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
1998-08-28
2001-05-01
Nelson, Amy J. (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
Reexamination Certificate
active
06225528
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the field of plant pathology and plant genetic transformation. In particular, the invention provides a method for producing transgenic plants having increased resistance to a wide variety of plant pathogens.
BACKGROUND OF THE INVENTION
Several publications are referenced throughout the specification to describe the state of the art to which this invention pertains. These publications are incorporated by reference herein.
Plant disease is a major cause of crop loss. Various strategies have been developed to control disease, one of the most common of which is the use of chemicals. This approach is usually expensive, not always effective, and often harmful to humans and the environment. A preferred approach is to develop, through breeding, genotypes resistant to diseases.
Conventional plant breeding has been based on genetic recombination through sexual crosses. Although nearly all of the current improved plants are developed through conventional breeding techniques, there are limitations to this approach. These include the lack of availability of appropriate germplasm and the incompatibility of certain crosses.
With the recent advances in molecular biology and gene transfer technology for plants, it has become possible to circumvent some of the limitations of conventional breeding by obtaining genes of interest from diverse sources and introducing them into crop plants by genetic transformation. Methods are now available in the art for transforming a wide variety of plant species, including both monocotyledonous and dicotyledonous flowering plants, which contain nearly all agronomically and horticulturally important crops.
The microbial causal agents of plant disease include fungi, bacteria and viruses. Plants have active mechanisms for defending themselves against microbial pathogen infection. For most resistant plants, challenge by a pathogen results in induction of a dual defense mechanisms, part of which is located at the site of infection, occurring as necrotic lesions resulting from host cell death (hypersensitive responses). Another part of the defense occurs in the surrounding, and even distal, uninfected parts of the plant (systemic acquired resistance). The HR is a highly regulated process involving cellular protein synthesis, increased cytosolic calcium ion levels, generation of reactive oxygen species, alteration of protein phosphorylation, among other signal responses. Both the hypersensitive response and systemic acquired resistance are associated with activated expression of a large number of defense or defense-related genes (such as PR genes), some of whose products may play important roles in the restriction of pathogen proliferation and spread by participating in strengthening host cellular structures or through their direct antimicrobial activities.
Salicylic acid has been identified as an important signalling factor in the induction of plant disease resistance. Evidence indicates that a systemic increase in salicylic acid is important for the induction of systemic acquired resistance. Salicylic acid also appears to play an important role in the primary, local defense associated with the hypersensitive cell death. One proposed mechanism of action of salicylic acid is to inhibit catalase activity, thereby elevating H
2
O
2
levels. These elevated levels of H
2
O
2
or other reactive oxygen species derived from H
2
O
2
may serve as a signal for activation of plant defenses such as the synthesis of PR proteins (Chen et al., 1993, Science 262: 1883-1886).
In some resistance responses, plants challenged with a pathogen produce their own anti-microbial substances, termed “phytoalexins.” The production of phytoalexins in the resistance response has been well characterized in several species. For instance, localized and systemic resistance of certain potato varieties to
Phytophthora infestans
may be elicited by several substances, including various portions of the fungus itself, and certain long-chain polyunsaturated fatty acids, such as arachidonic and eicosopentanoic acids. Systemic resistance of potato plants to
P. infestans
following surface applications of long-chain polyunsaturated fatty acids, eicosopentanoic (20:5), arachidonic (20:4), linolenic (18:3) and linoleic (18:2), has been reported (Cohen et al., 1991, Physiological and Molecular Plant Pathology 38: 255-263). However, systemic resistance was directly correlated with phytotoxicity of the fatty acids tested (oleic acid (18:1) was found to be neither phytotoxic nor an inducer of systemic resistance). Thus, it would appear from this report that long-chain polyunsaturated fatty acids are unsuitable for purposes of eliciting pathogen resistance in plants, due to phytotoxicity.
In plants, fatty acids are first produced in saturated forms. They are subsequently desaturated by a series of desaturases, the first of which is a &Dgr;-9 desaturase that produces monounsaturated fatty acids. Further desaturation results in the formation of polyunsaturated fatty acids. A &Dgr;-9 desaturase from yeast has been expressed in tobacco (Polashok et al., 1992, Plant Physiol. 100: 894-901) and tomato (Wang et al., 1996, J. Agric. Food Chem. 44: 3399-3402). Expression of yeast desaturase was reported to alter the fatty acid composition of both plants (increasing 16:1 and 18:1 monounsaturated fatty acids, as well as certain polyunsaturated fatty acids), with no apparent alteration in phenotype. However, no reference has been made to the relative pathogen resistance of the yeast desaturase-transgenic plants, as compared to non-transformed plants.
SUMMARY OF THE INVENTION
Surprisingly, it has now been discovered that transgenic plants expressing genes that lead to increased production of unsaturated fatty acids with no apparent phenotypic abnormalities also exhibit resistance to a wide variety of plant pathogens, including fungi, bacteria and viruses. Thus, according to one aspect of the present invention, a method is provided for making a pathogen-resistant plant, which comprises (1) transforming regenerable cells of the plant with a heterologous DNA, expressible in a plant, encoding an enzyme whose activity increases production of at least one unsaturated fatty acid; and (2) regenerating a transgenic plant from the cells which produces more unsaturated fatty acid than its untransformed counterpart, the production of the fatty acid causing the plant to be pathogen-resistant.
In a preferred embodiment of the invention, the transgenic plant comprises and expresses a DNA molecule encoding a &Dgr;-9 desaturase enzyme. Most preferably, the enzyme is a yeast &Dgr;-9 desaturase. In other embodiments, the transgenic plant comprises and expresses one or more DNA molecules encoding other desaturases, including, but not limited to (1) mammalian desaturases such as &Dgr;-4, &Dgr;-5 and &Dgr;-6 desaturase; (2) plant desaturases such as &Dgr;-9, &Dgr;-15 (omega-6) and &Dgr;-15 (omega-3); and (3) related fatty acid desaturases from other organisms, such as fish. Desaturases of non-plant origin may be modified for expression in plants, according to standard methods.
According to another aspect of the invention, transgenic plants produced by the foregoing methods are provided. In preferred embodiments, the plants are selected from the group consisting of tobacco, tomato and eggplant.
According to another aspect of the invention, the aforementioned method, and the transgenic plants produced by the method, further comprises transforming the plant with an expressible DNA encoding an enzyme selected from the group consisting of lipases and lipoxygenases. Preferably, the lipase is phospholipase A
2
.
Other features and advantages of the present invention will be understood by reference to the drawings, detailed description and examples that follow.
REFERENCES:
Stam M, et al. “The silence of genes in transgenic plants.” Ann. Bot. 79: 3-12, 1997.*
Koziel MG, et al. “Optimizing expression of transgenes with an emphasis on post-transcriptional events.” Plant Mol. Biol. 32: 393-405, 1996.*
Smith CJ
Chin Chee-Kok
Wang Chunlin
Xing Jinsong
Nelson Amy J.
Reed Janet E.
Rutgers The State University of New Jersey
Saul Ewing LLP
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