Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Plant proteins – e.g. – derived from legumes – algae or...
Reexamination Certificate
1996-11-20
2001-09-18
Saoud, Christine J. (Department: 1647)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Plant proteins, e.g., derived from legumes, algae or...
C435S418000, C435S419000, C530S350000, C530S300000
Reexamination Certificate
active
06291647
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to antifungal proteins, DNA coding therefor and hosts incorporating the DNA, as well as methods of combating fungal pathogens by causing said fungal pathogens to be contacted with said protein or proteins.
The invention further relates to plants, incorporating and expressing DNA coding for antifungal proteins, and to plants which as a result thereof show reduced susceptibility to fungal pathogens, in particular to the Oomycete
Phytophthora infestans.
BACKGROUND ART
Phytophthora infestans
belongs to the group of fungi referred to as Oomycetes.
Phytophthora infestans
infects various members of Solanaceae, such as potato, tomato and some ornamentals. It causes late blight of potatoes and tomatoes affecting all parts except roots. Geographically, the fungus is widely distributed, and it can be found in all potato-producing countries. Economically late blight in potatoes is of major importance, as infection early in the season can severely reduce crop yield. Currently the disease is controlled by spraying chemical fungicides (dithiocarbamates, such as mancozeb, manec and zineb) regularly. (For a review, vide: European Handbook of Plant Diseases, ed. by I. M. Smith et al., 1988, Blackwell Scientific Publications, Ch.8). Both from an environmental and economical point of view, biological control of diseases caused by
Phytophthora infestans
could have advantages over the use of chemical fungicides. A protein with antifungal activity, isolated from TMV-induced tobacco leaves, which is capable of causing lysis of germinating spores and hyphal tips of
Phytophthora infestans
and which causes the hyphae to grow at a reduced rate, was disclosed in WO91/18984 A1. This protein has an apparent molecular weight of about 24 kDa and was named AP24. Comparison of its complete amino acid sequence, as deduced from the nucleic acid sequence of the AP24 gene, with proteins known from a database revealed that the protein was an osmotin-like protein.
Experiments are in progress to evaluate plants which relatively over-express AP24 for resistance against various other fungal pathogens.
Despite initial success in combating fungal pathogens, such as
Phytophthora infestans
, and the genetic engineering of plants capable of producing these antifungal proteins with activity against this fungal pathogen there remains a need to identify and isolate other proteins with antifungal activity against this fungus.
SUMMARY OF THE INVENTION
The present invention provides an isolated protein obtainable from a plant source which has anti-Phytophthora activity and a molecular weight of about 60±5 kDa as judged by SDS PAGE-electrophoresis. A more preferred protein is one that is obtainable from tobacco or tomato plants. A still more preferred isolated protein is characterised in that it is selected from the group of proteins having the amino acid sequence extending from:
(a) amino adds 1 to 540 of SEQIDNO: 6, or
(b) amino adds 23 to 540 of SEQIDNO: 6,
as well as muteins thereof which have anti-Phytophthora activity. A still further preferred protein according to the invention is one characterised in that it is capable of being encoded by the open reading frame represented by SEQIDNO: 6, or by part of said open reading frame by virtue of an in-frame translational stop codon introduced in the 3′ end of said open reading frame, causing the protein encoded thereby to be C-terminally truncated.
The invention also embraces an isolated DNA sequence comprising an open reading frame capable of encoding a protein according to the invention, preferably characterised in that it comprises an open reading frame which is capable of encoding a protein as represented by amino acids 1 to 540 of SEQIDNO: 6, or the precursor of said protein, and DNA capable of hybridising therewith under stringent conditions.
The invention also provides a chimeric DNA sequence according to the invention further comprising a transcriptional initiation region and, optionally, a transcriptional termination region, so linked to said open reading frame as to enable the DNA to be transcribed in a living host cell when present therein, thereby producing RNA which comprises said open reading frame. A preferred chimeric DNA sequence according to the invention is one, wherein the RNA comprising said open reading frame is capable of being translated into protein in said host cell, when present therein, thereby producing said protein.
The invention also embraces a chimeric DNA sequence comprising a DNA sequence according to the invention, which may be selected from replicons, such as bacterial cloning plasmids and vectors, such as a bacterial expression vector, a (non-integrative) plant viral vector, a Ti-plasmid vector of Agrobacterium, such as a binary vector, and the like, as well as a host cell comprising a replicon or vector according to the invention, and which is capable of maintaining said replicon once present therein. Preferred according to that embodiment is a host cell which is a plant cell, said vector being a non-integrative viral vector.
The invention further provides a host cell stably incorporating in its genome a chimeric DNA sequence according to the invention, such as a plant cell, as well as multicellular hosts comprising such cells, or essentially consisting of such cells, such as plants. Especially preferred are plants characterised in that the chimeric DNA according to the invention is expressed in at least a number of the plant's cells causing the said anti-Phytophthora protein to be produced therein.
According to yet another embodiment of the invention a method for producing a protein with anti-Phytophthora activity is provided, characterised in that a host cell according to the invention is grown under conditions allowing the said protein to be produced by said host cell, optionally followed by the step of recovering the protein from the host cells.
The invention provides also for the use of a protein according to the invention for retarding the growth of
Phytophthora infestans
, preferably characterised in that spores of the said fungus are caused to be contacted with said protein. According to yet another embodiment, retarding the growth of the fungus
Phytophthora infestans
is on plant leaves, characterised in that hyphae thereof, or spores thereof, are caused to be contacted with a protein produced from a host cell according to the invention capable thereof.
The invention also provides a method for obtaining plants with reduced susceptibility to
Phytophthora infestans
, comprising the steps of
(a) introducing into ancestor cells which are susceptible of regeneration into a whole plant,
a chimeric DNA sequence comprising an open reading frame capable of encoding a protein according to claim
1
, said open reading frame being operatively linked to a transcriptional and translational region and, optionally, a transcriptional termination region, allowing the said protein to be produced in a plant cell that is susceptible to infection by
Phytophthora infestans
, and
a chimeric DNA sequence capable of encoding a plant selectable marker allowing selection of transformed ancestor cell's when said selectable marker is present therein, and
(b) regenerating said ancestor cells into plants under conditions favouring ancestor cells which have the said selectable marker, and
(c) identifying a plant which produces a protein according to claim
1
, thereby reducing the susceptibility of said plant to infection by
Phytophthora infestans
. Preferred according to the invention is a method characterised in that step (a) is performed using an
Agrobacterium tumefadens
strain capable of T-DNA transfer to plant cells and which harbours the said chimeric DNA on binary vector pMOG841, step (b) being performed in the presence of an antibiotic favouring cells which have a neomycin phosphotransferase.
The invention further provides an antifungal composition comprising a protein according to the invention and a suitable carrier.
An antibody, capable of reacting with an N-termin
Bres-Vloemans Alexandra Aleida
Kroon-Swart Saskia
Logemann Jurgen
Melchers Leo Sjoerd
Ohl Stephan Andreas
Ladas & Parry
Saoud Christine J.
Syngenta Mogen B.V.
Turner Sharon L.
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