Fungal target genes and methods

Details Fungal target genes and methods Fungal target genes and methods

2000-06-06

2001-09-18

Ketter, James (Department: 1636)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C435S252300, C435S254110, C435S320100

Reexamination Certificate

active

06291665

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to genes isolated from
Ashbya gossypii
that encode proteins essential for fungal growth and development. The invention also includes the methods of using these proteins as fungal targets, based on the essentiality of these genes for normal fungal growth and development. The invention is also useful as a screening assay to identify inhibitors that are potential 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 (Ashby S. F. and Nowell W. (1926) Ann. Botany 40: 69-84) and has also been isolated from tomatoes and citrus fruits (Phaff H. J. and Starmer W. T. (1987) In “The Yeasts”, Vol. I Rose A. H., Harrison, J. S. (eds), Academic Press, London, 123 ff; Dammer K. H. and Ravelo H. G. (1990). Arch. Phytopathol. Pflanzenschutz, Berlin 26: 71-78 Dammer and Ravelo, 1990). 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.
Studies characterising the karyotype of
A. gossypii
have been performed (Wright, 1990; Wendland, 1993; Gaudenz, 1994, “The small genome of the filamentous fungus
Ashbya gossypii
: Assessment of the karyotype”, Diploma Thesis, Department of Applied Microbiology, Biocenter, University Basel). It has been found using yeast chromosomes of precisely known length as size markers that the genome of
A. gossypii
has a total nuclear genome size of 8.85 Mb. Presently,
A. gossypii
represents the most compact eukaryotic genome, compared to genome sizes of 12.5 Mb for
Saccharomyces cerevisiae
(Chu et al. (1986) Science, 234:1582-1585), 31.0 Mb for
Aspergillus nidulans
(Brody and Carbon (1989) Proc Natl Acad Sci USA. 86:6260-6263), and 47.0 Mb for
Neurospora crassa
(Orbach et al.(1988) Mol Cell Biology, 8:1469-1473).
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 end products of meiosis.
Since
A. 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 fungicides. A feature of the invention is the identification of genes in
A. gossypii
having a putative biological activity based on their similarity to yeast genes. Genes of the invention comprise a putative serine/threonine protein kinase gene (herein referred to as AG007 gene), three genes of unknown function (AG008, AG009, AG010), and a putative serine/threonine protein kinase (AG011). Another feature of the invention is the discovery that the genes of the invention, AG007 (SEQ ID NO:1), AG008 (SEQ ID NO:3), AG009 (SEQ ID NO:5), AG010 (SEQ ID NO:7), and AG011 (SEQ ID NO:9) are essential for fungal growth and development. An advantage of the present invention is that the newly discovered essential genes provide the basis for identity of a novel fungicidal mode of action which enables one skilled in the art to easily and rapidly discover novel inhibitors of gene function useful as fungicides.
One object of the present invention is to provide an essential gene in fungi for assay development for inhibitory compounds with fungicidal activity. Genetic results show that when any of the genes described above are mutated in
A. gossypii
, the resulting phenotype ranges from suppressed growth to lethality. Suppressed growth as used herein results in a growth rate of half the growth rate observed in wild-type, or lower, e.g. 10% to 50% of the wild-type growth rate is observed, or no growth is detected at all macroscopically. Furthermore, when some of the genes described above are mutated in
A. gossypii
, abnormal filament development is observed. This demonstrates a critical role for the gene products encoded by these genes.
Using PCR-based gene disruption, the inventors of the present invention have demonstrated that the activities of these gene products are essential for
A. gossypii
growth. Thus, chemicals which inhibit the function of any of these gene products in 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 either the AG007, AG008, AG009, AG010, or AG011 gene, described below to identify inhibitors thereof, which can then be used as fungicides to suppress the growth of pathogenic fungi. Pathogenic fungi are 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
A. gossypii
, designated the AG007, AG008, AG009, AG010, and AG011 genes. The nucleotide sequence of the ORF in the genomic clones is set forth in SEQ ID NO:1 (AG007), SEQ ID NO:3 (AG008), SEQ ID NO:5 (AG009), SEQ ID NO:7 (AG010), and SEQ ID NO:9 (AG011). The amino acid sequences encoded by the above sequences are set forth in SEQ ID NO:2 (AG007), SEQ ID NO:4 (AG008), SEQ ID NO:6 (AG009), SEQ ID NO:8 (AG010), and SEQ ID NO:10 (AG011), respectively. 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, and SEQ ID NO:9. The present invention also encompasses fungal proteins whose amino acid sequences 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, and SEQ ID NO:10. The present invention also includes methods of using these gene products as fungal targets, based on the essentiality of the genes for normal fungal growth and development. Furthermore, the invention can be used in a screening assay to identify inhibitors of AG007, AG008, AG009, AG010, or AG011 gene function that are potential fungicides.
Other objects and advantages of the present invention will become apparent to those skilled in the art from a study of the following description of the invention and non-limiting examples.
DEFINITIONS
For clarity, certain terms used in the specification are defined and presented as follows:
Cofactor: natural reactant, such as an organic molecule or a metal ion, required in an enzyme-catalyzed reaction. A co-factor is e.g. NAD(P), riboflavin (including FAD and FMN), folate, molybdopterin, thiamin, biotin, lipoic acid, pantothenic acid and coenzyme A, S-adenosylmethionine, pyridoxal phosphate, ubiquinone, menaquinone. Optionally, a co-factor can be regenerated and reused.
DNA shuffling: DNA shuffling is a method to rapidly, easily and efficiently introduce mutations or rearrangements, preferably randomly, in a DNA molecule or to generate exchanges of DNA sequences between two or more DNA molecules, preferably randomly. The DNA molecule resulting from DNA shuffling is a shuffled DNA molecule that is a non-naturally occurring DNA molecule derived from at least one template DNA molecule. The shuffled DNA encodes an enzyme modified with respect to the enzyme encoded by the template DNA, and preferably has an altered biological activity with respect to the enzyme encoded by the template DNA.
Enzyme activity: means herein the ability of an enzyme to catalyze the conve

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