An Investigation of the Role of Amino Acids in Plant-Plant Parasitic Nematode Chemotaxis and Infestation

An Investigation of The Role of Amino Acids in Plant-Plant Parasitic Nematode Chemotaxis and Infestation

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

Timothy S. Frey

Graduate Program in Plant Pathology

The Ohio State University

2019

Dissertation Committee;

Professor Christopher G. Taylor, Advisor

Professor Soledad M. Benitez Ponce

Professor Pierluigi Bonello

Professor Joshua Blakelslee

2019

Abstract

Plant parasitic nematodes are economically devastating crop pests. They are responsible for billions of dollars in crop loss in all crop growing regions of the world. Management of these pests is difficult and involves many laborious, toxic or marginally effective measures that in the best of circumstances do not lead to complete control. Plant-parasitic nematodes are obligate biotrophic parasites and must obtain all of their nutrition from a living host. Plant parasitic nematodes lack the metabolic enzymes to synthesize certain amino acids, thus it is essential for them to obtain them from a plant. Because of the essential nature of amino acids for plant- parasitic nematodes the general aim of this study was to investigate their impact throughout nematode life cycles. This investigation examined the role of amino acids throughout the lifecycle of root-knot nematode, Meloidogyne incognita, and as a factor for chemotaxis of the sugar beet cyst nematode, Heterodera schactii, and the soybean cyst nematode, Heterodera glycines as well as the model nematode, Caenorhabditis elegans.

The role of amino acids in M. incognita infestation was investigated using amino acid homeostasis knockouts and overexpression lines. Changes in threonine homeostasis, particularly in the threonine catabolism overexpression mutant 35s omr1-7 were found to lead to decreased early infestation, late infestation, fecundity and increased male production in M. incognita. These effects were particularly pronounced at higher competition levels. Threonine homeostasis is important for successful nematode infestation and manipulation of host amino acid homeostasis should be further explored for its impacts on M. incognita parasitism

The role of amino acids in chemotaxis was investigated using choice assays. Threonine, aspartic acid, proline, and histidine were found to be the most attractive amino acids for M. incognita. Cysteine, phenylalanine and serine were the most repellant amino acids. The model nematode C. elegans showed different preferences for amino acids. Amino acids were present in soybean (Glycine max) root exudates, and a reconstructed blend of amino acids present in soybean root exudates was attractive to nematode species that can utilized soybean as a host, including M. incognita and H. glycines. These data could be utilized to create new integrated pest management strategies for nematode control.

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Acknowledgements

In order to make this research happen a lot of support, encouragement and assistance was required. First, the Ohio State Plant Pathology Department has an amazing atmosphere conducive to learning and collaboration. The wonderful logistical work put in by Monica

Lewandowski, Ken Nanes, Lori Jones and Lynn West, was crucial to my success. The Plant

Pathology Department faculty was warm, friendly, and always willing to give advice. Particularly,

I would like to thank Larry Madden, Mike Ellis, Peg Redinbaugh, Anne Dorrance, Pierce Paul, Sally

Miller and Soledad Benitez Ponce for their support. I would also like to thank the friendly people of Selby hall – Bob, Lee, Mark Jones, Jane Todd, Tea and many others for making this a wonderful work environment.

I would like to thank the wonderful Post. Docs who I have had a chance to work with, including Sawsan Elateek, Gina Pengue, and Wenshuang Xie. I have gleaned much from your technical and life wisdom.

I would like to acknowledge my fellow Taylor Lab graduate students – Ellie Walsh, Rebecca

Kimmelfield, Brittany Nauth, Dee Marty, Rachel Kauffman, Cecilia Chagas de Freitas, Krystel

Navarro Acevedo, Marlia Bosques Martinez, Donald Gillis, and Edwin Navarro – Your countless readings and criticism of my various ideas, presentations, and manuscripts were important for my success. Your support and friendship kept me sane while over the course of the degree. I

iv would particularly like to thank Ellie for teaching me many things in the lab over the years. Finally, a huge thanks is owed to Rebecca for countless hours editing my thesis chapters.

I would like to acknowledge all of the interns that I have had the pleasure of working directly or indirectly with over the years – Allison Grenell, Krystel Navarro Acevedo, Guillermo

Valero David, Madeline Horvath, Amanda Lietz, Gilbert Chen. You taught me so much. Your

Curiosity inspires me and I am proud to see where you are now. This work would not have been the same without you.

I must also acknowledge the following lab members –Leslie Taylor, for propagation of thousands of Arabidopsis plants, and for allowing me to fiddle around in your garden which helped keep me mentally sharp during grad school. Therese Miller, for the maintenance of the greenhouse nematode collections, and for your many contributions to making the Taylor Lab a fun place to work.

I would like to acknowledge OSU SROP program which I had the pleasure of working with for 2 summers. The leadership of this program Cyndi Freeman, Pamela Thomas, Ana’ Brown and

Jesse Goliath for inspiration and encouragement in my professional pursuits. All of my inspiring students in the program, I am overwhelmed with pride to see what you have achieved.

I would like to acknowledge PPGSA, and my fellow graduate student in Plant Pathology.

Thanks for all of the opportunities for leadership and to build relationships among fellow graduate students. I would like to acknowledge the amazing leadership the year I was President

– particularly the thoughtful guidance provided by Emile Gluck-Thaler and Coralie Farinas.

Another debt is owed to my current and former committee members, Dr. Ana Alonso, Dr.

Soledad Benitez Ponce, Dr. Pierluigi (Enrico) Bonello, and Dr. Joshua Blakeslee. Your questions, guidance and patience have been critical for the success of this dissertation.

A huge debt of gratitude is owed to my advisor Dr. Christopher G. Taylor – your deep patience, wise guidance, consistent cheerleading, bubbly passion, and enduring curiosity, made this Ph.D. one of the best experiences that I have ever had in my life.

I would like to acknowledge my parents, Charles and Susan Frey, for imbuing me with a love of learning and for always supporting me in all the crazy directions that life has taken me.

Finally, I would like to acknowledge my wife, Alessandra Frey, for all of your support, patience and curiosity about the research contained within; I could not have done it without you.

Vita

2011……………………………………………….B.S. Plant Health Management, minor in Horticulture, The

Ohio State University

2011-2012……………………………………….Research Assistant, Taylor Lab, The Ohio State University

2014…………………………………………………..Teaching Assistant, Plant Pathology 5040/41, Mycology,

The Ohio State University

2014,2015,2016,2018………………………..…………Guest Lecturer, Plant Disease Diagnostics Course,

The Ohio State university

2015-2016…………………………………………..Graduate Teaching Assistant, Summer Research

Opportunity Program, The Ohio State University

2015-2018………………………………………………..Guest Lecturer, Plant Pathology 5030, Nematology,

The Ohio State University

2012-Present……………………………………….Graduate Research Assistant, The Department of Plant

Pathology, The Ohio State University

Fields of Study

Major Field: Plant Pathology

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Table of Contents

Abstract……………………………………………………………………………………………………………………………………ii

Acknowledgements………………………………………………………………………………………………………………….iv

Vita…………………………………………………………………………………………………………………………………………vii

Table of Contents……………………………………………………………………………………………………………………viii

List of Tables……………………………………………………………………………………………………………………………xi

List of Figures…………………………………………………………………………………………………………………………xiii

Chapter 1: Literature Review…………………………………………………………………………………………….….……1

1.1 The Impact of Plant-Parasitic Nematodes on Agricultural Production……………………….…….……1

1.2 Lifecycles of Important Nematodes………………………………………………………………………………..……5

1.3 Current Options for Nematode Control………………………………………………………………………………10

1.4 Amino Acids; Their Influence on Nematode Parasitism………………………………………………………25

1.5 References…………………………………………………………………………………………………………………………35

Chapter 2 – Threonine Homeostasis Plays a Role in Successful Root-knot Nematode Infestation of Arabidopsis ……………………………………………………………………………………………………………….…..76

2.1Abstract………………………………………………………………………………….……………………………………….…76

2.2 Introduction………………………………………………………………………………………………………………………77

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2.3 Materials and Methods…………………………………………………………………………………………………….83

2.4 Results…………………………………………………………………………………………………………….………………..90

2.5 Discussion…………………………………………………………………………………………………….……….………….94

2.6 Acknowledgments………………………………………………………………………………………..…………………..99

2.7 References…………………………………………………………………………………………………..…….…………….100

Chapter 3 – Amino Acids Play a Role in Plant-Parasitic Nematode Chemotaxis……….…….……….128

3.1 Abstract……………………………………………………………………………………...... 128

3.2 Introduction…………………………………………………………………………………………………..…….…………..130

3.3 Materials and Methods……………………………………………………………………….………..……….……...…134

3.4 Results…………………………………………………………………………………………….………..………….………….145

3.5 Discussion……………………………………………………………………………………….………………….…………...149

3.6 Acknowledgements…………………………………………………………………………….………….……….……….155

3.7 References………………………………………………………………………………………..…………..…………..…….156

Chapter 4 – Amino Acid Changes in the Rhizosphere Impact Plant-parasitic Nematode

Chemotaxis…………………………………………………………………………………………….………………………………188

4.1 Abstract…………………………………………………………………………………………….………………..……………188

4.2 Introduction…………………………………………………………………………………..………….……..……………..189

4.3 Materials and Methods………………………………………………………………………..……….…..…………….193

4.4 Results………………………………………………………………………………..…..……………………….……………..200

4.5 Discussion……………………………………………………………………………………………………………………….203

4.6 Acknowledgements………………………………………………………………………..………………………………210

4.7 References……………………………………………………………………………………..………..…………….………211

Chapter 5 – CHALLENGES, IMPLICATIONS, QUESTIONS and FUTURE DIRECTIONS…………….…...231

Bibliography………………………………………………………………………………………………..…..…………………….238

Appendix A; Callose Deposition in Root-Knot Nematode Giant Cells is Affected by Plant Defense

Status ……………………………………………………………………………………………………….………………..…………301

Appendix B – Chemotaxis Raw Data……………………………………………………..…………………………………341

List of Tables

Table 2.1 Changes in aspartate-derived amino acid synthesis pathway gene expression observed during RKN infestation (Morse et al., 2010). Total RNA was collected from microdissected giant cells and surrounding root cells. Log2 fold changes in feeding site or infested treatment versus non-feeding site are presented with a false discovery rate of < 5% for giant cell(*). Threonine aldolase 1 and dihydropicolinate synthase 1 expression are increased at 14 days after infestation.

Threonine aldolase 1 and threonine synthase expression is increased at 21 days after infestation.…………………………………………………………………………….……………………………………………..112

Table 3.1 Amino acid composition of soybean root exudate (% of total free amino acids). Soybean plants were grown in sterile conditions for 14 days, after 14 days roots were washed with twice.

Collected root exudates were filtered and lyophyllized. Root exdates were then resuspended and run through LC/MS-MS analysis. Chromatograms were integrated using Analyst software. n=4.……………………………………………………………………………………………………………...…………………..…169

Table 3.2 Amino acid volumes added to make 100ml reconstructed amino acid root exudate stock. The final solution was then diluted 1:100 and 1:1000 to make test solutions.………………………………………………………………………….……………………………………………………171

Table A.1. Callose related genes are upregulated in nematode giant cells. Expression data was collected from Morse et al. (2010). The expression data is available at GEO Accession GSE21981

(http://www.ncbi.nlm.nih.gov/geo/). Total RNA was collected from microdissected giant cells and surrounding root cells. Log2 fold changes in feeding site or infested treatment versus non-

xi feeding site are presented with a false discovery rate of < 5% for giant cell(*). Significant differences are marked in yellow, red, or orange…………………………………….……………………………330

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List of Figures

Figure 2.1 The aspartate-derived amino acid pathway. Arrows indicate significant increases in gene expression in nematode giant cells compared to surrounding root cells at the 14 or 21 days post-infestation. Expression data can be accessed at GEO Accession GSE21981

(http://www.ncbi.nlm.nih.gov/geo/). ……………………………………………………………………………..114

Figure 2.2. Threonine catabolism mutants do not significantly impact plant root architecture.

Total length was measured at each time point from the base of the hypocotyl to the root tip.

Lateral root density was measured by dividing the length of root that contained lateral roots

(lateral root zone) by the total number of lateral roots. The threonine homeostasis mutants that we measured did not impact the total root length (A) or lateral root density (B). Error bars represent +/- standard deviation.……………………………………………………..………………………………………………………………………115

Figure 2.3 Threonine catabolism overexpression reduces egg mass numbers. Measurement of egg mass production in Columbia WT (Col - WT), threonine aldolase 1 (tha1-1), a threonine deaminase overexpression mutant (35s omr1-7) and a double mutant of threonine aldolase 1 and a threonine deaminase overexpression mutant (35s omr1-7/tha1-1). 3-week-old seedlings (5 per plate) were inoculated with 1000 RKN eggs. Seven weeks after inoculation, the number of females that successfully produced egg masses on each plate was counted. Significantly less xiii number of egg masses were produced on the 35s omr1-7 as compared with the WT. P values were calculated using the Students T-test (*P=<0.05). Error bars represent +/- standard error of the mean. Representative experiment of 5 total replications. ……………..………………………………117

Figure 2.4 Threonine catabolism overexpression reduces root-knot nematode early entry.

Measurement of nematode root penetration during early infestation in Columbia WT (Col - WT), threonine aldolase 1 (tha1-1), a threonine deaminase overexpression mutant (35s omr1-7) and a double mutant of threonine aldolase 1 and a threonine deaminase overexpression mutant (35s omr1-7/tha1-1). Ten seven day old seedlings were inoculated with 1000 RKN second-stage juveniles. After six days, plants were removed from the plates and stained with acid fuschin.

Significantly less number of juveniles were counted in the 35s omr1-7 root systems as compared with the WT control. n=15-25 plants. P values were calculated using the Students T-test

(*P=<0.05). Error bars represent +/- standard error of the mean. Representative experiment of 3 total replications.. ………………………………………………………………………….……………………………………..118

Figure 2.5. The addition of threonine restores threonine catabolism mutant to wild type infestation levels. Measurement of egg mass production in Columbia WT (WT - Col) and a threonine deaminase overexpression mutant (35s omr1-7) grown with or without supplementation with 5μM of L-threonine, D-threonine, L-aspartic acid or L-isoleucine in the growth media. 3-week-old seedlings (5 per plate) grown with or without threonine supplementation were inoculated with 1000 RKN eggs. Seven weeks after inoculation, the number of females that successfully produced egg masses on each plate was counted.

Significantly less number of egg masses were produced on the 35s omr1-7 as compared with the xiv

WT. However, 35s omr1-7 plants supplemented with 5μM of threonine produced egg masses at levels comparable to the WT - control. Differences were calculated using a Welch’s ANOVA followed by a Post-hoc Games-Howell test (*P=<0.05). Error bars represent +/- standard deviation. Data collected over at least three replications.. ……………………………………………………119

Figure 2.6. Range of concentrations used to test threonine rescuing effect on threonine catabolism mutant, 35s-omr1-7. Measurement of egg mass production in Columbia WT (WT -

Col) and a threonine deaminase overexpression mutant (35s omr1-7) grown with or without supplementation with 0.5 μM, 5μM, 50μM, or 50μM 0of L-threonine. 3-week-old seedlings (5 per plate) grown with or without threonine supplementation were inoculated with 1000 RKN eggs. Seven weeks after inoculation, the number of females that successfully produced egg masses on each plate was counted. Significant differences were measured with the control 35s omr1-7 plants (no threonine) and at the 0.5μM supplementation level. With the addition of 5 μM or higher of threonine to the media, differences between the Col Wt and 35s omr1-7 were no longer observed. P values were calculated using the Students T-test (*P=<0.05). Error bars represent +/- standard error of the mean. ………………………………………………………………………….121

Figure 2.7. The addition of threonine rescues early infstation in threonine catabolism mutant to wild type levels. Measurement of nematode root penetration during early infestation in

35s omr1-7 as compared with the WT, the addition of 5μM threonine to the media rescued resulted in 35s omr1-7 plants that are not different from the wild type controls. n=20-25 plants.

P values were calculated using the Students T-test (*P=<0.05). Error bars represent +/- standard error of the mean. Representative experiment of 3 total replications.………………………………………………………………..………………………………..……………………….122

Figure 2.8 Threonine catabolism overexpression reduces root-knot nematode fecundity.

Measurement of fecundity in Columbia WT (WT - Col), threonine aldolase 1 (tha1-1), a threonine deaminase overexpression mutant (35s omr1-7) and a double mutant of threonine aldolase 1 and a threonine deaminase overexpression mutant (35s omr1-7/tha1-1). 3-week-old seedlings (5 per plate) were inoculated with 1000 RKN eggs. Seven weeks after inoculation, the number of females that successfully produced egg masses on each plate was counted. After counting the number of egg masses, the agar from the plates was dissolved, and the number of the eggs of eggs on each plate was counted. Significantly less number of eggs per egg mass were observed on the 35s omr1-7 as compared with the WT. n=10-13 plates. P values were calculated using the

Students T-test (*P=<0.05). Error bars represent +/- standard error of the mean. Representative experiment of 3 total replications. …………………………………………………………………………………………123

Figure 2.9 Root-knot nematode competition impacts male production on a threonine catabolism mutant. Measurement of male production in Columbia WT (WT - Col), threonine aldolase 1

(tha1-1), a threonine deaminase overexpression mutant (35s omr1-7) and a double mutant of threonine aldolase 1 and a threonine deaminase overexpression mutant (tha1-1/35s omr1-7) exposed to different levels of competition. Three-week-old seedlings (5 per plate) were xvi inoculated with 500 (low competition), 1000 (average competition), or 2000 (high competition)

RKN eggs. Seven weeks after inoculation agar was dissolved and stained with acid fuchsin so males could be observed. Significantly increased male production was only measured at the highest competition level (2000 eggs at initial inoculation). n=10-13 plates. P values were calculated using the Students T-test (*P=<0.05). Error bars represent +/- standard error of the mean. Representative experiment of 3 replications.……………………………………………………………….124

Figure 2.10 Competition impacts root-knot nematode parasitism on a threonine overexpression mutant. Measurement of egg mass production in Columbia WT (Solid-triangles), threonine aldolase knockout, tha1-1 (dotted line, circles), a threonine deaminase overexpression mutant,

35s omr1-7 (Dashed-squares) and the tha1-1/35s omr1-7 double mutant (dash/dot-diamond) exposed to different levels of competition. Three-week-old seedlings (5 per plate) were inoculated with 500 (low competition), 1000 (average competition), or 2000 (high competition)

RKN eggs. Seven weeks after inoculation, the number of females that successfully produced egg masses on each plate was counted. Significantly less number of egg masses were produced on the 35s omr1-7 as compared with the WT, and the percentage of egg masses is less on the 35s omr1-7 with high competition. n=10-13 plates. P values were calculated using the Students T-test

(*=P<0.05, **=P<0.01). Error bars represent +/- standard error of the mean. Representative experiment of 3 total replications. ……………………………………………………………………….……………….125

Figure 2.11 Competition on the threonine catabolism mutant impacts root-knot nematode fecundity. Measurement of Eggs per Egg mass in Columbia WT (WT - Col), threonine aldolase 1

(tha1-1), a threonine deaminase overexpression mutant (35s omr1-7) and a double mutant of xvii threonine aldolase 1 and a threonine deaminase overexpression mutant (tha1-1/35s omr1-7) exposed to different levels of competition. Three-week-old seedlings (5 per plate) were inoculated with 500 (low competition), 1000 (average competition), or 2000 (high competition)

RKN eggs. Seven weeks after inoculation, the number of females that successfully produced egg masses on each plate was counted. After counting the egg masses, agar was dissolved in an autoclave, and the number of eggs per plate was counted. n=10-14 plates. P values were calculated using the Students T-test (*P=<0.05). Error bars represent +/- standard error of the mean. Representative experiment of 3 replications. …………………………………………….………………..127

Figure 3.1. Measurement of Soybean Amino Acid Root Exudate Profile. (A). Experimental Setup. Sterile soybean (Glycine max cv ‘Lee') seed was soaked overnight in ddH2O. One imbibed seed in each funnel, and then covered with sterile sand ¼ Murashige and Skoog media. The tubes were incubated for two weeks in a growth chamber. Funnels were then washed two times with ddH2O. Samples were filtered sterilized and lyophilized. Once dry, sterile samples were submitted for LC-MS/MS analysis. Data is the average of 4 replications (B). See Table 3.1 for variation.…………………………………………………………………………………………………………………………………173

Figure 3.2. Experimental setup for worm mazes. A. Top view of aluminum discs showing the channels extending from the center and the three wells at the end of the wings. B. Cross-section of one of the wings wells showing how the maze would be set up for an experiment. First, an aluminum plug was placed in the well and agar poured into the channel. Second, the plugs were removed from the wells, and an angle was cut in the agar starting 0.7cm away from the edge of

xviii the channel. Third, ddH2O was placed in the well for 24 hours for diffusion to equilibrate before adding the solution containing a solution.……………………………………………………….…………………….176

Figure 3.3. Nematode attraction to plant root exudates. Root exudates were collected from two- week-old soybean and corn plants, over a 48 hour period. 1.5ml of undiluted root exudate solution was placed in one of the wells on the worm mazes. Two-hundred juvenile nematodes were placed onto each plate, and the plates were incubated for 24 hours. After 24 hours, the number of juveniles in each well was counted under a dissecting microscope. n=9. Each experiment was repeated at least three times with similar results. Error bars represent standard error of the mean. …………………………………………………………………………………………………………………178

Figure 3.4. The attraction of M. incognita to L-threonine at various concentrations. Plates were set up with individual amino acid and control plugs. Solutions were allowed to diffuse for 24 hours. 100 RKN J2s were added and allowed to incubate on the plates for 24 hours. J2s that were within 5mm of the plug containing amino acids after 24 hours were counted as attracted. n=12.

A+B (C−( )) 2 Chemotaxis index was calculated using the following formula CI= A+B . P values were (C+( )) 2 calculated using the Students T-test (*P=<0.05), comparing each treatment to the water control.

Experiment was repeated twice with similar results. Error bars represent standard error of the mean.………………………………………………………………………………………………………………………….…………179

Figure 3.5. Measurement of root-knot nematode juvenile attraction to individual amino acids.

Mazes were made with two wells containing ddH2O and one well with 5mM amino acid solutions.

200 RKN J2s were inoculated onto the mazes and incubated for 24 hours. After 24 hours, wells xix containing amino acid or control solutions were removed, and the number of juveniles in each well was counted under a dissecting microscope. n=9. Each experiment was repeated at least three times with similar results. Chemotaxis index was calculated using the following formula

A+B (C−( )) 2 CI= A+B . P values were calculated using the Students T-test (*P=<0.05), comparing each test (C+( )) 2 solution to the water control. Error bars represent standard error of the mean.……………………………………………………………………………………………………………………………………180

Figure 3.6. Root-knot nematode juvenile attraction to individual amino acids does not follow a general pattern. Circles in the diagram represent various general characteristics of amino acids.

………………………………………………………………………………………………………………….…………………………..181

Figure 3.7. The attraction of Root-knot nematode to L-/D- Amino acids. Mazes were made with two wells containing ddH2O and one well with 5mm amino acid solutions. 200 RKN J2s were inoculated onto the mazes and incubated for 24 hours. After 24 hours, wells containing amino acid or control solutions were removed, and the number of juveniles in each well was counted under a dissecting microscope. Each experiment was repeated at least 3 times with similar

A+B (C−( )) 2 results. n=9. Chemotaxis index was calculated using the following formula CI= A+B . P values (C+( )) 2 were calculated using the Students T-test (*P=<0.05), comparing each test solution to the water control. Error bars represent standard error of the mean.…………………………………………………………………………………………………..………………………………182

Figure 3.8. C.elegans chemotaxis toward or away from amino acids. Two hundred synchronized

C. elegans juveniles (L1 stage) were plated onto mazes and incubated for 24 hours. After 24 hours, wells containing amino acid or control solutions were removed, and the number of juveniles in each well was counted under a dissecting microscope. n=9 Each experiment was repeated at least three times with similar results. Chemotaxis index was calculated using the

A+B (C−( )) 2 following formula CI= A+B . P values were calculated using the Students T-test (*P=<0.05) (C+( )) 2 comparing each test solution to the water control. Error bars represent standard error of the mean……………………………………………………………………………………………………………………………………183

Figure 3.9. C. elegans amino acid chemotaxis. C. elegans L1 stage juveniles are attracted to acidic and basic amino acids in general. Circles represent various general characteristics of amino acids.………………………..…………………………………………………………………………….……………………………184

Figure 3.10. C. elegans ionotropic glutamate receptor mutants affect chemotaxis phenotypes.

Two hundred synchronized C. elegans juveniles (L1 stage) were plated onto mazes and incubated for 24 hours. The number of juveniles that accumulated in each well was counted and the chemotaxis index was calculated. Each experiment was repeated at least two times with similar

A+B (C−( )) 2 results. n=18. Chemotaxis index was calculated using the following formula CI= A+B . P values (C+( )) 2 were calculated using the Students T-test (*P=<0.05). Error bars represent standard error of the mean……….…………………………………………………………………………………………………………………………….185

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Figure 3.11. Differential Attraction to Amino Acid Root Exudate Reconstructions. Mazes were made with two wells containing ddH2O and one well with reconstructed solutions of the amino acid root exudate mix that we found in soybean. The reconstructed solutions were diluted from

1:100 or 1:1000 times from a stock solution. 200 RKN J2s were inoculated onto the mazes and incubated for 24 hours. After 24 hours, wells containing amino acid or control solutions were removed and the number of juveniles in each well was counted under a dissecting microscope.

A+B (C−( )) 2 n=9. Chemotaxis index was calculated using the following formula CI= A+B . Each experiment (C+( )) 2 was repeated at least three times with similar results. Error bars represent standard error of the mean.. …………………………………………………………………………………………………….……………………………186

Figure 3.12. Contribution of most attractive and repellant amino acids to the overall attractiveness of the soybean root exudate amino acid profile. Mazes were made with two wells containing ddH2O and one well with reconstructed solutions of the amino acid root exudate mix that we found in soybean. The reconstructed solutions were diluted to 1:100, and in this experiment, the top attractants or repellants were replaced with their D-form equivalents in the same proportion as the L-form amino acid would have been added. 200 RKN J2s were inoculated onto the mazes and incubated for 24 hours. After 24 hours, wells containing amino acid or control solutions were removed, and the number of juveniles in each well was counted under a dissecting

A+B (C−( )) 2 microscope. n=9. Chemotaxis index was calculated using the following formula CI= A+B . Each (C+( )) 2 experiment was repeated at least three times with similar results. Error bars represent standard error of the mean………………………………………………………………………………………..………..…………..…187 xxii

Figure 4.1. Root-knot nematode chemotaxis towards or away from individual amino acids on agar plates. (A). Plates were set up with individual amino acid and control plugs. Solutions were allowed to diffuse for 24 hours. 100 RKN J2s were added and allowed to incubate on the plates for 24 hours. (B). J2s that were within 5mm of the plug containing amino acids after 24 hours were counted as attracted. n=12. P values were calculated using the Students T-test (*P=<0.05).

Error bars represent standard error of the mean.

………………………………………………………………………………………………………………………………………………222

Figure 4.2. Amino acids can distract nematodes from host roots. (A). Experiment Setup. Fifteen plates with a 7-day-old Arabidopsis thaliana ‘Columbia’ plant (Left) and either a Gamborg’s media or amino acid amended plug (Right) were inoculated with 100 M. incognita J2s (N). Plants were incubated for six days, and then roots were stained with acid fuschin. The number of J2s that were stained in each root system was counted under a dissecting microscope. (B). Results. n=15.

P values were calculated using the Students T-test (*P=<0.05). Error bars represent +/- standard error of the mean. Representative of 3 experiments with similar results.

………………………………………………………………………………………………………………………………………………224

Figure 4.3. Amino acids can repel nematodes from host roots. (A). Experimental setup. Fifteen plates with a Seven-day-old Arabidopsis thaliana ‘Columbia’ plant (Left) and Gamborg’s media plugs containing either an amino acid or no amino acid control (center) were inoculated with 100

M. incognita J2s (N). Plates were incubated for six days after which roots were stained with acid fuschin. The number of J2s in each root system were counted under a dissecting microscope. (B).

Results. P values were calculated using the Students T-test (*P=<0.05). n=15. Error bars represent xxiii

+/- standard error of the mean. Representative of 3 experiments with similar results.………………………………………………………………………………………..…………………………………………226

Figure 4.4. A push-pull setup is effective in keeping nematodes from plant roots. (A). Experimental setup.

A plate containing a seven-day-old Arabidopsis thaliana ‘Columbia’ plant and two rows of plugs was inoculated with 100 M. incognita juveniles. One row of plugs contained either a repellent amino acid or control (Center, P/S), the other row of plugs contained an attractive amino acid (T, right). Plates were incubated for six days and stained with acid fuschin. The number of J2s in each root system was counted under a dissecting microscope. (B). Results. n=15. P values were calculated using the Students T-test

(*P=<0.05). Error bars represent +/- standard error of the mean. Representative of 3 experiments with similar results…………………………………………………..………………………………………………………………………………228

Figure 4.5. Nematodes are attracted to different plant amino acid mutants more strongly than others. Plates with two Arabidopsis plants were inoculated with 200 M. incognita juveniles.

Plants were incubated for seven days and then root systems were stained with acid fuschin. The number of J2s in each root system was counted under a dissecting scope. The chemotaxis ratio was calculated. B. Results. n=12-15 P values were calculated using the Students T-test (*P=<0.05).

Error bars represent +/- standard error of the mean. Representative of 3 experiments with similar results.………………………………………………………………………………………….……………………………230

Figure A.1. Nematode infestations on jasmonic acid and salicylic acid mutants. Three-week-old

Arabidopsis plants, Col. WT, jar1-1 and sid2, were inoculated with 1000 M. incognita eggs. Plants were incubated in a growth chamber. After seven weeks, the number of females that had xxiv produced egg masses was counted under a dissecting microscope. Experiments were replicated five times each with similar results. n=12-15. P values were calculated using the Students T-test

(P=<0.05). Error bars represent +/- standard error of the mean……………………………………..……..332

Figure A.2. Jasmonic acid mutants affect M. incognita early infestation. Ten seven-day-old seedlings were inoculated with 1000 RKN second-stage juveniles. After six days, plants were removed from the plates and stained with acid fuschin. Significantly lower number of juveniles were counted in the jar1-1 root systems as compared with the WT control. n=15-25 plants. P values were calculated using the Students T-test (*P=<0.05). Error bars represent +/- standard error of the mean. Representative experiment of 3 total replications……………………………………333

Figure A.3 Nematode infestations on callose mutants. Three-week-old Arabidopsis plants (WT-

Columbia, gsl04, gsl07, gsl08, gsl11), were inoculated with 1000 M. incognita juveniles. Plants were incubated in a growth chamber. After seven weeks, the number of females that had produced egg masses was counted under a dissecting microscope. Experiments were repeated three times with similar results. n=12-15. P values were calculated using the Students T-test

(*P=<0.05). Error bars represent +/- standard error of the mean…………………….……………………334

Figure A. 4. Callose deposition in nematode feeding sites over time as measured with aniline blue staining. Three-week-old Arabidopsis thaliana ‘Columbia’ plants were inoculated with 500

xxv staining around feeding site, but vascular tissue stains brightly. B. fourteen days after inoculation

– no staining but area around nematode feeding site clearly visible in the center. C. twenty-one days after inoculation – clear staining of giant cells walls (bright blobs). D. twenty- eight days after inoculation – giant cell wall staining brightly. E. Three-week-old Arabidopsis thaliana Columbia wild-type plants were inoculated with 500 M. incognita juveniles. At, seven, fourteen, twenty- one and twenty-eight and thirty-five days past inoculation, fifteen knots were excised from roots and stained with aniline blue stain. The number of knots that was fluorescing was counted. n=15.

The experiment was repeated 4 times with similar results. Error bars represent standard error of the mean…………………………………………………………………………………………………………..………………….335

Figure A.5. Callose deposition over time in nematode feeding sites. Three-week-old Arabidopsis thaliana Col. WT plants were inoculated with 500 M. incognita juveniles. At seven, fourteen, twenty-one, twenty-eight and thirty-five days past infestation root systems were harvested. Root systems were weighed on an analytical balance. Callose was extracted from the roots using a method similar to Kohler et al. (2000). Callose deposition was measured on a Glomax fluorimeter, comparing to a curdlan standard curve. n=5 Experiment was repeated three times with similar results. Error bars represent +/- standard deviation……………………………………………………………339

Figure A.6. Callose deposition in JA/SA timing. Three-week-old Arabidopsis thaliana, jar1-1, sid2-

1 mutants were inoculated with 500 M. incognita juveniles. At, ten, fourteen, seventeen, twenty- one and twenty-four days post inoculation, 15 knots were excised from roots and stained with aniline blue stain. The number of knots that was fluorescing was counted. The experiment was repeated three times with similar results. . n=15. P values were calculated using the Students T- xxvi test (*P=<0.05), comparisons made to wt at each time point. Error bars represent +/- standard error of the mean…………………………………………………………………………………………………………..……..340

xxvii

CHAPTER 1: LITERATURE REVIEW

1.1 Impact of Plant-Parasitic Nematodes on Agricultural Production

Plant parasitic nematodes represent a great threat to agriculture worldwide (Allen et al.,

2017; Evans et al., 1993; Luc et al. 2005; Nicol et al., 2011). The first plant parasitic nematode to be described was the ‘ear cockle eelworm’, a seed galling nematode of wheat (Schrank, 1788).

Since that time over 4,100 plant parasitic nematode species have been described (Decraemer and Hunt, 2006; Nicol et al., 2011), although it is likely that many species are yet to be discovered and described (Palomares-Rius et al., 2014). Here we will focus on the most economically important sedentary endoparasitic nematodes; the root-knot nematodes (RKN), Meloidogyne spp., which were first observed on cucumber by Berkeley (1855) and first described by Goeldi

(1887), and the cyst nematodes, Heterodera and Globodera spp., first observed in the early 1800s on beets and first described by Schacht in 1859. These species of sedentary endoparasitic nematodes have an impact on the economic yield of many crops, and due to wide host ranges and a variety of successful feeding strategies are a threat to almost all crops (Nicol et al., 2011).

Managing nematode plant parasites is critical for producing and maintaining healthy crops worldwide.

World crop losses from plant parasitic nematodes have been estimated from $77 to 157 billion per year (Abad et al., 2008; Handoo, 1998; Jonathan, 2009). This represents an estimated

10% of world crop production (Whitehead and Turner, 1998) and up to 14.6% of total crop production in tropical and sub-tropical climates respectively (Nicol et al., 2011). The level of

1 economic impact varies widely by geographic region, crop and nematode species (Koenning et al., 1999). For example, the cereal cyst nematode, Heterodera avenae, can cause up to 23-50% losses in wheat production in Australia (Meagher, 1972). The southern root-knot nematode,

Meloidogyne incognita can completely destroy several vegetable crops including common bean, where impacts on the growth of the plant start at just 0.25 eggs/cm3 soil and completely inhibit plant growth at rates of 32 eggs/cm3 (Di Vito et al., 2004). On celery (Apium graveolens), M. incognita tolerance was measured to be at 0.15 eggs and juveniles/mL−1 of soil and at 16 eggs or juveniles/ml2 yield was reduced by ~70% (Vovlas et al. 2008). In some species the economic impact arises from the impact of nematode galls on marketability of the end product. Knots and swellings that can be seen by consumers are not marketable. In the example of carrot (Daucus carota), only 57% of the crop grown in the presence of Meloidogyne hapla was suitable for selling in a fresh market situation (Slinger and Bird, 1977). Initial densities of just 3 Meloidogyne chitwoodii juveniles/100g of soil were enough to cause 11% of the carrot crop to be damaged

(Wasemael and Moens, 2008). Nematodes can damage crops in a number of ways. First, nematodes create strong sink tissues that divert fixed carbon from seed and other areas of the plant leading to stunting and chlorosis (Carneiro et al., 1999). Nematodes can also interrupt the flow of water and minerals in the root system leading to increased sensitivity to drought and poor nutritional conditions (Kirkpatrick et al., 1991; Kirkpatrick et al., 1995; Wilhelm et al., 1985). In the roots nematodes can cause hypertrophy and hyperplasia of cells surrounding their feeding sites resulting in swelling and galling of the root (Caillud et al., 2008). These swollen and necrotic areas of roots lead to a very ugly and abnormal appearance, leading to reduced marketability, as

2 such the tolerance of this type of damage is very low. In other situations plant parasitic nematodes could also be described as ‘silent killers’ of crop yields. Due to their parasitic nature and presence beneath the soil surface, nematodes in crops where the root is not utilized directly can go undetected. As with other soil-borne diseases, nematode infections are easy to misdiagnose. As a result, the total impact of nematodes on agricultural production is probably underestimated.

Sedentary endoparasitic nematode species vary in the number of host species that they can infect. Tropical RKN species, M. incognita, M. hapla and M. javanica have a very wide host ranges encompassing most species of flowering plants (Taylor and Sasser, 1978; Trudgill, 1997).

It is hypothesized that this large host range is accomplished by suppression of host resistance and avoidance of host defense receptors (Trudgill, 1997). In contrast, other RKN species (M. pinus) have a very restricted host range of a few species within one or two plant families (Jepson,

1987; Riggs and Wrather, 1992; Stone, 1983). Cyst nematodes generally have narrower host ranges. The potato cyst nematode (Globodera pallida) can only infest a handful of species in the

Solanaceae family (Sullivan, 2007), while beet cyst nematode (Heterodera schactii) can infest around 200 crop species (Kahn et al., 2016) and the soybean cyst nematode (H. glycines) can infest several species of leguminous crops as well as about 60 other weedy species (Dorrance et al., 2013; Riggs and Wrather, 1992). While plant parasitic nematodes are present wherever plants are found, individual nematode species have more restricted geographical ranges. Meloidogyne incognita is found in most tropical, subtropical and warm temperate regions (Taylor et al. 1982).

The range of M. incognita is primarily restricted by low winter soil temperature, hatching is

3 limited at 120C and inhibited below 100C (Goodell and Ferris, 1989), M. incognita could not develop at temperatures of 10OC or less (Vrain et al. 1978). Increasing average winter low temperatures has allowed expansion of its range northward, and it is predicted to expand further

(Castagnone-Sereno, 2012; Ghini et al., 2008; Tsai, 2008). Although cyst nematodes such as

Heterodera and Globodera species have narrower host ranges many of these species can be found all over the world. Heterodera glycines, the soybean cyst nematode, is found most places where soybean is grown including China, Japan, Korea, Brazil, Argentina, Indonesia and the

Northern United States (CABI). Its range has spread quite rapidly in the last 50 years (Koenning and Wrather, 2010; Wrather et al., 2001) and its expansion continues to this day (Wang et al.,

2017).

Plant parasitic nematodes have a variety of successful strategies for obtaining nutrition from plant hosts (Bird et al. 2015). The stylet, a hypodermic needle-like tool, is present in all plant parasitic nematode species. This tool enables them to obtain food from their hosts and manipulate host gene expression and defense reactions (Baldwin et al. 2004). Feeding behavior strategies are more diverse. Some nematodes, such as Pratylenchus spp., remain mobile and feed from within the root throughout their life cycle. Other species are mobile as adults and feed from outside of the root using their stylet to penetrate deep into host tissue. In RKN and cyst nematodes and other sedentary endoparasitic nematodes, females lose their somatic musculature late in infestation, becoming immobile, and feeding from dedicated feeding sites made up of manipulated host cells. All plant parasitic nematode species are obligate biotrophic parasites, relying completely on a live host plant for all of their nutrition.

1.2 Nematode Lifecycles

Here we will briefly cover the lifecycles of the various nematodes that were used throughout the following dissertation research.

Caenorhabditis elegans

Caenorhabditis elegans (C. elegans) is a crucial model species for studying nematode biology as well as basic biology. C. elegans has been used to model many systems including the nervous system (Kraemer et al., 2003) and ageing (Van Ham et al., 2008). The genome of C. elegans was fully sequenced in 1998 (C. elegans Sequencing Consortium, 1998). Under well-fed conditions and at 25OC, C. elegans larva hatch from the egg as stage one juveniles at about 11 hours after fertilization (Wood et al., 1980). After hatching further development is dependent on feeding (Golden and Riddle, 1984). Development will proceed approximately 3 hours after in the presence of food, which for C. elegans is primarily bacteria. C. elegans will go through 4 juvenile stages (L1-L4) before achieving adulthood, the end of each of these stages is marked by a molt

(Félix and Braendle, 2010). In the absence of food the L1 larva can undergo developmental arrest for 6-10 days (Fukuyama et al., 2006). When food is introduced development will proceed. At the end of the second larval phase (L2) the larva can enter another arrested state called the dauer phase (Cassada and Russell, 1975; Golden and Riddle, 1984). This phase can be triggered by a number of different signals including lack of food, high temperatures as well as the presence of a crowding pheromone (Fielenbach and Antebi, 2008; Golden and Riddle, 1984). In good

5 conditions molts will continue until the adult hermaphrodites begin to lay their first eggs which usually happens 45-50 hrs after the hatch from the egg (Byerly et al., 1976). Hermaphroditic C. elegans can produce 300 eggs while sexual reproduction can increase this number to 1000-1200, although sexual reproduction is rare (Hodgkin and Barnes, 1991; Ward and Carrel, 1979).

Meloidogyne incognita

Meloidogyne incognita begins its lifecycle inside an environmentally resilient, chitinaceous egg in the soil (Bird and McClure, 1976). Juvenile nematodes undergo a molt from a stage-one juvenile to a stage-two juvenile (J2) inside the egg. RKN eggs are impenetrable even to very small molecules, such as alpha-amanitin, so it is unlikely that the eggs respond to external hatching signals (Edgar, 1994). Instead, hatching is accomplished with the help of enzymes, including lipases and proteases secreted by the J2 while it is still inside the egg (Perry et al., 1992).

After hatching, J2s use chemical signals from the host to locate a healthy root system (Čepulytė et al., 2018; Fudali et al., 2013; Perry, 2005). Upon arrival at the host root, RKN J2s use their stylet to physically soften cell walls (Wyss, 1992) while simultaneously secreting enzymes, including endoglucanases, cellulases, polygalacturonase, xylanases and pectate lyases that aid in the softening process (Béra‐Maillet et al., 2000; Huang et al., 2005; Jaubert et al., 2002; Mitreva-

Dautova et al., 2006; Rosso et al., 1999). With the cell walls softened, juvenile nematodes can navigate through the root. In the apoplast they cause minimal disturbance to the plant cells, attempting to avoid activation of plant defenses. Their final destination is the vascular cylinder, where 5-7 host cells will be selected for feeding site initiation (Berg et al., 2008). The juvenile uses its stylet to inject these cells with hundreds of protein effectors (Favery et al., 2016; Hewezi 6 and Baum, 2013; Nguyen et al., 2018). These effectors help reprogram the cells into feeding sites, termed giant cells, which serve as metabolic factories for the nutritional benefit of the developing nematode. Characteristics of giant cells include dense cytoplasm, endoreduplication without cytokinesis, multiplication of organelles, and the stretching of the cell membrane over a labyrinthine cell wall matrix to increase surface area for transfer of solutes (Berg et al., 2009; de

Almeida Engler and Gheyson, 2013; Huang and Maggenti, 1969). These are also the characteristics of plant transfer cells (Offler et al., 2004). After establishment of the feeding sites,

RKN has two more molts through juvenile stages 3 and 4 before molting into adults, a process that takes approximately 15 days but varies depending on host (Yu, 1995).

RKN species, including M. incognita, have distinct sexual dimorphism. Adult males retain their somatic musculature and vermiform shape. They will leave the root without feeding and return to the soil and die. Increased development of males is driven by nutrient deficiency in the host (Davide and Triantaphyllou, 1967). Adult females lose their somatic musculature and are thus sedentary. They are pyriform and feed from giant cells. They produce 200-2000 eggs, depending on the host plant and environment (Bhattarai et al., 2008; Javed et al., 2007; Wu et al., 1998). RKN females lay their eggs in a gelatinous, glycoprotein, egg matrix outside the root

(Sharon and Speigel, 1993). The gelatinous matrix protects the eggs from soil microorganisms

(Orion and Kritzman, 2001). The entire lifecycle takes around 20-30 days to complete under at

20-300C on tomato (Ploeg and Maris, 1999).

Heterodera glycines

Soybean cyst nematode begins its life in an egg in the soil or the cyst, the hardened, melanized cuticle of the female body. SCN undergoes one molt in the egg and hatches as a J2.

Hatching in cyst nematodes is known to be induced by a number of signals including temperature, host-derived molecules, including the small molecule glycinoeclepin A and time (Masamune et al., 1982; Tefft and Bone, 1985). Different eggs in a given cyst will be sensitive to different signals so that the timing of hatching is distributed, allowing more J2s the chance to find a healthy host root (Niblack et al., 2006; Yen et al., 1995). Chemotaxis in H. glycines is understudied, but probably involves movement to root exudates similar to other cyst nematodes such as H. schactii

(Bird, 1960). Upon entering a host root, they move intercellularly, using cell wall degrading enzymes to navigate to the vascular tissue. Like RKN, SCN has the capability of synthesizing and secreting these enzymes, including cellulases and pectate lyases (de Boer et al., 1999; de Boer et al., 2002; Smant et al., 1999). Once arrived at the vascular tissue, the SCN J2 will use stylet secretion of effectors to start the transformation of about one hundred root cells into a feeding site called a syncytium (Schmitt et al., 2004). During this process the cell walls between neighboring cells breakdown and are joined into the syncytia creating a large, multi-nucleate cell

(Jones and Northcote, 1972). The effectors responsible for virulence function through alterations in the shikimic acid pathway (Gao et al., 2003), alterations to plant developmental pathways using peptide hormones like CLE proteins (Mitchum et al., 2012) and other yet undiscovered or uncharacterized effectors that act on the cell wall, the cell cycle, the cytoskeleton and cellular metabolism. Similar to RKN giant cells, the syncytium resembles a plant transfer cell (Jones and

Dropkin, 1975). The syncytium is the sole source of nutrients for this obligate parasite. Three-to-

8 ten days after the initiation of the syncytia, the nematode undergoes 3 more molts through the final juvenile stages and adults.

SCN exhibits sexual dimorphism. Males maintain their vermiform shape, and females develop into a lemon-shaped adult stage that eventually protrudes from the root. Males do not feed and will leave the root to reproduce and die. Sex ratios can be changed under adverse conditions and host resistance (Colgrove and Niblack, 2005). Obligate sexual reproduction occurs between the motile males and the sedentary females. The number of eggs produced can range from 40 to over 600 depending on environmental conditions (Sipes et al., 1992). A few of the eggs are laid outside the female in a gelatinous matrix, but the majority is kept inside the female body which melanizes and hardens into the cyst. The SCN cycle can occur in as few as 21-28 days under ideal conditions (Alston and Schmitt, 1988). SCN eggs are notoriously resilient and can survive for up to 11 years under certain conditions (Inagaki and Tsutsumi, 1971).

Heterodera schachtii

As with SCN the lifecycle of the beet cyst nematode (Heterodera schachtii, SBCN) starts in the protective body of a female nematode, called a cyst. These cysts can contain 200-600 eggs and the cysts can protect the eggs in the soil for a very long time. About half of the eggs in a given cyst will hatch or die in a given year, but some eggs may still be viable for 10 years after the cyst was first produced. Soil temperatures are important for hatching as hatching is greatly reduced above 35°C. With increasing soil temperature the total generation time of the nematode decreases, meaning that crops planted in cooler weather will build up smaller populations that

9 those grown in warm summer seasons. As with SCN, hatching also responds to signals from plant root exudates. BCN finds its host using a blend of small molecule root exudates. Similar to RKN they enter the root near the root tips and navigate their way through the root a short distance before setting up a feeding site, in this case syncytia. Juveniles will molt 3 times before becoming adult males that leave the root or molt 4 times becoming sedentary adult females. The adult females are lemon-shaped and at least part of their body will be visible on the outside of the root. Females will produce a few eggs that are released into a gelatinous egg matrix in the soil, but the majority of which will stay inside the female body that will eventually become the cyst.

1.3 Current Options for Nematode Control

Controlling nematodes is important for most crop production around the world. Control options for nematodes are varied and strategies change depending on the economic value of the crop and threat represented by the species that may be present in a given field. Here are some of the options that are currently available, with reasons for their limited success are presented.

Prevention Strategies

Quarantines can be an important means of preventing nematode species from entering a country or area where susceptible crops are grown and are currently active for several different nematode species. An important quarantine for potato growers in the US, Canada and other nations prevents the movement of the cyst nematode, Globodera rostochiensis (Evans and Stone,

1977). When G. rostochiensis was found on Vancouver Island in British Columbia there was panic among seed potato growers that were located in that region, eventually leading to the blocking

10 of most agricultural exports from the island (Orchard, 1965). Upon closer inspections it was found that the nematode was actually only present in a small area of a few hundred acres and the quarantine was restricted to farms located in this smaller area (Rott et al, 2010). Attempts were made to fumigate the entire area but did not end up being successful in eliminating the nematode. The result of this was a complete ban on growing and selling anything outside this area that might lead to the spread of the nematode. The quarantine has been controversial through the years and led to financial ruin for farmers in the area of the quarantine. The quarantine seemed to be successful until the nematode was found near the village of Saint-

Amable in southern Quebec (Sun, 2007). This has led to a new quarantine of over 4000 hectares in this region. In the US, G. rostochiensis was discovered in the agriculture regions of Long Island,

New York, in 1948 (Mai and Lear, 1953). A quarantine was instituted for this area and was successful in keeping the nematode from spreading. The quarantine was lifted in February 2018, following successful eradication of the pathogen from Long Island. This nematode would be a huge problem for potato growers if it were to make its way into production areas in these countries.

The major nematode problems that we will discuss here are already widespread to most agricultural growing areas in the world, and thus implementation of a quarantine would likely be ineffective for these species. In addition, quarantines are very difficult to implement and maintain, and they can be a significant economic burden (Breukers et al., 2008). Diagnosing all plant and soil samples that enter into a country is impossible; therefore, quarantined nematodes have been introduced around the world.

Cultural Control Strategies

There are a number of different cultural control strategies that can be effective to reduce plant-parasitic nematode populations. Crop rotation is a system of using a set of specific crops planted in certain temporal patterns in order to improve soil health (Havlin et al., 1990), and help with fertility management (Edwards et al., 1992) and disease control (McMullen and Lamey,

1999). This practice has been an important part of agricultural production since ancient times.

Crop rotation can be an important practice for nematode control, particularly for nematode species that have a narrow host range (Trivedi and Barker, 1986). Crop rotation is also important in soybean production in the presence of SCN; current recommendations include rotating with non-host crops every other year and rotating resistance every year that soybean is planted (Tylka,

2018). For SBCN a 2-4 year rotation away from susceptible host is recommended; non-hosts include barley, corn, bean, potato and alfalfa (van der Woude, 2015). However, rotation to a non- host can take up to 2-8 consecutive years to reduce SBCN populations (Burt and Ferris, 1996).

This length of time rotating away from a susceptible host depends on how long a given species of nematode eggs, dauer-stage juveniles, or cysts can survive in the soil (Reynolds et al., 2000).

For control of SCN, the simple addition of a SCN resistant soybean cultivar to a soybean-maize rotation is sufficient to reduce inoculum density and increase yield of subsequent soybean growing years (Chen, 2007). One suggestion for enhancing crop rotation is to use cultivars of a given non-host crop that contain root exudates that will destroy non-host nematodes. In a condensed rotation, SCN control rotation of host soybeans with maize varieties that produce root exudates that are toxic to the nematodes showed that some maize varieties were able to reduce

12 nematode populations on soybean (Medina, 2017). For some nematode species the longevity of the inoculum or the breadth of the host range can make crop rotation almost impossible as a control strategy (Abawi and Widmer, 2000). Multiple species of nematode in a single field can make crop rotation nearly impossible.

Weed control is critically important for controlling nematode parasites, especially when combined with other control strategies (Norton, 1978; Rich et al., 2009). RKN, in particular, can use 100s of common weedy species as hosts (Belair and Benoit, 1996; Rich et al., 2009). When weeds are not controlled in fallow fields, nematode populations can increase (Schroeder et al.,

1993). Weedy fields can also allow nematodes to escape nematicide treatments or hostile environmental conditions, and change the ability of nematodes to infest other hosts in the future

(Duncan and Noling, 1998). Even if weeds are controlled in the cultivated portion of the field, nematodes can survive on weedy species at the margins of fields, which can be a source of inoculum for future infestations (Schroeder et al., 2005).

Cover crops are systems in which a crop is planted between cash crop seasons to provide a benefit to the farmer. Some of these crops produce compounds that can be detrimental to nematode growth and reduce populations at the start of a growing season. Sudangrass (Sorghum sudanese), castor bean (Ricinus communis), neem (Azadirachta indica) and various cruciferous vegetables (Johnson et al. 1992, Monfort et al. 2007) have been used experimentally in this role.

Some potential cover crops contain naturally containing nematidcidal or nematistatic products.

Brassica species work as a cover crop by production of glucosinolates, which may lead to biofumigation effects (Ploeg, 2008). Tagetes spp. contain α-terthienyl, a nematicide that has 13 been shown to be effective in reducing nematode populations in some fields (Goff, 1936;

Reynolds et al., 2000), an effect possibly produced by the creation of singlet oxygen in the presence of light or peroxides (Gommers, 1981, Gommers and Bakker, 1988). Tilling in

Chrysanthemum coronarium, another nematicide-containing plant, into the soil was effective in reducing root galling and increasing plant weight (Bar-Eyal, 2000), possibly using a similar mode- of-action to α-terthienyl (Bar-Eyal, 2006). Another benefit of cover crops is that they can help with weed control by decreasing the amount of time the soil is exposed to sunlight (Creamer et al., 1996). Choice of a cover crop should be made carefully in fields with high nematode populations. Choosing a nematode host species could cause further increasing the populations of nematodes that are present during the time of planting the crop following the cover crop

(Overman and Martin, 1979).

Trap cropping is cultural control system based on the fact that pests and pathogens may prefer different species of plants more than others. For nematode control these crops often allow nematodes to invade and perhaps develop, but to reproduce only in limited amounts. Solanum nigrum , Solanum sisymbriifolium (Scholte and Vos, 2000) Arugula (Eruca sativa) (Melakeberhan et al., 2006), Sunn hemp (Crotalaria juncea) (Kushida et al., 2003) and other species have been proposed and tested for their effectiveness as trap crops against various species of nematodes, but none of these options have seen much adoption.

Tillage has an impact on many aspects of plant pathogen interactions and nematodes are not excluded from this. In general, the trend seems to be that tillage in the spring or at planting were effective in bringing down nematode populations in general (Rahman et al., 2007). 14

However, the impacts of the individual tillage types on specific nematode species varies (Thomas,

1978). No tillage practice offers complete control.

Physical Control Strategies

Sanitation refers to cleanliness practices that may help the spread of contaminating organisms on a farm. Sanitation can prevent nematode infestation in new fields or prevent secondary infestation in fields that already contain nematodes (Collange et al. 2011). Some practices that fall under the umbrella of sanitation include thorough cleaning or flame sterilization of agricultural equipment between fields (Djian-Caporalino, 2009),for greenhouse- based production systems using sterilizing airlocks and pads at entrances (Collange et al., 2011), filtering of irrigation water (Hugo and Malan, 2010) and using nematode free planting stock

(Bridge, 1996). Sanitation is important to limit spread of nematodes to new fields but will not reduce populations that already exist in fields.

Soil solarization is a technique by which energy from the sun is used to heat up soil that is secured under plastic films (Katan et al., 1976). For RKN control, solarization temperatures must reach at least 38°C for up to 48 hrs in the top 15cm of soil to ensure the majority of juveniles and eggs are killed (Wang and McSorley, 2008). Solarization is not always effective because some nematodes can be resistant to very high temperatures (Ioannou, 2000). Nematodes can also survive at lower soil layers that maintain cooler temperatures (Chellemi et al., 1993).

Additionally, soil temperatures can vary over a small physical area, making it difficult to assure that whole plots have achieved the necessary temperature (Chellemi, 2002, Collange et al.,

2011). In systems where soil solarization has been used, it is often effective for a susceptible crop for one growing season, but populations will return to damaging levels in the second (Overman and Jones, 1986).

Flooding can be an effective way to control nematodes by creating anaerobic conditions that will kill juveniles and eggs. Total time of flooding required to reduce populations varies based on air temperature: at 20°C a reduction of nematode infestation was observed after 8 weeks of flooding (Rhoades, 1982). Alternating flooding with drying cycles over a few months can be even more effective at reducing nematode populations (Overman, 1964). However, flooding is difficult and expensive to implement and can cause populations of other pathogens that like flooded conditions to spike.

Chemical Control Strategies

Chemicals have been used to control nematodes since the introduction of the first fumigant, carbon bisulfide, in the late 1800s (Taylor, 2003). Control of various nematode species drives a $300 million nematicide industry in the US (Haydock, 2006). Nematicides are used most in vegetable, potato and banana production. They fall into several categories based on their mode of application. In fumigant application, a nematicide is applied as a gas to the soil, usually under a sealed plastic film. Liquid formulations are applied via drip irrigation. For dry granule application, microgranules of the nematicide are applied to the soil. Granular options are popular because they are easier to apply and require less specialized equipment than fumigants.

A few nematicide options are still on the market in some states for some vegetables.

Fumigant options include halogenated hydrocarbons such as dichloropropene (Telone II), chloropicrin (Larvacide) and isothiocyanate liberators such as sodium methyl dithiocarbamate

(Busan, Nemasol, Vapam). Most fumigants are applied before planting and must be applied under the appropriate conditions. The soil should be as clear of plant debris as possible and cultivated to a fairly fine texture with few large clods (Lembright, 1990). Soil temperature is also important and affects the diffusion and degradation rates of different nematicides (McKenry,

1974). Moisture levels should be fairly low, around the wiliting point in order to achieve proper penetration of the fumigant (Lembright, 1990). Granular options include organophosphates such as terbufos (Counter), thionazin (Nemafos) or ebufos (Rugby) as well as carbamates such as

Aldicarb (Temik) or Oxymyl (Vydate). Granular options are difficult to use on heavier soils because they do not always penetrate small soil pores. Soils high in organic matter can also make granular application difficult because organophosphate and carbamates can bind with the humus in organic matter and be taken out of the solution (Benson and Long, 1991). These nematicides are controversial and have been labeled for only a few crops over concerns about their half-life in soil and their inability to be broken down by microbes.

Nematicides act in different ways that all lead to changes in nematode behavior and survival. Many of the fumigant nematicides that contain halogenated hydrocarbons, for example methyl bromide, work as strong alkylating agents, leading to the methylation of many proteins

(Wright, 1981). Initially this causes hyperactivity in the nematodes (Thomason and McKenry,

1974) and quickly leads to the breakdown of respiration, killing the nematode (Castro and

Thomason, 1971). The mode-of-action of isothiocyanate fumigants is less understood.

Carbamates and organophosphates work by the inhibition of acetylcholinesterase leading a disturbance of the nervous system and many unusual behaviors including trembling, twisting, stylet thrusting, convulsions, coiling and uncoiling (Schneider, 1990). Fluensulfone (Nimitz) acts using a different, as of yet unclear mode of action than the nematcides listed above and is characterized by an early excitation of feeding and movement followed by wholesale inhibition of movement (Kearn et al., 2014). Practically, these behaviors adversely affect the ability of the nematode to hatch, locate and penetrate roots and develop properly.

Many of the most effective chemical control options for RKN have been removed from the market or are highly regulated due to toxicity concerns (Haydock, 2006; Zasada et al., 2010).

1,2-dibromo-3-chloropropane (DBCP), an effective nematicide, has a side effect that caused applicators to become sterile (Slutsky, 1999) and has been banned in many countries. Methyl bromide has also been tightly regulated or banned over threats to the atmospheric ozone layer

(Ristaino, 1997; Schneider, 2003). While withdrawal of these chemicals from the market is necessary for the future health of the environment, it has left a significant hole in the ability to control these persistent organisms.

Biocontrol

Biological control or biocontrol is a system of disease control that uses one biological species to control the population of another species. In the literature there are several well- studied examples of biocontrol organisms for nematode control, but few of these have achieved any commercial use. Pasteuria penetrans is an endospore forming, gram-positive bacterium, 18 related to Bacillus species. It is an obligate parasite on RKN and other nematodes. These bacteria have proven effective for nematode control under some high nematode populations (Chen et al.,

1996; Page and Bridge, 1985), and may be capable of bringing nematodes below economic thresholds (Trudgill et al., 2000). Similar to the nematicides above Pasteuria penetrans infection has an impact on nematode behavior. High levels of endospores led to RKN juveniles that were not capable of directional movement; even low levels of infection impaired directional movement in the juveniles (Vagelas et al., 2012). P. penetrans also has a large impact on the fecundity of females, with many infected females failing to produce eggs (Kariuki et al., 2006;

Mankau and Prasad, 1977). P. penetrans has proven to be difficult to propagate to levels that would be required to sell the bacteria on a commercial level (Bishop, 2011). Bacterial infection of the nematode relies upon very high soil bacteria populations and nematodes can often outgrow the parasite on more favorable crops (Ciancio and Bourijate, 1995).

A fungus, Paecilomyces lilacinus, is an effective pathogen of RKN, H.schatii and

Radopholus similis eggs. P. lilacinus has been shown to effectively reduce Meloidogyne incognita galling by 66% on tomatoes grown in growth chambers at 250C and 16hrs light (Kiewnick and

Sikora, 2006). These fungi are effective against nematode eggs; they use a battery of enzymes, including proteases, chitinases (Khan et al., 2004), leucinostatins (Park et al., 2004), to break down egg walls. P. lilacinus can also directly invade nematodes by forming an appressorium

(Holland et al., 1999). Effectiveness of P. lilacinus strain P251 for nematode control is variable depending on fermentation conditions, inoculation method of the fungus, soil texture and other

19 variables. Other problems for the application to P. lilacinus include incredibly high inoculation concentrations of 3 tons/ha (Carneiro and Cayrol, 1991).

Another fungal species, Pochonia chlamydosporia (formerly Verticillium chlamydosporia), was discovered as a part of root-knot nematode suppressive soil. P. chlamydosporia is a rhizosphere colonizer that infects nematode egg masses on the outside of roots (Palma-Guerrero et al., 2013). It infects nematode juveniles and eggs using appressoria (Lopez-Llorca and Claugher,

1990) as well as a suite of eggshell degrading enzymes including proteases and chitinases

(Tikhonov et al., 2002). It is an effective biocontrol agent under conditions where egg masses are not protected inside the root or when nematode infection is not extremely high (Hoedekie et al.,

2005; Vianene and Abawi, 2000). Several studies showed that Pochonia reduced nematode damage but did not affect overall densities of nematode populations over time (Tzortzakakis ,

2000; Verdejo‐Lucas et al., 2003)

An additional well-studied group are the nematophagous fungi including Arthrobotrys,

Dactylellina and Drechslerella. These species attack nematodes by forming hyphal loops that function as nematode traps (Xu et al., 2011). Evidence suggests formation of these traps may be triggered by nematode pheromones called ascarosides (Hsueh et al, 2013) and that the fungus may draw in nematode juveniles by using food and sex signals (Hsueh et al., 2017). After entering the trap the nematodes are immobilized and invaded by mechanical and enzymatic processes (Nordbring-Hertz, 2004; Veenhuis et al., 1985). There have been a few studies showing effective control of RKN in greenhouse studies (Bakr et al., 2014; Singh et al., 2013).

While other studies failed to demonstrate reduction of nematodes even in well-colonized rhizosperes of tomato and pineapple (Persson and Jansson, 1995; Wang et al., 2003).

As with other control methods there are concerns with biocontrol organisms and their potential impacts on soil health and the environment (Hajek et al., 2016). Any potential biocontrol agent must be thoroughly vetted for non-target effects on native fauna and potential effects on other native organisms in the environment. A biocontrol agent can impact native environments by direct effects, which involve targeting of native species that are present in the soil (Brimner and Boland, 2003). Biological agents can also have indirect effects, including shifting of community structure and alterations of food webs or ecosystem services (Pearson and

Callaway, 2003).

Host Resistance

Identification of host resistance genes can be a potent strategy for nematode management. Some sources of resistance have been found in germplasm of major crop species

(Cook and Starr, 2006; Starr and Mercer, 2009). However, very few of these cultivars have been widely adopted in crop production. In RKN management, an effective resistance gene, Mi, has been used extensively in peppers and tomatoes (Williamson, 2006). The Mi gene was originally found in populations of the wild tomato species Lycopersicon peruvianum (Medina Filho, 1980).

The Mi gene encodes a NBS-LRR protein, which activates the hypersensitive response in tomato and pepper plants infested with RKN (Milligan et al. 1998). The Mi-gene driven resistance requires the salicylic acid pathway (Branch et al., 2004), the MAP kinase pathway (Li et al., 2006), as well as HSP90-1-SGT1 chaperone complex (Bhattarai et al., 2007) in order to achieve a 21 resistance response. All of these components are a part of plant systemic acquired resistance

(SAR) signaling. Thus, it is likely that Mi-gene resistance works through the activation of this pathway. Adding evidence to this is the fact that Mi-gene-containing tomato had highly increased levels of PR-1 protein expression (Mollinari et al., 2014). Resistance breaking strains of RKN were found after the introduction of the Mi-gene in California (Kaloshian et al, 1996) and were found later in other areas including Greece (Tzortzakakis et al, 2005), Morocco (Eddaoudi et al, 1999) and Spain (Ornat et al., 2001).

Available germplasm with resistance to SCN usually comes from two sources, the soybean accessions, PI 88788, which harbors the rhg1 and rhg4 resistance loci, or Peking which has the rhg4 resistance locus. The mode of action for these resistance sources is unique and does not represent standard R gene-mediated resistance. The rhg1 was recently cloned and characterized

(Cook et al., 2012), and was revealed to have contributions from three genes; an amino acid transporter, an α-SNAP protein, and a WI12 (wound-inducible domain) protein. High copy numbers of these three genes in the soybean genome allows for a successful resistance response

(Cook et al., 2012). The gene responsible for resistance at the rhg4 locus has also been recently elucidated (Liu et al., 2012). A serine hydroxymethyl transferase was found to be responsible for the resistance, by a yet unknown mechanism (Liu et al., 2012). Germplasm with rhg1 is widely used throughout the soybean growing areas of the US; however, resistance-breaking strains of

SCN are becoming more common (Gardner et al., 2017; Niblack et al., 2008) and new sources of resistance germplasm have been slow to enter the market.

Resistance to PPNs and other pathogens can be induced by the addition of certain molecules, plant extracts, cell wall fragments and attack by various biotic and abiotic signals.

Collectively this induction has been termed Systemic Acquired Resistance (SAR). SAR is characterized by the activation of a plant-wide broad spectrum resistance wherein plants become more resistant to subsequent pathogen attacks (Durrant and Dong, 2004). This is achieved through activation of a resistance pathway mediated through the action of the hormone salicylic acid (SA) (Klessig et al., 2018). There have been a number of studies that have looked at SAR induction for the control of various species of nematode, with mixed results. Some studies have reported effective activation of SAR leading to nematode resistance, including greenhouse tomatoes (Molinari and Baser, 2010), pineapple (Chinnasri and Sipes, 2002), potato

(Molinari, 2016) and several other. Conversely SAR activation did not affect nematode populations in coffee (Salgado et al., 2007) or soybean (Puerari et al., 2013). This is an area of nematode control that remains an active area of research.

Need for new and novel control options

Almost all of the above control strategies have problems with their implementation or effectiveness. Quarantines can be effective where a nematode is not present, but require diligent and labor intensive inspections in order to be effective. Cultural controls can often be effective on small scales or under favorable environmental conditions but rarely offer good control. While biocontrol options are beginning to become available, they are still a long way off as a viable nematode control option. Chemical controls, while effective, can be toxic to the environment and the applicators, and expensive to apply. Additionally, they are labeled only for a few select

23 crops. While it is unlikely that we are to find a silver bullet solution for plant parasitic nematode control, it is important that we continue to look for new ways to control these damaging parasites.

There are certain categories of things that we are looking for any potential new control strategy. First, we looked for a strategy that will prove to be durable over a long period of time, one that will be difficult for nematodes to evolve resistance to. Secondly we looked for a strategy that will be viable with different crops and potentially different species of nematodes. Another consideration is cost which can prevent some types of growers from using a potential control method.

In this study I sought to look at the biology of various nematodes and identify parts of their life cycle that may leave an opening for us to control. An understudied portion of the nematode lifecycle centers on activities occurring soon after hatching, including host finding.

Nematodes, as obligate parasites, must efficiently find a living host cell to feed from with the limited resources that are available to them after they hatch. This represents a particularly vulnerable portion of the nematode lifecycle. They are also unprotected by the host root at this stage and are thus more vulnerable to changes in the soil environment. Juveniles in the soil are susceptibe to rapid changes in temperature, moisture and contact with potentially dangerous soil organisms. Any changes that are made to the soil, would potentially impact the ability of the nematode to find their host.

1.4 Amino Acids; Their Influence on Nematode Parasitism

Nematode Chemotaxis

Plant parasitic nematodes use chemotaxis to find plant roots and will move non- randomly through soil in the direction of roots (Perry and Wright, 1998; Reynolds et al. 2011).

Nematodes are attracted by molecules exuded into the soil by plants or by compounds that have been produced or modified by organisms in the rhizosphere (Bird, 1959; Dusenberry, 1987; Lee,

1973; Prot, 1980). The chemical cues used depend on the distance from the root. Long-distance chemoattractants allow nematodes to navigate to the general root zone, include gaseous chemicals such as CO2 (Dusenberry, 1987). Short-distance chemoattractants lead them to the area of the root they will invade (Čepulytė et al., 2018; Perry, 2005). Nematodes can also use physical cues to move toward the correct areas of a host root. For example, M. incognita juveniles are extremely sensitive to temperature gradients as low as 0.0010C (Dusenberry, 1988). This ability is useful over very short distances because of the rapid and large temperature changes that occur in the upper part of the soil over the course of a day (Curtis et al., 2009). It may allow

RKN to locate the metabolically active area of plant roots where juveniles are able to gain entrance (Perry, 2005; Wyss and Grundler, 1992).

Carbon dioxide has been shown to be an important component of plant attractants for

M. incognita and a few other nematodes (Robinson and Perry, 2006). CO2 represents an attractant in the long-distance category. In the soil, M. incognita can sense CO2 gradients over distances of up to a meter (Dusenberry, 1987). Robinson (1995) showed that RKN juveniles can be attracted to micromolar amounts of CO2 over centimeter distances in sandy soils.

As plant parasitic nematodes are obligate, biotrophic organisms it is essential that they obtain their nutrition from a living and healthy host cell. Since living or dead plant tissue as well as other organisms could be a source of CO2, it is important that nematodes be able to distinguish between live and dead food sources. Ammonia and inorganic ions are signals that could allow identification of decaying organic matter (Castro et al. 1991). It is possible that plant parasitic nematodes can detect these compounds in order to avoid spending energy moving toward decaying organic matter. In one example, Patil et al. (2013) showed that applying ammonia- containing fertilizer can interfere with M. graminicola attraction to rice seedlings. Ethylene is a plant hormone that acts in senescence. Fudali et al. (2013) showed that ethylene plays a role in

M. hapla chemotaxis, with nematodes being repulsed by high ethylene producing mutants and highly attracted to ethylene deficient mutants. M. hapla can detect high levels of ethylene, allowing the nematode to avoid a root that is in the process of dying.

Studies from the 1980s demonstrated that nematodes will move toward roots and places where roots had been removed (Prot, 1980; Prot and Van Gundy, 1981). In a study using pleuronic gel, M. incognita juveniles migrated toward several species of plants and aggregated near the root tip (Wang et al. 2009). In another pleuronic gel study using Y-tubes, Reynolds et al.

(2011) demonstrated that nematodes will choose the most efficient path to find suitable plant roots and that host specific species of nematodes can discern between host and non-hosts. Teillet et al. (2013) measured transcriptional changes in M. incognita and found that gene expression was altered when detecting exudates from Arabidopsis thaliana.

Few studies have addressed which plant exudates may be important for nematodes to find the root zone and the area behind the root cap. Wieser (1955) and Lee (1973) observed that root exudates from tomato and rice were attractive to M. hapla and Aphlenchoides besseyi. In

1959, Bird found that organic acids, in particular ascorbic acid and glutamic acid, were part of the attractive component of tomato root exudates. Recently, Čepulytė et al. (2018) discovered a potent short-range attractant for root-knot nematodes, a yet to be identified compound that is specific to the actively dividing area of root tips, a zone that is the usual target for invading RKN juveniles.

An aim of this thesis work was to investigate the chemotaxis behaviors of M. incognita and other nematodes toward various amino acids, particularly responses to individual amino acids, and amino acids that may be present in host root exudates. If M. incognita juveniles require amino aicds in their diet, I hypothesized that they will be able to detect and move towards these compounds in the rhizosphere as they will need to seek out a host that will be able to provide these compounds. Further, I sought to address the whether or not plant-parasitic nematode behavior be manipulated by the presence and application of amino acids.

Amino Acid Metabolism and Nematode Infestation

In addition to being important for the chemotaxis of some nematode species, amino acids are a critical part of the nematode diet. Nematodes, like other animals, lack the enzyme aspartate kinase that allows the synthesis of the amino acids threonine, methionine, isoleucine and lysine

(Balasubramanian and Myers, 1971). This means that nematodes must obtain these amino acids

27 from their feeding sites in plants. Manipulation of amino acid metabolism is an important part of parasitic infestations (Hofmann et al., 2010). Here we investigated the impact changes in homeostasis of the threonine catabolism pathway have on the chemotaxis, development, sex determination and reproduction of RKN.

Aspartic Acid Metabolism

In this research we focused on the aspartate-derived amino acids. Aspartate-derived amino acids are synthesized in plants but not in many of their parasites and pathogens.

Nematodes and other animals lack enzymes such as aspartate kinase (AK) that are involved in this pathway. Lacking these enzymes means that all of the aspartate-derived amino acids are considered to be essential amino acids in the diet of nematodes. In the case of an obligate plant parasite such as RKN or SCN, this means that these amino acids must be obtained from a plant host.

In order to understand how these essential amino acids affect nematode infestations it is important to understand the aspartate derived pathway in plants and the enzymes and metabolites that are involved in it. AK catalyzes the first reaction in the pathway that leads to the aspartate-derived amino acids isoleucine, lysine, methionine and threonine. Arabidopsis encodes two isoforms of AK. A monofunctional AK catalyzes the first committed step in the pathway, the phosphorylation of aspartic acid, producing aspartyl-4-phosphate (A4P) (Ghislain et al., 1994).

This monofunctional AK is subject to feedback inhibition by lysine and S-adenosylmethionine (Lea et al., 1979; Rognes et al., 1980). A bifunctional AK, which also has homoserine dehydrogenase

28 activity, can catalyze the same reaction and is feedback-inhibited by threonine (Paris et al.,

2002a). The second step of the pathway is catalyzed by aspartate semialdehyde dehydrogenase and involves the oxidation of A4P leading to the production of L-aspartate-4-semi-aldehyde

(LA4S) (Paris et al., 2002b). LA4S is another substrate for the bifunctional AK. The conversion of

A4P to LA4S is the first committed step leading to threonine, methionine or isoleucine biosynthesis. Alternatively, LA4S is also a substrate for dihydrodipicolinate synthase, the enzyme which catalyzes the first committed step in lysine synthesis (Frisch et al., 1991).

For threonine metabolism, homoserine dehydrogenase catalyzes oxidation and reduction of two different sites on LA4S producing homoserine (Ghislain et al., 1994). From there, homoserine kinase catalyzes the phosphorylation of homoserine to produce O- phosphohomoserine (Lee and Leustek, 1999). Finally, to produce threonine, threonine synthase catalyzes the removal of a phosphate group from O-phosphohomoserine to produce threonine

(Bartlem et al. 2000; Curian et al., 1996). Threonine synthase is feedback inhibited by S- adenosylmethionine, indicating a role of this enzyme in methionine production (Curian et al.,

1996). In plants, the breakdown of threonine can be catalyzed by two different enzymes, threonine deaminase, the rate-limiting enzyme in isoleucine production, and threonine aldolase, leading to the production of glycine (Joshi et al., 2006). The pathway is marked by feedback inhibition and feedforward activation in order to regulate the exact amounts of metabolites in the pathway.

The first molecule in the metabolism of isoleucine is 2-oxobutanoate, which is produced from catabolism of threonine or methionine. Most is generated by the action of threonine 29 deaminease on threonine and a little from the action of methionine γ-lyase (Joshi and Jander,

2009). Threonine deaminase is the main producer of 2-oxobutanoate for isoleucine biosynthesis, as evidenced by the fact that the action of threonine deaminase inhibiting herbicide, 2-(1- cyclohexen-3(R)-yl)-S-glycine, is lethal in Arabidopsis (Szamosi, 1994). Threonine deaminase experiences allosteric feedback regulation from isoleucine (Mourad and King, 1995), leucine, and valine (Wessel et al., 2000). Isoleucine and leucine are feedback inhibitive while valine promotes the activity of the enzyme (Halgand et al., 2002). Acetolactate synthase is the next enzyme in isoleucine production and catalyzes the condensation of two pyruvate molecules to form 2- aceto-2-hydroxybutarate (2A2H) from 2-oxobutanoate (Chaleff and Mauvais, 1984; Chipman et al., 1998). Acetolactate synthase undergoes allosteric regulation, its action is inhibited by amino acids in the branched chain pathway, valine, leucine, and isoleucine (Lee and Duggleby, 2001; Lee and Duggleby, 2002; Wu et al., 1994). Acetolactate synthase is the target of many herbicides including, sulfonylurea, triazolopyrimidine and imidazolinone (Jander et al., 2003). The next enzyme in the pathway is ketol-acid reductoisomerase which catalyzes the alkyl migration and reduction of 2A2H into 2,3-Dihydroxy-3-methylvalerate (23D3M)(Chunduru et al., 1989; Dumas et al., 1995). This enzyme is understudied but does seem to have some control over the levels of branched chain amino acids in tomatoes (Kovchenko and Fernie, 2011), and has also been studied as a potential site for herbicide action (Lee et al., 2005). Next dihydroxy-acid dehydratase catalyzes the conversion of 23D3M into 2-oxo-3-methylvalerate (Ray, 1984; Pirrung et al., 1991).

The final enzyme in isoleucine biosynthesis is branched-chain amino acid transferase (Diebold et al., 2002), it catalyzes the transamination of 2-oxo-3-methylvalerate into isoleucine. Isoleucine

30 catabolism is very complex and is not well studied in plants. Isoleucine is first transaminated, possibly by BCAT2 (Angelovici et al., 2013) and then decarboxylated by branched-chain α- ketoacid dehydrogenase (Mooney et al., 2002) and finally oxidized by isovaleryl-CoA dehydrogenase leading to the transfer of electrons in the electron transport chain (Däschner et al., 2001).

The first enzyme in lysine metabolism is dihydrodipicolinate synthase (DHDS) which was mentioned before as a competitor for homoserine (Sarrobert et al., 2000). There are two genes that encode dihydropicolinate synthase in Arabidopsis. They compete for a common substrate pool and are subject to feedback inhibition according to the lysine needs of the plant (Bryan et al., 1980; Tzchori et al., 1996). DHDS catalyzes the formation of dihydropicolinate. Next dihydropicolinate reductase catalyzes the converstion of dihydropicolinate into tetrahydrodipicolinate, a conversion which has been measured in plants but the enzyme has not been isolated (Tyagi et al., 1983). A series of enzymes that is not well studied in plants, diaminopimelate epimerase, diaminopemelate decarboxylase and finally diaminopimelate aminotransferase, complete the synthesis of lysine (Hudson et al., 2006).

The final aspartate-derived amino acid is methionine. The first step in methionine starts again, with the action of homoserine dehydrogenase on L-aspartate-4-semialdehyde resulting in a homoserine product. Homoserine is the substrate for homoserine kinase which is regulated by threonine, isoleucine, valine and possibly S-adenosylmethionine. O-phosphohomoserine is a substrate for cystathione γ-synthase the first committed step toward methionine synthesis.

Cystathione γ-synthase is regulated by S-adenosylmethionine, which blocks translation as well as 31 decay of cystathione γ-lyase mRNAs (Haraguchi et al., 2008; Onouchi et al., 2005). Transgenic plants without this feedback regulation accumulate large amounts of methionine-derived volatile compounds (Hachem et al., 2002). The next enzyme is cystathione β-lyase, it undergoes little regulation. The final enzyme is methionine synthase. There are two forms of methionine synthase, one expressed in the cytosol and another expressed in the plastids. The plastid form likely contributes to methionine synthesis while the cytosolic form functions in the S- adenylmethionine(SAM). SAM functions as a cofactor for many methylation reactions (Lu, 2000).

Although there is no established list of essential amino acids for root-knot nematodes and other plant parasitic nematodes, it is quite likely that they lack these pathways and must obtain them from their plant hosts. All nematodes that have been tested for essential amino acids have lacked the ability to synthesize threonine and most of the other aspartate-derived amino acids.

These have included free living species (Vanfleteren, 1973), animal-parasitic species (Brockelman and Jackson, 1978) and plant parasitic species (Balasubramanian and Myers, 1971). As such, these amino acids are an important aspect of nematode biology that should be explored.

Amino Acids in Plant-Microbe Interactions

Plants use chemistry to protect themselves in various ways. Some chemical compounds are synthesized constitutively as the plant grows; these are termed phytoanticipins and are a part of the plants preformed defenses. Other compounds are synthesized in response to pathogen attack and are termed phytoalexins. Amino acid metabolism is required for production of many of these different types of compounds.

Amino acids represent a source of nitrogen for infecting and infesting pathogens and mutualists. Biotrophs such as the rust fungus Uromyces fabae (Hahn and Mendgen, 1997) and the tomato leaf mold fungus, Cladosporium fulvum (Solomon and Oliver, 2001), have been shown to directly utilize host amino acids to meet at least a portion of their nitrogen needs. Particularly interesting is the corn smut pathogen, Ustilago maydis, which creates a strong sink tissue in corn leaves, driving the transport of amino acids into the infected leaves, where is can be utilized by the pathogen (Horst et al., 2010). In RKN interactions genetic studies suggest an upregulation of amino acid transporters in RKN induced giant cells, suggesting that the nematode probably imports amino acids into the feeding sites during infestation (Hammes et al., 2005; Hammes et al., 2006). Considering this evidence and the fact that PPNs are obligate parasites, it would not be surprising if access to particular amino acids were critical for success of PPN infestations.

Changes in amino acid homeostasis can also directly have an effect on pathogenic organisms. Mutations in Arabidopsis homoserine kinase, leading to increased homoserine concentrations, reduced downy mildew, Hyaloperonospora arabidopsidis (HPA), reproduction

(van Damme, 2009). A point mutation in one of the monofuntional aspartate kinases, AK2, inhibits feedback inhibition from lysine. This aberration in metabolism lead to increases in cellular levels of lysine, threonine, isoleucine and methionine and was found to lead to a reduction in spore production in HPA (Stuttman, 2011). Amino acids serve as important precursors to many plant defense compounds such as glucosinolates (Sønderby et al., 2010), alkaloids (Shoji, 2011) and others. The glucosinolates, which have nematicidal effects (Zasada and Ferris, 2004), are derived from the aspartate-derived amino acids isoleucine and methionine.

Changes in amino acid transport can also have an impact on plant pathogens. An

Arabidopsis knockout of the amino acid transporter, LYSINE HISTIDINE TRANSPORTER1 (lht1), was found to enhance defense by constitutively activating PR1 (Liu, 2010). The activation of PR1 leads to increased callose deposition, cell death and other functions and was mediated by a reduction of glutamine in lht1 plants. Application of glutamine to leaves of lht1 plants successfully rescued the normal defense phenotype (Liu, 2010). Marella et al (2013) investigated the role of amino acid transporters in RKN infestation and found that knocking out two amino acid permease transporters, AAP3 and AAP6, led to a reduction in M. incognita parasitism. AAP3 and AAP6 mutants produced more males, supported less egg hatching, and decreased the ability of the hatched juveniles to produce successful subsequent infestations (Marella, 2013).

Another aim of this research is to investigate how changes in the aspartate-derived amino acid pathway, particularly the catabolism of threonine, impact M. incognita. Particularly we looked at early infestation, late infestation, production of female and males, fecundity and how these are influenced by competition.

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Chapter 2; Threonine Homeostasis Plays a Role in Successful Root-knot Nematode Infestation of Arabidopsis.

2.1 ABSTRACT

Parasitic organisms that have an obligate biotrophic lifestyle, such as the root-knot nematode

(RKN, Meloidogyne spp.), require a living, compatible host plant from which to obtain nutrients required for their growth and development. Because of the dependence of a parasite on its host for its nutrition, small changes in host primary metabolism can lead to significant changes in parasite development and reproduction. For example, studies in plants have shown that changing amino acid homeostasis can impact the development of fungal biotrophic pathogens.

Our model animal parasite, RKN must obtain specific essential amino acids from its plant host.

We hypothesized that alteration of the content of one or more essential amino acids in the host could also lead to changes in parasitism rates. In this study, we used Arabidopsis threonine catabolism mutants to investigate the impact of changes in threonine metabolism on RKN infestation, development, and fecundity. We measured RKN infestation rates into Arabidopsis roots, female and male development, and fecundity at different parasite competition levels.

Our results reveal that disturbance of threonine homeostasis leads to a decreased ability of the nematode to effectively parasitize the host plant, resulting in reduced fecundity rates and increased male production, particularly under high competition levels. This study demonstrates that threonine homeostasis is important for the development and reproduction of RKN and

76 suggests that the manipulation of essential amino acids in the root could be useful in limiting plant-parasitic nematode interactions.

2.2 Introduction

Root-knot nematodes (RKN, Meloidogyne spp.) are a diverse genus of soil-dwelling roundworms responsible for devastating damage to crops worldwide. In one example, a 2001 survey of 207 vegetable crops in Ecuador found that RKN was responsible for up to 20% yield losses across different crops (Trudgill et al. 2000). The most economically important species of

RKN, M. incognita, and M. javanica, are limited in their geographic range by low winter temperatures (Daulton and Nusbaum 1961; Taylor et al. 1982). However, warming climates around the world have contributed to the range expansion of these and other RKN species (Djian‐

Caporalino 2012; Nicol et al. 2011). Another contributing factor to the economic damage caused by RKN is their extremely wide host range, being capable of infesting 1000s of plant species from many of the flowering plant families (Trudgill 1997). In the field, small initial populations can explode into damaging populations very quickly. On ideal hosts such as tomato, M. javanica can reproduce with up to 2000 eggs per female in as short as 29 days (Trudgill 1995; Trudgill and

Block 2001). In an experiment on potato (Solanum tuberosum cv. Spunta) initial populations of just four eggs and second-stage juveniles (J2s) per cubic centimeter of soil led to stunting and yellowing of the plants, with final nematode population levels reaching 2000 eggs and J2s per cubic centimeter (Vovlas et al. 2005).

Root-knot nematodes hatch as second-stage juveniles (J2s) from environmentally resilient eggs. The J2s detect root-secreted chemicals, CO2, and physical signals, such as temperature changes, to locate and migrate toward the host root (Rasmann et al. 2012; Wang et al. 2009). When the J2s reach a host root, they penetrate it using cell wall degrading enzymes and navigate toward the vascular cylinder (Wyss 1992). Once there, J2s will inject effector proteins into 5-7 host cells, reprogramming the cells into metabolically active feeding sites called giant cells (Hewezi and Baum 2013). These cells and the root cells surrounding them undergo hypertrophy and hyperplasia, leading to the development of the root gall or knot. Giant cells contain multiple nuclei, dense cytoplasm, and many organelles (Berg et al. 2009; de Almeida

Engler and Gheysen 2013). After undergoing two more additional molts, the juvenile nematodes develop into adult males or females. Females will lay approximately 300-1000 eggs depending on the environment and host (Trudgill 1995). Adult males will regain their somatic musculature, leave the root, do not feed, and can participate in sexual reproduction in some species

(Castagnone‐Sereno and Danchin 2014; Snyder et al. 2006). Interestingly, juveniles’ fate to become male or female occurs early (in J2s) and is determined by the available resources within the host with fewer resources there will be an increasing number of males (Davide and

Triantaphyllou 1967; Triantaphyllou 1960).

An established RKN feeding site is a strong metabolic sink, and nematodes force their hosts to allocate nutrients toward their feeding sites (Bird and Loveys, 1975; Hofmann and

Grundler 2007; Kaplan et al. 2011). Nutrients from the host fuel the dramatic restructuring of host cell architecture and the metabolic needs of the parasite (de Almeida Engler and Gheyson

2013; Wieczorek 2015), a result of increased gene expression and metabolic activity within the feeding site (Siddique and Grundler 2015). Carbon labeling experiments revealed that much of the carbon absorbed during nematode feeding was localized to the feeding site, while the nematode itself took up a smaller fraction (Böckenhoff et al. 1996). The level of nutrients available to nematodes in plant roots depends on rates of transport and the amounts and types of solutes contained in the phloem and xylem (Bartlem et al. 2013). Transcriptomic studies suggest that nematodes alter plant primary and secondary metabolism within feeding sites (Ithal et al. 2007; Kyndt et al. 2012; Szakasits et al. 2009). Primary metabolism includes the production of amino acids. Earlier studies noted increases in root amino acid content associated with RKN

(Hanounik and Osborne 1975; Hedin and Creech 1998; Meon et al. 1978) and other sedentary endoparasitic nematode infestations (Betka et al. 1991; Doney et al. 1970; Grundler et al. 1991;

Krauthausen and Wyss 1982). Metabolite profiling has revealed that some nematodes may specifically upregulate the synthesis of 14 amino acids, including threonine and other aspartate- derived amino acids (Hoffman et al. 2010). Nematodes also upregulate several genes in the branched-chain amino acid pathway, including threonine synthase and several other enzymes

(Anwar et al. 2016).

In this paper, we chose to investigate the impacts of changes in amino acid metabolism during RKN infestation by specifically looking at changes to the homeostasis of the amino acid threonine. Essential amino acids are defined as amino acids that cannot be synthesized by a given organism and being necessary parts of metabolism, must be obtained from a food source. There is no established list of essential amino acids for sedentary plant-parasitic nematodes (Goheen

79 et al. 2013). However, all nematodes that have been biochemically tested or bioinformatically examined lack the ability to synthesize at least 9 amino acids; histidine, isoleucine, leucine, lysine, methionine, phenylalanine, valine, threonine, and tryptophan (Cowey and Forster 1971;

Mazid et al. 1978; Mertz et al. 1952; Ogino 1980), with others becoming essential under stressful conditions (Lacey and Willmore 1990; Wakabayashi et al. 1994). Among nematodes that have been tested for their specific list of essential amino acids, threonine is required for the development of the free-living nematode, Caenorhabditis briggsae (Vanfleteren 1973), the animal parasitic nematode, Rhabditis maupasi (Brockelman and Jackson 1978), and the migratory plant-parasitic nematode Aphelenchoides spp. (Balasubramanian and Myers 1971). Threonine is essential in cell biology in several ways. First, it is a component of proteins and it is frequently found in active sites because of the reactivity of its hydroxyl group, which can form hydrogen bonds with a variety of substrates. Another critical role of threonine is the facilitation of phosphorylation as a part of serine/threonine kinases. Aspartate-derived amino acids, including threonine, are particularly crucial for parasitic interactions because animals, including RKN, lack the metabolic pathway necessary to synthesize these amino acids. RKN must then, as an obligate parasite, obtain threonine from a living plant host.

In Arabidopsis thaliana, the synthesis of aspartate-derived amino acids, including threonine, starts with aspartate kinase catalyzing the phosphorylation of aspartic acid and through several steps, eventually leading to the direct threonine precursor, O- phosphohomoserine (Fig. 2.1). In order to produce threonine, threonine synthase catalyzes the production of threonine from O-phosphohomoserine (Bartlem et al. 2000). In plants, the

80 catabolism of threonine can be catalyzed by two different enzymes, threonine deaminase, the rate-limiting enzyme in isoleucine production, and threonine aldolase, which leads to the production of glycine (Joshi et al. 2006). The metabolism of threonine, isoleucine hand methionine are intimately intertwined, and the aspartate-derived pathway is marked by feedback inhibition and feed-forward activation in order to regulate the exact amounts of metabolites in the pathway.

Changes in plant amino acid metabolism, including aspartate-derived amino acids, can influence plant immunity toward a variety of pathogens, especially biotrophic pathogens (Seifi et al. 2013; Zeier 2013). Amino acids can influence immunity via a number of factors including changes in redox potential in cells (Chen et al. 2011; Liu et al. 2010), regulation of systemic acquired resistance (Navarova et al. 2012), remobilization of nitrogen (Hwang et al. 2011; Olea et al. 2004), regulation of stomatal opening and closing (Macho et al. 2012), hormone crosstalk

(Song et al. 2004) and triggering of salicylic acid signaling (Chen et al. 2011). The aspartate pathway, specifically, has been reported to influence pathogens by several different research groups. In one example, the A. thaliana mutant, downy mildew resistant 1 (DMR1), a missense mutant of homoserine kinase, led to decreased conidiophore production by Hyaloperonospora arabidopsidis (van Damme et al. 2009). H. arabidopsidis also had reduced spore production on the aspartate kinase mutant, rsp1, that had increased levels of all of the aspartate-derived amino acids (Stuttman et al. 2011). In addition to changes in metabolism, changes in the transport of amino acids can also influence immunity toward pathogens (Sonawala et al. 2018). In a study with M. incognita, the amino acid transporter, AtCAT6, was specifically upregulated in infested

81 plant tissue (Hammes et al. 2006). Knockout mutants of two amino acid permease transporters,

AAP3, and AAP6, showed a reduction in RKN egg mass production (Marella et al. 2013).

Arabidopsis mutants of dihydroxyacid dehydratase, an enzyme in branched-chain amino acid synthesis were shown to support fewer cyst nematodes, more male nematodes, smaller cysts and a lower hatching rate (Anwar et al. 2016). The effects of altered amino acid biosynthesis and catabolism on root-knot nematode infestation remains unexplored.

A microarray study previously conducted in the Taylor lab revealed some genes in the aspartate derived pathway were altered in their expression in laser capture microdissected RKN giant cells as compared to surrounding root cells (Morse et al. 2010, Table 2.1). The expression data is available at GEO Accession GSE21981 (http://www.ncbi.nlm.nih.gov/geo/). In particular threonine aldolase 1, tha1 was significantly upregulated at 14 and 21 days after infestation compared to the surrounding root cells. Because of this, we decided to focus on genes that are involved in threonine homeostasis. The first objective of this study was to investigate the relationship between threonine metabolism and southern root-knot nematode (M. incognita) development, reproduction, and male production infestation in A. thaliana. RKN responses were tested in a threonine aldolase mutant, tha1-1 (Joshi et al., 2006), threonine deaminase overexpression mutant, 35s omr1-7 (Mourad and King, 1995) and a cross of the two mutants, tha1-1/35s omr1-7 (Joshi et al., 2006). The second objective was to investigate how competition among RKN impacts those phenotypes. We hypothesize that changes in threonine catabolism will impact the ability of the nematode to develop and reproduce successfully, and these effects will be exaggerated in the presence of competition.

2.3 MATERIALS AND METHODS

Plant Material

All mutants used in this study were generated from the A. thaliana Col-0 (hereafter, Col-

0) background. A threonine aldolase mutant, tha1-1 described in Joshi et al., (2006) is a knockout of threonine aldolase. The mutant 35s omr1-7 is a threonine deaminase overexpression mutant driven by the 35s cauliflower mosaic virus promoter and mutated to remove feedback inhibition by isoleucine (Mourad and King, 1995; Joshi et al. 2006). tha1-1/35s omr1-7 is a cross of the mutants described above and was made and described by Joshi et al. (2006). A. thaliana mutant, tha1-1, 35s omr1-7, and tha1-1/35s omr1-7 seeds, were a gift from G. Jander (Boyce Thompson

Institute, Ithaca, NY).

Plant Growth Conditions

Seeds were sterilized in 1ml of a 70% ethanol and 0.05% triton x-100 solution (Sigma-

Aldrich, St. Louis, MO, USA) and mixed at 15 rpm for thirty minutes on a roller drum (New

Brunswick TC-7, Fisher Scientific Hampton, NH, USA). The solution was then removed and replaced with 1ml of 100% ethanol. Seeds were then mixed for 15 more minutes on the roller drum and allowed to air dry in a laminar flow hood for approximately 2 hours. All seeds were germinated on Gamborg’s media plates [2% D-sucrose (Phyotech, Shawnee Mission, KS, USA),

0.3% Gamborg's basal salts (Phytotech, Shawnee Mission, KS, USA), 0.6% phytagel (Sigma-

Aldrich, St. Louis, MO, USA), pH 6.1] before being transferred to final Gamborg’s plates for root measurement, early infestation, late infestation, and male or fecundity assays. Plants for all

83 assays were grown in a Percival (Perry, IA, USA) growth chamber under the following conditions:

8 hours light/16 hours dark, 23°C. Plates were placed in clear polycarbonate boxes at an approximate 40° angle during the assays; sterile felt was placed on the bottom of the boxes to prevent the accumulation of water from condensation.

Root-knot Nematode Culture

A sterile culture of M. incognita was maintained in the lab using the following procedure

(Marella et al. 2013). Eggs were collected from plants grown in the conditions described above at 7-8 weeks after inoculation. Aerial portions of the plant were removed and discarded. Root material containing RKN egg masses was collected from approximately 60 plants and placed in two 50ml centrifuge tubes (Falcon, Corning, NY, USA). Forty-five ml of 5% bleach was added to the tubes, which were then shaken vigorously by hand for 2 minutes. The root material was poured over a 2mm coarse filter into a new 50ml centrifuge tube, which then contained the majority of the eggs. The tubes containing the eggs and solution were centrifuged for 5 minutes at 1000rpm. After centrifugation, approximately 40ml of the solution was poured out of the centrifuge tubes and replaced with approximately 40ml of ddH2O. The centrifugation and pouring off steps were then repeated three more times, to remove as much bleach as possible. The number of eggs was then counted under a Nikon SMZ645 dissecting microscope in three 10µl droplets to obtain an approximation of the total number collected.

In experiments where J2s were used, including the early infestation assay, we hatched

RKN juveniles from eggs under sterile conditions. After egg collection, the solution was poured

84 over a 500 µm filter to retain the eggs. Eggs were incubated in the dark for 6-10 days at 23°C.

Hatched J2s crawled through the filter and were collected in the water below the filter. The concentration of juveniles was estimated in three 10 µl droplets before inoculation.

Root Measurement Assay

Col-0 and mutant seeds were sterilized and germinated on agar plates as described above.

After two days, five germinated seedlings were transferred to a 100mm Petri dish with the upper fourth of the agar excised in order to avoid contact with the leaves (Dubrovsky and Forde 2012).

Ten total plates of seedlings were transferred for each mutant. Roots were then measured every three days for 21 days. Total root length was measured from the base of the hypocotyl to the root tip. The length of the lateral root zone was measured as length from the latest emerging lateral root to the first lateral root. Lateral root number was counted from the smallest observable emerging roots that could be seen in the Nikon SMZ645 dissecting microscope at 5X magnification. Lateral root density was calculated as the length of the lateral root zone divided by the number of lateral roots. Lateral root density was used to compare the differences between the available growing root tips of the root system. This assay was repeated at least three times with similar results.

Early Infestation Assay

Ten 7-day old seedlings were transferred in a circle approximately 6cm in diameter from leaf to leaf with roots facing the center of the plate on six 100x15mm Petri dishes. Col-0 and mutants were on separate plates. Twenty-four hours later plates were inoculated with 1000 RKN

J2s. Plates were incubated for six days in a Percival growth chamber as described above with the exception that plates were placed flat rather than at a 40° angle. After six days, seedlings were removed and placed in a 1.5% sodium hypochlorite solution for 4 minutes to clear the roots. This was followed by a 15-minute rinse in ddH20 and transfer to acid fuchsin staining solution (3.5g powdered acid fuchsin, 250ml Acetic Acid, 750ml ddH2O). Acid fuchsin solution and seedlings were brought to boiling in a microwave and then cooled to room temperature (~23°C). Seedlings were destained for 1 minute 30 seconds in ddH20 and then placed in acidified glycerol (250ml glycerol, 225ml ddH2O, 25ml 1% HCl). Nematode juveniles that were inside the root system were counted in each plant under a Nikon SMZ645 dissecting microscope. Early infestation assays were repeated at least three times.

Nematode Susceptibility Assay

Nematode susceptibility assays were performed similarly to Marella et al. (2013). Seven- day old seedlings of Col-0, tha1-1, 35s omr1-7, and tha1-1/35s omr1-7 were transferred onto fresh Gamborg’s media plates. There were five seedlings per plate with 14 plates for each line.

Fourteen days later, each plate was inoculated with 1000 sterile RKN eggs. The inoculated plates were placed in the growth chamber under the conditions listed above. Seven weeks after inoculation, the plates were removed, and females that have produced egg masses on each plate were counted under a Nikon SMZ645 dissecting microscope. Each assay was repeated at least three times.

Amino Acid Complementation Assay

Plant growing conditions and nematode inoculations were as described above with the following changes: Gamborg’s media was amended using filter-sterilized L-threonine to a final concentration of 5μM, 25μM, 50µM, and 5mM. L-glycine, L-isoleucine, and L-methionine were added to the media only at the 5μM concentration. This medium was used for plates that would be measured. Nematode counting was performed after seven weeks as described above. The experiment was repeated three times.

Male Assay

Each plate of 5 twenty-one-day-old Col-0 and mutant seedlings on a single plate were inoculated with 1000 eggs and incubated for seven weeks in the growth chamber in the conditions described above. The aerial portions of the plant were then excised and removed. The remaining agar and root material were heated to boiling with 50ml of ddH2O, 3ml of dilute acid fuschin stain and 3ml 0.5 EDTA (pH 8.0) to prevent re-solidification of the media. The material was filtered through a qualitative P8 filter paper (Fisher Scientific, Pittsburgh, PA, USA). Stained male worms from a whole plate that were collected on the filter were counted under a Nikon

SMZ645 dissecting microscope. Experiments were repeated five times with similar results.

Fecundity Assay

Each plate of 5 twenty-one-day-old Col-0 and mutant seedlings per plate were inoculated with 1000 eggs and incubated for seven weeks in the growth chamber at the conditions described above. After seven weeks, egg masses were counted, the aerial portions of the plant were excised, and the remaining agar and root material were placed in a 100 ml Erlenmeyer flask with

50 ml of ddH2O and 3ml 0.5M EDTA (pH 8.0). The flasks containing the agar and root systems were autoclaved to liquefy agar and allow for accurate counting of all of the eggs. After heating, the agar and root material were rinsed over a 2mm coarse screen to collect any root material.

The collected material was then rinsed through a 500μm mesh screen to collect eggs and egg masses. Eggs and egg mass-containing roots were then incubated for 10 minutes in 5% bleach to release eggs from the gelatinous matrix. Eggs were counted under a Nikon SMZ645 dissecting microscope, and the number of eggs was averaged from five 10μl droplets. The experiment was repeated four times with similar results.

Nematode Competition Assays

Plates of 5 twenty-one-day old Col-0 and threonine mutant plants were inoculated with three different levels of nematodes: low nematode pressure (500 eggs/plate), average nematode pressure (1000 eggs/plate) and high nematode pressure (2000 eggs/plate). Plants were grown in conditions as described under Plant Growth Conditions above. Mature nematode egg masses were counted seven weeks after inoculation under a Nikon SMZ645 dissecting microscope. Male competition and fecundity competition assays were also performed at the three nematode pressures described above. The experiments were repeated at least three times.

Data Analysis

Data analysis was performed in Microsoft Excel and Minitab, version 17.1.1 (2017; State College,

PA: Minitab, Inc. (www.minitab.com)). All experients were conducted using a completely randomized design. All data were analyzed for assumptions of normality and homoscedasticity,

88 using the Kolmogorov-Smirnov test and Levene's test, respectively. For normal data with equal variances T-test, ANOVA, and Tukey's means separations were used. For non-normal or tests without equal variance, Welch’s T-test, Games-Howell Pairwise comparisons or nonparametric

Kruskal-Wallis tests were used to compare treatments to controls.

2.4 RESULTS

Mutants in threonine catabolism lead to reduced root-knot nematode parasitism

In order to determine if changes in threonine catabolism have an impact on RKN parasitism, RKN egg mass production was tested in threonine catabolism mutants, tha1-1, 35s omr1-7 and tha1-1/35s omr1-7.

Because changes in root architecture can have an impact on root-knot nematode infestation, differences in root phenotypes were measured according to Dubruvsky and Forde

(2012). There were no significant differences in total root length or lateral root density of the mutants (Fig. 2.2 A,B).

In order to measure the impact of changes in threonine metabolism on RKN parasitism, all three homozygous mutants, tha1-1, 35s omr1-7 and tha1-1/35s omr1-7, and the Col-0 wild- type were inoculated with RKN eggs and measured for the number of adult females that produced egg masses after seven weeks. Tha1-1 showed no significant changes in the number of egg masses produced compared to the Col-0 wild type. 35s omr1-7 showed a significant 17% decrease in the number of egg masses that were produced as compared to the wild type. The double mutant tha1-1/35s omr1-7 also showed no significant changes in the number of egg masses as compared to the wild type (Fig. 2.3).

Changes in parasitism can be observed early in the infestation process

To measure the effect of changes in threonine homeostasis in the early days of RKN infestation, 7-day-old Arabidopsis seedlings were infested with 1000 sterile RKN J2s. Juveniles 90 that invaded the root, and stayed in the root were measured six days later after staining with acid fuschin. Similar to long infestation assays, roots of tha1-1 and tha1-1/35s omr1-7 mutants contained similar numbers of juveniles compared to the Columbia wild type. 35s omr1-7 mutant roots contained a significant, 47% fewer J2s than Col-WT plants, six days after inoculation (Fig.

2.4).

The effects on parasitism can be complemented with the addition of threonine to the growth media.

In order to complement threonine levels in the mutant, three different concentrations of threonine were added to the media. The addition of a minimum concentration of 5 μM L- threonine media was sufficient to restore 35s omr1-7 mutants to wild type egg mass concentrations (Fig. 2.5, Fig 2.6). The addition of 5μM D-threonine did not have the same effect.

The addition of L-aspartate also was unable to restore the number of egg masses to wild-type levels (Fig. 2.5). The addition of isoleucine further reduced egg mass numbers (Fig. 2.5). The addition of 50 μM threonine also restored the entry of juveniles into the 35s omr1-7 mutants during early infestation to wild type levels (Fig. 2.7). Addition of threonine at a 500μM caused detrimental effects to plant growth, but no changes were seen at lower concentrations that were tested (Fig. 2.6)

Threonine catabolism overexpression leads to decreased fecundity but does not significantly impact male production.

RKN fecundity and male development were investigated on threonine catabolism mutants. The number of eggs per egg mass was measured at seven weeks after infestation. Egg production in the threonine mutants was compared to production on the Col-0 wild type. 35s omr1-7 had an average of 9.8% decrease in the number of eggs per egg mass as compared to Col-

0 wild type eggs per egg mass; tha1-1 and tha1-1/35s omr1-7 were not significantly different from the wild-type (Fig.2.8).

Production of males in RKN is associated with a more stressful nutritional environment

(Davide and Triantophyllou 1967; Moura et al. 1993). Thus, male production was measured seven weeks after infestation. 35s omr1-7 mutant plants did not produce significantly more males than

Col-0, tha1-1, or tha1-1/35s omr1-7, although a trend for higher male production was present

(Fig.2.9).

Increasing competition on threonine deaminase mutant impacts root-knot nematode infestation.

To further test this idea, we measured the response of RKN egg mass production, male production, and fecundity on these mutants under different competition levels (Marella et al.

2013). Threonine catabolism mutants were inoculated with a gradient of nematode egg numbers;

500 eggs, representing a low level of competition, 1000 eggs, representing an average level of competition and 2000 eggs, representing a high level of competition.

At low competition levels, the number of egg mass, fecundity, and male production were similar to wild type levels (Fig 2.9, Fig 2.10, Fig 2.11). However, at average levels of competition,

35s omr1-7 displayed an average of 17% fewer egg masses (Fig. 2.10) and 8.3% fewer eggs per egg mass (Fig. 2.11) as well as a trend of producing more male nematodes (Fig. 2.9). At high levels of competition, the changes in 35s omr1-7, 21% fewer egg masses (Fig. 2.10) and 22% fewer eggs per egg mass were produced (Fig. 2.11), along with a significant 43.5% increase in the number of males produced (Fig.2.9).

2.5 DISCUSSION

There have been many reports of the impact of changes in amino acid homeostasis on the growth and reproduction of plant parasites (Liu et al. 2010; Stuttman et al. 2011; van Damme et al. 2009). The amino acid homeostasis of a given plant cell can be affected in a few different ways. Plants regulate the given concentration of an amino acid in a given cell, depending on the needs of that cell (Szabados et al. 2010). The concentration can be changed by altering amino acid biosynthesis or catabolism or by transporting amino acids from the vascular tissue or other parts of the plant (Joshi et al. 2010; Pratelli and Pilot 2014). Changes in amino acid transport, particularly from mutations in vascular transporters, have already been shown to negatively impact RKN parasitism, affecting many of the parameters that we tested in this paper (Marella et al. 2013). Here we investigated how changes to the catabolism of threonine impacts RKN parasitism. We investigated changes in RKN early infestation, late infestation, male development and fecundity in the threonine catabolism mutants, tha1-1, a knockout of threonine aldolase

(Joshi et al. 2006), 35s omr1-7, a threonine deaminase overexpression mutant (Mourad and King

1995; Joshi et al. 2006) and a cross of the previous two mutants tha1-1/35s omr1-7 (Joshi et al.

2006). All of the mutants were tested for root architecture phenotypes, including root length and lateral root density. We detected no differences in root measurements for the mutants listed above (Fig. 2.2). Therefore, we do not expect that root architecture plays a role in any of the phenotypes that we observed.

Threonine levels increased in root tissue infested with plant-parasitic nematodes

(Hoffman et al. 2010) and is likely an essential amino acid for RKN and other plant-parasitic 94 nematodes. Because of this fact, we expected that changes in threonine homeostasis would impact RKN infestation throughout their lifecycle. Changes in threonine homeostasis present in the threonine deaminase overexpression, 35s omr1-7 mutant led to a reduction of nematodes that entered the roots during early infestation (Fig. 2.4). This result suggests that threonine metabolism may be an important early trigger for determining whether a host is advantageous for RKN growth and reproduction. Changes in RKN were also observed at the late infestation stages in 35s omr1-7, which produced fewer egg masses than the Columbia control (Fig. 2.3).

Threonine deaminase is the first committed step in isoleucine metabolism, and the 35s omr1-7 mutant lacks feedback inhibition by isoleucine so the enzyme is continuously active and the mutant accumulates isoleucine (Joshi et al. 2006). The reduction of egg masses in the 35s omr1-

7 mutant indicates that altering threonine metabolism by pushing metabolism towards isoleucine may be deleterious to RKN infestation.

Tha1-1 has similar RKN infestation levels to the control (Fig. 2.3). In the double mutant, tha1-1/35s omr1-7 egg mass production is also at levels similar to the control (Fig. 2.3). This particular threonine aldolase, tha1-1, showed particularly strong expression in the seeds and other areas associated with transfer cells (Joshi et al. 2006). Transfer cells are a cell type that is responsible for moving nutrients across a symplastic barrier. This may indicate a role for this enzyme in transfer cells. RKN giant cells are often characterized as transfer cells (Rodiuc et al.

2014), and indeed, we see that tha1-1 is upregulated in giant cells (Table 2.1). This suggests that regulating levels of threonine and related compounds in the giant cell may be essential for normal

RKN infestation. This indication is strengthened by the fact that the addition of exogenous

95 threonine into the media was able to rescue the 35s omr1-7 mutant to wild type levels (Fig. 2.5).

Aspartate and isoleucine additions did not rescue the phenotype. The metabolism of this pathway is tightly controlled (Jander and Joshi 2009), and this suggests that it is difficult to add amino acids to other parts of the pathway in order to substitute for threonine. Also impacted were a trend for the production of more males (Fig 2.9) and a reduction in the number of eggs produced by individual females on 35s omr 1-7 (Fig. 2.11). Taken together, these results may indicate a limiting nature of threonine metabolism in the Arabidopsis/RKN parasitism interaction.

One explanation of the phenotypes that we see here is that threonine or a related compound is one of the molecules that may regulate competition in RKN. Competition between parasites can lead to negative impacts on parasite fitness, including size, sex ratios, and fecundity

(King 1987; Mueller et al. 1991; Pollitt et al. 2013; Poulin et al. 2003). In this study, competition had an impact on female development on the 35s omr1-7 mutant; as the rate of competition increased we saw a smaller percentage of females that were able to form egg masses (Fig. 2.10).

Competition also had an effect on sex ratio with higher rates of competition producing more males (Fig. 2.9). This result is consistent with other studies that have looked at plant-parasitic nematode male production under nutrient limiting conditions (Castagnone‐Sereno and Danchin

2014; Grundler et al. 1991). High levels of competition also impacted fecundity; 35s omr1-7 plants produced fewer numbers of eggs per egg mass (Fig. 2.11). The fitness of parasites is often impacted by different nutrient triggers that are present in the environment (Keymer et al. 1983;

Logan et al. 2005). Our data suggest that one of these triggers for RKN may be threonine.

Threonine is important as a dietary essential amino acid for nematodes and is in limited supply

96 in the plant host. Thus, it is plausible that threonine could be an important signal on whether the development of female and male root-knot nematodes is triggered (Snyder et al. 2006). This conclusion could be strengthened by looking at metabolite concentrations in roots and RKN knots to determine that threonine is indeed in short supply.

Another explanation for the changes in the phenotypes that we observed might be due to impacts of changes in amino acid homeostasis on the plant immune system (Liu et al. 2010;

Stuttman et al. 2011; Zeier 2014). Other amino acids, such as proline and glutamate, have been studied as factors in immunity against various parasites and pathogens (Cecchini et al. 2011; Seifi et al. 2013). An aspartate kinase mutant leading to the accumulation of threonine and other aspartate-derived amino acids caused decreased reproduction in the biotrophic pathogen,

Hyaloperonospora arabidopsidis (Stuttmann et al. 2011). It is possible that the alterations to threonine homeostasis in the 35s omr1-7 mutant are leading to activation of defense pathways and further to the changes in RKN parasitism that we see. Another explanation could be that the phenotypes that we see are due to the antifeedant nature of threonine deaminase and that overexpression of the enzyme has detrimental effects on RKN feeding. Threonine deaminase has been studied as an induced antifeedant compound in plant defense reactions against lepidopteran insects (Gonzales-Vigil et al. 2011). Tomato (Solanum lycopersicum) plants overexpressing this protein were shown to significantly decrease the weight of several lepidopteran species that fed on the overexpression mutant as compared to the wild type tomato

(Gonzales-Vigil et al. 2011). Toxicity caused by the high levels of isoleucine in the 35s omr1-7 mutant is also a possibility, given the extreme amount of isoleucine that accumulates in the

97 mutant (Joshi et al. 2006). A recent experiment where 5mM of L-isoleucine was added exogenously to H. schacttii juveniles before infestation, reported a 37% drop in the number of females, although a similar decrease was also seen with the addition of 5mM L-threonine (Blümel et al. 2018). The competition experiments that we conducted showed a pattern that was not consistent with a pattern of defense activation, threonine deaminase as an antifeedant or toxic levels of isoleucine (Fig. 2.9, 2.10, 2.11). If this were the case, we would expect to see a consistent decrease in nematode numbers at all competition levels, similar to the reaction that was seen to the aap3 mutant by Marella et al. (2013). However, more experiments are required to completely rule that these explanations are not responsible for all or part of the phenotypes that we observed in these tests.

In conclusion, we found that threonine deaminase overexpression was detrimental to

RKN development, male development, and fecundity, particularly at high levels of competition.

These results are consistent with the maintenance of threonine metabolism as a necessary element in the RKN lifecycle. These findings could potentially lead to novel methods for nematode control, by altering threonine homeostasis in plants or altering the amount of threonine that is released into the rhizosphere that could be detected early on by nematodes.

2.6 ACKNOWLEDGMENTS

The authors would like to thank Georg Jander for providing the Arabidopsis mutant lines, Marlia

Bosques Martinez, Cecilia Chagas de Freitas, Rebecca Kimmelfield, Amanda Lietz, Edwin Navarro and Anna Stasko for critically reviewing the manuscript and Leslie Taylor for the propagation of the Arabidopsis lines and general lab problem-solving.

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Zeier, J., 2013. New insights into the regulation of plant immunity by amino acid metabolic pathways. Plant, Cell & Environment, 36(12), pp.2085-2103.

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Table

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Table 2.1

Gene GeneID Fold Fold change change 14dai 21dai THREONINE ALDOLASE AT1G08630 2.26* 17.33* 1, THA1

THREONINE ALDOLASE AT3G04520 1.04 1.40 2, THA2

L‐O‐METHYLTHREONINE AT3G10050 2.02 2.65 RESISTANT 1, OMR1,

Threonine deaminase

METHIONINE OVER‐ AT4G29840 1.32 1.95* ACCUMULATOR 2, MTO2, THREONINE SYNTHASE ASPARTATE KINASE‐ AT1G31230 0.86 0.99 HOMOSERINE DEHYDROGENASE I, AK‐ HSDH I DIHYDRODIPICOLINATE AT3G60880 3.07* 1.26 SYNTHASE 1, DHDPS

DIHYDRODIPICOLINATE AT2G45440 0.54 0.87 SYNTHASE 2

CYSTATHIONINE AT3G01120 0.88 1.09 GAMMA‐SYNTHASE 1, METHIONINE OVERACCUMULATION 1, MTO1

113

Figures

(http://www.ncbi.nlm.nih.gov/geo/).

114

Figure 2.2. Threonine catabolism mutants do not significantly impact plant root architecture.

Measurement of the total root length and lateral root density of the threonine catabolism mutants, tha1-1, 35s omr1-7 (omr1), and tha1-1/35s omr1-7 (T/O). Plants were grown upright on agar plates with the top quarter of the plate excised in order to avoid agar contact to the leaves. Total length was measured at each time point from the base of the hypocotyl to the root tip. Lateral root density was measured by dividing the length of root that contained lateral roots (lateral root zone) by the total number of lateral roots. The threonine homeostasis mutants that we measured did not impact the total root length (A) or lateral root density (B).

Error bars represent +/- standard deviation.

WT 10 35s omr1-7 tha1-1 8 tha1-1/35s omr1-7

RootLength (cm) 4

0 Day 6 Day 11 Day 18 Day 21 Days after germination

115

Figure 2.2

1.2 WT 1 35s omr1-7 tha1-1 0.8 tha1-1/35s omr1-7 0.6

0.4

0.2

0 zone(cm)/#of lateral roots)

Day 6 Day 11 Day 18 Day 21 LateralRoot Density (Lateralroot -0.2

-0.4 Days after germination

116

Figure 2.3. Threonine catabolism overexpression reduces egg mass numbers. Measurement of egg mass production in Columbia WT (Col - WT), threonine aldolase 1 (tha1-1), a threonine deaminase overexpression mutant (35s omr1-7) and a double mutant of threonine aldolase 1 and a threonine deaminase overexpression mutant (35s omr1-7/tha1-1). 3-week-old seedlings

(5 per plate) were inoculated with 1000 RKN eggs. Seven weeks after inoculation, the number of females that successfully produced egg masses on each plate was counted. Significantly less number of egg masses were produced on the 35s omr1-7 as compared with the WT. P values were calculated using the Students T-test (*P=<0.05). Error bars represent +/- standard error of the mean. Representative experiment of 5 total replications.

200

* 150

100

50 Number of Eggof NumberMasses

0 Col - WT tha1-1 35s omr1-7 tha1-1/35s omr1-7 Arabidopsis Line

117

Figure 2.4. Threonine catabolism overexpression reduces root-knot nematode early entry.

Measurement of nematode root penetration during early infestation in Columbia WT (Col -

WT), threonine aldolase 1 (tha1-1), a threonine deaminase overexpression mutant (35s omr1-

7) and a double mutant of threonine aldolase 1 and a threonine deaminase overexpression mutant (35s omr1-7/tha1-1). Ten seven day old seedlings were inoculated with 1000 RKN second-stage juveniles. After six days, plants were removed from the plates and stained with acid fuschin. Significantly less number of juveniles were counted in the 35s omr1-7 root systems as compared with the WT control. n=15-25 plants. P values were calculated using the

Students T-test (*P=<0.05). Error bars represent +/- standard error of the mean. Representative experiment of 3 total replications.

8 *

2 Number of Juveniles NumberofJuveniles Root in System

0 Col - WT tha1-1 35s omr1-7 tha1-1/35s omr1-7 Arabidopsis Line

118

Significantly less number of egg masses were produced on the 35s omr1-7 as compared with the WT. However, 35s omr1-7 plants supplemented with 5μM of threonine produced egg masses at levels comparable to the WT - control. Differences were calculated using a Welch’s

ANOVA followed by a Post-hoc Games-Howell test (*P=<0.05). Error bars represent +/- standard deviation. Data collected over at least three replications.

119

Figure 2.5

120 A,B A A A,B,C A,B,C A,B,C 100 B,C,D C,D D 80 E 60 40

20 Egg Masses (%of WT) (%of Egg Masses

L-ile L-ile

L-thr

L-asp L-asp

D-thr D-thr

L-thr

Control 35s omr1-7 35s WT 35s omr1-7

120

Figure 2.6. Range of concentrations used to test threonine rescuing effect on threonine catabolism mutant, 35s-omr1-7. Measurement of egg mass production in Columbia WT (WT -

μM or higher of threonine to the media, differences between the Col Wt and 35s omr1-7 were no longer observed. P values were calculated using the Students T-test (*P=<0.05). Error bars represent +/- standard error of the mean.

200 180 160 * 140 * 120 100 80 60

Number of Egg NumberofEgg Masses 40 20 0 WT 35s WT 35s WT 35s WT 35s WT 35s omr1-7 omr1-7 omr1-7 omr1-7 omr1-7 Control 0.5μM L-thr 5μM L-thr 50μM L-thr 500μM L-thr

121

Figure 2.7. The addition of threonine rescues early infstation in threonine catabolism mutant to wild type levels. Measurement of nematode root penetration during early infestation in

Columbia WT (WT - Col), and threonine deaminase overexpression mutant (35s omr1-7). Ten seven day old seedlings grown on control media or media amended with 50μM threonine were inoculated with 1000 RKN second-stage juveniles. After six days, plants were removed from the plates and stained with acid fuschin. Significantly less number of juveniles were detected on the 35s omr1-7 as compared with the WT, the addition of 5μM threonine to the media rescued resulted in 35s omr1-7 plants that are not different from the wild type controls. n=20-

25 plants. P values were calculated using the Students T-test (*P=<0.05). Error bars represent

+/- standard error of the mean. Representative experiment of 3 total replications.

6 * 4

2 Number of Juveniles NumberofJuveniles Root in System 0 WT - Col. tha1-1 35s omr1-7 tha1-1/35s omr1-7 Arabidopsis Line

122

Figure 2.8. Threonine catabolism overexpression reduces root-knot nematode fecundity.

Measurement of fecundity in Columbia WT (WT - Col), threonine aldolase 1 (tha1-1), a threonine deaminase overexpression mutant (35s omr1-7) and a double mutant of threonine aldolase 1 and a threonine deaminase overexpression mutant (35s omr1-7/tha1-1). 3-week- old seedlings (5 per plate) were inoculated with 1000 RKN eggs. Seven weeks after inoculation, the number of females that successfully produced egg masses on each plate was counted. After counting the number of egg masses, the agar from the plates was dissolved, and the number of the eggs of eggs on each plate was counted. Significantly less number of eggs per egg mass were observed on the 35s omr1-7 as compared with the WT. n=10-13 plates. P values were calculated using the Students T-test (*P=<0.05). Error bars represent +/- standard error of the mean. Representative experiment of 3 total replications.

500 * 400

300

200

Number of Eggs NumberofEggs PerEgg Mass 100

0 WT - Col tha1-1 35s omr1-7 tha1-1/35s omr1-7 Arabidopsis Line

123

Figure 2.9. Root-knot nematode competition impacts male production on a threonine catabolism mutant. Measurement of male production in Columbia WT (WT - Col), threonine aldolase 1 (tha1-1), a threonine deaminase overexpression mutant (35s omr1-7) and a double mutant of threonine aldolase 1 and a threonine deaminase overexpression mutant (tha1-1/35s omr1-7) exposed to different levels of competition. Three-week-old seedlings (5 per plate) were inoculated with 500 (low competition), 1000 (average competition), or 2000 (high competition) RKN eggs. Seven weeks after inoculation agar was dissolved and stained with acid fuchsin so males could be observed. Significantly increased male production was only measured at the highest competition level (2000 eggs at initial inoculation). n=10-13 plates. P values were calculated using the Students T-test (*P=<0.05). Error bars represent +/- standard error of the mean. Representative experiment of 3 replications.

16 * WT 14 35s omr1-7 12 tha1-1 10 tha1-1/35s omr1-7 8

4 NumberofMales Plate on

0 500 1000 2000 Number of Eggs Inoculated

124

Figure 2.10. Competition impacts root-knot nematode parasitism on a threonine overexpression mutant. Measurement of egg mass production in Columbia WT (Solid- triangles), threonine aldolase knockout, tha1-1 (dotted line, circles), a threonine deaminase overexpression mutant, 35s omr1-7 (Dashed-squares) and the tha1-1/35s omr1-7 double mutant (dash/dot-diamond) exposed to different levels of competition. Three-week-old seedlings (5 per plate) were inoculated with 500 (low competition), 1000 (average competition), or 2000 (high competition) RKN eggs. Seven weeks after inoculation, the number of females that successfully produced egg masses on each plate was counted. Significantly less number of egg masses were produced on the 35s omr1-7 as compared with the WT, and the percentage of egg masses is less on the 35s omr1-7 with high competition. n=10-13 plates. P values were calculated using the Students T-test (*=P<0.05, **=P<0.01). Error bars represent

+/- standard error of the mean. Representative experiment of 3 total replications.

125

Figure 2.10

250 WT

35s omr1-7 200 tha1-1

150 tha1-1/35s omr1-7 **

100 * Number of Egg NumberofEgg Masses

0 500 1000 2000 Number of Eggs Inoculated

126

Figure 2.11. Competition on the threonine catabolism mutant impacts root-knot nematode fecundity. Measurement of Eggs per Egg mass in Columbia WT (WT - Col), threonine aldolase 1

(tha1-1), a threonine deaminase overexpression mutant (35s omr1-7) and a double mutant of threonine aldolase 1 and a threonine deaminase overexpression mutant (tha1-1/35s omr1-7) exposed to different levels of competition. Three-week-old seedlings (5 per plate) were inoculated with 500 (low competition), 1000 (average competition), or 2000 (high competition)

500

400 * 300 *

WT 200 35s omr1-7

Number of eggs Numberofeggs mass peregg tha1-1 100 tha1-1/35s omr1-7

0 500 1000 2000 Number of Eggs Inoculated

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Chapter 3: Amino Acids Play A Role in Plant-Parasitic Nematode Chemotaxis.

3.1 ABSTRACT

As obligate plant parasites, plant-parasitic nematode (PPN) species must efficiently navigate the soil environment to find a plant host. Plant roots exude a large number and significant concentrations of compounds into the rhizosphere, including mucilage, phenolics, organic and amino acids. PPNs use chemotaxis to move non-randomly through the soil toward plant roots. Specific compounds or combinations of compounds that are responsible for PPN chemotaxis remain understudied. Because nematodes require specific amino acids in their diet we hypothesized that the root-knot nematode (Meloidogyne incognita) would be attracted to the amino acid component of root exudates. In this study, we measured PPN chemotaxis toward plant root exudates, individual amino acids, and reconstructed blends of amino acids. We found that some amino acids, including threonine, aspartic acid, proline, and histidine, are highly attractive to PPNs while other amino acids including cysteine, serine, and phenylalanine were repellent. Additionally, specific attraction or repulsion was structure-dependent, in that nematodes responded to the L-form of amino acids but not the D-form. A reconstructed amino acid blend modeled after the amino acid content of soybean root exudates was attractive to

PPNs. These results represent an opportunity for novel means to control PPNs exploiting molecules that are perceived and alter the behavior of the nematode prior to entering a host.

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3.2 INTRODUCTION

Plant-parasitic nematodes (PPNs) are known to use a chemotaxis mechanism to find plant roots and will move non-randomly in the soil in the direction of roots (Perry and Wright, 1998).

The chemosensory organ responsible for environmental sensing in nematodes is the amphid.

Amphids are chemosensory sensilla located close to the mouth of the nematode contained in a pore that is open to the environment (Perry, 1996; Wright, 1983). Each nematode has two amphids, and they are located on either side of the mouth (Bird and Bird, 1991). PPN chemotaxis is driven by soluble or volatile molecules that are perceived by neurons located in the amphids.

In the soil, these molecules may be exuded or secreted into the soil by plants or by the organisms that live in the environment of the plant root zone (Ali et al., 2010; Bird, 1959; Dusenberry, 1987;

Lee and Evans, 1973; Prot, 1980; Rasmann et al., 2012). PPNs use different chemical cues depending on the distance from the root: long-distance chemoattractants generally attract them to the root zone, and short-distance chemoattractants lead them to the specific area of dividing cells in the root where they will invade the root system (Perry, 2005; Čepulytė et al., 2018).

Because plant-parasitic nematodes are obligate parasites, it is crucial for them to locate roots efficiently. This efficiency can be shown as they follow the shortest route to the root zone along a chemotactic gradient (Reynolds et al., 2010).

Nematode chemotaxis has been well studied in the model organism Caenorhabditis elegans. The first compounds discovered to be attractive to C. elegans included cyclic nucleotides

129

(cylic-AMP, cyclic-GMP), ions (Cl, Br,I), cations (Na+, Li+, K+, Mg2+ ) and the amino acids lysine, histidine, and cysteine (Ward, 1973). The perception of these compounds seems to be driven by detection through amphids, as the destruction of the chemo-sensing neurons in the amphids resulted in reduced chemotaxis. The specific neuron responsible for ionic and water-soluble chemotaxis is the ASE neuron (Bargmann and Horvitz, 1991). Another set of neurons, AWA, AWB, and AWC, are responsible for chemotaxis towards or away from volatile compounds (Bargmann et al., 1993; Troemel et al., 1997). C.elegans responds to increasing or decreasing gradients of compounds rather than absolute amounts (Pierce-Shinomura et al., 1999), and they continually evaluate the concentration of given attractants (Miller et al., 2006). Many chemoreceptors located in the neurons are likely G-protein coupled receptors, of which there are over 1000 in C. elegans (Chen et al., 2005). However, amino acid receptors recently identified in Drosophila melanogaster were ionotropic glutamate receptors (Croset et al., 2010; Croset et al., 2016). C. elegans appears to have several ionotropic glutamate receptors including, glr-7 and glr-8, that are expressed in similar tissues and could be related to food sensing, but this has not been fully explored (Brockie et al., 2001). Furthermore around 108 g-protein coupled receptors were identified in the genome of M. incognita neurons, the receptors they contain are understudied in PPNs (Abad et al., 2008). Virtually nothing is known about the specific neurons located in PPN amphids, the chemo sensing receptors they contain and the behavioral responses they regulate.

Plant root exudates include many different types of compounds including amino acids

(Carvalhais et al., 2011), CO2 (Dakora and Phillips, 2002), organic acids (Jones, 1998), phenolics

(Li et al., 2010), sugars (Kraffczyk et al., 1984), volatiles (Ens et al., 2009), mucilages (Czarnes et

130 al., 2000) and other specialized metabolites. These diverse compounds provide many different services for plants including curation of the rhizosphere microbiome (Sasse et al., 2018), acquisition of soil minerals (Dakora and Phillips, 2002), competition with other plants (Prati and

Bossdorf, 2004), facilitation of mutualistic symbioses (Peters et al., 1986), and protection from heavy metals (Horst et al., 1982), among other functions.

Among the various compounds that have been tested for PPN nematode chemotaxis, CO2 is an important attractant at centimeter distances for RKN and a few other nematodes (Robinson,

1995). Nematodes can theoretically sense CO2 gradients over distances as great as a 40cm

(Dusenberry, 1987). Robinson (1995) showed that RKN juveniles could be attracted to tiny amounts of CO2 over centimeter distances over a few hours in light, sandy soils. One of the problems for nematodes that move in response to CO2 is to know whether they are moving toward living or dead tissue as either could provide a CO2 gradient. PPNs may accomplish this through combinatorial responses, repulsing them from other compounds found in decaying matter such as ammonia and inorganic ions (Castro, 1991). Patil (2013) showed that applying an ammonia fertilizer such as ammonium nitrate can interfere with Meloidogyne graminicola attraction in rice production. Fudali (2013) showed that ethylene signaling, a hormonal modulator of plant senescence, plays a role in M. hapla chemotaxis, allowing the nematode to find a root that will be viable long term. Hu et al. (2017) confirmed a role for ethylene signaling in SCN chemotaxis as well.

Water-soluble root exudates have been noted to attract nematodes as well. Weiser

(1955) and Lee (1972) observed that root exudates from tomato and rice were attractive to M. 131 hapla and Aphlenchoides besseyi. In 1959, Bird found that organic acids from tomato roots, ascorbic acid, and glutamic acid, in particular, were responsible in part for the attractive component of tomato root exudates for M. hapla. Two studies from the 1980s demonstrated that nematodes would move toward roots and places where roots had been removed in sand and sand with small amounts of added clay (Prot, 1980; Prot and Van Gundy, 1980). In more recent studies using pluronic gel, M. incognita juveniles were observed to migrate toward the roots of several species of plants and aggregated near the root tip (Wang, 2009). In another pluronic gel-based study using Y-tubes, Reynolds (2010) demonstrated that nematodes will move along the most efficient path to find suitable plant roots and that host-specific species can discern between host and non-hosts. Teillet et al. (2013) measured transcriptional changes in M. incognita in response to Arabidopsis thaliana root exudates and found that expression of several genes, including genes with possible secretion signals and a gene involved in calcium signaling, increased in the nematode when root exudates were detected.

For signals that are active at a short distance, recent work by Čepulytė et al. (2018) discovered a novel compound that was specifically exuded at the area of dividing root cells and was extremely attractive to nematodes at distances of 10-20mm. PPNs are also extremely sensitive to extremely tiny gradients of temperature, calculated to be as low as 0.0010°C

(Dusenberry, 1988); this is likely useful over very short distances from the root because of the rapid and substantial temperature changes that occur in the upper part of the soil over the course of a day (Curtis, 2009). It has been suggested that this sensitivity may allow the juvenile stage of the nematode to locate the metabolically active area of plant roots behind the root cap where

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RKN juveniles can gain entrance, an area that would potentially have a higher temperature than surrounding root cells (Perry, 2005).

In this study, we examined whether amino acids can act as chemotactic signals to juveniles of a plant-parasitic (M. incognita) and a bacterial feeding nematode (C. elegans). We first determined the root exudate amino acid composition of a common crop, soybean, Glycine max, to determine which amino acids may be available to nematodes in the rhizosphere.

Secondly, we determined if the relative composition of the various sets of amino acids that are present in the soybean rhizosphere have any effect on host selection in RKN, SCN, and BCN.

Thirdly, to assess nematode responses, we used choice experiments with individual amino acids using either agar plugs or a manufactured nematode maze to determine behavioral responses

(i.e., attraction or repellence). Elucidating the response to amino acids by plant-parasitic nematodes may help us discover new and novel control techniques that exploit nematode chemotaxis in the rhizosphere.

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3.3 MATERIALS AND METHODS

Media

Experiments using PPNs were performed on Gamborg’s basal salts media, as described in Marella et al. (2013). Briefly, in 1L of ddH2O we added 20g D-sucrose, 3g Gamborg's basal salts, and 6g phytagel. We adjusted pH to 6.1 using 1M KOH. Media was autoclave for 30 minutes. Soybean plants were grown in ¼ Murashige and Skoog media (Murashige and Skoog, 1962; MS). ¼ MS is made by adding 1.08g of Murashige and Skoog basal salts to 1L of water, and adjusting pH to 6.1.

The medis is autoclaved for 30 minutes.

Experiments using C. elegans were performed using nematode growth media (NGM) and

M9 media, both of which are described in Stiernagle (1999). For NGM, we added 3g NaCl, 2.5g peptone, 17g agar to 975ml of ddH2O, and autoclaved for 30 minutes. After autoclaving, while the media is still warm, we added 1ml 5mg/ml cholesterol (dissolved in ethanol), 1ml 1M CaCl2,

1ml 1M Mg SO4 and 25ml of potassium phosphate buffer, pH 6. To make M9 media, we added 3g

KH2PO4, 6g Na2HPO4, 5gNaCl to 1L of ddH2O and autoclaved for 30 minutes. We allowed to cool briefly and then added 1ml of 1M MgSO4.

Nematode Culture

Root-knot Nematode: Meloidogyne incognita

M. incognita eggs were collected from sterile Arabidopsis thaliana (Columbia wild-type) culture plates (grown on Gamborg’s Basal Salts Media) using the following procedure, similar to

Marella et al. (2013). Stems and leaves of the plant were removed and discarded. The remaining

134 root material containing RKN egg masses was collected from approximately sixty plants and placed in two 50ml centrifuge tubes (Falcon, Corning, NY, USA). Forty-five ml of 5% bleach was added to the tubes, which were then shaken vigorously by hand for two minutes. The root material was poured over a 2mm coarse filter into a new 50ml centrifuge tube. The solution in the new tubes contained the majority of the eggs. The tubes containing the eggs and solution were centrifuged for 5 minutes at 1000rpm. After centrifugation, approximately 40ml of the solution was poured out of the centrifuge tubes and replaced with approximately 40ml of ddH20.

The centrifugation and pouring off steps were then repeated three more times, to remove as much bleach as possible. The number of eggs was then counted under a Nikon SMZ645 dissecting microscope in three 10µl droplets to obtain an approximation of the total number collected. Eggs were collected from plants grown in the conditions described above at 7-8 weeks after inoculation from previous egg hatches

In experiments where stage-two juveniles (J2s) were used, we conducted the following procedure to hatch J2s. After collection, the solutions containing eggs were poured onto a 25µm

(500 mesh) filter to capture the eggs. Eggs were incubated in the dark for ten days at 23°C. J2s that hatched from the eggs crawled through the filter and were collected in the water solution below the filter. Juveniles were counted in three 10 µl droplets prior to the infestation of plants or used on nematode mazes.

Beet cyst nematode: Heterodera schactii

BCN was propagated on cauliflower (Brassica oleracea cv ‘Snowball Early’ or ‘Snow

Crown’), which was maintained in a greenhouse in 1-gallon pots containing sand/turface mixture. 135

Greenhouse temperature conditions varied with an average low of 18°C to an average high of

30°C. Light supplementation was used to extend day length during the winter. BCN was extracted from cauliflower roots using the following method. First, the aboveground portions of the plant were removed. Roots and sand/turface media were placed in a large plastic Nalgene bin, the roots and media were washed by pouring tap water over the roots and agitating back and forth.

The water solution was poured over two sieves; first, a 595µm (30 mesh) sieve to remove soil particles and then a 177μm sieve (80 mesh) to collect cysts. This wash was repeated 5-8 times.

After each wash material on the 177µm sieve was collected into a beaker. To collect eggs, cysts were ground using a modified drill press with a rubber stopper attachment on a custom 250µM

(100 mesh) screen (Hershman et al., 2008), keeping the sample wet while grinding. The grinding was performed over two more sieves, a 74μm (200 mesh), and a 25μm (500 mesh), which collected the eggs. After collecting the eggs, the eggs were placed in a hatching chamber which consists of a 25µm screen (500 mesh), a diameter small enough to catch the eggs, but wide enough to allow the juveniles to get through over ddH20. The eggs were allowed to hatch for 4-

5 days before juveniles were used in the following experiments.

Soybean cyst nematode: Heterodera glycines

Soybean Cyst Nematodes Hg type 0 were propagated in a greenhouse on Glycine max cv.

‘Hutchinson’. SCN eggs were collected and hatched using the same method as the BCN eggs detailed above.

Caenorhabditis elegans

136

Strains

Escherichia coli strain ht115 (DE3) was obtained from the Ceanorhabditis Genetics Center

(https://cgc.umn.edu) and E.coli RNAi feeder strain F22A3.3 (glr-8) was obtained from

Dharmacon, Inc (https://dharmacon.horizondiscovery.com).

Propagation

C. elegans were maintained under the following conditions. Before the propagation of the

C. elegans, a culture of Escherichia coli strain op. 50 was started from a single colony grown on

LB plus Agar and spiked into 15ml of LB broth. This culture was grown overnight at 37°C on a

210rpm shaker. 250μl of this culture was then spread evenly on an NGM (see above) plate using sterile glass beads. After allowing the plates to dry in a hood for approx. 1 hr, an approximately

1cm square block containing C. elegans from a previous culture, was added to the plate. Plates were sealed with micropore tape (3M) and incubated on a lab bench at room temperature.

Synchronization

For all choice experiments, C. elegans were synchronized using the following method to achieve same-aged juveniles, similar to Stiernagle (1999). Ten ml of M9 media was pipetted onto each propagation plate and pipetted up and down several times to remove most of the nematode off of the agar. This solution was placed in a collection tube (50ml Falcon tube), on ice, until 5-7 plates worth of nematodes had been collected. Nematodes were allowed to settle at the bottom of the tube on ice for 15 minutes and most of the M9 solution was then removed, without disturbing the pellet. Then the following solutions were added to the tube 5.75ml of sterile

137 ddH2O, 750μl bleach, and 500μl of 10M NaOH. This solution was then shaken vigorously for 2 minutes. The solution was then allowed to rest for 7-9 minutes to allow the solution to dissolve the cuticles of the adult nematodes to expose the eggs. The solution was centrifuged for 5 minutes at 750 rpm. Most of the solution was then removed without disturbing the pellet. The solution was immediately replaced with 13ml of M9 media. The centrifugation step was then repeated 3 more times or until the bleach smell could no longer be detected in the tubes. Finally, the tubes were placed in a 15°C water bath for hatching. Hatching starts after 24 hours, and synchronized juveniles can be collected for several days afterward.

Root Exudate experiments

Soybean and corn seed sterilization

The following cultivars were used for the general root exudate experiments; Glycine max cv. ‘Lee’ and Zea mays cv. ‘B97’.

Soybean seed surface sterilization was carried out similarly to Kereszt et al. (2007).

Chlorine gas was created inside a vacuum chamber in a fume hood by adding ~15ml of 37% HCl to ~200ml of bleach. Soybean seeds were incubated overnight in a vacuum chamber with the gas. The gas was released, and the seeds were stored in sealed plates until use. For corn, we used a protocol from Schnable (2010): in a laminar flow hood, 10-15 corn seeds were placed in sterile

15ml tubes. Enough undiluted bleach was added to cover all of the seed. The seed was inverted in the bleach 3-4 times over a total of 1 minute. Bleach was removed and replaced with sterile ddH2O, and the seed was inverted again. This water was removed and replaced repeatedly until

138 there was no more bleach odor, usually 8-11 rinses. The seed was dried down in a laminar flow hood and was stored in 15ml tubes until use.

Soybean and corn growth

Soybean and corn seeds were germinated in the dark at 27OC, in a moist, sterile rolled towel. Seeds were incubated for four days. For corn and soybean, 50 ml Falcon tubes were filled with 40ml of ddH2O and a layer of sterile cheesecloth was placed over the top of the tube and held in place by a rubber band. After germination, the seedlings were suspended on the cheesecloth with the roots submerged in the ddH2O. Plants were placed in an acrylic box, sealed with parafilm, and with 12hr day/12 hr night conditions at 27°C. Plants were allowed to adjust to the new conditions, and the water was replaced after 24 hours. Compounds that were exuded into the water were collected after the next 48 hours and filter sterilized using a .22μm filter.

Exudates were stored in the fridge before use.

Collection and measurement of soybean root amino acid exudate to determine amino acid content

All non-seed materials for the experiment were autoclaved at least two times. The seed was sterilized as described above and soaked overnight (12-24hrs) in sterile ddH2O in a Petri dish inside of a laminar flow hood. Plastic funnels for growing the plants for root exudate collection were assembled. We used two-piece funnels, at the base of each funnel a disc of 33mm size one filter paper was placed with a wire mesh disc on top of that (Fig. 3.1a). In each funnel 50ml of sterile (3X autoclaved) play-sand was added. One imbibed seed was then placed into each funnel

139 and then covered with 30ml of sterile sand. Next, 35ml liquid ¼ Murashige and Skoog Media was added, to saturate the sand. The sand used in each chamber was tested for sterility on LBA plates.

The tubes containing the plants were placed in Nalgene chambers which were sealed tightly with micropore tape (3M). The tubes were incubated for two weeks in a growth chamber under the following conditions: 25°C, 16 hours light, 8 hours dark. After two weeks, chambers were opened in a laminar flow hood. Sand scrapings were taken from each plant sample and tested again for sterility on LBA plates. The root exudates were then washed into 50ml falcon tubes with 35ml of ddH2O. This was repeated once more for a total of 2 washes. If sand scrapings were sterile, each wash was filtered through a .22μm filter. Before filtering, samples were stored at 4°C. Samples were frozen at -80oC and lyophilized (Virtis 12L Freezemobile).

Once dry, sterile samples were submitted for LC-MS/MS analysis. Samples were resuspended in 5 ml ddH2O. Samples with collected amino acid extracts were analyzed using liquid chromatography (Agilent UHPLC 1290), with a UHPLC (Ultra-High Pressure Liquid

Chromatography) 1290 column (Agilent Technologies, Inc). This was coupled to a highly sensitive mass spectrometer (AB Sciex QTRAP 5500) at the Targeted Metabolomics Laboratory (TML) at the Ohio State University. LC-MS/MS analysis was performed as in Cocuron et al. (2014). Mass spectroscopy parameters are contained in Cocuron et al (2014), supplemental table 2. Data were acquired and chromatogram peaks were integrated using Analyst 1.6.1 software (Sciex, 500 Old

Connecticut Path, Framingham, MA 01701 U.S.A.). Amino acid content was analyzed as relative abundance. We conducted four replications of this experiment.

Agar Plug Choice Test 140

Amino acids

Gamborg’s media was used for preliminary agar plug assays. Twenty-five ml of the media was poured onto each plate. Plugs were created of each media, control, and amino acid amended

Gamborg’s media to a final concentration 0.25M, 5mM, 5µM and 5nM L-threonine. A number

7mm diameter bronze hole borer was used to create the plugs. Individual plugs were placed on a straight line across the plate using a ruler, 2cm from the edge of the plate. A dot was placed

2cm from the bottom of the plate to denote where the droplet of 500 M. incognita J2s would to be placed. Plates were incubated in the dark for 12 hours at 23°C. After 12 hours, juveniles within a 1.5cm radius of each plug were counted under a dissecting scope. Nematodes that were within a 1.5cm radius of each plug were counted as attracted.

Worm Maze Tests

Amino acids

In order to test for chemotaxis, worm mazes were created (Wayne Machine Inc, 532

County Road 1600 Ashland, OH 44805). Worm mazes were created with grooves cut into 2.4 cm high by 13 cm diameter aluminum discs. The grooves were cut in a Y-shaped pattern with a central section that had a diameter of 2.5 cm, three arms of 4cm and wells at the end of the arms that had a diameter of 1cm. The central circle and arm grooves had a depth of 1.3 cm, and the wells at the end had a depth of 1.5mm (Fig. 3.2). At the end of each arm, a well was pre-formed using a removable metal plug (5.2 cm long).

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Maze media was created using a solution of 3g of Gamborg’s basal salts for buffering and

6g of phytagel per liter, this solution adjusted to 6.1 pH using 1M KOH. Thirteen milliliters of maze media was pipetted into the mazes and allowed to solidify in a laminar flow hood. The metal plugs at the end of each arm were removed to create a hollow well which would trap any nematodes that swim into the well. A spatula with a flat end was used to make an angle at the end of each of the arms starting from 0.7cm away from the well and angling down into the well

(see Fig. 3.2). After the downward slopes were made, 1.5ml of ddH2O was pipetted into each of the wells. The mazes were incubated in humid boxes in the dark at 23OC for 24 hours to allow the solution to equilibrate. After 24 hours, water in one of the wells was removed and replaced with the root exudate solution or 5mM of each individual amino acid. After 24 hours, 250 nematode juveniles were inoculated into the central circle. These were incubated for 48 hours.

The solution in the individual wells was collected, and the number of nematode juveniles that fell into each well was counted under a dissecting microscope. Chemotaxis Index was calculated

A+B (C−( )) 2 using the following formula CI= A+B where C represents the well containing the test solution (C+( )) 2 and A and B are wells containing the control solution.

C. elegans tests

2-day-old synchronized C. elegans juveniles were used for testing chemotaxis.

Experiments were performed on worm mazes as with RKN juveniles.

For experiments involving the RNAi feeder strain, E. coli Ht115 (DE3) and F22A2.3 (glr-8) were grown overnight in 15ml LB culture. Two-hundred and fifty ml of each culture was then

142 spread on NGM plates and allowed to air dry for one hour in a hood. C. elegans L1 juveniles were added to the plates. C. elegans was propagated solely on the RNAi feeder strains for approximately three generations before experiments were started. Feeder strains and controls were synchronized to produce L1 juveniles as above for the chemotaxis experiments.

Soybean Cyst Nematode test

To synchronize the age of the juvenile SCNs, the solution containing juveniles was removed from the hatching chamber, and fresh ddH2O was added. SCN eggs were allowed to hatch for 24hours, and the juveniles that were used for the SCN tests were acquired from this mix. Mazes were inoculated with 200 SCN juveniles.

Beet cyst Nematode test

BCN juveniles were age synchronized as described under SCN above. Mazes were inoculated with 150 BCN juveniles. Maze tests were performed as described under RKN.

Tests of Amino Acid Root Exudate Profiles and their attractiveness to various nematodes.

A solution of the relative ratios of amino acids that we found in soybean root exudates was made (Table 3.2). Individual L-amino acids were obtained from Sigma-Aldritch (St. Louis,

MO). We incorporated the amino acids in ratios shown in Table 1. In brief, we started with stock solutions with 0.25M of each amino acid. Each amino acid was then added in the proportion that was found in the soybean amino acid root exudate. For example, 57.78 ml L-asparagine was added for a total concentration of 0.031M in the final stock. This stock was then further diluted

1:100 or 1:100 in sterile ddH2O to create the test solutions.

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Data Analysis (chemotaxis assay)

Data analysis was performed in Microsoft Excel and Minitab, version 17.1.1 (2017; State

College, PA: Minitab, Inc. (www.minitab.com)). All experiments in this chapeter utilized a completely randomized design. All data were analyzed for assumptions of normality and homoscedasticity, using the Kolmogorov-Smirnov test and Levene's test, respectively. All

A+B (C−( )) 2 chemotaxis experiments were analyzed using the chemotaxis index, CI= A+B modified from (C+( )) 2

Fleming et al., 2017.

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3.4 RESULTS

Root Exudate Chemotaxis

RKN juveniles were significantly attracted to Glycine max cv. ‘Lee' root exudates compared to water controls when exudates were collected from two-week-old plants over 24 hours. RKN was also significantly attracted to root exudates from Zea mays lines, ‘B73’. SCN was significantly attracted to Glycine max cv. ‘Lee’ root exudates but not Zea mays ‘B73’ exudates.

BCN was attracted to exudates from Zea mays ‘B73’ and Glycine max cv.’Lee’. Overall, RKN J2s showed the strongest response to maize and soybean root exudates and SCN J2s showed the weakest overall response (Fig. 3.3).

Soybean Root Exudate Profile

The Glycine max cv. ‘Lee’ amino acid root exudates were collected under sterile conditions. In the final analysis, the amino acid asparagine made up the largest portion of amino acid root exudates in this cultivar, under sterile conditions at 62.1% percent. Alanine, arginine, and asparagine made up the next largest percentage of the total amino acids at 13%, 4.6%, and

4.3% respectively. Most of the other amino acids that we found were present at levels detectable above the background. The smallest detectable amino acids were hydroxy-proline, tryptophan, and methionine at 0.05%, 0.04%, and 0.03% respectively. Cysteine, citrulline, and ornithine were not detectable above background (Table 1, Fig. 3.1b).

Amino Acid Chemotaxis

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RKN J2s were significantly attracted to L-threonine at concentrations of 5mM and 5μM, they were not attracted at concentrations of 0.25M or 5nM (Fig. 3.4). RKN J2s exhibited a variety of responses to the various amino acids that we tested. Eight amino acids were attractive, seven amino acids were repellant, and eight amino acids did not provoke a response from RKN J2s in our tests. Among the amino acids tested, 5mM solutions of L- threonine, L-aspartic acid, L- histidine, and L-proline were the most attractive compared to the water control. L-asparagine, L- arginine, L-tyrosine, and L-methionine were also significantly more attractive than the control. L- alanine, L-isoleucine, L-valine, L-tryptophan, L-lysine, and L-glutamine were not significantly attractive or repellant compared to the control. L-cysteine was the most repellant compared to the control with a chemotaxis index of -0.4. L-leucine, L-serine, L-phenylalanine, L-glutamine, glycine, and L-citrulline were also significantly repellant compared to the control, with chemotaxis indecies of -0.2 to -0.3 (Fig. 3.5). The essential amino acids L-threonine, L-histidine,

L-methionine, and L-arginine, were attractive to RKN J2's. In contrast, other amino acids, specifically, L-isoleucine, L-leucine, L-lysine, L-phenylalanine, L-valine, and L-tryptophan, were either not attractive or repellant to RKN J2's.

The attraction or repellant properties of the various amino acids did not fit a pattern that could be characterized by any basic characteristics of amino acids. For the acidic amino acids, L- asparagine was attractive, and L-glutamic acid was not attractive. For the aliphatic amino acids,

L-leucine was repellant and L-isoleucine, and L-valine were neither attractive nor repellant. The aromatic amino acids, L-tyrosine and L-histidine, were attractive, while L-phenylalanine was repellant and L-tryptophan was neither. The basic amino acids L-histidine and L-arginine were

146 attractive while L-lysine was not attractive. Five of the hydrophobic amino acids were attractive, three were repellant, and four were neither attractive or repellants. Six polar amino acids were attractive to RKN J2s, three were repellant, and three were neither attractive nor repellant. Of the sulfur-containing amino acids, L-methionine was attractive, and L-cysteine was repellant (Fig.

3.6).

The D-form amino acids, D-threonine, D-aspartic acid, and D-serine, did not exhibit chemotactic properties compared to the control. D-threonine and D-aspartic acid were not attractive, and D-serine was not repellant compared to the control (Fig. 3.7).

C. elegans L1 stage juveniles were attracted to the following amino acids; L-asparagine, L- lysine, L-serine, L-alanine, L-threonine, glycine, L-histidine, L-arginine, L-leucine, L-valine, and L- glutamine. Only two amino acids were found to be repellant compared to the water control, L- aspartic acid, and L-methionine (Fig. 3.8). Among the essential amino acids, C.elegans L1 juveniles were attracted to L-lysine, L-threonine, L-histidine, and L-leucine, one more than the four that were attractive to RKN J2s. C. elegans L1 juveniles were attracted to eleven amino acids compared to 8 that were attractive to RKN J2s. Fewer amino acids also repelled C. elegans L1 juveniles, two compared with seven for RKN J2s (Fig. 3.5, Fig 3.8). C. elegans L1 juveniles were overall attracted to both of the acidic amino acids, L-aspartic acid, and L-glutamic acid. C. elegans juveniles were also attracted to all of the basic amino acids, L-arginine, L-histidine and L-lysine

(Fig. 3.9)

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Attraction to L-threonine was ameliorated in the C.elegans ionotropic glutamine receptor

RNAi knockdown mutant glr-8. The attraction of C. elegans to glycine was also reduced.

Attraction to L-serine was not affected by the glr-8 mutant (Fig. 3.10).

Amino Acid Profile Reconstructed

Reconstructed amino acid profiles (Table 3.2) of soybean root exudate at a 1:100 dilution were attractive to SCN and RKN, but not BCN (Fig. 3.11). None of the 1:1000 diluted reconstructed profiles were attractive to any of the species that we tested. Replacing the top attractive amino acid L-threonine with its D-stereoisomer did not significantly change the overall attractiveness of the amino acid reconstruction. Likewise, taking out the top two attractants, L-threonine, and L- aspartic acid and replacing them with the corresponding D-amino acids did not change the overall attractiveness of the amino acid reconstruction. Exchanging two of the repellants with their D- form did not change the overall attractiveness of the amino acid reconstruction (Fig. 3.12).

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3.5 DISCUSSION

Root exudates represent a significant portion of plant fixed carbon with estimates ranging from 2-17% of fixed carbon (Kuzyakov et al., 1999; Nguyen, 2003). Earlier research found that nematodes were attracted to tomato plant roots (Wang et al., 2009), root leachates (Devine and

Jones, 2003) and to areas where plant roots had been (Prot, 1980; Prot and Van Gundy, 1981). In our tests, we found that the plant-parasitic nematodes, RKN, SCN, and BCN were attracted to root exudates that were collected from young maize and soy plants. SCN, a nematode with a relatively narrow host range showed signs of host specificity, being attracted to soybean root exudates but not maize root exudates. RKN and BCN were attracted to both maize and soy root exudates. The exudates were attractive to nematodes at ~5cm distance (Fig. 3.3). Root-knot nematodes are known to be attracted to low pH gradients (Wang et al. 2009). In our experiments, pH was adjusted to 6.1 in all solutions to attempt to avoid this effect. Interestingly, our results were confounded by the occasional clumping behavior of the nematode (~5% of the test). In these and other experiments, signals that lead to clumping of juveniles seemed to override other chemotaxic signals (Appendix B). This behavior has been observed in pluronic gel studies with several root-knot nematode species (Wang et al., 2009b; Wang et al., 2010) and suggests a hierarchical behavior pattern that controls chemotaxic behavior.

Amino acids are known to be a component of plant root exudates (Jones and Derrah,

1994), and the influx and efflux of amino acids is at least partially controlled by active transport

(Badri and Vivanco, 2009; Hirnir et al., 2006) and by proteins that facilitate passive transport

(Pratelli et al., 2010). In our study, we looked at amino acid exudates of Glycine max cv. Lee at 149 two weeks after germination. We chose soybean because it is a common crop and because in our tests it was the easiest to maintain under sterile conditions, compared to corn and tomato. It is also a common host of RKN and SCN. Early stages of the plant lifecycle are characterized by high root exudation (Pausch et al., 2013). LC-MS/MS analysis revealed that almost all of the amino acids that we tested for were present in the root-exudates of a 10-14 day old soybean plant (Fig.

3.1). One exception was cysteine, which was not well extracted from our containers using the ddH2O washes that we used.

Among the chemical compounds that are attractive to nematodes, they seem to fall into different effective distances from roots. CO2 could be detected as far away as several centimeters by RKN (Dusenberry, 1987). Other compounds have been tested at shorter distances, including the very powerful, as yet unidentified, attractant tested by Čepulytė et al. (2018). The amino acids that we tested here would fall under a medium distance attractant, as RKN juveniles were able to respond and move toward or away from the amino acids at a distance of 4-5 centimeters

(Fig. 3.5). There are a few reports of amino acids being attractive to plant-parasitic nematodes.

Bird (1960) was the first to identify an amino acid, in this case, glutamic acid, to be attractive to

M. javanica and M. hapla. Glutamic acid was not found to be attractive in our data (Fig. 3.5), but we are using M. incognita, so perhaps this difference comes down to species. Recently arginine and lysine were found to be attractive to nematodes over short distances on a pluronic gel

(Fleming et al., 2017). Arginine was found to be mildly attractive in our tests. However, lysine was not (Fig. 3.5), perhaps as a result of the greater distances we are dealing with in our experiments.

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RKN is attracted to some of the essential amino acid components of the nematode diet

(Balasubramanian and Myers, 1971; Brockelman and Jackson, 1978; Vanfletern, 1973). Essential amino acids that we found to be attractive to RKN J2s included threonine, histidine, proline, arginine, and methionine (Fig. 3.5). Essential amino acids must be obtained from the plant host, and thus it would make sense that a nematode would seek out a host that would be able to provide those essential nutrients. Aspartate can likely be lumped into this group as well, as the precursor for branched-chain amino acids, all of which are essential (Jander and Joshi, 2010).

Asparagine may be on the attractive list due to its prevalence in root exudates (Fig. 3.1) and in plant root systems in general as a vital transport amino acid (Lesuffleur and Cliquet, 2010; Lohaus et al., 1994). The repellency of phenylalanine probably derives from the fact that it is a precursor for compounds in the phenylpropanoid pathway and lignin biosynthesis, both of which have been implicated in host resistance against nematodes (Edens et al., 1995; Sirohi and Dasgupta, 1993;

Wuyts et al., 2006). Cysteine is also likely a repellant because of its involvement in the glucosinolate and hydrogen cyanide pathways, both of which have been shown to have adverse effects on plant-parasitic nematodes (Siddiqui et al., 2006; Zasada and Ferris, 2004).

The attractiveness or repellency of some of these compounds may be affected by diffusion rates as well as concentrations. RKN is known to be attracted or repelled to some compounds based upon concentration, in particular, lauric acid is attractive to nematodes at a low concentration but repellant at high concentrations (Dong et al., 2013). Some of the amino acids that we tested will likely exhibit different chemotaxis characteristics if we were to apply them at a range of concentrations. Indeed in preliminary experiments M. incognita juveniles

151 were attracted to L-threonine at millimolar and micromolar concentrations, but not at molar or nanomolar concentrations (Fig. 3.4) Also, the attractiveness or repellency of some amino acids may be affected by diffusion rates, which may change the ability of the nematode to detect the amino acid gradient over the defined period of time that we used in our assays.

There may be general characteristics of amino acids that might lead to their attractive or repellent qualities. However, when we look at the response of RKN J2s to amino acids, there is no consistent pattern that emerges (Fig. 3.6). No one category of amino acids contains solely attractive, repellant or neutral amino acids. In addition to this fact, RKN seems to specifically prefer the biological L-form amino acids to their D-form stereoisomers (Fig. 3.7). Taken together, these two points would seem to indicate that RKN can specifically detect different amino acids.

This interaction is likely facilitated by a receptor-mediated interaction of some kind, as has been detected in other organisms that can respond to amino acids (Croset et al., 2016). C. elegans is an excellent model to explore some of the receptors that may be responsible for amino acid chemotaxis in nematodes. The C. elegans genome is well-characterized, and there are mutant lines and RNAi knockdown lines for most genes in the C. elegans genomes. Although the genome contains over 1000 g-coupled protein receptors (C. elegans Sequencing Consortium, 1998), some candidates for an amino acid receptor are the ionotropic glutamate receptors glr-7 and glr-8

(Brockie et al., 2001; Croset et al., 2010). An RNAi knockdown of glr-8 was deficient in its abilities to tax toward L-threonine and glycine (Fig 3.10) but not L-serine, indicating there may be some specificity with these channels as well. More experiments will be required to confirm the validity of this result.

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C. elegans is attracted to a variety of compounds (Colbert et al., 1997; Komatsu et al.,

1997; Ortiz et al., 2009) including the amino acids, lysine, cysteine, and histidine (Ward, 1973).

In order to show how a bacteriovore may differ in its amino acid preferences compared with a plant parasite, we decided to test the amino acid preferences of C. elegans L1 stage juveniles. In our tests, synchronized C. elegans L1 juveniles were attracted to a greater variety of amino acids and repelled by fewer amino acids than M. incognita J2s. This higher attractiveness to amino acids may reflect the fact that C. elegans, as a bacterial feeder, needs to be attuned to a wider variety of compounds. The 1280 g-coupled protein receptors in C. elegans represent a massive amount compared with 108 that are found in M. incognita (Abad et al., 2008). The repellency of methionine may be explained as it is a precursor methane thiol synthesis and related volatile sulfur-containing compounds that can be toxic to C. elegans and other nematodes and is commonly produced in soil bacteria. The repellency of aspartic acid is less easy to explain (Fig.

3.8).

The specific combinations of amino acids in roots may play a role in the ability of a nematode juvenile to find a host species (Fig. 3.11). We found that the reconstructed amino acid profile that we determined from soybean was attractive to nematode species that can utilize soybean as a host, including RKN and SCN. In contrast, the reconstructed amino acid profile was not attractive to beet cyst nematode, which cannot utilize soybean as a host. None of the nematodes were attracted to the blend at a higher dilution, indicating that there may be a cut off at which nematodes no longer respond to certain rates of amino acids in the root systems

(Fig. 3.11). Likely, the concentrations that we used here do not accurately represent the

153 concentrations found in the soil. Many factors in the soil would cause these pools to be changed dramatically. Soil amino acids can be utilized (Halvorsen, 1972) and altered by the microbes that are present in the soil. In addition to direct metabolism of soil amino acids, levels of amino acids in the rhizosphere can also be influenced by microbes that can manipulate plants into exuding more amino acids into the soil (Phillips et al., 2004). The result of this is that rapid fluctuations likely occur in pools of free amino acids. This fluctuation means that the amino acid pool that the nematode encounters in the rhizosphere will vary significantly from the profile that is generated in a sterile environment, such as the one that we produced here. The contribution of individual amino acids seems to be minimal on the attractiveness of the amino acid mix as a whole, because removal of the top attractive or repellant amino acids and replacement with their D-form amino acid failed to significantly change the overall attractiveness of the soybean amino acid bled (Fig.

3.12). In many of the experiments listed here, the variation is quite large, one contributor to this variation is the fact that nematodes occasionally undergo a clumping phenotype (McBride and

Hollis, 1966; Wang et al., 2010). This clumping behavior has been observed occasionally, involving all of the species that we tested. When this phenomenon occurs it seems to overcome other chemotactic stimuli, leading to an accumulation of most of the nematode juveniles in a single well (Appendix B).

Overall, it would appear that amino acids elicit a variety of chemotactic responses from nematodes, and these responses are in a stereoisomer and nematode species-specific manner.

Amino acids may contribute to host-specific chemotaxis, but there are further experiments needed to look at how the amino acid profile may be affected in the soil. There may be potential

154 for altering amino acid profiles in the roots, blocking, or activating receptors that may be helpful for nematode control in the future.

3.6 ACKNOWLEDGMENTS

I would like to thank Rebecca Kimmelfield and Anna Stasko for critical reading of the document and Therese Miller for the maintenance and propagation of nematode populations. I would also like to thank Amanda Lietz for carrying out some of the receptor related experiments.

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Tables

Table 3.1. Amino acid composition of soybean root exudate (% of total free amino acids).

Soybean plants were grown in sterile conditions for 14 days, after 14 days roots were washed with twice. Collected root exudates were filtered and lyophyllized. Root exdates were then resuspended and run through LC/MS-MS analysis. Chromatograms were integrated using

Analyst software. n=4.

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Table 3.1

Average % total Standard error of the Amino Acid amino acids mean Alanine 13.6 5.49 Arginine 4.6 0.91 Asparagine 62.1 14.0 Aspartic Acid 4.3 2.24 Citrulline 0.02 0.003 Cystine 0.01 0.007 GABA 0.81 0.24 Glutamine 2.57 0.59 Glutamic Acid 0.63 0.22 Glycine 0.76 0.42 Histidine 0.54 0.11 Homoserine 0.03 0.01 Isoleucine 0.92 0.24 Leucine 0.72 0.22 Lysine 0.28 0.07 Methionine 0.03 0.008 OHPro 0.05 0.02 Ornithine 0.007 0.002 Phenylalanine 1.62 0.26 Proline 2.07 0.48

Serine 0.84 0.14 Threonine 0.92 0.16 Tryptophan 0.04 0.01 Tyrosine 0.2 0.09 Valine 2.01 0.54

170

Table 3.2. Amino acid volumes added to make 100ml reconstructed amino acid root exudate stock. The final solution was then diluted 1:100 and 1:1000 to make test solutions.

171

Table 3.2

Amino acid Vol. of 0.25M stock added to 100ml reconstructed RE mix

L-alanine 13.6ml L- arginine 4.6ml

L-asparagine 62.1ml

L- aspartic acid 4.3ml

L-citrulline 0.02ml

L-cysteine 0.01ml

L-GABA 0.81ml

L-glutamine 2.57ml

L-glutamic acid 0.63ml

glycine 0.76ml

L-histidine 0.54ml

Homoserine 0.03ml

L-isoleucine 0.92ml

L-leucine 0.72ml

L-lysine 0.28ml

L-methionine 0.03ml

L-ornithine 0.007ml

L-phenylalanine 1.62ml

L-proline 2.07ml

L-serine 0.84ml

L-threonine 0.92ml

L-tryptophan 0.04ml

L-tyrosine 0.2ml

L-valine 2.01ml 172

Figures

Figure 3.1. Measurement of Soybean Amino Acid Root Exudate Profile. (A). Experimental Setup.

Sterile soybean (Glycine max cv ‘Lee') seed was soaked overnight in ddH2O. One imbibed seed in each funnel, and then covered with sterile sand ¼ Murashige and Skoog media. The tubes were incubated for two weeks in a growth chamber. Funnels were then washed two times with ddH2O. Samples were filtered sterilized and lyophilized. Once dry, sterile samples were submitted for LC-MS/MS analysis.

Data is the average of 4 replications (B). See Table 3.1 for variation.

173

Figure 3.1

174

Figure 3.1

175

Figure 3.2 Experimental setup for worm mazes. A. Top view of aluminum discs showing the channels extending from the center and the three wells at the end of the wings. B. Cross-section of one of the wings wells showing how the maze would be set up for an experiment. First, an aluminum plug was placed in the well and agar poured into the channel. Second, the plugs were removed from the wells, and an angle was cut in the agar starting 0.7cm away from the edge of the channel. Third, ddH2O was placed in the well for 24 hours for diffusion to equilibrate before adding the solution containing a solution.

176

Figure 3.2

177

Each experiment was repeated at least three times with similar results. Error bars represent standard error of the mean.

0.6

0.5

0.4

0.3 Maize 'B73'

0.2 Soy 'Lee' ChemotaxisIndex 0.1

-0.1 SCN RKN BCN

178

퐀+퐁 (퐂−( )) ퟐ n=12. Chemotaxis index was calculated using the following formula CI= 퐀+퐁 . P values were (퐂+( )) ퟐ calculated using the Students T-test (*P=<0.05), comparing each treatment to the water control. Experiment was repeated twice with similar results. Error bars represent standard error of the mean.

0.7 * 0.6

0.5

0.4 * 0.3

0.2

ChemotaxisRatio 0.1

-0.1

-0.2 Water L-thr 0.25M L-thr 5mM L-thr 5uM L-thr 5nm

179

Figure 3.5. Measurement of root-knot nematode juvenile attraction to individual amino acids.

Mazes were made with two wells containing ddH2O and one well with 5mM amino acid solutions. 200 RKN J2s were inoculated onto the mazes and incubated for 24 hours. After 24 hours, wells containing amino acid or control solutions were removed, and the number of juveniles in each well was counted under a dissecting microscope. n=9. Each experiment was repeated at least three times with similar results. Chemotaxis index was calculated using the

퐀+퐁 (퐂−( )) ퟐ following formula CI= 퐀+퐁 . P values were calculated using the Students T-test (*P=<0.05), (퐂+( )) ퟐ comparing each test solution to the water control. Error bars represent standard error of the mean.

0.8 ** ** ** 0.6 **

0.4 * * * * 0.2 0 -0.2 * * * * Chemotaxis Index Chemotaxis -0.4 * * -0.6 -0.8 *

-1

L-ile

L-lys

L-tyr

L-his

L-gly

L-val

L-ala

L-thr L-trp

L-glu L-gln

L-ser

L-leu

L-cys

L-arg

L-asp L-asn

L-pro

L-phe

GABA

L-met citrulline ornithine

180

Figure 3.6. Root-knot nematode juvenile attraction to individual amino acids does not follow a general pattern. Circles in the diagram represent various general characteristics of amino acids.

181

퐀+퐁 (퐂−( )) ퟐ results. n=9. Chemotaxis index was calculated using the following formula CI= 퐀+퐁 . P (퐂+( )) ퟐ values were calculated using the Students T-test (*P=<0.05), comparing each test solution to the water control. Error bars represent standard error of the mean.

0.8 * *

0.6

0.4

0.2

0 ChemotaxisIndex

-0.2

-0.4

-0.6 * L-thr D-thr L-asp D-asp L-ser D-ser

182

Figure 3.8. C.elegans chemotaxis toward or away from amino acids. Two hundred synchronized

퐀+퐁 (퐂−( )) ퟐ following formula CI= 퐀+퐁 . P values were calculated using the Students T-test (*P=<0.05) (퐂+( )) ퟐ comparing each test solution to the water control. Error bars represent standard error of the mean.

0.8 **

0.6 * * * * * * * * * 0.4 *

0.2

0 ChemotaxisIndex -0.2

-0.4 * * -0.6

183

Figure 3.9. C. elegans amino acid chemotaxis. C. elegans L1 stage juveniles are attracted to acidic and basic amino acids in general. Circles represent various general characteristics of amino acids.

184

Figure 3.10. C. elegans ionotropic glutamate receptor mutants affect chemotaxis phenotypes.

퐀+퐁 (퐂−( )) ퟐ CI= 퐀+퐁 . P values were calculated using the Students T-test (*P=<0.05). Error bars (퐂+( )) ퟐ represent standard error of the mean.

0.8 * 0.7

0.6 *

0.5

0.4

0.3 Control - Ht115 (DE3)

0.2 glr-8 ChemotaxisIndex 0.1

-0.1

-0.2

-0.3 L-glycine L-serine L-threonine

185

퐀+퐁 (퐂−( )) ퟐ n=9. Chemotaxis index was calculated using the following formula CI= 퐀+퐁 . Each (퐂+( )) ퟐ experiment was repeated at least three times with similar results. Error bars represent standard error of the mean.

0.5

0.4

0.3

0.2 RE 1:100 RE 1:1000

ChemotaxisIndex 0.1

-0.1 SCN RKN BCN

186

J2s were inoculated onto the mazes and incubated for 24 hours. After 24 hours, wells containing amino acid or control solutions were removed, and the number of juveniles in each well was counted under a dissecting microscope. n=9. Chemotaxis index was calculated using

퐀+퐁 (퐂−( )) ퟐ the following formula CI= 퐀+퐁 . Each experiment was repeated at least three times with (퐂+( )) ퟐ similar results. Error bars represent standard error of the mean.

0.9

0.8

0.7

0.6

0.5

0.4

ChemotaxisIndex 0.3

0.2

0.1

0 RE 1:100 RE 1:100 Minus RE 1:100 Minus Top RE 1:100 Minus Top Threonine (D-thr) Two Attractants (D-thr, Two Repellants (D-ser, D-Asp) D-phe)

187

Chapter 4: Changes to Amino Acids in the Rhizosphere Lead to Alteration of Nematode Chemotaxis

4.1 ABSTRACT

Targeting nematodes before they reach the root has been a major goal for many who wish to control plant-parasitic nematodes. Chemical and biological controls have successfully targeted this stage by restricting the movement of nematodes. Because amino acids are a component of root-knot nematode chemotaxis, we hypothesized that changes in rhizosphere amino acids might be able to prevent nematode juveniles from reaching host root systems. Here we look at the potential to use the attractive (L-threonine, L-aspartic) or repellant (L- phenylalanine and L-serine) amino acids to control root-knot nematode juveniles on plants in vitro. We also tested the root-knot nematode juveniles’ ability to distinguish between different amino acid metabolism and transporter mutants in Arabidopsis in choice experiments. We found that applications of agar plugs containing L-threonine or L-aspartic acids in choice experiments reduced RKN juveniles ability to infest host roots. Application of agar plugs containing L-serine or L-phenylalanine between the host plant and the nematode resulted in reduced infestation of the plant. A push-pull combination of attractive and repellant amino acids was even more successful in reducing nematode infestation in plants. We also found that RKN juveniles were able to distinguish between amino acid biosynthesis or transport host mutants. These experiments represent a starting point for testing new potential nematode control measures on a larger scale.

188

4.2 INTRODUCTION

As an obligate parasite, the root-knot nematode, (Meloidogyne incognita; RKN) must navigate the rhizosphere environment to find a plant host. The rhizosphere can, in many ways, be a challenging environment, with rapid changes in temperature (Hu and Feng, 2003), moisture

(Van de Griend et al., 1985), and antagonistic microbes (Li et al., 2015). Although RKN and other nematodes are well adapted to this environment, it represents a vulnerable portion of the lifecycle for an endoparasitic nematode. The stage responsible for this navigation in RKN is the second-stage juvenile (J2), which hatches from eggs in the soil. Once inside a host plant root, the nematode is buffered from many of these environmental variables that may impact its survival.

RKN relies on chemotaxis to find a suitable plant host in an efficient manner (Reynolds et al., 2010). RKN perceives its environment using chemosensory sensilla called amphids. These amphids are located in two pores bilaterally on either side of the oral opening of the nematodes, and consist of a glandular sheath cell, a socket cell and dendritic processes (Perry, 1996). These sensilla can detect changes in temperature (Dusenberry, 1988), pH (Wang et al., 2009), chemical compounds(Fleming et al., 2017, Čepulytė et al., 2018) and other environmental cues (Rasmann et al., 2012). RKN is known to respond to a variety of chemical signals in the rhizosphere including

CO2 (Pline and Dusenbery, 1987; Robinson, 1995), amino acids, organic acids (Fleming et al.,

2017), host ethylene status (Fudali et al., 2013) and inorganic salts (Castro et al., 1991; Hida et al., 2015). Nematode chemotactic behavior has been proposed as an area that could be exploited for nematode control. These behaviors include reactions to stress in the form of inorganic ions

(Castro et al., 1991), reactions to pathogens in the form of hyphal secretions of nematophagous 189 fungi (Robinson and Jaffe, 1996) and reactions to host root exudates (Hiltpold and Turling, 2012).

There have been limited control strategies proposed that involved use of chemoattractant molecules, such as CO2, to lure nematodes toward a nematicidal compound, but this has not been pursued further (Robinson, 1995).

RKN is currently managed using chemical controls, host resistance, and cultural practices.

Many of the nematicide options for RKN are limited due to price, toxicity, and/or environmental concerns (Haydock, 2006; Ristaino and Thomas, 1997; Pope et al., 2005). Many nematicides are effective because they have a mode of action that leads to behavioral changes in the nematode, often impacting its ability to follow chemotactic gradients. Methyl bromide (Brom-o-Gas) causes hyperactivity (Thomason and McKenry 1974) and eventual death of the nematode (Castro and

Thomason, 1971). The nematicides, Temik (carbamate) and Nemacur (organophosphate), which act on acetylcholine esterase, can affect nematode behavior (Opperman and Chang, 1990), causing an inability for the nematode to move toward roots (Wright et al., 1980). Newer nematicides such as fluensulfone (Nimitz) also are effective because of behavioral changes, in this case, an initial rapid movement followed by complete paralysis (Kearn et al., 2014). Alteration of nematode chemotaxis may be defense strategy employed by plants. Some plants employ compounds in root exudates from root borders cause a reduction in nematode motility and ability to enter the root (Zhao et al., 2000). When M. incognita juveniles were exposed to 1-2 mm apical sections of pea roots with border cells, the juveniles accumulated and entered into a quiescent state with minimal activity (Zhao et al., 2000). However, subsequent experiments

190 revealed that M. incognita juveniles that regained mobility after exposure to these compounds actually became more virulent (Hawes et al., 2005).

In Chapter 3 of this thesis we demonstrated that certain amino acids can be used by RKN as chemotaxis signals (Bird, 1959; Fleming et al., 2017), with different amino acids acting as attractants or repellants. We found that the most potent attractant amino acids were threonine, aspartic acid, histidine, and proline. Among the most potent repellants were cysteine, phenylalanine, and serine. Isoleucine was consistently neutral in our experiments. For the amino acids that we tested individually, threonine, aspartic acid, and phenylalanine, we found that RKN

J2s responded to them in a stereoisomer specific manner. In this chapter, we investigated whether these amino acids can be used to manipulate nematode behavior in in vitro experiments involving plants. We tested a setup utilizing agar plugs to see if it would result in similar outcomes to those observed when we utilized the mazes described in Chapter 3 (Fig. 4.1).

Since nematodes can sense different amino acids and potentially different amino acid profiles, it is possible that nematode juveniles could detect and respond to plant mutants affected in their amino acid homeostasis and/or amino acid transport. We chose a set of seven

Arabidopsis thaliana mutants to explore this possibility. The first set of mutants have changes in threonine homeostasis, a knockout of the threonine catabolism protein, threonine aldolase, tha1-1 (Joshi et al., 2006); an overexpression of the threonine catabolism protein, threonine deaminase, 35s omr1-7 (Mourad and King, 1995); and a cross of the two mutants, tha1-1/35s omr1-7 (Joshi et al. 2006). A second set is a group of amino acid transporters, including a knockout of lysine-histidine transporter 1, localized to the root epidermis, lht1-1 (Hirnir et al., 2006) and 191 knockouts of two amino acid permeases (AAP3 and AAP6), which are vascular system-localized transporters which that previously been shown to be involved in RKN infestations (Marella et al.,

2013). Here we explore the ability of the nematode to distinguish between mutants that are likely to have different amino acid profiles in the rhizosphere.

192

4.3 MATERIALS AND METHODS

Media

Gamborg’s Basal Salts Media

We added 20g D-sucrose (Phyotech, Shawnee Mission, KS, USA), 3g Gamborg's basal salts (Phytotech, Shawnee Mission, KS, USA), and 6g phytagel (Sigma-Aldrich, St. Louis, MO, USA) to 1L of ddH2O. We adjusted pH to 6.1 using 1M KOH. We autoclaved for 30 minutes on a liquied cycle (Marella et al., 2013).

Amino acid amendments

For experiments involving amino acid plugs, 500ml of the above media was amended with individual amino acids, including L-aspartic acid, L-isoleucine, L-phenylalanine, L-serine or L- threonine, or D-threonine before autoclaving. Individual amino acids were obtained from Sigma-

Aldrich (St. Louis, MO, USA). Individual amino acids were added at a final concentration of 5mM.

Arabidopsis Culture

Arabidopsis thaliana ‘Columbia’, tha1-1, 35s omr1-7, tha1-1/35s omr1-7, lht1-1, aap3, and aap6 seeds were sterilized in 1.7ml Eppendorf tubes in 1ml of a 70% ethanol and 0.05% triton x-100 solution (Sigma-Aldrich, St. Louis, MO, USA) and agitated at 15 rpm for 30 minutes on a roller drum (New Brunswick TC-7, Fisher Scientific Hampton, NH, USA). The solution was then removed and replaced with 1ml of 100% ethanol. Seeds were then mixed for 15 more minutes on the roller drum and allowed to air dry in a laminar flow hood for approximately 2 hours. All

193 seeds were germinated on Gamborg’s media (30ml/media per plate). Plates were sealed with parafilm before being transferred to final Gamborg’s plates and sealed with micropore tape (3M) for root measurement and early infestation assays. Plants for all assays were grown in a Percival

(Perry, IA, USA) growth chamber under the following conditions: 8-h light/16-h dark, 23°C. Plates were placed in clear polycarbonate boxes during the assays; felt was placed on the bottom of the boxes to prevent the collection of water (Marella et al, 2013).

Root-Knot Nematode Culture

M. incognita eggs were collected from sterile Arabidopsis thaliana (Columbia wild-type) culture plates using the following procedure, similar to Marella et al, 2013. Stems and leaves of the plant were removed and discarded. The remaining root material containing RKN egg masses was collected from approximately 30 plates and placed in two 50ml centrifuge tubes (Falcon,

Corning, NY, USA). Forty-five ml of 5% bleach was added to the tubes, which were then shaken vigorously by hand for two minutes. The root material was poured over a 2mm coarse filter into a new 50ml centrifuge tube. The solution in the new tubes contained the majority of the eggs.

The tubes containing the eggs and solution were centrifuged for five minutes at 1000rpm. After centrifugation, approximately 40ml of the solution was poured out of the centrifuge tubes and replaced with approximately 40ml of ddH2O. The centrifugation and pouring off steps were then repeated three more times, to remove as much bleach as possible. The number of eggs was then counted under a Nikon SMZ645 dissecting microscope in three 10µl droplets to obtain an

194 approximation of the total number collected. Eggs were collected from plants grown in the conditions described above at 7-8 weeks after inoculation from previous egg hatches

In experiments where J2s were used, we used the following procedure to hatch juveniles.

After collection, the solutions containing eggs were poured onto a 25 µm (500mesh) filter. Eggs were incubated in the dark for ten days at 23°C. J2s that hatched from the eggs crawled through the filter and were collected in the water solution below the filter. Juveniles were counted in three 10 µl droplets prior to infestation of plates.

Agar Plug Choice Test

Media for the choice assays were made as follows; 6g/1L phytagel, 3g/L Gamborg’s basal salts, and if amending with amino acids, the volume require to adjust the solution to 5mM of the amino acid in the final volume. This concentration was chosen because it was the most responsive in the experiments that we conducted to measure M. incognita juveniles response to various concentrations of amino acids (Fig 3.4). The pH was adjusted to 6.1 and the preperation was autoclaved for 30 minutes. Twenty-five ml of the media was poured onto each plate. Plugs were created of each media, control and amino acid amended, using a 7mm diameter bronze hole borer. Two plugs were placed on straight line across the top of the plate, with the plug 2cm from the edge of the plate. A dot was placed 2cm from the bottom of the plate to denote where the droplet of worms was to be placed (Fig. 4.1A).

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500 M. incognita J2s were inoculated on the bottom of the plate on the 2cm dot.

Nematodes were incubated in the dark for 12 hrs at 23OC. After 12 hours juveniles within a 2cm radius of each plug were counted under a dissecting scope.

Agar Plug Distraction test

Fifteen plates were made using Gamborg’s media, as described above. Two dots were placed on a clean 100x15 mm Petri dish containing new media, on a straight line 2cm from the edge of the plate as described above. On one dot we placed a 7mm diameter agar plug on the other dot we placed a one-week old A. thalania ‘Columbia' plant (Fig. 4.1A). Plates were incubated for 12 hours to allow the plant to adjust to the new agar and for the amino acid solution to diffuse into the plate. After 12 hours, 100 M. incognita J2s were inoculated 2cm from the bottom of the plate. Plates were then incubated at 8-h light/16-h dark, 23°C for six days. After six days, plants were harvested, and acid fuschin staining was performed.

First, plants were placed in 5% bleach to allow for clearing of roots for 10min. Then plants were rinsed for 15 min in ddH2O. After the rinse plants were placed in a 3:5 solution of acid fuschin (1.4g Acid fuschin, 100ml Acetic acid, 300mlddH2O):ddH2O. Plants were brought to a boil in a microwave to allow the adherence of the stain to the cuticle of the nematode. The solution containing the plants was allowed to cool to room temperature, and then the plants were removed and allowed to destain for 10 minutes in ddH2O. Plants were then placed in acidified glycerol until observation. Juveniles present in the root system were counted under a dissecting microscope. The experiment was repeated four times.

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Agar Plug Repulsion Test

On fifteen plates, five dots were drawn on an agar starting in the middle of the plate 3cm from the edge and two on either side 2cm apart (Fig. 4.3A). A final dot was drawn on the opposite side of the plate 2cm from the edge of the plate. Seven-millimeter diameter agar plugs, containing either L-phenylalanine, L-serine or Gamborg’s media control, were placed on the line of 5 dots, and an A. thaliana ‘Columbia’ plant was placed on the lone dot on the other side of the plate. Plates were incubated at the above conditions for 12 hours to allow the plant to acclimate to the plate.

After 12 hours 100 M. incognita juveniles were inoculated onto the plate on the side of the plate opposite the plants and the plugs. Plates were incubated for six days, and acid fuschin staining was performed as listed under the "Amino Acid Distraction test". Stained juveniles in root systems were counted under a dissecting microscope. The experiment was repeated three times.

Agar plug Push-Pull test

For the push-pull test, two rows of five dots each were drawn on the bottom of fifteen

Petri dishes (Figure 4.4A). The distance between the lines was 4cm and each dot was 1.5cm apart.

A 2cm agar plug with amino acids or control solution was placed on each dot. Looking down at the top of the agar to the right of the second row of dots, a seven-day-old Arabidopsis thaliana

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‘Columbia' seedling was placed. Plates were incubated for 24hours to allow diffusion gradients to set up and for the plant to establish itself. After 24 hours, 500 RKN juveniles were inoculated between the two rows of plugs. Plates were incubated for seven days with the nematodes in the conditions described above. After this time, plants were removed and stained with acid fuschin, as described above. The experiment was repeated three times.

Arabidopsis choice tests

For choice experiments between different Arabidopsis mutants, the mutants were sterilized as described under plant culture above. The following amino acid mutants were used, tha1-1, 35s omr1-7, tha1-1/35s omr1-7 (Joshi et al., 2006; Mourad and King, 1995), lht1-1 (Chen and Bush, 1997; Hirnir et al., 2006), ANT1 (Chen et al., 2001), aap3-3, aap6-1 (Marella et al., 2013;

Okumoto et al., 2002). Mutants and wild type Columbia controls were germinated for a week on

Gamborg’s media. Two dots were placed on the bottom of a Petri dish in a straight line using a ruler, and each dot was 1 cm from the edge. Seven-day old germinated seedlings were placed on each of the dots, either a mutant or control plant. The seedlings were allowed to adapt to the new plates and were incubated at 230C for 24 hours (8hrs light:16hrs dark). 200 RKN juveniles were inoculated at the bottom of the plate between the seedlings. After inoculation plates were incubated for a further seven days at the conditions described above. After seven days, the plants were removed, acid fuschin staining was performed, and the number of nematodes in each root system was counted using a dissecting microscope. Each experiment was repeated three times.

Data Analysis

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Data analysis was performed in Microsoft Excel and Minitab, version 17.1.1 (2017; State

College, PA: Minitab, Inc. (www.minitab.com)). All experiments in this chapter utilized a completely randomized design. All data were analyzed for assumptions of normality and homoscedasticity, using the Kolmogorov-Smirnov test and Levene's test, respectively. All chemotaxis experiments were analyzed using the chemotaxis ratio, as described in Fleming et al.,

2017. The chemotaxis index (CI) for the choices featured in this chapter used the following formula CI=(A-B)/(A+B), where A is the number of nematode that moved in the attractant zone and B is the number of nematodes that moved into the control zone.

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4.4 Results

Amino Acid Plugs

To see if the agar plate setup would produce similar results to the worm mazes described in Chapter 3 of this thesis, we set up a series of choice tests involving all of the amino acids.

Similar to the worm mazes, L-threonine, L-aspartic acid, L-proline, L-histidine and L-asparagine were found to be attractive to RKN juveniles. L-tryptophan was attractive in the plug experiments

(Fig 4.1), even though it was not in the worm maze assays (Fig. 3.5). L-arginine, L- tyrosine and

L-methionine were not different than the control, in contrast to the maze experiments. L- cysteine, L-glutamic acid, L-alanine, L-leucine, L-serine and L-phenylalanine were repellant (Fig.

4.1). All of these, with the exception of L-glutamic acid, were also repellant in the maze experiements (Fig. 3.5)

In the experiments testing host attraction, six days after inoculation, A. thaliana plants on plates containing a plug amended with 5mM L-threonine plugs, contained 49% less RKN J2s than plants that were grown on plates with Gamborg’s agar plug without amino acid amendments. A. thaliana plants on plates with plugs of 5mM L-aspartic acid also contained 49% less RKN J2s than plates with a Gamborg’s agar plug. Plants on plates with a 5mM L-isoleucine plug contained similar numbers of RKN J2s to the control, as did plates with 5mM D-threonine plugs (Fig. 4.2B).

In plates containing a line of 5mM L-phenylalanine plugs wall, A. thaliana seedlings contained 47% less RKN J2s than in plates with a wall of Gamborg’s media plugs. Seedlings grown on plates with a line of 5mM L-serine plugs saw a 50% reduction in the number of RKN J2s as

200 compared to plates with the Gamborg’s media plugs. Seedlings grown on plates with a line of

5mM L-isoleucine plugs contained similar amounts of RKN J2s as the Gamborg’s control plates, as did plates with 5mM D-threonine plugs (Fig. 4.3B).

In the push-pull experiments, A. thaliana plants on plates with a line of attractive 5mM L- threonine plugs contained 43 percent fewer J2s than the controls. The addition of a row of 5mM

L-serine plugs resulted in 41 percent fewer juveniles than control plates. Plants with a row of 5M

L-serine plugs and a row of 5mM L-threonine plugs reduced the number of J2s in the roots by 72 percent. Finally, plants on plates with a row of 5mM L-serine/L-phenylalanine repellant plugs combined with a row of 5mM L-threonine plates had an 88 percent reduction in the number of juveniles in the root system (Fig. 4.4B).

Amino Acid Mutants

When given a choice between two wild type A. thaliana plants, RKN J2s showed no preference between the two plants. RKN juveniles were preferentially attracted to two of the threonine metabolism mutants, tha1-1 and tha1-1 /35s omr1-7 when compared to wild-type A. thaliana. Mutant tha1-1 plants contained 51 percent more RKN J2s than wild type plants, and tha1-1/35s omr1-7 plants contained 42% more RKN J2s than wild type plants. RKN juveniles also showed a preference for the root epidermal amino acid transporter mutant, lht1-1, which attracted 24% more juveniles than wild type plants. RKN juveniles also showed a preference for the amino acid permease mutant, aap6 over wild type plants. aap6 plants contained 34 percent more RKN J2s than wild type plants. RKN juveniles showed no preference for the threonine

201 deaminase overexpression mutant, 35s omr1-7, or the amino acid permease mutant, aap3 plants over wild type plants (Fig. 4.5).

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4.5 DISSCUSSION

RKN pressure on cropping systems continues to grow (Allen et al., 2017; Djian‐Caporalino,

2012). Chemical nematicides are expensive and heavily regulated in the face of increasing environmental and health concerns. Sources of resistance have only been identified for a few crops of the 1000s of species that are affected by RKN (Djian-Caporalino et al., 2001; Milligan et al., 1998). These resistance sources are also not durable in all situations (Kaloshian et al., 1996).

There are a few approaches for cultural control of plant-parasitic nematodes, but none work under all circumstances (Butler et al., 2012; Scholte and Vos, 2000; Wang and McSorley, 2008).

Thus it is essential to continue to pursue alternative control options.

Plates with a plug containing L-threonine or L-aspartic acid significantly reduced the number of RKN juveniles that infested root systems after six days. This likely represents nematodes that were distracted or confused by the presence of the strong attractants. This distraction does not seem to be a general amino acid effect because plates with L-isoleucine in the plugs were unable to cause the same reduction in RKN numbers after six days. Further experiments are needed to see if these numbers would continue to decrease over a more extended period. There have been a few successful attempts to use attractants as a way to manipulate the direction a pathogen or insect takes toward a crop. Many of these examples take the form of trap crops, where the pest or pathogen is drawn to a non-crop plant, or concentrating them in certain sections of the field to decrease infestation in the remainder of the field (Shelton and Badenes-Perez, 2006). Another common example are pheromone traps, which are used to attract insects, and draw them away from a host plant. The oldest successful example is probably 203 the use of trap trees or logs to control the European bark beatle, Ips typographus, in forest settings, a practice that has been in place for over 200 years (Bakke and Reige, 1982). Other examples include use of snap beans to attract Mexican bean beatles, Epilachna varivestis, to attract them away from a soybean crop (Rust, 1977). In a technique in use since the 1960s, Alfalfa is often interplanted with cotton in California to attract the lygus bug, Lygus hesperus (Godfrey and Leigh, 1994). Effective trap crops for nematodes have been observed: lauric acid from

Chrysanthemum coronarium, which can be intercropped with tomatoes, lures M. incognita juveniles in and reduces infestation on adjacent tomato plants (Dong et al., 2013). Use of an attractant to confuse a pathogen has been proposed in the past as well for control of fire blight,

Erwinia amylovora, on apples. The attractants sodium malate or sodium tartarate were applied to apple blossoms but the technique proved to be unsuccessful (Bayot and Ries, 1986).

Repellant amino acids were set up as a barrier on agar plates to see if they could mask the attractiveness of an A. thaliana plant. L-phenylalanine and L-serine plugs were both able to successfully reduce RKN numbers in the root systems of plates that contained those plugs. This does not seem to be a general amino acid effect as L-isoleucine was unable to cause the same reduction in nematode numbers. An alternative explanation lies in the possibility that one or both of these compounds may activate or prime plant defense in some way leading to a reduction of nematodes that gained entrance or stayed in the host root system. Other repellants for M. incognita have been found in root exudates through fractionation (Diez and Dusenbery, 1989), but their exact chemical makeup has not been elucidated. Repellants have been used to control pest and pathogens in agriculture in the past. An early example was the use of diesel or kerosene

204 as a seed coat to prevent the removal of freshly seeded pasture by ants (Russel et al., 1967).

There has been at least one attempt to engineer control of nematodes using a repellant. In this case a peptide that was repellant to Heterodera schachtii was specifically expressed in root cap cells, the area of the root where nematodes usually enter. Some of the lines used in this study reduced nematode infestation by over 90% (Lilley et al., 2011). Use of a repellant compound for the control of the parasitic plant dodder (Cuscuta spp.) has been proposed but has not been pursued further (Runyon et al. 2009).

To see if we could achieve an even more significant effect, we looked at a combination of attractive and repellant amino acids setup in a push-pull experiment. In this experiment, there was a line of repellant amino acids in between the RKN inoculation point and a line of attractive amino acids, either L-threonine or L-aspartic acid pulling the nematodes farther away from the

Arabidopsis seedling. An individual L-threonine plug on the outside of the plant was enough to reduce the number of nematodes that were able to enter root systems. The addition of a repellent line reduced the number of nematodes in the root system even further. A plug with a combination of phenylalanine and serine with a threonine attractant further reduced numbers.

This combinatorial approach produced the most substantial effects in reducing nematode numbers. Push-pull approaches have been used in control of pests, pathogens and parasitic plants in agriculture. The term was coined by Pyke et al. (1987) to define a system where they used attractive and repellant stimuli together to push Helicoperva spp. and pull them towards attractive traps. They used an application of neem oil, an anti-feedant, as the “push” component on host plants and used an intercropped species, pigeon pea or maize as the “pull” component.

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This reduced egg production on host plants by up to 92% compared with untreated controls. This idea was expanded upon and retermed “Stimulo-deterrent diversion” by Miller and Cowles

(1990) although the original name has persisted. They used a push-pull system for the control of the onion maggot, Delia antiqua. They used oviposition deterrants, such as cinnamaldehyde derivatives as the “push” and onion culls positioned close to the field as the “pull” component.

Another well-known system has been used for control of cereal stem borers (including Busseola fusca, Chilo partellus and others) as well as the parasitic plant, Striga hermonthica (Kahn et al.,

2000; Kahn et al., 2008). In this system maize is intercropped with a plant that is repellant to the borers, Desmodium uncinatum, a plant that also has allelopathic properties toward the Striga weeds. At the borders of the field Napier grass (Pennisetum purpureum) is planted and “pulls” the borers out of the field. This practice has been used in Kenya and other areas of East Africa

(Kahn et al., 2008). With a change of the border crop to Brachiaria ‘Mulato II’ the system becomes effective for fall armyworm as well (Midega et al., 2018)

The experiments described in here represent a first look at how some amino acids might be used in crop systems. Hypothetically, distractants, such as L-threonine or L-aspartic acid could be applied similarly to slow-release fertilizer in rows to confuse nematode chemotaxis. Also, drenches could be used to confuse nematode juveniles as they navigate the soil environment.

Repellants could be added to seed coats to protect the germinating seedling, which is one of the most vulnerable stages of plant development and the stage where many root exudates are deposited into the soil.

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Even with the potential demonstrated here, some pitfalls are already apparent that would make it challenging to employ amino acids in a rhizosphere environment. Amino acids are under severe competition in the rhizosphere. The amino acid composition of the rhizosphere is competed for and regulated by plants (Ganetag et al., 2017; Pratelli et al., 2010). The amino acid composition can also be modified by microorganisms, which may use them to navigate the rhizosphere environment themselves (Allard-Massicotte et al., 2016) or utilize them directly as a carbon or nitrogen source (Sivolodskii et al., 2009). Indeed it seems that our two most attractive amino acids, threonine and aspartic acid are some of the easiest amino acids for soil bacteria to use as a carbon source (Halvorsen, 1972). All of this means that any amino acids that enter the soil will be quickly acted upon by microorganisms that are present there. Indeed, one study calculated the half-life of amino acids in the soil to be around 1-6 hours (Jones and Hodge, 1999).

Another potential pitfall is the age of the plants that we dealt with in this experiment. These plants are very young and do not have very many branches on their roots. Because of the lack of branching, there are fewer sites for the RKN J2s to enter the roots, probably causing the small overall percentage of nematodes that were able to enter the roots compared to the number of

J2s that were inoculated onto the plate.

RKN can also detect differences in amino acid mutants. Arabidopsis mutants tha1-1, tha1-

1/35s omr1-7, lht1-1, and aap6 were all more attractive to RKN juveniles that wild type plants.

Mutants tha1-1 and tha1-1/35s omr1-7 are threonine catabolism mutants. This is a system that we have shown to have an impact on RKN parasitism (Chapter 2), so it is perhaps not a surprise that nematodes would be able to differentiate these mutants. Interestingly 35somr1-7, a mutant

207 which showed significant differences in female number, male development, and fecundity (Ch.

2), was not able to be distinguished by the RKN J2s (Fig. 4.5). lht1-1, a mutant of an epidermal amino acid uptake transporter likely accumulates more amino acids in general in the rhizosphere, representing a rich target for RKN juveniles (Ganetag et al., 2017). Plants with altered amino acid profiles could be made to select for profiles that were not as attractive to RKN or other nematodes, although these plants would have to be tested for non-target effects on the host due to amino acid changes or bacterial or fungal changes in the rhizobiome. There are examples in the literature where the attractiveness of a host has been altered in order to change pest behavior. Intercropping with coriander to mask the odor of host tomato was effective to reduce numbers of adults and nymphs of the silverleaf whitefly, Bemisia tabaci, as compared with a tomato monoculture (Togni et al., 2010). As mentioned above there are examples in the literature where M. incognita has been lured to a less attractive host in a trap cropping situation

(Dong et al., 2013), but to our knowledge there have been no attempts to change the host exudates or odors to alter M. incognita behavior.

The fact that nematodes are strongly attracted or repelled by amino acids could be used to create other kinds of control solutions in roots as well. The fact that nematodes seem to detect the L-form of the amino acids specifically is an indication that nematode juveniles may use receptors to detect amino acids. Using RNAi or another strategy to block amino acid receptors could be a potential avenue for control. Another strategy would be to use a large library of small molecules to identify compounds that are structurally similar to amino acids. These compounds could then be tested for nematode chemotaxis and characteristics in the soil environment.

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This work represents an initial thrust into the use of amino acids for control of RKN in plant systems. In a controlled system, amino acids could distract nematodes from plant roots systems and block nematodes from entering root systems. Root-knot nematodes could also distinguish between plants that were mutated in different parts of amino acid synthesis or transport. In the future, these strategies could be tested on a larger scale for nematode control under more practical conditions.

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4.6 AKNOWLEDGEMENTS

The authors would like to thank Krystel Navarro, Guillermo Valero, Amanda Lietz, and Gilbert

Chen for performing repetitions of experiments. Thanks to Rebecca Kimmelfield for critical reading of the document. Thanks to Leslie Taylor for the propagation of Arabidopsis plants.

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FIGURES

Error bars represent standard error of the mean.

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Figure 4.1

0.5

0.4 * * * 0.3 * * * 0.2 0.1 0 -0.1 *

-0.2 ChemotaxisIndex -0.3 * * * -0.4 *

-0.5 * -0.6

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Plants were incubated for six days, and then roots were stained with acid fuschin. The number of J2s that were stained in each root system was counted under a dissecting microscope. (B).

Results. n=15. P values were calculated using the Students T-test (*P=<0.05). Error bars represent +/- standard error of the mean. Representative of 3 experiments with similar results.

224

Figure 4.2

* 10 *

5 Number of Juveniles NumberofJuveniles root in system

0 Control 5mM L-asp 5mM L-thr 5mM L-ile 5mM D-thr

225

100 M. incognita J2s (N). Plates were incubated for six days after which roots were stained with acid fuschin. The number of J2s in each root system were counted under a dissecting microscope. (B). Results. P values were calculated using the Students T-test (*P=<0.05). n=15.

Error bars represent +/- standard error of the mean. Representative of 3 experiments with similar results.

226

Figure 4.3

8 * * 6

2 Number of Juveniles NumberofJuveniles Root in System

0 Control 5mM L-phe 5mM L-ser 5mM L-Ile 5mM D-thr

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Figure 4.4. A push-pull setup is effective in keeping nematodes from plant roots. (A).

Experimental setup. A plate containing a seven-day-old Arabidopsis thaliana ‘Columbia’ plant and two rows of plugs was inoculated with 100 M. incognita juveniles. One row of plugs contained either a repellent amino acid or control (Center, P/S), the other row of plugs contained an attractive amino acid (T, right). Plates were incubated for six days and stained with acid fuschin. The number of J2s in each root system was counted under a dissecting microscope. (B). Results. n=15. P values were calculated using the Students T-test (*P=<0.05).

Error bars represent +/- standard error of the mean. Representative of 3 experiments with similar results.

A. 228

Figure 4.4

10 9 8 7 6 * * 5 4 * 3 2 *

1 Number ofjuveniles system root in 0 Control 5mM L-thr 5mM L-ser 5mM L-ser + 5mM L- L-thr ser/L-phe + L-thr

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Figure 4.5. Nematodes are attracted to different plant amino acid mutants more strongly than others. Plates with two Arabidopsis plants were inoculated with 200 M. incognita juveniles.

Plants were incubated for seven days and then root systems were stained with acid fuschin.

The number of J2s in each root system was counted under a dissecting scope. The chemotaxis ratio was calculated. B. Results. n=12-15 P values were calculated using the Students T-test

(*P=<0.05). Error bars represent +/- standard error of the mean. Representative of 3 experiments with similar results.

0.8

* * 0.6 *

* 0.4

0.2

ChemotaxisIndex 0

-0.2

-0.4 WT tha1-1 35s omr1-7 tha1-1/35s lht1-1 aap3 aap6 omr1-7

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Chapter 5: CHALLENGES, IMPLICATIONS, QUESTIONS and FUTURE DIRECTIONS

Biological Implications and Challenges

Throughout my research, I have examined the importance of plant amino acid biosynthesis, metabolism and transport throughout the lifecycle of obligate parasitic plant-parasitic nematodes. In this thesis I particularly focused on the role of amino acids in root-knot nematode (Meloidogyne incognita) and measured impacts of plant-produced amino acids on nematode chemotaxis, early infestation, development, and fecundity.

I demonstrated that plant-parasitic nematodes and the model bacterial consuming nematode,

Caenorhabditis elegans, chemotax toward and away from specific amino acids. Amino acids that are attractive to a nematode varied depending on species. The attraction of a given species is affected by distance and concentration and is driven by chemical gradients. Nematodes are animals with a need for essential amino acids in their diets (a total of nine are required) therefore I hypothesized that amino acids would be at least partially required in chemotactic behavior required for their survival. However, it is clear that in both of the species that I tested the essential nature of each amino acid is not the only factor in the determination of whether a given amino acid is attractive or not. Both M. incognita and

Caenorhabditis elegans were attracted to some essential amino acids but not all. Interestingly, both species were also attracted to several amino acids that are not considered essential as nematodes can biosynthesize them. Taken together, it is likely that evolutionary forces that drive chemotactic behavior affected by the need to obtain essential food components with those that may represent either food source indicators and those that are repellent. These behavioral reactions are likely driven by biological interactions, including evaluation of the nutritional status of the plant, the defensive posture of the plant

231 immune system, and the presence of other organisms (i.e. competitors and/or pathogens of nematodes) within the rhizosphere.

Additional questions need to be addressed before we can provide a clear conclusion as to the contribution of amino acids to nematode chemotaxis. First, what concentrations of amino acids are present in the soil? In Chapter 3 I observed that nematodes behave differently when exposed to varying concentrations of amino acids. The volume of soil water can be quite variable, so it is possible that gradients of amino acids may be steeper due to water absorption by the roots. On the other hand, soil amino acids are a desirable commodity in the rhizosphere, and the half-life of amino acids could be short in soil depending on how quickly they are acquired and/or metabolized by biotic (rhizosphere microorganisms) and absorbed onto soil particles. This means that the amino acid profile that is present in root exudates may change before a host-seeking nematode has a chance to encounter them. In my experiments the attraction and repulsion responses that I observed may be an artifact of the highly controlled environment in which the experiments were conducted as compared to the complex soil environment where amino acid concentrations could be modified by other soil environmental factors. An understanding of amino acid flux in the rhizosphere would involve accounting for several factors that were not adequately addressed by this study: 1) The size, age, and the number of roots, and how roots of different ages may exude different amino acids at different rates. 2) How far do amino acids travel from roots, and are they detectable at distances where nematodes can utilize them? If amino acids are not present at the distances that nematodes must travel to find a host, why does it matter that they attract them? 3) How do changes in the rhizosphere environment impact the exudation of various amino acids?

Among the factors that would be important to address would be; timing and cycles of root exudation, including those dictated by day/night cycles and seasonal rhythms. Additionally, how do changes in soil moisture impact exudation of amino acids? How does the level of soil moisture alter the attractiveness of 232 a given plant? And 4) What is the influence of the microbiome of a given plant from which the amino acids are exuded? How quickly do rhizosphere microbes utilize the available amino acids? Does this utilization occur before they could to be detected by a nematode in the soil environment? Are plant amino acid profiles impacted by the specific makeup of species that are present in the rhizosphere? Answering some of these difficult questions would give us a clearer picture of the chemical cues that are available to nematode juveniles in the soil.

In addition to their role in chemotaxis mechanisms, I revealed that plant amino acid homeostasis is critical for the success of plant-parasitic nematode infestation. In this work, I looked at the example of threonine metabolism, which seems to play a role in the infestation process for M. incognita. Changes in the threonine pathway had an impact on M. incognita infestation, from early infection through the lifecycle. As obligate and sedentary parasites M. incognita juveniles must commit to a plant host. After they commit they lose their somatic musculature rendering it impossible for them to leave for another host. As a result, it is critical that M. incognita and other sedentary nematodes ascertain the suitability of a host before making this commitment. A suitable host plant provides essential nutrients that the obligate parasitic nematode is reliant on the host to synthesize, an example of which is the essential amino acids.

I saw that changes in threonine homeostasis led to decreased nematode presence, even from an early stage. It is plausible then that a compound from this pathway acts as a signal for nematodes to ascertain whether a host is suitable or not. As an organism with an extremely wide-host range, M. incognita can infest plants with diverse chemical makeup. Because of this, it is plausible that M. incognita would utilize a ubiquitous signal, like amino acids or another diet-related compound, for the determination of the desirability of a given host, rather than a species-specific compound that might be expected in the case of other nematode species. However, the identity of the triggers that plant-parasitic nematodes use to evaluate their plant hosts is yet to be ascertained. Answering this question will involve the identification 233 of the receptors mentioned in previous chapters as well as extensive testing of how juveniles act in response to having these receptors knocked out or knocked down.

The fact that changes in threonine metabolism led to impacts on late infestation implicate that either the threonine pathway or its derivatives are important in the completion of the nematode lifecycle.

The nematode may rely upon particular concentrations of some of the metabolites in this pathway to trigger various events later in the lifecycle. These events include whether or not nematodes develop into males or females, how many females develop overall, and how many eggs each female can develop. In order to maximize the number of successful offspring, it may be imperative for nematodes to be sensitive to triggers such as changes in amino acid homeostasis. These factors may be important for nematode control when we consider that this is a pathogen that can grow exponentially.

Another question that needs to be eventually addressed is what is the free amino acid content, as well as related metabolites of the roots, giant cells, and other parts of the mutants that I tested? The lack of data on this point leaves open the possibility of other explanations for the phenotypes that I observed. The intricacy of plant metabolism means that altering metabolism at one site could potentially lead to wide-ranging metabolic changes through the plant. These changes may mean fluctuations in the concentrations of many different molecules in related pathways. It is possible that these changes lead to the decreased female production, increased male production, and other phenotypic changes that I observed. It is crucial for us to pursue at the very least a profile of the root amino acid composition of these root systems, to see what changes might have occurred during these infestations. Another question is how the nematode perceives the changes in the concentration of these molecules? I presented preliminary data in this thesis that nematodes may use ionotropic glutamate receptors to sense amino acids, but where these receptors are located, how is the signal triggered and transduced and what are the

234 responses of the nematode on the molecular level remains to be elucidated. It would also be interesting to explore the gene expression profiles or metabolome of M. incognita females and eggs grown on amino acid mutants. Gene expression profiles or metabolomes could also give insight into whether the nematodes growing on these plants are exhibiting starvation or other indicators of nutrient stress. Further bioassays could be conducted to determine whether nematodes grown on threonine mutants are less virulent than those grown on wild-type plants.

Implications and future directions for plant-parasitic nematode control

Although there is no direct product that can be patented or recommended from this research, I believe that the data presented here is sufficient to warrant further exploration of the use of amino acid root exudates for nematode control, particularly in highly managed crops. Further exploration is required to ascertain the viability of the following potential strategies. One approach to be explored is the use of a push-pull strategy utilizing amino acids for nematode control. Push-pull mechanisms have been used effectively over the years for insect control, as detailed in Chapter 4. This approach might involve the application of repellant amino acids as a part of a seed coating regime and attractive amino acids to areas adjacent to plants. To make this strategy more viable in a soil environment, it may be necessary to look at a small molecule library and identify molecules that have structural similarity to attractive or repellant amino acids, and could be produced in commercially relevant amounts. We would then identify candidate compounds that may be more resistant to microbial breakdown in the soil and perhaps respond at lower concentrations or have higher affinities thus overriding their chemotaxic signaling system. Another potential strategy that warrants exploration is the deployment of a gene edited plant or traditionally bred plants that are altered in their root amino acid exudation profile. Such a plant could potentially be less attractive to plant-parasitic nematodes. A final idea for further exploration is to look at the application of

235 an RNAi molecule to knockdown the receptor responsible for chemotaxis to specific amino acids. There is much work needed to accomplish this, including, critically, the identification of the receptor that is responsible for these behaviors in plant-parasitic nematode species.

In the introduction, I mentioned different qualities that would make a good control strategy.

Nematode control via amino acid chemotaxis could be employed with a variety of different crop species, and it also seems likely that multiple species of nematodes would be affected by altering the rhizosphere amino acid environment. When it comes to the durability of the control strategy, amino acids strategies have positive and negative attributes. As an essential part of the nematode diet they represent compounds that are necessary, and thus would be difficult to evolve around. However, if chemotaxis toward amino acids is receptor-mediated, as the early signs of specificity that the research indicates, a single mutation in the receptor may allow the nematode to overcome this very quickly, but this may also produce a nematode that can no longer detect the target amino acid. Another unknown is whether redundancy is built in to chemotaxis strategies. Because of this any strategy using a receptor knockdown may also need to target multiple receptors. Finally, amino acids are somewhat expensive, so this may disqualify their use as control agents, unless production could be scaled up. There are general drawbacks in these control strategies; as pointed out above and in previous chapters, amino acids are acted upon dynamically in soil. Any control strategy employing amino acids would have to take into account the biotic and abiotic conditions that lead to their turnover. Any approach using GMOs or gene edited products or the application of RNAi molecules to the soil would have public perception and regulatory challenges to overcome.

236

A deeper understanding of the biology described in this work will help obtain a clear picture of plant-nematode interactions, to increase the tool kit of management options to control these economically devastating organisms.

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