CN1286979C - Cell specific necrosis - Google Patents

Abstract

Plants are transformed with two genes whose promoters work together to respond to stimuli applied to the plants, including attack by pathogens. The necrotic effect of the first molecule encoded by the first gene on plant-specific cells is not inhibited by the molecule encoded by the second gene, while in other cells the necrotic effect of the first molecule mentioned is suppressed The second molecule inhibits. Transformed plants exhibit resistance to nematode infestation.

Description

cell-specific necrosis

The present invention relates to the induction of cell-specific necrosis as a means, for example, of increasing the resistance of plants to pathogenic agents.

It has been mentioned in WO 89/10396 that the transformation of plants with a construct containing a tissue-specific promoter and a gene encoding an RNA or polypeptide can severely interfere with cellular metabolism. In EP412911 a procedure to counteract metabolic interference by expressing another RNA or polypeptide is published. Similar techniques have been proposed on the subject of hindering nematode resistance in plants, as disclosed in WO 92/21757. EP 537399 also proposes a destruction/inhibition mechanism.

The present invention provides a method of inducing necrosis in specific cells of an organism. Plants are transformed with two chimeric genes, one of which has a coding sequence encoding a first necrosis molecule and the other gene has a coding sequence encoding a second molecule. The second molecule is said to be an inhibitor of necrosis of the first molecule. Each of these two genes contains a promoter which together responds to a specific stimulus applied to the organism such that ① the necrotic effect of the first molecule on the cell is not inhibited; ② in the other The expression of the first and second molecules in the cells (the organism needs other cells in order to have a healthy condition and not to die) in said other cells in which the necrotic effect of the first molecule on other cells is inhibited.

An advantage of the present invention is that cell-specific necrosis can be achieved without the need for a cell-specific promoter. That is, use of both promoters results in cell-specific necrosis, while use of either of the two does not direct cell-specific expression. However, the overlapping expression properties of the two promoters and the differential responses to applied stimuli affect the direction of expression of the effector and repressor genes, so that the two promoters are produced in (and only in) specific cells. A molecular lethal imbalance.

Such organisms may be plants.

It is possible that the first molecule in a specific cell is expressed only when the organism, such as the specific cell, is stimulated. In this case, depending on the stimulus, the promoters act together to ensure the relative levels of the first and second molecules in specific cells, leaving uninhibited necrosis of the first molecule. Similarly, it is possible that there is no expression of the first molecule in other cells prior to administration of the stimulus. In this case, the two promoters act together to ensure the relative levels of the first and second molecule in other cells following administration of a stimulus such that necrosis of the first molecule is inhibited.

If the first molecule is already expressed in a particular cell before the stimulus is applied, the two promoters work together to ensure that the relative levels of the first and second molecule in the specific cell change according to the stimulus used . This shifts it from a state in which the necrosis of the first molecule is inhibited to a state in which it is no longer inhibited.

If the first molecule is already expressed in other cells before the stimulus is applied, then the relative levels of the first and second molecules in the other cells before and after stimulation are such that the first molecule is The necrosis of other cells was inhibited.

It will be apparent to those skilled in the art that, in response to the applied stimulus, changes in the relative levels of the first and second molecules in specific cells will be induced by increased activity of the promoter of the chimeric gene containing the gene encoding the first molecule level and/or the effect of reduced levels of promoter activity of another gene in the chimeric gene. It is naturally also possible that the levels of both the first molecule and the second molecule are both increased or decreased, so long as the change in the relative levels results in the necrosis of the first molecule in specific cells being no longer inhibited by the second molecule.

Panels A-F of Figure 1 in the drawings herein represent by way of illustration the expression levels (Y-axis) of the first molecule (shaded bars) and the second molecule (unshaded bars). Panel A represents the relative expression levels in target cells transformed according to the invention (specificity), which may have been present before the administration of the stimulus. Panel A may also represent the relative levels of other non-target cells before and after stimulus administration. Panels B-F show different relative levels of the first molecule and its inhibitor, the second molecule, in the target cells following said stimulation. Arrows indicate an increase or decrease in expression levels corresponding to each level in panel A. Notably, in any of panels B-F, the first molecule is expressed in the presence of excess inhibitor. Thus, necrosis of the target cells occurs in each case.

In practicing the invention, the first molecule can be an RNase and the second can be an RNase inhibitor.

Other first and second molecules are: restriction endonucleases and corresponding DNA modifying enzymes; proteases and corresponding protease inhibitors; antisense and sense RNAs of key regulatory or structural genes.

For the purposes of the present invention, the said stimulus applied to the plants may consist of an attack by a pathogen.

Those skilled in the art will recognize that the present invention has wide applicability in the plant kingdom for protection against infections such as fungi, nematodes, bacteria and viruses, among other purposes.

The invention can be used to selectively destroy or inhibit the development of stable plant organs such as spines, buds, flowers or setae. In this case, the stimulus may be the result of the natural development of the plant or an artificial stimulus such as a chemical agent applied to the plant.

It will be understood that, as used herein, "necrosis" includes the concept of substantial damage to metabolism such that the objectives of the application of the present invention, such as disease protection, can be achieved.

Those skilled in the art will recognize that the concepts pertaining to the present invention can be applied to non-plant kingdoms.

A preferred procedure of implementing the present invention will be described below with the example of root-knot nematode infecting tobacco plants:

Tobacco Growth and Infection

Tobacco C319 seeds were germinated in FisonsF1 mixed fertilizer under the following conditions: light intensity of 4500-5000Lux, 16 hours of light interval and 8 hours of darkness, and temperature between 20-25°C. Three weeks later, the seedlings were washed gently under tap water, removed the soil, and were transferred to bags (2 plants per bag, Northrupking), at 25°C, light intensity 5500Lux, 16 hours light interval 8 hours dark Conviron growth box Grow for another week. Elevate the roots on the back of the bag, and support the root tips with Whatman GF/A glass fiber paper, release three-day-old nematodes to these root tips with a small aliquot of 10 μl (50 nematodes), and then put a sheet on the root tips GF/A paper, wrapping the root tip completely. The GF/A paper was removed 24 hours after nematode infestation to ensure synchronous nematode infestation. After 3 days of infection, the root knots were excised (leaving the root tip and healthy root tissue) and immediately frozen in liquid nitrogen. About 0.5-1.0 g of infected root tissue could be harvested from 80 nematode-inoculated plants.

Stain to visualize nematodes in infected roots

To determine the quality of infection, the number of infected nematodes in each root tip was determined. Roots were harvested from plants 3 days post-infection, immersed in lactophenol solution containing 0.1% Gossypium indigo at 95°C for 90 seconds, washed with water for 5 seconds, and soaked in lactophenol at room temperature for 3-4 days , to make the root transparent. The stained nematodes can be observed with a light microscope.

Extraction of RNA from Healthy and Infected Root Tissues

Root tissues were ground to a fine powder in a mortar precooled with liquid nitrogen. Aliquots of 100 mg were added to similarly pre-chilled Eppendorf centrifuge tubes. Add 300 μl of hot phenol extraction buffer (50% phenol, 50% extraction buffer: 0.1M lithium chloride. 0.1M Tris-HCL pH8.0 (room temperature), 10mM EDTA, 1% SDS), incubate at 80°C for 5 minutes, and then An equal volume of chloroform was added, and the homogenate was micro-ultracentrifuged at 4°C for 15 minutes. The aqueous phase was then extracted with 600 μl of phenol/chloroform and micro-ultracentrifuged as above. Subsequently, the aqueous phase was removed, and an equal volume of lithium chloride was added to precipitate the RNA at 4°C, and micro-ultracentrifuged at room temperature for 15 minutes to obtain a precipitated cake. Wash the precipitate with 70% ethanol, freeze-dry the precipitate cake, suspend the precipitate with DEPC-treated water, and test it with a spectrophotometer. The quality of RNA was determined by denaturing gel electrophoresis (modified from the method of Shirzadegan et al. 1991).

Subtractive cloning of infection-specific cDNA

Poly(A) ⁺ -RNA (mRNA) was isolated from 200 μg total RNA samples from C319 healthy and infected root tissues using magnetic oligo dTDynabeads according to the manufacturer's protocol. First-strand cDNA synthesis is accomplished in situ on Dynabeads bound to Poly(A) ⁺ components obtained from healthy tissue. This is the driver DNA (DriverDNA). Poly(A) ⁺ RNA extracted from infected tissue is bound to the Dynabead column for in situ synthesis of first and second strands, which is the target DNA (TargetDNA). All cDNA synthesis reactions were carried out using the cDNA synthesis kit from Pharmacia according to the manufacturer's instructions. Three oligonucleotides, SUB21 (5'CTCTTGCTTGAATTCGGACTA3'), SUB25 (5'TAGTCCGAATTCAAGCAAGAGCACA3') (sequence from Duguid and Dinauer, 1990) were treated with _T4 polynucleotide kinase using the method of Maniatis et al. (1982). Kinase treatment with LDT15 (5'GACAGAAGCGGATCCd(T) ₁₅ 3') (O'Reilly et al., 1991). SUB21 and SUB25 were then annealed to form an adapter, which was then ligated to the target DNA using _T4 DNA ligase according to the method of King and Blakesley (1986). Beads carrying target DNA were extensively eluted with TE, while the second strand of cDNA was eluted with 5×SSC at 95°C.

RNA bound to Dynabeads containing driver DNA can be removed by heating. The eluted target DNA was hybridized with driver DNA in 5×SSC at 55°C for 5 hours. According to the method provided by the manufacturer, unhybridized target DNA was isolated from the beads bound to the driver DNA at room temperature, and subsequently, the hybridized target DNA was isolated from the beads bound to the driver DNA at 95°C using a similar method. The target DNA eluted at room temperature is then added to the driver DNA, and the hybridization is repeated. This process is repeated until the amount of target DNA hybridized to the driver DNA no longer exceeds the amount of target DNA that does not hybridize. The concentration of DNA was determined using Invitrogen's DNA-Dipstick instrument according to the method provided by the manufacturer.

A small aliquot of the final room temperature eluted fraction was subjected to PCR amplification (Eckert et al., 1990) to generate double-stranded cDNA cloned into a plasmid vector. According to the conditions proposed by Frohman et al. (1988), the target DNA was amplified with SUB21 and LDT15 primers and hybridization thermal cycler. The PCR product was then ligated into the Smal digested pBluescript vector (King and Blakesley, 1986).

Screening of subtracted libraries by reverse Northern analysis

Recombinants were identified by colony PCR. (Gussow and Clackson, 1989). Southern blots of the amplified inserts were performed in triplicate on Pall Biodyne membranes using the protocol provided by the membrane manufacturer. Prehybridization and hybridization were performed at the same temperature and buffer: 42°C, 5XSSPE, 0.05% BLOTTO, 50% formamide. Membranes were hybridized to cDNA probes from healthy tissue, infected tissue (see below) and probes containing target DNA amplified from the final subtraction. Clones showing only hybridization to cDNA probes from infected tissue and clones showing hybridization signals to subtracted probes but not cDNA probes were selected for further analysis.

Generation of cDNA probes

In order to obtain highly specific probes for differential screening, isotope-free cDNA was synthesized from total RNA. The synthesized product is then labeled with an oligolabel. 10 μg of total RNA samples from healthy and infected tissues were treated with 2.5 units of DNase I for 15 minutes at 37°C, followed by denaturation of DNase at 95°C for 10 minutes before cDNA synthesis proceeded (standard Pharmacia procedure). RNA was removed by treating at room temperature for 10 minutes in the presence of 0.4M NaOH. The DNA was purified by a rotating Sephacryl 400HR column, and the amount and concentration of the DNA were measured with a DNA Dipstick instrument (Invitrogen), followed by labeling the cDNA product (concentration, 35ng/probe) according to the Pharmacia oligolabeling standard procedure.

Northern blot

In order to determine the expression characteristics of cDNA selected by reverse Northern technology from different plant tissues, total RNA or Poly(A) ⁺ -RNA obtained from healthy and infected roots, stems, leaves, and flowers These clones were used as probes for Northern analysis. Total Northern blots contained 25 μg RNA per lane; and Poly(A) ⁺ - Northern blots contained 0.5 μg-1 μg RNA per lane. RNA electrophoresis was performed on formaldehyde gels and RNA was blotted onto Pall Biodyne B membranes as described by Fourney et al. (1988). Probes were labeled and hybridized to blots as previously described.

Southern blot

To determine whether the cDNA was of plant or nematode origin, DNA from C319 and M. Javanica were prepared according to the method of Gawel and Jarret (1991). For Southern blotting, each lane contained 10 μg of EcoRI and HindIII digested DNA. Blots were hybridized to oligolabeled probes as described previously.

in situ hybridization

In situ hybridization was performed to determine the site of expression of our cDNA of interest at the feeding site. According to the method of Jackson (1991), the healthy root tissue and the infected root tissue were embedded in wax, sectioned, and then hybridized with the probe.

Isolation of mRNA 5′ ends

Before isolating the mRNA promoter sequence, determine the 5'-end of the RNA of interest. This can be done with 5'-RACE as described by Frohman et al. (1988).

Isolation of the promoter region

The promoter region of the gene of interest was isolated using a process called vector ligation PCR. C319 genomic DNA samples digested with 100 ng of restriction endonucleases were ligated with 100 ng of pBluescript samples (digested with restriction enzymes that generate compatible ends) for 4 hours at room temperature (King & Blakesley, 1986). Typical usable enzymes include EcoRI, BamHI, HindIII, BGlII, XhoI, ClaI, SalI, KpnI, PstI and SstI. Use carrier primers such as -40 sequencing primers and primers complementary to the 5' end of mRNA to carry out PCR reaction on the ligated products, and then clone and sequence the PCR products. If necessary, repeat PCR with a new primer complementary to the 5' end of the promoter fragment to ensure isolation of the promoter control sequence.

Construction of binary plant transformation vector pStarnase

Figure 2 is a schematic representation of the T-DNA segment of the binary plant transformation vector pStarnase. This region includes the NPTII gene (transgenic plants can be selected with Kanamycin). A barstar ORF controlled by the CaMV35S promoter and a barnase ORF with a NotI restriction site in between for insertion proximal to the stimulus-responsive promoter. This vector has been deposited with the National Collection of Industrial and Marine Bacteria, Aberdeen, UK, on May 5, 1994, under the accession number NCIMB40634, and is protected by the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

On May 11, 1994, this vector was deposited in the China Center for Type Culture Collection, and its registration number is CCTCC94206.

Illustration of Figure 2:

A-right border

B-NPT II

C-Nos terminator

D-Barnase ORF

E-CaMV 35S promoter

F-Barstar ORF

G-Nos terminator

H-left border

I-NotI site

Production of transgenic plants

Transgenic plants, such as tobacco, can be produced by the standard Agrobacterium-mediated leaf disc method as described by Horsch et al. (1985). In this way, root-knot nematode-resistant plants can be obtained. The plant seeds or other propagules obtained in the present invention can be preserved for further use.

Those skilled in the art will recognize that, for certain classes of plants, it may be appropriate or necessary to transform plants using non-Agrobacterium-mediated methods.

Example

KNTI: Examples of plant-parasitic nematode-inducible genes and the use of their promoters according to the present invention to produce plants resistant to plant-parasitic nematodes.

Using the above steps, a KNTI gene was isolated and identified from tobacco. Although its expression level in healthy plants is very low, Northern blotting shows that infection of root-knot nematode MJavanica strongly induces its expression. An approximately 0.8 kb KNTI promoter fragment starting from the transcription initiation site was isolated as described above and inserted into the Gus-reporter vector pBI101 (Jefferson et al. 1987). This resulted in a construct called pG21.08, which can be used to transform tobacco plants. It can be seen that in healthy plants, this promoter sequence directs the expression of the GUS gene only in the apical and lateral meristems. When infected by M. Javanica, the expression of Gus gene in these tissues did not change, while strong expression of Gus gene was seen in the feeding site. Then insert this promoter sequence into the NotI site of pStarnase, so that it can control the expression of barnaseORF. This resulted in build pS21.08. It can be seen that plants transformed with this construct are resistant to root-knot nematode infection.

The KNTI gene shows homology to other non-tobacco plant species. These types include, but are not limited to potatoes, tomatoes and beets. We can also see that the KNTI gene can be induced by root-knot nematodes and cyst nematodes. On May 5, 1994, the constructed vector pS21.08 has been preserved by the National Collection of Industrial and Marine Bacteria in Aberdeen, UK, with the accession number NCIMB40635, and is subject to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedures Protect.

The vector was deposited in the China Center for Type Culture Collection on May 11, 1994, and its registration number is CCTCC M 94027.

references:

DUGUID, J.R. & DINAUER, M.C. (1990) Nucleic Acids Research 18(9): 2789-2792.

ECKERT, K.A. & KUNKEL, T.A. (1990) Nucleic Acids Research 18(13): 3737-3744.

FOURNEY, R.M., MIYAKOSHI, J., DAY III, R.S. & PATERSON, M.C. (1988) Focus 10(1): 5-7.

FROHMAN, M.A., DUSH, M.K. & MARTIN, G.R. (1988) Proceedings of the National Academy of Sciences USA 85: 8998-9002.

GAWEL, N.J. & JARRET, R.L. (1991). Plant Molecular Biology Reporter 9(3): 262-266.

GUSSOW, D., & CLACKSON, T. (1989). Nucleic Acids Research 17: 4000-4008.

HORSCH, R.B.; FRY, J.E.; HOFFMANN, N.L.; EICHOLTZ, D.;

ROGERS, S.G. & FRALEY, R.T. (1985). Science 227:1229-1231.

JACKSON, D. (1991). Molecular Plant Plant Pathology: APractical Approach. IRL Press, Oxford.

JEFFERSON, R.A.; KAVANAGH, T.A. & BEVAN, M.W. (1987) EMBOJ 6(13):3901-3907.

KING, P.V. & BLAKESLEY, R.W. (1986) Focus 8(1): 1-3. MANIATIS, T.; FRITSCH, E.F. & SAMBROOK, J. (1982) Molecular Cloning; A Laboratory Manual. N.Y. Cold Spring HarbourLaboratory.

O'REILLY, D.; THOMAS, C.J.R. & COUTTS, R.H.A. (1991) Journal of General Virology 72:1-7.

SHIRZADEGAN, M.; CHRISTIE, P. & SEEMANN, J.R. (1991). Nucleic Acids Research 19(21): 6055.

Claims (11)

1. A method of inducing necrosis in plant-specific cells, wherein the plant is transformed with two chimeric genes, one of which has a coding sequence encoding a first cell necrosis molecule and the other of said genes The coding sequence encodes a second molecule, the second molecule is an inhibitor of necrosis of said first molecule, said two genes each comprise a promoter, and their promoters work together to induce a negative effect on said plant Responses to specific stimuli such that i) said first molecule does not inhibit necrosis of said cell, ii) expression of said first and second molecule in other cells results in expression of said first molecule in said cell Inhibiting the necrotic effect of the first molecule on said other cells which are required by the plant for a healthy condition without necrosis. 2. The method according to claim 1, wherein said first molecule is RNase and the second molecule is an RNase inhibitor. 3. The method according to claim 2, wherein said first molecule is barnase and said second molecule is barstar. 4. The method according to claim 1, wherein said first molecule is a restriction enzyme and said second molecule is a corresponding DNA modifying enzyme. 5. The method according to claim 1, wherein said first molecule is a protease and said second molecule is a corresponding protease inhibitor. 6. The method according to claim 1, wherein said second molecule is the mRNA of a regulatory or structural gene and said first molecule is the corresponding antisense RNA. 7. The method according to any one of claims 1-6, wherein said stimulus consists of a pathogenic infestation of said plant. 8. The method according to claim 7, wherein the pathogen includes fungi, nematodes, bacteria and viruses. 9. The method according to any one of claims 1-6, wherein said stimulus is the result of natural development of said plant or of an artificial stimulus applied to the plant. 10. A necrotic plant cell produced by the method of claim 1. 11. The plant cell according to claim 10, which is a tobacco plant cell.

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