Note: Descriptions are shown in the official language in which they were submitted.
~"092t21757 ~ ~ _ G 1 S ~ P`CI`/EP92/01214
NEHATODE--RESPONSIVE PI.ANT PROMOTERS
This invention relates to nematode-responsive
promoter~ which can be isolated from plants infected ~y
nematodes and which can either induce (i.e., stimulate) or
repress the expression of genes or DNA fragments, under
their control, at least substantially selectively in
specific cells (e.g., fixed feeding site, pericycle,
endodermis, cortex or vascular cells) of the plants' roots,
preferably in cells of the plants' fixed feeding sites, in
response to the nematode infection. The nematode-induced
promoters of this invention are especially useful in
transgenic plants for controlling foreign DNAs that are to
be expressed selectively in the specific root cells of the
plants, 80 as to render the plants resistant to nematodes,
particularly to sedentary endoparisitic nematodes.
This invenSion also relates to a first ~r nematode-
induced chimaeric gene that can be used to transform a cell
of a plant and that contains a first fo~eign gene or DNA
fragment that: a) encodes a product which, when expressed
in specific cells of the plant's roots, preferably in cells
of fixed feeding sites of the plant, can eithar kill or at
least disturb significantly the specific root cells of the
plant, preferably the cells of the plant's fixed feeding
sites, or kill, disable or repel nematodes feeding at fixed
feeding sites; and b) is under the control of a nematode~
induced promoter of this invention.
This invention further relates to a cell of a plant,
the genome of which is transformed to contain the first
chimaeric gene and optionally a second or restorer
chimaeric gene that contains a second promoter controlling
W09~217S7 PCT/EP92/0]21~
! f ` ~
~ 'U 1 d J
a second foreign gene or DNA fragment encoding a product
that is expressed so as to inhibit or inactivate the first
foreign gene or DNA fragment or the product encoded thereby
in cells other than the specific cells of the plant's
roots, preferably in cells other than fixed feeding site
cells of the plant.
This invention yet further relates to: a) a nematode-
resistant plant (such as tomato or potato) which is
regenerated from the plant cell of this invention and is
transformed with the first and optionally the second
chimaeric genes, b) nematode-resistant plants derived from
the regenerated plant and seeds of such plants, and c)
plant cell cultures, all of which consist essentially of
the transformed plant cells of this invention. The plants
of this invention are characterized by the nematode-induced
expression of the first chimaeric gene of this invention in
their specific root cells, preferably their fixed feeding
site cells, and either a) the substa~tial, preferably
complete, absence of expression of the first chimaeric gene
in all other plant cells or b) the substantial absence~ and
preferably the complete absence, by expression of the
second chimaeric gene of this invention, of the effects of
any expression of the first chimaeric gene in all other
plant cells ---thereby rendering the plants resistant to
nematode infec~ions.
This invention still further relates to a process of
rendering a plant resistant to plant-parasitic nematodes by
transforming the plant with the first and optionally the
second chimaeric gene(s~ of this invention.
~.~",,.,,~,.s,,Z,~ ,,,""",;,;~
092~21757 ~ 11 a 1 ~ 9 PCT/EP92/01214
Backaround of the Invention
Plant-parasitic nematodes are small (generally 100-300
~m long but up to 4 mm long, and 15-35 ~m wide) worm-like
animals which feed on root, stem or leaf tissues of living
plants. Nematodes are present wherever plants are
cultivated. Ectoparasitic nematodes, such as the dagger
~Xi~hinema and Lonaidorus spp.), stubby-root (Trichodorus
and Paratrichodorus spp.) and spiral (Scutellonema and
Helicotvlenchus spp.) nematodes, live outside the plant and
pierce the plant cells with their stylet in order to feed.
Migratory endoparasitic nematodes, such as the lesion
(Pratylenchus spp.), stem and bulb (Ditvlenchus spp.) and
burrowing (RadoDholus spp.) nematodes, live and feed inside
the plant, migrating through the plant tissues. Sedentary
endoparasitic nematodes, such as the root-knot (Meloidoqvne
spp.), cyst (Globodera and Heterodera æpp.), citrus
(Tyl~nchulus spp.) and reniform (Rot~lenchulus spp.)
nematodes, live and feed inside the plant, indusing
specialized fixed feeding sites called giant cells,
syncytia or nurse cell~ in susceptible plants. Such fix~d
feeding sites serve as food transfer cells for the various
developmental stages of the nematodes. Syncytia originate
in the pericycle, endodermis or adjacent cortex (Jones,
981).
Parasitic nematodes can cauce significant plant yield
losses. The most striking effect of n~matode infection is a
general reduction in plant growth. Nematodes can act
directly as plant pathogens that predispose plants to
bacterial or fungal infections or as vectors of plant
viruses. Plant diseases caused by nematodes include root
galling, root lesions, root rot, stubby roots, stunting and
WO9V2175~ PCT/EP92/0121~
1G~
wilting. Overall average annual yield loss of the world's
major crops due to damage by plant-parasitic nematodes is
estimated at 12.3 % (Sasser and Freckman, 1987). Monetary
losses, when all crops are considered, exceed US ~ 100
billion annually (1984 production figures and prices;
Sasser & Freckman, 1987).
On a worldwide basis, the ten most significant
nematode genera are the ectoparasitic Xi~hinema spp., the
migratory endoparasitic Pratvlenchus spp., Dit~lenchus
spp., RadoPholus spp. and HelicotYlenchus spp., the
~edentary endoparasitic MeloidooYne spp., Heterodera spp.,
Globodera spp., Tvlenchulus spp. and RotYlenchulus spp.
(Sasser and Freckman, 1987). Especially significant are the
sedentary endoparasitic nematodes, comprising the genera
Heterodera, Globodera and MeloidoaYne which cause severe
damRge to many crops and are of major economic importance.
For example, cyst nematodes (e.g~, Heterodera and
Globodera) cause great problems in the production of
potatoes, soybeans, sugar beets, and wheat. Once cyst
ne~atodes have infested a field, it is practically
impossible to.eliminate them. MeloidoqYne spp. affect many
species of plants (over 2,000), including most of the major
crops of the world. In the tropics, root-knot is often the
limiting factor in crop production. In contrast to cyst
nematode infections, extensive gall formation accompanie~
the infection with root-knot nematodes.
Various methods have been used to control plant
parasitic nematodes (Brown and ~erry, 1987). They include
quarantine measures, manipulation of planting and
harYesting dates, improved fertilization and irrigation
programs that lessen plant stresses, crop rotation and
~92J21757 2 ~ 1 ~ 1 S 3 PCT/EP92/01214
fallowing, use of resistant and tolerant cultivars and
rootstocks, organic soil amendments, and physical (e.g.,
solarization), biological and chemical control. Although
quarantines are useful, especially when an infestation is
first discovered, they are very expensive measures and
usually cannot prevent the spread of nematodes (Dropkin,
1980). Furthermore, biological control is difficult to
manage, and high quantities and repeated additions of
agents are required.
Today, control of plant-parasitic nematodes relies
mainly on chemical control. Nematicides used commercially
are generally either fumigants (e.g., halogenated aliphatic
hydrocarbons and methyl isothiocyanate precursor compounds)
or non-fumigants (e.g., organophosphates and oxime-
carbamates). However, the use of chemical nematicides poses
an increasing number of difficulties:
A. Developing a new nematicide requires a high
financial inYestment, and very few new nematicidal
compounds have been discovered despite intensive
screening efforts (Morton, 1987)~ As a result,~ only
a limited number of nematicides are currently
a~ailable, and new ones are increasingly expensi~e.
, . .
B~ Nematocides are only efficient under ~ertain
agronomical and environmental conditions (Bunt,
1987), and soil and roots act as barriers,
protecting plant-parasitic nematodes from
nematicides.
C. Nematodes are known to rapidly invade fields, and
phytonematodes are easily distributed with soi~ and
plants. As a result, nematode control with
nematicides does not persist for long but must be
WO9~21757 PCT/EP92/0121
repeated frequently and with relatively high
concentrations of nematicides to keep nematode
populations at low levels.
D~ All nematicides are highly toxic. They are
therefore hazardous not only to the user but also to
the en~ironment. In the USA, several nematicides
(e.g., DBCP, EDB, D-D, aldicar~ and carbofuran) have
already been found in the groundwater (Thomason,
1987). Due to their harmful effects on humans and
non-target organisms, their persistence in the soil,
and their concentration in ground water, nematicides
are being withdrawn from the market worldwide. As a
result, there is today a real need to have new, more
effective, and safe me~ns to control plant-parasitic
nematodes.
In susceptible plants, the infective second-stage
juveniles of the sedentary endoparasitic nematode species
invade the roots and migrate inter- and-intra-cellularly
through the cortex to specific regions, usually close to
the pericycle, within the roots. Once the nematode~ has
traveræed the cortex, it can initiate feeding in a cortical
cell, but in most cases, feeding is initiated in endodermal
or pericyclic cells (Endo, l987). Here, the juveniles
settle and begin to puncture the cells surrounding heads.
Then, the juveniles introduce their secretions into these
cells which induces changes in the cells, thereby forming
fixed feeding sites in which a ~arge vol~me of cytoplasm is
available to the juveniles (Endo, 1987). The growth cycle
of fixed feeding sites is directly related to the further
develop~ent of the nematodes. After establishment of fixed
WO92/21757 ~ 6 ~ PCT/EP92/01214
feeding sites, the second-stage juveniles increase steadily
in size and undergo three moults in quick succession. The
third- and fourth-stage juveniles cannot feed, but the
young females resume feeding. Thus, further changes in the
cells of the fixed feeding sites are controlled by
secretions produced by the females and maintained by
removal of cytoplasm by the feeding females. The
initiation, development and maintenance of fixed feeding
sites is essential for the establishment, development and
reproduction of the nematodes. Tbe fixed feeding sites are
thus critical for the survival of the sedentary
endoparasitic nematodes. Without the fixed feeding sites,
the nematodes would be unable to feed and reproduce and
would die. The sedentary endoparasitic nematodes thus
illustrate an important principle -- that the relationship
between parasite and plant host depends on a continuing
exchange of information by the two organisms. WAen the
nematodes are killed, fixed feeding sites degenerate,
leading to the conclusion that the maintenance of fixed
feeding sites depends on the continued presence of a
functional parasite. Each of the sedentary phytonematodes
induces the development of fixed feeding sites upon which
it feeds.
SummarY of the Invention
In accordance with this invention are provided
nematode-responsive cDNA sequences isolated from tomato
plants comprising the sequences, SEQ ID no. 1, SEQ ID no.
2, SEQ ID ns. 3, SEQ ID no. 4, SEQ ID no. 5, SEQ ID no. 6,
SEQ ID no. 7 and SEQ ID no. 8 described in the Sequence
Listing.
WO92/21757 PCT/EP92~0121~
1 6 3 8
Also in accordance with this invention are provided
nematode-responsive promoters of the tomato genes
corresponding to the cDNA sequences of SEQ ID nos. 1-8,
particularly:
a) nematode-induced promoters of the genes
corresponding to the cDNA sequences of SEQ ID nos.
2 8, more particularly of: i) the gene corresponding
to the cDNA sequence of SEQ ID no. 7 which gene is
substantially selectively expressed in fixed feeding
site cells, particularly in cells within galls, and
ii) tbe genes corresponding to the cDNA sequences of
SEQ ID nos. 4 and 6 which genes are substantially
selectively expressed in pericycle cells; and
b) a nematode-repressed promoter of the gene
corresponding to the cDNA sequence of SEQ ID no. l.
Further in accordance with this invention is provided
the first or nematode-induced chimaeric gene that comprises
the following, operably linked, DNA sequences:
l) a nematode-induced promoter that is suitable to
direct transcription of a foreign DNA at least
substantially selectively, pre~erably selectively,
in the sp~cific root cells, preferably in the cells of
fixed feeding sites, of a plant (at whose fixed
feeding sites nematodes would feed);
2) a first foreign DNA that encodes a first RNA
and/or protein or polypeptide which, when produced
or overproduced in the specifio root cells, preferably
the cells of the fixed feeding sites, of the plant,
either a) kills, disables or repels the nematodes when
the nematodes feed from the fixed feeding sites or b)
~o 9~21757 2 1 1 0 1 ~ ~ PCT/EP92/01214
kills the specific root cells, preferably the cells of
the fixed feeding sites, or at least disturbs
significantly their metabolism, functioning and/or
development, thereby at least disturbing
significantly, and preferably ending, the ability of
the nematodes to feed from the fixed feeding sites of
the plant; and
3) suitable 3' transcription termination signals
(i.e., 3'end) for expressing the first foreign DNA in
the cells of the specific root cells, preferably the
fixed feeding sites.
Still further in accordance with this invention is
provided a cell of a plant, in which the nuclear genome is
transformed to contain the first chimaeric gene of this
invention and preferably, when the nematode-induced
promoter directs transcription of the first foreign DNA
only substantially selectively in the specific root cells,
preferably the fixed feeding site cells, of the plant, to
also contain the second or restorer chimaeric gene,
preferably in the same ~enetic locus; the second chimaeric
gene comprises the following, operably linked, DNA
sequences:
1) a second promoter, such as a nematode-repressed
promoter, which can direct transcription of a foreign
DNA in cells of the plant where the ~irst foreign DNA
is expressed, preferably at least substantially
selectively in cells other than the specific root
cells, particularly in cells other than the ixed
feedinq site cells, of the plant;
2) a second foreign DNA that encodes a second RNA
and/or protein or polypeptide which, when produced
WO9~21757 PCT/EP92/01214.
9 l o
or overproduced in cells of the plant, inhibits or
inactivates the first foreign DNA or the first RNA or
protein or polypeptide; and
3) suitable 3' transcription termination signals for
expressing the second foreign DNA in cell~ of the
plant.
Still further in accordance with this invention are
prov~ded the nematode-resistant plant regenerated from the
transformed plant cell of this invention, nematode-
resistant plants derived therefrom and their seeds, and
plant cell cultures, each of which consists essentially of
the plant cells of this invention.
Yet further in accordance with this invention is
provided a process for rendering a plant resistant to
nematodes, particularly sedentary endoparisitic nematodes,
comprising the step of transforming the plant's nuclear
genome with the first chimaeric gene and optionally the
second chimaeric gene of this invention.
Detailed DescriPtion of the Invention
Throughout this Description, the following defini~ions
apply:
"Fixed feeding sites" should ~é und~rstood as
specialized feeding sites (such as giant cells,
syncytia and nurse cells and, if pr~sent, galls), the
formation of which is induced by sedentary
endoparasitic nematodes in susceptible plants. The
plant cells of such sites serve as food transfer cells
for ~he various developmental stages of the nematodes.
WO9~21757 ~ PCT~EP92/01214
"Nematode-infected plant" means a plant in which a
nematode has entered.
"Giant cells" should be understood as the
multinucleate plant root cells induced by nematodes
such as root-knot nematodes. The multinucleate
condition of each giant cell is believed to r~sult
from multiple mitosis in the absence of cytokinesis.
~Syncytium" refers to multinucleate plant root cells
induced by nematodes such as cyst nematodes. The
multinucleate condition of each syncytium results
from cell wall dissolution between contiguous cells
with preexisting nuclei.
"Nurse cells" refers to a group of six to ten
uninucleated plant root cells, induced by
T~lenchulus spp., which have a dense cytoplasm
without a vacuole and a much enlarged nucleus a~d
nucleolus.
"Galls" ref~r to a proliferation of cortical p~ant
cells/tissue induced by nematodes. Typically, giant
cells reside within galls.
"Nematode-responsive promoter" means a promoter
whose action in controlling transcxiption of a DNA
sequence (e.g., gene) in a plant is influenced -- that
is, either induced (i.e., stimulated) or repressed --
by infection of the plant by nematodes and preferably
is influenced selectively in specific cells of the
plant's roots, particularly in cells of the plant's
fixed feeding sites. A "nematode-responsive promoter"
W09~21757 PCT/EP92JO1214~
, . . , ,,, ,,~ ;~
~ tJ~ 12
can be either a "nematode-induced promoter" or a
"nematode-repressed promoter".
"Specific cel~s of a plant's roots" or "specific root
cells of a plant" means cells of a root tissue such as
the fixed feeding sites, the pericycle, the
endodermis, the cortex or the vascular tissue,
preferably a) cells of the ~ixed feeding sites or b)
cells of tissue (e.g., pericycle cells) which i) will
differentiate into fixed feeding site cells upon
infection of the plant by nematodes or ii~ can be
altered to reduce the ability of nematodes to feed at
fixed feeding sites of the plant. Particularly
preferred specific root cells of a plant are fixed
feeding site cells~
nHomologous" refers to proteins or nucleic acids
having similar sequences of amîno acids or
nucleotides, respectively, and thus having
substantially the same structural and/or functional
properties.
"Expression" means transcription and translation to
a product from a DNA encoding the product.
"Foreign" with regard to a DNA sequence, such as a
first or second foreign DNA of this invention, means
that such a DNA is not in the same genomic
environment (e.~., not operably linked to the same
promoter and/or 3' ~nd) in a plant cell, transformed
with such a DNA in accordanc~ with this invention,
as is such a DNA when it is naturally found in a
~09~21757 PCT/EP92/01214
OlG~
13
cell of the plant, bacteria, animal, fungus, virus,
or the like, from which such a DNA originates.
In accordance with this invention, a nematode-
resistant plant can be produced from a ~ingle cell of a
plant by transforming the plant cell in a known manner to
stably insert, into its nuclear genome, the first chimaeric
gene of this invention which comprises at least one first
foreign DNA that is: under the control of, and fused in
frame at its upstream (i.e., 5') end to, one of the
nematode-induced promoters of this invention; and fused at
its downstream (i.e., 3') end to suitable transcription
termination (or regulation) signals, including a
polyadenylation signal. Thereby, the first RNA and/or
protein or polypeptide is produced or overproduced at least
predo~inantly, preferably exclusively, in the specific root
cells, preferably cells of the fixed feeding sites, of the
plant. optionally the plant cell genome can also be stably
transformed with the second chimaeric gene, comprising at
least one second foreign DNA that is: under the control of,
and is fused at its ~' end to, the second promoter whidh is
capable of directing expression of the second foreign DNA
in cells of the plant where the first foreign DNA is
expressed, preferably substantially selectively in plant
ce~ls other than the specific root cells, particularly in
cells other than the fixed feeding site cells; and fused at
its 3'end to suitable transcription termination signals,
including a polyadenylation ~ignal. The second chimaeri
gene is preferably in the same geneti~ locus as the first
chimaeric gene, so as to ~uarantee, with a high degree of
certainty, the joint segregation of both the first and
WO92~21757 PCT/EP92/0121~.
~ 14
second chimaeric genes into offspring of the plant
regnerated from the transformed plant cell. However in some
cases, such joint segregation is not desirable, and the
second chimaeric gene should be in a different genetic
locus from the first chimaeric gene.
In accordance with this invention, the first foreign
DNA, controlled by the nematode-induced promoter, encodes a
first RNA and/or protein or polypeptide which, when
produced or over-produced in the specific root cells,
preferably the cells of the fixed feeding sites, of the
plant, either: a~ kills such cells or at least disturbs
significantly their me~abolism, functioning and/or
development 80 as to at ~east disturb significantly, and
preferably end, the ability of nematodes to feed from the
fixed feeding sites; and/or b) kil~s, disables or repels
any nematode(s) feeding at the fixed feeding sites. First
foreign DNAs preferably encode, for example, the following
which can kill the specific root cells, preferably fixed
feeding site cells, or at least distur~ significantly their
~ metabolism, functioning and/or development: ~Nases such as
RNase Tl or barnase: DNases such as endonucleases (e.g.
EcoRI); proteases such as papain; enzymes which catalyze
the synthesis of phytohormones, such as isopentenyl
transferase or the gene p~oducts of gene l and gene 2 of
the T-DNA of qrobacterium: glucanases; lipases; lipid
peroxidases; plant cell wall inhibitors; or toxins such as
~the A- f ragment of diphtheria toxin or botulin. Other
preferred examples of such first foreign DNAs are antisense
DNAs complementary to genes encoding products essentiaI for
~he metabolism, functioning and/or development of the
specific root cells, preferably the fixed feeding site
~09~217~7 ~ ~ i 0 1~3 PCT/EP92/01214
cells. First foreign DNAs preferably encode, for example,
the following first polypeptides or ~roteins which can kill
or disable nematodes: the Bacillus thurinqiensis toxins
descr$bed in European patent publication ("EP") 303426
(which is incorporated herein by reference), collagenases,
chitinases, glucanases, peroxidases, superoxide dismutases,
lectins, glycosidases, antibacterial peptides (e.g.,
maga~nins, cecropins and apidaecins), gelatinases, enzyme
inhibitors or neurotoxins. When the nematode-induced
promoter is a pericycle-specific promoter, such as the
promoter of the gene corresponding to the cDNA of SEQ ID
no. 4 or 6, the first foreign DNA under the control of such
a promoter preferably encodes either: a) a material such as
callose or lignin which, when produced in the pericycle
cells, will make the pericycle substantially impenetrable
to nematodes, so as to prevent the nematodes from feeding
at the fixed feeding sites or establishing other fixed
feeding sites and thereby repel the nematodes from the
fixed feeding sites. Plants transformed w;th such a first
foreign DNA in a first chimaeric gene of this invention
will be resistant to nematode infections either because of
a nematode-induced breakdown of their fixed feeding sites,
which are essential for the survi~al of nematodes, or
because nematodes, feeding on the fixed ~eeding sites, will
be killed, repelled or disabled by, for example, a nematode
toxin produced in situ by their fixed feeding site cells.
Each of the nematode-induced promoters of this
invention, particularly the promoter of the gene
corresponding to the cDNA of SEQ ID no. 7, which can be
used to control expression of the first foreign DNA of this
invention substantiaily exclusively, preferably
W092/21757 PCT/EP92/0121~
f~ 9
16
exclusively, in the specific root cells, particularly fixed
feeding site cells, of a plant, and each of the nematode-
repressed promoters of this invention, which can be used to
control expression of the second foreign DNA of this
invention predominantly, preferably substantially
exclusively, in cells other than the specific root cells,
particularly cells other than the fixed feeding site cells
of a plant, can be identified and isolated in a well known
~anner in the specific root cells, particularly the fixed
feeding site cells, of the plant. For example, a suitable
nematode-induced or nematode-repressed promoter can be
identified and isolated in one or more plants, preferably
two or more plants (e.g., tomato and potato~, infected with
nematodes by the following process steps:
1. searching for an mRNA which is, respectively,
~ubstantially present or substantially absent in the
cells of the roots of the plant(s) after nematode
infection thereof by construction of a cDNA library
and differential screening;
2. isolating the cDNA that corresponds to the
nematode-responsive mRNA;
3. using these cDNA as a probe to identify the region~
in the plant(s) gPnome(s3 which contain DNA coding for
the nematode-responsive mRNA; and then
4. identifying the portion of the plant genome(s) that
is upstream ti.e.~ 5') from this DNA and that codes
for the nematode-responsive promoter of this DNA.
The nematode-responsive cDNA clones of step 3 of this
process can also be isolated by other methods ~Hodge et al,
1990). Examples of nematode-responsive ~romoters, which
W~09~21757 ~ 0~ PCT/EP92/01214
17
can be obtained by this process, are the promoters of this
invention which can be identified using the cDNAs of SEQ ID
nos. 1-8, particularly the nematode-induced promoters which
can be identified with the cDNAs of SEQ ID nos. 2-8 and the
nematode-repressed promoter which can be identified with
the cDNA of SEQ ID no.l. Certain of the nematode-induced
promoters of this inention, such as that which can be
identified with the cDNA sequence of SEQ ID no~ 6, causes
expression of the first chimaeric gene in all cells of a
nematode-infected plant, transformed with the first
chimaeric gene, but is believed to cauæe expression at
substantially higher levels in fixed feeding site cells.
For this reason, at least certain of the nematode-induced
promoters are preferably combined in the first chimaeric
gene with a first foreign DNA selected ~o that its
differential expression in the specific root cells,
particularly fixed feeding site cells (as compared to the
other cells of the infected plant), has the desired
selective effect on the specific root cells, preferably the
fixed feeding site cells. Other promoters of this
invention, such as those which can be identified by means
of the cDNA sequences of SEQ ID no. 4 and SEQ ID no. 6,
cause expression of the first foreign DNA predominantly in
pericycle cells.
When the nematode-induced promoter in the first
chimaeric gene of this invention is not l00% specifis for
the specific root cells, preferably the fixed feeding site
cells, of a plant transformed thsrewith, it is preferred
that the plant be further transformed so that its nuclear
genome contains, stably integrated therein, the second
chimaeric gene of this invention. The second promoter of
WO9~21757 ~ PCT/EP92/0l21~--
~ 18
the second chimaeric gene is selected so that it is capable
of directing transcription of the second foreign DNA to
provide sufficiently high expression levels of the second
RNA or protein or polypeptide to inhibit or preferably
inactivate the first RNA or protein or polypeptide in all
plant cells, with the exception of the specific root cells,
preferably the fixed feeding site cells. An example of the
second promoter is a nematode-repressed promoter of this
invention, such as the promoter of the gene which can be
identified with the cDNA of SEQ ID no. 1. Other examples
of second promoters are: the strong constitutive 35S
promoters of the cauliflower mosaic virus of iæolates CM
1841 (Gardner et al, 1981), CabbB-S (Franck et al, 1980)
and Cab~B-JI (Hull and Howell, 1987); and the TRl' and TR2'
promoters which drive the expression of the 1' and 2'
genes, respectively, of the T-DNA (Velten et al, 1984).
Alternatively, a second promoter can be utilized which is
specific for one or more plant tissues or organs, such as
roots, whereby the second chimaeric gene is expressed only
in cells of the specific tissue(s) or organ(s). Another
alternative is to use a promoter whose expression is
inducible (e.g., by temperature or chemical factors~. To
control root-knot nematodes, it may be preferred that the
second chimaeric gene be under the control of a gall-
specific promoter.
In accordance with this invention, the --econd foreign
DNA, controlled by the second promoter, encodes a second
RNA and/or protein or polypeptide which, when produced or
overproduced in cells of a plant, inhibits or preferably
inactiva~es the first RNA, protein or polypeptide in such
cells. Second foreign DNAs preferably encode, for example,
'VO9V2l757 ~ PCT/EP92/01~14
the following: barstar which neutralizes the activity of
barnase (which degrades RNA molecules by hydrolyzing the
bond after any guanine residue); EcoRI methylase which
would prevent the activity of the endonuclease EcoRI; or a
protease inhibitor which would neutralize the activity of a
protease, such as papain (e.g., papain zymogen and papain
active protein). Another preferred example of a second
foreign DNA is a DNA which encodes a strand of antisense
RNA which would be complementary to a strand of sense first
RNA.
In the first and second chimaeric genes of this
invention, 3' transcription termination signals or 3'ends
can be selected from among those which are capable of
providinq correct transcription termination and
polyadenylation of mRNA in plant cells. The transcription
termination si~nals can be the natural ones of the first
and second foreign DNAs, to be transcribed, or can be
foreign. Examples of foreign 3' transcription termination
signals are those of the octopine synthase gene (Gielen et
al, 1984) and of the T-DNA gene 7 (Velten and Schell,
1985)~-
The cell of a plant, particularly a plant capable ofbeing in~ected with A~robacterium, can be transformed using
a vector that is a disarmed Ti-plas~id containing the first
chimaeric gene and optionally the second chimaeric gene of
this invention and carried by A~robacterium. This
transformation can be carried out using the procedures
described, for example, in EP 116,718 (29 August 1984), EP
270,822 (15 June 1988) and Gould et al (1991) [which are
also incorporated herein by reference]. Preferred Ti-
plasmid vectors contain the forei~n DNA sequences between
WO 92t21757 PCI/EP92/0121~*~
the border sequences, or at lea~t located to the left of
the right border sequence, of the T-DNA of the Ti-plasmid.
of course, other types of vectors can be used to transform
the plant cell, using procedures such as direct gene
transfer (as described, for example, in EP 233,247),
pollen-mediated transformation (as described, for example,
in EP 270,356, PCT publication WO 85/01856, and US patent
4,684,611),plant RNA virus-mediated transformation (as
described,for example, in EP 67,553 and US patent
4,407,956)and liposome-mediated transformation (as
described,for example, in US patent 4,536,475). In case
the plantto be transformed is corn, rice or another
monocot, it is preferred that more xecently developed
methods be used such as, for example, the methods described
for certain lines of corn by Fromm et al (l990) and
Gordon-Xamm et al (l990), the methods described for rice by
Datta et al (l990) and Shimamoto et al tl9B9) and the more
recently described method for transforming monocots
generally of PCT patent appli ation no~ PCT/EP 9102198.
The first and second chimaeric genes of this invention
are preferably inserted in the same genetic locus in the
plant genome. Therefsre, it is preferred that the first
and second chimaeric genes be transferred to the plant
genome as a single piece of DNA, so as to lead to their
insertion in a single 1DCUS in the genome of the plant.
However, plants containing the two chimaeric genes can also
be obtained in the following ways:
1. The chimaeric genes can be separately transferred
to the nuclear genomes of separate plants in
independent transformation events and can subsequently
be combined in a sin~le plant genome throus~h crosses.
~vog~21757 ( i ~ B 1 ~ 3 PCT/EP92/01214
2. The chimaeric genes can be separately transferred
to the genome of a single plant in the same
transformation procedure, leading to the insertion of
the respective chimaeric genes at multiple loci
(cotransformation).
3. One of the two chimaeric genes can be transferred
to the genome of a plant already transformed with the
other chimaeric gene.
The resulting transformed plant can be used in a
conventional breeding scheme to produce more transformed
plants with the same characteristics or to introduce the
first chimaeric gene and optionally the second chimaeric
gene in other vzrieties of the æame or related plant
species. Seeds obtained from the transformed plants contain
the chimaeric gene(s) of this invention as a stable genomic
insert.
The Examples, which follow, describe the isolation and
characterization of nematode-responsive cDNA sequences of
this invention of SEQ ID nos. 1-8 and their use as
molecular pro~es for isolating and identifying t~e
corrssponding genomic sequences. Once the corresponding
genomic sequences have been identified, the promoter
regions are isolated according to well-known methods as
described, for example, in European patent applications
~"EPA") 89401194.9 and 90402281.l.
Unless stated otherwise in the Examples, all nucleic
acid manipulations are done by the standard procedures
described in Sambrook et al, Molecular Cloninq A
aboratorY Manual, Second Edition, Cold Spring Harbor
Laboratory Press, N.Y. (198g). Oligonucleotides are
WO9V217~7 PCT/EP92/0121~-
2~
designed according to the general rules outlined by Kramer
and Fritz (1988) and synthesized by the phosphoramidite
method (Beaucage and Caruthers, 1981) on an Applied
Biosystems 380A DNA synthesizer (Applied Biosystems B.V.,
Maarssen, Netherlands).
In the following Examples, reference is made to the
following Sequence Listing (SEQ ID nos. 1-8~ :
SEQUENCE LISTING
SEQ ID no. 1 : LEMMI 1 cDNA (Between brackets)
SEQ ID no. 2 : LEMMI 2 cDNA (Between brackets)
SEQ ID no. 3 : LEMMI 4 cDNA (Between brackets)
SEQ ID no. 4 : LEMMI 7 cDNA (Between brackets)
SEQ ID no. 5 : LEMMI 8 cDNA (Between brackets)
SEQ ID no. 6 : LEMMI 9 cDNA (Between brackets)
SEQ ID no. 7 : LEMMI 10 cDNA (Between brackets)
SEQ ID no. 8 : LEMMI 11 cDNA (Between brackets)
ExamPle 1 : ISOLATION AND CHARACTERIZATION OF NEMATODE-
RESPONSIVE cDNAs FROM TOMATO
Young tomato plants (LvcoPersicon esculentum cv.
Marmande) were each grown at 20- C in industrial pots under
semi-sterile c,onditions in a 1:1 sand:soil mixture, whieh
was ~terilized by irradiation and watered daily with a
filtered sterilized nutrient solution (Cooper, 1976).
Plants were infected by inoculation with about 6,000
MeloidoaYne incoqnita race 1 eggs per pot~ The nematode
~noculum was obtained as described by Hussey and Barker
(1973). Infected and control plants were grown under
identical conditions. Five weeks after inoculation, plant
m~terial was harvested from both infected and control
plants, frozen under liquid nitrogen and stored at - 80C
for further processing. Total RNA was prepared from frozen
92/2l757 ~ PCT/EP92/01214
tissue (-70- C) according to Jones et al (19B5). Poly
(A)~RNA was isolated by oligo - dT cellulose affinity
chromatography as described by Slater (1984).
In order to construct a c~NA library from infected
tomato plants, mRNA was extracted from hundreds of
MeloidooYne incoqnita race 1 induced root-knots. cDNA was
~ynthesized using the cDNA synthesis system plus (Amersham
Intl. PLC, Buckinghamshire, England). The cDNA library was
constructed in plasmid pUCl9 (Yanisch-Perron et al, 1985)
which was electroporated in E. coli. About 3,000 randomly
selected clones were individually grown in the wells of
microtiter plates containing LB medium (Niller, 1972)
~upplemented with 100 ~g/ml ampicillin. Replicas of the
~ublibr~ry were made with a replica block on Hybond N nylon
membranes (Amersham) which were further treated according
to Sambrook et al (19891.
First strand cDNAs, reverse transcribed from total RNA
of root-knots and of control roots, were used as probes for
differential screening~ To this end, reproducible replicas
of 3,000 individual cDNA clones were hybridized overnight
at 68-C with 32P-labeled probes. Subsequently, the
hybridization patterns obtained with cDNA probes fr~m
root-knots were compared to those obtained with cDNA probes
from ~ontrol roots. Ninety-three ~93) clones gave a
stronger hybridization signal with the "infected" probes
than with the "control" probes. These clones were labeled
as "nemat~de-stimulated" or "nematode-induced" clones and
were subjected to a second screening. Several clones gave a
weaker hybridization signal with the "in~ected" probes than
with the "control" probe, and one of these clones was
W092/21757 PCT/EP92/0121
~ 24
labeled as a "nematode-repressed" clone and subjected to a
second screening.
The one "nematode-repressed" and eight (8) of the
"nematode-stimulated" LEMMI (Lvcopersicon esculentum cv.
Marmande - MeloidoqYne incoqnita race l) cDNA clones showed
pronounced differential hybridization patterns and were
selected for further analysi~. The following cDNA clones
showed a nematode-stimulated hybridization pattern: LEMMI
2, LEMMI 4, LEMMI 6, LEMMI 7, LEMMI 8, LEMMI 9, LEMMI lO
and LEMMI ll. The following cDNA clone showed a nematode-
repressed hybridization pattern: T-~NMI l~ Cross-
hybridization performed under high stringency conditions
showed that LEMMI 6 and LEMMI 9 most likely correspond to
the ~ame mRNA.
The different cDNA clones were sequenced. According to
a database search, LEMMI 8 and LEMMI ll appeared to be an
extensin. Southern blot analysis of toma~o DNA performed
under high stringency conditions pro~ed the plant origin of
these clones. Since root-knots contain nematodes, some of
the nematode-stimulated clones could have been from
nematode origin. Southern blot and northern blot analysis
further showed that _EMMI 4 and LE~MI 8 belong to different
multigene families. The differential hybridization patterns
of the cDNA clones were confirmed by Northern blot
analysis. In situ hybridization experiments on tissue
sections of nematode-infected _n vitro grown tomato plants
using ~EMMI 7, LEMMI 9 and LEMMI lO as probes showed that:
both LEMMI 7 and LEMMI 9 are predominantly expressed in
peri~ycle cells and T~MMI I0 has high specificity for fixed
feeding site cells.
W09~21757 ~ .; 0 1 ~ 3 PCT/EP92/01214
Exa~Dle 2 : ISOLATION AND CHARACTERIZATION OF NEMATODE -
RESPONSIVE eDNAs FROM POTATo
Potato plants (SolAnum tuberosum cv Bintje) were
infeeted by inoeulation with the potato eyst nematode,
Globodera Dallida. Infected and control plants were grown
under identical conditions. Eight weeks after inoeulation,
infected roots were harvested, and RNA was prepared as
descr~bed in Example l. S ~g of poly (A)~RNA was used as a
starting material for the construction of a cDNA library. A
cDNA library of 40,000 reeombinant elones was obtained
a~ter l~gation in the plasmid vector pUCl8 (Norrander et
al, 1983) and electroporation in E. eoli. 3,700 of these
clones were isolated and grown in mierotiter we~ls.
Sub~-qu-ntly, tbese clones were ~ubjected to a differential
screening procedure as described in Example l to identify
n~Jatode-repre~sed cDNAs and nematode-stimulated eDNAs of
pot~to.
E1ou~Dle 3: ID~NTIFICATION AND~CHARACTERIZATION OF OTHER
EMATODE-RESPONSIVE GENES FROM~PLANTS
For the purpose of identifying pl~nt genes whieh ,are
~indueed by-nematodes, tobaeeo and Meloidoqvne iavaniea were
u~ed as a model system. An in vitro system has been
developed whieh allows synehronized infeetion by a number
of nematodes and immediate anaIysis of resulting proteins.
The system used in vitro grown SRl tobaceo plants as a
startinq material.
Explants eonsisting of an internode and a leaf were
eut off tobaeco plants. The explants were then put into
Petri dishes (13.5 em diameter); which eontain the normal
eulture medium~ used for SRl~tobaeeo plants. The explants
started rooting after about 5 to 7~days. After lO days, the
.., " ~ "
~,. .~ ,
.~ f.~~~f ---~r.~.~ 7~~ 7~f~ 7,~ ,r~ .s~ r~Q~ f~.3.-rrrr=.. r~ S.,: ~:'':~~'~'
WO92/21757 PCT/EP92/012~
~ ~ lJi~9 26
roots were infected in the following way: the culture
medium is carefully lifted and a solution containing
approximately 1000-2000 nematode larvae (2nd larval instar)
is added. This in vitro system had several advantages,
including the synchronicity of the infection, the easy
scoring and the possibility of stage-specific observations.
Several pathogen-induced and pathogenesis-related proteins
were tested using this system. A very strong induction of
extensin (8-fold higher than in control roots) was
observed. Subsequently, the promoter of the unique extensin
gene of Nicotiana Plumbaqinifolia (De Loose et al, 1991)
was fused to a reporter gene, B-glucuronidase (Jefferson et
al, 1986), and transformed into tobacco. These transformed
plants were analysed by means of histochemical A-
glucuronidase assays (Peleman et al, 1989). A very
localized and strong Gus-activity was observed around the
fixed feeding sites.
EXAMPLE 4 : ISOLATION OF NEMATODE-RESPONSIVE GENES
CORRESP9NDING TO THE NEMATODE-RESPONSIYE cDNA CLONES OF
EXAMPLES 1 AN~ 2
In order .to isolate the genomic DNA clones carrying
the regulatory sequences of the genes corresponding to the
~elected cDNA clones of Examples 1 and 2, ~ genomic library
is constructed. To this endl total genomic DNA of tomato is
digested with a tetra-cutter restriction enzyme in order to
obtain approximately 20 kb DNA fragments. These genomic DNA
fragments are then cloned in the phage vector, Charon 35
(Rimm et al, 1980).
The nematode-responsive cDNAs of Examples 1 and 2 are
used as probes for screening the library. Genomic clones,
which hybridize to the probes, are selected and sequenced.
wog~21757 2 1 1 0 1 6 ~ PCT/EP92/01214
Comparison of the sequences from the cDNA clones of this
invention with those of the genomic clones leads to the
identification of the homologous regions. At the 5' end of
the homologous region of each genomic clone, the ATG
translation initiation codon and TATA consensus sequence
are identified in order to locate the nematode-responsive
promoter region. The fact that the "TATA-box" is part of
the promoter region is confirmed by primer extension.
Confirmation of the nematode-responsive promoter
regions is made by use of the "inverse PCR" technology as
described by Ochman et al (1988, l989). By this method, the
DNA sequences flanking a well-defined core region of each
nematode-respor.sive gene sequence, which corresponds to the
seguence of a nematode-responsive cDNA, are amplified.
ExamPle 5 : CONSTRUCTION OF NEMATODE-RESPONSIVE PROMOTER
CASSETTES DERIYED FROM THE NEMATODE-RESPONSIVE GENES OF
EXAMPLE 4
The 5' regulatory sequences, including the nematode-
responsive promoter, of each of the nematode-responsive
genes of Example 4 are subcloned into the polylinker of
pMAC 5-8 (EPA 87402348.4). This produces vectors which can
be used to isolate single stranded ~NA for use in site-
directed mutagenesis. Using site-directed mutagenesis
(EPA 87402348.4), sequences surrounding the ATG translation
initiation codon of the 5' regulatory sequences of each of
the nematode-responsive genes are modified to create a
unique recognition site for a restriction enzyme, for which
there is a corresponding recognition site at the 5' end of
the first foreign DNA of this invention (that is to be
fused to the 5' regulatory sequences in Example 6, below).
The resulting plas~ids each contain the newly created
WO92/21757 PCT/EP92~0121~
` ' J ll ~ 69
28
restriction site. The precise nucleotide sequence spanning
each newly created restriction site is determined in order
to confirm that it only differs from the 5' regulatory
sequences of the corresponding nematode-responsive gene by
the substitution, creating the new restriction site.
ExamDle 6 : CONSTRUCTION OF PLANT TRANSFORMATION VECTORS
FROM THE PROMOTER CASSETTES OF EXAMPLE 5
Using the procedures described in EPA 89401194.9 and
90402281.2, the promoter cassettes of Example 5 are used to
construct plant transformation vectors comprising first
chima,eric genes of this invention, each of which contains
the 5' regulatory sequences, including the nematode-
responsive promoter, of one of the nematode-responsive
genes isolated in Example 4. Each of these 5' regulatory
~equences is upstream of, is in the same transcriptional
unit as, and controls a first foreign DNA (from EPA
89401194.9) encoding barnase from Bacillus
am~loliauefaciens (Hartley and Rogerson, 19~2). Downstream
of the first foreign DNA is the 3' end of the octopine
synthase gene (Gielen et al, 1984). Each chimaeric gene
also comprises the 35 S'3 promoter (Hull and Howell, 1987)
fused in frame with the neo gene encoding kanamycin
resistance (EPA 84900782.8), as a marker, and $he 3' end of
the octopine synthase gene.
ExamPle 7 : TRANSFOR~ATION OF TOMATO AN~ PO,TATO WITH THE
PLANT TRANSFORMATION Y~CTORS OF EXAMPLE 6
To obtain transformation of, and major expression in,
tomato and potato by the plant transformation vectors of
Example 6, each vector is inserted between the T-DNA border
sequences of a Ti-plasmid carried by Aqrobacterium
~EPA 89401194.9 and EPA g0402281.1). In this regard, the
`~09~21757 ~ PCT/EP92/01214
29
vectors from Example 6 are each mobilized into
Aqrobacterium tumefaciens C58Cl RifR containing pMP90
(Koncz and Schell, 1986). The resulting recombinant
Aarobacterium strains are used to transform tomato leaf
discs using the standard procedures described in
EPA 87400544Ø The result ng recombined Aqrobacterium
strains are also used to transform potato plants (Solanum
tuberosum cv. Bintje) by means of tuber disc infection as
described by Deblock et al (1987). Transformed calli are
selected on a substrate containing lO0 ~g/ml kanamycin, and
resistant calli are regenerated into plants.
Plants transformed with the nematode-induced chimaeric
genes of this invention containing nematode-induced
promoters, partic~larly the nematode-induced promoters of
Ex~mple 4 identified with the cDNAs of SEQ ID nos. 2-8,
qulte particularly the promoter identified with the cDNA of
SEQ ID no. 7, show a significantly higher degree of
resistance to s~dentary endoparasitic nematode infection,
such as Meloidoavne incoqnita infection, than do non-
transformed control plants. As a result, the transformed
plants have significantly lower yield losses than do the
control plants.
Needless -to say, the use of the nsmatode-responsive
promoters of this invention is not limited to the
transformation of any specific plant(s). Such promoters can
be useful in transforming any crop, such as rapeseed,
alfalfa, corn, cotton, sugar beets, brassica vegetables,
tomato, potato, soybeans, wheat or tobacco where the
promoters can control gene expression, preferably where
such expression is to occur abundantly in specific root
cells, preferably in fixed feeding site cells.
WO92~21757 PCT/EP92/0121~
21iOi~ 30
Also, the use of the nematode-responsive promoters of
this invention is not limited to the control of particular
foreign DNAs but can be used to control expression of any
gene or DNA fragment in a plant.
Furthermore, this invention is not limited to the
specific nematode-responsive, preferably nematode-induced,
promoters described in the foregoing Examples. Rather,
this invention encompasses promoters equivalent to those of
the Examples which can be used to control the expression of
a structural gene, such as a first foreign DNA, at least
substantially selectively in specific root cells,
preferably fixed feeding site cells, of a plant. Indeed,
it is believed that the DNA sequences of the promoters of
the Examples can be modified by replacing some of their
codons with other codons, provided that such modifications
do not alter su~stantially the ability of polymerase
complexes, including transcription activators, of specific
root cells, particularly fixed feeding site cells, to
recognize the promoters, as modified.
WO9~2175~ 6 9 PCT~EP92/01214
REFERENCES
BEAUCAGE AND CARUTHERS, Tetrahedron Letters 22, 1859
(198~).
BROWN, R.H. and KERRY, B. eds. Principles and Practice
of Nematode Control in Crops. Academic Press, N.Y.
(1987).
BUNT, J.A. Mode of Action of Nematodes. pp. 461-468 in
J.A. Veech and D.W. Dickson, eds. Vistas on
Nematology. Society of Nematologists (1987).
COOPER, in : Co~position of Nutrient Solution for
Tomato and Butterhead Lettuce (1976).
DATTA ET AL, Bio/Technology 8, 736 (1990)
DE BLOCK ET AL, EMBO J. 6, 2513-2518 (1987).
DELLAPORTE ET AL, Plant Molecular Biology Reporter I,
19-21 (1983).
DE LOOSE M., GHEYSEN G., TIRE C, GIELEN J., VILLAROEL
R~, GENETELLO C., VAN MO~TAGU M., DEPICKER A. and INZE
D., Gsne 99, 95-100 (1991).
DROPKIN, Introduction to Plant Nematology, Wiley &
Sons, NY, p. 293 (1980).
~NDO, B.Y, Histopathology and Ultrastructure of Crops
Invaded by Certain 5edentary Endoparasitic Nematodes.
pp. 196-210 in J.A. Veech and D.W. Dickson, eds.
Vistas on Nematology. Society of Nematologists (19%7).
FRANCK, GUILLEY, JONARD, RICHARDS and HIRTH, Cell 21,
285-294 tl980).
~ROMM M., MORRISH F., ARMSTRONG C., WILLIAMS R.,
THOMAS J. and KLEIN T., Bio/Technolo~y 8, 833-839
(1990) -
WO9~21757 PCT/EP92/0121~
2 1 1 ~ 32
GARDNER, HOWARD, HAHN, BROWN-LUEDI, SHEPARD and
MESSING, Nucleic Acids Research 9, 2871-2887 (1981).
GIELEN J., DE BEUCKELEER M., SEURINCK J., DEBOECK F.,
DE GREVE H., LEMMERS M., VAN MONTAGU M. and SCHELL J.,
E~BO J. 3, 835-845 (1984).
GORDON-KAMM W., SPENCER M., MAGANO M., ADAMS T.,
DAINES R., START W., O'BRIEN J., CHAMBERS S., ADAMS
W., WILLETS N., RICE T., MACKEY C., KRUEGER R., KAUSCH
A. and LEMAUX P., The Plant Cell 2, 603-618 (1990).
GOULD ET AL, Plant Physiology 95, 426-434 (19gl).
HARTLEY AND ROGERSON, Preparative Biochemistry 2,
243-250 (1972).
HODGE ET AL, Glasgow Symposium, p. 33 (Sept. 1990~.
HULL and HOWELL, Virology 86, 482-493 (1987).
HUSSEY R. and BARKER K., Plant Dis. Reptr. 57,
1025-102~ (1973).
JEFFERSON R.A., BURGESS S. and HIRSH D., Proc. Natl.
Acad. Sci. U.S.A. 83, 8447-8451 (1986)~
JONES J., PUNSMUIR P. and BEDBROOK J., EMBO J. 4,
2411-2418 (1985).
JONES, M., The Development and Function of Plant cells
Modified by Endoparasitic Nematodes, pp. 225 279 in
ZUCKERMAN B. and ROHDE R., eds. Plant Parasitic
Nematodes, vol. III, Academic Press, N.Y. (1981).
KONCZ and SCHELL, Mol. Gen. Genetics 204, 383-396
(1986).
KRAMER and FRITZ, Methods in Enzymology 154, 350
(1988).
MILLER J.H., Experiments in Molecular Genetics, N.Y.,
Cold Spring Harbor La~oratory Press, N.Y., p. 466
(1972).
~"092t21757 2 ~ 1IJ1 ~ PCT/EP92/01214
MORTON, H.V. Industry Perspectives in Nematology. pp.
47-51 in J.A. Veech and D.W. Dickson, eds. Vistas on
Nematology. Society of Nematologists (1987).
NORRANDER ET AL, Gene 26, 101-106 (1983).
OCHMAN H., AJIOXA J.W., GARZA D. and HARTL D.L.,
Inverse PCR, in Erlich A., ed. PCR Technology,
Principles and Applications for DNA amplification, M.
Stockton Press, N.Y., (1989).
OCHMAN H., GERBER A.S~ and HARTL D.L., Genetics 120,
621-623 (1988).
PELEMAN J., BOERJAN W., ENGLER G., SEURINCK J.,
BOTTERMAN J., ALLIOTTE T., VAN MONTAGU M. and INZE D.,
The Plant Cell 1, 81-93 (1989).
RIMM D., ~ORNERS D., KUCERA J. and BL~TTNER F., Gene
12, 301-309, (1980).
SASSER, J.N. and FRECKMAN, D.W., A Warld Perspective
on Nematology: the Role of the Society. pp. 7-14 in
J.A. Veech and D.W. Dickson, eds. Vista~ on
Nematology. Society of Nematologists (1987).
SHIMAMOTO ET AL, Nature 338, 274 ~1989).
SLATER R., The Purification of Poly(A)-containing RNA
~y Affinity Chromatography. pp~ 117-120 ln: Methods in
Molecular Biology, Vol. 2, J.M. Walker, ed. Humana
Press, Clifton (1984).
THOMASON, I.J., Challenges Facing Nematology:
Environmental Risks with Nematicides and the Need for
New Approaches. pp. 469-476 in Veech and D.W. Dickson,
eds. Vistas on Nematology. Society of Nematologists
(1987).
VELTEN J. and SCHELL J., Nucleic Acids Research 13,
6981-6998 (1985).
WO 92~21757 PCI`/EP92/0121~
9 34
- VELTEN J., VELTEN L., HAIN R. and SCHELL J., EMBO J.
3, 2723-2730 (~984).
- YANISCH-PERRON C ., VIERRA J . AND MESSING J ., Gene 3 3,
103-119 ( 1985 ) .
~0 92~2175~ 2 ~ PCT/EP92/01214
SEQUE~CE LISTING
1. G~er~ or~-tlo~
i) APPLICANT : PLAN~ GENETIC SYSTEM N.V.
ii) TITLE OF INVENTION: N~natode-Resp~nsive Plant Pr~ters
iii) NUMBER OF SEQUENCES: 8
iv) CO~RESPONDENCE ADDRESS;
A. ADI)RESSEE: Plant Genetic Systems N.V.
B. STRE~? : Plateaustraat 22,
C. POSTAL CODE AND CSTY : 9000 Ghent,
D. COUNTRY : Belgium
v) CCMPUT~ READA8LE ~ORM :
A. MEDIUM TYPE 5.25 inch, double sided, hig~ density
1.2 Mb floppy d~sk
a. COMPV?ER : IBM PVAT
C. OPE~ATI~G SYSTEM : DOS version 3.3
D. SOFTW ME : WordPerfect 5.1
vi) C~RRE~T APPLIC~SION DATA : Not Available
Ivii) PRIOR APPLICATION DATA : EPA 914014Zl.2
W09~217S7 ~ 7~ ~1 6 9 36 PCT~EP92/0121
SEQ ID NO. l
SEQUENCE TYPE: nucleotide
SEQUENCE LENGTH: 1193 bp
STRANDEDNESS: double-stranded
TOPOLOGY: linear
MOLECULAR TYPE: cDNA to mRNA
ORIGINAL SOURCE: pl~nt
ORGANISM: to~to
PROPERTIES: Ne~tode-re~pon~ive cDNA ~
MIS OE LLANEOUS: cDNA desinated ~s LEMMI 1
TGTGNGTTGC TCNCTCNTTG GCNCNCCGGC TTTNCNCTTT NTGCTTCCGG 50
CTCGTNTGTT GTGTGGNNTT GTGTGNGNTT NNCNNTTTCN CNCNGGNNNC lOO
GCTATGNCCA ,TGATTACGCA AGCTTGCATT CCTGCAGGTC GACTCTAGAG l50
GATCCCCGGG TACC~aGCTC GCCATGGTAG GCGGATC~TC GAATTCGAGG 200
ATCCGGGTAC Q TGqAGAAA CTAGAACTCA AATACCAAGA GTCAG m CT 250
GAAGAATATC AATG~GTCA CTTCAGTGCA AAAGCCAGTT GCACAACCAA 300
CTGTCTGCCA AAAAACCACC AACACTGTCA CTTGCCACAA GGCGAATGAG 350
CACCAC$TGG CTGACAAAAT GAAACAGATG ACAACCAAGA TGTTGCACCA 400
CGACAACCAT~ GGTCAGCAAT CTGCCTGCCA TGGAGCCAAA ACTCAACATT 450
CAGCAGGCCA TGGCTCCACT GCTAT,TCACG GAAATCATGG TAGCCACTGC soo'
CAGCAGACTG CATGCCATGG TGCAAAAACT CAACATTCAG CATGCCATGG 550
CTCCACTGCT ATGCATGGAA ATCATGCTAA GGGACACAAC ACTGCATGCC 600
: AS Q SGCSAA ANCTQ A Q S TCAGCNGGCC ATGGCTCCAC TGTTGGGCAT 650
GGAAATCATG:CCAAGG`GCAC AACNCTGCAT GCCATGGMAC CAAAWCTCAA 700
QTTCAACAS GCCATGGTCC ACCGCTACTC ATCGAAAYAT GCTAWKN.NNA 750
GA,TNACTGNA:TGCATGGCAN NAAAACTCAA NATTCAANAG GCCATGGCTC 800
QCTGCTATG~CATGGAGN~T ATGCTAACCA~CGGACAG~AC AC~GCAAGTC 850
ATGGCTCCAC TGCTGTGCAT GGAAGTCATG GTAGCCACAA CCAGCAGACT 900
GTGTCCAT~GG:CTCAAAGAAG GAAGGGGGCA TCATGCATAA GATAGGTAGT 950
CAGC~GAAGA:CCA,TCGGGAA AAAGAAGAAC AAAGATGGAC ACTGCAGAGA Iooo
TGGCAGTGAC AGCAGCGACA GCAG AGCAG CAGCGATGAT GAGAGCGACA 1050
ATGAGAhTTG'TGGAAAAAAA ~ CATG GTACCCGGAT CCTCGAATTC llOO
ACTGGCCGTC GT m ACAAC TTCGTGACTG GGAAANCCCT NGCGTTACCC 1150
AACTTAATNG CCTTGCNGCA CATCCCCCTT NNNCCAGCTG GGT 1193
`"09~21757 ~ . PCT/EP92/01214
37
SEQ ID NO. 2
SEQUENCE TYPE: nucleotide
SEQUENCE LENGTH: 305 bp
STRANDEDNESS: double-stranded
TOPOL0GY: linear
MOLECULAR TYPE: cDNA to mRNA
ORIGINAL SOURCE: pl~nt
ORGANISM: tomato
PROPERTIES: Nematode-responsive cDNA
MISCELLANOUS: cDNA designated ~s LENMI 2
~GAATGNNNNN NNGGGGNNNN NNAAGNGNNA AAGNNNAAAG GAGAGAAAGA 50
AGTCAAAG NGGAGTCAGN AGAAGAGAAG GATATTGNNN AAGNNNAGAA l00
GGATAAAGAG AAGAAAGACA AGAAAAAAGG GGCATCAGAC GAAGAGAACG 150
AGCGCGAAGA AGAGAATGAT GAAAAAGGTG TGAAAAAAAA AAAAAA~CCA 200
TGGTACCCGG ATCCTCGAAT CGAGGATCCG NNNNNNATNG CGAGCTCGGT 250
ACCCGGGGAT CCTCTAGAGT CGACCTGCAA ACATGCAAGC TTGGNGTAAT 300
CATGT
wos~217S7 PCT/EP92/01214
211~ 38
SEQ ID NO 3
SEQ~ENCE TYPE: nucleotide
SEQUENCE LENGTH: 543 bp
STRANDEDNESS: double-stranded
T~POLOGY: linear
MOLECULAR TYPE: cDNA to mRNA
ORIGINAL SOURCE: pl~nt
ORGANISM: tom~to
FEATVRES: Nuc~eotide l to 64: cloning vector sequence
Nucleotide 6S to 88: cloning adaptor sequence
PROPERTIES: Nematode-responsive cDNA
MISCELLANEOUS: cDNA designated as LEMMI 4
ACATGATTAC NCCAAGCTTN CATGCCTGCA GGTCGAC~CT AGAGGATCCC 50
CGGGTACGAG CNCNAATTCG AGGATCCGGG TACA ~ TG GTTTTCTACG l00
AAATATA m TAGTAAAGCA A m TTAGAA A MCAA~FGAA GTAGAGATAC l50
X ~CCGAG~T AAAGTM TTT CATTAACTCA TCITTnGAAA CTCACAAAGC 200
TTATG~rl~ A ACAAGCATCA GACAGACATA AAGCNCGAAG G~TTrAT~TC 250
TAAAGGGAAA CAGCCCAGAT GAATAGATAA GGTCCCAAAT AG~TATTAAA 300
M TAAAAT M GTT~TCA~TT AAAAGATTAA AAGGTATGCT TGZAAGCAGC 350
CAGCCTTT~A AGAAAGCGTT GCAGCTCATT AATTGAATAA TAANAACTTA 400
TTGAGAATr.T ACGGGAATGA M TAACTCAC AAAAAACCAA CCGAA~C m 450
CAATATG~-.~ ATAAAAAGTA TGTCATATGG TAGTAGAATA TNTTGTCANA S00
AAAATA~;C N ~AGAGAAA ATCTNTTGGT ATAAGTAGCG AA~ 543
WO9~217~ PCT/EP92/01214
~ 2~lOlG~
SEQ ID NO.4
SEQVENCE TYPE: nucleotide
SEQ~ENCE LENGTX: 482 bp
STRANDEDNESS: double-str~nded
~DPOLOGY: line~r
MK~LECULAR qYPE: cDNA to m~NA
ORIGINAL SOURCE: pl~nt
ORGANISM: tomat~
FEATURES: Nucleotide 1 to 8: c~oning vector sequence
Nuc~eotide 9 to 32: cloning adaptor sequence
Nucleotide 33 to 482: putative Open Reading F~ame
~-ORF~)
PROPERTIES: Nematode-responsive cDNA
~ISCELLANEOVS: cDNA designated as LEMMI 7
GGCCA ~ A Tq~-GAGGATC CGGGTACCAT ~ TTATTC TCAACCAATG 50
GælGAAAAAA TCAAAGSqGA AGGAGCAGAG AA~AAGAACG AAAGTTCAAT l00
qGTlqTAA~A ClGGATTTGC ATTGTGAAGG ~TGTGCACAA AAAC~CAGAC lS0
GATTCATTCG CCATACTCAT GGIGIGGAAA AAGTGAAATC GGA~TGTGAA 200
ACTGGAAJU~C T~AC ~ TA~ AGGTGACGTT GACCCTlCAT GGCTCCGGGA 250
GAGAGTGG~G ATCAAAACCA AAAAGAAGGT GGAGCTTATA lCATCGC~-GC 300
CCAAAAAGr~A CNCCGGAGAT AAAAAGAGCG GCGGAGATAA AAAGTCGGlG 350
AAAAAACA-~A GGACAAGAAG GAAGACGAGA AGAAACOCAA AGAGGCTCAA 400
GTAACAGT S GGTGGCA~TA AAGATTCGGG ~T~GTGA~ GG~TGTGCAC 450
ATAAAATC;~ ACGAGTTATT AAAAAGAT~A A~ 482
WO9~21757 PCT/EPg2/01214
h 11(31
SEQ. ID. NO. 5
SEQUENCE TYPE: nucleotide
SEQUENCE LENGTH: 2610 bp
STRANDEDNESS: double-stranded
TOPOL0GY: l~near
MOLECULAR TYPE: cDNA to mRNA
ORIGINAL SOURCE: plant
ORGANISM: tom~to
FEATURES: nucleotides 1878 to 2434 constitute nematode-
re~pon~-ive cDNA
PROPERTIES: Ne~tode-responcive cDNA
MISCELLANEOUS: cDNA de~ignated ~ LEMMI 8
A~AACAATTT CACACAGGAA ACAGCTATGA CCGATGATTA CGCCAAGCTT 50
GCATGCCTGC AGGTCGACTC TAGAGGATCC CCGGGTACCG AGCTCGAATT l0
CGAGGA~CCG GGTACCATGG ACTCCGAAAA TAT~CAGGAAT AGCAGTGAAT l50
CGAGCGGGAG GGCGAGCGAG TAAGCGAGAT CGGAGATCGG AAGATGTCGT 2~0
CGGAACCACC GCCATTTCAA GAAGCTTCAC GTTGTGAT~T CTGCAATTGC 250
AGCTTCAA~A CTTTCCGGCG ACGGCACCAT TGCAGATGTT GCGGCCGAAC 300
~TTATGTGCT GAACATTCAG CAAATCAGAT GGCCT~GCCA CAATTTGGTC 350
TTCACTCA~G TGTGAGAGTT TGTGGAGATT GTTTTAATAA CTCCTCTCGG 400
TAAATTTC~G CCACTATTAT CAATGATATA TCGTATACTT CTCAATAT~G 450
ATGCCAA~:A TGCT~TTTAT TTTGCCCAAA TTCTAAAGTC AGAGATGCTG 500
TCATAGAG T ATGAGGAATT TATTCTTGGA ATTCCATCTT CTCTCACCCT 550
ATTCAATC.T AACCTCC M T TTTTCAGTCA TTAAGAATCT CTTrTTGTAT 600
GACAAAG~ ACCCGCAGCC GCTACCC m GGG~CACAC AAGGCAAGTC 650
ATTAAGA~X r~TTATCTAG GAAGTAATAT TATACTCTTA TTAGCTCA~G 700
GGTGATT~. AACTAGTACA TGAAAGACAA AAAAACTGCC ATCAAGCAGG 750
AAACACTT-A ATATATAAAC ACTGTTCTCA AAACTAAAAT AATAATAAAT 800
AAAAACCC~T GAAAATGTTG AAGCCCTTAT ~GCTTCATCA ~ AGAA '850
~AGAAGCT~G AGAGTAGCAT CCTCCCT~AT TTGTT~AAGA ATGTAGGGAG 900
TTGGAAACA~ TCTGAAGTGC T~GTGATACC ATCTGTAAGC ACTTGGATTC g50
AGGACAT~C AGAAATGGGT AAATTTTGAA AAGTAGCCAG TTrTTGCTCT l000
GT~TGG~G~T TCAT~GATCG ~AGGATCATC ATATrTCAGT ACTGCAATAT 1050
GTTGGAA~.~G TA~TACTGTT TCATCTTTGA GGTTTGAGGC CATAGATTTA ll00
GTGCCCG~G GATTAAC~CA CTTTTAGGTG CTTTTGTCTT TTAAGCA m ll50
TA~AAGTT~T GGAGGTGTTT GGAAAGGTTA AAAAGTGCTT CTAAGCATTC 120~
- ACT~TG~C CAAAAAAGTT TTAAAATAAG qCAAAAGTCG AATGTAGGGT 1250
A~CA~CTA~T TATGACTT~T AGCGTTTTGA CTTATAAATT ACTTTTATAA 1300
GCTCA~CC.~A ACAGGCCCTT GTrCATTTAT ACCTCCCTAA AGATCTTIGA 1350
CGAGACTC-AA GGCTATTCTG CATATCGGTG TTGATAG~CT GAAGAAAATA 1400
ATC~GCCT;T ATACATCGAG cTTcTTrTcA ATTTATACAT CATrTAACAG 1450
AATTGCT~.T TAGTATCTTT AATTCTTTTT AATGACACTT AAAATGTTTT 1500
ACATCTTT~T ACTAATCTGA TTCTTAGTGG ACCCGTTGGA GATGGCGTGA 1550
TGGCTTCT~C AAGTGAAGTC AATGCCCTGA AAGATTCATT TTCAGCTTTA 1600
GA~GTTGGTG TCGTGGCAGA TATCAAAACT GAAGACACTG TCAAGCAGAC 1650
~CCTGCTG-.A GGCATCACAG ACTGCAAATG TGGGATGCCT TTGTGTATCT 1700
CCAAGTGTC AGCTACACCA ACAACATCCA TqGCTTCACA GGAAAGGACA 1750
~ATTTACTGG CC~TCAAATG CTTTrTGAGC AAATCACAAT TCCTTTATAT 1800
lq~rrrrrT~A TTCCAGCAGG GAATTATTAT GCCAAATCCA ATTGTAAACA 1850
TAAATCCAhA ACCAAAAAAA ~ TC TACAAGTCAC CACCACCACC 1900
AGTGAAGCCA TACCATCCTA CACCCGTATA CMGTCTCCA CCACCACCAA 1950
CTCCCGTT-A CAAGTCACCA CCATCACCAG TGAAGCCATA TCATCCCTCA 2000
WO9~17S7 2:i~ 01 ~'~ PCT/EP92/01214
CCAACACCAT ACCACCCTAC ACCAGCATAC AAGTCTCCAC CArCACCAAC 2050
TCCAGTCTAC AAGT,CTrCAC CACCAACCCA CTATGTT~CC ~CCTCTCCCC 2100
CTCCTCCCTA CCATTACTAA ~AAGTGAG~ TACTATA~CT GAGGAAAAGC 2150
CTAATGTrGA GCTGAAAGAA AGGCATTTTC CATTTTCAAG AAGAAAATTA 2200
TAGTAAATAA TM GGC~TAC AGAAGATCAG ACGAAGTTCT TTTGTAGCTT 2250
CATGTTATCT AACTAGTCTT AGTGATATAT TG m TTGTA CTCTATTTTT 2300
ATATATTACT m ATGTGTC TTTGTGTATG mGcTcAcT TTcAATcTrc 2350
qTGCAAAA~G CAGAGATTAA TTATGAGATT ATCATGAATA AAATAAGTTA 2400
q~ACTACTCC CATAT~TTTT AAAAAAAAAA A~AACCATGG TKCCGGACC 2qS0
TCGAGGATCC GGG`TACCATG GCACTG&CCG TC ~ TACA ACGrCG~GAC 2500
q~GGAAAACC ClGGCGTTAC CCAACTTAAT CGC~TTGCAG CACA~CCCCC 2550
~NNCGCCAGC TGGGCTAATA GCGAAGAGGC CCGCACCGAT CGCOCTTCCA 2600
ACAGTToOGC 2610
WO9~217S7 211 01 ~ 9 4 2 PCT/EP92~01214
SEQ ID NO.6
SEQUENCE qYPE: nucleotide
SEQUENCE LENGTH: l004 bp
ST,RANDEDNESS: doub~e-stranded
TOPOLOGY: linea~
MOLECULAR 5YPE: cDNA to m~NA
ORIGINAL SOURCE: pl~nt
ORGANISM: tomato
PROPERTIES: Nematode-responsi~e c~NA
MISCELLA~OUS: cDNA designated as LEMMI 9
ACCATGATTA CNCCAAGCTT GCATGCCTGC AGGTCGACTC TAGAGGATCC 50
CCGGGTACCG AGCTCGAAT~,CGAGGATCCG GGTACCATGC CGGTATGGTA l00
CCCGGATCCT CGATTCGAGG ATCCGGGTAC CAT ~ CGTT TAGCACAAAA 15D
CAGGCAT~_T ATTCAATTCC CTTTCGTTCC AGAA~CATGG ATCTAAT~GA 200
CAAGGCGA;G AATTTTGTGT CGGAGAAGAT AGCCAACATG GAGAAACCGIG 250
AGGCAACC~T CACCGACGTC GATCTTAAGG GGATCGGTrT CGACGGCCT,T 300
GCITTqCA_G CTAAAGTCTC CGTTAAGAAC CCTTACTCTG TTCCTA~TCC 350
AATCAT~K~G ATCGATTACG TCCTCAAAAG CGCCACCAGG GTAATCGCAT 400
CAGGAAG~;T TCCAGACCCA GGGAGCA~AA AGGCAAATGA CTCAACCATG 450
TTAGATGT~C CAGTGAAGGT TCCTCACAGT GTGCTAGTGA GTTTGGTTAG 500
GGACATTC-^-A GGAGATTGGG ACGTCGATTA TACCCTGGAA TTGGGTCTCA SS0
TTATTGA.~S TCCGGTCATT GGCAACATCA CCATTCCCCT CTCT,~ATAGC 600
AGGCGAG..sT AAGCT~CCTA CATTGTCAGA m ATGGAAG G6TGGAAAAG 650
AAGAAGAC~ A AAAAGAAGAT GAAGAGGAGA AAGAAGATCC ATCAAAGGTT 700
GTTGAGAT.~T GAAGAGTTAT ACCTA,TCTAA TAATGlGGCT TTAATATGCC 750
TAGTTTCT_T TCTGTTGm TAATAACATA AAGTTqG~rT ACCTTATAAG .800
ThTCAT~A AGGATACAAA ATGCACAACT TTATGAAACT CACATTACTC 850
TTATC~CA-~ TGATTTGATG A~ATGAAGAT TTGATGATGT TAGGT~TAAA 900
AAUUUUUU~A A ~ CATGGT ACCCGGATCC qCGAAT~CAC TG~CCGTCGT 950
m ACAAC~T CGTGACTGGG AAAACCC~NN NGTNAhCCCA ACTTAATCGC l000
C~TG 1004
WO9~217S7 4 ~ 2 1 1 0 1 ~ ~ PcT/En2/0l2l4
SEQ ID NO.7
SEQUENCE ~YPE: nuc~eotide
SEQUENCE LENGTH: 507 bp
SlRANDEDNESS: double-stranded
TOPOL0GY: linear
MOLECULAR ~YPE: CDNA tO mRNA
ORlG$NAL SOVRCE: pl~nt
ORGANISM: tomato
PROPERTIES: Nem~tode-responsi~e CDNA
MISCELLANE W S: CDNA design~ted ~s LENMI 10
GGCCAGTGAA TnCGAGGATC CGGGTACATG ~ ATGAAAAA GGTGTGAAGA 50
AGGATAAGGA ~AAG M ACCC AATAAGGAAA A~AAAGAGAA AAAGGATAAA 100
GGAAAGAAAG ATAAGAGCAA AGAGGAGTCG GA~GAAGAAG AGAAGGATGA 150
TGTAAAAGGG AAGAAGAAGG ATAAAGAGAA GAAAGATAAG AATAAAGAG? 20~
~GTCGGAAGA AGAAGATAAT GAAGAGAAGG ATGATAAAGT AGG~CAAGAAG 250
AAGGATAAAG AAAAGAAAGr~CAAGGCGAAT GCGG~TGAAG TCGCCACAAG 300
AGAGCTAG~A GTTGAGGAAG ACAAGAAAGT ATCCGACGA~ GAATCAGAAG 350
AGAAAAGTAA AAGCAA~CA TGGTACCCGG AT0CTCGAAT TQGAGCT0GG 400
TASCCGGGGA ~CCTCTAGAG TCGACCTGCA GGCATGCAAG CTTAANATAA 450
~CATGGTCAS AGCIGTqnCT GTGTGAAATT GTTATCNTCA CAATT`CACAC 500
AACATAC 507
WO 92~21757 PCI`/EI'92/01214
~ i L a 1 ~ ~ 4 4
SEQ ID NO. 8
SEQUENCE TYPE: nucleotide
SEQUENCE LENG~H: 731 bp
STRANDEDNESS: double-stranded
T~POLOGY: ~inear
MOLECULAR TYPE: cDNA to mRNA
ORIGINAL SOURCE: pl~nt
ORGANISM: tomato
PROPERTIES: Nematode-responsive cDNA
MISCELLANEOUS: cDNA designated as LEMMI ll
CGGCCAGTGA AT~CGAGGNT NCGGGTACAT G ~ CCCA~CC TACAAGTTAC 50
CACC~CCACC AACTCCCATT TACAAGTCGC CACCACCACC AACTCCnGCC 100
TACAAC~CTC CTCCACCACC TTACTATCTT TACACCTCTC CCCCTMNGGG 150
.CTACCATTAC TAAAAAGCCA CqTCATTATA TCGAGGTAAT TAAGACAAAC 200
TAAAACTATT ACTMTBATG TGTCTAAAAT GCAAAGCAAT MGTTGCTCA 250
ATTTCC~CAT GCAAATCATT AATATATAGA CCTATGCAGT CTATATTCTT 300
ATTrGGATAA CTTTAATTAC ATGT~CrCAT TCACAAAATT ATA~CTrAT 350
GCAGGAAAAG TAAAATB~TG AGCTTAAAGA AAGGCA m T GCATTTTCGA 400
GAGACGA$GA AAAAAGAAGA AAACTATAGT AAATAATAAG CCCCACATAA 450
GTCGAAG$TC TTTTGTAGCT TCATGTTATC TAAGCTAGTG ATATTGTTNG 500
TACTCTATTA TTTATATTTG TATTTTTACT TTTATGTCTT T~l'GTATGT~ 550
TCCTCAG m TAATATCCTA GCA~AATGCA M GATTAATT ATGAGATGCA 600
TAAAATAAGT TATTACTATT A~JU~ ~AAA A ~ CAT~G TACCCGGAGG 650
ATCCGNNNW~ NNTNGNGAGC TCGGTACCCG GGGATCCT~T AGAGTCGACC 700
TGCAGGCATG CAAGCTTGGC GTAATCATGG T 731
