Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
2000-05-17
2001-06-05
Davenport, Avis M. (Department: 1653)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C530S300000, C530S326000
Reexamination Certificate
active
06242420
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to a novel elicitor protein for use in stimulating defense responses in plants.
2. Description of the Prior Art
Trichoderma virens
(formerly known as
Gliocladium virens
) has been recognized as a mycoparasite and antibiotic-producing antagonist of plant pathogens, and has been used as an effective biocontrol agent of several soilborne root or seedling diseases [Aluko and Hering, 1970, Trans. Br. Mycol. Soc., 55:173-179; Beagle-Ristaino and Papavizas, 1985, Phytopathology, 75:560-564; Howell, 1982, Phytopathology, 72:496-498; Howell and Stipanovic, 1983, Can. J. Microbiol., 29:321-324; Weindling and Fawcett, 1936, Hilgardia, 10:1-16; and Wright, 1956, Plant Soil, 8:132-140
]. T. virens
produces gliotoxin and gliovirin, which are particularly effective antifungal antibiotics, as well as the antibacterial compound heptelidic acid and the antifungal compound viridin.
More recently, efforts have focused on techniques for improving the efficacy of
T. virens
as an antifungal biocontrol agent. These techniques have targeted reducing the production of the phytotoxic agent viridiol by
T. virens
, which may limit its use on valuable crops. Howell has described the addition of sterol inhibiting fungicides to the developing fungus cultures to reduce the production of viridiol (U.S. Pat. No. 5,268,173). Howell has also described the production of mutant strains of T. virens which are deficient for production of viridiol.
SUMMARY OF THE INVENTION
We have now discovered a novel fungal protein which is effective for inducing or stimulating the defense responses of plants against disease. This protein, which is also referred to as an elicitor protein, may be used for the treatment or prevention of fungal infections in plants. The protein is produced by culture of
Trichoderma virens
, and may be subsequently recovered from the culture medium and purified.
In accordance with this discovery, it is an object of this invention to provide a novel elicitor protein for controlling or reducing the severity of plant diseases.
Another object of this invention is to provide a naturally occurring compound which may be used to stimulate the defense responses of plants against disease.
Other objects and advantages of this invention will become readily apparent from the ensuing description.
DETAILED DESCRIPTION OF THE INVENTION
Previously, the antimicrobial activity of
Trichoderma virens
has been attributed to the production of compounds such as gliotoxin, gliovirin, heptelidic acid, and viridin. More recently, chitinase has also been implicated in the biocontrol of cotton seedling disease by
T. virens
(Baek et al., 1999, Curr. Genet., 35:41-50). However, we have unexpectedly discovered an elicitor protein produced by
T. virens
which induces production or activity of defense-related compounds, such as phytoalexins and peroxidase, in plants.
In the following description, the nomenclature used to define the proteins is that specified by Schroder and Lubke [“The Peptides,” Academic Press (1965)] wherein, in accordance with conventional representation, the N-terminal appears to the left and the C-terminal to the right. Where the amino acid residue has isomeric forms, it is the L-form of the amino acid that is represented herein unless otherwise expressly indicated.
The mature, native form of the elicitor protein of the invention has a molecular weight of about 18 kDa as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The protein is heat-stable, insoluble in chloroform, and sensitive to treatment with proteinase K but not &bgr;-glucosidase. The N-terminal amino acid sequence of the mature form of the natural protein has been determined as DTVSYDTGYDNGSRSLNDV. It is understood that some forms of the protein may have additional N-terminal amino acids which may occur, for example, as a result of incomplete processing or hydrolysis of the signal sequence. The protein may also be covalently bonded to a non-related fusion protein. Further, the protein may be glycosylated. The invention also encompasses substantial equivalents of this protein which retain the ability to elicit production of defense-related compounds in a plant against disease. The practitioner of ordinary skill in the art will recognize that slight deviations of the amino acid sequences may be made without significantly affecting the efficacy of the protein. Substantial equivalents of the above protein include conservative substitutions of amino acids with other amino acids, including either naturally occurring or non-conventional amino acids, which maintain substantially the same charge and hydrophobicity as the original amino acid. Conservative substitutions include for example, replacement of glycine for alanine, valine for isoleucine, leucine for isoleucine, aspartic acid for glutamic acid, lysine for arginine, asparagine for glutamine, phenylalanine for tryptophan, and tryptophan for tyrosine. Examples of conservative substitutions with non-conventional amino acids are described in Rosenberg et al. (U.S. Pat. No. 5,679,782) the contents of which are incorporated by reference herein.
The elicitor protein is produced by a variety of strains of
Trichoderma virens
. Strains of
T. virens
suitable for use herein are typically selected for their ability to increase the synthesis of defense-related compounds in plants. In the preferred embodiment, the strains are screened for their ability to increase synthesis of phytoalexins or to increase peroxidase activity or both. As described in the Examples, seeds or seedlings treated with the strain of interest or its culture supernatant or filtrate, are assayed for production of phytoalexins, such as the terpenoid phytoalexins desoxyhemigossypol and hemigossypol in cotton, or for peroxidase activity. Strains eliciting an increase in the production of one or both of these defense-related compounds in plants treated therewith, relative to untreated controls, are retained as presumptive producers of the elicitor protein. Alternatively, or to confirm presumptive positive strains, the strains may be screened for production of the above-mentioned elicitor protein per se, by assay of the culture media following growth therein. In this embodiment, production of the 18 kDa protein may be confirmed, for example, by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
Optionally, treated seeds or seedlings may also be planted in soil contaminated with a fungal pathogen such as
Rhizoctonia solani
or
Pythium ultimum
. After incubation, the seeds or seedlings are examined for evidence of disease. Strains which are effective to prevent or significantly reduce the incidence or degree of disease (in comparison to untreated controls) are retained for subsequent use in the production of the elicitor protein.
Suitable strains of
T. virens
may be obtained from a wide variety of sources, including but not limited to naturally occurring strains from soil, plant debris, or fungus propagules, or purified isolates. Traditionally, strains of this organism have been separated into one of two broad groups, designated P and Q, based upon gliovirin or gliotoxin production (Howell and Stipanovic, 1991, Petria, 1:129-130, the contents of which are incorporated by reference herein). It is envisioned that strains from either group may be selected for use in this invention. A preferred strain of
T. virens
which produces and secretes extracellular elicitor protein has been retained and has been deposited under the Budapest Treaty in the United States Department of Agriculture Agricultural Research Service Culture Collection (1815 N. University St., Peoria, Ill.), on May 3, 2000, and has been assigned deposit accession number NRRL 30286.
Production of the elicitor protein may be accomplished by culture of any of the aforementioned strains of
T. virens
, isolates or subcultures having the identifying characteristics of those strains, mutants of those strains capable of producing the
Hanson Linda E.
Howell Charles R.
Davenport Avis M.
Deck Randall E.
Fado John D.
Silverstein M. Howard
The United States of America as represented by the Secretary of
LandOfFree
Elicitor protein produced by Trichoderma virens that induces... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Elicitor protein produced by Trichoderma virens that induces..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Elicitor protein produced by Trichoderma virens that induces... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2436454