Nucleic acid sequences encoding cell wall-degrading enzymes...

Details Nucleic acid sequences encoding cell wall-degrading enzymes... Nucleic acid sequences encoding cell wall-degrading enzymes...

2000-08-28

2003-02-18

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

C435S183000, C435S200000, C435S252300, C435S320100, C435S419000, C435S468000, C435S069100, C536S023200, C800S295000, C800S298000, C800S320300

Reexamination Certificate

active

06521435

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to nucleic acid sequences derived from fungal genes which encode polypeptides having cell wall-degrading activity and isolated polypeptides having cell wall-degrading activity. The invention also relates to recombinant nucleic acid molecules, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing and using the polypeptides, including expression in plant cells to confer or enhance a plant's resistance to Fusarium and other pathogens.
2. Description of the Art
Overview
Wheat is one of the most important food crops for both domestic and export markets. The United States produces about 2.4 billion bushels of wheat per year with a value of over 7 billion dollars. Fusarium head blight or scab is a ftngal disease of wheat, barley, oats, rye, and wheatgrasses that affects both grain yield and quality. It occurs worldwide, particularly when temperatures and humidity favor the proliferation of the causal agent,
Fusarium graminearum
, at the time of heading. Head blight has caused losses in the billions of dollars to United States and Canadian growers and processors within this decade. Yield and grain quality losses of wheat due to Fusarium head blight approached one billion dollars in Minnesota, North Dakota, and South Dakota in 1993 and 200-400 million dollars across the region in subsequent years. Losses were in excess of 300 million dollars in Ohio, Michigan, Indiana, and Illinois in 1995 and 1996. Quality of grain is also compromised since infected grain is usually contaminated with a mycotoxin, vomitoxin or DON, produced by the fungus that is detrimental to humans and livestock. In addition, the disease has threatened barley production in the upper Midwest because brewers have imposed zero tolerance limits for vomitoxin in grain.
The Head Blight Disease
Cereal crops, including wheat, maize, barley, oats, and rye, are susceptible to infection by many types of fungi and by many species of the pathogenic fungus Fusarium.
Fusarium graminearum
(Schwabe) and
Fusarium culmorum
are the primary causal agents of a disease known as Fusarium head blight, head blight, or scab, of wheat, barley, oats and rye (reviewed in Bai and Shaner 1994; Parry et al. 1995). The life cycle of the pathogen alternates between two hosts, wheat and maize. The teliomorph (sexual stage) of
F. graminearum
, called
Gibberella zeae
, causes stalk rot, ear rot and seedling blight of maize. Head blight is a problem of wheat and barley worldwide, and has occurred in epidemic proportions in the United States since 1991. Thus far, no effective resistance genes for Fusarium head blight have been identified in wheat, barley or their sexually-compatible relatives. Due to this deficiency, and as a consequence of no-till agriculture and unfavorable climate patterns, the wheat and barley industries in 12 states have sustained direct losses totaling 2.6 billion dollars. Increased accumulation of corn and wheat stubble has resulted from no-till agriculture and contributes to the propagation of spores and conidia in the field. Moreover, periods of rainfall and warm temperatures at the time of anthesis (pollen shed) favor germination and growth of the pathogen.
F. graminearum
causes death of the floral organs (florets) that harbor the developing grain, giving the head a bleached and “scabby” appearance, and resulting in moderate to severe reductions in grain yield. In addition,
F. graminearum
and other Fusarium species produce trichothecene mycotoxins, such as deoxynivalenol (DON), that exacerbate disease severity and pose a health threat to humans and livestock that ingest contaminated cereal products.
The marked susceptibility of the wheat head to Fusarium was noted as early as 1891 by Arthur (Parry et al. 1995). Pugh and coworkers (1933) reported that wheat heads inoculated with cultured conidia of
F. roseum
(
culmorum
) were most susceptible to infection during a 20-day window, from anthesis to the soft dough stage, depending upon the cultivar. Dehisced anthers and other degenerating tissue appeared to serve as foci for the proliferation of hyphae into the phloem and throughout the rest of the head. In histological studies, Pugh noted that hyphae usually invade intercellularly but can penetrate the thin walls of the inner parts of the floret. Although
F. graminearum
can become established on various parts of the flower, rapid spread of infection was correlated with the presence of extruded anthers both in laboratory and field studies (Andersen 1948, McKay and Loughnane 1945). Aqueous extracts of anthers were found to stimulate growth of hyphae in vitro (Strange and Smith 1971), whereas germination of macroconidia appeared to be unaffected (Strange and Smith 1978). Hyphal growth stimulation was almost entirely attributable to two quaternary ammonium compounds, glycinebetaine and its precursor choline (Strange et al. 1974). These compounds are present in other organs of the floret, but are most abundant in pollen (Pearce et al. 1976). Glycinebetaine, an osmoprotectant, accumulates over the normal course of pollen development during desiccation (reviewed in McCue and Hanson 1990). Along with choline, it is postulated to serve as a fortuitous source of carbon and nitrogen for
F. graminearum
and for other fungal pathogens. Whether fungal hyphae readily access these compounds at the pollen surface or must invade the pollen cytoplasm is not known at this time.
Identification of genes that confer resistance to Fusarium head blight is essential if wheat and barley are to remain in production in Minnesota, North Dakota, and other states. If Fusarium infection can be curtailed by even 10%, the economic impact is expected to be millions of dollars saved by producers, processors and, ultimately, consumers. Multigenic loci for resistance to scab have been identified in germplasms of wheat and other cereals, but at present, the degree of tolerance obtained in adapted cultivars through traditional breeding is inadequate for control of the pathogen. Since no-till agriculture offers clear benefits for soil and water conservation and since Fusarium is so abundant in wheat-growing regions, alternative sources for host resistance are needed.
There are no effective control measures for the disease. Resistance genes for Fusarium head blight have not been identified in wheat, barley or other sexually-compatible species, limiting the efforts of wheat breeders to develop resistant varieties. What are needed are ways to obtain crop varieties that are resistant to Fusarium head blight.
Roles of Fungal Glucanases and Chitinases
In many fungi, including species of Fusarium, glucan and chitin are principle components of the cell wall. The outer wall layer of Fusarium is comprised of polymers of glucose (1,3/1,6-&bgr;-D-glucan) with &bgr;-1,3 and &bgr;-1,6 linkages. The basal-most inner layer consists primarily of chitin microfibrils, linear polymers of &bgr;-1,4-N-acetylglucosamine that account for about one-third of the mass of the hyphal wall (Barbosa and Kemmelmeier 1993). Fusarium cell walls appear to be more refractory to the action of hydrolytic enzymes, possibly because of high levels of protein and chitin (Sivan and Chet 1989a), and clustering of acetylated glucosamine residues (Fukamizo et al. 1992).
Chitinases and glucanases are produced by naturally occurring bacteria, fungi and plants. In the fungi, these proteins have roles in the self-hydrolysis of cell wall chitin and glucan, respectively, and in the hydrolysis of cell wall components of other microorganisms (Srivastava et al. 1985; Sivan and Chet 1989b, Chérif and Benhamou 1990, Vázquez-Garcidueñas et al. 1998). This latter feature has been applied to the discovery of anti-microbial biocontrol agents. Chitin is also found in the cuticles of insects and in nematode egg shells. For fungi that can utilize chitin as a carbon source, chitinase might have a role in fungal metabolism (Flach et al. 1992). Endochitinases cleave randomly between C1 and C4 lin

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