Date of Award:

5-1-1992

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Biology

Department name when degree awarded

Biology

Committee Chair(s)

Nabil N. Youssef

Committee

Nabil N. Youssef

Committee

Elizabeth Hood

Committee

Jack R. Lancaster

Committee

Joseph K. K. Li

Committee

Gregory J. Podgorski

Committee

Thomas Grover

Abstract

Two secretory proteins associated with sexual reproduction m the bee pathogenic fungus Ascosphaera proliperda Skou were identified. In order to obtain sufficient quantities of homogeneous cells for this study, a minimal medium (MM) was developed to synchronously induce sporulation of A. proliperda. By culturing a mixture of sexually compatible mycelial fragments on this MM, a high degree of synchronous sporulation of the fungus was obtained. Monoclonal antibodies (McA) produced against secretory vesicles isolated from the synchronously sporulating cultures of A. proliperda were used to study antigens associated with sexual reproduction of this fungus. Two secretory proteins (SSF3 and SSE4) specific for sexual reproduction were characterized. SSF3, a glycoprotein, is estimated to be 65 kD by SDS-PAGE, or 65-85 kD by gel chromatography. It is packaged in small cytoplasmic vesicles which accumulate at edges of initial protubrance buds and the developing ascogonial primordia. Surface immunolabelling of the SSF3-specific McA F3 was also present in these areas, indicating discharge of SSF3 by these accumulated vesicles. This observation was further supported by immunoelectron microscopy, which demonstrated displacement of these small vesicles and SSF3 secretion. Neither specific surface labelling of McA F3 nor accumulation of these small vesicles was observed on non-contacted hyphae and mature ascogonia. This finding suggests that SSF3 secretion depends upon contact between two compatible hyphae, and that SSF3 is associated with early sexual development. The presence of continuous immunogold labelling around the periphery of two closing hyphae suggests involvement of SSF3 in the interaction between such hyphae. Additionally, SSF3 was purified from synchronously sporulating cultures of A. proliperda by using McA F3. Protein SSE4 was observed at later stages of development. This protein, with a molecular weight of approximately 43 kD, was found only on the surface of and inside ascogonia. No SSE4 was present on the surface of non-contacted hyphae and ascogonial primordia. A large amount of SSE4 was found primarily in large cytoplasmic vesicles that transport the protein between and along hyphae. Synthesis and secretion of SSF3 and SSE4 are independently regulated events. Unlike their secretion, synthesis did not depend on contact between two compatible hyphae, as shown by the similar levels of SSF3 or SSE4 in the homogenates of contacted and non-contacted cultures of A. proliperda. Synthesis of these two proteins appears to be triggered by starvation. This is suggested by a higher reactivity of McAs F3 or E4 in the cultures grown with a solid or liquid minimal medium relative to those grown with a solid or liquid rich medium (Sab' s with 1% yeast extract). SSF3 and SSE4 levels increased significantly with the age of cultures.

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