Biology Ph.D. Dissertations

Characterization of PUT1, A Polyamine Transporter from Phytophthora parasitica

Date of Award

2020

Document Type

Dissertation

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Biological Sciences

First Advisor

Vipaporn Phuntumart (Advisor)

Second Advisor

James C. Foust (Other)

Third Advisor

George Bullerjahn (Committee Member)

Fourth Advisor

Paul F. Morris (Committee Member)

Fifth Advisor

Scott O. Rogers (Committee Member)

Abstract

Phytophthora is a genus of oomycetes that causes devastating diseases in both natural and agricultural ecosystems. Phytophthora parasitica is a root rot pathogen that can infect over 72 plant genera and thus serves as a model organism for genome study. I chose to investigate polyamine metabolism and transport in this group of plant pathogens, because polyamines are signaling compounds that have critical roles in growth and development in all organisms. BLAST analysis of the P. parasitica genome using the polyamine uptake transporter PUT5 of Arabidopsis thaliana identified eight homologues. Similar numbers of this family were found in other Phytophthora genomes. The phylogenetic analysis indicates that the PUT family in Phytophthora is surprisingly more diverse than plant PUTs given that the oomycete morphology is less complex than that of plants. PPTG_00424 (PUT1) was chosen for further analysis since its transcript was highly expressed in swimming zoospores which are the primary dispersal propagules of oomycetes. To determine the subcellular localization of this protein, a plasmid vector containing a PPTG_00424 fused with green fluorescent protein (GFP) was constructed and transformed into P. sojae protoplasts. Confocal microscopy showed that PUT1 is localized to the endoplasmic reticulum (ER). To investigate if PUT1 could account for the exogenous uptake of polyamines from the environment, the gene was heterologously expressed in yeast, Saccharomyces cerevisiae. In yeast complementation assays, PUT1 protected yeast cells from toxic accumulation of spermidine (Spd) indicating that this protein is involved in the cellular efflux of Spd. In yeast, TPO5 is required for export of excess polyamines and expression of PUT1 in TPO5 mutants partially complemented the WT phenotype. Collectively, our data indicates that PUT1 codes for an ER-resident PA transporter that is involved in the transport of spermidine. Its high level of expression hints at the importance of polyamine homeostasis in zoospores. Polyamines may be present in the soil environment and zoospore actively uptake polyamines. PUT1 is thus the first characterized polyamine transporter in Phytophthora.

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