Nucleic acid comprising the sequence of a promoter...

Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part

Reexamination Certificate

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C800S278000, C536S023600, C435S419000, C435S320100

Reexamination Certificate

active

06800794

ABSTRACT:

BACKGROUND OF THE INVENTION
The present invention relates to plants which exhibit improved resistance to certain pathogenic agents which are sensitive to stilbenes, and relates, more specifically, to a set of constructs which combine a plant promoter which can be induced by a biotic stress, which stress is engendered, in particular, by the said pathogens, with (a) gene(s) encoding a stilbene synthetase.
A large part of the world harvest of cultivated plants is regularly destroyed by parasites and pathogens. Among the possible options for decreasing or preventing the attack by these parasites on cultivated plants, chemical control (plant protection treatments) is the method which is most used. Nevertheless, the application of chemical products is not without consequences for the environment and sometimes presents technological problems as, for example, the appearance of new resistant pathogenic strains or, in the field of oenology, the difficulties which can arise during fermentations (the use of inhibitors of sterol biosynthesis can block yeast growth at the end of fermentation) or the presence of chemical products, such as procymidone, an anti-Botrytis product, which are sometimes found in wine.
The control method which consists in improving the resistance of cultivated plants to the diseases which are caused by these pathogens has been envisaged as a way of overcoming the drawbacks associated with chemical control. It is possible, for example, in a first approach, to achieve this improvement by the sexual route, i.e. using classical genetics, by hybridizing the plants whose resistance is to be improved with tolerant varieties. Nevertheless, this approach is not always feasible (tolerant natural variety not known) or is not permitted by legislation such as, for example, in viticulture as a result of French legislation on Appellations d'Origine Controlée (A.O.C.) (registered designations of origin) which limits the grapevine varieties which are to be used for a given appellation (designation).
In a second approach, it is possible, using the modern techniques of cell and molecular biology, to integrate, into the genome of the plant, one or more homologous or heterologous genes which make it possible to overexpress or express a molecule of interest, which is of protein nature, in order to increase the production of a metabolite, or a metabolic pathway, or to open a new biosynthetic pathway or to synthesize a novel molecule for example for increasing the opening of a new biosynthetic pathway, for example increasing the resistance of the plant by reinforcing its defence mechanisms with regard to the pathogens in question.
There are several different defence mechanisms of this type in plants. Some can be regarded as being passive and are linked to the physicochemical characteristics of the cells, the epidermal tissues and/or the organs of the plant. Others belong to the dynamics of gene/gene interactions (plant resistance genes and pathogen avirulence genes, mechanisms of host/pathogen interactions). While these interactions can lead to the development of a hypersensitivity reaction (rapid death of the cells of the plant around the point of infection in order to block colonization of the plant by the microorganism), they can also lead to the synthesis and accumulation of a whole series of compounds. Of these, some can be parietal constituents which are involved in the formation of a “physical” barrier around the point of infection (callose, lignin, hydroxyproline-rich protein: HRGP, etc.), and other compounds can be molecules having antimicrobial functions which are more or less well defined (phytoalexins, pathogen-associated proteins: PR proteins (pathogenesis-related proteins), etc.). The molecules of the phytoalexin type which are synthesized and accumulated by plants during, for example, host/pathogen interactions include, in particular the stilbenes, which are toxic, in particular for microorganisms. The term stilbene designates a group of chemical substances which possess the trans-diphenyl-1,2-ethylene skeleton as the common basal structure, with resveratrol and pinosylvine being among the simplest. This basal skeleton is synthesized in plants by a stilbene synthetase or related enzymes from substrates such as malonyl-CoA, cinnamoyl-CoA or coumaroyl-Coa, which are substances which are present in all plants (flavonoid precursors). Genes for stilbene synthetase or related enzymes have been isolated, sequenced and cloned, in particular from groundnut, orchid and grapevine. Using these genes, it has been possible to transform plants such as potato, lucerne or tobacco, with these plants then exhibiting greater resistance than untransformed plants to pathogen attack (EP-309862; EP-648839; MELCHIOR, F. et al., Arch. Biochem. Biophys. 1991, 288, 2, 552-557; WIESE, W. et al., Plant Mol. Biol. 1994, 26,2,667-677; HAIN, R. et al., Nature 1993, 361, 153-156).
The expression or overexpression of these molecules having antimicrobial functions can provide plants with a “natural” resistance in response to stresses, in particular stresses of the microbial type. However, constitutive overexpression of this type of protein necessarily has disadvantages for the plant (energy cost, slowing down of growth, etc.) (FISCHER, R. et al., The Plant Journal 1997, 11, 3, 489-498).
On the other hand, in some plants, such as grapevine or herbaceous plants, stilbenes are only found in some healthy tissues and at very low concentrations. Conversely following an infection or a lesion, these stilbenes increase strongly at the infected or damaged site, since the stilbene synthetase genes are inducible under conditions of biotic or abiotic stress (for example wounds, ultraviolet rays, etc.).
Nevertheless, this regulation is rarely present in plants of agricultural interest or, when it is present, it can be insufficiently effective. For example, studies on phytoalexin synthesis in the grapevine have demonstrated that the only healthy tissue in which stilbenes, including resveratrol, are present is healthy wood tissue. Stilbene is found to be present in the tissues of the grape berry when, on the one hand, the berry has been subjected to a stress such as attack by a pathogen (
Botrytis cinerea
for grey mould or
Plasmopora viticola
for grape downy mildew) and, on the other hand, only during the period up to the incipient ripening of the young fruit. By contrast, the concentration of the stilbene decreases strongly from incipient ripening to maturation. However, damage due, for example, to Botrytis is rarely encountered during the period up to incipient ripening but rather during the period close to maturation of the berry. For this reason, expression of the stilbene synthetase gene has to be controlled with strong promoters which escape the natural regulation of the gene and which should be inducible, in particular by the stress itself. The present invention specifically relates to a promoter of this nature.
BRIEF SUMMARY OF THE INVENTION
The present invention relates to nucleic acids which comprise the sequence of the promoter for a lucerne PR protein linked to at least one sequence of a gene encoding a stilbene synthetase.
The invention relates, in particular, to nucleic acids according to the invention, characterized in that the promoter for a lucerne PR protein is a promoter which can be induced in plants, in a tissue-specific manner or not, by a biotic or abiotic stress.
The invention also relates to nucleic acids according to the invention, characterized in that the sequence of the promoter for a lucerne PR protein is selected from the group comprising:
a) the IND S1 sequence,
b) any sequence corresponding to a fragment of the IND S1 sequence and having a promoter sequence effect in plants.
The sequences of the promoter for a lucerne PR protein are preferred which exhibit at least 80% homology with the IND S1 sequence. Those sequences are particularly preferred which exhibit at least 90% or 95% homology with the said sequence.
The sequences of promoters for lucerne PR proteins according to the invent

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