WO2019077477A1 - Biological fungicide - Google Patents

Abstract

An object of the present invention is a method for inducing or increasing resistance to one or more pathogens in a plant, plant part, or plant cell wherein said method comprises introducing a nucleotide sequence coding for the PGIP polygalacturonase inhibitor and a nucleotide sequence coding for the stilbene synthase in said plant, plant part, or plant cell.

Description

Description

"Biological fungicide"

An object of the present invention is a method for inducing or increasing resistance to one or more pathogens in a plant, plant part, or plant cell, wherein said method comprises introducing a nucleotide sequence coding for the PGIP polygalacturonase inhibitor and a nucleotide sequence coding for the stilbene synthase in said plant, plant part, or plant cell.

Background

Plasmopara viticola is a microorganism belonging to the class of oomycetes causing the downy mildew of the vine. The downy mildew is one of the most widespread and dangerous diseases of the vine in many European and Italian regions.

Botrytis cinerea is a fungus of the Sclerotiniaceae family, a parasite that attacks many varieties of plants. In viticulture it is commonly known as grey rot or grey mould. This species is endowed with a wide biological diversity, which manifests itself in numerous morphological phenotypes and in an early acquisition of resistance characters when subjected to fungicidal pressure. The presence of at least two subpopulations of botrytis, called vacuma and transposa, which are present in field with different incidence during the season has been demonstrated: the first is found in the vineyard only l at the beginning of the vegetative period, where it attacks the senescent inflorescences, while the second is present all year round, but above all on the berries during ripening.

Given the economic importance, over the years many products have been developed to counteract the attack of the above-mentioned microorganisms.

Synthetic products are currently the most commonly used methods of fighting the disease. They can be basically classified in 5 groups:

1 - contacting or covering products (copper products, dithiocarbamates : mancozeb, propineb, metiram and dithianon) , act by contact through a multi-site action mechanism.

2 - translaminar cytotropics (cymoxanil, dimetomorf, zoxamide, cyazofamid, mandipropamid, fluopicolide and amisulbrom) , penetrate the tissues and reach the parenchyma;

3 - systemic agents (metalaxyl and benalaxyl and iprovalicarb) , absorbed and translocated inside the plant ensuring the protection of the growing vegetation as well;

4 - mitochondrial respiration inhibitors (Qoi) (pyraclostrobin, famoxadone, fenamidone) , active at cellular respiration level through a single site mechanism; 5 - resistance inducers, active directly on the fungus through the activation of the plant natural defences with the particularity of fosetyl-Al being considered as the reference standard for the protection of the apical leaves of buds and secondary shoots.

The fungicides currently in use, although proven to be effective, do not meet the current needs of viticulture, attentive not only to the health of operators exposed to the substances in use, but also to residual concentrations of fungicides that, although within the levels permitted by law, remain however unwanted.

It should also be considered that pesticides give rise to the early development of resistance, which makes them ineffective after some years of use. Finally, when there is more need for botryticide treatments, i.e. near the harvest, not all products can be used, in order to avoid the presence of toxic residues in the final product

The need to have new methods to counteract Plasmopara viticola and Botrytis cinerea, environmentally friendly methods, not favouring the onset of resistance and usable even near the harvest, is particularly strongly felt, since harvest is a particularly favourable moment for the development of fungi and phytoplasmas.

Polygalacturonase (PG) is an enzyme belonging to the class of glycosidases , which catalyzes the reaction of hydrolysis: Polygalacturonic acid + H2O τ Polygalacturonic acid (shortened) + Galacturonic acids. PG plays an essential role in the fruit ripening process. During ripening, protopectins are degraded to pectic acids, due to the action of pectinesterase . Subsequently, pectic acids, which are galacturonic acid polymers, are hydrolyzed and solubilized by the PG, resulting in pulp softening .

The pathogens in object, producing PG, arrive in plants to demolish the cell wall. Plants possess a defence mechanism consisting of a PG inhibitor (PGIP) . The numerous crossbreeds to which the Vitis vinifera plants have been exposed over time have led to changes in the PGIP. Typically, in plants are found PGIP without myristic acid, myristic acid capable to anchor the inhibitor to the cell membrane, making the defence against the pathogenic attacks more effective. The plants are thus strongly exposed to the pathogenic attacks.

Another important enzyme implemented by Vitis vinifera for the defence against pathogens is the stilbene synthase. The stilbene synthase induces the production of phytoalexins . The biosynthesis of phytoalexins is induced by the presence of molecules, called elicitors, signalling the pathogenic attack to the plant. An example of phytoalexin is represented by resveratrol, a stilbene produced by some plant species, including Vitis vinifera, in response to a fungal attack. The multiple selection steps to which Vitis vinifera has been exposed over time have transformed PGIP and stilbene synthase considerably decreasing their protective capacity against pathogens (Jaillon 0, et al . , Nature, 2007 Sep 27. PMID 17721507) .

Agrobacterium tumefaciens is a gram-negative bacterium capable of infecting plants through the transmission of a DNA segment, called T-DNA, carried on the "Ti plasmid" . Agrobacterium tumefaciens is the instrument of choice for the generation of transgenic plants (Tzfira T, Citovsky V Agrobacterium-mediated genetic transformation of plants: biology and biotechnology. Curr Opin Biotechnol, 2006, 17 (2) : 147- 54), a variant thereof being available without its oncogenic functions but maintaining the intrinsic infectious capacity.

Typically, the bacterial strains in use contain a T-DNA modified natural plasmid, providing only vir genes and a binary plasmid that can be manipulated in E. coli. Once replicated in E. coli, after digestion and re^¬ circulation through the DNA ligase, the binary vector is extracted and used to transform Agrobacterium tumefaciens by electroporation, making the bacterium capable of carrying out the viral infection in the plant by transporting the binary vector and the gene of interest into it. A method of infection of a plant of particular interest is agroinfiltration, i.e. the infiltration of a suspension of Agrobacterium tumefaciens into the intercellular spaces of the leaves by means of a plastic syringe without a needle.

In Vitis vinifera, it has been shown that good levels of transient gene expression are obtained with the agroinfiltration method (Santos-Rosa et al . 2008; Zottini et al., 2008; Bertazzon et al . 2011) .

Description of the invention

An object of the present invention is a biological method to counteract the attack of pathogens such as Plasmopara viticola and Botrytis cinerea in plants, in particular in Vitis vinifera.

The first step was selecting Vitis vinifera varieties wherein the PGIP and stilbene synthase enzymes were naturally particularly effective in defending the plant from the pathogenic attack. The selection work involved first the isolation of Vitis vinifera strains particularly resistant in field to the attack of Plasmopara viticola and Botrytis cinerea pathogens. From the selected strains, through methods known in the background art, the total RNA was extracted. This was retro-transcribed and amplified using primers specific for the PGIP and stilbene synthase. The gene sequences found in the different strains were compared to each other and the following sequences were identified:

PGIP (SEQ ID no. 1)

ATGGAGACTTCAAAACTTTTTCTTCTCTCCTCCTCTCTCCTCCTAGTCTTACTCGC CACTCGTCCATGTCCTTCTCTCTCTGAACGTTGCAACCCAAAAGACAAAAAAGTTC TCCTTCAAATCAAAAAAGCCCTAGACAATCCCTACATTCTAGCTTCGTGGAATCCC AACACCGATTGCTGCGGATGGTACTGCGTCGAATGTGACCTCACCACCCACCGCAT CAACTCGCTCACCATCTTCTCCGGCCAGCTATCCGGCCAGATTCCCGACGCTGTTG GTGACCTTCCGTTCCTCGAGACCCTCATCTTCCGCAAGCTCTCTAACCTCACCGGT CAGATCCCGCCGGCGATTGCCAAACTCAAGCACCTAAAAATGGTTCGCCTTAGCTG GACCAACCTCTCCGGTCCCGTGCCGGCGTTCTTCAGCGAGCTTAAGAACCTCACGT ACCTCGACCTCTCCTTCAATAACCTATCTGGACCCATTCCCGGCAGCCTCTCTCTC CTCCCCAACCTCGGCGCACTCCATCTCGACCGGAACCACCTCACAGGCCCAATCCC TGACTCCTTCGGAAAATTCGCCGGCTCTACCCCAGGTCTACACCTCTCACACAACC AACTTTCCGGGAAAATCCCATATTCTTTCAGAGGATTCGACCCCAATGTCATGGAC TTATCGCGTAACAAGCTTGAGGGTGACCTGTCAATATTCTTCAATGCCAATAAGTC AACACAGATCGTTGACTTCTCACGGAACTTGTTCCAGTTTGATCTTTCGAGAGTGG AATTCCCGAAGAGTTTGACGTCGTTGGACCTTTCGCATAACAAGATCGCCGGGAGC CTGCCGGAGATGATGACTTCTCTGGATTTACAGTTCCTGAACGTGAGTTACAATCG TTTGTGTGGTAAGATTCCGGTGGGTGGGAAGTTGCAGAGCTTCGATTACGACTCCT ACTTTCACAATCGGTGCTTGTGTGGTGCTCCACTCCAGAGCTGCAAGTGA

Stilbene synthase (SEQ ID no. 2)

GCTTCAATTTCATTACGTATCTAGCATCCATGGCTTCAGTTGAGGAATTTAGAAAC GCTCAACGTGCCAAGGGTCCGGCCACTATCCTAGCCATTGGCACAGCTACTCCTGA CCACTGTGTCTACCAGTCTGATTATGCTGATTACTATTTCAGGGTCACTAAGAGCG AG C AC AT GAC T GAG T T GAAGAAGAAG T T C AAT C G C AT AT G T AAG TATATTCATGCA T T AAT T C T TAT AT AC AT AAC AT TTGTATGCATC TAAGAG TGTGTGCTATTAGGTGA GGCTCACCTCCAAGCGAATGAATGTTCCAACCTTTCTAGAGTAAAGCTTTTAGATA AAT T AC T T C AG GAAAC T T GAAAAT C AT T T T AC T T C AG T AAC C AAT AT TCCTTTCAT T T GAC TAT AAT T GC T T GAAAAGC T GT T T T T T GAAT CATATAGCAT T GC TAGC TATA ATTAAGAATCCCTTTTATACTTTCTTCAATGTTAAATGCATGTTGATCATCTTGAA C AAT AT AC CAT AT GAC TTGTCGATTGG TAAAAC TAATGTGTTCATGTTACCTCATT T AC AG G T GAC AAAT C AAT GAT CAAGAAG C G T T AC AT T CAC T T GAC C GAAGAAAT G C T T GAG GAG C AC C C AAAC AT TGGTGCTTATATGGCTC CAT C T C T T AAC AT AC G C C AA GAGAT TAT CAC T G C T GAG G T AC C T AGAC T T G G TAG G GAT G C AG CAT T GAAG G C T C T TAAAGAG T G G G G C C AAC CAAAG T C CAAGAT CAC CCATCTTGTATTTTG T AC AAC C T CCGGTGTAGAAATGCCCGGTGCGGATTACAAACTCGCTAATCTCTTAGGTCTTGAA ACATCGGTTAGAAGGGTGATGTTGTACCATCAAGGGTGCTATGCAGGTGGAACTGT CCTTCGAACTGCTAAGGATCTTGCAGAAAATAATGCAGGAGCACGAGTTCTTGTGG TGTGCTCTGAGATCACTGTTGTTACATTCCGTGGCCCTTCCGAAGATGCTTTGGAC TCTTTAGTTGGCCAAGCCCTTTTTGGTGATGGGTCTTCAGCTGTGATTGTTGGATC AGATCCAGATGTCTCGATTGAACGACCACTCTTCCAACTTGTTTCAGCAGCCCAAA CATTTATTCCTAATTCAGCAGGAGCCATTGCCGGAAACTTACGTGAGGTGGGGCTC ACCTTTCATTTGTGGCCCAATGTGCCTACTTTGATTTCTGAGAACATAGAGAAATG CTTGACCCAGGCTTTTGACCCACTTGGTATTAGCGATTGGAACTCGTTATTTTGGA TTGCTCACCCAGGTGGCCCTGCAATTCTCGATGCAGTTGAAGCAAAACTCAATTTA GAGAAAAAGAAAC T C GAAG C AAC TAG G CAT G T G T T AAG T GAG T AC G G T AAC AT G T C AAGTGCATGTGTGTTGTTTATTCTGGATGAGATGAGAAAGAAATCCTTGAAGGGGG AAAAGGCTACCACAGGTGAAGGATTGGATTGGGGAGTATTATTTGGTTTTGGGCCG GGCTTGACCATCGAAACTGTTGTGCTGCATAGCGTTCCTACAGTTACAAATTAAGA GAAAT AAAAGAGAAT G G T T GAC C C T T C AAT GGCGTAATGTAT C AAAT AG The coding sequence for the enzymes of interest PGIP and/or stilbene synthase was cloned into an expression vector with which Agrobacterium tumefaciens was engineered .

In a preferred embodiment, the hypervirulent , non- tumorigenic strain GV3101 of Agrobacterium was selected.

In one embodiment, the applied method was as follows .

1) RNA extraction from Vitis vinifera;

2) RT-PCR for the amplification of the stilbene synthase and PGIP genes, preferably performed using modified primers so as to provide the amplificated with the desired sequence for the subsequent digestion with restriction enzymes;

3) elution, purification, digestion and transformation into E. coli;

4) insertion into the vector which is preferably PRI-101-AN, selected for the capacity of expression exclusively in dicotyledonous plants and to possess a strong promoter such as CAMV 35 S, preferably through the restriction endonucleases BamHI-Kpnl, and Smal and Sail;

5) electroporation in Agrobacterium tumefaciens;

6) multiplication in LB soil;

7) application on the crop. In one embodiment, said coding sequence for the stilbene synthase is cloned in 5 '-3', in an alternative embodiment, in 3 '-5'.

In one embodiment, the plant is treated with Agrobacterium tumefaciens expressing PGIP and stilbene synthase cloned in 5 '-3'. In a further embodiment the plant is treated with Agrobacterium tumefaciens expressing PGIP and stilbene synthase inverted-cloned in 3'-5' .

The expression of the parallel- and antiparallel- cloned proteins has proved to be particularly advantageous. In both cases, in fact, proteins compete for the substrate which is the botrytis polygalacturonase. Said competition was shown to reduce the negative impact on the leaf tissue by the fungal infection, to the benefit of the inhibition effect from botrytis and/or downy mildew .

In one embodiment, said application is for agroinfiltration . An alternative for direct application is for example by spraying, preferably with an adhesive.

One aspect of the present invention is a method of protecting Vitis vinifera against the attacks of Plasmopara viticola and/or Botrytis cinerea including the expression in Vitis vinifera of stilbene synthase and PGIP. In a preferred embodiment, said method comprises the expression of one or more nucleic acid molecules comprising the nucleotide sequences selected from the group consisting of:

a) the nucleotide sequence coding for PGIP, SEQ ID no. 1, cloned in 3 '-5' and/or 5 '-3';

b) the nucleotide sequence coding for stilbene synthase, SEQ ID no. 2, cloned in 3'-5' and/or 5'-3'; c) the nucleotide sequence having at least 80% sequence identity with SEQ ID no. 1 ;

d) the nucleotide sequence having at least 80% sequence identity with SEQ ID no. 2.

Said nucleotide sequences SEQ ID no. 1 and SEQ ID no. 2 were selected as particularly effective in inducing resistance against Plasmopara viticola and/or Botrytis cinerea in Vitis vinifera.

Said nucleotide sequences are under the control of an expression promoter in a plant cell.

The used vector is preferably pR101-AN.

Said vector, advantageously, is expressed only in dicotyledonous plants and has as promoter a strong promoter such as CAMV 35S. In a preferred embodiment, the synthesis terminator is Nos-ter (nopaline synthase terminator) .

In one embodiment, the engineering of Agrobacterium tumefaciens was obtained by electroporation. After amplification, Agrobacterium tumefaciens is applied in Vitis vinifera according to methods of the prior art.

Following treatment, exogenous gene expression was evaluated in some samples.

The protection against downy mildew and botrytis was then monitored.

Description of the Figures

Figure 1: one embodiment of an expression vector according to the present invention. Highlighted in an oval, the coding sequences for the enzymes of interest PGIP and RESV.

Figure 2: expression levels of stilbene synthase and PGIP in an infected leaf and after agroinfiltration of stilbene synthase and PGIP.

Figure 3: expression levels of stilbene synthase and PGIP in an infected and non-agroinfiltered leaf.

Figure 4: examplary photos of a Vitis vinifera plant at 72 h from contamination with Botrytis cinerea (10,000 cfu/leaf) . (A) untreated plant. (B) plant treated with agroinfiltration with Agrobacterium tumefaciens expressing PGIP and stilbene synthase cloned in 3 '-5', leaf indicated by the arrow. (C) plant treated with agroinfiltration with Agrobacterium tumefaciens expressing PGIP and stilbene synthase cloned in 5' -3', leaf indicated by the arrow. Figure 5: expression levels of the GAPDH gene (·) and of the hyper-expressed PGIP + Stilbene synthase construct .

Figure 6: exemplary photos of Vitis vinifera leaves contaminated with Plasmopara viticola treated with Agrobacterium tumefaciens expressing PGIP and stilbene synthase cloned in 3'-5', sprayed on a single leaf. (A) time 0; (B) 1 day after treatment; (C) 2 days after treatment; (D) 3 days after treatment. The arrows highlight the treated area.

Figure 7: exemplary photos of Vitis vinifera leaves contaminated with Plasmopara viticola treated with Agrobacterium tumefaciens expressing PGIP and stilbene synthase cloned in 3'-5', sprayed on a single leaf. (A) time 0; (B) 2 days after treatment; (C) 3 days after treatment; (D) 6 days after treatment; (E) 10 days after treatment. The arrows highlight the treated area.

Figure 8: exemplary photos of Vitis vinifera leaves contaminated with Plasmopara viticola treated with Agrobacterium tumefaciens expressing PGIP and stilbene synthase cloned in 3'-5', sprayed on a single leaf. (A) time 0; (B) 1 day after treatment; (C) 10 days after treatment. The arrows highlight the treated area.

Detailed description of the invention:

The method according to the present invention has surprisingly eliminated the effects observed at level of the Vitis vinifera leaves and berries caused by fungal contamination thereof.

In one embodiment, Vitis vinifera was exposed by agroinfiltration to PGIP SEQ ID no. 1 and to Stilbene Synthase SEQ ID. no. 2. In a further experiment, Vitis vinifera was exposed by agroinfiltration to PGIP SEQ ID no. 1 and to inverted Stilbene Synthase SEQ ID. no. 2. The enzymes of interest were expressed in the plant after contamination with Botrytis cinerea.

In one embodiment, said treatment is obtained with

Agrobacterium tumefaciens wherein 1 μg of the Prl 101-AN vector comprising the enzymes of interest had previously been electroporated . The electroporation was conducted at a power of 2.5 kV 25 iF, 200 Ω.

Preferably, said treatment takes place in silwet velonex (heptamethyltrisiloxane, polyalkylene oxide- modified) wherein said vehicle was shown to be surprisingly effective in spreading the cure and stabilization of the disease.

The 24-hour effect from the agroinfiltration of PGIP and stilbene synthase is shown in Figure 4. The effect is such that it nullifies the attack of the exogenous polygalacturonases of botrytis on the leaf. On the same leaf no shrinkage, atresia of phloem transport systems, or rottenness are observed. This is due to the activation of a cascade effect of phytoalexins and polygalacturonase inhibitors. With reference to the leaves indicated by the arrows in Figure 4 (B) , the leaf is stable, vital, very moist and curled on itself, due to the massive presence of fluids recalled by the combined effect of PGIP and Resveratrol synthase.

In a further embodiment, wherein a construct coding for inverted stilbene synthase is used in combination with PGIP, the effect obtained is shown in Figure 4 (C) . The leaf, 24 hours after treatment, has grey plates keeping the leaf vital even in the infection margin until an effective regeneration of the underlying plant tissue is obtained .

The combined action of PGIP and stilbene synthase has an immediate effect on the leaf such to inhibit the development of the disease which is no longer present on the plant.

What is incredible is the reaction time of only 24h from exposure to agroinfiltration, exposure that follows of 48h the infection by Botrytis cinerea.

At 72h from the agroinfiltration, the treated leaves are vital, with absence of rottenness and atresia of the lymphatic vessels and in the process of regeneration both where said agroinfiltration takes place with PGIP and stilbene synthase, and where the treatment comprises the inverted stilbene synthase. As a further check, treatments with the individual culture media and/or the tested leaf adhesives were performed, including the preferred Velonex. No recovery effect has been observed. Velonex accelerates the disease but localized in plant tissues, preventing the spread in other rows, and avoiding the use of copper and other metals with oxidation-reductive action to stop the infection .

Figure 2 shows the expression levels of the Stilbene synthase gene (continuous line) and PGIP (dashed line) in Vitis vinifera at 24h from the agroinfiltration, obtained by RT-PCR assay.

Figure 3 shows how, in the absence of agroinfiltration, the PGIP and stilbene synthase levels of the leaf are below the detection threshold.

At phenotypic level, the method according to the present invention has proved capable of circumscribing the disease in Vitis vinifera to a necrosis area limited in at least 80% of the cases, or in at least 85%, or in at least 90%.

Where said plants are exposed to the treatment according to the present invention before the pathogenic attack, in at least 80% of the samples, or in at least 85%, or in at least 85%, a complete resistance of the plant that is not attacked has been observed. Figure 5 shows the expression levels, in the treated plant, of the control DNA (GAPDH) and of the DNA coding for the enzymes of interest. It is important to note that the expression levels indicate a massive presence in the plant of the DNA of interest. After a first phase in which the two curves overlap, at 120 hours the two curves are well separated, demonstrating that the construct is hyper- expressed with respect to the GAPDH gene. The method according to the present invention allows a correct transcription of PGIP and Stilbene Synthase.

The herein-presented data therefore show how the expression in Vitis Vinifera of PGIP SEQ ID no. 1 and/or stilbene synthase SEQ ID no. 2 and/or inverted stilbene synthase is able to decrease the effects induced by fungal attacks on the plant.

Example 1: treatment in field

Rows of Vitis vinifera contaminated with Plasmopara viticola were treated in the spring season with Agrobacterium tumefaciens expressing PGIP and stilbene synthase cloned in 3 '-5', sprayed on a single leaf in the presence of an adhesive.

The photos shown in Figures 6, 7 and 8 refer to three replicates, experiments conducted in parallel on different plants belonging to different rows. Panel A is an example of a leaf immediately after treatment. In panel B, the same leaf after 24 hours from treatment with PGIP + resvetrarol synthase. The blockage of the disease is evident. In panel C and panel D, respectively at 48h and 72h from treatment, the oxidation is indicative of a complete blockage of the disease. The arrows highlight the area affected and recovered with the treatment.

Figure 7 shows the chronology of the experiment on a different leaf, belonging to a different row. Panel (A) : immediately after treatment. At panel (B) there is shown how the same leaf, with infection also under the leaf page, after 2 days from treatment exhibits a blockage of the disease. At panel (C) , the same leaf after 3 days from treatment. After 6 days, panel (D) , the disease is completely oxidized and blocked. The blockage of the disease is persistent, as shown by the photo of the panel (E) obtained 10 days after treatment. It is important to note that the application was single at day 0. After the application, rains have occurred that have not diminished the effectiveness of the treatment.

In Figure 8, the exemplary results of the third replicate. Panel (A) : an exemplary leaf immediately after treatment; at the panel (B) the same leaf after one day from the treatment, where the blockage of the disease is already evident. The blockage is persistent, as observed at 10 days from treatment (panel C) .

Experimental :

Ampli ication of pRlOl-AN vector: 1) Stirring gently E. coli DH5 competent fresh cells and transfer 100 μΐ into a polypropylene tube.

2) Adding to 100 μΐ of pR101-AN cells in amounts <

10 ng .

3) Incubating in an ice bath for 30' .

4) Incubating at 42 °C for 45' ' .

5) Returning to the ice bath for 1-2 minutes.

6) Adding the SOC medium, pre-incubated at 37 °C up to a final volume of 1 ml.

7) Incubating by stirring at 160-225 rpm for 1 hour at 37 °C.

9) Plating on selective media, typically less than 100 μΐ for each 9 cm diameter plate.

10) Incubating overnight at 37 °C.

11) Selection of the colonies and amplification thereof by incubation overnight at 37 °C in liquid LB with resistance .

Extraction of the amplified plasmid

Once cloned after growth at +37 °C, the replicated plasmid is extracted from E. Coli according to methods known to those skilled in the art.

Enzymatic cut and linearization

Multiple reaction of enzymatic digestion and linearization.

In a final volume of 20 μΐ, the following are mixed:

Buffer 10X 2 μΐ DNA Prl01-An Da in an amount from 0.2 to 1 μg Restriction enzymes as follows:

1 μΐ KpNI

1 μΐ BamHI

1 ul Smal

1 ul Sal I

Nuclease-free water q.s.

The reaction proceeds according to the protocol known to those skilled in the art.

Product ligation

In a final volume of 20 μΐ, the following are mixed: DNA of the linearized vector as above

DNA insert from 10 to 100 ng, molar excess 3:1 with respect to the vector DNA

T4 DNA Ligase Master Mix 5 μΐ

Nuclease-free water q.s.

Cloning in E. coli

The plasmid comprising the genes of interest following the ligation is replicated to obtain the necessary amounts using DH5 cells according to methods known to those skilled in the art.

Plasmid purification

The amplified plasmid is purified according to methods known to those skilled in the art.

Electroporation in Agrobacterium tumefaciens 1. Placing 1.5 ml tubes containing competent cells of Agrobacterium tumefaciens LBA4404 in ice.

2. Adding 1 μΐ (1 ng) of binary vector plasmid DNA to 20 μΐ of competent cells and stirring gently.

3. Placing the 0.1 cm electroporation cuvette on ice .

4. Setting the Gene Pulser II to 25 iF, 200 Ω and 2 - 2.5 kV. * 1

5. Transferring the cells and DNA prepared in step 2 into the electroporation cuvette and electroporation.

6. Removing the cuvette from the electroporator , adding 1 ml of SOC * 2 media and transferring into a 14 ml round bottom tube.

7. Incubating for 1 hour at 30 °C, stirring at 100 rpm.

8. Plating 50 - 100 μΐ of cells onto LB agar plates with 50 g/ml kanamycin * 3 and incubating for up to 48 hours at 30 °C.

9. Amplifying in liquid LB with Kanamycin at 30 °C. Agroin iltration/adhesion

The bacteria are filtered on leaves by insulin syringe and/or by spraying on the leaf the compound containing 0.005% of adjuvant for agro-pharmaceuticals. A preferred aspect is the use of a leaf adhesive, in a preferred embodiment said co-adjuvant is "Silwet Velonex" for a broad-spectrum application.

Claims

1. A method for inducing or increasing resistance to one or more pathogens in a plant, plant part, or plant cell wherein said method comprises introducing a nucleotide sequence coding for the PGIP polygalacturonidase inhibitor and a nucleotide sequence coding for the stilbene synthase in a plant, plant part, or plant cell and expressing said nucleotide sequence in the plant, plant part, or plant cell, wherein said nucleotide sequence coding for PGIP and said nucleotide sequence coding for stilbene synthase is increased in the plant, plant part, or plant cell with respect to the original plant, plant part, plant cell, which has not received said sequence .

2. A method according to claim 1, wherein said plant is Vitis vinifera, Prunus persica, Fragaria sp.

3. A method according to claims 1 or 2, comprising the expression of one or more nucleic acid molecules comprising the nucleotide sequences selected from the group consisting of:

a) the nucleotide sequence coding for PGIP, SEQ ID no. 1, cloned in 3 '-5' and/or 5 '-3';

b) the nucleotide sequence coding for stilbene synthase, SEQ ID no. 2, cloned in 3 '-5' and/or 5 '-3';

c) the nucleotide sequence having at least 80% sequence identity with SEQ ID no. 1 ; d) the nucleotide sequence having at least 80% sequence identity with SEQ ID no. 2.

4. A method according to one of claims 1 to 3, wherein said one or more pathogens belong to the class of oomycetes or Sclerotiniaceae .

5. A method according to one of claims 1 to 4, wherein said pathogens are selected from Plasmopara viticola and Botrytis cinerea.

6. A method according to one of claims 1 to 5, wherein said plant part is the leaf.

7. A method according to one of claims 1 to 6 comprising introducing into said plant an expression vector coding for said nucleotide sequence (s) under the control of a promoter that is active in plants.

8. A method according to claim 7, wherein said vector is introduced by means of agroinfiltration .

9. A method according to claim 7, wherein said vector is introduced by application, preferably by spray application .

10. A method according to one of claims 7 to 9, wherein said vector is introduced with the aid of an adhesive.

11. An expression vector comprising at least one fragment of a nucleic acid molecule comprising SEQ ID no. 1 and at least one fragment of a nucleic acid molecule comprising SEQ ID no. 2.

12. A DNA expression cassette comprising a nucleic acid molecule that is essentially identical to the nucleic acid molecule of SEQ ID no. 1 and/or a nucleic acid molecule that is essentially identical to the nucleic acid molecule of SEQ ID no. 2.

13. An expression vector according to claim 11 which is PRI-101-AN.

14. A method according to one of claims 1 to 10, wherein said carrier is carried by Agrobacterium tumefaciens .