WO2011107537A1

WO2011107537A1 - New plants with modified expression or activity of ethylene response factors

Info

Publication number: WO2011107537A1
Application number: PCT/EP2011/053168
Authority: WO
Inventors: Mondher Bouzayen; Alain Latche; Jean-Claude Pech; Julien Pirrello; Farid Regad
Original assignee: Institut National Polytechnique De Toulouse
Priority date: 2010-03-05
Filing date: 2011-03-03
Publication date: 2011-09-09

Abstract

The present invention concerns a Solanacea plant, having a genetic alteration in the regulatory or in the coding sequence of a gene encoding an Ethylene Responsive Factor (ERF), said ERF being a functional homologous of the sequences SEQ ID NO 1 or SEQ ID NO 2, wherein said genetic alteration confers at least one of the following properties to the Solanaceae plant: resistance to insects, modified internodes length, modified ripening time, and modified fruit shelf-life.

Description

NEW PLANTS WITH MODIFIED EXPRESSION OR ACTIVITY OF

ETHYLENE RESPONSE FACTORS INTRODUCTION

Solanaceae is a family of flowering plants that contains a number of important agricultural plants. The family includes Datura (Jimson weed), Mandragora (mandrake), belladonna (deadly nightshade), Capsicum (paprika, chili pepper), Solanum (potato, tomato, eggplant), Nicotiana (tobacco), and Petunia (petunia). With the exception of tobacco (Nicotianoideae) and petunia (Petunioideae) most of the economically-important genera are contained in the sub- family Solanoideae.

As for all economically-important plants, there is a need to generate plants of this family with new phenotypic properties including, but not limited to, increased resistance to pests, insects and diseases, modified internodes length, modified properties of the fruit.

The plant hormone ethylene is involved in the control of a myriad of plant developmental processes and is also known to play an active role in plant responses to biotic and abiotic stresses. It is well established that this plant hormone exerts its effects by modulating the expression of a large number of genes through the activation of specific members of the Ethylene Response Factor (ERF) gene family. ERFs are plant specific transcriptional regulators encoded by one of the largest gene family known in plant kingdom. Because ERFs are down-stream mediators of ethylene action, they are the main actors for the amplification and diversification of plant responses to ethylene.

The making of a fruit is a developmental process unique to plants, involving a complex network of interacting genes and signalling pathways. In the case of fleshy fruit, this process involves three main stages : (a) fruit set, (b) fruit development, and (c) fruit ripening. This latter stage is crucial for fruit quality and most of the sensory and health promoting compounds accumulate during the ripening step.

Ripening is a genetically programmed process orchestrated by complex interplay between endogenous hormones and environmental cues. The regulated changes at the level of gene expression are the first steps leading to the metabolic changes associated with fruit ripening. In climacteric fruit type like tomato, the phytohormone ethylene is a key regulator of this process.

Transcription factors from the ERF type are thought to be, at least partly, responsible of the complex network of metabolic activations associated with the ripening process, nevertheless, the mechanism by which they select ripening-specific genes remains largely unknown. Moreover, ERFs are involved in numerous developmental processes of the plant. ERFs have been shown to be involved in normal and abnormal plant processes such as plant defense, abiotic stress tolerance, and seed germination. PRIOR ART

At first, Ethylene Responsive Factors were isolated as GCC box binding proteins from tobacco (Ohme-Takagi and Shinshi, 1995). Later, ERFs have been identified in numerous plants including Arabidopsis Thaliana and Solarium lycopersicum (tomato).

A highly conserved DNA binding domain, known as the "AP2/ERF domain", consisting of 58 to 59 amino acids, is the unique structural feature of common to all factors belonging to this protein family. This ERF domain binds to DNA as a monomer, with high affinity.

ERFs can act as either transcriptional activators or repressors for GCC box- dependant gene expression (Fujimoto et al, Plant Cell, 2000).

In tomato, new members of the ERF family were identified in 2003 and their respective role and expression pattern were studied (Tournier et al, FEBS Letters, 2003). Four distinct classes of ERF were described, based on functionalities and structures of each subtype.

Later, the physiological function of one of the identified ERF was extensively studied. In particular, overexpression of said ERF gene in transgenic tomato line results in premature seed germination and increased ethylene sensitivity (Pirrello et al, 2006).

These data indicate that each ERF controls a specific subset of ethylene-regulated genes. Therefore, identifying the genes that are under the regulation of a given ERF will open new prospects towards the targeted control of genes involved in specific metabolic & developmental pathways. The modification of the expression and/or activity of each ERF can induce new and interesting phenotypic properties in plants.

DESCRIPTION OF THE INVENTION

Inventors have discovered that a Solanaceae plant having specific genetic alterations modifying the expression and/or the activity of at least one Ethylene Response Factor, shows some modifications of its phenotype, in particular in its resistance to insects, internodes length, fruit ripening time and/or shelf life.

Inventors have seleted two ERFs of primary importance, wherein the modification of the expression or activity of one of these ERFs in a Solanaceae plant results in a modification of these phenotypic properties. DETAILLED DESCRIPTION OF THE INVENTION

The present invention is related to a Solanacea plant, having a genetic alteration in the regulatory or in the coding sequence of a gene encoding an Ethylene Responsive Factor (ERF), said ERF being a functional homologous of the sequences SEQ ID NO 1 or SEQ ID NO 2, wherein said genetic alteration confers at least one of the following properties to the Solanaceae plant: resistance to insects, modified internodes length, modified ripening time, and modified fruit shelf-life.

The term of "Ethylene Responsive Factor" or "ERF" designates a transcription factor having as DNA binding domain, a domain called "AP2/ERF" consisting of 58 to 59 amino acids, highly conserved among all the factors of the family.

The term "functional homologous of a sequence" means according to the invention that the sequence of the polypeptide may include less or not exactly the same amino-acid than shown in SEQ ID NO 1 or NO 2, but still enough or equivalent amino acids to confer the Ethylene Responsive Factor activity. It is well known in the art that a polypeptide can be modified by substitution, insertion, deletion and/or addition of one or more amino acids while retaining its DNA binding activity. For example, substitutions of one amino-acid at a given position by a chemically equivalent amino-acid that do not affect the functional properties of a protein are common. For the purposes of the present invention, substitutions are defined as exchanges within one of the following groups:

^■ Small aliphatic, non-polar or slightly polar residues : Ala, Ser, Thr, Pro, Gly

^■ Polar, negatively charged residues and their amides : Asp, Asn, Glu, Gin

^■ Polar, positively charged residues : His, Arg, Lys

^■ Large aliphatic, non-polar residues : Met, Leu, He, Val, Cys

■ Large aromatic residues : Phe, Tyr, Trp.

Thus, changes which result in substitution of one negatively charged residue for another (such as glutamic acid for aspartic acid) or one positively charged residue for another (such as lysine for arginine) can be expected to produce a functionally equivalent product.

The positions where the amino acids are modified and the number of amino acids subject to modification in the amino acid sequence are not particularly limited. The man skilled in the art is able to recognize the modifications that can be introduced without affecting the activity of the protein. For example, modifications in the N- or C-terminal portion of a protein would not be expected to alter the activity of a protein.

In a specific embodiment of the invention, the polypeptide of the present invention have at least 70% identity with the sequences shown as SEQ ID NO 1 or SEQ ID NO 2, preferentially at least 80% identity and more preferentially at least 90% identity. Methods for determination of the percentage of identity between two protein sequences are known from the man skilled in the art. For example, it can be made after alignment of the sequences by using the software CLUSTALW available on the website http://www.ebi.ac.uk/clustalw/ with the default parameters indicated on the website. From the alignment, calculation of the percentage of identity can be made easily by recording the number of identical residues at the same position compared to the total number of residues. Alternatively, automatic calculation can be made by using for example the BLAST programs available on the website http://www.ncbi.nlm.nih.gov/BLAST/ with the default parameters indicated on the website.

In a particular embodiment of the invention, ERFS are selected among the proteins having an amino acid sequence as shown in SEQ ID NO 1 or 2.

The ERFs having the amino acids sequence as shown in SEQ ID NO 1 and in SEQ ID NO 2 have a constitutive expression in the plant, and are transcriptional activators of responsive genes.

The term "genetic alteration" designates a modification in the genome of the plant; said genetic alteration resulting from :

- a selection process of naturally occuring mutations, or

- a genetic manipulation of the plant.

The genetic alteration may be a mutation, a deletion or an overexpression of the gene encoding the ERF according to the invention. The genetic alteration is present in the regulatory or in the coding sequence of a gene.

To obtain an increase of the expression of a gene encoding a specific ERF, the man skilled in the art knows different methods, and for example:

Replacement of the endogenous promoter of said gene encoding an ERF with a stronger promoter;

Introduction into the cell of an expression vector carrying said gene encoding a specific ERF; the term « vector » refers to an extra-chromosomal element carrying genes or cassettes, that is usually in the form of a circular double-stranded DNA molecules, but may be a single strand DNA molecule, too. Both terms "vector" and "plasmid" are used indifferently.

Introduction of additional copies of said gene encoding a specific ERF into the chromosome;

stabilization of the encoded RNA messenger.

The modulation of the activity of a protein can also be achieved by various means known by the man skilled in the art, including but not limited to :

introduction of point mutations into the gene leading to the translation of a protein with an increased level of activity than the native protein;

stabilization i.e. the increase of life of the encoded protein. To obtain the attenuation of the expression of a gene, different methods exist, and for example:

Introduction of a mutation into the gene, decreasing the expression level of this gene, or decreasing the level of activity of the encoded protein;

- Replacement of the natural promoter of the gene by a low strength promoter, resulting in a lower expression;

Use of elements destabilizing the corresponding messenger RNA or the protein; Deletion of the gene if no expression is needed.

To obtain a partial or total inhibition of the transcriptional activity of one ERF, a classical method is the expression of the dominant negative (repressor) form of said ERF, inducing the suppression of the expression of the target genes. Construction of dominant negative forms are fully explained in (Hiratsu et al, 2003).

Genetic transformations of plants are now well known in the art, comprising introducing a new gene fragment in a plant cell and then regenerating a plant form the cell. The new gene fragment is preferably introduced with known techniques of particle bombardment and/or infection with a transformed Agrobacterium. Regeneration procedure are also well known in the art and documented for numerous plant today. The new gene fragment is preferably integrated into the plant cell genome. New techniques of homologous recombination and gene replacement are also known today to be effective in plant cells. The term « transformation » refers to the incorporation of exogenous nucleic acid by a cell, this acquisition of new genes being transitory (if the vector carrying genes is cured) or permanent (in the case the exogenous DNA is integrated chromosomally).

The term "resistance to insects" designates an increased tolerance to insects attacks. Preferentially, the genetic alteration in the regulatory or coding sequence of a member of the ERF family induces an increased plant tolerance to insects attacks such as: acarians, aphids, white flies, trips, caterpillar, leaf miner.

The man skilled in the art knows how to determine the resistance of a plant to a specific insect using screening method well known to the one skilled in the art of agronomy, agrochemistry and agriculture. Classical methods are described in manuals such as "Techniques for Evaluating Insect Resistance in Crop Plants" (from Charles M. Smith, Kansas State University; Z. R. Khan; Mano Dutta Pathak) or "Plant Resistance to Insects: A Fundamental Approach" (from C. Michael Smith).

The term "modified internodes length" means that the interval between two nodes (the internode) in the plant is modified, decreased or increased. This modification induces a modified size and/or architecture of the plant. Plants having a decreased internodes length present a plant architecture more compact.

The term "ripening time" designates the time necessary for the fruit to reach the breaker stage, meaning when it starts to turn from green to yellow/orange. The term "shelf life" designates the length of time that a fruit is given before it is considered unsuitable for sale or consumption. Shelf life is the recommendation of time that products can be stored, during which the defined quality of a specified proportion of the goods remains acceptable under expected conditions of distribution, storage and display.

Shelf life is most influenced by several factors: exposure to light and heat, transmission of gases (including humidity), mechanical stresses, and contamination by things such as micro-organisms. Product quality is often mathematically modelled around the fruit firmness/softness parameter.

In a specific embodiment of the invention, the Solanacea plant has a genetic alteration in the regulatory or in the coding sequence of the gene encoding the ERF being a functional homologous of the sequence SEQ ID NO 1, and said genetic alteration confers to the Solanaceae plant: resistance to insects and/or a modified internodes length.

In a first aspect of this embodiment, the genetic alteration induces an overexpression of said gene or an increase of the activity of said ERF protein, and said alteration confers an increased internodes length to the plant.

In a specific embodiment of the invention, the Solanacea plant has a genetic alteration that induces an inhibition of the expression of said gene, or a reduction of the activity of said ERF protein, and said alteration confers resistance to insects and/or a decreased internodes length to the plant.

In a preferred embodiment of the invention, the Solanacea plant expresses a dominant negative version of the wild type ERF (see above), and the plant has a dwarf phenotype, characterized by shorter internodes and a compact plant architecture.

In another embodiment of the invention, the Solanacea plant has a genetic alteration in the regulatory or in the coding sequence of the gene encoding the ERF being a functional homologous of the sequence SEQ ID NO 2, and said alteration confers to the Solanaceae plant a modified ripening time and/or a modified shelf life of the fruit.

In a specific aspect of said embodiment, the genetic alteration is an inhibition of the expression of said gene, or a reduction of the activity of said ERF protein, and said alteration confers a delayed ripening time and/or an extended shelf life to the fruit of the plant.

In a preferred embodiment of the invention, said Solanacea plant expresses a dominant negative version of the wild type ERF, and the fruit of the plant has a delayed ripening time.

In another embodiment of the invention, the Solanacea plant contains at least two genetic alterations:

one in the regulatory or in the coding sequence of the gene encoding the ERF being a functional homologous of the sequence SEQ ID NO 1 and one in the regulatory or in the coding sequence of the gene encoding the ERF being a functional homologous of the sequence SEQ ID NO 2,

and said genetic alterations confer at least two of the following properties to the Solanaceae plant: resistance to insects, modified internodes length, modified ripening time, and modified fruit shelf-life.

In a specific aspect of the invention, the genetic alteration in the regulatory or in the coding sequence of a gene encoding an Ethylene Response Factor is obtained by chemical mutagenesis. Chemical mutagens creating primarily point mutations and short deletions, insertions, transversions, and/or transitions (about 1 to about 5 nucleotides) can be used to generate mutations into the plants described in the present invention. For example, mutagens such as ethyl methanesulfonate (EMS), methylmethane sulfonate (MMS), N- ethyl-N-nitrosurea (ENU), triethylmelamine (TEM), N-methyl-N-nitrosourea (MNU), procarbazine, chlorambucil, cyclophosphamide, diethyl sulfate, acrylamide monomer, melphalan, nitrogen mustard, vincristine, dimethylnitosamine, N-methyl-N'-nitro- Nitrosoguanidine (MNNG), nitrosoguanidine, 2-aminopurine, 7,12 dimethyl- benz(a)anthracene (DMBA), ethylene oxide, hexamethylphosphoramide, bisulfan, diepoxyalkanes (diepoxyoctane (DEO), diepoxybutane (BEB), and the like), 2-methoxy-6- chloro-9[3-(ethyl-2-chloro-ethyl)aminopropylamino] acridine dihydrochloride (ICR- 170), formaldehyde, and the like may be used to mutagenize the plant tissue in order to generate plants having a genetic alteration in a gene encoding an ERF.

Preferentially, seeds from the Solanaceae plant are mutagenized and then grown into Ml plants. The Ml plants are then allowed to self-pollinate and seeds from the Ml plant are grown into M2 plants, which are then screened for mutations in their ERF genes.

Preferentially, seeds are placed on a shaker in a fume hood at ambient temperature. The mutagen ethyl methanesulfonate (EMS) is added to the imbibing seeds for final concentrations ranging from about 0.1% to about 1.6% (v/v). Following a 24-hours incubation, the EMS solution was replaced with fresh H₂0. Finally, the mutagenized seeds are planted in potting soil and allowed to germinate in the greenhouse. Four to six week old surviving plants are transferred to the field to grow to fully mature Ml plants. The mature Ml plants are allowed to self-pollinate and then seeds from the Ml plant are collected and planted to produce M2 plants.

In another aspect of the invention, the genetic alteration is present in all cells of the plant, or only in part of them. This genetic alteration may be constitutive or induced at a specific time, by addition of inducers.

In a preferred aspect of the invention, the Solanacea plant belongs to the genus Solarium or to the genus Capsicum. Preferentially, the plant is chosen from the group consisting of : Bell Pepper (Capsicum annuum), Eggplant (Solarium melongena), Potato (Solarium tuberosum), Tomato (Solarium ly coper sicum), and more preferably tomato plants. DRAWINGS

Figure 1: Plant size of lines LI, L2, L3 expressing a dominant negative version of the ERF having the SEQ ID NO 1, and of the wild type plant (WT). Plant sizes have been measured in centimeter on 5 weeks old plant. The presented data correspond to the average of the size of 10 plants for each independent line. Mann whitney or t test have been used according the data. Bars represent standard deviation of 10 independent plants.

Figure 2: Intemodes length of lines LI, L2, L3 expressing a dominant negative version of the ERF having the SEQ ID NO 1, and of the wild type plant (WT). Intemodes length have been measured in centimeter on 5 weeks old plant. The presented data correspond to the average of the intemode length between the 4^th and the 5^th leaves on 10 plants for each independent line. Mann whitney or t test have been used according the data. Bars represent standard deviation of 10 independent plants.

Figure 3: Intemodes length of lines LI, L2, L3 expressing a dominant negative version of the ERF having the SEQ ID NO 1, and of the wild type plant (WT). Intemodes length have been measured in centimeter on 5 weeks old plant. The presented data correspond to the average of the intemode length between the 3^th and the 4^th leaves on 10 plants for each independent line. Mann whitney or t test have been used according the data. Bars represent standard deviation of 10 independent plants.

Figure 4: Number of globular trichomes per 10 mm², for each line LI, L2, L3 expressing a dominant negative version of the ERF having the SEQ ID NO 1, and for the wild type plant (WT). The presented data correspond to the average of measures obtained on leaflet harvested on 10 plants 5 weeks old. Mann whitney or t test have been used according the data. Bars represent standard deviation of 10 independent plants.

Figure 5: Time to reach breaker stage is expressed in days. It corresponds to the time between the anthesis stage corresponding to the day of pollination and the moment where breaker stage is reach. The presented data correspond to the average of the number of day to reach the breaker stage of 60 fruits for each independent line LI, L2 expressing a dominant negative version of the ERF having the SEQ ID NO 2, and of the wild type plant (WT). Mann whitney or t test have been used according the data. Bars represent standard deviation of 60 fruits.

Figure 6: Firmness of fruit is measured in lines LI, L2, expressing a dominant negative version of the ERF having the SEQ ID NO 2, and in the wild type plant (WT). Fruit firmness is expressed in N/mm. The presented data correspond to the average of firmness measured on 60 fruits at Beaker stage plus 10 days for each independent line. Mann whitney or t test have been used according the data. Bars represent standard deviation of 60 fruits. EXAMPLES

Example 1 : Transformation of tomato plants with Agrobacterium tumefaciens Plant regeneration

Lycopersicon esculentum cv. microtom seeds were sterilized by shaking in 50% bleach for 10 min and wash 4 times with sterilized water. Surface-sterilized seeds were placed on 50 ml of MS medium (Murashige and Skoog) (table 1). Seedlings were grown at 25 °C and 70% relative humidity for 8 days, 8 h dark and 16 h light. Cotyledons of the sterile tomato seedlings were cut off, and the tips were removed and sectioned transversely with a scalpel in two fragments. Cotyledon pieces were placed upside down in 90x 15 mm Petri dishes containing KCMS medium (Table 1) and incubated for 24 hours at 25 °C in the dark.

Plant transformation

Agrobacterium tumefaciens strain C58 harbouring the binary vector pBCKH carrying the hygromycin resistance gene, was grown to 2 OD600. The bacteria are centrifuge 10 minutes at 3000 rpm. The bacterial pellet is diluted in KCMS liquid to obtain 0.5-0.8 OD600 prior to cocultivation. Tomato cotyledon explants were removed from the KCMS solid plates and transferred to the bacterial suspension for 30 min. Then, cotyledon explants were placed on KCMS medium (Table 1), for 2 days at 25 °C in the dark. After 2 days cotyledon explants were transferred to shoot regeneration medium (2Z) containing Augmentin 800 (Table 1) in plates at 25 °C and 70% relative humidity under fluorescent light (90 umol m-2 seg-1), 8 h dark and 16 h light. Two weeks later, when callus have been generated they were transferred on 2Z medium containing Augmentin 500. When plantlet are regenerated they are cut off and placed on a rooting medium (ENR) (Table 1) in sterile plant containers (90^χ90^χ90 mm). Transgenic plant selection

Dominant repressor constructs (SRDX) were under the transcriptional control of the cauliflower mosaic virus 35S promoter (CaMV 35S) and the nopaline synthase (Nos) terminator. Transformed lines were first selected on hygromycin (25 mg L-l) and then analyzed by semi quantitative PCR to check the expression of the transgene in the various transgenic lines obtained.

Example 2 : Phenotypic characteristics of new plants expressing a dominant negative version (SRDX) of the ERF having the SEQ ID NO 1 : ERF.B3::SRDX

Three transgenic lines expressing ERF.B3::SRDX have been generated as previously described. Plants are cultivated as well known by the man skilled in the art.

Measure of the plant size and internodes length on 5 weeks-old plant

• Plant sizes have been measured in centimeter on 5 weeks old plant. The presented data in figure 1 correspond to the average of the size of 10 plants for each independent line. Although the average size of the wild-type plant is about 15 centimeters, transgenic lines LI, L2 and L3 are under 10 centimeters.

• Internodes length have been measured in centimeter on 5 weeks old plant. Nodes correspond to the leaf insertion point. Nodes 3-4 correspond to the distance between the insertion point of the 3rd and the 4th true leaves, whereas nodes 4-5 correspond to the distance between the insertion point of the 4th and the 5th true leaves. The presented data in figures 2 and 3 correspond to the average of the internodes length of 10 plants for each independent line. Although the internodes length (node 4-5) of the wild-type plant is about 2,5 to 3 centimeters, those of transgenic lines LI, L2 and L3 are under 2 centimeters. The same difference is observed in figure 3.

Measure of trichome number on leaves

Number of globular trichomes (insects) per 10 mm², have been measured on 10 plants, 5 weeks old, for each line LI, L2, L3 expressing ERF.B3::SRDX, and for the wild type plant (WT). The presented data in figure 4 correspond to the average of measures obtained on leaflet harvested.

Trichomes are appendixes that secrete molecules involved in insects resistance. Their presence indicates a good defense reaction against insects. As shown in figure 4, number of trichomes is increased in new plants, suggesting that their defense reaction against insects is increased. Example 3 : Phenotypic characteristics of new plants expressing a dominant negative version of the ERF having the SEQ ID NO 2 (ERF.C2::SRDX)

Two transgenic lines expressing ERF.C2::SRDX have been generated as previously described. Plants are cultivated as well known by the man skilled in the art.

Measure of ripening parameters on fruits

The "time to get breaker stage" corresponds to the time between the anthesis stage and the moment when the fruit starts to turn from green to yellow/orange (previously described as breaker stage). The presented data in figure 5 correspond to the average of the number of day to reach the breaker stage of 60 fruits for each independent line LI, L2 and for the wild type plant (WT).

An average of about 45 days is observed for the fruits of the wild type plant; for the fruits of new lines LI and L2, the ripening time is longer, more than fifty days for LI .

Measurement of tomato firmness have been done on 60 fruits, at Beaker stage plus 10 days, for each independent line. A non destructive method adapted from Lesage et al. (1995) and Coombe (1992) has been used, based on the following mathematical formula: Fruit firmness (N.mm-1) = [Displacement of spring attachment (mm) x Spring stiffness (N.mm-1 )]/Displacement of sensor in fruit (mm)

Results are presented on figure 6 : at breaker stage + 10 days, fruits from the wild-type plants have a firmness of about 0.3 N/mm; at the same date, fruits from the transgenic lines LI, L2 have a stronger firmness, of about 0.35 to more than 0.4; fruits are more firm, and therefore are considered having an extended shelf life in compared to the wild type fruit.

These data indicate that fruits from the transgenic lines expressing ERF.C2::SRDX have a delayed ripeting time and an extended shelf life compared to the fruits from wild type plants.

References :

Lesage P., Destain M.-F. (1996) : Measurement of tomato firmness by using a non destructive mechanical sensor. Postharverst Biology and technology, 8 : 45-55.

Coommbe B.G (1992) : Research on development and ripening of the grape berry. American Journal of enology and viticulture. 43 : 101-110.

Charles M. Smith, Kansas State University; Z. R. Khan; Mano Dutta Pathak "Techniques for Evaluating Insect Resistance in Crop Plants" , CRC Press, December 17, 1993.

C. Michael Smith "Plant Resistance to Insects: A Fundamental Approach" Wiley- Interscience (September 1989).

Claims

1. A Solanacea plant, having a genetic alteration in the regulatory or in the coding sequence of a gene encoding an Ethylene Responsive Factor (ERF), said ERF being a functional homologous of the sequences SEQ ID NO 1 or SEQ ID NO 2, wherein said genetic alteration confers at least one of the following properties to the Solanaceae plant: resistance to insects, modified internodes length, modified ripening time, and modified fruit shelf- life.

2. The Solanacea plant of claim 1 , wherein the genetic alteration is in the regulatory or in the coding sequence of the gene encoding the ERF being a functional homologous of the sequence SEQ ID NO 1, and wherein said genetic alteration confers to the Solanaceae plant: resistance to insects and/or a modified internodes length.

3. The Solanacea plant of claim 2, wherein the genetic alteration induces an overexpression of said gene or an increase of the activity of said ERF protein, and wherein said alteration confers an increased internodes length to the plant.

4. The Solanacea plant of claim 2, wherein the genetic alteration induces an inhibition of the expression of said gene, or a reduction of the activity of said ERF protein, and wherein said alteration confers resistance to insects and/or a decreased internodes length to the plant.

5. The Solanacea plant of claim 4, wherein the encoded ERF protein is a dominant negative version of the wild type ERF, and wherein the plant has a dwarf phenotype, characterized by shorter internodes and a compact plant architecture.

6. The Solanacea plant of claim 1 , wherein the genetic alteration is in the regulatory or in the coding sequence of the gene encoding the ERF being a functional homologous of the sequence SEQ ID NO 2, and wherein said alteration confers to the Solanaceae plant : modified ripening time and/or modified shelf life of the fruit.

7. The Solanacea plant of claim 6, wherein the genetic alteration is an inhibition of the expression of said gene, or a reduction of the activity of said ERF protein, and wherein said alteration confers a delayed ripening time and/or an extended shelf life to the fruit of the plant.

8. The Solanacea plant of claim 7, wherein the encoded ERF protein is a dominant negative version of the wild type ERF, and wherein the fruit of the plant has a delayed ripening time.

9. The Solanacea plant of claim 1 , wherein it contains at least two genetic alterations, one in the regulatory or in the coding sequence of the gene encoding the ERF being a functional homologous of the sequence SEQ ID NO 1 and one in the regulatory or in the coding sequence of the gene encoding the ERF being a functional homologous of the sequence SEQ ID NO 2, and wherein said genetic alterations confer at least two of the following properties to the Solanaceae plant: resistance to insects, modified internodes length, modified ripening time, and modified fruit shelf-life.

10. The Solanacea plant of claim 1, wherein the genetic alteration in the regulatory or in the coding sequence of a gene encoding an Ethylene Responsive Factor (ERF) is obtained by chemical mutagenesis.

11. The Solanacea plant of claim 1, wherein the plant belongs to the genus Solanum or the genus Capsicum.

12. The Solanacea plant of claim 11 wherein the plant is chosen from the group consisting of: Solanum tuberosum, Solanum lycopersicum, Solanum melongena, Capsicum annuum.