Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part
Reexamination Certificate
2001-03-21
2003-10-07
Nelson, Amy J. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
Higher plant, seedling, plant seed, or plant part
C800S317000, C800S279000
Reexamination Certificate
active
06630618
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to nucleic acid molecules useful for producing plants having enhanced pathogen resistance characteristics, transgenic plants expressing these nucleic acid molecules, methods of making such plants, and methods for increasing the resistance of plants to pathogens.
BACKGROUND OF THE INVENTION
Plants are hosts to thousands of infectious diseases caused by a vast array of phytopathogenic fungi, bacteria, viruses, and nematodes. These pathogens are responsible for significant crop losses worldwide, resulting from both infection of growing plants and destruction of harvested crops (Baker et al., 1997).
Plants recognize and resist many invading phytopathogens by inducing a rapid defense response, termed the hypersensitive response (HR). HR results in localized cell and tissue death at the site of infection, which constrains further spread of the infection. This local response often triggers non-specific resistance throughout the plant, a phenomenon known as systemic acquired resistance (SAR). Once triggered, SAR provides resistance for days to a wide range of pathogens. The generation of the HR and SAR in a plant depends upon the interaction between a dominant or semi-dominant resistance (R) gene product in the plant and a corresponding dominant avirulence (Avr) gene product expressed by the invading phytopathogen. It has been proposed that phytopathogen Avr products function as ligands, and that plant R products function as receptors. Thus, in the widely accepted model of phytopathogen/plant interaction, binding of the Avr product of an invading pathogen to a corresponding R product in the plant initiates the chain of events within the plant that produces HR and SAR and ultimately leads to disease resistance. A detailed review of the current understanding of Avr/R gene product interactions is presented in Baker et al. (1997).
Because the systemic acquired resistance (SAR) response acts nonspecifically throughout the plant to provide enhanced resistance to many pathogens, it has been extensively studied as a possible mechanism for conferring broad-spectrum pathogen resistance. The SAR response is characterized by the induction of at least nine gene families (known as SAR genes) in uninfected leaves of the plant. Some of the SAR genes encode proteins that have antimicrobial activity, including glucanases, chitinases and the pathogenesis-related (PR) proteins such as PR-1. A number of chemicals are known to trigger SAR, including salicylic acid, 2,6-dichloroisonicotinic acid (INA), 1,2,3-benzothiadiazole-7-thiocarboxylic acid-S-methyl ester, and arachidonic acid when applied through the roots, sprayed onto leaves or injected into stems. These and other chemicals are being investigated as candidate agents for inducing SAR in crop plants on an agricultural scale. For a more detailed discussion of the SAR response, see Agrios (1997), Chapter 5.
While efforts to utilize the SAR response to enhance resistance to phytopathogens have largely been based on the external application of inducing agents, molecular genetic research has focused on isolating and manipulating plant resistance (R) genes. Since the cloning of the first R gene, Pto from tomato, which confers resistance to
Pseudomonas syringae
pv. tomato (Martin et al., 1993), a number of other R genes have been reported (for reviews see Hammond-Kosack and Jones, 1997 and Baker et al., 1997). Sequence analyses of the proteins encoded by these R genes have revealed a number of motifs that are conserved between various R proteins; including leucine rich repeats (LRR), nucleotide binding site (NBS), Toll-IL-1R homology (TIR), leucine zipper (LZ) and transmembrane (TM) domains (Baker et al., 1997).
Much effort is currently being directed towards using R genes to engineer pathogen resistance in plants. The production of transgenic plants carrying a heterologous gene sequence is now routinely practiced by plant molecular biologists, and methods for incorporating an isolated gene (such as an R gene) into an expression cassette, producing plant transformation vectors, and transforming many types of plants are well known. Examples of the production of transgenic plants having modified characteristics as a result of the introduction of a heterologous transgene include: U.S. Pat. No. 5,719,046 to Guerineau (production of herbicide resistant plants by introduction of bacterial dihydropteroate synthase gene); U.S. Pat. No. 5,231,020 to Jorgensen (modification of flavenoids in plants); U.S. Pat. No. 5,583,021 to Dougherty (production of virus resistant plants); and U.S. Pat. No. 5,767,372 to De Greve and U.S. Pat. No. 5,500,365 to Fischoff (production of insect resistant plants by introducing
Bacillus thuringiensis
genes).
In conjunction with such techniques, the isolation of plant R genes has similarly permitted the production of plants having enhanced resistance to certain pathogens. A number of these genes have been used to introduce the encoded resistance characteristic into plant lines that were previously susceptible to the corresponding pathogen. For example, U.S. Pat. No. 5,571,706 to Baker and Whitham describes the introduction of the N gene into tobacco lines that are susceptible to Tobacco Mosaic Virus (TMV) in order to produce TMV-resistant tobacco plants. WO 95/28423 describes the creation of transgenic plants carrying the Rps2 gene from
Arabidopsis thaliana
, as a means of creating resistance to bacterial pathogens including
Pseudomonas syringae
, and WO 98/02545 describes the introduction of the Prf gene into plants to obtain enhanced pathogen resistance. Cao et al. (1998) describes the introduction into Arabidopsis of the NPR1 cDNA expressed under the control of the 35S promoter to produce enhanced resistance to multiple bacterial pathogens.
The first R gene conferring virus resistance to be isolated from plants was the N gene of
Nicotiana glutinosa
tobacco (Whitham et al., 1994). The N gene (or homologs of this gene) is present in some but not all types of tobacco, and confers resistance to Tobacco Mosaic Virus (TMV). TMV is an important pathogen of not only tobacco, but also of other crop plants including tomato (Lycopersicon sp.) and pepper (Capsicum sp.). A review of the wide range of host species that serve as hosts to TMV is presented in Holmes (1946). TMV is the type virus of the genus Tobamovirus, which includes a number of closely related viral pathogens of commercially important plants. For example, the Tobamovirus group includes tomato mosaic virus, pepper green mottle virus and ondontoglossum ringspot virus, which is a pathogen of orchids (Agrios, 1997).
The
N. glutinosa
N gene is described in detail in U.S. Pat. No. 5,571,706 (“Plant Virus Resistance Gene and Methods”) to Baker & Whitham, which is incorporated herein by reference. The sequence of this gene is available on GenBank under accession number U558886. U.S. Pat. No. 5,571,706 discloses the sequence of the N gene, as well as two cDNAs corresponding to the gene. The N gene (including the 5′ and 3′ regulatory regions) is over 12 kb in length and comprises five exons and four introns, encoding a full-length N protein of 1144 amino acids, with a deduced molecular mass of 131.4 kDa. cDNA-N is a cDNA encoded by the N gene; it is approximately 3.7 kb in length and encodes the full-length N protein. A second cDNA, cDNA-N-tr, is approximately 3.8 kb in length. It results from an alternative splicing pattern and encodes a truncated protein, N-tr, that is 652 amino acids in length and has a deduced molecular mass of 75.3 kDa. The N protein include TIR, NBS and LRR domains (Whitham et al., 1994, Baker et al., 1997). U.S. Pat. No. 5,571,706, and Whitham et al. (1994) describe the production of transgenic tobacco plants carrying a full-length N transgene; these plants show the HR response following TMV challenge.
While molecular genetic approaches that focus on individual R genes are promising, such approaches may be somewhat limited since each individual R gene will likely confer resistance to a relatively narro
Baker Barbara J.
Dinesh-Kumar Savithramma Puttaswamy
Alston & Bird LLP
Kubelik Anne
Nelson Amy J.
The United States of America as represented by the Secretary of
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