Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide alters fat – fatty oil – ester-type wax – or...
Patent
1994-09-19
1998-04-28
Fox, David T.
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide alters fat, fatty oil, ester-type wax, or...
800DIG25, 435 692, 4351723, 4352523, 43525233, 4352542, 4353201, 435419, 536 236, 536 241, A01H 500, C12N 1529, C12N 1584, C12N 1570, C12N 1581
Patent
active
057446920
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
This invention relates to nucleotide sequences coding a endopolygalacturonase inhibitor.
More particularly, this invention relates to a gene coding a new protein having an inhibiting activity against the fungal endopolygalacturonase enzyme, or fractions thereof, and to the protein itself or fractions thereof. The invention relates also to recombinant vectors comprising such sequences, to cells and plants transformed by means of such recombinant vectors and to the use of such vectors and cells to produce the protein.
References are listed at the end of the specification in the order cited.
The fungal endo alpha-1,4-D-polygalactorunase enzyme (EC 3.21.15) is an important pathogenic factor for plants (1,2). The enzyme catalyzes the scission reaction of polygalacturonic acid and induces the subsequent solubilization of plant cell wall homo-alpha-1,4-D-galactorunanes, thus facilitating the penetration of fungi into plant tissues and then supplying the same fungi with nutritional substrates.
Moreover, the activity of such an enzyme could, if controlled, produce non-virulence factors, i.e. factors able to activate some plant defence mechanisms by converting plant cell wall homogalactorunanes into oligogalacturonides, which are known to stimulate phytoalexin synthesis and other defence mechanisms as well (2).
An inhibiting activity of fungal endopolygalacturonase, associated with the cell wall, named PGIP, has been detected in dicot plants.
The authors of the present invention have already detected such activity in different plant tissues of Phaseolus vulgaris L. (3), showing that it is specific for fungal endopolygalacturonases, with no activity against either bacterial or plant enzymes, or other pectic enzymes of microbial origin (4), Moreover, the authors found that the PGIP is able to stabilize in vitro and oligogalacturonide mixture which stimulates phytoalexins (5).
Therefore it is evident the need to purify and isolate the molecule responsible for such activity by means of cloning coding PGIP sequences. Said sequences may be inserted in suitable vectors to transform plants that are sensitive to fungi or other microorganism pathogens, into plants producing the PGIP protein on large scale. The protein could be then used with many advantages in the food industry.
SUMMARY OF THE INVENTION
Prior attempts to isolate the PGIP gene have been unsuccessful, essentially due to low levels of the protein in plant tissues.
The authors of the invention have now isolated and sequenced the PGIP protein coding DNA, both from a genomic and cDNA library, and have also identified the amino acid sequence of the PGIP protein.
An object of the present invention is a DNA fragment comprising a sequence coding a PGIP protein having an inhibiting activity of the fungal endo-alpha-1,4-D-polygalacturonase enzyme (PG), or parts thereof. Preferably said DNA fragment is of plant origin, more preferably isolated either from plants, or parts of plants, or in vitro cultured plant cells of the Phaseolus genus, most preferably of the Phaseolus vulgaris species.
According to a preferred embodiment said DNA fragment codes for a PGIP protein having the amino acid sequence of SEQ ID N15, or parts thereof. Alternatively said amino acid sequence SEQ ID N15 lacks or is substituted of one or more amino acids, preferably is a variant of a PGIP protein, more preferably said variant of a PGIP protein comprises the amino acid sequence SEQ ID N17, or parts thereof.
According to another embodiment said DNA fragment comprises the nucleotide sequence of SEQ ID N14, consisting of:
a coding region from nucleotide 1 to nucleotide 1026;
a 3'-end untranslated sequence from nucleotide 1027 to nucleotide 1116.
Alternatively said nucleotide sequence lacks or is substituted of one or more nucleotides, preferably comprising the nucleotide sequence of SEQ ID N16.
Another object of the invention is a DNA fragment hybridizing to at least one of DNA fragments having the sequences above described, preferably complementary to them.
Another obje
REFERENCES:
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Brown, A.E. and Adikaram, N.K.B. (1982), "The Differential Inhibition of Pectic Enzymes from Glomerella cingulata and Botrytis cinerea by a Cell Wall Protein from Capsicum annuum Fruit," Phytopath. Z. 105:27-38.
Favaron, F. et al. (1994), "Purification and molecular characterization of a soybean polygalacturonase-inhibiting protein," Planta 195:80-87.
Fielding, A. (1981), "Natural Inhibitors of Fungal Polygalacturonases in Infected Fruit Tissues," J. Gen. Microbiol. 123:377-381.
Johnston, D.J. et al. (1993), "A Protein from Immature Raspberry Fruits which Inhibits Endopolygalacturonases from Botrytis cinerea and other Micro-organisms,"J. Exp. Bot. 44(262):971-976.
Powell, A.L.T. (1994), "Glycoprotein Inhibitors of Fungal Polygalacturonases: Expression of Pear PGIP Improves Resistance in Transgenic Tomatoes," Plant Physiol. Suppl. 105:159.
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Stotz, H.U. et al. (1993), "Molecular Characterization of a Polygalacturonase Inhibitor from Pyrus communis L. cv Bartlett," Plant Physiol. 102:133-138.
Stotz, H.U. et al. (1994), "Structure and expression of an inhibitor of fungal polygalacturonases from tomato," Plant Mol. Biol. 25:607-617.
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De Lorenzo et al. (1991) "Cloning of the Polygalacturonase-inhibiting Protein (PGIP) of Phaseolus Vulgaris L.", J. Cell Biochem. Suppl., Keystone Symposium, The Genetic Dissection of Plant Cell Processes, Jan. 10-17, 1991 vol. 15A, pp. 54, Abstract A208.
Toubart et al. "Cloning and Expression of a Polygalacturonase-inhibiting Protein PGIP of Phaseolus Vulgaris L.", Biological Abstracts BR43:11387 and G. Bot. Ital. 124(4):151-152 1990.
Cervone et al. (1987) "Purification and Characterization of a Polygalacturonase-inhibiting Protein from Phaseolus Vulgaris L.", Plant Physiol. 85:631-637.
Salvi et al. (1990) "A Polygalacturonase-inhibiting Protein in the Flowers of Phaseolus Vulgaris L.", J. Plant Physiol. 136:513-518.
De Lorenzo et al. (1990) "Host-pathogen Interactions. XXXVII. Abilities of the Polygalacturonase-inhibiting Proteins from Four Cultivars of Phaseolus Vulgaris to inhibit the Endopolygalacturonases from Three Races of Collectotrichum lindemuthianum", Phys. and Mol. Plant Path. 36:421-435.
Toubart et al. (1992) "Cloning and Characterization of the Gene Encoding the Endopolygalacturonase-inhibiting Protein (PGIP) of Phaseolus Vulgaris L.", The Plant Journal 2(3):367-373.
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Cervone et al. (1990) "Can Phaseolus PGIP Inhibit Pectic Enzymes from Microbes and Plants?", Phytochemistry 29:447-449 No. 2.
Albersheim Peter
Bergmann Carl
Cervone Felice
Darvill Alan
De Lorenzo Giulia
Fox David T.
University of Georgia Research Foundation Inc.
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