Pathogen resistance in plants using CDNA-N/intron constructs

Details Pathogen resistance in plants using CDNA-N/intron constructs Pathogen resistance in plants using CDNA-N/intron constructs

1999-07-20

2002-04-16

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

C435S320100, C435S468000, C536S023600, C536S024100, C800S301000, C800S317100, C800S317300, C800S317400

Reexamination Certificate

active

06372962

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to nucleic acid molecule useful for producing plants having virus resistance characteristics, and transgenic plants expressing these nucleic acid molecules.
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.
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.
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 (Hammond-Kosack and Jones, 1997). Much effort is currently being directed towards using these 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. Methods for incorporating an isolated gene sequence 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 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 broad-spectrum 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. 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.
SUMMARY OF THE INVENTION
The inventors have discovered that while the introduction of the full length N gene into a plant results in TMV resistance, introduction of the full length N cDNA (cDNA-N) does not. Neither, it has been discovered, does introduction of cDNA-N-tr or the combination of cDNA-N-tr and cDNA-N. In particular, while plants containing the cDNA sequences exhibit HR in response to a TMV infection, the virus spreads systemically throughout the plants, suggesting that the normal SAR is not triggered.
Use of the shorter cDNA sequences rather than the full gene sequence would be advantageous because the shorter length makes manipulating the sequence easier, and reduces the likelihood that errors will be introduced into the sequence either during laboratory manipulation, or in the plant transformation process. To that end, the inventors have produced a form of the cDNA that does produce TMV resistance when introduced into plants. In this context, TMV resistance refers to the ability of a plant to resist systemic spread of the virus.
The inventors have identified a critical intron region of the N gene that is required for TMV resistance. cDNA-N constructs including this intron region (termed cDNA-N/intron constructs) are able to confer TMV resistance on otherwise susceptible plants. The intron region that is required for a cDNA-N to confer TMV resistance is contained within intron 3 (I13) of the N gene, and includes the 70 base pair alternative exon (AE) that is included within cDNA-N-tr and encodes part of the N-tr protein.
The structural region of the N gene (the sequence of which is shown in Seq. ID No. 1) comprises a series of exons (E) and introns (I) that may be schematically illustrated as follows:
E1-I1-E2-I2-E3-I3-E4-I4-E5
cDNA-N comprises the structural N gene sequence with the introns omitted, and may therefore be represented as:
E1-E2-E3-E4-E5.
The inventors have discovered that inclusion of I13 in the cDNA-N sequence in its naturally occurring position (i.e., between E3 and E4) restores the ability to encode TMV resistance. Thus, one possible cDNA-N/intron construct that may be employed is represented as:
E1-E2-E3-I3-E4-E5 (SEQ I

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