CA2110169A1 - Nematode-responsive plant promoters - Google Patents

Abstract

Nematode-responsive plant promoters, particularly useful in the production of transgenic plants which can produce fixed feeding site cells that become capable of killing, disabling or repelling nematodes or that are themselves killed or rendered unsuitable for nematodes to feed upon when nematodes infect the plants.

Description

~"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~
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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.

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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~
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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~

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~

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~ 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~

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 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 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 ~ 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

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 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

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 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

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~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).
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WO 92~21757 PCI`/EP92/0121~

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~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 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 CACCAC$TGG CTGACAAAAT GAAACAGATG ACAACCAAGA TGTTGCACCA 400 CGACAACCAT~ GGTCAGCAAT CTGCCTGCCA TGGAGCCAAA ACTCAACATT 450 CAGCAGGCCA TGGCTCCACT GCTAT,TCACG GAAATCATGG TAGCCACTGC soo' : 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 GTGTCCAT~GG:CTCAAAGAAG GAAGGGGGCA TCATGCATAA GATAGGTAGT 950 CAGC~GAAGA:CCA,TCGGGAA AAAGAAGAAC AAAGATGGAC ACTGCAGAGA Iooo ATGAGAhTTG'TGGAAAAAAA ~ CATG GTACCCGGAT CCTCGAATTC llOO
ACTGGCCGTC GT m ACAAC TTCGTGACTG GGAAANCCCT NGCGTTACCC 1150 `"09~21757 ~ . PCT/EP92/01214 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 AGCGCGAAGA AGAGAATGAT GAAAAAGGTG TGAAAAAAAA AAAAAA~CCA 200 CATGT

wos~217S7 PCT/EP92/01214 211~ 38 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 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 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 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 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$NAL SOVRCE: pl~nt ORGANISM: tomato PROPERTIES: Nem~tode-responsi~e CDNA
MISCELLANE W S: CDNA design~ted ~s LENMI 10 GGCCAGTGAA TnCGAGGATC CGGGTACATG ~ ATGAAAAA GGTGTGAAGA 50 AGGATAAGGA ~AAG M ACCC AATAAGGAAA A~AAAGAGAA AAAGGATAAA 100 GGAAAGAAAG ATAAGAGCAA AGAGGAGTCG GA~GAAGAAG AGAAGGATGA 150 TGTAAAAGGG AAGAAGAAGG ATAAAGAGAA GAAAGATAAG AATAAAGAG? 20~
~GTCGGAAGA AGAAGATAAT GAAGAGAAGG ATGATAAAGT AGG~CAAGAAG 250 AAGGATAAAG AAAAGAAAGr~CAAGGCGAAT GCGG~TGAAG TCGCCACAAG 300 AGAGCTAG~A GTTGAGGAAG ACAAGAAAGT ATCCGACGA~ GAATCAGAAG 350 AGAAAAGTAA AAGCAA~CA TGGTACCCGG AT0CTCGAAT TQGAGCT0GG 400 TASCCGGGGA ~CCTCTAGAG TCGACCTGCA GGCATGCAAG CTTAANATAA 450 ~CATGGTCAS AGCIGTqnCT GTGTGAAATT GTTATCNTCA CAATT`CACAC 500 WO 92~21757 PCI`/EI'92/01214 ~ i L a 1 ~ ~ 4 4 SEQ ID NO. 8 SEQUENCE TYPE: nucleotide SEQUENCE LENG~H: 731 bp STRANDEDNESS: double-stranded T~POLOGY: ~inear MOLECULAR TYPE: cDNA to mRNA
ORIGINAL SOURCE: pl~nt ORGANISM: tomato PROPERTIES: Nematode-responsive cDNA
MISCELLANEOUS: cDNA designated as LEMMI ll CGGCCAGTGA AT~CGAGGNT NCGGGTACAT G ~ CCCA~CC TACAAGTTAC 50 CACC~CCACC AACTCCCATT TACAAGTCGC CACCACCACC AACTCCnGCC 100 TACAAC~CTC CTCCACCACC TTACTATCTT TACACCTCTC CCCCTMNGGG 150 .CTACCATTAC TAAAAAGCCA CqTCATTATA TCGAGGTAAT TAAGACAAAC 200 ATTTCC~CAT GCAAATCATT AATATATAGA CCTATGCAGT CTATATTCTT 300 ATTrGGATAA CTTTAATTAC ATGT~CrCAT TCACAAAATT ATA~CTrAT 350 GCAGGAAAAG TAAAATB~TG AGCTTAAAGA AAGGCA m T GCATTTTCGA 400 GAGACGA$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

Claims (8)

WO 92/21757 PCT/EP92/0121?

1. A nematode-responsive, preferably nematode-induced, plant promoter which can be isolated from genomic tomato DNA, upstream of a gene thereof having a DNA
sequence which corresponds to a cDNA selected from the group consisting of SEQ ID nos. 1-8, preferably SEQ ID
nos. 2-8, particularly SEQ ID nos. 4, 6 and 7, quite particularly SEQ ID no. 7.

2. A nematode-induced chimaeric gene, suitable for transforming a plant to protect it against nematode infection, which comprises the following operably linked, DNA sequences:
a nematode-induced promoter, preferably the nematode-induced promoter of claim 1, that is suitable to direct transcription of a foreign DNA
at least substantially selectively, preferably selectively, in specific cells of the roots, preferably in the cells of fixed feeding sites, of the plant;
a first foreign DNA that encodes a first RNA
and/or protein or polypeptide which, when produced or overproduced in the specific cells of the roots, preferably in 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) kills the specific cells of the roots, preferably the cells of the fixed feeding sites, or a 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 suitable 3' transcription termination signals for expressing the first foreign DNA in the specific root cells, preferably the fixed feeding site cells.

3. The nematode-induced chimaeric gene of claim 2 wherein the nematode-induced promoter is suitable to control transcription of the foreign DNA at least substantially selectively in the cells of the fixed feeding sites of the plant.

4. A plant cell or plant cell culture transformed with the nematode-induced chimaeric gene of claim 2 or 3.

5. A plant or its seeds consisting essentially of the plant cells of claim 4.

6. The plant of claim 5 or its seeds, in which 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 and which also contains a restorer chimaeric gene, preferably in the same genetic locus as the nematode induced chimaeric gene;
the restorer chimaeric gene having the following, operably linked, DNA sequences:
a second promoter, such as the nematode-repressed promoter of claim 1, which can direct transcription of a second foreign DNA in cells of the plant where the first foreign DNA is expressed, preferably substantially selectively in cells other than the specific root cells, preferably in cells other than the fixed feeding site cells, of the plant;
a second foreign DNA that encodes a second RNA
and/or protein or polypeptide which, when produced or overproduced in cells of the plant, inhibits or inactivates the first foreign DNA or the first RNA or protein or polypeptide; and suitable 3' transcription termination signals for expressing the second foreign DNA in plant cells.

7. A cell of the plant of claim 6 or a cell culture consisting essentially of the cells.

8. A process for rendering a plant resistant to nematodes, particularly sedentary endoparasitic nematodes, comprising the step of transforming the plant's nuclear genome with the nematode-induced chimaeric gene of claim 2 or 3 and optionally the restorer chimaeric gene of claim 6.