Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide confers pathogen or pest resistance
Reexamination Certificate
1998-08-24
2001-08-07
Prouty, Rebecca E. (Department: 1652)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide confers pathogen or pest resistance
C800S301000, C435S200000, C435S209000, C435S069100, C435S320100, C536S023200, C424S094610, C424S094200, C514S012200
Reexamination Certificate
active
06271438
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to a pathogen-resistant organism and to a process for generating it.
BACKGROUND OF THE INVENTION
It is known in the state of the art that infestation of a plant by pathogens causes a series of different reactions. These include, for example, changes in the cell wall structure, the synthesis of phytoalexins which have antimicrobial activity, the accumulation of so-called PR proteins (pathogenesis-related), protease inhibitors and enzymes with hydrolytic functions (Hahlbrock and Grisebach in Ann. Rev. Plant. Physiol., 30 (1979), 105-130).
Many pathogens (fungi and insects) have chitin as a constituent of their cell wall. By contrast, plants possess no chitin. It has now been demonstrated in some cases that there is enhanced production of chitinases in plants after infestation by pathogens. Chitinases are among the enzymes with hydrolytic functions and they catalyze chitin breakdown. It has now been possible to show that plants acquire an increased resistance to pathogens by the production of chitinases.
It is furthermore known to use a gene from barley plants whose gene product codes for an inhibitor of fungal protein synthesis. The incorporation of a corresponding inhibitor gene in transgenic plants led to improved resistance to fungi.
Finally, it has also been disclosed that the use of a polypeptide from Aspergillus giganteus is able to protect, by virtue of its antifungal activity, plants from infestation by fungi.
However, given this state of the art there is a need to provide further transgenic pathogen-resistant organisms. Moreover, the organisms which are particularly desired are those whose resistance is increased overall by comparison with the known organisms or is extended with respect to the number of possible pathogens.
This problem is solved by a transgenic pathogen-resistant organism having the features of the present invention.
The invention is based on the surprising finding that the incorporation of at least two different genes with pathogen-inhibiting action into the genome of an organism assists the latter to resistant pathogens to an extent going far beyond an additive effect of each of the genes on its own.
The dependent claims indicate further embodiments of the invention.
The genes can code for gene products which reduce the vitality of fungi. In particular, the genes can be of fungal, bacterial and plant, animal or viral origin. In particular, the gene products have properties which promote resistance to fungi. The gene products are chitinase (ChiS, ChiG), glucanase (GluG), protein synthesis inhibitor (PSI) and antifungal protein (AFP).
The transgenic pathogen-resistant organism can be a plant, and tobacco, potato, strawberry, corn, rape or tomato plants are preferred.
The invention also relates to DNA-transfer vectors with inserted DNA sequences as are indicated in detail in this description.
The invention furthermore relates to a process for the generation of pathogen-resistant organisms as are described herein, wherein at least 1 gene with pathogen-inhibiting action is transferred into the genome of an organism, and the pathogen-resistant organism is obtained
(a) by crossing the organism with another, optionally transgenic, organism which contains at least one other gene with pathogen-inhibiting action, and subsequently selecting, and/or.
(b) by transformation of this other gene with pathogen-inhibiting action into the organism. The process can be used with DNA-transfer vectors with inserted DNA sequences corresponding to a gene with pathogen-inhibiting action as described herein.
Finally, the invention relates to a process for the generation of pathogen-resistant organisms, wherein vectors which comprise more than one gene with pathogen-inhibiting action are used for the transformation into the genome of an organism.
The invention also relates to a process for ensuring the resistance of organisms to pathogens, characterized in that the organism used is a transgenic pathogen-resistant organism according to the present invention or an organism whose genome contains at least one gene complying with the definitions used herein and at least one substance which is not expressed by the organism but corresponds to any other one of the gene products complying with the definitions given in this application is applied to the organism.
It was possible to achieve the synergistic effects very particularly with transgenic pathogen-resistant organisms to which the gene sequences which coded for proteins of the attached sequence listings A to E, or corresponded to the latter, were transferred or transfected. ChiS:
A DNA fragment is 1.8 Kb in size, that codes for a chitinase called ChiS (EQ ID NO:8) was isolated from the soil bacterium Serratia marcescens. In vitro investigations with purified ChiS protein sowed that it is able effectively to inhibit the growth of fungi, even in low concentrations. The reason for the inhibition is that the ChiS protein has a chitinase activity which is able to damage the tips of the fungal hyphae. In this way the fungus is unable to grow further and is inhibited. PSI:
The PSI gene originates from barley and codes for a protein which inhibits protein synthesis by fungi. In vitro tests show that even low concentrations of PSI are sufficient to inhibit various fungi such as, for example, Rhizoctonia solani.
AFP:
It is possible for a polypeptide which has antifungal activity to be isolated from the fermentation broth of Aspergillus giganteus and to be sequenced. This polypeptide is suitable as antifungal agent, for example as spraying agent and as preservative for industrial products and human and animal foods. It can furthermore be combined with other substances which have pesticidal activity, fertilizers or growth regulators. Inhibitory activities against fungi were detectable inter alia against various Aspergillus, Fusaria, Phytophthora and Trichlophyton species.
ChiG and GluG:
Two genes which code, respectively, for a chitinase (ChiG) and glucanase (GluG) can be isolated from certain types of barley. Purified ChiG protein or GluG protein inhibits various phytopathogenic fungi in virto (inter alia Rhizoctonia solani) (see R. Leah et al., Journal of Biological Chemistry, Vol. 266, No. 3 (1991), pages 1564-1573).
SUMMARY OF THE INVENTION
The inventors have now found, completely, surprisingly, that an at least binary combination of expression of PSI, AFP, ChiS, ChiG or GluG leads to synergistic effects in respect of the acquired resistance to fungi in transgenic plants. In particular, the effects of the individual substances in the combination are markedly exceeded. These include resistance to the fungus Rhizoctonia solani, Sclerotinia infestation, Botrytis infestation, etc.
Combinations according to the invention are (DNA and/or polypeptides):
(binary combinations)
ChiS, GluG; ChiS, PSI; ChiS, ChiG; ChiS, AFP; GluG, PSI; GluG, ChiG; luG, AFP; PSI, ChiG; PSI, AFP;
(ternary combinations)
ChiS, GluG, PSI; ChiS, GluG, ChiG; ChiS, GluG, AFP; GluG, PSI, ChiG; GluG, PSI, AFP; PSI, ChiG, AFP; ChiG, AFP, GluG
(quaternary combinations) ChiS, GluG, PSI, AFP; ChiS, GluG, PSI, ChiG;
(quinary combination)
ChiS, GluG, PSI, AFP, ChiG
The invention furthermore relates to the combined use of the proteins with pathogen-inhibiting action, preferably ChiS, PSI, AFP, ChiG and GluG, against pathogens. Combined use also means in this context that at least a first pathogen-inhibiting substance is expressed by the organism and at least a second substance which has pathogen-inhibiting action is applied to the organism from outside.
The agents according to the invention also include those which contain the abovemented proteins in at least binary combination. The agents according to the invention can contain other active substances besides the proteins. The other active substances can be pesticides, fertilizers and/or growth regulators, and the agents according to the invention can be prepared in various formulations such as concentrates, emulsions, powders, formulations carriers, mixtures with other active substances, et
Chet Ilan
Eckes Peter
Gornhardt Birgit
Jach Guido
Logemann Jurgen
Baker & Botts LLP
Prouty Rebecca E.
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