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
1998-07-31
2001-05-22
Benzion, Gary (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide confers pathogen or pest resistance
C435S069100, C435S419000, C435S468000, C800S288000, C800S301000, C800S323100
Reexamination Certificate
active
06235973
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to transgenic plants having surprisingly improved resistance to plant pathogens due to expressed antimicrobial peptide genes, particularly Magainin and PGL classes of peptide genes, in the plants.
2. Description of the Related Art
In recent years, it has become widely recognized that many organisms, including plants, utilize peptides as a component of their host defense strategies (for review see Hancock and Lehrer.
Trends In Biotechnology
16:82-88 (1998) or Broekaert et al., Critical Reviews in Plant Sciences 16:297-323 (1997)). These broad-spectrum antibiotic peptides have been shown to be active against Gram-negative and Gram-positive bacteria, fungi and protozoa. Overexpression of both plant- and non-plant-derived (e.g., amphibian and insect) antimicrobial peptides with antimicrobial activity in transgenic plants has been touted by some as a means to confer pest resistance in crop plants. However, published reports describing transgenic tobacco plants expressing antimicrobial peptides revealed generally disappointing results (Florak et al.,
Transgenic Res.
4:132-141 (1995)). In several cases, the antimicrobial peptides failed to accumulate to significant amounts within the plant cell as rapid degradation of the peptide was observed. For this reason, and also due to serious concerns about potentially phytotoxic effects exerted by the antimicrobial peptides when expressed in plants, plant scientists have not aggressively pursued this technology.
Antimicrobial peptides can be classified into many categories based upon their structure (e.g., linear vs. cyclic), their size (20-45 amino acids) and their source (e.g., insect, amphibian, plant). However, despite their apparent diversity, numerous defense-related peptides have the common features of being highly basic and being capable of forming amphipathic structures. These unifying features suggest that most peptides appear to act by a direct lysis of the pathogenic cell membrane. Their basic structure facilitates their interaction with the cell membrane, and their amphipathic nature allow them to be incorporated into the membrane ultimately disrupting its structure.
Frog skin secretions of the African clawed frog,
Xenopus laevis
, have been discovered to be a particularly rich source of antibiotic peptides (Bevins and Zasloff
Ann. Rev. Biochem.
59:395-414 (1990)). Known peptides include magainins, PGL
a
, xenopsin and caerulein. Magainins 1 and 2 are very closely related; each are 23 residues in length, contain no cysteine, and form an amphipathic &agr; helix. PGL
a
is a small peptide processed from a larger precursor and is both cationic and amphipathic in nature (Andreu et al.,
Eur. J. Biochem.
149:531-535 (1985)). It has the somewhat unusual feature of containing a COOH-terminal amide group rather than the expected carboxyl group. Moreover, it has been reported that magainin 2 (but not magainin 1) and PGL
a
can interact synergistically with one another to exert enhanced levels of antibacterial activity (U.S. Pat. No. 5,254,537). Magainin/PGL peptides co-evolved in the frog, which may explain the synergy. Maloy and Kari,
Biopolymers
37, 105-122 (1995) describe, inter alia, the magainin and PGL classes of peptides.
Insects have also been demonstrated to possess a variety of defense-related peptides (Boman and Hultmark.
Ann. Rev. Biochem.
41:103-126 (1987)). Cecropins from moths and flies are slightly larger than the frog-derived peptides (31-39 residues), are basic due to the presence of multiple arginine and lysine residues, and therefore interact strongly with the negatively charged lipid bilayers. Studies of these peptides have shown that they form an N-terminal &agr;-helical region connected by a hinge region to a C-terminal &agr;-helical domain.
Other antimicrobial peptides, termed defensins (for review, see Broekaert et al.,
Plant Physiol.
108:1353-1358 (1995)) have been isolated from radish (Terras et al.,
Plant Cell.
7:573-588 (1995)) and barley (Mendenez et al.,
Eur. J. Biochem.
194:533-539 (1990)), and feature a more complex three-dimensional structure which includes cysteine-stablized triple anti-parallel &bgr; sheets with an &agr;-helix. Terras et al., (1995) reported very good levels of protection against infection by Alternaria in transgenic tobacco which overexpressed the radish AFP2 protein. However, a threshold level of AFP2 peptide (which was not easily obtained) in the transgenic plants was required to detect any significant level of disease resistance.
In addition to the naturally-occurring peptides, a wide array of synthetic analogs representing deletion, substitution and variable chain length derivatives have been generated for structure/activity relationship studies. Not unexpectedly, a number of these synthetic variants exhibit increased antimicrobial activity against bacteria and fungi. Moreover, in some cases, not only has the potency of the synthetic antimicrobial peptides to microbes increased dramatically, but their spectrum of antimicrobial activity has also broadened.
Reports of expression of antimicrobial peptides in transgenic plants is rather limited, and the conclusions which have been reached are inconsistent. Montanelli and Nascari,
J. Genetic Breed.
45:307-316 (1991) introduced the cecropin gene into potato and showed antibacterial activity associated with extracts prepared from fresh tissue, but no demonstration of resistance of the whole plant. Hightower et al.,
Plant Cell Report
13:295-299 (1994). reported similar disappointing results against a bacterial pathogen after introducing the cecropin gene into tobacco. In contrast, Carmona et al.,
Plant J.
3:457-462 (1993) and Jaynes et al.,
Plant Sci.
89:43-53 (1993) observed that transgenic tobacco plants expressing &agr;-thionin and Shiva-1 (a modified cecropin) were more resistant to infection by bacterial pathogens. Clearly, a need exists for transgenic plants having improved resistance to plant pathogens due to expressed antimicrobial peptide genes therein. Specifically, peptides belonging to the magainin and PGL
a
classes needed to be tested in this regard.
SUMMARY OF THE INVENTION
The present invention provides antimicrobial peptides, particularly Magainin and PGL classes of peptides, and the genes and other nucleic acid constructs that encode for the peptides. In particular, the peptides according to the invention are effective against phytopathogenic microbes including bacteria, fungi, and phytoplasma. The invention also provides methods of conferring antimicrobial resistance on plant tissue plants by transforming the plant tissue with a nucleic acid construct encoding such peptides. Further provided by the invention are transgenic plant tissues that are resistant to a broad range of microbes. Essentially any plant transformed to express one or more of the antimicrobial peptides according to the invention will manifest resistance to a broad range of microbes, particularly bacteria and fungi.
Prior to the invention, it was uncertain whether the Magainin and PGL classes of antimicrobial peptides would confer resistance in plants to fungal pathogens as well as bacterial pathogens. As shown herein, this class of peptides does have impressive anti-fungal properties. An important advantage provided by the invention results from the fact that Magainin and PGL classes of peptides are derived from frogs and are known to have low vertebrate and mammalian toxicity, and Magainin class peptides have a history of pharmaceutical use.
The representative, non-limiting, discoveries of the present invention include:
1) antimicrobial peptides that possess potent anti-phytopathogenic fungal activity (particularly the Magainin and PGL classes of peptides Magainin 2, MSI-99, MSI-55, and PGL, but also D5-C) in addition to anti-phytopathogenic bacterial activity;
2) antimicrobial peptides that are particularly effective against specific pathogens (e.g., MSI-99 and D5-C are especially active against the fungus, Phytophthora);
3) synthetic genes
Blowers Alan D.
Sanford John
Smith Franzine
Van Eck Joyce
Benzion Gary
McDonnell & Boehnen Hulbert & Berghoff
Mehta Ashwin D.
Sanford Scientific, Inc.
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