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FUNCTIONAL ANALYSIS AND CHARACTERIZATION OF TRANSPORTER OF PUTRESCINE AND (TOPAS1) IN PHYTOPHTHORA PARASITICA

Nilanjana Chakrabarti

A Thesis

Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE

August 2017

Committee:

Paul Morris, Advisor

Vipaporn Phuntumart

George Bullerjahn

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ABSTRACT

Paul Morris, Advisor

Sequence data and gene models are now available for 19 oomycete genomes. Only a very small number of oomycete genes have been functionally characterized, because oomycetes are in the Kingdom Stramenopila, and many of the genes have no close homologues in fungi, , or . Membrane transporters are particularly challenging to characterize because these proteins may be part of large families, and homology is not particularly useful in predicting substrate specificity. Here we show by heterologous expression in yeast that the P. parasitica protein PPTG_16698, Transporter of Putrescine and Spermidine (TOPAS1) is a transporter. Heterologous expression of this gene in yeast protects cells from the toxic effects of high levels of putrescine in the media, suggesting that TOPAS1 also acts to sequester within the . TOPAS1 has the fifth highest level of expression of all membrane transporters in the zoosporic stage of this pathogen. Since zoospores take up polyamines form the media this high level of expression hints at the importance of polyamine acquisition and compartmentation for this developmental stage. TOPAS1 is one of 13 highly conserved membrane transporters in

P. parasitica, and orthologs to these proteins are found in other Phytophthora species. Members of this family are also found in other oomycetes, although there are no homologues to these proteins in other .

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I dedicate this work to my parents and

my family iv

ACKNOWLEDGEMENTS I am deeply indebted to my advisor, Dr. Paul Morris, Department of Biological Sciences,

Bowling Green State University and wish to thank him for all his support and supervision throughout my study. I am deeply grateful to my committee members Dr. Vipaporn Phuntumart and Dr. George Bullerjahn, Department of Biological Sciences, Bowling Green State University for their invaluable assistance provided throughout my study and also for understanding the issues and delay I faced during my experiments.

I thank Dr. Hans Wildschute, Department of Biological Sciences, Bowling Green State

University for providing the plate reader; the department of biological sciences for providing the scintillation counter for my isotope assays.

I thank my lab members Dr. Sheaza Ahmed, Menaka Ariyaratne and Chandra Sarkar for their support. Finally, I thank my wonderful family for their constant support and encouragement.

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TABLE OF CONTENTS

CHAPTER I. INTRODUCTION………………………… ...... 1

Evolutionary history of oomycetes…………………………………………...... 1

Life cycle of Phytophthora ……………………………………………………… ... 2

Mechanism of infection in oomycetes…………………………………………...... 3

Role of polyamines and the need to study about its transport in cells……...... 4

Goals of the study……………………………………………………...... 5

CHAPTER II. FUNCTIONAL ANALYSIS AND CHARACTERIZATION OF

TRANSPORTER OF PUTRESCINE AND SPREMIDINE (TOPAS1) IN PHYTOPHTHORA

PARASITICA………………………...... 6

Introduction and overview …………………………………………………………. 6

Materials and methods …………………………………………………………...... 8

Chemicals and reagents and cell strains …………………………… ...... …. 8

Preparation for transformation of yeast cells ………………………… ...... 10

Growth assay of yeast strains...... 10

Preparation of buffer and chemicals for polyamine transport assay for

TPO5Δ…………………………………… ...... 11

Preparation of buffer and chemicals for polyamine transport assay for

AGP2Δ…………………… ...... 11

Phylogenetic tree generation and alignment of similar sequences

in other oomycetes……………………...... 13

Results……………………………………………… ...... 13 vi

Heterologous expression of PPTG_16698 in yeast…...... 22

Growth Curve……………...... 23

Uptake of Polyamines………………...... 25

Excretion of Polyamines……………………...... 26

Discussion…………… ...... 27

LITERATURE CITATIONS ...... ……………………………. 29

APPENDIX A. MEDIA…………………… ...... ………………………………… 37

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LIST OF FIGURES

Figure Page

1 Genetic map of p_YES_DEST52 vector, showing ampicillin resistance marker, uracil

biosynthesis marker and GAL1 promotor ...... 9

2 RNA sequence of six highly expressed predicted transporters in

P. parasitica zoospores ...... 13

3 Seaview showing the aligned sequences of proteins that are a part

of the TOPAS1 family ...... 14

4 Phylogenetic tree showing with the 13 members of the TOPAS family ...... 15

5 The RNA-sequence of a conserved clade of predicted transporters related

to PPTG_16698 in P parasitica ...... 17

6 Phylogenetic tree showing the proteins that are conserved outside

Phytophthora but within the other oomycetes ...... 17

7 Phylogenetic analysis of sequences related to PPTG_16698 in oomycetes

within Phytophthora ...... 19

8 The hydropathy plot showing the predicted transmembrane regions ...... 20

9 The uptake and export data of the AGP2∆ cells expressing TOPAS ...... 21

10 Growth curve data for TPO5∆ cells ...... 23

11 Uptake data of polyamines for TPO5∆ cells ...... 24

12 Export data of polyamines for TPO5∆ cells ...... 25

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CHAPTER I. INTRODUCTION

Since plants are sessile, they are challenged by many environmental factors that include both biotic and abiotic stresses. These include water stress, adverse temperatures, high soil alkalinity, or mineral toxicity, negatively impact growth, development, and seed quality

(Luigi et al., 2003). Similarly, plant growth and development is challenged by pathogen attack.

Host-specific and opportunistic pathogens of plants include, bacteria, fungi, insects, nematodes, oomycetes, and viruses. While fewer in number, oomycetes play a very important role in causing some of the major plant diseases around the world (Kamoun et al., 2014). The pathogens causing these diseases, include the major worldwide pathogen of potatoes and cocoa, a major pathogen of soybeans and lettuce, soil borne pathogens of citrus and apple trees, an introduced species that threatens the eucalyptus forests of Australia, and another introduced pathogen that threatens oak forests on the coastal range of California, and forests in the British Isles (Vallance et al., 2009;

Kamoun et al., 2015). Based on its economic importance, the oomycete research community has identified the top 10 species of oomycetes in the world which include, Phytophthora infestans,

Hyaloperenospora arabidopsidis, Phytophthora ramoram, Phytophthora sojae, Phytophthora capsica, Plasmopara viticola, Phytophthora cinnamon, Phytophthora parasitica, ultimum and Albugo candida respectively (Kamoun et al., 2014).

Evolutionary history of oomycetes

Oomycetes are classified within the kingdom Stramenopila (Baldauff et al., 2000) which also includes the golden-brown , kelps and diatoms. Members of this Kingdom share a common ancestor of a plastid that engulfed a red alga and in the case of oomycetes the plastid has been lost (Cavalier-Smith, 1982; Bhattacharya and Medlin, 1995). There is also evidence that the common ancestor of Stramenopiles include a primary endosymbiotic event involving a 2 engulfing a green alga, and the subsequent transfer of nuclear and plastid genes to the host nuclear genome (Moustafa et al, 2009, Keeling et al., 2013). Molecular time-scale studies have estimated that oomycetes diverged from other Stramenopiles at least 400 million years ago

(Matari and Blair, 2014). Horizontal transfer events from both fungi and bacteria have also contributed to the evolution of oomycetes as plant pathogens (Savory et al, 2015; Morris et al.,

2009). Oomycetes have acquired 48 families of genes that facilitate carbohydrate metabolism and plant degradation (Savory et al, 2015). Acquisition of bacterial genes have contributed to novel biosynthetic pathways for and (Morris et al, 2009). Oomycete hyphae are not septate, leading to the formation of a multinuclear structure called the coenocyte

(Kortekamp 2005). While the cell walls of fungi are composed of chitin, in oomycetes, the major carbohydrates are composed in cell walls are β-1, 3 and β-1, 6 glucans (Melida et al.2013).

However, chitin is a component of the cell wall in at least some of the oomycete lineages (Jiang et al 2013).

Life cycle of Phytophthora

Phytophthora has both sexual and asexual modes of reproduction. Aseptate, hyaline hyphae are often observed with occasional hyphal swellings, producing sporangia, zoospores and chlamydospores. Zoospores are wall-less bi- cells that are the principal dispersal mechanism (Erwin and Ribeiro, 1996). Phytophthora species can be either homogametic or heterogametic. Species like P. sojae are homothalic and outcrossing is rare. However, in P. infestans, oospores only form when both mating types A1 and A2 are present (Ko, 1981, Fry

2008).

Successful infection requires spore production and dispersal of them to the plant tissues.

Soil borne pathogens use zoospores as primary agents of dispersal (Duniway 1976); while, in P.

3 infestans the spores are spread by wind (Erwin and Ribeiro, 1996). Zoospores, which are the main agents of infection in most instances, convert into immobile cysts on reaching the plant surfaces.

Specialized structures called appressorium are formed at the tip of the germ tubes that aid in penetration of the leaves and roots (Kebdani et al., 2010; Wang et al., 2011). This is followed by the production of invasive hyphae and haustoria-like structures, and eventually several sporangia are formed on the infected surface.

Mechanism of infection in oomycetes

Colonization by oomycetes in plant tissues is achieved by manipulating the host cell mechanism, a common strategy used by other (Wang et al., 2016; Boevink et al., 2016; Tyler et al., 2009). Oomycetes manipulate the host cell mechanism by secreting large numbers of effector proteins (Kamoun et. al 2006 Amaro et al., 2017; Wang et al., 2017; Olivera et al., 2016). These include effectors that are secreted in the extracellular space are called apoplastic effectors, while proteins delivered into the cell are called cytoplasmic effectors.

Apoplastic effectors such as cell wall hydrolases, enzyme inhibitors and small rich residues target the cell wall matrix and plant cell receptors. Cytoplasmic effectors include a diverse set of proteins sharing a common RXLR sequence and a second diverse family of genes called Crinklers (CRNs) which have an n-Terminal motif of LXLFLAK (Stam et al., 2013; Petre and Kamoun, 2014). While effectors are delivered into the host cells, there is another class of molecules called the elicitors, also called the Avr genes, that stimulate the host immune system.

They have pathogen-associated microbial patterns and are identified by the plant immune system

(Staskawicz, 1995). One example of such a class of elicitors are Pep-13, isolated from

Phytophthora sojae (Tyler et al., 2008). The plants have developed the “R -genes”, or resistance genes which are activated upon host invasion. The R genes encodes specific receptors which

4 interacts with the Avr genes (Kamoun et al.,1999). This initiates a signal transduction pathway that leads to the activation of the Host Response (HR) pathway (Dangl et al., 1996)

Role of polyamines and the need to study about its transport in cells

Polyamines are small ubiquitous cations that are essential for life (Cohen et al., 1998;

Igarashi and Kashiwagi, 2000) Antony van Leeuwenhoek observed them as stellate crystals in aging sperm while viewing under a microscope in 1678. One of the earliest molecules to be discovered, they have been widely studied and characterized in mammals, plants, and microbes. They are low molecular weight compounds, and the names of them (putrescine, spermidine, , and thermospermine) reflect where they were first isolated from. In plants, four major polyamines are found that include putrescence, spermidine, spermine and thermospermine. They play major roles in regulating processes like , replication and transcription and protein synthesis (Igarashi 2015; Tabor and Tabor, 1999). In plants, polyamines have been found to play roles in developmental processes such as embryogenesis, fruit ripening, root formation and flower initiation (Kumar et al., 1997; Walden et al., 1997;

Cohen, 1998; Malmberg et al., 1998). Polyamines also function as signaling molecules, and enable plants to respond to abiotic stresses (Alcázar et al., 2010).

Swimming zoospores are the primary dispersal agent of soil-borne oomycetes (Duniway,

1976). Zoospores use root exudates such as ethanol produced by roots under flooding conditions and species specific signaling compounds such as the isoflavonoids of soybeans (Morris et al.,1998; Morris et al. 1992). Uptake experiments with radiolabeled polyamines has also shown the presence of two high affinity transporters for both putrescine and spermidine expressed in

5 swimming zoospores of P. sojae (Chibucos and Morris 2006). Putrescine is excreted by roots into the rhizosphere but these compounds are not chemotactic (Chibucos and Morris 2006).

RNA sequence data for the P. parasitica genome annotation project included a novel predicted transporter that was highly expressed in swimming zoospores. PPTG_16698, was found to be a member of a highly-conserved clade of 13 proteins in P. parasitica. Closely related orthologs of this protein family were found in all of the oomycetes. Thus, functional analysis of this protein could provide insights on the role of this and other transporters in this family.

Goals of the study

The major goal of this study was to characterize a class of novel transporter proteins in

Phytophthora parasitica. We hypothesized that this protein might play a role in polyamine transport, since P. sojae zoospores have high concentrations of intracellular polyamines, and polyamines are actively acquired from the media by the swimming stage of this pathogen

(Chibucos and Morris, 2006).

Specific goals were,

• To develop radioisotope assays to determine the role of PPTG_16698 in uptake and export

of polyamines

• To use PPTG_16698 as a model to identify the roles of conserved orthologs to this protein

in P. parasitica and other oomycetes.

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CHAPTER II. FUNCTIONAL ANALYSIS AND CHARACTERIZATION OF

TRANSPORTER OF PUTRESCINE AND SPREMIDINE (TOPAS1) IN PHYTOPHTHORA

PARASITICA

Introduction and overview

Oomycetes are plant pathogens that resemble fungi, because they have a hyphal growth form, but were later found to be related to brown algae and diatoms (Gunderson et al., 1987;

Jiang and Tyler, 2012; Lamour and Kamoun, 2009; Thines 2014). Phytophthora is a large genus consisting of many species classified under the kingdom Stramenopila (Baldauff et al., 2000).

Together with other oomycetes, they represent a major class of plant pathogens that affect a wide range of crop species.

Phytophthora parasitica is a commonly known pathogen found all over the world (Erwin and Ribeiro, 1996). It causes diseases like citrus-root rot, gummosis, foliar and fruit diseases; and crown rots on herbaceous plants in over 250 genera (Cline et al., 2008). Although it has a broad host range at a species level, P. parasitica isolates were found to be host specific (Erwin and

Ribeiro, 1996). The display of differential virulence has been observed in several hosts (Colas et al., 1998) and thus it is important to know the genetic structure of P. parasitica. Because P. parasitica can also infect the model plant Arabidopsis, the genetic targets of oomycete effectors can be more efficiently identified using the genetic resources available for A. thaliana.

In response to environmental cues, oomycete hyphae produce sporangia which release uninucleate biflagellate zoospores. Zoospores use the flagella to swim in the soil water and are the primary agents of dispersal for these soil pathogens (Duniway 1976). Zoospores move towards the plant tissues by swimming; they have two unequal flagella, one whiplash and the

7 other tinsel (Erwin and Ribeiro, 1996). Studies have shown that negative geotaxis of

Phytophthora zoospores might play an important role in helping them to migrate upward in flooded soil (Cameron and Charlie, 1977). They use the two flagella to swim in a helical path

(Ho and Hickman, 1967). Zoospores are attracted to chemo-attractants like isoflavone secreted by the plants in the rhizosphere (Morris and Ward, 1992; Tyler et al., 2002; Judelson et al.,

2005). They swim at a speed of 121-182 µm/s (Allen and Newhook, 1973). The flagella produce a thrust by undulation from base to tip and the pressure exerted by the anterior flagella is more than the posterior one which helps to maintain direction (Erwin and Ribeiro, 1996).

Previous studies in our lab has shown that zoospores have a high concentration of polyamines in their cells which is approximately about 38nM/1e5 of cells (Chibucos, 2004).

PPTG_16698 was found to have the fifth highest expression in zoospores (Phytophthora parasitica Assembly Dev initiative, Broad Institute (broadinstitute.org)). RNA sequence data together with predicted gene models suggest that P. parasitica is a potential candidate to study the transport of polyamine in swimming zoospores. PPTG_16698 is a part of the AAAP superfamily and we hypothesized its role in polyamine transport. To test our hypothesis, we first tried searched for proteins that were similar to PPTG_16698 within P. parasitica. This approach enabled us to find a family of 13 proteins having highly conserved sequences, but differential expression in different tissues. Heterologous expression of this protein in WT and mutant strains that are deficient in polyamine transport indicate that PPTG_16698 functions as a putrescine and spermidine transporter. Members of this clade of transporters are predicted to play essential roles in polyamine homeostasis.

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Materials and methods

Chemicals and reagents and cell strains

The experiments were carried out using a variety of chemicals that we obtained from several companies. Media reagents (Appendix A) included Tryptone, Peptone, Granulated Yeast Extract,

Agar, D-Galactose and Dextrose, were obtained from VWR International LLC, Radnor PA; sodium chloride and glucose were obtained from Fisher Scientific, Hampton NH. SC minimal media (Appendix A) was prepared using Yeast Base and Agar obtained from VWR

International LLC. Sodium citrate was obtained from Mallinckrodt Chemical Works, United

Kingdom, and HEPES was obtained from Research Organics, Cuyahoga Heights, Ohio.

Antibiotic stocks were prepared by dissolving Ampicillin obtained from Sigma-Aldrich (St.

Louis, MO), in sterile water at a concentration of 100 mg/ml. The pH of the buffers was adjusted using 12M HCL diluted to 6M and sodium hydroxide pellets obtained from Mallinckrodt

Chemical Works and Fischer Scientific respectively. The polyamines were obtained as

Putrescine Dihydrochloride and Spermidine Trihydrochloride and stock cultures of 100 mM were prepared in sterile distilled water. The radioisotopes were obtained from PerkinElmer,

Waltham MA, with a specific activity of 80 X 3.7 X 1010 and 62 X 3.7 X 1010 for putrescine and spermidine respectively. Scintillation counts were measured using Ecoscint H (Scintillation liquid) obtained from National Diagnostics, Atlanta, GA.

The nucleotide sequence of PPTG_16698 was codon optimized for its expression in yeast cells and obtained from GeneScript, China in a puc57 vector. They were transferred into an entry vector followed by transformation in E. coli cells in a Gateway vector p-YES-DEST52 using a

LR cloning reaction (Frueler et al., 2008). The yeast knockout TPO5∆ gene was obtained from

GE Dharmacon, Lafayette, CO. The AGP2∆ knockout strain was as a gift from Dr. Dindial

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Ramotar, University of Montreal, Canada (Aouida et al., 2005). The plasmid from E. coli cells were isolated using AxyPrep Plasmid Isolation Miniprep Kit from Genscript. following the kit instruction manual. The important properties of this plasmid included a GAL1 promoter, to induce expression of the gene in the presence of galactose; an ampicillin resistance marker gene for selection of the bacterial transformants, and a selectable marker for the biosynthesis of uracil to select the yeast transformants.

Figure 1: Genetic map of p_YES_DEST52 vector, showing ampicillin resistance marker, uracil biosynthesis marker and GAL1 promotor.

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Preparation for transformation of yeast cells

E. coli cells containing the plasmid p-YES-DEST52 were grown overnight in a volume of

5 ml of Luria Broth (Appendix A) containing ampicillin was kept at shaking conditions at 37ºC.

The plasmid was isolated using AxyPrep plasmid isolation kit and was checked for its DNA concentration using NanoDrop 1000 spectrophotometer from Thermo Scientific, Waltham MA.

To get better transformation efficiency, an improved method of yeast transformation was followed (Lorenzo et al., 2010). For transformation of the yeast cells, BY4741, AGP2 knockout and TPO5 knockout cells was grown overnight in Yeast Peptone Dextrose (YPD) until OD600 =

0.5. These growth phase cells were centrifuged at a 58xg and re-suspended twice in ice-cold 1M sorbitol respectively. 50 µl of competent cells were mixed with 5µl of plasmid DNA of a concentration of 30 ng/ml. An electric field of 1.5KV was applied at a rate of 5 millisecond pulse using the yeast transformation settings for the Bio-Rad Gene Pulser XCell. Transformants were selected on CSM-uracil plates grown at 30 °C. Transformed colonies were re-streaked on selection plates, and glycerol stocks were prepared.

Growth assay of yeast strains

Strains were grown overnight in Yeast Peptone Galactose (YPG) media. A 500mM stock concentration of putrescine was made by dissolving putrescine in 20 ml of sterile water. This stock was further diluted into concentrations of 50mM, 75mM and 100mM of putrescine. The cells were grown overnight and then diluted to 0.1 OD540 into fresh media and 2 ml aliquots were added to 24-well tissue culture plates. Growth of the cells was monitored using a Synergy HT microplate Reader (BioTek Instruments Inc, Winooski, VT) at 30 °C in the presence of 0, 50, 75,

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100 mM putrescine over 24h. The experiment was done with three replicates for each condition and strain.

Preparation of buffer and chemicals for polyamine transport assay for TPO5Δ

A pilot culture of 5 ml was grown overnight in YPG media. Then 5 µl of culture was added to 50 ml of fresh media and grown to OD600 0.6. These cells were harvested by centrifugation

(1060xg) washed three times in 20mM Na-HEPES (pH 7.2) buffer containing 2% galactose.

They were re-suspended in the same buffer at a concentration of 1.2 µg/ml of protein and kept at

30 ºC for 5min. 1µl of labelled putrescine of 2.22x106 dpm was added to 8µl of 0.5mM cold putrescine for each condition in a microfuge tube. The reaction was started by adding 791 µl of cells. At each of the time points, (0, 5, 10 and 20 min) 0.20 ml of cells were removed and filtered through Whatman™ 25 mm GF/A filters. Cells were washed with 3 ml of ice cold 20 mM Na-

HEPES, 2% galactose, pH 7.2 containing 500 µM putrescine. The radioactivity of the filters was determined by liquid scintillation counting. As a control, pre-chilled cells were treated with

0.5mM[3H] putrescine ice for 0, 5, 10 and 20 mins. Counts from these washed filters were subtracted from experimental samples for 5, 10, and 20 min to correct for nonspecific binding.

The same process was repeated with labelled spermidine.

For the export assay, 791 µl of cells were resuspended in 20 mM Na-MES, 2% galactose buffer with a pH of 5.5 and incubated with [3H] of putrescine for 90 mins. Then cells were then filtered through Whatman™ 25 mm GF/A filters, and washed with 20 mM Na-MES, 2% galactose, pH 5.5 containing 0.5mM putrescine to stop the reaction and reduce non-specific binding. The filter was transferred to vial and washed with 1 ml of 20 mM Na-MES buffer of pH

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5.5. To measure export of polyamines from the cells 0.20 ml aliquots were removed, centrifuged and the supernatant was transferred to a scintillation vial for counting.

Preparation of buffer and chemicals for polyamine transport assay for AGP2Δ

A pilot culture of 5ml was grown overnight in YPG media. Then 5 µl of culture was added to 50 ml of fresh media and grown to OD 0.6. These cells were harvested by centrifugation

(1060xg) washed three times in 20 mM Na-HEPES (pH 7.2) buffer containing 2% galactose.

They were re-suspended in the same buffer at a concentration of 1.2 µg/ml of protein and kept at

30 ºC for 5min. A labelled stock of putrescine was made by prepared by adding 2.22x106 dpm to

5 ml of 100 mM putrescine. To start the reaction 5 µl labeled putrescine was added to 995 µl of cells. At each of the time points time points (0, 5, 10 and 20mins) 0.25ml of cells were filtered through a Whatman™ 25 mm GF/A filters. Cells were washed with 3 ml of ice cold 20mM Na-

HEPES, 2% galactose, pH 7.2 containing 500 µM putrescine. The radioactivity of the filters was counted by placing them in a liquid scintillation counter. As control, pre-chilled cells were treated with 0.5 mM[3H] putrescine ice for 0, 5, 10 and 20 mins. The same process was repeated with labelled spermidine.

For the export assay, 995 µl of cells were resuspended in 20mM Na-MES, 2% galactose buffer with a pH of 5.5 and incubated with [3H] of putrescine for 90 mins. Then cells were then filtered through a Whatman™ 25 mm GF/A filters; washed with 20mM Na-MES, 2% galactose, pH 5.5 containing 0.5mM putrescine to stop the reaction and correct for non-specific binding.

The filter was transferred to a vial and washed with 1 ml of 20mM Na-MES, 2% galactose, pH

5.5. Aliquots of 0.25 ml were removed centrifuged and the supernatant was transferred to a scintillation vial for counting. 13

Phylogenetic tree generation and alignment of similar sequences in other oomycetes

The protein sequence of PPTG_16698 was obtained from FungiDb and used in a BLAST analysis to retrieve similar sequences from other sequenced oomycete genomes in this database.

The same sequence was used to perform a BLAST to check for similarity in sequences outside the Phytophthora genus. Sequenced were aligned using the CLUSTAL option of Seaview (Gouy et al., 2009). Aligned sequences were subjected to phylogenetic analysis using PhyML; a phylogeny software program based on maximum likelihood principle (Guindon et al., 2003). The tree was built using the LG model and default settings, with 100 bootstrap replications. The tree was visualized using iTOL tree-building program. (Letunic, 2016). The hydropathy plots were constructed using TMPred, a program that predicts the membrane spanning regions and their orientation (Hoffman and Stoffel, 1993).

Results

Figure 2: RNA sequence of six highly expressed predicted transporters in P. parasitica zoospores (Phytophthora parasitica Assembly Dev Initiative, Broad Institute

(broadinstitute.org)). Expression reads were calculated as median centered log2 (RPKM) reads per kilobase, per million mapped reads.

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To improve the quality of predicted gene models for P. parasitica, the genome annotation program of the Broad institute, MA conducted a RNA sequence analysis of transcripts from hyphae, swimming zoospores, and infected tomato tissues. We first examined the expression of highly expressed transporters in swimming zoospores (Fig. 2) using RNA-Seq data generated by the Broad Institute, to improve the quality of predicted gene models. The sequence with the highest expression (PPTG_18357) is orthologous to PDR1 a drug resistance transporter from P. sojae (Connolly et al. 2005). The other two ABC transporters were incomplete models. The

RNA sequence data shows the expression of PPTG_16698 a predicted transmembrane transporter of 494 amino acids, to be the 5th highest of all transporters expressed by swimming zoospores of P. parasitica.

To identify related sequences in P. parasitica, we performed a BLAST analysis using the protein sequence of PPTG_16698. BLAST analysis returned 52 related protein sequences, 13 of which were found to share a highly conserved sequence of amino acids (Fig. 1 and Fig. S1). The majority of the variability in these sequences was confined to a short region of l5-20 amino acids

(Fig 1 B) that is predicted to be a loop between the 341 and 367 residues.

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Figure 3 (A): Conservation of amino acids in a novel clade of transporters in P. parastica. A high level of conservation is observed along the majority of the length of the proteins (see also

Fig. S1). (B): Highlighted region of the alignment showing the domain that is most variable for this clade of proteins.

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Figure 4: Mid-point rooted tree showing maximum likelihood phylogeny of the TOPAS family of proteins in P. parastica. The tree was generated using PhyML with amino acid equilibrium frequencies were defined by the LG model with 100 bootstrap replications.

The RNA-sequence data produced by the Broad Institute has been used to see the differential expression of the proteins at various stages of its life cycle, and its expression in the affected tissues of the plant. Sequences in this clade of proteins that were most closely related

(Fig. 3 (the tree) showed the most divergence in expression. For example, PPTG_16697 which is the closest sequence to PPRG_16698 is significantly downregulated in zoospores. The

17 sequence PPTG_08976 shows the highest expression in mycelia, while PPTG_08979 is down regulated in this tissue and exhibits a higher level of expression in zoospores.

Figure 5: The RNA-sequence of a conserved clade of predicted transporters related to

PPTG_16698 in P parasitica. Expression reads were calculated as median centered log2(RPKM) reads per kilobase per million mapped reads.

To determine how this family of proteins expanded in Phytophthora species,

PPTG_16698 was used to retrieve related sequences in other oomycetes. These sequences were aligned using CLUSTALW and used to assess phylogenetic relationships using PhyML (Fig. S2;

Fig. 5). Next, we used PPTG_16698 to retrieve all the related sequences outside Phytophthora sp. in oomycetes. These sequences along with the other members of the clade from P. parasitica were then aligned and subjected to phylogenetic analysis using PhyML resulted in the retrieval of 100 closely related sequences in other oomycetes outside Phytophthora. In other genera, the

18 closest match to the P. parasitica gene was found in Pythium vexans with an identity of 66% and a similarity of 80%. Other close matches were found in species of Albugo, Saprolegnia, and

Hyaloperonospora. We also note that even outside the Phytophthora genus there is an elevated level of sequence conservation in this clade of proteins suggesting that they transport similar compounds.

Figure 6: Phylogenetic analysis of sequences related to PPTG_16698 in oomycetes other than

Phytophthora. The tree was generated using PhyML with amino acid equilibrium frequencies were defined by the LG model using the default conditions with 100 bootstrap replicates. The

100 organisms are mainly from Pythium, Saprolegnia, Hyaloperonospora and Albugo. They

19 have similar conserved amino acid sequences with fewer variations giving rise to two primary branches.

To determine how this family of proteins expanded in Phytophthora species,

PPTG_16698 was used to retrieve related sequences within Phytophthora sp. These sequences were aligned using CLUSTALW and used to assess phylogenetic relationships using PhyML

(Fig S3; Fig. 5). Next, we used PPTG_16698 to retrieve all the related sequences within

Phytophthora sp. in oomycetes. These sequences along with the other members of the clade form

P. parasitica were then aligned and subjected to phylogenetic analysis using PhyML. resulted in the retrieval of 99 closely related sequences in other oomycetes within Phytophthora. The closest related protein sequence was found in P. infestans. Other related sequences were found in P. ramoram, P. sojae, and P. capsici. 20

Figure 7: Phylogenetic analysis of sequences related to PPTG_16698 in oomycetes within

Phytophthora. The tree was generated using PhyML with amino acid equilibrium frequencies were defined by the LG model using the default conditions with 100 bootstrap replicates. The 99 organisms are mainly from P. infestans, P. ramoram, and P. capsici. They have similar conserved amino acid sequences with fewer variations giving rise to two primary branches.

To summarize, PPTG_16698 is member of a large but very conserved clade of transporters in oomycetes. There are no significant orthologs to this family of proteins outside of oomycetes, thus no inferences can me made about the possible function of these proteins.

21

However, based on the elevated level of expression of PPTG_16698 in swimming zoospores, we hypothesized that it might function in polyamine transport.

TmPred predicts that PPRG_16698and the other 12 genes in this cluster have 11 TM domains. The hypervariable region seen in the alignment of these proteins Fig. S1 is a loop between TM8 and TM9.

Figure 8: The hydropathy plot showing the predicted transmembrane regions generated by

TMPred. The graph represents the prediction of both the inside and outside domains. This prediction was identical to all the 13 protein sequences in P. parasitica, that were found to be highly conserved.

22

Heterologous expression of PPTG_16698 in yeast

To evaluate the direct involvement of PPTG_16698 in transport of polyamines, we incubated the wild type cells and the mutants (AGP2Δ and AGP2Δ + PPTG_16698) in 0.5 mM of labelled putrescine and spermidine (Figure 7). As expected, wild type cells showed uptake of putrescine and spermidine over a time of 20 mins (Fig.8 (A and B); (Aouida et al., 2005).

AGP2∆ cells did not uptake putrescine or spermidine (Fig 8 (A and B)). We also noted that

AGP2∆ cells expressing PPTG_16698 did not acquire polyamines from the media. Thus, this gene does not function as a plasma membrane-localized polyamine transporter in yeast.

Figure 9: The uptake and export data for wild type cells (BY4741), AGP2 knockout cells and

AGP2-16698 cells; A and C: The wild type cells shows uptake of putrescine and spermidine respectively, while the AGP2-KO and AGP2-KO+ PPTG_16698 cells do not show any uptake even after 20 mins; B and D: The wild type cells shows export of putrescine and spermidine respectively, while we don’t see any export for the other two mutants.

23

Growth Curve

TPO5 is a gene in yeast cells and it has a role in polyamine export (Igarashi et al., 2010);

TPO5∆ cells are deficient in the export of polyamines, and are thus more sensitive than WT cells to toxic levels of polyamines in the media (Igarashi et al., 2010). To evaluate the impact of the expression of this transporter in yeast cells, WT, WT + PPTG_16698, TPO5∆ and TPO5∆

+PPTG_16698 cells were grown overnight and then diluted into fresh minimal media supplemented with 50, 75, and 100 mm putrescine (Fig. 8). In both the wild type cells, and the

TPO5∆ cells, the initial growth of cells in media supplemented with putrescine was greater than controls. This was not however the case with WT or TPO5∆ cells expressing PPTG_16698.

After 16h of growth, there was a sharp drop in the OD of WT cells, indicating that cell death and lysis had occurred. The TPO5∆ show a similar pattern of growth till about 16 hours, and a subsequent decline in the OD of the culture. However, in WT cells expressing PPTG_16698, there was a decline in the growth rate of cells but not a decrease in OD that might be indicative of cell lysis. After 12-14h, TPO5∆ expressing PPTG_16698 were found to be inhibited at concentration of 50mM with higher concentrations resulting in cell lysis.

24

Figure 10: Growth of yeast strains in response to exogenous putrescine. A: WT: Putrescine initially promotes growth but a decline in OD is seen after 14-16 h. C: TPO5∆ cells show similar patterns in growth inhibition as the wild type cells; B: WT + PPTG_16698: Growth of cell is inhibited after 14-16h with exogenous putrescine D: TPO5∆ + PPTG_16698: Inhibition of growth occurs after 16h, and a decline in OD is seen in the presence of exogenous 75 and 100 mM putrescine.

Collectively, the analysis of these growth experiments suggested that the expression of

PPTG_16698 altered the yeast cell responses to exogenous putrescine. To determine if the cellular response were related to net uptake of exogenous putrescine we followed the acquisition of radiolabeled putrescine in the four cell lines.

25

Uptake of Polyamines

An uptake experiment was done to measure the net accumulation of putrescine and spermidine of a concentration of 0.5mM at 5, 10, and 20 min. The wild type cells showed a lower uptake of polyamines compared to TPO5∆ cells. Although the net uptake for the WT with

PPTG_16698 increased over time, if we compare it with the WT cells, we see that their net uptake was lower than the net uptake of the wild type cells. The net uptake in the TPO5∆ with transformed PPTG_16698 was found to be the higher than all the other cell types. Taken as whole, these data suggested that the expression of PPTG_16698 enabled the rapid excretion of putrescine and spermidine in WT cells. However, in TPO5∆ cells, excretion of polyamines was inhibited, and TPO5∆ cells accumulated the most polyamines.

Figure 11: Uptake of preloaded [3H] putrescine and [3H] spermidine by yeast strains. Yeast strains were preloaded with radiolabeled 0.5 mM putrescine (A) or spermidine 0.5mM (B) for 20 min. Release of isotope into the media was assessed as described in Materials and Methods. WT

+ PPTG_16698 cells showed the highest export over time relative to TPO5∆ cells; the excretion of polyamines for the TPO5∆ containing PPTG_16698 was found to be the lowest.

26

Excretion of Polyamines

To evaluate the role of PPTG_16698 in polyamine excretion, we first incubated the yeast strains in labelled polyamines for 90 min, and then followed the excretion of polyamines form labelled cells into the media. Polyamine excretion in all cell lines increased over 60 min following the labelling treatment (Fig. 10). TPO5∆ cells were found to have lower excretion of polyamines than the wild type cells. The wild type cells expressing PPTG_16698 showed the highest excretion of polyamines while the TPO5∆ cells expressing PPTG_16698 were found to have the lowest excretion of polyamines. These data indicate that PPTG_16698 is a polyamine transporter and can be given the name Transporter Of Putrescine And Spermidine (TOPAS1).

Figure 12: Excretion of preloaded [3H] putrescine and [3H] spermidine by yeast strains. Yeast strains were preloaded with radiolabeled 0.5 mM putrescine (A) or spermidine 0.5mM (B) for 90

WT + PPTG_16698 cells showed the highest export relative to TPO5∆ cells; the excretion of polyamines for the TPO5∆ containing PPTG_16698 was found to be the lowest.

27

Discussion

Our data demonstrate that PPTG_16698 is a transporter of putrescine and spermidine.

Heterologous expression of this gene in yeast strains facilitates the export of putrescine and spermidine from the cell. These results confirm that it aids in polyamine export and hence can be named Transporter of Putrescine and Spermidine (TOPAS1). Analysis of the transport data of

AGP2∆ cells expressing PPTG_16698 confirms the fact that TOPAS1 is not directly involved in uptake. We therefore hypothesize that it is an internal transporter that could be localized to the

ER, Golgi or the vacuole. Relatively few polyamine transporters have been characterized in eukaryotes. In yeast, TPO5 is localized to Golgi or Golgi secretory vesicles (Tachihara et al.

2005) and UGA4 is localized to the vacuole (Uemura et al 2004). In A. thaliana, PUT5 has been localized to the ER (Ahmed et al. 2017).

Our yeast growth assays provide insight on how exogenous expression of TOPAS1 in yeast cells may function to protect cells from toxic accumulation of exogenous polyamines. In the growth assays, for both wild type cells and TPO5Δ, we noted that low levels of exogenous putrescine promoted growth of the cells in the first 14-16h. The decline in OD in WT and

TPO5∆ cells after 14-16h suggests that the levels of putrescine in the media resulted in cell lysis

(Fig 9A, C). In contrast, expression of this gene in WT cells enabled them to continue to grow

(Fig. 9B). The absence of a decline in OD in WT cells expressing PPTG_16698 suggest that lysis of cells did not occur. A possible explanation for this could be that this gene enabled the cells to more efficiently excrete putrescine, or concentrate putrescine within the cell. TPO5Δ cells were found to be inhibited in a similar pattern as the wild type after continued growth in the presence of exogenous putrescine (Fig 9C). However, in TPO5Δ cells expressing PPTG_16698, an inhibition of growth in the presence was observed in the presence of 50 mM putrescine, and a

28 drop in the OD of the culture occurred after 16 h in the presence of 75 and 100 mM putrescine.

We hypothesize that the absence of a functional TPO5 gene in these cells didn’t allow

PPTG_16698 excrete putrescine, as it did in the wild type cells. Thus, cell death also occurred in the presence of high levels of putrescine.

In our transport assays, WT cells were found to have higher net uptake than the wild type cells expressing PPTG_16698 after 20 min (Fig 10). When these data are assessed together with our growth assays, they suggested that continued growth of yeast cells expressing TOPAS1 in the presence of exogenous putrescine was due to the ability of these cells to excrete putrescine efficiently back into the media. We showed that excretion of putrescine and spermidine was highest in WT cells expressing TOPAS1 (Fig 11). The net uptake of polyamines is highest in

TPO5Δ cells expressing TOPAS1. We also noted that TPO5Δ cells expressing TOPAS1 had the lowest export of polyamines over a span of 60 mins. Yet expression of TOPAS1 conferred protection from the toxicity of 50 mM putrescine in our growth assays. These results suggest that the reduction of toxicity is due to the ability of cells to sequester putrescine in the cell.

Our alignment data for the 13 gene models of TOPAS1 family in P. parasitica (Fig S1; Fig

3) shows that the amino acid sequences is highly conserved along the entire length of the protein except for a variable region between residues 343 and 367. This hypervariable region might enable members of this family to be targeted to different membranes, as is the case for some members of the PIN family of auxin transporters (Krecek et al 2009). TOPAS protein are found in all oomycete species and are strongly conserved (Fig. S2; Fig. S3). Based on the level of sequence conservation, we hypothesize that all members of this family play a role in polyamine transport. This large family of conserved transporters hints at the importance of differential regulation and transport of polyamines in oomycetes.

29

TOPAS1 has the highest expression of all member of this family and exhibits the highest expression in swimming zoospores. Based on its expression in yeast we hypothesize that

TOPAS1 acts to sequester putrescine and spermidine in a subcellular compartment of swimming zoospores. Zoospores have relatively high levels of putrescine and spermidine (Chibucos and

Morris 2006) but whether these high protein polyamine levels contribute to the infectivity of encysted zoospores has yet to be established.

Our future experiments will start with the sub-cellular localization of the protein

PPTG_16698. Our approach will be to generate a GFP fusion protein, and transform it in P. sojae. GFP-tagged markers to the ER, Golgi, mitochondria, and nucleus are already available to aid in this analysis (Judelson et al., 2011). However, the identification of co-transformed genes is more laborious than single constructs. In P. parasitica the 13 members of this clade show differential expression in mycelia, zoospores and infection. Now that a CRISPR editing strategy for oomycete has been implemented for oomycetes (Fang et al., 2017) we are in a position to target members of the TOPAS family in P. sojae and P. parasitica.

30

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38

APPENDIX A: MEDIA

LB Medium (1L)

10g tryptone

5g Yeast extract

10g NaCl

950 ml of deionized water

Final Ph 7

15g Agar

SC Minimal Medium

0.67% Yeast nitrogen base

2% glucose or raffinose

0.01% (adenine, arginine, cysteine, leucine, lysine, , , uracil)

0.005% (, , , , , , serine,

, )

2% agar

YPD (Yeast Extract Peptone Dextrose Medium)

1% yeast extract

2% peptone

2% dextrose

2% agar