Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
: ~ WO 94/10320 ~ PCl/EP93/03091
PI~T5 WITH REDl~CED ~:IJSCEPTIBILITY TO PLANT-P~R~SITIC l.
NE~ATODES
TECHNICAL FIELD
This invention concerns plants with reduced susceptibility to ¦ -
plant~parasitic nematodes and methods for obtaining same
involving recombinant DNA technology.
1 0 , ,
BACKGROIJND OF_THE INVENTION
Plant-parasitic nematodes worldwide cause diseases of
nearly all crop plants of economic importance with estimated
losses of about $ 5.8 billion/yr in the Unites States alone
(Sasser and Freckman, ls87, World prospective on nematology
In: Vistas on Nematology, Eds. Veech & Dickson. Hyatts Will,
Maryland. pp. 7-14~. ~hile in tropical regions losses caused
by nematodes are due mainly to root-knot nematodes ~Meloido-
qYne), in Europe cyst nematodes of the genera Globodera and
Heterodera are regarded as serious pests and important
limitinq factors in e.q. potato, rapeseed and sugarbeet
cultivation, respectively. Only a small number of resistant
crop varieties have emerged from breeding programmes for e a.
potato, sugarbeet, tomato, soybean and oil radish (Dropkin,
lg8~, Ann. Rev. Phytopath. 26, 145-161; Trudgill, 1991, Ann.
Rev. Phytopath. ~9, 167-192). The resistance is often based
on single R-genes (Rick ~ Fobes, 1974, Tomato Gen. Coop. 24,
25; Baron~ et al. 1990, Mol. Gen. Genet. 224, 177-182) and
leads to breakdown of resistance aftPr several generations
(Shidu & Webster, in: Plant Parasitic Nematodes, Vol. III,
1981, Zuckerman et al. (eds.) Acad Press, New York, pp 61-
87; ~urner, 1990, Ann. Appl. Biol. 117, 385-397).
Plant parasitic nematodes are obliga~e parasites.
Nematodes such as cyst and root-knot nematodes are completely }
35 dependent on the formation of proper feeding structur s
inside the plant root. These feeding structures arise from
single r~ot c~lls that are selected by the nematode after j -
invasion of the root. In the case of cyst nematodes, the IFC ~` -
(initial feeding cell) develops into a syncytiu~ through
40 digestion of c~ll walls and hypertrophy. After infection with
a root-kno~ nemat~de, the IFC develops into a giant cell
through several nuclear divisions without cytokinesis and
SU~3STJTUTE S~E I .- `
.. , .<, . .. , . .,., .. , . . . , .. , , . , . . ,., . .. , . , , .. . , .. , . ~ .. ... .......... .. .. ... . .
. .
W094/10320 21~ (3 ~ i PCT/EP93/03091;,; ~-
~ 2 ~
becomes metabolically very active. During establishment of J
the fe~ding structure, the infective juvenile nematode
becomes immobile and loses the ability to move to other }
feeding sites, illustrating their dependence on the induced
5 nematode feeding structure (NFS).
Recent published methods to engineer nematode resistance
~PCT W092/04453) involve the modification of a gene that is s
specifically induced in the NFS after nematode infection. The
local expression of phytotoxic genes would inhibit thP
lO development of the feeding structures, thus making a plant
essentially resistant~ This approach is strictly dependent on
the availability of a promoter that is highly specific for
nematode-induced feeding structures (NFS). Any promoter
activity outside this structure will hav~ adverse effects on
15 plant development and crop yield. A highly NFS-specific
promoter has been disclosed by Taylor et al. (1992, Proc.
3lst Annual Meeting Amer. Soc. Nematologists, Vancouver
Canada) and involves a truncated version of a root specific
regulatory sequence (~0.3 TobRB7 as described in Yamamoto,
l99l Plant Ce}l 3: 371-382).
SUMMARY~OF THE INVENTION
The invention provides recombinant DNA whlch comprises
in sequence:
-a promoter that is capable o~ driving expression o~ a
downstream gene specifically in an initial feeding cell
and/or a nematode feeding structure,
-a gene encoding a product that is inhibitory to an endoge-
nous gene encoding a protein or polypeptide salected from the
group consisting of ATP synthase, adenine nucleotide trans-
locator, tricarboxylate translocator, dicarboxylate trans-
locator, 2-oxo-glutarate translocator, cytochrome C, pyruvate
kinase, glyceraldehyde-3P-dehydrogenase, NADPH-cytochrome
p4~0 reductase, fatty acid synthase complex, glycerol-3P -
35 acyltransferase, hydroxymethyl-glutaryl CoA reductase, '`
aminoacyl transferase, a transcription initiation factor, and
a transcription elongation factor, and optionally
-a transcription terminator and a polyadenylation signal
sequence,
SU~3STI~UT~ Stt~E~
1~ ~ WO94/10320 ~ P~71 ~ PCT/EP93/03091
- 3 - .
and wherein the said gene is expressed in said initial ~-
feeding cell or nematode feeding structure upon infection by
the said nematode. Preferred according to the invention is a ¦
recombinant ~NA according to the inYention, wherein said ¦
5 product comprises a RNA transcript that is complementary or '
partially complementary to the said endogenous gene tr~n-
script. A preferred nematode feeding structure-specific
promoter according to the invention is one obtainable from
the Delta~0.3TobRB7-SA promoter, joined to said inhibitory
gene such that, upon infection of a plant parasitic nematode,
the inhibitory gene is expressed specifically or predominant-
ly in said nematode feeding structure. -
The invention further provides a replicon comprising a
recombinant DNA according to the invention, such as a Ti- or
Ri-plasmid of an Aqrobacterium species or a replicon capable
of replication in E. coli and Aqrobacterium species, a so- ;
called binary vector system, as well as bacterium species,
such as, Aqrobacterium species, comprising a said replicon
according to the invention.
Another embodiment of the invention is a plant genome
which comprises a recombinant DNA according to the invention,
as well as plant cells comprising same. Also preferred L:,
embodiments are plant~ comprising a cell or cells according
to the invention. More preferred are plants regenerated from
a cell according to the invention.
An especially preferred embodiment is a plant which, as
a result of expression of a gene encoding a product that is
inhibitory to an endogenous gene, shows reduced susceptibil-
ity to a plant parasitic nematode, preferably one which
belongs to the family Solanaceae, more preferably the one is
Sol~num tuberosum~ as well as plant material, such as ~ `
flowers, fruit, leaYes, pollen, seeds, or tubers, obtainab}e
from a plant according to the invention.
The invention also-provides a method for obtaining a ~-
plant with reduced s~1sceptibility to a plant parasitic
nematode, comprising the steps of
(1) transforming a recipient plant cell with recombinant DNA
according to the invention, '
(2) generating a plant from a transformed plant cell,
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WO94/10320 PCT/EP93/03091 -- ~
21 ~13 !1 5 :' 4 - `
. . .
(3) identifying a transformed plant with reduced susceptibil-
ity to said plant parasitic nematode.
According to another aspect of the invention a method i5
provided for reducing damage to a crop due to plant parasitic
nematodes, by growing plants according to the invention.
The meaning of the expressions used herein, as well as '!
the application and the advantages of the invention will ;
become clear from the following detailed description of the
invention.
BRIEF DESCRIPTION OF THE FIGURES .:
Figure 1. Binary vector pMOG23
Figure 2. Plasmid pMOG707, intermediate vector constructed
for cloning purposes.
15 Figure 3. Restriction map of fragment from Ti-plasmid pTiB6 .
Figure 4. Intermediate vector pMOG579
Figure 5. Binary vectors pMOG711 - pMOG715. These plasmids
are derivatives of pMOG23 and contain a truncated .-
~0.3 TobRB7 promoter and an antisense construct of .
a gene that is essential for cell viability.
:
DETAILED DESCRlPTION
In the examples accompanying this description of the
invention reduced susceptibility to plant parasitic nematodes
25 is engineered in tobacco, potato and Arabido~sis plants by -
interfering with the autonomous, prima~y metabolism of cells
comprislng the feeding structure. In particular, the inven-
tion is outlined in somewhat more detail through antisense
expression of the homologous gene coding for essential steps
in primary metabolic pathways. For potato, the example is
given for a gene coding for tha mitochondrial adenine
nucleotide translocator (Winning et al. 1992 Plant J. 2; 763
773). For ArabidoPsis, the example is described or NADPH- -
cytochrome P450 reductase ATR2 (Mignote-Vieux et al. 1992
EMBL accession nu~ber X66017). For tobacco, the example i5 ` '-
described using a gene coding for the beta subunit of the
mitochondrial ATP-synthase (Boutry & Chua, 1985 EMBO J. 4; . .
2159-2165). The examples are described using the regulatory
promoter se~uence ~O.3 (Taylor et al. 1932, Proc. 31st Ann~
,.
SW13STITUT~ SHFET
.~
`"~`. WO 94/10320 ~ PCT/EP93/03091 1~
- 5 - ~.
Meeting Amer. Soc. Nematologists, Vancouver Canada) of the
TobRB7 gene (Y~mamoto et al. 1991, Plant Cell 3i 371-382) to
ensure that expression is limited to the feeding structure.
Although exemplified in somewhat more detail for three
plant species, the invention is not limited to any plant or
nematode species.
Interference with autonomous primary metabolism brought '~
about by disrupting genes inhibitory to an endogenous gene
that encodes a protein or polypeptide product that is essen-
tial for cell viability. Disrupter gen~s according to the
invention may be selected from such genes as formed by the
group consisting of (a) genes encoding ribozymes against an
endogenous RNA transcript, ~b) genes which when transcribed
produce RNA transcripts that are complementary cr at least -
partially complementary to RNA transcripts of endogenous
genes that are essential for cell viability, a method known -
as antisense inhibition of gene expression ~disclosed in EP-A
240 208), or (c) genes that when transcribed produce RNA
transcripts that are identical or at least very similar to ~`
transcripts of endogenous genes that are essential for cell
viability, an as yet unknown way of inhibition of gene
expression referred to as co-suppression (disclosed by Napoli
C. et al., 1990, The Plant Cell 2, 279-289).
According to a preferred embodiment of the invention use
is made of antisense genes to inhibit expression of endoge-
nous genes essential ~or cell viability, which genes are
expressed in the nematode feeding structures by virtue of a `
suitable nematode-specific promoter fused upstream to the~~~
said antisense gene. -~
Target genes for antisense disrupter genes are selected
from those coding for enzymes that are essential for cell~ :
viability, also called housekeeping enzymes, and should be
nuclear encoded, preferably as single copy genes, although a
small size gene family would also be suitable for the purpose
of the invention. Furthermore, the effect of antisense
expression of said genes must not be nullified by diffusion
- or translocation from other cells or organelles of enzyme
products normally synthesized by such enzymes. Preferably,
genes coding for membrane-translocating enzymes ~xe chosen as
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WO94/10320 2 i ~ 3 ~ ~ ~ PCT/EP93/03091
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these are lnvolved in establishing chemical yradients across
organellar membranes. Inhibition of such proteins by anti-
sense expression can not, by definition, be cancelled by
diffusion of substrates across the membrane in which these ',-
proteins reside. The translocated compound is not limite~d to
organic molecules but can be of inorganic nature; e.q. P, H,
OH or electrons~
Preferably, the membrane-translocating enzymes should be
present in organelles that increase in numbers during para-
sitism, thereby illustrating the essential role that suchorganelles have in cells comprising the NFS. Specific
examples for such organelles are mitochondria, endoplasmic
reticulum and plasmodesmata (Hussey et al. 1992 Protoplasma
167;55-65, Magnusson & Golinowski 1991 Can. J. Botany 69;44- ;
15 52). A list of ~arget enzymes is given in Table 1 by way of -
example but the invention is not limited to the enzymes
mentioned in this table. More detailed listings ca~ be
assembled from series as Biochemistry of Plants (Eds. Stumpf -
& Conn, 1988-1991, Vols. 1-16 Academic Press) or Encyclopedia
20 of Plant Physiology (New Series, 1976, Springer-Verlag, !'".'.'
Berlin).
Although only in some cases, the genes coding for these ~;
enzymes have been isolated and, therefore, the number of gene
copies are not known, the criteria that have to be met are
described in this invention.
, ~
``' ~`~
~' :
SU~3STIT(IT~ S~EF~
',~ WO94/10320 ~ PCT/EP93/03091
- 7 -
TABLE l ! :
EXAMPLE~ OF TARGET ENZYMES FOR ANTISENSE EXPRESSION
-
enz~ Pathway~orqanelle
ATP synthase mitochondrion
adenine nucleotide translocator mitochondrion
phosphate translocator mitochondrion
l0 tricarboxylate translocator mitochondrion '
dicarboxylate translocator mitochondrion
2-oxo-glutarate translocator mitochondrion ~ :
cytochrome C mitochondrion
15 pyruvate kinase glycolysis -
glyceraldehyde-3P-dehydrogenase glycolysis
NADPH-cytochrome P450 reductase lipid metabolism
fatty acid synthase complex lipid metabolism
20 glycerol-3P-acyltransferase lipid metabolism
hydroxymethyl-glutaryl CoA reductase mevalonic acid pathway
aminoacyl transferase nucleic acid metabolism
transcription factors nucleic acid metabolism
elongation factors nucleic acid metabolism
To maximize the antisense effects in a plant host, the
use of homologous genes is preferred. With homologous is
meant genes obtainable from the same plant species as the
plant host. Heterologous, for the purpose of this specication
shall mean obtainable from a different plant or non-plant
species. Heterologous shall also comprise synthetic analogs
of genes, modified in their mRNA encoding nucleic acid
sequence to diverge at least 5% of the host gene. As house-
keeping genes are in general highly conserved, heterologous
probes from other ~plant) species can be used to isolate the
corresponding gene from the crop species that is to be made
40 resistant. Such gene isolations ~re well within reach of ~-~
those skilled in the art and, in ~iew of the present teaching
require no undue experimentation.
To differentiate between possible target genes and
se}ect favourable candidates to engineer nematode resistance,
the following procedure can be appli~d by those skilled in
the ar~: via the gene of interest, promoter-sequences can be
isolated from genomic DNA and u5ed for cloning in front of a
SUB~;TlTlJTc S~EE ~ - `
WO94~10320 ~ PCT/EP93/03091-
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marker gene such as GUS ~Jefferson et al. 1987 EMBO J.
6~3901-3~07;. This expression construct can then be and
integrated into the plant genome. Re~enerated plants can then
be infected with PPN and used for histochemical GUS analysis
5 of entire plants and the feeding structures in particular. `
Alternative disrupter genes may be selected on the basis ~ -~
of the availability of mutants in unicellular eukaryotes such
as yeast or ChlamYdomonas can be used as indication. If for a
particular enzyme, a large number of mutants are available
then it is likely that thi~ enzyme is redundant, present as
multi-copy gene families, or that alternative pathways are
available to circumvent the mutated enzyme (Strathern, Jones
& Broach (Eds.) 1981 The molecular biology of the yeast
Saccharomvces cerevisiae. Cold Spring Harbor Laboratory
Press, New York). Such genes are less suitable for the
methods described in this invention. By contrast, mutations
in enzymes that are usually lethal for the recipient cell and
therefor rarely available, indicate that an antisense deregu-
lation of such genes will inhibit the proper development of
20 that cell and can be used for the approach to engineer -~
reduced susceptibility to PPN as disclosed in this invention.
Gene disruption methods are available to test if a gene is
essential for cell v~ability in which case the disruption
e~ent will be lethal (Rothst~in, 1983 Methods Enzym. 101;
202-211). The homologous gene can then be isolated from the
target crop with the yeast gene as a probe. Alterna-
tively, the following promoter sequence can be used as
nematode feeding site specific promoter; a truncated version
of a tobacco root-specific promoter ~0~3TobRB7 (Yamamoto et
al. 1991 Plant Cell 3; 371~382). The full length sequence of
tha TobRB7 promoter is hi~hly active inside NFS and this
activity becomes more specific for the ~FS when the truncated
~0.3 version of the promoter is used (Taylor et al. 1~92,
Proc. 3lst Ann. Meeting Amer. Soc. Nematologists, Vancou~er i-
35 Canada). '~
Other regulatory sequences such as terminator se~uences
and polyadenylation signals include any such sequence func-
tioning as such in plants, the choice of which is within the
level of -Ckill of the average s~illed person in the art. An
"~
SU8STJ~UT~ SHEE~
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,~ WO94/10320 2 ~ PCT/EP93/03091 ¦-
_ g _ ;~
example of such sequences is the 3' flanking region of the
nopaline synthase (nos) gene of Aqrobacterium tumefaciens
(Bevan, 1984, Nucl. Acids Res. 12, 8711-8721).A suitable
promoter can be isolated via genes that are expressed at
5 increased le~els inside the NFS during nematode infectiQn. ! ;
Such genes can be isolated through differential screening of
cDNA clones made from ~RNA extracted from infected andj ,~;
healthy roots as was demonstrated for potato (Gurr S.J. et
al. 1931, Mol. Gen. Genet. 226, 361-366). Although such
promoters have never been described in detail, they can be
selected and isolated in a well known manner from a plant by:
1. searching for a mRNA which is present primarily
talthough not necessarily exclusively) in infected root
tissue,
2. isolating this mRNA
3. preparing a cDNA from this mRNA
4. using this cDNA as a probe to identify the regions in
the plant genome which contain DNA coding for this
specific mRNA
5. identifying and isolating the upstream (5') sequences
from the DNA coding for this specific mRNA and that
contains the promoter region.
Preferably, the infected roots used for mRNA isolation should
be enriched for NFS e.a. by synchronous infection (Hammond-
Kosack et al. 1989 Physiol. Mol. Plant. Pathol. 35, 495~506) -~
or through direct isolation of feeding structures from plants
in which NFS are visible at low magnification. For example
fe~ding-structures that develop inside Arabido~sis roots can -~
b~ seen at low magnification and are easy to isolate with a
minimum of contaminating cells tsijmons et al. 1991, Plant J.
1, 245-254). This al}ows the isolation, preferably using
molecular enrichment procedures (Dickinson et al., 1991 Adv.
Mol. Gen. Plant-Microbe Interact. 1 276-279) of genes corre-
sponding to these RNA's and suhsequent isolation of upstreampromoter elements. Once identified, similar genes can be
isolated from other plant species when the identified gene ic
used as a probe as in step 4. Species-speCific upstream
se`quences can than be isolated from these othPr plan~ species
SUBSTI~ c S'~.~ET
WO94/10320 ~ PCT/~P93/0309l, ,
- 1 0 ~
for use in a similar strategy as described in this invention. ~`
Upstream sequences of identified genomic clones can be fused
to a qene for insertion in a suitable expression vector for
plant transformation such as pMOG22 or pMOG23.
Alternatively, suitable promoters for expression of can
be isolated via interposon tagging (Topping et`al., 1991,
Developm. 112, 1009-101~). In this approach, a number of
different transgenic plants are regenerated after transform-
ation with T-DNA from Aqrobacterium carrying promoterless GUS
constructs such as described by Topping et al. (1991, De-
velopm. 112, 1009-1019) or pMOG452 as described in the
Examples. After infection with a root-knot or cyst nematode
and allowing some development of the NFS, roots can be
stained for GUS activity. The random integration of the T-DNA ~--
enables the identification of promoter sequences that are
active exclusively in the NFS. This type of interposon
tagging of promoter sequences is especially well established
in Arabido~sis (Kertbundit et al., 1991, Proc. Nat. Acad.
Sci. USA 88, 5212-5216) and tobacco (Topping et al., 1991,
Developm. 112, 1009-1019). The 5' upstream sequences respon-
sible for GUS expression can be isolated with inver~ed
polymerase chain reaction (inverted PCR) (Does et al. 1991,
Plant Mol. Biol. 17, 151-153). Once suitable regulatory
se~uences are identified or genes that are transcribed inside ;`
NFS, they can be used as probes for the isolation of
homologous sequences from other plant species. In turn, these
sequences from other species can be fused to a disrup~er gene
for lnsertion in a suitable ~ector for plant transformation.
The application o~ this invention is not restricted to ~
30 the plant species that are shown by way of demonstration. The ~;
choice of the plant species is primarily determined by the
amount of damage through PPN infections çstimated to occur in
agriculture and the amenability o~ the plant species to `
transformation. Plant genera which are damaged during agri~
cultural practice by PPN and which can be made significantly
less susceptible to PPN by ways of the present invention
include but are not limited to the genera mentioned in Table
2.
Nematode species as defined in the context of the ~-
SUBSTI~UTZ= S~!EE i - ~
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WO 94/10320 f~J ~ ~ ~J ~ 3 i PCr/EP93/03091
~ 11 ~ `~
present invention belong tb the superfamily Heteroderoidea
and are divided among the families Heteroderidae and Meloi-
doqYnidae and include, but are not limited to the species
mentioned in Table 2.
The choice of the plant species is primarily determined 1
by the amount of damage through PPN in~ections estimated to
occur in agriculture and the amenability of the plant species i
to transformation. Plant genera which are damaged during
agricultural practice by PPN and which can be made signifi-
cantly less susceptible to PPN by ways of the present inven-
tion include but are not limited to the genera mentioned in
Table 2.
Nematode species as defined in the context of the
present invention include all plant-parasitic nematodes that
modify host cells into specially adapted feeding structures
which range from migratory ectoparasites (e~q. Xiphlnema
spp.) to the more evolved sedentary endoparasites (eOq.
Heteroderidae, MeloidoqYnae or Rotylenchulinae). A list of
parasitic nematodes are given in Table 2, but the invention
is not limited to the species mentioned in this table. More
detailed listings are presented in Zuckerman et al. (eds.,
in: Plant Parasitic Nematodes, Vol. I 1971, New York, pp.
139-162).
The methods according to the invention to combat damage
to crops due to nematode invasion is likewise applicable with
non-nematode pests and pathogens, whenever said pathogen or
pest locally down-regulates plant promoters at the site o~
infestation (e a. in fungi-induced haustoria or aphid-induced
galling). The principle of effecting the production of a
neutralizing substance in all or most of the non-infestated
plant parts to neutralize a cell disruptive substance the
production of which is effected in at least the site of
infestation, is independent of the type or species of the
pathogen or pest.
- '
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~.
TABLE 2
EXAMPLES OF PLANT-PARASITIC NEMATODES AND THEIR
~ PRINCIPAL HOST PLANTS
Nematode Species Principal Host Plants
Meloido~Yne
M. hapla wide range
10 M. incognita wide range ~ -
M. exigua coffee, tea, Capsicum, Citrullus
M. indica Citrus
M. javanica wide range
M. africana coffee
15 M. graminis cereals, grasses
M. graminicola rice
M. arenaria wide range
_eterodera & Globodera
20 H. mexicana Lycopersicon esculentum, Solanum spp.
H. punctata cereals, grasses
G. rostochiensis Solanum tuberosum, Solanum spp, Lycoper-
sicon esculentum
G. pallida Solanum tuberosum
25 G. tabacum Nicotiana tabacu~, Nicotiana spp.
H. cajani Cajanus cajan, Vigna sinensis
H. glycines Glycine max, Glycine spp.
H. oryzae Oryza sativa
H. schachtii Beta spp, Brassica spp,
30 H. trifolii TrifoIium spp.
H. avenae cereals, grasses
H. carotae Daucus carota
H. cruciferae Cruciferae
H. goett ngiana Pisum sativum, Vicia spp.
Within the context o~ this invention, a plant is said to
show reduced susceptibility to PPN if a statistically signi-
f.icant decrease in the number of mature females developing at
the surface of plant roots can be observed as compared to
control plants. Susceptible / resistance classification
according to the number of maturinq females is standard
practice both for cyst- and root-knot nematodes (~ LaMon-
dia, 1991, Plant Disease 75, 453-454; Omwega et al., 1990,
Phytopathol. 80, 745-748).
The ~asic principle of reducin~ the plant`s susceptibi-
lity to plant para~itic nematodes according to the invention
is the manipulation of the nematode feeding structure.
Manipulation of the ne.matode feeding structure for the `~
purpose of this description of the invention shall include
both preventing or retarding NFS formation as well as disxup- : i
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WO94~10320 ~ PCT/EP93/03091 ~
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tion once formation of the NFS is in an advanced stage. ¦-
It is ~referred to prevent or retard formation of the NFS,
i.e. during the first stages of nematode invasion; to that
end the NFS disruptive gene must be under the control of a
promoter that drives expre~sion at the onset of NFS formati-
on.
However, in principle, it will also be acceptable if a
disruptive gene is under the control of a promoter that
drives expression of the disrupter gene in a more advanced
stage of NFS formation causing the NFS to decline or to
collapse. Either of these two extremes will provide the
infected plant with decreased susceptibility towards the
invading nematode. For the purpose of this invention the
expression "disruption of the NFS" shall include retardation
lS of NFS formation, decline of NFS formation once formed, or in
the process of being formed, as well as total collapse of the
NFS formed.
Reduced susceptibility to a plant parasitic nematode may --
be the resu}t of a reduction of the number of NFS of the
infected plant root, a reduction in the advancement of NFS
formation, or a combination of both effects.
A nematode feeding structure according to the present
invention shall include an initial feeding cell, which shall
mean the cell or a very limited number of cells destined to
become a nematode feeding structure, upon induction of the
invading nematode.
A NFS disruptive effect accordiny to the invention is
not limited to adverse effPcts on the NFS only; also disrup-
tive effects are contemplated that in addition have an
ad~erse effect on nematod~ development by way of direct
interaction.
Several techniques are available for the introduction of
recombinant DNA containing the D~A sequences as described in , ~-;
the present invention into plant hosts~ Such techniques ~-
include but are not limited to transformation of protoplasts
using the calcium/polyethylene giycol method, electroporation `
and microinjection or ~coated) particle bombardment ~Potry- . -
kus, 1990, Bio/Technol~ 8, 535-542).
In addition to these so-called direct DNA transformation
SlJÇ3STlr~T_ S~.E~, -
WO 94/10320 ~ i Li ~ '~ ~ i PCr/EP93/0309l " .
-- 14 -- t.- ~`
methods, transformation systems involving vectors are widely
availa~le, su~h as viral vectors (e.g. from the Cauliflower
Mosaic Virus (CaMV) and bacterial vectors (e.g. from the
qenus Aqrobacterium) (Potrykus, 1990, Bio/Technol. 8, 535- s
5 542). After selection and/or screening, the protoplastsr
cells or plant parts that have been transformed can be
regenerated into whole plants, using methods known in the art
(Horsch et al., 1985, Science 225, 1229-1231). The choice of
the transformation and/or regeneration techniques is not
critical for this invention.
According to a preferred embodiment of the present
invention use is made of so-called binary vector system
(disclosed in EP-A 120 516) in which Aqrobacterium strains
are used which contain a helper plasmid with the virulence
genes and a compatible plasmid, the binary vector, containing
the gene construct to be transferred. This vector can repli-
cate in both E.coli and in Aqrobacterium; the one used here
is derived from the binary vector Binl9 (Bevan, 1984, Nucl.
Acids Res. 12, 8711-8721). The binary vectors as used in this
example contain between the left- and right-border se~uences -
of the T-DNA, an identical NPTII-gene coding for kanamycin
resistance (Bevan, 1984, Nucl. Acids Res. 12, 8711-8721) and
a multiple cloning site to clone in the required gene con-
structs.
The transformation and regeneration of monocotyledonous
crops is not a standard procedure. However, recent scientific
progress shows that in principle monocots are amenable to
transformation and that fertile transgenic plants can be -~
regensrated from transformed cells. The development of
reproducible tissue culture systems for these crops, together
with the powerful methods for introduction of genetic materi-
al into plant cells has facilitated transformation~ Present-
ly, preferred methods for trans~ormation of monocots are
microprojectile bombardment of explants or suspension cells, 3.`
and direct DNA uptake or electroporation (Shimamoto, et al,
1989, Nature 338, 274-276). Transgenic maize plants have been
obtained by introducing the Streptomyces hygroscopicus bar
gene, which encodes phosphinothricin acetyltransferase ~an
enzyme which inacti~ates the herbicide phosphinothricin)~
,.
SUBSTI~UT-~ Sk ~ ~
W094/10320 ~) ' ` '^ ~ PCT/EP93/030gl
- 15 -
into embryogenic cells of a maize suspension culture by t
microp~rticle.bombardment (Gordon-Kamm, 1990, Plant Cell, 2,
603-618). The introduction of genetic material into aleurone
protoplasts of other monocot crops such as wheat and barley
has been reported (~ee, 1989, Plant Mol. Biol. 13, 21-30~.
Wheat plants have been regenerated from embryogenic suspensi-
on culture by selection only the aged compact and nodular
embryosenic callus tissues for the establishment of the
embryogenic suspension cultures ~Vasil, l9gO BioJTechnol. 8,
429-43~). The combination with transformation systems for
these crops enables the application of the present invention
to monocots. These methods may also be applied for the
transformation and regeneration of dicots.
Suitable selectable marker genes that can be usPd to
15 select or screen for transformed cells, may be selected from
any one of the following non-limitative list: neomycin
phosphotranspherase genes conferring resistance to kanamycin
(EP-B 131 623), the hygromycin resistance gene (EP 186 425
A2) the Glutathione-S~transferas~ gene from rat liver confer- :
20 ring resistance to glutathione derived herbicides (EP-A 256 `
223), glutamine synthetase conferring upon overexpression :
resistance to glutamine synthetase inhibitors such as phosp-
hinothricin (W087/05327), the acetyl transferase gene from
strePtomyces viridochromoqenes conferring resistance to the
selective agent phosphinothricin (EP-A 275 957), the gene -~.
encoding a 5-enolshikimate-3-phosphate synthase (EPSPS) ;.
conferring tolerance to N-phosphonomethylglycine, the bar
gene conferring resistance against Bialaphos -(e.q.
W09}/02071), and the like. The actual choice of the marker is
not crucial as long as it is functional (i.e. selective) in
combination with the plant cells of choice.
The marker gene and the gene of interest do not necessa-
rily have to be linked, since co transformation of unlinked
genes (U.S, Patent 4,399,216) is also an efficient process in
35 plant transformation. ~`
The following examples are given only for purposes of
illustration and:do not intend to limit the scope of the ~ -
invention. Unless otherwise stated in the Examp~es, all . :
procedures for manipulatiny recombinant DNA were earried out
SU~3STI~UT~ S~T
WO94/10320 iSi~ ~ 51 PC~tEP93/03091 -
- 16 -
by using standard procedures as described in Sambrook et al.
(Molecula~ Clonin~, A laboratory Manual 2nd Edition, Cold ~ -~
Spring Harbor Laboratory (1990).
r
EXAMPLE I
Construction of cloninq vectors
a) Construction_of binarv vector PMOG23
In this example the construction of the binary vector
pMOG23 (in E. coli K-12 strain DH5, deposited at the Centraal
Bureau ~oor Schimmel-cu}tures on January 29, l99G under
accession number CBS 10~.90) is descxibed.
The binary vector pMOG23 is a derivative of vector Binl9
(Bevan, 1984, Nucl. Acids Res. 12, 8711-8721). To obtain
pMOG23, the vector Binl9 is changed in a way not essential
15 for the present invention, using techniques familiar to those -
skilled in the art of molecular biology. First, the positions
of the left border (LB) and the right border (R~) are swit~
ched with reference to the neomycine phosphotransferase gene
II (NPTII gene). Secondly, the orientation of the NPTII gene
is reversed giving transcription in the direction of LB
Finally the polylinker of Binl9 is replaced by a polylinker
with the following restriction enzyme recognition sites: -
EcoRI, SmaI, BamHI, XbaI, SacI, XhoI and HindIII (Figure 1).
.
b~ Construction of cloninq vector ~MOG707
A cloning vector pMOG707 is constructed, containing a
rlght border T-DNA sequence, a multiple cloning site and a
t~rminator for the purpose of cloning different promoter~gene
combinations on a suitable fragment. This vector is construc
30 ted in the following manner: in the cloning vector pMTL26 ~
(Chambers et alO 198~ Gene 68, 139-149) the XhoI site is 1 -
removed by XhoI digestion, blunt-ended with Klenow polymerase
followed by religation, resulting in pMTL26~2. This modified
pMTL vector is used to clone the EcoRI - BqlII fragment from T '`
35 p~QG23j cQntaining the multiple cloning site and the right J~`- .''
border sequences, resul~ing in pMOG584bis. The polylinker
sequence is extended by inserting a synthetic linker between ~`
the BamHI and XhoI site, thus creating additional NcoI, XhoI
and XbaI sites. Subsequently, the nopaline synthase ~rans~
,:~
SUBSTI~T,_ S~ET ` .
'VO 94/lû320 ~ i Lf ;'.) '-' 3 ~ PCl`lEP93/03091
- 17 -
cription terminator is isolated as a BamHI/HindIII fragment
from the plasmud ROK1 (Baulcombe et al. 1986, Nature 321; ¦
446), ligated to a synthetic adaptor such that the HindIII
site is not recovered and an EcoRI site is introduced and
5 subsequently cloned into the extended pMOG584bis as a BamHI - ¦
EcoRI fragment, resulting in plasmid pMOG707(Figure 2).
c~ Mobilisation of binary vectors into Aqro~acterium tumefa-
ciens
The binary vectors described in Example IV-VIII are
mobilized in a triparental mating with E. coli K-12 strain
HB101 (containing plasmid RK2013) (Ditta et al., 1980, Proc.
Nat. Acad. Sci. USA 77, 7347-7351), into Aqrobacterium ~--
tumefaciens strains MOGlQl (Example II) or LBA4404 (Hoekema
et al. 1983, Nature 303, 179-180) that contains a plasmid
with the virulence genes necessary for T-DNA transfer to -
plants.
EX~MPLE II
Construction of AqE~bacterium straln MOG101
A binary vector system was used to transfer gene constructs
into ArabidoPsis plants. The helper plasmid conferring the
A~robacterium tumefaciens virulence functions was derived
2S from the octopine Ti-plasmid pTiB6. MOG101 is a Aqrobacterium
tumefaciens strain carrying a non-oncogenic Ti-plasmid
(Koekman et al. 1982, Plasmid 7, 119-1323 from which the
entire T-region was deleted and substituted by a bacterial
Spectinomycin resistance marker from tran~poson Tn 1831
(Hooykaas et al., 1980 Plasmid 4, 64-75).`
The Ti-plasmid pTiB6 contains two adjacent T-regions, TL j
(T-left) and TR (T-right). To obtain a derivative lacking the
TL- and TR-regions, we constructed in~ermediate vector
pMOG579. Plasmid pMOG579 i5 a pBR322 derivative, which
contains the 2 Ti-plasmid fragments that are located to the
left and right, outside the T-regions (Figure 3). The 2
fragments (shown in dark) are seperated in pMOG579 by a 2.5
kb ~amHI HindIII fragment from transposon Tnl831 (Hooykaas
et al., 1980 Plas~id 4, 6~75~ carrying the spectinomycin
.
SUBSTI~UT~ S~ET - :
W094/10320 PCT/EP93/03091~ ~
- 18 - ~-
resistance marker (Figure 4). The plasmid was introduced into
Aqrobac~erium tumefaciens strain LBA1010 [C58-C9 (pTiB6) - a
cured CS8 strain in which pTiB6 was introduced (Koekman et ¦ -
al. 1982, Plasmid 7, 119-132), by triparental mating from ~ -
5 E.coli, containing pRK2013 as a helper. Transconjugants ~ere t
selected for resistance tQ Rifampicin (20 mg/l) and spectino-
mycin (250 mg/l). A double recombination between pMOG579 and
pTiB6 resulted in loss of carbenicillin resistance (the
pBR322 marker) and deletion of the entire T-region. Of 5000 ,~
10 spectinomycin resistant transconjugants replica plated onto
carbenicillin (100 mg/l) 2 were found sensitive. Southern
analysis showed that a double crossing over event had deleted
the entire T-region (not shown). The resulting strain was
called MOG101. This strain and its construction is analogous
to strain GV2260 (Deblaere et al. 1985, Nucl. Acid Res. 13,
4777-4788).
EXAMPLE III
Isolation_of a promoter fraqment DeltaO.3TobRB7 from tobacco
The DeltaO.3TobRB7-5A promoter sequence (Yamamoto et al.
1991, Plant Cell 3: 371-382) was isolated by a two-step PCR
on genomic DNA isolated from tobacco. In the first PCR -
reaction, part of the TobRB7-5A gene is being isolate~ using
the following pri~ers:
5' primer: 5' CTCCAAATACTAGCTCAAAACC 3' (SEQIDNO:1)
3' primer: 5' CCTCACCATGGTTAGTTCTC 3' (SEQIDNO:2).
The resulting PCR product is used to isolate the DeltaO.3To~-
RB7-5A fragment using the following primers:
5' primer: 5' CTTGAATTCTAGAT~AGCTTATCTAAAC 3'
(S~QIDNO:3)
3' primer: 5' CCTCACCATGGTTAGTTCTC 37 ~SEQIDNO:4).
The resulting PCR pro~uct is purified out of gel, blunt ended
and subcloned into pUC9 ~Vieira & Messing 1982 Gene 19; 259
268) which is then linearised with SmaI. Digestion of the .-
rasulting plasmid with XbaI and partially with NcoI yields
the correct DeltaO.3TobRB7-5A fragment for cloning in
Examples IV-VIII.
.
SUE3gT~TUT_ S~ET
~ ! WO 94tlO320 ~ 3 ~ PCT/EP93/03091
-- 19 -- 1'""
EXAMPLE IV
Cloninq of chimaeric DNA sequences of the DeltaO.3 TobRB7
Promoter and the antisense NADPH-CYtochrome P450 ATR1 qene
for specific rePression of the nematode-induced feedinq ! `
5 structures in Arabidopsis. ~ ¦ ;
a) cloninq antisense NADPH-C~to~hrome P450 ATR1 and construc-
tion o~ binarv vector pMOG711 -~
The clone for NADPH-cytochrome P450 reductase ATR1 (EMBL
accession number X66016) is isolated from Arabido~sis tha- `
liana var. Landsberg erecta using PCR technology on cDNA made
of mRNA from this species. The primer set 5' GGCGGATCGGAGCGG-
GGAGCTGAAG 3' (SEQIDNO:5) and 5' GATACCATGGATCACCAGACATCTCTG
3' (SEQIDNO:6) is used to amplify the sequence of interest.
lS This introduces a NcoI site on the N-terminus of the PCR
fragment. Subsequently, the PCR fragment is digested with
BamHI - NcoI and cloned antisense before the nopaline
synthase terminator into pMOG707. The truncated promoter - -
se~uence DeltaO.3TobR~7-5A (Yamamoto et al. 1991, Plant Cell
20 3; 371-382), isolated as described in Example IIIm, can then
be inserted as a XbaI - NcoI fragment. The entire sequence is
then cloned into the binary vector pMOG23 after digestion
wlth EcoRI and one of the remaining unique restriction
enzymes, resulting in binary vector pMOG711 (Figure 5). ~-
b) expression of the DeltaO.3 TobRB7/antisense NADPH-
Cvtochrome P450 ATRl construct in ArabidoPsis ;;
Arabidopsi$ is transformed by cocultivation of plant
tissue with qrobacterium tumefaciens strain MOG101 contain-
ing the binary vector pMOG711. Transformation is carried out
using cocultivation of Ara~idoDsis thaliana (ecotype C24~ j
root segments as described by Valvekens et al. (1988, Proc.
Nat. Acàd. Sci. USA 85, 5536-5S40). Transgenic plants are 7
regenerated from shoots that grow on selection medium (50
mg/l kanamycin), rooted and transferred to germination medium
or soil. Young plants can be grown to maturity and allowed to
self-pollinate and set seed. .
'
;.
SU~3STI~U~-~ S~EET
W094/10320 ~ iS ~ PCT/EP93~03091
- 20 -
EXAMPLE V
Clonin~ of chimaeric DNA sequences of the DeltaO.3 TobRB7
promoter and the antisense NADPH-CYtochrome P450 ATR2 qene
for specific re~ression of the nematode-induced feedinq
S structures in Arabidopsis.
a) cloninq antisense NADPH-CYtochrome P450 ATR2 and construc-
tion of binarY vector ~MOG712
The clone for NADPH-cytochrome P450 reductase ATR2 (EMBL
accession number X66017) is isolated from Arabid~sis tha-
liana var. Landsberg erecta using PCR technology on cDNA made
o~ mRNA from this species. The primer set 5' GGTTCTGGGGATCCA-
AAACGTGTCGAG 3' (SEQIDMO:7) and 5'
~GCTTCCATGGTTTCGTTACCATACATC 3' (SEQIDNO:8) is used for
amplification. This introduces both a BamHI and a NcoI
flanking the PCR fragment. Subsequently, the PCR fragment is -
digested with BamHI - NcoI and cloned antisense before the ~
nopaline synthase terminator into pMOG707. The truncated ;~-
promoter sequence DeltaO.3TobRB7-5A (Yamamoto et alO 1991,
Plant Cell 3; 371-382), isolated as described in Example
IIIm, can then be inserted as a XbaI - NcoI fragment. The
entire sequence is then cloned into the binary vector pMOG23
after digestion with XhoI and partial digestion with EcoRI
or, alternatively, after digestion with XbaI and partial
digestion with EcoRI, resulting in binary vector pMOG712
(Fiqure S~.
b~ exDression o~ the DeltaO.3 TobRB7~antisense NADPH~
~~tochrome P450 ~TR2_construct in ArabidoP-sis
ArabidoPsis is transformed by cocultivation of piant
tissue with Aarobacterium tumefaciens strain MOG101 contain ~ ~`
ing the binary ~ector pMOG712. Transformation is carried out ^.
using cocultivation of ArabidoPsis thaliana ~ecotype C24) '~
root segment~ as described by Valvekens et al. ~1988, Proc.
Nat. Acad. Sci. USA 85, 5536-5540). Transgenic plants are
regenerated from shoots that grow on selectiQn medium (~0
mg/l kanamycin), rooted and transferred to germination medium
or soil. Young plants can be grown to maturity and allowed to ,
self-pollinate and set seed.
SUE~STITUT~ S~EE ~ --
; wo g4,l0320 ~ i '. 3 i ~ ~ PCT/EP93/03091 ~,
- 21 ~
EX~MPLE VI
Clonin~ of chimaeric DNA seauences of the DeltaO.3 TobRB7
Eromoter and the antisense ql~erol-3-phosphate 1 -
acy,ltransferase qene for specific rePression of the nematode- ¦
5 induced feedinq structures in Arabidopsis. ~,
a) cloninq anti$ense qlYcerol-3-PhosPhate acyltransferase and
construction o~ binar~ vector PMOG713
The clone for glycerol-3-phosphate acyltransferase
ATSl(EMBL accession number D00673) is isolated from
Ar,abidopsis thaliana using PCR technoloqy on cDNA made of
mRNA from this species. The primer set 5'
GCCCGGGATCCGGTTTATCCACTCG 3' (SEQIDNO:9) and 5'
GAGTATTTTCCATGGATTGTGTTTGTG 3' (SEQIDNO:10) is used for
amplification. This introduces both a SmaI, Ba~I and a NcoI
flanking the ATS1 clone. Subsequently, the PCR fragment is
digested with SmaI - NcoI and as such subcloned into pMOG445.
(pMOG445 is a pUC18 derivative that contains, by insertion of
an oligo adaptor in the multiple cloning site, thP extra ,
restriction sites ClaI, NcoI and BqlII between EcoRI and
SstI). Subsequently, the ATS1 clone is isolated after NcoI
and partial BamHI digestion and subcloned antisense before
the nopaline synthase terminator into pMOG707. The truncated
promoter sequence DeltaO.3TobRB7-SA (Yamamoto et_al. 1991,
2S Plant Cell 3; 371-382), isolated as described in Example
IIIm, is then inserted as a XbaI NcoI fragment. The entire
sequence is then cloned into the binary vector pMOG23 after
digestion with EcoRI and one of the remaining unique restric-
tion enzymes, resulting in binary vector pMOG713 (Figure 5).
P ~exPression o~ the_DeltaO 3 TobRB7 ~ tisense NADPH- ,-
Cvtochrome P450 ~TR2 construct in Arabidopsis /-
ArabidoPsis is transformed by cocultivation of plant
tissue with A~robacter,ium tu,mefaciens strain MQG101 contain~
35 ing the binary ~ector pMOG712. Transformation is carried out
using cocultivatiorl of ArabidoPsis thaliana (ecotype C24)
root segments as described ~y Valvekens et,al. (1988, Proc.
Nat. Acad. Sci. USA 85, 5536-5540)O Transgenic plants are
regenerated from shoots that grow on selection m~dium (50
SUE~STIT'JT~ S~c~T
W094/10320 PCT/EP93/03091,
~ c~15 l - 22 - ~
mg/l kanamycin), rooted and transferred to germination medium
or soil. Young plants can be grown to maturity and allowed to
self-pollinate and set seed.
`' '.
EXAMPLE VII ~ ;
Cloninq of chimaeric DNA sequences of the DeltaO.3 TobRB7
Promoter and the antisense adenine nucleotide translocator
qene for sPecific rePression of the nematode-induced feedin~
structures in ~otato.
}O :
a) cloninq antisense adenine nucleotide translocator and
construction of binarY vector PMOG714
The clone for the mitochondrial adenine nucleotide
translocator (PANT1, EMBL accession number X57557; Winning et
al. 1992 Plant J, 2; 763-773)-is isolated from Solanum
tuberosum using PCR technology on cDMA made of mRNA from this
species. The primer set 5' GCTAGCCGGATCCATCTGAGCTCCAG 3'
(SEQIDNO:11) and 5' GACGTCCATGGCTGAATTAGCCACCACCG3'
(SEQIDNO:12) is used for amplification. This introduces both
20 a BamHI and a NcoI flanking the PANT1 clone. Subsequently,
the PCR fragment is digested with BamHI - NcoI and cloned
antisense before the nopaline synthase terminator into
pMOG707. The truncated promoter sequence DeltaO.3TobRB7-5A
(Yamamoto et al. 1991, Plant Cell 3; 371-382), isolated as
25 described in Example IIIm, is then be inserted as a XbaI -
NcoI fragment. The entire sequence is then cloned into the
binary vector pMOG23 after digestion with ~RI and one of
the remaining unique restriction sites, resulting in hinary
vector pMOG714 (Figure 5).
b) expressi_n Qf the DeltaO.3 TobRB7/antisense adenine
nucleotide translocator construct in Potato
Potato is transformed by cocultivation of plant tissue
with Aqrobacterium_tumefaciens strain LBA4404 containing th~
35 binary vector pMOG714. Transformation is carried out using
cocultivation of potato (Solanum tuberosum ~ar. Desiree) 7
tuber disks as described by Hoekema et_al. 1989, Bio/Techn.
7, 273-278). Transgenic plants are regenerated from shoots
that grow on selection medium (100 mg/l kanamycin), roo~ed,
SlJ~3$T3~Ur~ S~.EET `- I
' ~ W094/10320 ~ l~-J~ i PCT/EP93/D309l
- 23 -
multiplied axenically by meristem cuttings and transferred to
soil to produce tubers.
EXAMPLE VIII
Cloninq of chimaeric DNA seq~ences of the DeltaO.3 TobRB7
promoter~and thQ antisense ATP svnthase aene for specific
repression of the nematode-induced feedinq structures in ?~'
tobacco.
a) clonln~ antisense ATP synthase and construction of binar~
vector PMOG715
The clone for the beta subunit of ATP synthase (Boutry &
Chua 1985 EMBO J. 4; 2159-2165) is isolated from tobacco
(Nicotiana Plumbaqlnifolia) using PCR technology on cDNA made
~S of mRNA from this species. The primer set 5'
CCCTCCAGGATCCCTTCTCGGAGGCTTC 3' ~SEQIDNO:13) and 5'
GAAAAGAAAGCCATGGAACTTTATAATC 3' (SEQIDNO:14) is used for
amplification. This introduces both a BamHI and a NcoI
flanking the ATP synthase clone. Subsequently, the PCR
fragment is digested with BamHI - NcoI and cloned antisense
before the nopaline synthase terminator into pMOG707. The
truncated promoter sequence DeltaO.3TobRB7-5A (Yamamoto et
al. 1991, Plant Cell 3; 371-382), isolated as described in
Example IIIm, is inserted as a XbaI - NcoI fragment. The
entire sequence is then cloned into the binary vector pMOG23
after digestion with EcoRI and one of the remaining unique
restriction sites, resulting in binary vector pMOG715 (Figure
5).
b) expression of the Del aO 3 TobRB7 antisense ATP sYnthase
construct in tobacco
Tobacco is transformed by cocultivation of plant tissue
with A~rob3cterium tumefaciens strain LBA4404 (Hoekema et al. '~ ~;
1983, Nature 303, 179 180) containing the binary vector
pMOG715 Transformation is carried out using cocultivation of
tobacco ~Nicotiana tabacum SR1) leaf disks as described by
Horsch et al. 1985, Science 2~7, 1229-1231). Transgenic j;
plants are regenerated from shoQts that grow on selection
medium (100 mg/l kanamycin), rooted and transferred to soil.
S ~ S ~
WO94/10320 2 1 , i~ .51 - 24 - PCT/EP93/03091~, ~
EXAMPLE IX ',
Analy.sis of transqenic Arabidopsis p~lants for susceptibility
to SPPN
Transgenic Arabidopsis plants can be assayed both in vitro
or in soil for resistance against M. incoqnita or the cyst
nematode H. schachtii. For in vitro analysis, seeds are
surfaee sterilized, grown and inoculated as described by ~ -~
Sijmons et al. (l~9l, Plant J. ~; 245-254). For soil-grown
plants, seedlings are germinated on kanamycin-containing
medium (lO mg/ml) and kanamycin-resistant seedlings are
transferred to soil/sand mixtures (l:3 v/v) in lxlx6 cm ~
transparent plastic tubes. Once the rozettes are well devel- ~-
oped (ca. 14 days~ the containers are inoculated with ca. 300
hatched J2 of H. schachtii each. Eighteen days after inocula-
tion, the roots are carefully removed from the soil/sand
mixture and stained with acid fuchsin (Dropkin, 1989 in:
Introduction to plant nematology, 2nd edition, Wiley & Sons,
New York). In this assay, susceptible plants score a mean of
17 cysts per root system (range 4-40 cyst per xoot system).
Similarly, plants can be inoculated with hatched J2 of M.
incoqnita or with egg-masses that are mixed through the
soil/sand mixture. The plants can than be scored for the
presence of galls which are clearly visible once the roots
are washed clear of the soil/sand mixture.
EXAMPLE X
Analysis of trans~enic ~otato Plants for susceptibility to
SPPN
Transgenic potato plants can be assayed for resistance
against M. inco~nita using soil that is preinfected with M.
incoqnlta egg masses mixed with sand (l:3 w/w), growing the
po~ato plants in that soil mixture for 6 weeks and , after
removing the soil, count the developed number of galls on a
root system. Alternatively, to assay for resistance against ~;>
Globodera ssp. a closed container is used. For khis assay,
three replicate 2-4 cm tu~ers are transferred to soil which
is pre inoculated with cysts from G rostochiensis or GL
pallida in transparent containers. The peripheral root
systems can be analyzed visually 7-8 weeks after germination
SVB~TlTU~ SHEET .`
WO94/10320 ~ l PCT/EP93/0309
- 25 -
for the presence of cysts. A genotype will be scored as t
resistant if none of the three rPplicates had cysts and
susceptible if at least one of the three replicates shows t
cysts.
S ~ '
EXAMP~E XI
Analysis of transqenic tobacco Plants ~or susceetibil itY to
SPPN
For anlysis of nematode resistance, the soil is preinfected
with M. incoanita egg masses. This inoculum can be produced
by maintaining a stock culture of M. incoanita on soil grown
celery plants (APium araveolens) under standard ~reenhouse
conditions, belaw 25C. Mature celery root systems, contain-
ing a high number of root knots and mature females of M.
incoqnita, are carefully dusted off to remove the soil,
homogenized briefly in a Waring blendor (2 seconds) and ~-
weighed in portions of 60 gram. These root samples are mixed
with 1 Xg sand:potting soil (l:l) mixtures and used for
growth of transgenic tobacco transformants. As control
plants, primary kanamycin resistant transformants (transgenic
for pMOG23) are used. Per construct; 100 primary
transformants are grown in infected soil for 6 weeks. The
soil/sand mixture is washed away carefully and the number of
galls / root system is counted with a binocular. Control
25 plants have a mean o~ 25 l ll galls. A genotype is considered --
resistant when the mean number of galls is reduced to 2 per
root system. The primary transformants meet this requirement,
can than be used for a rapid multiplication cycle by placing
transformed lea~es again on media that allows shoot re~ene-
30 ration (Horsch et al. 1985, Science 22?, 1229-1231~ or the `,
plant~ ~an be grown to maturity and allowed to flower and `-
` ` seed setting and used for more extensive testing of nematode
resistanc~ using 100 plants of each genotype. `
'''' ~';
",
. .
SU~ Ti~T~ .EET
.
W O 94/10320 PCT/EP93/03091
;3.1 - 26 - :
S~ENCE LISIING
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SU~3STITUT~ S~ET
WO 94/10320 PCr/EP93/03091
- 27 - 1 ~
(iii) ~ICi~L: YES "'
(xi) SEQI~CE DESCRIPrION: S~Q ID NO: 2: l
CCI~CCA~ ~ITA~C ~ 20 ~ :
~2) INE~IC~ F~R SEQ ID NO: 3:
( i) S~CE a~ACrE~S~ CS:
(A) IEI~: 28 base pairs
(B) TYPE: nucleic acid
(C) S~ANDE~NESS: sir~3le
(D) IOPOIDGY: linear
~xi) SE~NOE DESCRIPrION: SEQ ID NO: 3: :.
C~rI~ A~A~ ~C 28
(2) ~lION FOR SEQ ID NO: 4:
(i) S~tOE CH~CI~;: ~
(A) ~: 20 base pairs .
(B) TY~E: nucleic acid
(C) S~RANDI~ESS: single
(D) I~PO~GY: l~near
(ii) ~LECUL~ ~ (genamic) `.
~0 ` ::
txi) S~CE DESCRE~5QN: S~Q ID NO: 4:
C~CC~G ~Grl~C ~ 2~ ..
(2) ~FO~XCN F~R S1~ ): 5: 3 ,
(i) S~OE C~RP.CI~ISFICS~
(A) IEN~: 25 base pai:r., ` .
(B) q~: ~n~cleic acid
(C) SI~S : sir~le ~; -
55 (iii) HYE~C~
-~
Sl 3 ~35T~TlJTE S~
WO 94/1032Q PCI/EP93/03091~ b~ ;
-- 28
(x~) SE~UENOE DESCRIPlION: S~Q ID NO: 5:
GG~;GAI'~X;G ~C 1~ 25
(2) INF~ FION F~R SE)Q ID NO: 6: ¦
(A) LE~: 27 ~ase p~rs
(B) qYP~: nucleic acid
(C) SrRANDE[~ s~ngle
(iii) HYFaI~IC~1: YES
:
(x~) S~UENCE DESCRIPIION: S}~ ID NO: 6:
G~CC~I~G AICAC~C ~ 27
(2) INF~RM~LION F~R SEQ ID NO: 7:
(i) SEQ~ENCE CH~RACI~ISrICS:
(A) IE~: 27 base pa~rs .
(B) TY~: nucleic acid .
(C) SrRANDE~ESS: single ~ -
(D) I~FO~DGY: linear . ...
3Q ;
(ii) ~IECULE TYPE: c~ . :
(iii) HY~I~CAL: YES ':
(xi) S~OE I~ESCRIPIION: S}3Q ID NO: 7: ~ `:
G~C~ P:~A~CG ~ 27
(2) INE~ION F~}? SE~2 ID NO: 8:
tA) I~: 28 base pa~rs
tB) T~E: m~cleic acid ..
(~) S~: s~le ,. ~.
(D) TOPOI~Y: linear
. .
~ . .
(xi) S~aOE ~ESCRI~: S~ 8: j ~.
~CII~ Gr~l~C C~C~c 28 ,-
SUBSTITUT~: SHEE~
WO 94/ 1 0320 21~ PCI /EP93/0309 1
-- 29 --
(2) ~ON F~:)R SEQ ID NO: 9:
(i) SE~ENOE ~RACn~ISIICS: j
(P,) IEt~:I~: 25 base pa~rs
S (B) TY~: nucleic acid
(C) S~ANDE~ESS: s~ngle ~.-
1 :
, ,,, ,
(xi) SEQUENCE DESCRIE~ON: S~2 ID NO: 9: ';
GCCC~I~ ~OEI~ ACI~ 25
(2) ~ ON F~R SEQ ID NO: lO:
(i) S~QU~OE CH~RACnSRISl'ICS:
(A) LENGI~: 27 base pairs
(B) TYE~ cleic acid :~
tc) SI~NDE~;S: sir~}e
(D) IOPO~: linear
:
(~i) S~ENCE ~S~ION: S~Q Il) NO: 10:
35 GA~lTrI~ C~I~ G-lll~IG 27
:,...
(2) INF~ION F~:)R SEY2 ID N~
(A) ~: 26 base paiB :
(B) TYPE~ cleic acid
(C) Sl~: sirlgle ,`~`
(D) IOPOI~Y: linear ~ , -
(ii) ~LECSJIE ~Y~E: c~.
.
a~ c~; 26
55 (2) ~T~ ~R S~ ID ~
(A) IENG~l: 29 base p~s
. ~
S~IBSTITUT~ ET
.i~
WO 94/10320 h i i!' 3 1 5 ~ PCI/EP93/03091 . .
- 30 - 1:
(B) T~: Ilucleic acid
(C) SIRANDEI~ sir~le
(D) I~POLC~Y: l~near .
5 (ii) MI~LEaJI~ 1~: cl~
~ ",
~xi) Si3Qt~OE DESCRIPIION: SEQ ID NO: 12: ,;
29
15 (2) :~RMPlION F`OR SE~Q ID N~:): 13:
(i) SE~OE CH~ACI~RISTICS: : ~.
(A) IENGIH: 28 base pa~rs
(B) TYE~: nucleic acid -
(C) SrRANDE~NESS: s~le
(D) IOPOLOGY: linear ~-.
(iii) }~I~C~L: YES
(xi) SE~OE DESCRIPIION: ~ ID NO: 13:
30 CCC~ ~C 28
(2) ~}~IION ~R SEQ ID NO: 14: ~ ~
(A) IENGI~: 28 base pairs .
(B) TYP~: nucleic acid
~C) SrR~NDE[~ESS: s~le
(ii) ~9DLE~IE qY~: c~
(xi) S~NOE DESCI~I: SI~Q ID N~: 14:
SU~3STITUTE SHEET - -
'~:
.. `. - . , , , ~ .. .......... .
