Doctoral DISSERTATION ABSTRACT


Biogenic Polyamines in the Plant-Pathogenic Oomycete Phytophthora sojaeFirst Functional Analysis of Polyamine Transport

Marcus Constantine Chibucos

Paul F. Morris, Advisor


Abstract. Because zoospores are the predominant dispersal mechanism of the plant-pathogenic oomycete Phytophthora sojae, understanding zoospore biology may facilitate development of new control mechanisms. Polyamines are ubiquitous biologically active aliphatic cations, which have been detected in the soil. The purpose of this study was to identify and quantify endogenous polyamines in P. sojae hyphae and zoospores and to characterize polyamine uptake kinetics in zoospores. Endogenous polyamines were assayed in P. sojae hyphae and zoospores, media, and soybean roots and root secretions using acid extraction, benzoylation, HPLC, and GC-MS. P. sojae contained high levels of putrescine and spermidine, and hyphae and zoospores had different ratios of free and conjugated polyamines. While large quantities of cadaverine were detected inside roots, only putrescine root efflux was detected. (This is the first report of polyamine efflux from intact soybean roots.) Hyphae were unable to utilize polyamines as a sole nitrogen source, and hyphae grown in the presence of the putrescine biosynthesis inhibitor α-difluoromethylornithine had lower endogenous polyamine levels than non-inhibited counterparts. Zoospores supplemented with polyamines had different polyamine titers, providing evidence of transporter-mediated uptake. 14C-labeled putrescine & spermidine uptake experiments confirmed the existence of high-affinity polyamine transport: for putrescine, a KM of 5.39 µM and Vmax of 112.0 picomoles•[3e5 zoospores]-1•[5 min]-1 were observed. Spermidine transport was similar, exhibiting a KM of 4.59 µM and Vmax of 36.0 picomoles•[3e5 zoospores]-1•[5 min]-1. Putrescine uptake was competitively inhibited by cadaverine. Finally, because genome sequence data suggest the likelihood of multiple transporters, radiolabeled substrate uptake was measured in the presence of inhibitory concentrations of non-radiolabeled putrescine and spermidine in order to assess crosstalk between putrescine and spermidine transporters. The existence of at least two high-affinity polyamine transporters is postulated: (i) a spermidine-specific transporter, and (ii) a putrescine-specific or putrescine-preferential transporter. The results of this work provide initial evidence that polyamines play an important role in the biology of Phytophthora sojae and will be useful in planned future studies involving heterologous expression in bacteria to enable functional identification of polyamine permeases.

 

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Phytophthora sojae zoospore, zoosporangium, zoosporangia, hyphae on V8 medium.

 

Polyamines

 

Extracting polyamines from biological samples and preparing them for identification & quantification by HPLC/GC-MS 

 

Schotten-Baumann

 

Thank You for Reading My Dissertation Work On The Plant-Pathogenic Oomycete Phytophthora sojae.

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Mushroom nestled in the moss after a rain. Photo by Marcus Chibucos, Ph.D.