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
2000-11-09
2004-10-19
Mehta, Ashwin (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
C800S265000
Reexamination Certificate
active
06806400
ABSTRACT:
The present invention is directed to the field of inducing resistance in plants to a broad range of pathogens. Specifically, the invention relates to methods of inducing resistance in plants against a variety of viruses, bacteria, and fungal pathogens by expression of a viral polyprotein encoded by members of the potyvirus group of plant viruses or by expression of other suppressors of gene silencing.
BACKGROUND OF THE INVENTION
The survival of a plant attacked by an invading pathogen depends upon a number of factors. One important factor is how quickly the plant responds and mounts a defense to the pathogen, including the induction of both local and systemic pathways for resistance. Local responses to pathogen attack include callose deposition, physical thickening of cell walls (lignification), synthesis of compounds which exhibit antibiotic activity (e.g., phytoalexins), and the production of protein molecules like cell wall hydrolases (Ward et al., “Coordinate Gene Activity in Response to Agents that Induce Systemic Acquired Resistance,”
The Plant Cell
3: 1085-1094, 1991; the contents of which is incorporated herein in its entirety).
In addition to mounting a local response, plants may also employ general resistance mechanisms to prevent an infection from spreading or to fight secondary infections from a broad array of pathogens. For example, induction of a hypersensitive response (HR), resulting from the interaction of a specific resistance gene from the plant with the corresponding avirulence gene from the pathogen, results in localized cell death and permits a plant to mount a rapid defense against a pathogenic organism (Dong, Xinnian, “SA, JA, Ethylene, and Disease Resistance in Plants,”
Current Opinion in Plant Biology
1: 316-323, 1998; the contents of which is incorporated herein in its entirety).
Another general mechanism of resistance, which may be induced either after an HR response or during an active infection, is known as systemic acquired resistance (SAR). SAR refers to a distinct signal transduction pathway which results in the development of a broad-spectrum resistance (for a general review of the SAR pathway, see Chen et al., “Induction, Modification, and Transduction of the Salicyclic Acid Signal in Plant Defense Responses,”
Proc. Natl. Acad. Sci.
USA 92: 4134-4137, 1995; Hunt and Ryals, “Systemic Acquired Resistance Signal Transduction,”
Crit. Rev. Plant Sci.
15: 583-606, 1996; Neuenschwander et al., “Systemic Acquired Resistance,” pgs. 81-106 in
Plant
-
Microbe Interactions
, Vol. 1, Chapman and Hall, New York, 1996; and Shirasu et al., “Signal Transduction in Plant Immunity,”
Curr. Opin. Immunol.
8: in press, 1996; the contents of which are incorporated herein in their entirety).
The accumulation of salicylic acid (SA) in plants is believed to serve as one signal for the onset of SAR, since the removal of SA in transgenic plants expressing salicylate hydroxylase prevents SAR from being established (Dong, 1998). In some plants, studies have demonstrated that SA alone is sufficient for SAR induction. For example, treatment of plants such as tobacco, cucumber, and Arabidopsis with SA, or its functional analogues, induces SAR (Metraux el al., “Induced Resistance in Cucumber in Response to 2,6-Dichloroisonicotinic Acid and Pathogens,” pgs. 432-439 in
Advances in Molecular Genetics of Plant
-
Microbe Interactions,
Kluwer Academic Publishers, The Netherlands, 1991; the contents of which is incorporated herein in its entirety.)
Tobacco remains one of the best characterized models of SAR. In tobacco, SAR has been shown to provide resistance against seven of nine tobacco pathogens, including infection with fungal, bacterial, and viral isolates (Ryals et al., “Systemic Acquired Resistance,”
The Plant Cell
8:1809-1819, 1996; the contents of which is incorporated herein in its entirety).
SAR is not the only mechanism used by plants to induce a broad-spectrum resistance. Evidence indicates that other signals may play a role in the induction of resistance against microbial pathogens. For example, two alternative signal molecules, jasmonic acid and ethylene, have been shown to induce resistance, as well as mediate the wounding response in plants (Dong, 1998).
In addition to signal molecules which induce resistance pathways, it is known that plants contain many genes which encode for defense-related proteins. In addition to the genes involved in mediating HR, and encoding signal transduction molecules like SA, genes encoding pathogenesis-related (PR) proteins, such as phytoalexins, and enzymes involved in providing stress protection and repairing tissue damage are important mediators of protection in plants (P. Reymond and E. E. Farmer, “Jasmonate and Salicylate as Global Signals for Defense Gene Expression,”
Current Opinion in Plant Biology,
1: 404-411, 1998; the contents of which is incorporated herein in its entirety).
Although it was previously known that exposure to certain pathogens could induce both local protective responses as well as elicit a state of general resistance to a broad range of plant pathogens, the present invention represents the first example of inducing such general resistance by ectopic expression of a viral polyprotein encoded by members of the potyvirus group of plant viruses. The nucleotide sequence encoding this polyprotein, and responsible for the induction of this generalized resistance in plants, has been identified as the P1/HC-Pro sequence.
Previously, the present inventors demonstrated that the potyvirus P1/HC-Pro sequence could function to suppress post-transcriptional gene silencing in plants, thus providing a method to enhance the expression of either foreign or endogenous genes introduced into plants transformed with this “booster” sequence (U.S. Pat. No. 5,939,541 to Vance et al., the contents of which is incorporated herein in its entirety).
For purposes of this specification, the term “booster sequence” refers to the P1/HC-Pro sequence of a potyvirus, or at least the part of the P1/HC-Pro coding sequence able to induce a generalized resistance in plants transformed with that sequence.
The term “gene” or “genes” is used to mean nucleic acid sequences (including both RNA or DNA) that encode genetic information for the synthesis of a whole RNA, a whole protein, or any functional portion of such whole RNA or whole protein sufficient to possess a desired characteristic. Genes that are not part of a particular plant's genome are referred to as “foreign genes” and genes that are a part of a particular plant's genome are referred to as “endogenous genes.” The term “gene products” refers to RNAs or proteins that are encoded by the gene. “Foreign gene products” are RNA or proteins encoded by foreign genes and “endogenous gene products” are RNA or proteins encoded by endogenous genes.
The initial step in the present discovery of the viral booster sequence was the finding that PVX/potyviral synergistic disease syndrome, characterized by increases in symptom severity and in accumulation of the PVX pathogen, does not require infection with both viruses. This was reported by Vance, et al. in “5′ Proximal Potyviral Sequences Mediate Potato Virus X/Potyviral Synergistic Disease in Transgenic Tobacco.” 206
Virology,
583-590 (1995). The synergistic disease is mimicked in plants expressing only a subset of the potyviral genomic RNA and infected singly with PVX. The potyviral region shown to mediate the synergistic disease comprises the 5′-proximal 2780 nucleotides of the genomic RNA, including the 5′-untranslated region (5′-UTR) and the region encoding the potyviral gene products P1, helper component-proteinase (HC-Pro), and a portion of P3. This described potyviral region is referred to herein as the “P1/HC-Pro sequence.”
Thus, Vance et al. (1995), identified a disease determinant carried by the potyvirus genome (the P1/HC-Pro sequence), and this disease determinant was shown to mediate the well-known PVX/potyviral synergistic disease. Although the mechanism by which this potyviral sequence med
Pruss Gail J.
Vance Vicki B.
Alston & Bird LLP
Mehta Ashwin
University of South Carolina
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