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
1997-04-15
2001-09-18
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
Reexamination Certificate
active
06291743
ABSTRACT:
DESCRIPTION OF INVENTION
A variety of geminivirus genes and mutant derivatives were generated and transferred to plant cells. Transgenic plants containing these genes were produced. Transgenic plants containing trans-dominant mutations developed resistance to geminivirus infection.
BACKGROUND OF THE INVENTION
Geminiviruses present the most serious disease problem in many vegetable crops in tropical and subtropical regions. For example, major epidemics of geminivirus infections of beans and tomatoes have occurred in Florida, the Caribbean Basin, Mexico, and Central America. In the past, traditional breeding methods failed to produce cultivars with significant levels of resistance to geminiviruses. An alternative approach lies in producing virus-resistant transgenic plants according to the present invention.
The geminivirus group are single stranded DNA viruses that infect both monocotyledonous (monocot) and dicotyledonous (dicot) plants. A common feature among all gemini viruses is the mode of genomic replication, which involves a rolling circle mechanism.
Tomato mottle virus (ToMoV) is one example of a geminivirus. It has a two component (bipartite) genome, an ability to infect dicot plants and is transmitted by whitefly. The DNA of its two genomic components, DNA-A and DNA-B, has previously been cloned and sequenced. Isolated clones of DNA-A and DNA-B of ToMoV are themselves infectious when mechanically inoculated into tomato and
N. benthamiana
, or when delivered to either host by agroinoculation. An invariant geminiviral DNA sequence required for replication is present in an intergenic, common region (CR) in each genomic component.
The ToMoV DNA-A genomic component has four ORF, one of which, AC1, must be expressed for efficient replication of both A and B components. The AC1 ORF encodes a protein having several functional activities: a DNA binding site specific to the DNA-A CR; a DNA nicking activity; and a NTP binding activity. The DNA binding region mediates an initiator protein function during rolling circle replication.
AC3 protein is a second ToMoV-coded function involved in DNA replication and production of single-stranded circular DNA.
Tomato yellow leaf curl virus (TYLCV) is another example of a geminivirus. TYLCV has a monopartite genome organization, infects monocot plants, and is leaf-hopper transmitted. The TYLCV Cl protein is required for replication, encoded by the C1 ORF.
Being DNA viruses, geminiviruses offer advantages for antiviral strategies. Several geminiviruses have been cloned and sequenced. Transgenic plants having mutant viral genes can be produced, e.g., by introducing expression cassettes comprising mututated virus genes directly into plants with a particle gun, or into plant suspension cells or protoplasts by electroporation, or by Agrobacterium transfection.
SUMMARY OF THE INVENTION
The invention involves production of transgenic plants containing DNA encoding AC1/C1 wildtype and mutant sequences that negatively interfere in trans with geminiviral replication during infection. The resulting transgenic plants are resistant to viral infection.
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Ahlquist Paul G.
Gilbertson Robert L.
Hanson Steven F.
Luu Hang T.
Maxwell Douglas P.
Nelson Amy J.
Rockey Milnamow & Katz Ltd.
Seminis Vegetable Seeds Inc.
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