Date of Award

2013

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biotechnology Science and Engineering

Committee Chair

Lynn Boyd

Committee Member

Joseph Ng

Committee Member

Leland Cseke

Committee Member

Luciano Matzkin

Committee Member

Khairy Soliman

Subject(s)

Botrytis cinerea, Pathogenic fungi, Tomatoes, Gene expression

Abstract

Botrytis cinerea is a severe plant pathogen that destroys more than 200 crop species worldwide. During early infection, B. cinerea secretes a large store of proteins onto the host surface, many of which cause acute necrosis, senescence, and the loss of cell wall integrity. Recently, cerato-platanin (CP) family member BCSNOD1 was found among fungal secretory products. A subsequent expression profile of B. cinerea-infected strawberry showed the early induction of BCSNOD1 and the CP homologue BCSNOD2. The function of CPs is poorly understood, although evidence suggests a role in plant-pathogen interactions. To characterize CP during B. cinerea infection, the interactions of BCSNOD1 and BCSNOD2 were studied in vitro. The primary objectives were to (a) isolate cerato-platanins in soluble form, (b) assess protein activity on the B. cinerea host Solanum lycopersicum cv Moneymaker (heirloom tomato), and (c) analyze the transcriptome of the BCSNOD-treated tissues. A bacterial expression system was utilized for the isolation, purification, and solubilization of BCSNOD1 and BCSNOD2. Detergent-induced extraction yielded 110-150 mg/L of BCSNOD1 and BCSNOD2, respectively. Application of 50-200 nmol of purified proteins to tomato leaves resulted in severe necrosis within 7 days. For transcription profiling of treated tissues, RNA transcripts were analyzed via next generation sequencing. Both BCSNOD1 and BCSNOD2 show significant regulation of hormonally-induced genes associated with growth, ripening, and defense response. Most notably, BCSNOD2 suppresses the wound-responsive abscisic acid (ABA) signaling genes, while BCSNOD1 is involved with DNA synthesis and transcriptional regulation. These findings demonstrate a significant role for cerato-platanins in B. cinerea pathogenicity.

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