undertaken by direct gene transfer techniquesutilizing protoplasts or particle bombardment. Protoplast-mediated transformation has been
described for Japonica-types and Indica-types (Zhang et al., Plant Cell Rep 7: 379-384
(1988); Shimamoto et al. Nature 338: 274-277 (1989); Datta et al. Biotechnology 8: 736-740
(1990)). Both types are also routinely transformable using particle bombardment (Christou
et al. Biotechnology 9: 957-962 (1991)).
Patent Application EP O 332 581 describes techniques for the generation,
transformation and regeneration of Pooideae protopl~sh. These techniques allow the
transformation of Pooideae such as Dactylis and wheat. Furthermore, wheat transformation
has been described by Vasil et al., Biotechnology 10: 667-674 (1992)) using particle
bombardment into cells of type C long-term regenerable callus, and also by Vasil et al.,
Biotechnology 11: 1553-1558 (1g93)) and Weeks et al., PlantPhysiol. 102: 1077-1084
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(1993) using particle bombardment of immature embryos and immature embryo-derived
callus. A prel~n~d technique for wheat translor",~l;on, however, involves the
- transformation of wheat by particle bor,lbard,l,enl of immature embryos and includes either
a high sucrose or a high maltose step prior to gene delivery. Prior to bombardment, any
number of embryos (0.75-1 mm in length) are plated onto MS medium with 3% sucrose
(Murashige & Skoog, Physiologia Plantarum 1~: 473-497 (1962)) and 3 mg/l 2,4-D for
induction of somatic embryos which is allowed to proceed in the dark. On the chosen day
of bombardment, embryos are removed from the induction medium and placed onto the
osmoticum (i.e. induction medium with sucrose or maltose added at the desired
concentration, typically 15%). The embryos are allowed to plasmolyze for 2-3 h and are
then bombarded. Twenty embryos per target plate is typical, although not critical. An
appropriate gene-carrying plasmid (such as pClB3064 or pSG35) is precipitated onto
micrometer size gold particles using standard procedures. Each plate of embryos is shot
with the DuPont Biolistics~ helium device using a burst pressure of -1000 psi using a
standard 80 mesh screen. After bombardment, the embryos are placed back into the dark
to recover for about 24 h (still on osmoticum). After 24 hrs, the embryos are removed from
the osmoticum and placed back onto induction medium where they stay for about a month
before regeneration. Approximately one month later the embryo explants with developing
embryogenic callus are transferred to regeneration medium (MS + 1 mg/liter NAA, 5 mglliter
GA), further containing the appropriate selection agent (10 mg/l basta in the case of
pClB3064 and 2 mg/l methotrexate in the case of pSOG35). After approximately onemonth, developed shoots are transferred to larger sterile containers known as "GA7s" which
contained half-strength MS, 2% sucrose, and the same concentration of selection agent.
Lactoferricin when expressed in plants controls or inhibits a broad spectrum of plant
pathogens, including viral, bacterial and fungal plant pathogens. Commercially important
plant diseases suitable for control by ex,uression of lactoferricin in plants include the
following:
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Corn ~athogens
Fungal: Gibberella zeae
Aspergillus flavus
A. parasiticus
Fusarium moniliforme
Diplodia maydis
Collelc,l"~l-um graminicola
Cephalosporium acremonium
Macrophomina phaseolina
Cercospora zeae-maydis
Exserohilum turcicvm
Elipolaris maydis
Kabatiella zeae
Puccinia sorghi
Puccinia polysora
Sphacelotheca reiliana
Ustilago maydis
Colletotrichum graminicola
Helminthosporium carbonum
Peronosclerospora sorghl
Sclerophthora rayssiae
Peronosclerospora sacchari
Peronoscler. philippinensis
Peronosclerospora maydis
Peronosc/erospora spontanea
Peronosclerospora heteropogoni
Sclerospora graminicola
Bacterial: Erwinia stewartii
Corynebacterium nebrasi~ense
Virat: Maize dwarf mosaic virus
Maize rough dwarf (Maize rio cuarto) virus
Maize streak virus
Maize chlorotic dwarf virus
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Maize chlorotic mottle virus
Barley yellow dwarf virus
Corn lethal necrosis virus
High plains virus
Maize stripe virus
Sweet corn Pathogens:
A. Bacterial Diseases
Erwinia stewartii
Pseudomonas avenae
Erwinia chrysanthemi
B. Fungal Diseases
Bipolaris maydis
Exserohilum turcicvm
Colletotrichum graminicola
Phyllosticta maydis
Kabatiella zeae
Cercospora zeae-maydis
Sclerophthora spp.
Peronosclerospora spp.
Ustilago maydis
Sphacelotheca reiliana
Puccinia sorghi
Puccinia polysora
Physopella zeae
Fvsarivm moniliforme
Pythium spp.
Penicillium oxalicum
Fusarium spp.
Diplodia maydis
Gibberella zeae
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C. Viral Dise~ses
Sugarcane Mosaic
Maize Dwarf Mosaic
Maize Chlorotic Dwar~
High Plains Virus
Maize Chlorotic MotUe
Wheat Streak Mosaic
Barley Yellow Dwarf
Wheat Pathoqens
- Fungal pathogens:
Puccinia graminis f.sp. tritici
Puccinia recondita f.sp. trftici
Puccinia striiformis
Tilletia tritici
Tilletia controversa
Tilletia indica
Vstilago tritici
Urocystis tritici
Gaeumannomyces graminis
Pythium spp.
Fusarium culmorum
Fusarium graminaervm
Fusarivm avenaceum
Drechslere tritici-repentis
Rhizoctonia spp.
Colletot,i~hum gra",i,-icola
Helminthosporium spp.
Microdochium nivale
Pseudocercosporella herpotrichoides
~rysiphe graminis f.sp. trifici
Sclerophthora macrospora
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Septoria tritici
Septoria nodorum (Stagonospora nodorumJ
Bipolaris sorokiniana
Fusarium roseum
Cephalosporivm gramineum
Cochliobolus sativus
Claviceps purpurea
Bacterial pathogens:
Pseudomonas syringae pv. atrofaciens
Pseudomonas syringae pv. syringae
Xanthomonas campestris pv. transJucens
Viral pathogens:
Barley Yellow Dwarf virus
Soilborne Wheat Mosaic virus
Wheat Spindle Streak Mosaic virus
Wheat Yellow Mosaic virus
Soybean Pathoqens
1. Fungal
Phytophthora megasperma var. sojae
Colletotrichum truncatvm
Septoria glycines
Cercospora kikuchii
Cercospora sojina
Peronospora manshvrica
Microsphaera diffusa
Rhizoctonia solani
Phakopsora pachyrhizi
Corynespora cassico!-
Calonectria crotalariae
Fusarium so/ani
Fusarium oxysporum
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Macrophomina phaseolina
Diaporthe phaseolorum var caulivora-& meridionalis
Diaporthe phaseolorum var. sojae
Phialophora gregata
Pythium spp
Sclerotinia sclerotiorum
Sclerotiun rolfsii
Thielaviopsis basicola
2. Bacterial
Pseudomonas syringae pv. glycinea
Xanthomonas campestris pv. phaseoli
3. Viral
Soybean mosaic
Bean pod monle
Bud blight
Peanut mottle
Cowpea Chlorotic Mottle
Yellow Mosaic
Pea Pathogens
1. Seed & Seedling Diseases
Pythium ultimum
Rhizoctonia solanl
Il. Diseases Caused ~y Bacteria
Pseudomonas syringae pv. pisi
Pseudomonas syringae pv. syringae
111. Foliar Diseases Caused by Fungi
Ascochyta pisi
Mycosphaerella pinodes
Ascochyta pinodella
Sclerotinia sclerotiorum
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Peronospora pisi
Erys)phe pisi
Colletol,i~hum pisi
Altemaria alternata
Botrytis cinerea
IV. Root Dise~-ses Caused by Fungi
Fusarium oxysporum f. sp. pisi
Aphanomyces euteiches f. sp. pisi
Pythium ultimum
Fusarium solani f. sp. pisi
Thielaviopsis basicola
V. Diseases Caused by Viruses
Pea Enation Mosaic
Bean (Pea) Leaf Roll
Pea Seedborne Mosaic
Pea Streak
Pea Mosaic
Cucumber Mosaic
Pea Early Browning Virus
Red Clover Vein Mosaic
Bean Patho~ens
1. Root Diseases
Aphanomyces euteiches f. sp. phaseoli
Pythium ultimum
Rhizoctonia solani
~usarium solani f. sp. phaseoli
Jhielaviopsis bacicol~
ScleroUum rolfsii
Il. Fungal Diseases of Aerial Parts
Colletotrichum lindemuthianum
Uromyces appendiculatus
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Sclerotinia sclerotiorum
Fusarium oxysporum f. sp. phaseoli
Phoma exigua var exigua
Cercospora canescens
Chaetoseptoria wellmanii
Phytophthora nico~ianae
Erysiphe polygoni
Pythium ultimum & debaryanum
Altemaria altemata, various species
Phaeoisariopsis griseola
Macrophomina phaseolina
Botrytis cinerea
Rhizoctonia solani
111. Diseases Caused by Bacteria
Pseudomonas syringae pv. phaseolico.
Pseudomonas syringae pv. syringae
Xanthomonas phaseoli
IV. Diseases Caused by Nematodes
Meloidogyne incognita
Pratylenchus species
V. Diseases Caused by Viruses
Bean Common Mosaic
Bean Golden Mosaic
Curly Top
Bean Curly Dwarf Mosaic
Bean Pod Mottle
Clover Yellow Vein
Red Node
Cucumber Mosaic
Peanut Stunt
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Suaar beet Dathogens
1. Fungal:
Cercospora beticola leaf spot disease
Aphanomyces cochlioides
Rhizoctonia solani damping off, root rot
Erysiphe betae powdery mildew
Rhizoctonia violacea (Helicob~ m purpureum)
Ramularia beticola
Pythium spp.
Phoma betae
Uromyces betae
Peronospora farinosa
Alternaria tenuis
Fusarium oxysporium
Verticillium dahliae
Scierotium rolfsii
- Erysiphe polygoni powderymildew
Polymyxa betae
2. Bacterial:
Pseudomonas syringae
Erwinia carotovora Erwinia root rot
3. Viral:
Rhizomania Beet necrotic yellow vein virus (BNYVV)
Beet mild yellowing virus (BMYV)
Beet yellows virus (BYV)
Beet curly top virus
Beet mosaic virus
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The invention provides plants and seed, which when germinated and cultivated,
express la~lofer~i~in. Preferably, lactoferricin is expressed from recombinant DNA stably
integrated into the genome. The transgenic plants or seed are either monocotyledoneous or
dicotyledoneous and preferably selected from the group consisting of maize, rice, wheat,
barley, sorghum, rye, oats, millet, cotton, soybeans, peas, beans, sunflower, grasses and oil
seed rape. The trangenic seed can be packaged into a bag preferably containing lable
instructions for the use thereof to control plant pathogens.
The genetic properties engineered into the transgenic seeds and plants describedabove are passed on by sexual reproduction or vegetative growth and can thus be
maintained and propagated in progeny plants. Generally said maintenance and propagation
make use of known agricultural methods developed to fit specific purposes such as tilling,
sowing or harvesting. Specialized processes such as hydroponics or greenhouse
technologies can also be applied. As the growing crop is vulnerable to attack and damages
caused by insects or infections as well as to competition by weed plants, measures are
undertaken to control weeds, plant diseases, insects, nematodes, and other adverse
conditions to improve yield. These include mechanical measures such a tillage of the soil or
removal of weeds and infected plants, as well as the application of agrochemicals such as
herbicides, fungicides, gametocides, nematicides, growth regulants, ripening agents and
insecticides.
Use of the advantageous genetic properties ot the transgenic plants and seeds
according to the invention can further be made in plant breeding which aims at the
development of plants with improved properties such as tolerance of pests, herbicides, or
stress, improved nutritional value, increased yield, or improved structure causing less loss
from lodging or shattering. The various breeding steps are characterized by well-defined
human intervention such as selecting the lines to be crossed, directing pollination of the
parental lines, or selecting appropriate progeny plants. Depending on the desired properties
different breeding measures are taken. The relevant techniques are well known in the art
and include but are not limited to hybridization, inbreeding, backcross breeding, multiline
breeding, variety blend, interspecific hybridization, aneuploid techniques, etc. Hybridization
techniques also include the sterilization of plants to yield male or female sterile plants by
mechanical, chemical or biochemical means. Cross pollination of a male sterile plant with
pollen of a different line assures that the genome of the male sterile but female fertile plant
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will uniformly obtain properties of both parental lines. Thus, the transgenic seeds and plants
according to the invention can be used for the breeding of improved plant lines which for
example increase the effectiveness of conventional methods such as herbicide or pestidice
treatment or allow to dispense with said methods due to their modified genetic properties.
Alternatively new crops with improved stress tolerance can be obtained which, due to their
optimized genetic "equipment", yield harvested product of better quality than products which
were not able to tolerate comparable adverse developmental conditions.
In seeds production germination quality and uniformity of seeds are essential product
characteristics, whereas germination quality and uniformity of seeds harvested and sold by
the farmer is not important. As it is difficult to keep a crop free from other crop and weed
seeds, to control seedborne diseases, and to produce seed with good germination, fairly
extensive and well-defined seed production practices have been developed by seedproducers, who are experienced in the art of growing, conditioning and marketing of pure
seed. Thus, it is common practice for the farmer to buy certified seed meeting specific
quality standards instead of using seed harvested from his own crop. Propagation material
to be used as seeds is customarily treated with a protectant coating comprising herbicides,
insecticides, fungicides, bactericides, nematicides, molluscicides or mixtures thereof.
Customarily used protectant coatings comprise compounds such as captan, carboxin,
thiram (TMTD~), methalaxyl (Apron ), and pirimiphos-methyl (Actellic~). If desired these
compounds are formulated together with further carriers, surfactants or application-
promoting adjuvants customarily employed in the art of formulation to provide protection
against damage caused by bacterial, fungal or animal pests. The protectant coatings may
be applied by impregnating propagation material with a liquid formulation or by coating with
a combined wet or dry formulation. Other methods of application are also possible such as
treatment directed at the buds or the fruit.
It is a further aspect of the present invention to provide new agricultural methods such
as the methods examplified above which are characterized by the use of transgenic plants,
transgenic plant material, or transgenic seed according to the present invention to provide
control against plant diseases
To breed progeny from plants transformed according to the method of the present
invention, a method such as that which follows may be used: plants produced as described
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in the examples set forth below are grown in pots in a greenhouse or in soil, as is known in
the art, and permitted to flower. Pollen is obtained and used to pollinate the same plant,
sibling plants, or any desirable plant. Similarly, the transformed plant may be pcl':natecl by
pollen obtained from the same plant, sibling plants, or any desirable maize plant.
Transformed progeny obtained by this method may be distinguished from non-transformed
progeny by the presence of the introduced gene(s) and/or accompanying DNA (genotype),
or the phenotype conferred. The transformed progeny may similarly be selfed or ctossed to
other plants, as is normally done with any plant carrying a desirable trait. Similarly other
transformed plants produced by this method may be selfed or cfossed as is known in the art
in order to produce progeny with desired characteristics.
The invention thus provides, in a first embodiment,
1. A recombinant DNA molecule comprising in operative sequence:
(a) a promoter which functions in plant cells to cause the production of an RNA
sequence, e.g., a promoter as described above, for example the maize ubiquitin promoter;
(b) a structural coding sequence that encodes for production of lactoferricin, e.g.,
Iactoferricin B or lactoferricin H, e.g., a peptide of SEQ ID NO:1 or 2 optionally having an N-
terminal methionine; preferably a plant optimized coding sequence, e.g., of SEQ ID NO:3,
4, 5, or 6; and
(c) a 3' non-translated region which functions in plant cells to cause the addition of
polyadenylate nucleotides to the 3' end of the RNA sequence, e.g., a transcriptional
terminator as described above, for example, the nopaline synthase transcriptional
terminator from Agrobacterivm tumifaciens;
and optionally further comprising one or more enhancers or targeting sequences, as
described herein.
In a second embodiment. the invention provides
2. A method of producing transgenic disease resistant plants, e.g., plants as described
above, for example maize or wheat plants capable of expressing lacto~erricin, comprising
the steps of:
(a) inserting into the genome ot a plant cell, a recombinant DNA molecule as
described above, e.g., comprising (i) a promoter which functions in plant cells to cause the
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production of an RNA sequence; (ii) a structural coding sequence that encodes for the
production of lactoferricin; and (iii) a 3' non-translated region which functions in plant cells to
cause the addition of polyadenylate nucleotides to the 3' end of the RNA sequence;
(b) obtaining transformed plant cells, and
(c) regenerating from the transformed plant cells genetically transformed plants which
express lactoferricin in an amount effective to reduce damage from infection by a pathogen.
In a third embodiment, the invention provides
3. A plant (e.g., a maize, wheat or sugar beet plant) comprised of cells which express
lactoferricin,
for example, a plant having stably integrated in its genome recombinant DNA comprising in
operative sequence:
(a) a promoter which functions in plant cells to cause the production of an RNA
sequence;
(b) a structural coding sequence that encodes for production of lactoferricin; and
(c) a 3' non-translated region which functions in plant cells to cause the addition of
polyadenylate nucleotides to the 3' end of the RNA sequence;
e.g., wherein the plant is developmentally and morphologically normal.
In a fourth embodiment, the invention provides
4. Seed which when germinated and cultivated will produce a plant comprised of cells
which express lactoferricin, e.g., a plant as described above, e.g., wherein the seed is
optionally coated (e.g., with polymers, fungicides, insecticides, dyes or other seed coatings)
and/or packaged, e.g., in a bag or other container for sale or transport.
In a fitth embodiment, the invention provides
5. Use of lactoferricin to control plant pathogens, e.g., plant bacterial, fungal or viral
pathogens, e.g., as described above;
and a method of controlling plant pathogens, e.g., as described above, comprising e)~osi"g
the pathogens to a plant which expresses lactoferricin.
The following description further exempiifies the methods and compositions of this
invention. However, it will be understood that that other methods, known by those ot
ordinary skill in the art to be equivalent, can also be employed.
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ExamPles
Example 1: Anli~icrobial activity of Lactoferricin B on plant pathogenic fungi and other
micro-organisms .
~. Inhibition of SDore Germination
The activity of lactoferricin B was tested in spore germination inhibition assays using
Diplodia maydis and Colletotrichum graminicola spores. The fungal strains were obtained
from Loral Castor, CIBA-GEIGY, Illinois. The lactoferricin B peptide and la~tofer,in were
obtained from Morinaga Milk Industry Co, LTD, Tokyo, Japan.
Fungal spores were harvested from sporulating cultures grown on potato dextrose agar
(PDA, Difco) plates. D. maydis spores were used fresh, or stored at -80~C in 25% glycerol.
C. graminicola spores were used fresh.
For the assay, 10 ml molten medium ~1.5% agar, containing 8% carrot extract) wascooled to approximately 50~C. Ten microliter sl~eptc",ycin (250 mg/ml) was added, followed
by 100 microliter of either D. maydis or C. graminicola spores, at concentrations of
approximately 106/ml(D. maydls) and 1071ml (C. graminicola). The solutions were mixed by
gentle swirling and poured into sterile petridishes. After the medium solidified, sterile 1/4
inch diameter filter discs were placed on top of the medium and test solutions were pipetted
onto these disks. Test solutions were lactoferrin (10 microgram) lactoferricin B (10
microgram), purothionin (the positive control, at 0, 5, 10 and 20 microgram; purchased from
Calbiochem) and buffer (20 mM Tris pH7.5).
After incubation at roomtemperature for 2-5 days, fungal growth was clearly visible on the
plates, except around the filterdiscs containing purothionin and lactoferricin B. The sizes of
the inhibition zones are shown in 1. In the D. maydis experiment, the filterdisk with 10
microgram lactoferricin B showed a zone of inhibition of 4.95 mm, whereas 20 microgram
purothionin showed an inhibition zone of 4.5 mm. In the C. graminicola experiment,
lactoferricin B produced a zone of inhibition of 1.7 mm, whereas pu~othiGni~ produced an
inhibition zone of 2.1 mm. Lactoferrin did not cause inhibition of fungal germination and
growth.
~ rwinia stewartiiwas also tested in this system and it was shown that lactoferricin B
also inhibits growth of this bacterium.
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Table 1: Inhibition of spore germination by lactoferricin B
zone of inhibition
Fungus BufferPurothioninLactoferricin B
20~1g 1 Ollg
D. maydis Omm 4.5 mm 4.95 mm
C. graminicoJa O mm 2.1 mm 1.7 mm
b. Inhibition of mycelial growth
A second method used to evaluate antifungal activity involved measuring inhibition of
mycelial growth. In this assay, wells were punched with the broad end of a Pasteur pipette
at the perimeter of 1.5% agarose plates containing 4% carrot extract. The bottom of the
wells was sealed with a drop of the molten agarose solution. Individual fungal pluys were
taken from a master plate of either Bipolaris maydis, Fusarium monoliforme, Gibberella
zeae, Pythium aphanidermatum, or Rhizoctonia solani and cenl.~lly placed on each test
plate. Test solutions placed in the wells (40 microliter) were lactoferricin B (100 microgram),
lactoferrin (100 microgram) or buffer (20 mM Tris ptl7.5). After incubation at
roomtemperature for the time required for the fungi to have grown from the inoculated plug
to the welts, inhibition of fungal growth was scored. All fungi showed inhibition of growth
near the wells containing lactoferricin B, but not near the lactoferrin or buffer control well.
The results are shown in table 2.
Table 2: Inhibition of fungal growth by lactoferricin B.
growth inhibition (mm)
Fungus Buffer Lactoferricin B
(100 1l9)
Bipolaris maydis 0 8
Fusariummonoliforme o 9.7
Gibberella zeae 0 9.8
Pythium aphanidermatum 0 20.9
~hizoctonia solani 0 1.6
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c. Minimum Inhibitory Conce~ alion in liauid culture
The activity of lactoferricin B and 2 derivatives on Fusarium culmorum and Septoria
nodorum was tested in liquid inhibition assays. The peptides were lactoferricin B (as
described above, Met-lactoferricin B (lactof~nici" B with an additional N-terminal
methionine), and Gln-lactoferricin B (lactoferricin B with an additional N-terminal glutamine).
All peptides were purchased from the W,M. Keck Biotechnology Resource Center, New
Haven, CT. Peptides were disolved in double distilled sterile water at 1.5 mg/ml.
Fungal spores were harvested from sporulating cultures grown on potato dextrose
agar (PDA, Difco) plates as descibed above and diluted to 15,000/ml in 1/2 strength PDA.
To 50 microliter spores, 25 microliter peptide of a dilution series was added, all in 96 well
plates. After mixing the wells, the OD at 590 nm was measured (a measure for the growth
of the fungi). The plates were next incubated at 28~C and the OD at 590 nm was measured
at intervals over the next 2 days. The minimum concentration of the lactoterricin B peptides
that totally inhibited growth was expressed as the Minimum Inhibitory Concentration (MIC).
These values are shown in Table 3.
Pichia pastoris was grown on YPD plates and Pseudomonas syringae BL882 and E.
coli DH5a in L broth. A small amount of P. pastoris cells was resuspended in YPD, the cell
concentration was counted and adjusted to 30,000 cells/ml with YPD. The OD at 600 nm
was measured of cultures of DH5a and BL882. Cell concentrations were adjusted to30,000, cells/ml in L broth, assuming that an OD=1 at 600nm for DH5~ corresponded to
109 cells/ml and 5x1Q~ cells/ml for BL882.
To 80 microliter cells, 20 microliter of a dilution series of lactoferricin B peptides was
added, all in 96 well plates. The OD at 590 nm of the plates was next read and next the
plates were incubated at 28~C. The OD at ~90 was read regularly over the next 2 days. The
minimum concentration of the lactoferricin B peptides that totally inhibited growth was
expressed as the Minimum Inhibitory Concentration (MIC). These values are shown in
Table 3.
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Table 3: Minimum Inhibitory Concentrations of Lactoferricin B
O,~ani_." Peptide
~ la~;lofer,i-,in B Met-lactoferricin B Gln-lactoferricin B
F. culmorum 20 20 20
S. nodorum 100 ~00 100
C. y~ n~cota 100 100 100
P. syringaeBL882 20 20 20
E. coliDH5a 100 100 100
P~chia pastoris300 300 300
Example 2: Stability of the Gln-lactoferricin B peptide in plant extracellular fluid
The lactoferricin B peptide is expressed in plants using control sequences that direct
the peptide to several different subcellular locations (described in detail in Examples 3-6,
below). For extracellular expression, the coding sequence of la.;Lofer,ici" B is fused to the
leader sequence of the maize PR-1 gene, which causes secretion of the peptide into the
extracellular space. The leader sequence used for this purpose includes the glutamine
residue at the start of the mature, secreted PR-1 protein and thus results in secretion of a
lactoferricin B peptide with an additional N-terminal glutamine residue (Gln-lactoferricin B).
A synthetic Gln-lactoferricin B peptide was tested for its stability in extracellular wash fluid of
wheat, corn and tobacco.
Extracellular wash fluid (ECF) was obtained by infiltrating leaf tissue with sterile, double
distilled water, which was recovered by centrifugation of the infiltrated tissue. For wheat, a
leaf of a UC703 ptant was used, and for corn, a leaf of a 6N615 plant. These leaves were
cut into small pieces that were placed in the barrel of a 20 ml syringe, which contained
cottonwool in the bottom. The cuttings were washed with water to remove cellular exud~te
from the cut surfaces. Next, water was added and the plunger placed on the syringe barrel.
The syringe was placed upside down in a Iyophilizer vessel, which was then connected to
the Iyophilizer. After applying a gentle vacuum for 30-40 seconds, the vacuum was
released. This was repeated once. The leaf pieces were next placed in the barrel of a 10 ml
syringe, which was placed in a Centrifuge tube. This was spun at 1000 to 2000 rpm in a
clinical centrifuge, to recover the extracellular wash fluid, which was next frozen.
For tobacco (cultivar Xanthi.nc), a leaf was injected with water, using a 10 ml syringe.
The leaf was next removed from the plant, blotted dry, rolled up and cut into short cylinders
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that were placed in a 10 ml syringe barrel. The syringe was placed in a centrifuge tube and
next spun in a clincal centrifuge at 1000 rpm. The wash fluid that collected in the tube was
collected and frozen.
Aliquots of the ECF were run on a 10% Tricine gel according to the manufacturersinstructions (Novex). An estimate of the relative protein concentration was made for each
plant ECF. This was used to make an estimate of the amount of ECF needed from each
collected fluid to have approximately equal amounts of total ECF protein in the subsequent
incubation. Thus, 18.~ microliter ECF from wheat, 31 microliter corn ECF and 60 microliter
tobacco ECF were each incubated with 11.25 microgram Gln-lactoferricin B peptide, in a
total volume of 75 microliter, containing 10 mM Tris pH7.5, ~0 mM NaCI, 2.5 mM MgCI2 and
2.5 mM CaCI2. The reactions were incubated at 37~C and 1~ microliter aliquots were taken
at 0, 15, 30, 60, 90, 120 and 180 minutes. These samples were immediately frozen.
The samples were analyzed for the presence of Gln-lactoferricin B by gel
electrophoresis, after heat inactivation and alkylation. Following a 10 minute incubation at
1 00~C, the samples were cooled and quick spun in an eppendorf centrifuge. The alkylation
of each sample was accomplished by first reducing the samples with 2 microliter 1 mM Dl~
in an N2 atmosphere for 10 minutes at 1 00~C. Next, iodoacetamide was added (2 microliter
of 10 mM) and 1.4 microliter 1 M Tris (pH 8.5), to make the solution more basic. After
flushing the tubes with N2, the samples were incubated in the dark at 50~C for 50 minutes.
Control peptide samples (Gln-lactoferricin B peptide without buffer or ECF) were alkylated in
a similar manner. The samples were stored frozen until analyzed by gel electrophoresis.
To each sample, an equal amount of 2xTricine/SDS sample buffer was added (Novex)after which they were incubated at 1 00~C for ~ minutes. The samptes were next run on a
10-20% Tricine/SDS gel (Novex~, according to the manufacturer's instructions. The gel was
stained with Coomassie (Pharmacia) and destained in order to visualize the peptide bands.
The Gln-lactoferricin B peptide was quite stable in wheat ECF. After a 3 hour
incubation, substantial amounts of peptide are still present, indicating that the degradation
of the peptide is relatively slow in this ECF. In corn and to~Acco ECF, Gln-lactoferricin B
peptide is also still present after the 3 hour incubation, albeit at decreased levels.
The results indicate that the Gln-lactoferricin B peptide is only slowly degraded in the
ECF of these 3 plant species. Degradation of the peptide in ECF takes over 3 hours under
these conditions.
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Example 3: Construction of a gene for cytoplasmic expression of lactoferricin B in plants
The coding and noncoding DNA sequences for the cytoplasmically expressed
synthetic lactofe,.ic;.~ B gene were obtained by synthesis of 2 partly complementary
~ oligonucleotides. The oligonucleotides encoded (from 5' to 3' on the coding strand) a BamHI
restriction enzyme end, a Kozak consensus site, including a meti,.cnine start codon, 75
base pairs encoding the amino acid sequence of the lactoferricin B peptide, a stop codon,
and a Notl restriction enzyme end.
The nucleotide sequences of the oligos were:
Oliqo 1 (SEQ ID NO:7):
5'-GATCCACCATGTTCAAGTGCCGCCGCTGGCAGTGGCGCATGAAGAAGCTGGGCGCCCCCAGC-
ATCACCTGCGTGCGCAGGGCCTTCTAAGC-3'
Oliqo 2 (SEQ ID NO:8):
~'-GGCCGCTTAGAAGGCCCTGCGCACGCAGGTGATGCTGGGGGCGCCCAGCTTCTTCATGCGCC-
ACTGCCAGCGGCGGCACTTGAACATGGTG-3'
~hese synthetic oligonucleotides were purchased and electrophoresed on a 10%
urea/polyacrylamide gel and the bands containing the full length oligonucleotides were
excised. The oligonucleotides were eluted, annealed and used for PCR amplification using
oligos 1-1 and 2-1.
Oliqo 1-1 (SEQ ID NO:9):
5'-AGTAGGATCCACCATGTTCAAGTGCC-3'
Oliqo 2-1 (SEQ ID NO:10):
5'-TACTGCGGCCGCTTAGAAGGCCCTGCGC-3'
The PCR product was ligated into pCRII, clones containing the insert were identified
and inserts were sequenced to confirm the absence of introduced mutations. The BamHI-
Notl insert of a clone with the correct sequence was cloned into BamHI and Notl
predigested Bluescript. A 2 kb Hindlll-BamHI fragment containing the ubiquitin promoter
(Toki, et al. Plant Physiol. 100: 1503) was cloned into the Hindlll - BamHI sites of this clone.
The nos terminator was ligated into the Notl-Sacl sites to generate pClB7703 (shown in
figure 1), a plasmid with the ubiquitin-lactoferricin B-nos gene cassette. A DNA fragment
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containing ubi-SynPAT-nos was cloned into the Hindlll site of this plasmid. The resulting
plasmid, pClB7704 ~shown in figure 2), contained the ubi-SynPAT-nos and ubi-lacto-nos
gene cassettes. This plasmid contains the SynPAT gene ~or selection in plants and is useful
for transformation of plants with the lactoferricin B gene under control of the maize ubiquitin
promoter.
Plasmid pClB7704 is cleaved with Kpnl and Sacl to release the frayment containing
the ubi-SynPAT-nos -- ubi-lacto-nos gene fusions. This fragment is cloned into Kpnl, Sacl
digested pClB6848 to form pClB7705 (shown in figure 3). Plasmid pClB7705 contains the
NPTII (kanamycin resistance) gene for selection in bacteria and the SynPAT gene for
selection in plants, and is useful for transformation of plants with the lactoferricin B gene
under control of the maize ubiquitin promoter.
The Kpnl-Sacl fragment from pClB7703, containing the ubi-lacto-nos construct is
cloned into plasmid pClB6848, to generate pClB7706 (shown in figure 4). This places the
ubi-lacto-nos gene construction in a kanamycin resistance gene containing plasmid.
~xample 4: Construction of a synthetic gene for extracellular expression of lactoferricin B
in plants
For extracellular expression of lactoferricin B, a leader sequence was placed at the 5'
end of lhe lactoferricin B gene. The leader sequence was derived from a maize PR1 cDNA
(PR-1 mz) that was cloned from a maize cDNA library (using a barley PR-1 gene probe) and
sequenced (patent publication WO 95/19443). Synthetic oligonucleotides and PCR
amplification are used to make this gene construct, in 2 steps. Oligonucleotide 3 was
identical to the PR-1 leader and the start of the lactoferricin B coding sequence, with the
exception of a single silent nucleotide change (to prevent potential secondary structure
problems). Oligo 4 was complementary to the lactoferricin B coding sequence (the design of
which is described in the detailed description), and the 3' end of the PR-1 leader sequence.
Oligo 3-1 was identical to the 5' end of the PR-1 coding sequence.
The nucleotide sequences of the synthetic oligonucleotides were:
Oliao 3 (SFQ ID NO~
5'-GATCCACCATGGCACCGAGGCTAGCGTGCCTCCTAGCTCTGGCCATGGCAGCCATCGTCGTGG-
CGCCATGCACGGCCTTCAAGTGCCG-3'
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Oliqo 4 (SEQ ID N0:12):
S'-GGCCGCTTAGAA~GCCCTGCGCACGCAGGTGATGCTGGGGGCGCCCAGCTTCTTCATGCGCCA-
CTGCCAGCGGCGGCACTTGAAGGCC-3'
Oliqo 3-1 (SEQ ID N0:13):
5'-AGTAGGATCCACCATGGCACCGAGGCTAG-3'
Oliqo 5 (SEQ ID N0:14):
5'-AGTACCATCGTCGTGGCGCCATGCACGGCCCAGTTCAAG-3'
First, the maize PR1 leader sequence was PCR amplified using oligos 3-1 and 4, with
the maize PR-1 cDNA clone as template. This generated a PCR fragment containing an
intact BamHI restriction site, the PR-1 leader, lactoferricin B and part of the Notl restriction
site. The lactoferricin B template was used in a PCR reaction, using as primers oligo 2-1
(described in Example 3 above) and oligo 3. These 2 PCR fragments were next mixed,
denatured at 94~C and extended with Taq DNA polymerase for 5 minutes at 72~C. Next,
this reaction was PCR amplified using oligos 2-1 and 3-1 for 10 cycles.
The PCR fragment was cloned into PCRII. Clones containing the insert were identified and
the inserts sequenced to confirm the absence of introduced mutations. A plasmid, #10,
containing an insert with one mutation in the leader sequence was isolated (GCC changed
to a GAC at the twelfth amino acid). In a repeat of the above experiment, a plasmid, #11,
with one mutation in the lactoferricin B gene sequence was isolated (GCC changed to an
ACC at the fifteenth amino acid). These two plasmids, each containing a different mutation,
were used to produce a corrected sequence. The BamHI-Notl inserts were cloned into
pBluescript as BamHI-Notl inserts. A BstXI fragment containing the mutation was removed
from the Bluescript plasmid containing insert #11 and replaced with the corresponding BstXI
fragment from the Bluescript plasmid containing insert#10. A 2 kb Hindlll-BamHI fragment
containing the ubiquitin promoter (Toki, et al. Plant Physiol. 100: 1503) was cloned into the
Hindlll - BamHI sites of this clone and the nos terminator was ligated into the Notl-Sacl
sites. The resulting plasmid was called p#10/11.
This corrected plasmid was next used as a template in a PCR reaction, using as primers
oligo 5 and oligo 2-1. Oligo 5 added one amino acid, a glutamine, before the first amino
acid of lactoferricin B. This was done to generate an authentic leader cleavage site at the
junction of the PR1 leader and the lactoferricin B sequence. Removal of the PR1 leader in
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plant cells is thus expected to generate a lactoferricin B peptide with an added N-terminal
glutamine.
The resulting PCR fragments were cloned into pCRII and clones were sequenced. A
BstXI-Notl insert with the correct sequence (containing the additional glutamine) was
isolated and next cloned into BstXI, Notl digested p#10/11 to generate pClB7707. This
placed the maize PR1 leader-lactoferricin B coding sequence under control of the ubiquitin
promoter and the nos terminator (ubi-PR1-lactoferricin B-nos). A DNA fragment containing
ubi-SynPAT-nos was cloned into the Hindlll site of this plasmid, resulting in plasmid
pClB7708, which contains the SynPat and PR1-lactoferricin B gene cassettes.
This plasmid is cleaved with Kpnl and Sacl to release the insert containing the ubi-
SynPAT-nos -- ubi-P~1-lacto-nos gene fusions. This fragment is cloned into Kpnl, Sacl
digested pClB6848 to form pClB7709. This plasmid contains the NPTII (kanamycin
resistance) gene for selection in bacteria and the SynPAT gene for selection in plants, and
is useful for transformation of plants with the lactoferricin B gene under control of the maize
ubiquitin promoter.
The Kpnl-Sacl fragment from pClB7707, containing the ubi-lacto-nos cassette is
cloned into plasmid pClB6848, to generate pClB7710. This places the ubi-lacto-nos gene
constrl~ction in a kanamycin resistance gene containing plasmid.
Example 5: Construction of a gene for vacuolar expression of lactoferricin B in plants
For vacuolar expression of lactoferricin B, a sequence for vacuolar targeting was
added to the 3' end of the PR1 leader-lactoferricin B gene cassette described in Example 4
(Construction of a synthetic gene for extracellular expression of lactoferricin B in plants)
above. This additional 18 base pair sequence encoded a C-terminal Val-Phe-Ala-Glu-Ala-lle
vacuole targeting sequence (Dombrowski et al., Plant Cell 5, 587-596,1993) and was
followed by a stop codon. In addition, restriction ends were incorporated at the 5' and 3'
ends of the synthetic gene, to facilitate cloning.
Two oligonucleotides were used to construct this synthetic gene. The sequence ofoligo 3-1 is described in Example 4 above. The sequence of oligo 6 contains the
complement of the 3' end of the lactoferricin B coding sequence, the codons for the vacuoJe
targetting sequence, a stopcodon and a Notl restriction enzyme site. Its sequence is listed
below.
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Oliao 6 (SEQ ID NO:15):
5'-AGTAGCGGCCGC-TTA-GATGGCCTCGGCGAACAC-GAAGGCCCTGCGCACGCA-3'
Oligo 3-1 and oligo 6 were used to PCR amplify the PR1 leader-lactoferricin B gene
construct in plasmid pClB7707, described in Example 4 above. The PCR fragment was
cloned into pCRII and clones with insert were selected. The inserts were sequenced and a
clone with the correct sequence was used to excise the BamHI-Notl fragment. Thisfragment was cloned into Bluescript, and a 2 kb Hindlll-BamHI fragment containing the
ubiquitin promoter (Toki, et al. Plant Physiol. 100: 1503) was cloned into the Hindlll - BamHI
sites of this clone and the nos terminator was ligated into the Notl-Sacl sites. This
generated plasmid pClB7712. This placed the maize PR1 leader-lactoferricin B-vacuole
target sequence under control of the ubiquitin promoter and the nos terminator (ubi-PR1-
lactoB-vac-nos). A DNA fragment comprised of ubi-SynPAT-nos was ligated into the Hindlll
site of this plasmid, resulting in plasmid pClB7713.
pClB7713 is cleaved with Kpnl and Sacl to release the insert containing the ubi-SynPAT-nos -- ubi-PR1-lacto-vac-nos gene fusions. This fragment is cloned into Kpnl, Sacl
digested pClB6848 to form pClB7714. This plasmid contains the NPTII (kanamycin
resistance) gene for selection in bacteria and the SynPAT gene for selection in plants, and
is useful for transformation of plants with the lactoferricin B gene under control of the maize
ubiquitin promoter.
The Kpnl-Sacl fragment from pClB7712, containing the ubi-lacto-nos cassette is
cloned into plasmid pClB6848, to generate pClB771015. This places the ubi-lacto-nos gene
construction in a kanamycin resistance gene containing plasmid.
Example 6: Construction of a gene for plastid expression of lactoferricin B in plants
For plastid expression of lactoferricin B, the chloroplast target sequence of the
ribulose-1,5-biphosphate carboxylase small subunit (ssu) gene is cloned and placed 5' to
the lactoferricin B gene sequence. This transit peptide sequence directs the small subunit
protein to the chloroplast, where it is cleaved off, to release the mature protein. The
designed transit peptide-lactoferricin B gene constructs contain (5' to 3') a BamHI restriction
site, a Kozak consensus sequence, including the methionine start codon, a chloroplast
target sequence fused to the lactoferricin B coding sequence, a stop codon and a Notl
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restriction site. Lactoferricin B gene constructs are made with the chloroplast target
sequence of the ssu gene from Arabidopsis (atslA; Wong et al. Plant Mol. Biol. 20, 81-g3,
1992) and from maize (Matsuoka et al., J. Biochem. 102, 673-676, 1987). The coding
sequence of the transit peptide of these ssu genes (Wong et al., Plant Mol. Biol., 20, 81-g3,
1992) is cloned by PCR amplification from genomic DNA, cDNA or a cDNA library. A series
of three transit peptide sequences (TP1, TP2, and TP3) are constructed from both the
Arabidopsis and maize ssu genes, following the design of Wong et al. (Plant Mol. Biol. 20,
81-93, 1992). Thus, TP1 contains the coding sequence of the transit peptide only (up to the
cleavage site), whereas TP 2 and 3 have the entire coding sequence for the transit peptide
and part of the coding sequence of the mature ssu protein, with a duplicated cleavage site.
TP2 has an additional 2 amino acid codons past the duplicated cleavage site. TP3 ends
exactly at the duplicated cleavage site.
a. Arabidopsis chloroolast target sequence-lactoferricin B gene constructs
The first transit peptide sequence was cloned by using oligonucleotides ATP-5 'and
ATP1-3' for the amplification. The sequences for these oligonucleotides were:
ATP-5' (SEQ ID NO:16):
5'-A~TAGGATCCACCATG~CTTCCTCTATGCTC-3'
ATP1-3' (SFQ ID NO:t7):
5'-GCAGTTAACTCTTCCGCCGTT-3'
The ATP-5' oligonucleotide contained the BamHI restriction site to facilitate cloning, a
Kozak consensus sequence ~' to the initiation codon and the first 18 nucleotides of the
coding sequence of the atslA gene. Genomic DNA was used as template. The amplified
product was cloned into pCR-Script(SK+) and clones were sequenced to verify that the
insert contained 165 bp of the atslA transit peptide coding sequence, extending from +1 to
+165 bp. A clone with the correct sequence was called pATP1.
Fusion of the transit peptide sequence to lactoferricin B was achieved by PCR using 4
primers:
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ATP-LF 5' (SEQID NO:18): 5'-GTAGGATCCACCAT~GCT-3', from the 5' end of the ATP1
al"F';'ication product;
ATP-LF 3': identical to oligo 2-1 described in Example 3 above;
and 2 bridge primers spanning the ats1A/lactoferricin B fusion and consisling of the 18
nucleotides at the 3' end of ATP1 followed by the 18 nucleotides at the 5' end of the
lactoferricin B coding sequence. The sequence of 5' bridge primer was:
ATP1-LFB 5 (SEQ ID NO:19): 5'-GGCGGAAGAGTTAACTGCTTCAAGTGCCGCCGCTGG-3'
The sequence of 3' bridge primer (the complement of the 5' bridge primer) was:
ATP1-LFB 3 (SEQ ID NO:20): 5'-CCAGCGGCGGCACTTGAAGCAGTTAACTCTTCCGCC-3'
For each transit fusion construct, two rounds of amplification were performed. The first
round involved two sets of reactions. Reaction 1 generated specific ATP1 PCR fragments
with a short lactoferricin B sequence extension. This reaction included primers ATP-LF 5'
and ATP1-LFB 3' and plasmid pATP1 DNA. Reaction 2 generated lactoferricin B PCR
fragments with a short 5' ATP1 sequence extension. This reaction included primers ATP-LF
3' and either ATP1-LFB 5' using cloned lactoferricin B as template. The second round of
amplification was performed by mixing the products of reactions 1 and 2 in equal volumes,
denaturing for 1 min at 94~C, then extending the annealed products for 5 min. at 72~C in the
presence of Taq DNA polymerase and nucleotides to form "full-lengthr template. This was
followed by addition of primers ATP-LF 5' and ATP-LF 3' and PCR amplification.
This generated the ATP1-lactoferricin B gene fusion. The PCR products were cloned into
the PCRII vector.
Clones are isolated and sequenced. Plasmids with the correct sequence are digested with
BamHI and Not I and the insert cloned into BamHI, Notl predigested Bluescript. A 2 kb
Hindlll-BamHI fragment containing the ubiquitin promoter (Toki, et al. Plant Physiol. 100:
1503) is cloned into the Hindlll - BamHI sites of this clone and the nos terminator is ligated
into the Notl-Sacl sites. The resulting plasmid contains the ATP1 chloroplast target
sequences upstream of the lactoferricin B coding sequence. This gene construct is
positioned between the ubiquitin promoter and the nos terminator.
A DNA fragment containing the ubi-SynPAT-nos gene construct is cloned into the Hindlll
site of these plasmids, to generate pClB7721. The resulting plasmids are cleaved with Kpnl
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and Sacl to release the insert containing the ubi-SynPAT-nos -- ubi-ATP1/213-lacto-nos
gene fusions. These fragments are cloned into Kpnl, Sacl digested pClB6848 to form
pClB7722. This plasmid contains the NPTII (kanamycin resi~ ce) gene for selection in
bacteria and the SynPAT gene for selection in plants, and is useful for transfor~"ation of
plants with the lactoferricin B gene under control of the maize ubiquitin promoter.
The Kpnl-Sacl fragment from pClB7720, cor)tair,i"g ubi-ATP1-lacto-nos gene fusions are
cloned into plasmid pClB6848, to generate pClB7723. This places ubi-ATP1-lacto-nos gene
fusions in a kanamycin resistance gene containing plasmid.
The second transit peptide coding sequence, ATP~, is cloned in a similar manner as ATP1,
using oligonucleotides ATP-5' and ATP2-3', using genomic DNA or oligo dT-primed, reverse
transcribed RNA as template.
Oligo ATP2-3' sequence (SEQ ID NO:21):
5'-CTGCATGCAGTTGACGCGACCACCGGAATCGGTAAGGTCAGG-3'
The PCR products are cloned and clones with insert are sequenced to verify that it contains
261 bp of the ats1A transit peptide coding sequence, extending from +1 to +261. A clone
with the correct sequence is called plasmid pATP2.
The third transit peptide coding sequence, ATP3, is cloned in a similar manner as ATP2,
using oligonucleotides ATP-5' and ATP3-3'.
Oligo ATP3-3' sequence (SEQ ID NO:22):
5'-GCAGTTGACGCGACCACCGGAATCGGTAAGGTCAGG-3'.
The PCR products are cloned and a clone with the correct sequence is called pATP3,
containing 255 bp ot the atslA transit peptide coding sequence, extending from +1 to +255.
These transit peptide sequences are fused to the lactoferricin B gene sequences as
described above for ATP1.
The sequences of the set of 5' bridge primers are:
ATP2-LFB 5' (SEQ ID NO:23):
5'-CGCGTCAACTGCATGCAGTTCAAGTGCCGCCGCTGG-3'
ATP3-LFB 5' (SEQ ID NO:24):
5'-GGTGGTCGCGTCAACTGCTTCAAGTGCCGCCGCTGG-3'.
The sequences of the set of 3' bridge primers (the complement of the 5' bridge primers) are:
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ATP2-LFB 3' (SEQ ID NO:25):
5'-CCAGCGGCGGCACTTGAACTGCATGCAGTTGACGCG-3'
ATP3-LFB 3' (SEQ ID NO:26):
5'-CCAGCGGCGGCACTTGAAGCAGTTGACGCGACCACC-3'.
The resulting PCR fusion products are cloned. Clones with the correct sequence have
ATP2-lactoferricin B and ATP3-la~;lof~rlici" B gene fusions. These fragments are cloned
into Bluescript and the ubiquitin promoter and nos terminator are added as described above
to generate pClB7730 and pClB7740. The ATP~/2/3-lactoferricin B gene constructs are
positioned between the ubiquitin promoter and the nos terminator. New constructs with the
ubiquitin-SynPat-nos fragment are generated as described above for pClB7720, resulting in
plasmids pClB7731 and pClB7741.
b. Maize chloroPlast taraet sequence-lactoferricin B qene constructs
Additional transit peptide-lactoferricin B fusion constructs are synthesized using transit
peptide sequences of a maize ssu gene (Matsuoka et al., J. Biochem.102, 673-676,1987)
in the same manner as described for the Arabidopsis ssu transit peptide-lactoferricin B gene
constructs. The only differences are the source of template DNA for the generation of the
transit peptides and the oligonucleotide sequences.
The template DNA for the PCR amplification of the maize ssu transit peptide sequence is
maize genomic DNA, cDNA or a cDNA library. The oiigonucleotide sequences (5' to 3') are
as follows:
MTP-5' (SEQ ID NO:27):
5'-AGTAGGATCCACCATGGCGCCCACCGTGATG-3'
MTP1-3' (SEQ ID NO:28):
5'-GCACCGGATTCTTCCGCCGTT-3
MTP2-3' (SEQ ID NO:29):
~ 5'-CTGCATGCACCGTATGCGACCACCGTCCGTCGACAGCGGCGG-3'
MTP3-3' (SEQ ID NO:30):
5'-GCACCGTATGCGACCACCGTCCGTCGACAGCGGCGG-3'
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MTP-LF 5' (SEQ ID NO:31):
5'-GTAGGATCCACCATGGCG-3'
MTP-L~ 3' (SEQ ID NO:32):
5'-GCGGCCGCTTAGAAGGC-3'
MTP1-LFB 5' (S~Q ID NO:33):
5'-GGCGGAAGAATCCGGTGCTTCAAGTGCCGCCGCTGG-3'
MTP2-LFB 5' (SEQ ID NO:34):
5'-CGCATACGGTGCATGCAGTTCAAGTGCCGCCGCTGG-3'
MTP3-LFB 5' (SEQ Ir) NO:35):
5'-GGTGGTCGCATACGGTGCTTCAAGTGCCGCCGCTGG-3'
MTF1-LFB 3' (SEQ ID NO:36):
5'-CCAGCGGCGGCACTTGAAGCACCGGATTCTTCCGCC-3'
MTF2-LFB 3' (SEQ ID NO:37):
5'-CCAGCGGCGGCACTTGAACTGCATGCACCGTATGCG-3'
MTF3-LFB 3' (SEQ ID NO:38):
5'-CCAGCGGCGGCACTTGAAGCACCGTATGCGACCACC-3'
The plasmids that are generated contain the maize transit peptide sequences (MTP1, 2 and
3) fused to the lactoferricin B coding sequence.
Example 7: Lactoferricin B gene expression in maize protoplasls
Protoplasts which were known to allow transient expression were used for transient
expression of lactoferricin B containing plasmids. ~lotoplAsts were isolated from susl~ension
cultured cells as described in Shillitto et al. (US patent 5,350,6B9: Zea mays plants and
transgenic Zea mays plants regenerated from protoplasts or protoplast derived cells. 1989)
and resuspended in 0.5M mannitol/1 5mM MgCI2 at a concentration of 20x1 o6 per ml.
Plasmid DNA used for transformations were: pUbi-bar, containing the Ubiquitin promoter
plus first exon and intron, the bar gene and the nos ten"inato~, pUbi-GUS, containing the
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Ubiquitin promoter plus first exon and intron, the GUS gene and the nos terminator; and
pClB7703.
Plasmid DNAs were introduced into the prDtopl~stc by PEG preG,pit~lion (Kramer et al.,
Planta 190, 454-458,1993) as follows: protoFI2sts were heat-shocked by gently agitating
the tube in a 45~C water bath for 4 min. Aliquots of 500 microliter, containing 10 million
protoplasts were l,ansfer,ed to sterile tubes and the following additions made to each tube:
25-50 ~Lg of each plasmid and 450 microliter 40% PEG. The PEG was prepared from PEG
8000 (Sigma Chemical Co., St. Louis, M0) dissolved in a sotution of 0.4M mannitol and
0.1 M Ca(N03)2-4H20, with the pH adjusted to 8Ø Tubes were allowed to stand for 20 min.
with occasional gentle swirling, then diluted at 5 min. intervals with 1 ml, 2 ml and 5 ml
volumes of W6 solution (5mM KCI, 61mM CaCI2-H20,154mM NaCI,51mM glucose,0.1%
2-(N-morpholino) ethanesulfonic acid, pH 6.0). After the final dilution, protoplasts were
centrifuged for 10 min at 609-1009 and most of the supernatant removed, leaving about 0.5
ml of liquid over the protopl-st~. Protoplasts were resuspended in the remaining liquid by
gentle shaking. Protoplasts were resuspended at a density of two million/ml in FW medium
and cultured at room temperature in the dark for 18 h after DNA introduction for analysis of
RNA and protein expression.
Protein extracts were prepared from aliqouts of all transformations for subsequent GUS
activity determinations, according to the manufacturer's specifications (Tropix). The results
showed that cells transformed with a combination of pClB7703 and ubi-GUS had abundant
GUS activity (10x106 counts/min/1x106 protopl~sts), comparable to the GUS activity in
cells transformed with ubi-bar and ubi-GUS ~10x106 counts/min/1x106 protoplasts). This
indicated that expression of the lactoferricin B gene construct had no effect on GUS gene
expression in the transiently expressing cells, i.e. no cell toxicity was evident. Cells
transformed with ubi-bar and pClB7703 showed only background levels of GUS activity
(5000 counts/min/1 X106 protoplasts).
RNA samples were prepared for reverse transcription-PCR (RT-PCR) analysis to
evaluate the presence of lactoferricin B mRNA. For this purpose, total and poly(A) RNA was
isolated from transfected protoplasts. RT-PCR was performed using a commercial kit
(Stratagene), per manufacturer's instructions. For detection of ubi-lactoferricin B-nos cDNA,
PCR primers were used that were complementary to the sequence just 5' to the
polyadenylation signal of the nos terminator and to the first exon in the ubiquitin proi"oter.
Since there was an intron in the pClB7703 construct between this exon and the lactoferricin
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B coding sequence/nos terminator, the resulting PCR band could be verified as being
derived from RNA, based on its size.
For the RT-PCR reaction, 1 ~11 of total RNA was reverse l,~,-s~ ed according to the
manufacturer's instruction, in a 50 ~11 reaction volume. Of this, 1 1ll was used for PCR
amplification using primers 176 (SEQ ID NO:39: 5'--ll'CCCCAAC~ 3') and
179 (SE~ ID NO:40: 5'-CCAAATGTTTGAACGATCGCG-3'), or primers 177 (SEQ ID NO:41:
5'-CACAACCAGATCTCCCCCAAA-3') and 180 (SEQ ID NO:42:
5'-AAATTCGCGGCCGC~TAGAAG-3'). Reaction condilions were: 45 sec. at 94~C, 45 sec. at
60~C and 2 min. at 72~C, for 43 cycles. The samples were size separated on agarose gel.
The primers 176 and 179 yielded a PCR fragment of approximately 220 bp, as expected if
template DNA was cDNA derived from cells that were transformed with the pClB7703plasmid. A PCR band derived from plasmid was expected to be 1.3 kb in size.
For northern analysis, RNA samples are size separated on an agarose gel and blotted
onto a nylon membrane. This blot is subsequently hybridized to a radioactive probe
containing lactoferricin B gene construct sequences.
Protein extracts for immunoblotting are prepared by making a protoplast suspension
in an appropriate buffer. The suspension is microcentrifuged for five minutes and the
supernatant and pellets are suspended in Laemmli sample buffer and subjected to Laemmli
SDS gel electrophoresis. Since disulfide linkage of lactoferricin B peptide to proteins may
be a problem, reduction with DTT or 2-ME, followed by iodoacetamide blockage of protein -
Stl groups in extracts may be required before electrophoresis.
Electrophoresed gels are electroblotted to nitrocellulose for Westem analysis, using an
anti-lactoferricin B antiserum.
Protein extracts for mass spectroscopy are prepared by vortexing the proloplast
suspension vigorously in a buffer, microcenl,il.lging twice for 10 minutes after which the
supernatant is collected each time. An appropriate dilution is analyzed by MALDI-MS
(Voyager, Perceptive Biosystems), using standard protocols for peptide analysis. If the
samples need to be further purified, extracts can be fraclionated on a C18 column.
Fractions are then analyzed for the presence of the lactoferricin B peptide by MALDI-MS.
Protein extracts are also prepared for evaluation of antifungal activity. In this case,
the protoplasts are homogenized in 20mM Tris buffer, pH 7.5, containing 1.5% PVPP, 5mM
Drr, and 10 !lM AEBSF (4 - (2-Aminoethyl) - benzenesulfonyl fluoride, h~,cl,ocl,loride). The
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extracts are evaluated for antifungai activity based on in vitro fungal inhibition assays as
described in Example 1 above.
Transient expression analysis of other la~,t~fer,i..;n B gene containing constructs is
performed using methods described in this Example.
.
Example 8: Plant transformation, selection and regeneration
a. Description of plasmids and selectable markers
The lactoferricin B gene constructs are under control of the ubiquitin promoter and the
nos terminator. For corn transformation, a select~hle marker is cloned into these plasmids,
consisting of the synthetic phosphinothricin acetyltransferase gene (designated SynPAT),
under control of the maize ubiquitin promoter and the nos terminator (ubi-SynPAT-nos).
SynPA~ is a synthetic version of the bar gene from a Streptomyces strain (Thompson et al.,
EMBO 6:2519-2523,1987), that was optimized for expression in plants (US patent
5,276,268: Phosphinotricin-resistance gene and its use). The plasmid containing the ubi-
SynPAT-nos cassette (pUBlAc) was obtained from Hoechst Aktiengesellschaft, Frankfurt,
Germany. The plasmids containing ubi-lactoferricin B-nos and ubi-SynPAT-nos cassettes
are used to transform corn. The presence of the SynPAT gene cassette allows selection of
transformed plants using the herbicide BASTA.
For wheat transformation, plasmids containing ubi-lacto-nos gene cassettes are co-
bombarded with a plasmid (pUBA or pUBA/kan) containing the bar selectable marker gene
under control of the ubiquitin promoter and nos terminator (Toki et al,1992 Piant Physiol.
100:1503-1506). The presence of the bar gene cassette allows selection of transformed
plants with the herbicide BASTA. The pUBA plasmid conhins ubi-bar-nos and the ampicillin
resistance gene for selectioin in E. coli. Plasmid pUBAJkan contains a kanamycin resisldnce
gene instead of the ampicillin resistance gene for selection in E. coli.
A hygromycin gene from E. coli (Gritz and Davies, Gene 25,179-188,1983) was
isolated, Bgl ll linkers were ligated onto the ends and this fragment was next cloned into the
BamHI site of vector pClB710, to generate pClB712. Two oligonucleotides
~5'-AGTAGGATCCATGAAAAAGCCTGAACTC-3' (SEQ ID NO:43) and
5'-TACTGGATCCCTATTCCTTTGCCCTC-3' (SEQ ID NO:44)) were used to PCR amplify
the gene from pClB712. The PCR fragment was digested with BamHI and cloned into the
BamHI site of pPEH3. Clones with insert were sequenced and one with the correct
sequence and the correct orientation of the gene was named pClB7613. This plasmid
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contained the hygromycin resistance gene under control of the ubiquitin promoter and the
nos terminator. rel'~ .ing bombardment of plants with this plasmid, translor",ed plants can
be selected using hygromycin as the selection agent.
The acetohydroxyacid synthase (AtlAS) gene was cloned from a Lambda-Zap
genomic library derived from sulfonylurea (Beacon) resistant maize tissue, using as a probe
the Ar~.dopsis gene (K. Sathasivan et al., Nucl. Acid Res. t8, 2188). A G to A transition
was introduced at position 1862 in this gene, by PCR amplilic~lion, using one
oligonucleotide with the appropriate single base change. This nucleotide change resulted in
a Ser to Asn change. This gene was cloned into a vector to generate plasmid pClB4247.
PCR amplification using 2 oligonucleotides specific for the start and end of the maize AHAS
gene in pClB4247 (5'-TATCTCTCTCTATAAGGATCCATGGTCACC-3' (SEQ ID NO:45) and
5~-TAcTG~ATccTcAGTAcAcAGTccTGcc-3~(sEQ ID NO:46)) generated fragments that
were cloned into pCRII. Inserts were sequenced to verify the absence of mutations and a
plasmid with a correct insert was used to isolate the BamHI fragment with the AHAS
sequence. This fragment was cloned into the BamHI site of pPEH3, to generate pClB7612.
This plasmid contains a mutated maize AHAS gene under control of the maize ubiquitin
promoter and the nos terminator. This gene confers resistance to imidazolinone
- (imazaquin/Scepter) when transformed into plants.
The protoporphyrinogen oxidase (protox) cDNAs from Arab.~'~psis and maize and the
protox gene promoter from Arabidopsis were isotated. The protox cDNAs from Ar~hidopsis
and maize were cloned by complementation of a protox deficient E. coli strain. Mutant
Arabidopsis and maize protox genes were selected that resulted in increased resistance to
the protox inhibiting herbicide. The herbicide resistant Arabidopsis protox cDNA was cloned
into a plasmid between the double 35S promoter and the tml terminator.
The Arabidopsis protox promoter was isolated from a genomic library, using a
fragment from the 5' end of the Arabidopsis protox cDNA as a probe. The plasmid with the
promoter was digested with Ncol and BamHI, leaving the promoter attached to the vector,
but removing the downstream sequences. The Ncol-BamHI fragment containing the
herbicide resistant protox cDNA and tml terminator was cloned into it. This created a
plasmid with approximately 600 nucleotides of the Ardbidopsis protox promoter, the coding
sequence of the herbicide resistant Arab protox gene and the tml ten.,inator. Ptants
transformed with this plasmid can be selcted on medium containing the herbicide.
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b. Transformation of lactoferricin B gene constructs into maize
The method used for maize transformation has been described by Koziel et al.
(Biolecl-nology 11, 194-200,1993) using particle bG~ arJulent into cells of immature
embryos. Immature embryos of maize inbred CGA00526 were isolated aseptically
approximately 10 days after pollination, and plated scutellum side up on modified Duncan's
"D" medium (Duncan et al., Planta 165, 322-332, 1985). The medium was supplemented
with Chloramben (5mg/L) instead of dicamba. App~oxi",ately 2 weeks later, callus was
isolated from the embryos and plated on media supplemented with 2,4-dichlorophenoxy
acetic acid (5mgtL) instead of Chloramben and subsequently subcultured at 10 - 14 day
intervals. Four to six hours prior to transformation, 1 - 4 month old callus was transferred to
fresh medium containing 12% sucrose as an osmoticum.
Plasmid DNA, or isolated fragment DNA was precipitated onto gold particles as described in
the DuPont Biolistic manual. Each plate of tissue was shot twice with the DuPont Biolistics
helium device. A rupture pressure of 600-1100psi and a standard 80-100 ~lm mesh
screen were used. After bombardment, the tissue was placed in the dark for 12 - 24 hours.
Following this time period, the tissue was transferred back to modified Duncan's "D"
medium with the addition of the selection agent BASTA at a minimum of 20 mg/L and grown
in the dark. The modified Duncan's "D" medium has the amino acid supplement replaced
with modified Koa's amino acid supplement (K. W. Kao and M. R. Michayluk, Planta 126,
105-110, 1975), except that glutamine and asparagine are omitted completely. The tissue
was transferred to fresh media containing BASTA at a minimum of 20 mg/L, at no less than
14 day intervals tor 60 - 80 days. During this subculture period nonembryogenic callus was
removed.
After this period of selection in the dark, the tissue was transfer,ecl to an MS based
medium (Murashige and Skoog, Physiol. Plant 15, 473-439, 1962) containing the folloY,~;ng
hormones to induce germination and development of somatic embryos: ancimidol
(0.25mglL), kinetin (0.5mglL), napthaline acetic acid (1 mg/L), and BASTA at a minimum of
5 mg/L. The tissue was cultured on this medium in a 16 hour light regime for 10 - 14 days,
then subcultured to MS based media with BASTA (3 mglL) minus the plant hormones to
allow plantlet development. As plantlets developed, they were transferred to a 314 sl,erlgll,
MS medium and allowed to develop roots. Rooted plants were transferred to soil and grown
in the greenhouse.
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c. Transformation of lactoferricin B gene constructs into wheat
Transformation of immature embryos and immature embryo-derived callus using
particle bombardment has been described by Vasil et. al. (Biotechnology 11: 1553-1558,
1g93) and Weeks et. al. (Plant Physiology 102: 1077-1084,1993).
Immature emb~yos of spring or winter wheat are i50'~tecl aseplically appro~ ,ately 2 weeks
after pollination, and cultured scutellum side uppermost on MS based medium (Mu,dsh ~e
and Skoog,1962 Physiol. Piant 15:473-439) supplemented with 2,4-D
(dichloropheoxyacetic acid), glutamine and asparagine for callus induction for 6-10 days.
Four to six hours prior to transformation embryos are selected for an embryogenic response
and transferred to fresh medium also containing 15% maltose as an osmoticum. Constructs
used for transformation include a plasmid containing the ubi-bar-nos casseme (pUBA or
pUBA/kan) allowing selection of transformed tissues with BASTA. Alternatively, constructs
containing genes conferring resistance to other herbicides (e.g., sulfonylureas,imidazolinones) or antibiotics (e.g., hygromycin), can be used. This construct is co-
transformed with one of the lactoferricin B containing gene constructs described in
Examples 2-6 above, including pClB7706, pClB7710, pClB7715, pClB7723, pClB7733 and
pClB7743. For this, plasmid DNA is precipitated onto gold particles as described in the
Dupont Biolistic manual. Each plate of embryos is shot with the DuPont PDS1000 helium
device using a burst pressure of 1100psi and a standard 80 mesh screen. After 24 hours,
the embryos are removed frorn the osmoticum and placed back onto induction media.
Approximately one month later, the embryo explants with developing embryogenic callus
are transferred to regeneration medium (MS + 1mglL napthaline acetic acid and 5mg/L
gibberellic acid), containing 1 mg/L BASTA for two weeks, then transferred to hormone free
regeneration media containing 3 mg/L BASTA for 2 to 6 weeks, until shoots develop. The
embryos with shoots are transferred to half strength MS with 0.5mglL NAA and 3 mg/L
BASTA until roots develop, at which time the plantlets are transferred to soil and grown to
maturity. Plant tissue is harvested for analysis (described in Example 10) and plants are
selfed for production of seed.
d. Transformation of lactoferricin B gene constructs into sugar beet
Guard cells are isolated from sugarbeet leaf sections without midribs, as described in Hall et
al, Nature Biotechnology, vol.14, pages 1133-1138,1996. The leaf sections are
homogenized with a blender (23,000 rpm for 60 seconds) in 50 ml Ficoll medium (100g/l
Ficoll,735 mg/l calcium chloride ~ 2 H20 and 1 g/l PVP40) while kept on ice. Following
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dlion through a nylon mesh (300 micrometer~, the homogenate is washed with ~00 ml of
cold water and transferred to 10 ml CPWgM containing 3.8% (w/v) calciumchloride ~ 2 H20.
The epidermis is then recovered by centrifugation and digested with cell wall degrading
enzymes (0.5% cellulase and 3% macerozyme, Yakult Honsha, Japan) for 16 hours torelease individual cells. Following filllation through 55 micrometer filters, the suspension is
mixed with an equal volume of Percoll containing 15% sucrose. In tubes, 1 ml CPW15S is
layered on top of the protoplast suspension, f~ wed by 0.5 ml 9% mannitol/l mM
calciumchloride. The guard cells are recovered from the top layer after a 10 minute spin at
~5g and reisolated once again in the same way.
DNA (50 microgram per one million cells) is added to guard cells resuspended in 0.75 ml
9% mannitol/1 mM calciumchloride (mannitol solution), and this is followed by the addition
of 0.7~ ml 40% PEG 6000. After 30 minutes at room temperature, 2 ml aliquots of F
medium is added every 5 minutes, for a total of 4 aliquots. Protoplasts are washed with the
mannitol solution and then embedded in Ca alginate. They are cultured in modified K8P
medium and selection with ~ialaphos is started one week later at 200 microgramlliter.
Eleven days later, small alginate sections are embedded in agarose, and cultured in PG1B
medium with 250 microgram bialaphos/liter. Calli that develop are transferred to fresh plates
with bialophos and cultured for 2 weeks. After this they are cultured without selection, at
25~C; with a lighUdark regimen (1 6hl8h). Subculturing is performed every 2 weeks and
plantlets are regenerated in 4 weeks. They are rooted and planted in soil.
Fxample 9: Analyses of transgenic plants expressing lactoferricin B
Tissue from transformed plants that survive the selection process (Example 8) are
analyzed for the presence of the lactoferricin B gene construct (PCR), RNA derived from
this transgene (northern blot hybridization, RT-PCR) and protein derived from this transgene
(Western).
a. PCR analysis
DNA is extracted from transformed plant tissue using the IsoQuick nucleic acid
extraction kit (ORCA Research Inc.) according to instructions provided with the kit. PCR
analyses are performed according to standard protocols. The primers used for a"~pli~ic~t;on
of the lactoferricin B gene constructs are designed from the known sequences of the
transgene constructs.
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b. Northern analysis
Transformed plants were analyzed for the presence of RNA by northem blot
hyb,idi~dlion. For northern blot analysis, RNA was extracted from leaf tissue. r~'lc.~i"g
grinding of the frozen tissue, extraction buffer was added (0.1 M LiCI, 100 mM Tris pH 8, 10
mM EDTA, 1% SDS), followed by equal volumes of water-saturated phenol and chl~n~fc ~
The RNA was prec;~ilated from the aqueous phase with sodium acetate/ethanol and size
separted on a formaidehyge containing agarose gel. The gel was blotted to GeneScreen
Plus nylon, which was hybridized to a probe derived from the lactoferricin B gene. Following
overnight hybridization, the blot was washed at high stringency (65~C) and exposed to X-ray
film.
c. RT-PCR analysis
RNA samples are also analyzed by RT-PCR for the presence of lactoferricin B RNA,as described in example 7 above.
d. Western analysis
Transformed plants were tested for the presence of lactoferricin B or lactoferricin B-
derivative proteins encoded by the transgene. For western analysis, fresh leaves were
snap frozen in liquid nitrogen and ground to a fine powder. The samples were then treated
with cold 10% trichloroacetic acid in acetone at -20~C for 1 hr, spun down, washed with cold
acetone and air-dried. The dried samples were then incubated with 0.05 M sulfuric acid (3
mllg fresh weight) for 1 hr on ice. The extracts were neutralized with 0.01 N sodium
hydroxide, and dried. Samples were run on 10%-20% Tricine SDS gels (Novex) according
to manufacturer's instructions. Proteins were transferred to nitrocellulose at a consld,)l
current of 200 mA for 3 hr in a wet cell, then stained with Ponceau Red to visualize
membrane-bound protein.
Proteins transferred to nitrocellulose were probed with immuno-affinity purified goat anti-
lactoferricin-B antibodies, followed by secondary and alkaline phosphatase-conjugated
tertiary antibodies. Anti-lactoferricin B reactive bands were detected after addition of a nitro
blue tetrazolium / bromo-chloro-indolyl phosphate substrate solution.
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e. Mass sPectroscoDy
Protein extracts prepared from llari:jlur.,,ed plants are also analyzed for the presence
of the lactoferricin B peptide, using a MALDI-MS, as described in Example 7 above.
f. Evaluation of antimicrobial activity in lactoferricin B transgenic Plant extracts
Protein extracts are prepared from la-,1Ofer,ic;n B transgenic plants by homogeni2i,)g
tissue in 20mM Tris buffer, ptl 7.5, containing 1.5% PVPP, 5mM DTT, and 10 ~M AEBSF (4
- (2-aminoethyl) - benzenesulfonyl fluoride, hydrochloride). Protein extracts are further
purified by est~hlished protocols such as ammonium sulfate preciriPtion, HPLC
chromatography and immuno purification using an anti-lactoferricin B antiserum. The
protein concentration is determined in each extract and aliquots are used in antifungal
assays.
The three assays described in Example 1 above are used to evaluate the presence of
antimicrobial activity in the crude and partially purified protein extracts. The organisms used
to test for the presence of antimicrobial activity include those listed in Example 1, as well as
other important fungal pathogens of corn and wheat and lactoferricin B sensitive bacterial
lab strains, such as E. coli and Staphylococcus aureus (W. Bellamy et al., J. Appl. Bacteriol.
73, 472-479, 1992).
Example 10: Testing Lactoferricin B transgenic plants for increased disease resistance
a. Testinq of transgenic maize Plants
1 Southern corn leaf blight (SCLB) assay
SCLB is caused by Bipolaris maydis, formerly named Cochliobolus heteroslruphus
and I lel""nll,osporium maydis. B. maydis cultures are grown on PDA (potato dextrose agar)
under continuous near-UV light to induce sporulation. Spores are harvested by gently
scraping the plate using a glass rod in 1% Tween-20. The solution is collected and the
spore concentration ad~usted to 500-1000 spores/ml with double distilled, sterile water.
Transgenic plants and wild type control plants are grown in a greenhouse to 3-5 weeks of
age. The 3 upper fully expanded leaves are sprayed with the spore suspension, byapplying
a fine mist of the spore suspension to each leaf, using an aerosol sprayer. Next, the plants
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are placed in a closed chamber and incubated overnight (16-20 hours) in a mist chsmber,
under high humidity conditions. The plants are moved from the mist chamber into a growth
chamber. Appro~-i"lately one week after inocul~tion, lesions that develop are counted and
the size of 10-15 representative lesions are measured and compared between the control
and transgenic plants. Plants are also visually rated for dise~se progression, at 3-4 day
intervals over a 14 day period following inoculation.
Plants from a T1 line, E~10b, that expressed Met-Lactoferricin B mRNA showed
increased disease resistance, as disease symptoms that were less than those of the
transgenic controls and wild type controls.
2. Anthracnose leaf blight
Colletotrichum graminicola is the causal agent of anthracnose leaf blight. It causes
necrosis of infected leaves. For the disease assays, spores were collected from sporulating
agar plates as described for B. maydis. Leaves of 3 to ~ week old plants were sprayed with
a spore suspension at approximately 10E6 sporesJml concentration. The inoculated plants
were incubated overnight in the mist chamber, after which they were moved to a growth
chamber, as described for B. maydis infected plants. Over a 2 week period, at 3-4 day
inte~als, the percentage of leaf area that was necrotic was determined and compared
between transgenic and non transgenic controls.
3. Carbonum leaf spot assay
Helminthosporium carbonum is the causal agent of maize carbonum leaf spot. For
the disease assay, spores were harvested from sporulating cultures, as described for B.
maydis. Leaves of 3-5 week old corn plants were sprayed with the spore suspension at an
concentration of 2.5 to 10 x 10E4/ml, and the plants were then incubated in the mist
chamber overniyht. Next, the plants were grown for approximately 14 days in the growth
chamber. Disease progression was followed, as described for anthracnose leaf blight.
Plants from T1 line B10b that expressed RNA for Met-lactofelricin B showed
increased disease resistance. In the ex~uressi"g plants, decreased dise~se s~"l,,~tor,ls were
observed following carbonum leaf spot inoculation, compared to transgenic controls and
wild type controls.
, ... .. . . .
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4. FusariL~m root assay
Fusarium moniliforme causes Fusarium ear mold on corn ears. F. moniloforme is
grown on PDA plates and spores harvested from the plates as described for B. maydis.
Perforated filter paper is folded, placed in a pouch, and surface sterilized corn seeds placed
in the fold, such that the emerging roots grow through the pe~ ~orQtions to the bottom of the
pouch. A 12 ml spore suspension of F. moniliforme is placed in the bottom of the pouch
and the pouch incubated in a growth chamber at 2~-30~C. Inhibition of root growth and root
necrosis due to fungal infection is assayed 7-14 days later and compared betweentransgenic and non transgenic controls.
5. Pythium root assay
Pythium aphinadermatum is one of the causal organisms involved in maize stalk rot
and root rot. For disease assays, a pouch format is used, as described for F. Inoculum is
prepared from zoospores or mycelium. For zoospore formation, mycelia are placed in
contact with leaf pieces of tall fescue grass, incubated under continuous fluorescent light
and zoospores that develop are harvested. Alternatively, potato dextrose medium is
inoculated with a mycelial plug from a Pythium PDA plate and allowed to grow for 2-3
weeks. The mycelia are removed from the culture and dispersed using a blender, in sterile
double distilled water.
Corn seeds are placed in the filter paper fo1d, as described and inoculum (zoospores
or dispersed mycelium, at an appropriate concentration) added to the bottom of the pouch.
As described for F. moniliforme, after incubation for 1-2 weeks, root length and necrosis is
assayed and compared between transgenic and non transgenic controls.
6. Stewalt's bacterial wilt
Erwinia stewartii causes Stewart's wilt on corn plants. For ~isease assays, bacteria
are grown to high density in Nutrient medium and diluted to approximately 107 bacteria/ml.
Leaves of corn plants are infected with the bacteria by pricking the leaves with needles that
are dipped in the bacterial suspension. Lesions are measured 1, 2 and 3 weeks after
inoculation.
7. Leaf disc assays
The whole-plant disease assays described above can also be performed on leaf discs
that are placed in moist petridishes.
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b. Testin~ of transgenic wheat Plants
1. Pseudo")onas ~ eAse assay
Pseudomonas syringae pv. syringae Van Hall causes bacterial leaf blight of wheat.
For the dise~se assay in which visual disease s~""pto",s are scored, the bacteria (strain
BL882) are grown overnight on L-broth agar plates. A small swath of cells is scraped from
the plate with a toothpick and resuspended into a solution of 10 mM MgCI2. The
absorbance of the suspension at 600 nm is taken in order to determine the concentration of
bacteria in the suspension, which is calculated as 1 x toB cfu/ml per OD600 of 0.1. The
culture is diluted to 1X107 bacteria/ml and small amounts are drawn by suction into a 1 ml
plastic pasteur pipet. Approximately 50 ul of bacteria are injected into the leaves of 3 week
old wheat plants. The extent of the inoculated area is lightly marked using a black felt-tip
pen. The plants are placed in a plastic box with the internal environment humidified by
heavy application of water to the plants using a common garden sprayer. The box is closed
tighUy and placed in a Percival chamber at 1 8-20~C and given 16 hours of light per day.
After five days, the severity of disease is scored by calculating the percentage of lesioned
area, as evidenced by dark necrotic patches surrounded by a thin layer of chlorosis,
occurring within the inoculated zone.
For more precise quantification of the degree of disease development, leaves areevenly inoculated with B~882 containing a plasmid with the kanamycin resistance gene, by
infiltration of bacteria at a concentration of 1 x105 cfu/ml. On the day of infiltration and on
consecutive days, the bacterial titer in the injected area is determined. This is accomplished
by combining leaf punches from infiltrated areas that collectively cover a ~ cm space,
grinding them in 10 mM MgCI2, and spreading dilutions of the extract on LB plates
supplemented with 50 microgramlml kanamycin ~this BL882 isolate has been transformed
with a kanamycin resistance gene). After three days of growth, single bacteria from the leaf
grow into colonies which are easily counted. In this manner, the growth rate of the bacteria
in transformed plants can be quantified and compared to that of control, untransformed
plants.
2. Septoria nodorum assay
S. nodorum causes causes leaf blotch on wheat leaves and glume blotch on wheat
ears. For the disease assay, spore are collected from sporuiating plates or liquid culture,
and sprayed onto the leaves of 2 week old plants, at an appropriate concentration. The
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inoculated plants are placed in a high humidity mistchamber for 2-3 days and subsequently
moved to a growth chamber. The percent diseased leaf area is determined over the next 3
weeks and the presence or absence of pycnidia is noted.
3. Septoria tritici assay
S. tritici causes a leaf blotch ~Jiseace on wheat. For the disease assays, spores are
isolated from sporulating plates or liquid cultures, and sprayed, at an appropriate
concentration, onto leaves of 1-2 week old wheat plants. The inoculated plants are placed
in a high humidity mistchamber for 3 days and subsequently moved to a growth chamber.
The percent diseased leaf area is determined 2-3 weeks after inoculation and the presence
and absence of pycnidia is noted.
4. Leaf disc assays
The whole-plant disease assays described above can also be performed on leaf discs
that are placed in moist petridishes.
5. Fusarium pouch assay
This assay is similar to the F. moniliforme and P. aphinadermata assays for maize
described above. Spores from F. culmorum are harvested from plates as described for B.
maydis. Perforated filter paper is folded, placed in a pouch, and surface sterilized wheat
seeds are placed in the fold, such that the emerging roots grow through the perforations to
the bottom ot the pouch. A 12 ml spore suspension of F. culmorum in Hewitt's medium (a
nutrient medium to allow the wheat seeds to germinate and grow) is placed in the bottom of
the pouch and the pouch is incubated in a growth chamber at 10-t5~C for about 1 week,
after which the incubation is continued at 15-1 8~C. About four days later rootlength and
rootbrowning is scored.
6. Powdery mildew leaf assay
Erysiphe graminis f. sp.tritici causes powdery mildew on wheat leaves. It is an obligate
biotroph and hence inoculum is maintained on wheat leaves. Plants for disease testing are
grown to 12-14 days of age, 2.5 cm sections are cut from the leaves and placed on agar
plates containing 50 mg/ml benzimidazole. Spores collected from infected leaves are used
to inoculate the leaf pieces on the agar plate, at an appropriate concentration. Next, the
plates are incubated at 17~C under low light conditions. Disease sy",pto",s on the leaf
pieces are evaluated approximately 10 days after inoculation.
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Fxample 11: Human Lactoferricin (Lacto H)
An antibacterial and antifungal peptide has also been isolated from human lactoferrin,
referred to as lactoferricin H. This peptide corresponds to aminoacids t-33 of the mature
protein. A synthetic gene is generated that codes for the lactoferricin H peptide (SEQ ID
NO:2), with codons optimized for expression in maize (SEQ ID NO:5):
G R R R R S V Q W C A
GGC-CGC-CGC-CGC-CGC-AGC-GTG-CAG-TGG-TGC-GCC-
V S Q P E A T K C F Q
GTG-AGC-CAG-CCC-GAG-GCC-ACC-AAG-TGC-TTC-CAG-
W Q R N M R K V R G P
TGG-CAG-CGC-AAC-ATG-CGC-AAG-GTG-CGC-GGC-CCC
To allow transcription and translation of this gene sequence, a methionine start codon and a
stopcodon are added. In addition, the ACC sequence from the Kozak consensus sequence
is added to the beginning of the sequence, to improve the likelihood of efficient translation.
This generates the Met-Lactoferricin H gene sequence as shown below, with the start and
stop codons underlined.
5'-ACCATGGGCCGCCGCCGCCGCAGCGTGCAGTGGTGCGCCGTGAGCCAGCCCGAGGCCACCAAG-
TGCTTCCAGTGGCAGCGCAACATGCGCAAGGTGCGCGGCCCCTAG-3'~SEQID NO:6)
This gene sequence is expressed in plant cells under the control of an appropriate
promoter, using methods analogous to those descibed in the preceeding examples for
lactoferricin B.
.. . . .
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~UU~NL~ LISTING
(1) GENERAL INFORM~TION:
(i) APPLICANT:
(A) NAME: NOV~RTIS AG
~B) ST~Ekl: SChWar~A~1 ~A1 1 ee 215
~C) CITY: Basel
~E) COUNTRY: Switzerland
~F) POSTAL CODE tZIP): 4058
(G) TELE~HoNE: +4161 324 11 11
(H) TF~FFAX: + 4161 322 75 32
(ii) TITLE OF INVENTION: Peptide with inhibitory activity towards
plant pathogenic fungi
(iii) N WBER OF SEOUENCES: 46
(iv) C0MPUTER REACABLE FORM:
(A) MEDIUM TYPE: Floppy disk
~B) COMPVTER: I~M PC compatible
(C) OPERATING SYST~M: PC-DOS/MS-DOS
~D) SOFTW~RE: PatentIn Release #1.0, Version #1.25 (EPO)
(2) rNFORMATION FOR SEQ ID NO: 1:
(i) SEOUENCE CHARACTERISTICS:
tA) LENGTH: 25 amino acids
(B) TYPE: amino acid
(D) TOPOL0GY: linear
~ F~ F TYPE: peptide
(iii) H~ul~kllCAL: NO
(iii) ANTI-SENSE: NO
(v) FRAEPENT TYPE: N-t~
(vi) ORIGIN~L SOUROE:
(A) ORGANISM: Bovine Lactoferricin
(xi) SEOUENCE DESCRIPTION: S_Q ID NO: 1:
Phe Lys Cys Arg Arg Trp Gln Trp Arg Met Lys Lys Leu Gly Ala Pro
1 5 10 15
Ser Ile Thr Cys Val Arg Arg Ala Phe
~2) INFORM~TION FOR S_Q ID NO: 2:
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-~4-
(i) ~Uk~ C~PRA~qERISTICS:
(A) LEN~TH: 33 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) M~T.FCUT.E TYPE: peptide
~iii) H~J~ CAL: NO
~iii) ANTI-SENSE: NO
~v) FR~EeENT TYPE: N-t~rm;
(vi) ORIGIN~L SOURCE:
(A) ORGANISM: Human Lactoferricin
~xi) SEQUENCE DESCRIPTION: SEO ID NO: 2:
Gly Arg Arg Arg Arg Ser Val Gln Trp Cys Ala Val Ser Gln Pro Glu
l 5 10 . 15
Ala Thr Lys Cys Phe Gln Trp Gln Arg Asn Met Arg Lys Val Arg Gly
Pro
(2) rN~ORMATION EOR SEQ ID NO: 3:
(i) SEOUENCE CH2RACTERISTICS:
(A) LEN~TH: 75 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDMESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: ~NA (genomic~
(iii) HYFCTHETICAL: NO
(iii) ANTI-SENSE: NO
(vi) ORIGlN~L SOURCE:
(A) ORGANISM: Plant optimized bov me lactoferricin gene
~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
TTCAAGTGCC ~C~l~G~A ~l~GCG~ATG AA~AAGCDGG G~ CC~ ~G CAICA~CTGC 60
~l~CGCAGGG CCTTC 75
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
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~A) LEN~TH: 78 base pairs
~B) TYPE: nucleic acid
~C) STRA~nT~nNF~: double
tD) TOPOLOGY: linear
~ii) ~TT~T~ TYPE: DNA (genomic)
(iii) ANTI-SENSE: NO
(vi) ORIGIN~L SOURCE:
(A) ORGANISM: Plant opt;m-~e~ bovine lactoferricin gene
~xi) ~U~N~ DESCRIPTION: SE0 ID NO: 4:
TTCAAGr~CC GCC~C~l~G~A ~ C~ATG AA~AAaCTaG ~ C~G CATCACCTGC 60
GTGCGCAGGG C~ ~lAA 78
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 99 base pairs
(B) TYPE: nucleic acid
(C) STR~N3EDMESS: double
(D) TOPOLOGY: linear
(ii~ MOLECULE TYPE: ~NA (genomic)
(iii) HyForHErIcAL: NO
(iii) ANTI-SENSE: NO
(vi) ORIGIN~L SOURCE:
(A) ORGANISM: Plant optimized human lactoferricin gene
(xi) SEQUENCE DESCRI~TION: SEQ ID NO: 5:
~GCCGC~GCC GCCGCAGC~T GCA~ ~C G~C~l~AGCC AGCCCGAGZ~ CACCAA~IGC 60
TTCCAGTGGC AGCGCAACAT GCGC~hGGI3 cr~ccc 99
~2) INF0RMATION F0R S_Q ID N0: 6:
(i) SEQUENCE CHARACq~ISTICS:
~A) LENGTH: 108 base pairs
(B) TYPE: nucleic acid
~C) STRANDE~NESS: double
~D) T0POLOGY: linear
~ii) MOLECULE TYPE: DNA ~genomic)
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~iii) H~uln~llcAL: NO
(iii) ANTI-SENSE: NO
(vi) ORIGIN~L SOUROE:
(A) OR&ANISM: Plant opt;~;~ human lactofericin gene
(xi) SEQUENCE DESCRIPTICN: SEQ ID NO: 6:
ACCAIGGGCC G~l~'CGCC~ CAk~ ~A~ C~C~ TGA~CCAGCC C~GGCCACC 60
AA~l~ U-1~ A~n~X~GC3 CAACATGCGC AA~ C~C~ GG~1AG 108
(2) INFORMATI0N FOR SEQ ID N~: 7:
(i) SEQUENCE CEURA~E~ISTICS:
(A) LEN~TH: 91 b2se pairs
(B) TYPE: nucleic acid
(C) STRANDE~NESS: single
~D) TOPOLOGY: linear
~ nr,~ ~,~ TYPE: r~rA (genomic~
(iii) H~Ul'~'l'lCAL: NO
(iil) ANTI-SENSE: NO
(Vi~ ORIGrN~L SOURCE:
(A) ORGANISM: Oligo 1
~xi) SEQUENCE ~ lON: SEQ ID NO: 7:
GATCCACCAT GrICAAGTGC C~CC~ A~l~G~AT GAAGAAGCTG G~C~ -C~A 60
GCATCACCTG C~ iAGG ~C~ l'AAG C 91
(2) INFORM~TION FOR SEQ ID NO: 8:
(i) SEÇUENCE CHARACTERISTICS:
(A~ LENGT~: 91 base pairs
~B) TYPE: nucleic acid
(C) STRA~nT~NF~S: single
(D~ TOPOLCGY: linear
( i i ) ~)T T~rUT ~T;~ TrYPE: ~ ( g~nr~ml c )
(iii) H~ul~llCAL: NO
(iii) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Oligo 2
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(X1) ~F~T~G~ DESCRIPTION: SEQ ID NO: 8:
G~C~ AG AAGGCC~l~C GC;O~CA3GT GA~ G~ n~CCr~GCT TCTTCATGCG 60
CCPCTGCCA~ C~G~ ~CT T~AACAT~Gl G 91
(2) INFORMATION FOR SEO ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 26 base pairs
~B) TYPE: nucleic acid
~C) STRA~nFnNF~S: single
(D) TOPOLOGY: linear
~ii) MDLECULE TYPE: DNA (genomic)
(iii) H~u~ lCAL: NO
~iii) ANTI-SENSE: NO
~vi) ORIGrN~L SOUROE:
(A) ORGANISM: Oligo 1-1
(Xl~ SEQUENCE DESCRIPTION: SEO ID NO: 9:
AGTAGGATCC ACCATGTTCA AGTGCC 26
(2) INFORMATION FOR SEO ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LEN~TH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEnNESS: single
~D) TOPOLOGY: linear
~ii) M~rF~TITF TYPE: nNA ~genomic)
~iii) EYPOnHErICAL: NO
~iii) ANTI-SENSE: NO
~vi) ORIGIN~L SO~ROE:
(A) ORGAN19M: Oligo 2-1
~xi) SEQUENCE DESCRIPTION: SEO ID NO: 10:
TA~-lGC~CC GCTTAGAAGG C~ iC 28
(2) INFORM~TION FOR SEO ID NO: 11:
~i) SEQUENCE CHARACTERISTICS:
~A) LENeTX: 88 base pairs
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(B) TYPE: nucleic acid
~C) STRANDECNESS: single
(D~ TOPOLOGY: linear
(ii) Mnr-Fr~ TYPE: DNA (genomic)
(iii) H~u~ ~AL: NO
(iii) ANTI-SENSE: NO
(vi) ORIGIN~L SOURCE:
(A) ORGANISM: Oligo 3
~xi) SEQUEN OE ~S~l~llON: SEO ID NO: 11:
GATCCACCAT GGCACCGA~G CTA~ C TCCTAGCTCT GGCCATGGCA GCCATCGTCG 60
CG~AIG CAC~GC'~ AG5aCCG 88
(2) INFORMATION FOR SEO ID NO: 12:
(i) SEOUENOE C~RACIERISTICS:
(A) LENGTH: 88 base pairs
(B) TYPE: nucleic acid
(C) STRANDEnNESS: single
(DJ TOPOLOGY: linear
~ii) MOLECULE TYPE: DNA (genomic)
(iii) H~ n~llCAL: NO
(iii) ANTI-SENSE: NO
(vi) ORIGIN~L SOURCE:
(A) ORGANISM: Oligo 4
(xi) SEOUENCE DESCRIPTION: SEO ID NO: 12:
~GC~ll~G A~CC~l~C GCACGCA~GT GAlG~l~GG GC~C~A~CT TCTTCATGOG 60
CCAC~CCA~ ~GC~ACT TGAAGGCC 88
(2) IMFORMATION FOR SEQ ID NO: 13:
(i) SEOUENCE CHARACTERISTICS:
(A) LENGTH: 29 h~se pairs
(B) TYPE: nucleic acid
(C) STRA~v~LN~:~S: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA ~genomic)
~iii) HY~ulh~llCAL: NO
. . .
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(iii) ANTI-SENSE: NO
(vi) ORIGIN~L SOVRCE:
(A) ORGANISM: Oligo 3-1
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
AGTAGGATCC ACCATGGCAC CGAQGCTAG 29
(2) INFORM~TION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
~A) LENaTH: 39 base pairs
~B) TYPE: nucleic acid
~C) STRA~LN~SS: single
~D) TOPOLOGY: linear
(ii) Mnrr~l~r~' TYPE: DNA (genomic)
~iii) H~ul~kllCAL: NO
(iii) ANTI-SENSE: NO
~vi) ORIGIN~L SOVRCE:
(A) ORGANISM: Oligo 5
~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
AGTACCATC5 ~l~l~GCGCC AIGCA~CCCC CAÇTICAA~ 3g
~2) INFORMATION FOR SEQ ID NO: 15:
~i) SEOUENCE CHARACTERISTICS:
~A) LENGTH: 51 base pairs
~B) TYPE: nucleic acid
~C) STRANDEDNESS: single
tD) TOPOLOGY: linear
~ii) Mnr~Jr~ TYPE: nNA (genomic)
(iii) HNPCnEErICAL: NO
(iii) ANTI-SENSE: NO
(vi) ORIGIN~L SOURCE:
(A) ORGANISM: Oligo 6
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
AGTAGCGGCC GCTTAGAT~G C~-l~L~A CA~GAA3aCC ~l~CG~ACGC A 51
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(2) INFORMATION FOR SEO ID NO: 16:
(i) ~U~N~: CEAR~CTERISTICS:
(A) LEN~T~: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDE~NESS: single
~D) TOPOLOGY: linear
(ii) ~nT.T~T.T~ TYPE: ~NA (genomic)
tiii) H~U~ lCAL: NO
(iii) ANTI-SENSE: NO
(vi) ORIGIN~L SOVR OE:
(A) ORGANISM: ATP-5~
(xi) SEQUENCE DESCRIPTION: SEO ID NO: 16:
AGTAGGATCC ACCATGGCIT CCTCTATGCT C 31
(2) INFORMATION FOR SEO ID NO: 17:
(i) SEQUENCE CHARAC~E~ISTICS:
(A) LEN~TH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEnNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iii) ANTI-SENSE: NO
~vi) ORIGDN~L SOURCE:
(A~ ORGANISM: ATPl-3~ -
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
GCAGTTAACT ~ l~C~C~l T 21
(2) IN~ORMATION FOR SEQ ID NO: 18:
(i) SEQVEN OE CHARACIE~ISTICS:
(A) LEN~TH: 18 base pairs
(B) TYPE: nucleic acid
tC) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) ~T,F~T~T-T~' TYPE: ~NA (genomic)
(iii) HYPOTEETICAL: NO
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(iii) ANTI-SENSE: NO
~vi) ~nI~LNAL SOUROE:
(A) ORGANISM: ATP-LF 5'
~xi) SEQUENCE DESCRIPTION: SEO ID NO: 18:
GTAGGAT~CA CCATGGCT 18
(2) rNFORMATION FOR SEQ ID NO: l9:
(i) SEOUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STR~NDEDNESS: single
(D) TOPOLOGY: linear
(ii) ~nT,F~T.T~. TYPE: ~NA (genomic)
(iii) H~ul'~llCAL: NO
(iii) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: ATP-LFB 5'
(xi) SEOU~NCE DESCRIPTION: SEO ID NO: l9:
GGCGGAAGAG TTAACTGCTT CAA~ C~C CGCTGG 36
(2) INFORMATION FOR SEO ID NO: 20:
~i~ SEOUENCE CHARA~l~KISTICS:
(A) LENGT~: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) ~T~rUT~T~ TYPE: DNA (genomic)
~iii) HYPOr~ErICAL: NO
~iii) ANTI-SENSE: NO
(vi) ORIGIN~L SOURC'E:
~A) ORGANISM: ATP-LFB 3'
(xi) SEOUENCE DESCRIPTION: SEQ ID NO: 20:
CCA~CG~G CACTTGAAGC AGTTAACTCT TCCGCC 36
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(2~ INFORMATION FOR SE~ ID NO: 21:
(i) ~yu~L~ C~ARACT}RISTICS:
~A) LENGTH: 42 ~ase pairs
(B) TYPE: nucleic acid
tC) S~RP~L~ 5~: single
~D) TOPOL0GY: linear
(ii) MnTT~rVT~ TYPE: DNA (genomic)
~iii) H~Ol~ lCAL: NO
~iii) ANTI-SENSE: NO
~vi) ORIGINAL SOURCE:
~A) ORGANI5M: ATP2-3'
~xi) SEQUENCE ~k~K~ ON: SEQ ID NO: 21:
CT~CATGCA~ TTGACGOG~C CACCGGAATC GGTAAG~TCA GG 42
(2) rNFORM~TION FOR SEQ ID NO: 22:
(i) SEOUENOE C~A~AC TE~ISTICS:
(A) LENGTX: 36 ~ase pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii~ MOLECULE TYPE: ~N~ (genomic)
(iii) H~J~ CAL: NO
(iii) ANTI-SENSE: NO
(vi) ORI OENAL SOURCE:
~A) ORGANISM: ATP3-3'
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:
GCAGTTGACG CGACCACCGG AATCGGTAAG G~CA~G 36
~2) rNFORM~TION FOR SEO ID NO: 23:
(i) SEQUENCE CHARAC5}FISTICS:
(A) LEN~TH: 36 base pairs
(3) TYPE: nucleic acid
(C) STRANDE~NESS: single
(D) TOPOLOGY: linear
(ii) MDLECULE TYPE: DN~ (genomic)
(iii) H~uln~l~lCAL: NO
. . .. .
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(iii) ANTI-SENSE: NO
(vi) ORIGIN~L SOUROE:
(A) ORGANISM: ATP2-LE~3 5'
(xi) SE~OUENCE DESCRIPTION: SEO ID NO: 23:
w~ ACT GCATGCAGTT CAA~l~C~C C~CIGG 36
(2) rNFORMATION FOR SEO ID NO: 24:
(i) SEOUEN~E CXARPCIERISTICS:
(A) LEN~TH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) ~nT.F~lTT.F TYPE: DN~ (genomic)
(iii) HYPOrHETICAL: NO
(iii) ANTI-S_NSE: NO
(vi) ORIGIN~L SOVROE:
~A) ORGANISM: ATP3-LFB 5'
(xi~ SEQUENCE DESC~IPTIQN: SEQ ID N~: 24:
w~l~Gl~'GC~ TCAAC~TT CM ~l~CC~C CGCTGG 36
(2) INFORM~TION FOR SEO ID NO: 25:
(i) SEOUENOE CHARACTERISTICS:
~A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) ~nT,Ff~.F. TYPE: ~NA (genomic)
(iii) HYPOqHErICAL: NO
~iii) ANTI-SENSE: NO
(vi) ORIGIN~L SOURCE:
(A) ORGANISM: ATP2-LFB 3'
(Xl) SEOUENCE DESC~IPTION: SEQ ID NO: 25:
cc~ cr~ CACTT&~ACT GCATGCAGTT GACGCG 36
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(2) INPORMATION FOR SEQ ID NO: 26:
(i) .~QUF~r~ C~RPCl~RISTICS:
(A) LENGTH: 36 ~Qe pairs
(B) TYPE: nucleic acid
(C) STR~NDEnNESS: single
(D) TOPOLOGY: linear
(ii) M~T~FrUT~ TYPE: DN~ (genomic)
~iii) H~O~ ~AL: NO
(iii) ANTI-SENSE: NO
(vi) ORIGIN~L SOUROE:
~A) ORGANISM: ATP3-LFB 3'
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:
CCA~C~GC~ CACTTGAA~C AGTTGACGCG ACCACC 36
(2) INFORMATION FOR SEQ ID NO: 27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGT~: 31 h~ce pairs
(B) TYPE: nucleic acid
(C) STRANDE~NESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DN~ (genomic)
(iii) HYPOIEE~ICAL: NO
(iii) ANTI-SENSE: NO
(vi) ORIGIN~L SOUROE:
(A) ORGANISM: MTP-5'
(xi) SEQUENGE D~:5~m ~llON: SEQ ID NO: 27:
A~TAG5ATCC ACCATGGCGC CCACCGTGAT G 31
(2) INFORMATION FOR SEQ ID NO: 28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENaTH: 21 kase pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYpO~HETIC~L: NO
.
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~iii) ANTI-SENSE: NO
(vi) ORIGIN~L SOUROE:
(A) ORGANISM: MTP1-3'
(Xi) ~QTJ~r~ UKl~l'lU~: SEQ ID NO: 28:
GCACCGGATT ul~l~G~C~l~ T 21
(2) IMFORMATION FOR SEO ID NO: 29:
(i) SEOUENCE CHARA{IERISTICS:
(A) LENGTH: 42 base pairs
(B) TYPE: nucleic acid
(C) STRANDE~NESS: single
~D) TOPOLOGY: 1inear
~ ~T~~JTT' TYPE: DNA ~genomic)
(iii) ~UlH~ll~AL: NO
(iii) ANTI-SENSE: NO
~vi) ORIGINAL SOUROE:
~A) ORGANISM: MTP2-3'
(xi) SEOUENCE DESCRIPTION: SEO ID NO: 29:
CT~CA~GCAC CGTATGCGAC CAC~-l~C~l CGA~AECGGC GG 42
(2) INFORMATION FOR SEO ID NO: 30:
(i) SE2UENKE CHARACIERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
~D) TOPOLOGY: linear
(ii) ~nT.T~T.T~. TYPE: DNA (genomic)
(iii) H~uln~llCAL: NO
~iii) ANTI-SENSE: NO
~vi) ORIGINAL SOUROE:
(A) ORGANISM: MTP3-3'
~xi) SEQUENCE DESCRIPTICN: SEQ ID NO: 30:
GCACCGTATG CGACCACCGT CC~-l~A~AG CGGCGG 36
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t2) INFORMATION FOR SEQ ID NO: 31:
U~N~: CHARACTERISTICS:
(A) LENaTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
( i i ) ~nT .FrlTT ~T~ TYPE: nNA (genomic)
(iii) H~ CAL: NO
(iii) ANTI-SENSE: NO
(vi) ORIGIN~L SO~ROE:
(A) ORGANISM: MIP-LF 5'
~xi) SEQUENCE DESCRIPTION: SEO ID NO: 31:
GTAGGATCCA CCATGGCG 18
(2) INFORMATION FOR SEQ ID NO: 32:
(i) SEOUENCE CHP~ACTE~ISTICS:
(A~ LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDE~NESS: single
(D) T~POLOGY: linear
(ii) MOLECULE TYPE: DN~ (genomic)
(iii) HYPOIHETICAL: NO
(iii) ANTI-SENSE: NO
(vi) ORIGIN~L SOURCE:
(A) ORGANISM: MTP-LF 3'
(xi) SEÇUEN~E DESCRIPTION: SEQ ID NO: 32:
~C~GCC~ll~ AGAAGGC 17
(2) INFORMATION FOR SEQ ID NO: 33:
(i) SEQUENCE CEURA~TERISTICS:
(A) LEN~TH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDELNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: ~NA (genomic)
(iii) HyporHErIcAL: NO
. , ~.,. .,.. ~ .... ..
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~iii) ANTI-SENSE: NO
(vi) ORIGIN~L SOUROE:
(A) ORGANISM: MTP1-LFB 5'
(xi) SE0UENGE DESC~IPTION: SEQ ID N0: 33:
GGCGGPAGAA ~ W ~ CAA~l~CC~C CGCTGG 36
(2) INFORMATION FOR SEO ID NO: 34:
(i) SEQUENOE CHARACIERISTICS:
(A) LEN~IH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDE~NESS: single
(D) TOPOLOGY: linear
(ii) ~T.F~UT.F. TYPE: nN~ (genomic)
(iii) H~ CAL: NO
(iii) ANTI-SENSE: NO
~vi) ORIGrN~L SOUROE:
(A) ORGANISM: MTP2-LFB 5'
(xi) SEOUENCE DESCRIPTION: SEO ID NO: 34:
CGCATACGGT GCATGCA~ C'AA~ C~C' CGKTGG 36
(2) rNFORMATION FOR SEQ ID NO: 35:
(i) SEOUENCE CHARAC~ERISTICS:
(A) LEN~IH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEnMESS: single
(D) TOPOLOGY: linear
(ii) ~nT.~TF TYPE: ~N~ (~enomic)
(iii) H~ llCAL: NO
(iii) A~TI-SENSE: NO
(vi) ORIGIN~L S0URC'E:
(A) ORGANISM: MTP3-LFB 5'
(xi) SEOUENCE DESCRIPTION: SEQ ID NO: 35:
TAC~ CAA~l~CC~C CGCTGG 36
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~2) INFORMATION FOR SE0 ID NO: 36:
u~ ~: CHPFACTERISTICS:
~A) LENGrH: 36 kase pairs
(B) TYPE: nucleic acid
~C) STRA~LN~S: single
(D) TOPOLOGY: linear
nTT~-UTT~ TYPE: DNA (genomic)
H~ CAL: NO
~iii) ANTI-SENSE: N~
(vi) ORIGIN~L SOURCE:
~A) ORGANISM: MTF-LFB 3'
(xi) SEOUENCE ~S~KI~liON: SEQ ID N~: 36:
CCA~Y~r~G CACTTaAAEC ACCGGATTCT TCC~CC 36
(2) IMFORMATION FOR SEO ID NO: 37:
(i) S_QTJENCE CBPRA~TERISTICS:
~A) LENGTH: 36 kase pairs
~B) TYPE: nucleic acid
~C) STRAM~E~NESS: single
~D) TOPOLOGY: linear
(ii) ~TT~UTF TYPE: DNA ~genomic)
~iii) EYPOIHETICAL: NO
~iii) ANTI-S_NSE: N0
(vi) ORIGIN~L SOURCE:
~A) ORGANISM: MTF2-L~B 3~
~xi) SE2UENCE ~ K~ ON: SEQ ID NO: 37:
CCAG-~GCGG CACTTGAACT GCATGCACCG TAT~CG 36
~2) INFORMATI0N FOR SEQ ID NO: 38:
(i) SEQUENCE CBARACr~RISTICS:
~A) LEN~TH: 36 base pairs
~B) TYPE: nucleic acid
(C) STRA~wkLN~:SS: single
(D) TOPOLOGY: linear
~ii) MOLECULE TYPE: DN~ ~genamic)
(iii) BypcrHETIcAL NO
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(iii) ANTI-SENSE: NO
(~i~ ORIGIN~L SOURCE:
(A) ORGANISM: MTF3-LFB 3'
(xi) SEQUEN~E DESCRIPTION: SEQ ID ND: 38:
CC~ CG~ CACIqGAAEC ACCGTATGCG ACCACC 36
(2) INFORMATICN FOR SEQ ID NO: 39:
(i ) SF~TJ~rF CHARACTERISTICS:
(A) LEN~TH: 21 ~ase pairs
(B) TY~PE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) M~T~T~TT' TYPE: DN~ (genomic)
(iii) HYPOTXETICAL: NO
(iii) ANTI-SENSE: NO
(vi) ORIGIN~L SOUROE:
~A) ORGANISM: Primer 176
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:
C~AACC l~~ C 21
(2) IN~ORMATION FOR SEO ID NO: 40:
(i) S_QUENCE CHARACTERISTICS:
(A) LEN~TH: 21 ~ase pairs
(B) TYPE: nucleic acid
(C) STRANDEnNESS: single
(D) TOPOLOGY: linear
(ii) M~TFr~TF TYPE: nNA (genomic)
tiii) HYP{n~ETICAL: NO
~iii) ANTI-SENSE: NO
(vi) ORIGIN~L SOUROE:
(A) ORGANISM: Primer 179
(xi) SEQUEN~E DESCRIPTION: SEO ID NO: 40:
CCAAATGTTT GAACGATCGC G 21
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(2) INFORMATION FOR SEQ ID NO: 41:
(i) SEQUENCE CH~RACTERISTICS:
~A) LENGTH: 21 kase pairs
~B) TYPE: nucleic acid
(C) STRA~u~LN~SS: single
~D) TOPOLOGY: line~
~ii) ~nT,T~IlT.T~. TYPE: DNA ~genomic)
(iii) HY~ lCAL: NO
~iii) ~NTI-SENSE: NO
~vi) ORIGIN~L SOURCE:
~A) ORGANISM: Primer 177
~xi) SEOUENCE DESCRIPTION: SEQ ID NO: 4}:
CACAACCAGA ~ ~CCC~AA A 21
~2) INFORMATION FOR SEO ID NO: 42:
(i) SEOUENCE CHAR~qEFISTICS:
(A) LENGTH: 21 base pairs
~B) TYPE: nucleic acid
(C) STRANDE3NESS: single
(D) TOPOLOGY: linear
(ii) MOLE~ULE TYPE: ~NA (genomic)
(iii) HYPCrHErICAL: NO
(iii) ANTI-SENSE: NO
(vi) ORIGIN~L SOURCE:
(A) ORGANISM: Primer 180
(xi) SEOUENKE DESCRIPTION: SE~ ID NO: 42:
AAAl~l~ CC~C-llAGAA G 21
(2) IMFORMATION FOR SEQ I~ NO: 43:
(i) SEQUENCE CXARACqER5STICS:
(A) LEN~TH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDE3NESS: single
(D) TOPOLOGY: linear
(ii) MnT,~ ~TF TYPE: DNA (genomic)
(iii) H~Jl~llC'AL: NO
... . ..... ..... .
CA 02262429 1999-02-05
WO ~8/~e~0 PCTtEP97tO4438
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~iii) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43:
AGTAG&ATCC ATGAAAAAGC CTGAACTC 28
(2) INFORMATION FOR SEQ ID NO: 44:
(i) SEQUENCE CHARACTERISTICS:
~A) LEN~TH: 26 ~ase pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) ~nT-T~lT-~ TYPE: ~NA (gen~mic)
(iii) H~ul~kl~lCAL: NO
(iii) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44:
TACTGGATCC CTA~ GCCCTC 26
(2~ INFORMATION FOR SEQ ID NO: 45:
(i) SEQUENCE CHARACTERISTICS:
(A) LEN~TH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEnNESS: single
(D) TOPOLOGY: linear
(ii) MOLECVLE TYPE: ~NA ~genomic)
(iii) HYEOIHErICAL: NO
(iii) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:
TAl~ TATA~GGATC CA~GGTCa~C 30
(2) INFORMATICN FOR SEQ ID NO: 46:
(i) SEQUENCE CXA~AC~ERISTICS:
(A) LEN~TH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
CA 02262429 1999-02-05
W O 9~06S'O
- PCT~r97/04438
-72-
(D) TOPOLOGY: linear
~ ii ) ~T-T;~Tr T~'. TYPE: ~NA (~nf~n; c )
(iii) ANI'I-SENSE: NO
(xi) SEOUENCE DESCRIPI'ION: SEQ ID NO: 46:
TACq~GATCC TCAGIACACA ~il~C 1~: 28
.
