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
1999-02-08
2001-10-02
McElwain, Elizabeth F. (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
C435S417000, C435S419000, C435S468000, C800S287000, C800S301000, C800S315000, C800S317200
Reexamination Certificate
active
06297428
ABSTRACT:
This is the U.S. national phase under 35 U.S.C. § 371 of International Application PCT/BE97/00092, filed Aug. 18, 1997.
1. Field of the invention
The present invention is related to a method for inducing viral resistance into a cell and plant, especially BNYVV-resistance into a sugar beet cell and plant and the viral resistant cell and plant obtained.
2. Background of the invention and state of the art
The widespread viral disease of the sugar beet plant (
Beta vulgaris
), called Rhizomania is caused by a furovirus (as used herein, the term “furovirus” is used in the sense that term was used at the time of filing, which was prior to the taxonomic reclassification effected by the Seventh Report of the International Committee on Taxonomy of Viruses in 2000), the beet necrotic yellow vein virus (BNYVV)(23, 24) which is transmitted to the root of the beet by the soilborne fungus
Polymixa betae
(25).
The disease affects significantly acreages of the area where the sugar beet plant is grown for industrial use in Europe, USA and Japan and is still in extension in several places in Western Europe (26, 27). As there exists no practical method to effectively control the spread of the virus at a large scale by chemical or physical means (28), neither in the plants nor in the soil, the main focus has been to identify natural sources of resistance within the sugar beet germplasm and to develop by breeding, varieties of sugar beet plants expressing the resistance genes. A variety of such tolerance genes to the virus has been identified and, some have been successfully used in the breeding of commercial sugar beet varieties (29, 30, 31).
Only the use of BNYVV-resistant or tolerant varieties will enable farmers to grow sugar beet plants in BNYVV-infected areas where sugar beet plant is an essential component of the crop rotation and contributes significantly to the grower's income.
A number of detailed studies have shown that a difference in susceptibility to the BNYVV-infection among sugar beet genotypes or varieties, generally reflect difference in the diffusion or translocation of the virus in the root tissues (32).
However, there are still few reports which indicate clearly that the tolerance genes, even from differing sources of sugar beet germplasm or wild relatives germplasm (33), would provide distinct mechanisms of resistance. Such a situation would represent a more manageable situation to design long lasting BNYVV-resistance strategies.
Since 1986, a number of reports and publications have described the use of isolated viral gene sequences expressed in plant to confer a high level of tolerance against the virus or even to confer a broad spectrum type of resistance against a number of related viruses (34, 35, 36). One of the most documented viral resistance strategy based on genetic engineering, in many cultivated species such as potato, squash, cucumber or tomato, is the use of the viral gene sequence encoding the coat-protein of the target virus (37) which under the control of plant regulatory elements, will be expressed in the plant.
However in the case of the coat-protein mediated resistance, the expression of a certain level of resistance in the transgenic plant might be attributed to different mechanisms such as RNA co-suppression and not necessarily to the production of the protein sequence.
In general, the virus sequence will be transformed in an appropriated cell or a tissue culture of the plant species using an Acrobacterium mediated transformation system or a direct gene transfer method according to the constraints of the tissue culture or cell culture method which can be successfully applied in a given species. A whole plant will be regenerated and the expression of the transgene will be characterized.
Though sugar beet is known as a recalcitrant species in cell culture, limiting the extent of practical genetic engineering applications in that species, there are number of isolated reports of successful transformation and regeneration of whole plants (38). A few examples of engineering tolerance to the BNYVV by transforming and expressing the BNYVV coat-protein sequence in the sugar beet genome have also been published (39, WO91/13159) though they rarely report data on whole functional transgenic sugar beet plants (40). In particular, reports show limited data on the level of resistance observed in infected conditions with transgenic sugar beet plants transformed with a gene encoding a BNYVV coat-protein sequence (41, 42).
A complete technology package including a sugar beet transformation method and the use of the expression of the BNYVV coat-protein sequence as resistance source in the transgenic sugar beet plant obtained by said transformation method has been described in the Patent Application WO91/13159.
Based on the information published, it can not be concluded that the coat-protein mediated resistance mechanism provides any potential for conferring to the sugar beet plant a total immunity to the BNYVV-infection by inhibiting completely the virus multiplication and diffusion mechanisms. To identify a resistance mechanism which enables to block significantly the spread of the virus at the early stage of the infection process would be a major criteria of success to develop such a transgenic resistance, in addition to the fact that even a level of resistance comparable to those known from the genes of resistance identified within the sugar beet germplasm would diversify the mechanisms of resistance available.
Because the disease is shown to expand in many countries or areas, at a speed depending upon the combination of numerous local environmental and agricultural factors, there is a major interest to diversify the sources of genetic resistance mechanisms which may, alone or in combination, confer a stable and long lasting resistance strategy in the current and future varieties of sugar beet plants which are grown for industrial use.
The publication of Xu H. et al. (Plant Cell Report, Vol. 15, pp. 91-96 (1995)) describes genetically engineering resistance construct to potato virus X in four commercial potato cultivars. However, said document states that transgenic potato clones which have included the 8KG gene (the TGB3 construct). However, when these transgenic plants were challenged with PVX, there was no protection against PVX suggesting that the OK protein does not play a role in the protection against PVX.
AIMS OF THE INVENTION
The present invention aims to provide a new method for introducing various viral resistances into a cell and a plant and the viral resistant cell and plant obtained.
A main aim of the invention is to provide a new method for introducing BNYVV resistance into a cell and a plant and the BNYVV-resistant cell and plant, in particular a sugar beet cell and plant (
Beta vulgaris
ssp.), obtained.
SUMMARY OF THE INVENTION
The present invention provides the use of an alternative sequence of plant virus, especially the BNYVV, to obtain a high degree of tolerance to the viral infection, in particular to ensure a rapid and total blocking of virus multiplication and diffusion mechanisms in a plant, especially in the sugar beet plant (
Beta vulgaris
), including fodder beet, Swiss Whard and table beet, which may also be subject to this viral infection. Expression of the resistance will be obtained in transgenic cell and plant, especially sugar beet cells and plants produced by the transformation method subject to the Patent Application WO95/10178 or by other transformation methods based on
Agrobacterium tumefaciens
or direct gene transfer. Because of its high efficiency, the transformation method as described in WO95/10178 enables the production of large numbers of transformated plants, especially sugar beet plants, and will be preferred to develop transgenic plants which may be analysed and characterized for their level of viral resistance, especially BNYVV Resistance, including their field evaluation.
The genome of beet necrotic yellow vein furovirus (BNYVV) consists of five plus-sense RNAs, two of which (RNAs 1 and 2) encode functions essen
Bleykasten-Grosshans Claudine
Bouzoubaa Salah
Guilley Hubert
Jonard Gerard
Lefebvre Marc
Knobbe Martens Olson & Bear LLP
McElwain Elizabeth F.
Mehta Ashwin D.
SES Europe N.V./S.A.
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