Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-10-08
2001-09-18
McKelvey, Terry (Department: 1636)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
Reexamination Certificate
active
06291660
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to nucleic acid sequences isolated from
Ashbya gossypii
that encode proteins essential for fungal growth. The invention also includes the methods of using these proteins pesticide targets, particularly fungicide targets, based on the essentiality of the gene for normal growth and development. The invention is also useful as a screening assay to identify inhibitors that are potential pesticides, particularly fungicides.
BACKGROUND OF THE INVENTION
The phytopathogenic fungus
Ashbya gossypii
is a filamentously growing ascomycete that was first isolated as a plant pathogen in tropical and sub-tropical regions. It infects the seed capsule of cotton plants and has also been isolated from tomatoes and citrus fruits. The infection of the seed capsule is caused by transmission of
A. gossypii
mycelium pieces or spores by stinging-sucking insects and causes a disease called stigmatomycosis. Presently,
A. gossypii
represents the most compact eukaryotic genome, compared to genome sizes of 12.5 Mb for
S. cerevisiae
(Chu et al., 1986), 31.0 Mb for
Aspergillus nidulans
(Brody and Carbon, 1989) and 47.0 Mb for
Neurospora crassa
(Orbach et al., 1988).
A. gossypii
is systematically grouped to the endomycetales belonging to the family of spermophthoraceae. This classification is based on the observation that the spores that develop in hyphal compartments called sporangia look like ascospores, which are defined as endproducts of meiosis.
Since
Ashbya gossypii
is a filamentous ascomycete, and is capable of growing only by filamentous (hyphal) growth, fungal targets found in this model organism are predictive of targets which will be found in other pathogens, the vast majority of which grow in a filamentous fashion.
SUMMARY OF THE INVENTION
It is an object of the invention to provide an effective and beneficial method to identify novel pesticides, particularly fungicides. A feature of the invention is the identification of genes having a putative activity based on their homology to yeast genes. Genes of the invention comprise a putative GTP binding protein genes (herein referred to as AG001 and AG002 genes), putative GTPase activating protein genes(AG003 and AG004), putative phosphatidylinositol-4 kinase protein gene (AG005) and putative cytokinesis gene (AG006). Another feature of the invention is the discovery that the genes of the invention, AG001 (SEQ ID. NO: 1), AG002 (SEQ Id. NO 3):, AG003 (SEQ ID. NO: 5), AG004 (SEQ ID. NO: 7), AG005 (SEQ Id. NO: 9) and AG006(SEQ ID. NO: 11) are essential for fungal growth and development. An advantage of the present invention is that the newly discovered essential genes containing a novel fungicidal mode of action enables one skilled in the art to easily and rapidly identify novel fungicides.
One object of the present invention is to provide essential genes in fungi for assay development to detect inhibitory compounds with pesticidal, particularly fungicidal activity. Genetic results show that when AG001, AG002, AG003, AG004, AG005 and AG006 are mutated in
Ashbya gossypii
, the resulting phenotype is at best suppressed growth and at worst lethal. Suppressed growth as used herein results in a growth rate of half the growth rate observed in wild type or lower where 10% that of the wild-type growth rate was observed or no growth was macroscopically detected at all. Applicants further observed that when AG001, AG002, AG003, AG004, AG005 and AG006 are mutated in
Ashbya gossypii
abnormal filament development was observed. This suggests a critical role for the gene products encoded by the mutated genes.
The inventors of the present invention have demonstrated that the gene products of the invention are essential in
Ashbya gossypii
. This implies that chemicals which inhibit the function of the protein in fungi, particularly, filamentous fungi, are likely to have detrimental effects on fungi and are potentially good fungicide candidates. The present invention therefore provides methods of using a purified protein encoded by the gene sequence described below to identify inhibitors thereof, which can then be used as fungicides to suppress the growth of pathogenic fungi.
Pathogenic fungi is defined as those capable of colonizing a host and causing disease. Examples of fungal pathogens include plant pathogens such as
Septoria tritici, Stagnospora nodorum, Botrytis cinerea, Fusarium graminearum, Magnaporthe grisea, Cochliobolus heterostrophus, Colletotrichum heterostrophus, Ustilago maydis, Erisyphe graminis
, plant pathogenic oomycetes such as
Pythium ultimum
and
Phytophthora infestans
, and human pathogens such as
Candida albicans
and
Aspergillus fumigatus
The present invention discloses novel nucleotide sequences derived from
Ashbya gossypii
designated as the AG001 gene, the AG002 gene, the AG003 gene, the AG004 gene, the AG005 gene and the AG006 gene. The nucleotide sequence of the genomic clones are set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 respectively. The amino acid sequence encoded by the above sequences are set forth in SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO: 8, SEQ ID NO:10 or SEQ ID NO:12 . The present invention also includes nucleotide sequences substantially similar to those set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 OR SEQ ID NO: 11 and amino acid sequences substantially similar to those set out in SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO: 8, SEQ ID NO:10 or SEQ ID NO:12
The present invention also encompasses fungal proteins whose amino acid sequence are substantially similar to the amino acid sequences set forth in SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO: 8, SEQ ID NO:10 or SEQ ID NO:12. In a particular embodiment, the present invention encompasses nucleic acid sequences and amino acid sequences of filamentous fungi. The present invention also includes methods of using the AG001 to AG006 gene products as fungicide targets, based on the essentiality of the genes for normal growth and development. Normal growth and development is defined as a growth rate substantially similar to that observed in wild type fungus, preferably greater than at least 50% the growth rate observed in wild type fungus and particularly greater than 10% the growth rate obeserved in wild type fungus. Normal growth and development may also be defined, when used in relation to filamentous fungi, as normal filament development development (including normal septation and normal nuclear migration and distribution), normal sporulation, and normal production of any infection structures (e.g. appressoria). Conversely suppressed or inhibited growth as used herein is defined as less than half the growth rate observed in wild type or lower where 10% that of the wild-type growth rate was observed or no growth was macroscopically detected at all or abnormal filament development.
Furthermore, the invention can be used in screening assays to identify inhibitors that are potential pesticides, particularly fungicides. Encompassed by the present invention is the use of sequences selected from the attached Sequence Listing to identify substances having antifungal activity; the use of sequences selected from the attached Sequence Listing to identify substances having pesticidal, particularly fungicidal, activity.
Further comprised is the use of an a DNA sequence selected from the Sequence Listing and variants thereof in a screening method for identifying compounds capable of inducing broad spectrum disease resistance in plants.
In a further embodiment according to the invention, a DNA sequence selected from the Sequence Listing may also be used for distinguishing among different species of plant pathogenic fungi and for distinguishing fungal pathogens from other pathogens such as bacteria. In another preferred embodiment, the present invention describes a method for identifying chemicals having the ability to inhibit any one or more of AG001, AG002, AG003, AG004, AG005 and AG006 activity in fungi prefera
Dietrich Fred
Gaffney Thomas Deane
Goff Stephen Arthur
Philippsen Peter
Wendland Juergen
McKelvey Terry
Sandals William
Syngenta Participations (AG)
Vrana Bruce
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