Characterization of a Spontaneous Phaseolus Vulgaris Mutant

CHARACTERIZATION OF A SPONTANEOUS PHASEOLUS VULGARIS MUTANT

WITH THE ABILITY TO SELECTIVELY RESTRICT NODULATION

A dissertation presented to

the faculty of

the College of Arts and Sciences of Ohio University

In partial fulfillment

of the requirements for the degree

Doctor of Philosophy

Sarah Laity Bashore

August 2006

This dissertation entitled

CHARACTERIZATION OF A SPONTANEOUS PHASEOLUS VULGARIS MUTANT

WITH THE ABILITY TO SELECTIVELY RESTRICT NODULATION

SARAH LAITY BASHORE

has been approved for

the Department of Biological Sciences

and the College of Arts and Sciences by

Allan M. Showalter

Professor of Environmental and Plant Biology

Benjamin M. Ogles

Dean, College of Arts and Sciences

Abstract

BASHORE, SARAH, Ph.D., August 2006. Molecular and Cellular Biology

CHARACTERIZATION OF A SPONTANEOUS PHASEOLUS VULGARIS MUTANT

WITH THE ABILITY TO SELECTIVELY RESTRICT NODULATION (195 pp.)

Director of Dissertation: Allan M. Showalter

A spontaneous Phaseolus vulgaris mutant was isolated that selectively restricts

nodulation. This recessive mutation exhibits a phenotype that has never been seen before

with beans and is a perfect tool to study the symbiotic relationship and the associated

signaling molecules between legumes and Rhizobia. The goal of this dissertation was to

characterize the mutant bean’s morphology and nodulation capacity. Rhizobial screening

was done to examine how many different strains of Rhizobia were able to nodulate the

mutant bean. The bean was examined for phenotypic characteristics and then examined

for how the mutation was affecting nodulation. This was done by using green

fluorescently labeled bacteria to visualize steps in the nodulation process and by chemical isolation and characterization of the signals involved in forming the symbiosis. This research also examined the overall competitiveness of strains with the ability to nodulate the mutant bean. A final experiment used Tn5 mutagenesis of USDA 2669 to determine if any novel signaling molecules were present in the excluded strain. It was determined that the mutant P. vulgaris had no deleterious phenotypic characteristics and that three strains of Rhizobia, USDA 9017, USDA 9032 and USDA 9041, had the ability to nodulate the mutant. It was also demonstrated that the mutation blocked nodulation before the formation of infection threads and therefore was affecting the plant's perception of the bacterial signal.

Approved:

Allan M. Showalter

Professor of Environmental and Plant Biology

Acknowledgments

I would like to express my sincere gratitude to Dr. Art. T. Trese, my advisor, my mentor and my friend, who has always stood by me in times of doubt. I would also like to acknowledge Dr. Allan Showalter who served as my dissertation advisor and has helped me through a strange and difficult time. I would also like to acknowledge Dr.

Ivan K. Smith and Dr. Marcia J. Kieliszewski for their encouragement and support.

I would like to thank Harold Blazier as well as Aaron Matthers, for their technical assistance, and Kurt Hartman for his review of my statistical analysis. I am also grateful for the help and support of the faculty and staff in the Molecular and Cellular Biology

Interdisciplinary Program as well as the Plant Biology Department at Ohio University.

Financial support from the Molecular and Cellular Biology Program as well as

Sigma Xi, Houk Grant and Graduate Student Senate was greatly appreciated.

Special thanks goes to my husband, Paul D. Meiss, III and my family, Sam and

Marion Bashore, Melissa and Doug Blacksmith, Elizabeth Bashore, Zach Meiss and Lily

Meiss who have suffered through this almost as much as I have. They have never stopped supporting and loving me and for that I am eternally grateful and full of love.

Table of Contents Page

Abstract...... 3

Acknowledgements...... 5

List of Tables ...... 9

List of Figures...... 10

List of Abbreviations ...... 12

Chapter 1. Introduction ...... 13 1.1 The Symbiotic relationship between Phaseolus vulgaris and Rhizobium sp...... 14 1.2 Phaseolus vulgaris L. phaseoli ...... 15 1.3 Genetics of P. vulgaris...... 19 1.4 Rhizobia sp...... 20 1.5 Rhizosphere as a habitat for Rhizobia ...... 24 1.6 Genetics of Rhizobia...... 26 1.7 Flavonoids...... 28 1.8 Lipo-chitin oligosaccharides...... 30 1.9 Plant response to LCOs ...... 35 1.10 Plant recognition ...... 37 1.11 Nodule formation...... 40 1.12 Nitrogen cycle...... 44 1.13 Nitrogen fixation by Rhizobia ...... 49 1.14 Mutants that restrict nodulation ...... 50 1.15 The mutant P.vulgaris...... 51 1.16 Objective...... 52

Chapter 2. Rhizobial Screening to Examine Nodulation Capacity of Nod- Mutant Phaseolus vulgaris...... 56 Summary...... 57 Introduction...... 58 Materials and Methods...... 62 Growth of P. vulgaris ...... 62 Growth of Rhizobium species ...... 63 Inoculation of P. vulgaris ...... 63 Jensen’s reagent ...... 63 Plant growth...... 65 Harvest and identification of positive inoculation...... 65 Results...... 66 Discussion...... 71

Chapter 3. Phenotypic, Developmental and Morphological Differences between a Nodulation of the Nod- P. vulgaris and the Wild-type...... 74 Summary...... 75 Introduction...... 76 Materials and Methods...... 78 TY media ...... 78 Analysis of nitrogen content: Elementar C:N Analyzer ...... 79 Results...... 79 Discussion...... 85

Chapter 4. Mechanism of Nodulation in Nod- P. vulgaris...... 89 Summary...... 90 Introduction...... 91 Materials and Methods...... 94 GFP transformation...... 94 Results...... 96 Discussion...... 102

Chapter 5. LCOs Derived from Overcoming and Excluded Rhizobial Strains...... 110 Summary...... 111 Introduction...... 112 Materials and Methods...... 115 Isolation of LCOs...... 115 High performance liquid chromatography...... 116 Results...... 117 Discussion...... 125

Chapter 6. Competitiveness of Overcoming strains ...... 130 Summary...... 131 Introduction...... 132 Materials and Methods...... 137 Nodule occupancy in growth chambers and under greenhouse conditions ...... 137 Statistical analyses ...... 138 Results...... 138 Discussion...... 141

Chapter 7. Tn5 Mutagenesis of USDA 2669...... 148 Summary...... 149 Introduction...... 150 Materials and Methods...... 151 Results...... 152 Discussion...... 152

Chapter 8. Conclusions ...... 156

Literature Cited ...... 166

List of Tables

Page

Table 1-1. Common LCO structures and modifications of different bacteria in the Rhizobiaceae...... 34

Table 1-2. Rates of nitrogen fixation from natural systems...... 46

-1 Table 1-3. N2 fixed ha per season median value (kg) by a variety of legumes ...... 55

Table 2-1. Rhizobium strains used in this study...... 61

Table 2-2. Inoculation results of forty strains of Rhizobia on wild-type and mutant P. vulgaris...... 68

Table 2-3. Inoculation success of excluded Rhizobia strains on the mutant P. vulgaris when grown in proximity to wild-type host roots in a single pot...... 69

Table 2-4. Inoculation success of excluded Rhizobia strains on the mutant P. vulgaris with the addition of the plant flavonoid naringenin...... 70

Table 4-1. Mean number of infection threads found on 1 cm lengths of roots isolated from both wild-type and mutant P. vulgaris...... 98

Table 4-2. Mean number of infection threads per 1 cm section on plants inoculated with GFP transformed bacteria...... 104

Table 5-1. Curled root hairs seen in LCO inoculated P. vulgaris 2, 3 and 5 days after addition of crude extract (daa)...... 124

Table 7-1. Number of nodules that formed on the wild-type and mutant P. vulgaris after inoculation with Tn5 mutated USDA 2669...... 154

List of Figures

Page

Figure 1-1. Overview of chemical exchange between soil Rhizobia and the host legume...... 16

Figure 1-2. Phaseolus vulgaris (Common bean) plant...... 17

Figure 1-3. Common bean (P. vulgaris) seeds...... 18

Figure 1-4. Typical backbone structure of plant flavonoids excreted into the rhizosphere...... 29

Figure 1-5. Basic backbone structure of LCOs...... 32

Figure 1-6. Basic LCO structure showing possible R groups for Rhizobium sp. NGR234 and Bradyrhizobium japonicum...... 33

Figure 1-7. The first three steps involved in initiation of nodulation...... 36

Figure 1-8. Last three steps in nodulation initiation...... 38

Figure 1-9. Plant cell with bacteroids formed and inhabiting the cell...... 41

Figure 1-10. Pink nodules formed on P. vulgaris...... 42

Figure 1-11. The Nitrogen cycle...... 45

Fig. 2.1: Arrangement of wild-type and mutant P. vulgaris plants in 6" azalea pots. ....64

Figure 2-2. Chlorotic leaves of P. vulgaris from lack of nodulation...... 67

Figure 3-1. Mean heights of both wild-type and mutant P. vulgaris with and without inoculation...... 80

Figure 3-2. Mean weights of both wild-type and mutant P. vulgaris with and without inoculation...... 81

Figure 3-3. Percent nitrogen isolated from wild-type and mutant P. vulgaris with and without inoculation ...... 83

Figure 3-4. Number of nodules formed on wild-type and mutant P. vulgaris with and without inoculation ...... 84

Figure 4-1. GFP transformed USDA 9032 ...... 97

Figure 4-2. Root hairs on uninoculated P. vulgaris...... 99

Figure 4-3. Wild-type P. vulgaris response to inoculation with USDA 2669...... 100

Figure 4-4. Root hairs of inoculated P.vulgaris...... 101

Figure 4-5. Nod- mutant P. vulgaris inoculated with excluded USDA 2669...... 103

Figure 4-6. Mean number of infection threads found on roots of wild-type and Nod- mutant P. vulgaris when inoculated with USDA 2669 or 9032...... 105

Figure 5-1. Baseline of HPLC with naringenin ...... 118

Figure 5-2. HPLC of USDA 2669 with and without naringenin induction...... 119

Figure 5-3. HPLC of USDA 9017 with and without naringenin induction...... 120

Figure 5-4. HPLC of USDA 9032 with and without naringenin induction...... 121

Figure 5-5. HPLC of USDA 9041 with and without naringenin induction...... 122

Figure 5-6. Root hair response to inoculant with LCOs isolated from HPLC after induction with naringenin ...... 123

Figure 5-7. HPLC profile of extracts from R. giardinii bv. giardinii H152 ...... 126

Figure 6-1. Three nodules under UV light...... 139

Figure 6-2. Mean number of nodules formed on both wild-type and mutant P. vulgaris when inoculated with individual Rhizobium strains ...... 140

Figure 6-3. Nodule occupancy of both wild-type (WT) and Nod- mutant (M) P. vulgaris...... 142

Figure 6-4. Nodule occupancy of both wild-type (WT) and Nod- mutant (M) P. vulgaris...... 143

List of Abbreviations

GFP Green Fluorescent Protein

RFP Red Fluorescent Protein

YFP Yellow Fluorescent Protein

LCO Lipo-chitin oligosaccharides

HPLC High Pressure Liquid Chromatography

SYM Symbiotic plasmid

Dai Days after inoculation

Daa Days after addition

MAG Modified arabinose-gluconate media

TY Tryptone yeast agar

Tn5 Transposable element 5 nif nitrogen fixation genes (Rhizobial) nod nodulation genes (Rhizobial)

WT Wild-type P. vulgaris

M Nod- mutant P. vulgaris

NH3 ammonia nodABC nodulation genes ABC in Rhizobia sp.

Lb Leghemaglobin

Chapter 1 Introduction

The Symbiotic relationship between Phaseolus vulgaris and Rhizobium sp.

Nitrogen is commonly a limiting element in most agricultural systems (Newbould

1989). It is the fourth most common element required by cells and is a major component

of cellular protein and DNA. One mechanism living organisms have evolved to obtain

nitrogen is by forming symbiotic relationships with bacteria that have the ability to fix

atmospheric nitrogen. The most prevalent and important of these symbiotic relationships

is formed between legume plants and bacteria in the family Rhizobiaceae, or more commonly Rhizobia.

The relationship is formed by a process called nodulation, a highly sophisticated

relationship that is dependent on the concerted exchange of chemical signals between the

host plant and the soil bacteria (Fisher and Long 1992, Hirsch 1992). The relationship

culminates in the formation of a tumor-like growth on the roots of the host plant termed a

nodule. The soil bacteria, Rhizobium sp., reside inside the nodule and use photosynthate

from the plant to acquire the energy necessary to drive nitrogen fixation. Nitrogen

fixation has a high demand for energy; however, the benefit of having a ready source of

nitrogen compensates the legume partner for the investment in carbohydrates (Hirsch

1992, Fraysse et al. 2003).

Nodulation is a multi-step process where the initiation is dependent on molecular

signals being exchanged between the bacteria and the plant. The success of the symbiosis is dependent on the sequential activation of both plant and bacterial genes, both of which are highly specific and only certain combinations will result in nodulation.

The two organisms signal and respond until the Rhizobial endosymbiont is an inhabitant

15 of the root nodule (Figure 1-1) (Hungria 1997, Fraysse et al. 2003, Hungria and Stacey

1997, Fisher and Long 1992).

1.2 Phaseolus vulgaris L. phaseoli

Phaseolus vulgaris is a tropical plant in the subfamily Faboideae (429 genera,

12,615 species) of the family Fabaceae (Figure 1-2). The plant family Fabaceae

(formally Legumaceae) is the third largest angiosperm family containing three subfamilies including Faboideae. Faboideae not only contains P. vulgaris but other genera such as Astragalus, Desmodium, Glycine, Pisum, Wisteria, and Trifolium

(Gleason 1981, Walters 1996, Gunn et al. 1992). They are herbs to trees with zygomorphic floral symmetry and white, pink or purplish flowers. P. vulgaris is a highly polymorphic annual herb, 20-60 cm tall or twinning with stems 2-3 m long. The leaves are alternate, green or purple, trifoliolate, stipulate, petiolate, with a marked pulvinus at base. The leaflets are ovate, entire, acuminate, 6-15 cm long and 3-11 cm wide. Legume pods are slender, cylindrical and may be green, yellow, black or purple in color. Each pod has 4-6 seeds that are oblong and can be white, red, tan, purple, gray, black, or variegated (Figure 1-2). There are 14,000 cultivars of P. vulgaris known including kidney, field, pea, navy, black, dwarf, snap, and wax (Mabberley 1997). My research uses beans that are 1 to 1.5 cm long and pinkish buff with brown spots (Figure 1-3)

(Gleason 1981).

P. vulgaris is native to the new world and probably originated in Central Mexico

(Guatemala) and was domesticated in the Andes (Argentina) (Mabberley 1997). It is a widely cultivated crop in tropical, subtropical and temperate zones. The optimum

Figure 1-1. Overview of chemical exchange between soil Rhizobia and the host legume. The plant exudes flavonoids which act as co-inducers to upregulate the Rhizobial nod genes. The nod genes encode enzymes that synthesize a bacterial nod factor signal which initiates plant root hair deformation and ultimately the formation of the root nodule (.http://www.glycoforum.gr.jp/science/word/saccharide/SA-A02E.html).

Figure 1-2. Phaseolus vulgaris (Common bean) plant. P. vulgaris has trifoliate and alternate arrangement leaves.

Figure 1-3. Common bean (P. vulgaris variety) seeds. Note the pinkish buff color with brown spots. Seeds tend to be 1- 1.5 cm in length (photo courtesy of Art Trese).

growing conditions for a yield of 400-5000 kg/ha are: a temperature range of 14-26 °C, a

precipitation of 400-1600 mm/year, a growth cycle 70-330 days (Mabberley 1997).

Plants in the subfamily Faboideae are of particular interest to humans because the

fruit, pulses, are an important world source of vegetable protein. In addition to the common bean, other important pulse crops are Arachis hypogaea (peanuts), Glycine max

(soybean), and Pisum sativum (pea), and additional plants in the family are important forage crops such as Medicago sativa (alfalfa) and Trifolium spp. (clover). The value in

pulse crops resides in their protein content which arises from the subfamily's unique

capability of forming symbioses with soil bacteria from the family Rhizobiaceae. (Conn

1938). This relationship not only provides a source of nitrogen influx for the host plant

resulting in high levels of protein in the fruit and seed, but also provides a way for humans to restore the environment in agricultural systems (Knősel 1984).

1.3 Genetics of P. vulgaris

Legumes are not as well characterized on the molecular level because of the difficulty in creating and isolating insertion mutants (Vance et al. 1988, Phillips and

Teuber 1992, Purdom and Trese 1995). There has been some success in identifying genes involved in nodulation and nitrogen fixation through plant breeding. Among 8

different legume species, scientists have identified 45 loci involved in nitrogen fixation or nodulation (Vance et al. 1988, Phillips and Teuber 1992). The phenotypes exhibited by these legumes range from non-nodulation, ineffective nodulation (low nitrogen fixation capability) to early senescence and supernodulation. Of the total 45 loci found in legumes, 4 are associated with P. vulgaris (Pedalino et al. 1992, Park and Buttery 1989,

Park and Buttery 1994). The four plant genes known to be involved in the nodulation

process are; nie: an induced mutation for ineffective nodulation by Rhizobium sp.(Park

and Buttery 1994), nnd (sym-1): an induced mutation for non-nodulation by Rhizobium

sp., i.e., lacking capacity for symbiosis (Pedalino et al. 1992), nnd-2: an induced mutation

for non-nodulation by Rhizobium sp. (Park and Buttery 1994), and nts (nod): nitrogen

tolerant supernodulation: an induced mutation that permits abundant nodulation in the

presence of high nitrogen (Park and Buttery 1989).

1.4 Rhizobia sp.

Rhizobiaceae is a bacterial family comprised of medium sized rods ranging from

0.5-0.9 μm in width and 1.2-3.0 μm in length. The bacteria do not form endospores and are Gram-negative mobile rods with single flagella or 2-6 peritrichous flagella. The family contains 7 genera; Agrobacterium, Azorhizobium, Bradyrhizobium,

Mesorhizobium, Phyllobacterium, Rhizobium, and Sinorhizobium (Conn 1942, Holmes

1988).

Agrobacterium are classified based on their phytopathogenic characteristics. At one point the genera contained three separate species; Agrobacterium tumefaciens, A.

radiobacter, and A. rhizogenes. Each species was classified on the symptoms produced

in the infected host. A. tumefaciens causes pathogenic symptoms resulting in tumor like

growths on the crown of the plant (Conn 1942, Holmes 1988, Skerman et al. 1980). A.

rhizogenes (Riker et al. 1930) causes roots of the infected host to become hairy and A. radiobacter infection is asymptomatic (Beijerinck 1902). Further research into the phylogeny of the three species using 16S rRNA homology and plasmid isolation resulted

21 in the discovery of the pTi plasmid and pRi plasmid, which when transformed into any of the three species will convey pathogenicity in the form of tumor formation or hairy roots respectively (Holmes 1988). All three species have now been characterized as one A. tumefaciens (Holmes 1988).

Azorhizobium is the only genus that exhibits stem nodulation. The genus was identified in 1988 and only one species has been classified, A. caulinodans (Dreyfus et al.

1988). The genus is clearly defined based on molecular phylogeny including 16S rRNA sequence homology and DNA-DNA reassociation analysis (Dreyfus et al. 1988, Sawada et al. 1993, Willems and Collins 1993).

Bradyrhizobium are slow growing bacteria (Jordan 1984) that quite possibly arose from photosynthetic ancestry (Jarvis et al. 1986, van Berkum et al. 1995). They are classified based on their 16S rRNA and their ability to nodulate Glycine max (soybean).

The genus is heterogeneous with over 17 distinct serogroups but only 2 recognized species, B. japonicum and B. elkanii (Jordan 1984, Kuykendall et al. 1992).

The fourth genus, Mesorhizobium, is distinct based on its fast growth rate and the fact that its symbiotic genes are integrated into the genome rather than carried on a plasmid as seen in the other genera (Jarvis et al. 1997).

Phyllobacterium was first identified in 1962 (Knösel 1962) and classified as it was found on surfaces and hypertrophies of leaves of tropical plants. It has also been isolated from roots of the sugar beet and classified as its own genus in 1990 based on 16S rRNA homology and DNA-DNA reassociation determination (Lambert et al. 1990).

De Lajudie et al. (1994) isolated a fifth genus, Sinorhizobium, based on a

discovery of a species of bacteria that was similar to Bradyrhizobium by nodulating

soybeans, but were fast growing bacteria. Other scientists isolated a genetically distinct

genus (group 3) that was able to nodulate Medicago and Trigonella spp. (Crow et al.

1981). Further molecular analysis determined that these two genera were actually one

and it was named Sinorhizobium. The genus now consists of 4 recognized species; S.

meliloti, S. fredii, S. saheli and S. terangae (De Lajudie et al. 1994).

All bacteria in the genus Rhizobium fix atmospheric nitrogen when in symbiosis

with a plant from the family Fabaceae (Leguminosae). The bacteria are facultative

microsymbionts that live as normal occupants of the soil (Conn 1942). Currently, there

are 9 species of Rhizobium, 5 having the ability to nodulate Phaseolus vulgaris. Species

identification and classification are based on host range, DNA-DNA relatedness, a 260 bp

region of the 16S rRNA genes, and geographical locality (Frank 1889, Jordan 1984,

Martinez-Romero et al. 1991, Segovia et al.. 1993, Lindstrom 1989, Amarger et al.. 1997,

Chen et al. 1997, van Berkum et al. 1997, Eckhardt et al.. 1931, and Schroeter 1886,

Hernandez-Lucas et al. 1995, Herrera-Cervera et al. 1999).

Rhizobium leguminosarum was the first species isolated from nodules on P.

vulgaris (Frank 1889). The species was also found to nodulate plants in the tribe Viciae

and genus Trifolium, therefore, the species was divided into three biovars; biovar viciae

for species that nodulate the tribe Viciae, bv. trifolii for species that nodulate plants in the genus Trifolium and bv. phaseoli for species that nodulate Phaseolus sp. (Frank 1889).

Scientists have since isolated 4 additional species that nodulate P. vulgaris: R. tropici,

(Martinez-Romero et al.. 1991) R. etli, (Segovia et al. 1993) R. gallicum and R. giardinii

(Amarger et al. 1997).

The four other species when isolated were placed in their respective species classification based on 16S rRNA sequence homology, DNA-DNA relatedness, and geographic location. The Western hemisphere (American) types, R. leguminosarum, R. etli and R. tropici were first all classified as R. etli and had the following characteristics:

1) have multiple copies of nifH genes (Quinto et al. 1982; Martinez et al.. 1985, Aquilar et al. 2006), 2) their DNA hybridizes with the psi (polysaccharide inhibition) gene

(Borthakur et al. 1985) and 3) their nodABC genes are not organized in a single operon

(Davis and Johnston 1990; Vazquez et al. 1991). The three strains have 97.8-99% 16S rRNA sequence homology (van Berkum et al. 1996, Willems and Collins 1993). R. etli and R. leguminosarum have even been shown to exhibit recombination events (Eardly et al. 1995, 1996). All American isolates with these characteristics were classified as R. etli while R. leguminosarum bv. phaseoli is considered to have originated from the addition of the symbiotic R. etli plasmid to a R. leguminosarum background (Segovia et al. 1993).

The R. tropici species is phylogenetically different from the four other species and was isolated in Mexico (Martinez-Romero et al. 1991, Willems and Collins 1993, Geniaux et al. 1995). It was previously referred to as R. leguminosarum bv. phaseoli type II, has a single copy of the nifH gene and its DNA does not hybridize with the psi gene (Martinez et al. 1985, Martinez et al. 1988).

Laguerre et al. (1993) isolated two new species in soils in France. R. gallicum sp. nov. and R. giardinii sp. nov., were officially made separate species based on RFLP

analysis, partial 16S rRNA sequence analyses, and phenotypic differentiation (Geniaux et

al. 1993, Laguerre et al.. 1993a, Amarger et al. 1997). These two species nodulate P.

vulgaris but differ from the other three species with only 95.5-96.2% 16S rRNA

sequence homology (Amarger et al. 1997).

1.5 Rhizosphere as a habitat for Rhizobia:

Rhizobial populations tend to be extremely low in the soil accounting for only 0.1

- 8% of the resident bacteria. Little attention has been paid to their growth patterns without legumes and all that has been found is that they are slow growing bacteria and poor competitors with the faster growing bacteria in the same habitat (Bottomley 1992,

Brockwell et al. 1995).

The plant root and surrounding soil, the rhizosphere, is a unique environment

providing Rhizobia with a competitive niche. It is very favorable to rhizobium as well as

many other bacterial species, exhibiting a 100-fold increase in species number relative to

free soil (Rovira 1961, Pena-Cabriales and Alexander 1983a.). The plant host dramatically influences the resident bacterial soil population. Studies have shown that not only do Rhizobial numbers increase in soil with a plant present, but there is an even

greater increase in numbers of specific strains when they are in the rhizosphere of their

specific host (Woomer et al. 1988). Bottomley et al. (1992) suggest three possible

explanations for this phenomenon; 1) the host plant has already formed a symbiosis with specific Rhizobial strains and is constantly forming and terminating nodulation releasing increased numbers of rhizobium into the soil, 2) the root exudates serve as metabolic nutrient source for its specific Rhizobial symbiont, similar to the specificity seen in

Agrobacterium opine production or 3) the ability of the host legume to select specific

populations and/or species of bacteria out of a mixture and positively influence their

growth and occupation of the soil rhizosphere.

Soil bacterial populations will inhabit the growing root and developing

rhizosphere as soon as germination begins with a seed assimilating water and the young

roots beginning to grow. At this point Rhizobia will go from being primarily dormant in

soil to exhibiting rapid proliferation and development (Ramirez and Alexander 1980,

Lennox and Alexander 1981).

The Rhizobial bacteria migrate to the root hairs and enter the symbiosis forming

and living inside a tumor-like growth called a nodule. While inside, the bacteria divide until millions of individuals exist inside each nodule. Once the nodule senesces or the plant dies, the Rhizobia are released back into the soil where they do not transition well and most end up dying.

There are only three situations where increased numbers of Rhizobia are found; 1)

initial phases of plant growth as the roots are colonized, 2) as nodules decay and release

the bacteria contained within, and 3) following the addition of commercial inoculants to

seeds sown in the soil. At the termination of all three of these there is a rapid die-off of

bacteria (Bottomley et al. 1992)

The reason for rapid declines in Rhizobial populations is the protozoan

community found in soil. Protozoan organisms are known predators of Rhizobia sp. and

will hunt the bacteria until a steady state is reached. Only once the steady state is reached

can the bacteria multiply and survive in the soil by slow grazing (Bottomley et al. 1992).

Overall, Rhizobia are not particularly good competitors, and they rarely, if ever,

grow in circumstances typical of normal farming practices. The bacteria proliferate

chiefly or solely in the presence of germinating seeds and developing roots, and

thereafter, populations are subjected to a variety of biological and non-biological stresses.

The organisms are quite susceptible to extremes of acidity and temperature, and their

numbers are greatly reduced when soils are dried (Bottomley et al. 1992).

The nodulation and concurrent symbiotic relationship is beneficial to not only the

plant host, but to humans and animals as well. The ideal strain of rhizobium is one that is

able to survive well in the soil, to grow at the opportune moment, to colonize the roots

readily and in the presence of organisms that are antagonistic to it, and then to initiate the

nitrogen-fixing symbiosis. Similarly, the ideal strain for seed inoculation has these

properties but also possesses the capacity to survive in large numbers on seeds that it

becomes established on, by inoculation, once the seed is planted.

1.6 Genetics of Rhizobia

Successful nodulation of a legume host requires concerted gene expression in

both the host and the symbiont. Genes isolated in Rhizobia sp. that are involved in

nodulation are designated nod genes and abbreviated by nod, noe or nol (Gordon et al.

1995).

Early research reported that when a strain of R. leguminosarum had a large

extrachromosomal plasmid removed the resulting derivative could no longer nodulate

peas (Casse et al. 1979). Similar observations were made by Zurkowski and Lorkiewicz

(1978) and Lie and Winarno (1979), who were able to restore nodulating ability to Nod-

strains of R. leguminsarum by the introduction of the proper plasmid. Johnston and

Beringer (1977) localized the nod genes on this large plasmid that was designated the

Symbiotic (Sym) plasmid. The SYM plasmid controls infectivity and host range and

when inserted into other non nodule forming genera, such as Agrobacterium or

Phyllobacterium, will induce root hair curling and nodule initiation, but not true nodules as signified by the lack of any bacteria in curled root hairs (Hooykasas et al. 1981,

Martinez et al. 1987, Rodriquez-Quinones et al. 1989, van Veen et al. 1988). Geniaux et al. (1995) isolated a megaplasmid (Sym plasmid) in R. tropici and stated that it was the defining characteristic of that species.

The nodABC genes, which encode a 3 to 5 β 1-4 lipo-chitin oligosaccharide

(LCO) signal molecule, have been mapped to the Sym plasmid, as well as accessory

genes that encode modifications to the basic LCO backbone structure, also termed nod

genes (van Kammen 1984). The nodABC genes isolated are located on a single operon

termed the nod box (Rostas et al. 1986). Studies done with a lacZ fusion protein resulted

in the identification of a regulatory activator nodD protein that works as a coinducer with

plant flavonoids to upregulate the transcription of the nod box (Miller 1992, Spaink et al.

1986, Spaink et al. 1989). Other nod genes have been isolated and found to be associated

with the nod box, but not part of it (Baev et al. 1991, van Kammen 1984), although they

are still clustered in a small region.

Expression of the nodD gene is low in wild-type Rhizobia and becomes increasingly upregulated in the presence of certain plant flavonoids (Schlaman et al..

1989, Davis and Johnston 1990, Freiberg et al. 1997, Wang and Stacey 1991). The

combination of plant flavonoids and nodD gene product induce transcription of the nod

box, which results in production of LCOs (Broughton et al. 1991, Gottfert et al. 1992,

Van Rhijn et al. 1993).

Nitrogen fixation is essential to the legume/rhizobium symbiosis, but it is not

known what part of the nitrogen-fixing system of Rhizobia is encoded for by plasmids.

Research by Ruvkun et al. (1980) and Nuti et al. (1979) has shown that some of the

structural nitrogenase genes are plasmid borne.

1.7 Flavonoids

Virtually all plants excrete a range of sugars, organic acids, and amino acids into

the rhizosphere. Each of these chemicals has a specific reason for being exuded into the

rhizosphere, whether as a waste or a chemical signal. Specifically, legumes are found to

excrete thousands of chemicals many of which have been characterized as flavonoids

(Figure 1-4). Flavonoids all have a basic 3-ring backbone and vary with additional

functional groups (Stafford 1990, Hartwig et al. 1990, Maxwell and Phillips 1990).

Flavonoids are plant exudates that act as co-inducers of nod box transcription in

conjunction with nodD gene product (Innes et al.1995, Stafford 1990, Hartwig et al.

1990, Maxwell and Phillips 1990, Hungria et al. 1991, Phillips 1992, Phillips et al. 1994).

Many flavonoids have been isolated from P. vulgaris including

Figure 1-4. Typical backbone structure of plant flavonoids excreted into the rhizosphere. All flavonoids have a 3 ring backbone with additional functional groups (R) (Stafford 1990).

delphinidin, malvidin, eriodictyol and naringenin (Hungria et al. 1991, Phillips 1992).

Research has shown that low nitrogen in soil stimulates flavonoid production and

therefore nod box transcription (Coronado et al. 1995, Bongue-Bartelsman and Phillips

1995).

1.8 Lipo-chitin oligosaccharides

The successful formation of the legume-rhizobium symbiosis is dependent on the

highly specific exchange of chemical signals between the host plant and soil Rhizobia

(Fisher and Long 1992, Hungria and Stacey 1997, Long 2001, Mathesius 2003). The

plant exudes flavonoids which act as co-transcriptional regulators of Rhizobial nod genes by binding to the Rhizobial nodD gene product. These co-transcriptional proteins work together to upregulate transcription of the nodABC genes (Innes et al. 1985, Phillips

1992, John et al. 1993, Rossen et al. 1985, Schlaman et al. 1992). The product of transcription, lipo-chitin oligosaccharides (LCOs), or nod factors, are encoded by the nod box genes, nodABC, located on the large Sym plasmid in Rhizobia sp. are the chemical signals excreted by bacteria to signal the host plant to begin forming the symbiosis. When

LCOs are isolated and applied to their specific host, the plant will respond by producing root-hair deformation and nodule initiation/cortical cell division (Denarie et al. 1992,

Denarie et al. 1996, Long 1996), both are plant responses to the initiation of nodulation

(Fraysse et al. 2003).

Characterization of LCOs from 7 species of Rhizobia (Rhizobia meliloti, R.

leguminosarum bv. viciae, R. NGR234, BradyRhizobia japonicum, AzoRhizobia

31 caulinodans, R. fredii, and R. tropicii) shows that all LCOs have the same basic backbone structure. LCOs consist of β-1,4 linked N-acetylglucosamine (chitin) residues with an amide linked fatty acid moiety on the non-reducing terminal residue (Figures 1-5,

1-6) (Carlson et al. 1994, Inon de Iannino et al. 1995, Thomas-Oates et al. 1996). The common nodulation genes, nodA, nodB and nodC encode the basic backbone structure found in LCOs (Cohn et al. 1998, Hungria et al. 1997, Peters and Verma 1990, Phillips

1992, Schlaman et al. 1992, Hungria 1994). All three genes are necessary for LCO production.

All LCOs have various chemical modifications that are believed to play the major role in determining host specificity (Table 1-1, Figure 1-5) (Vargas et al. 1990, Voets et al. 1995, Denarie et al. 1996, Long 1996, Cardenas et al. 1995, Geremia et al. 1994,

Perret et al. 2000). Modifications to the LCOs such as sulfation of the reduced terminal residue, degree of unsaturation of the fatty acid, O-acetylation of the non-reducing terminal residue, and variations in length of the oligosaccharide backbone all play a part in determining host specificity (Carlson et al. 1994, Downie 1994, Lerouge 1994,

Martinez-Romero 1994, Schultze et al. 1994, Spaink 1995, Denarie et al.1996).

Figure 1-5. Basic backbone structure of LCOs. The figure shows the β-1,4 linked glucosamine residues where n = 3, 4 or 5 (Carlson et al. 1994, Inon de Iannino et al. 1995, Thomas-Oates et al. 1996).

Figure 1-6. Basic LCO structure showing possible R groups for Rhizobium sp. NGR234 and Bradyrhizobium japonicum (Carlson et al. 1994, Inon de Iannino et al. 1995).

Table 1-1: Common LCO structures and modifications of different bacteria in the Rhizobiaceae .

a Rhizobium Host plant R1 R5 n

Bradyrhizobium Soybean japonicum USDA110 2-O-methyl fucose C18:1/C16:0 3

USDA135 2-O-methyl fucose C18:1/C16:0 3

R. meliloti Alfalfa Sulfate C16:1/C16:2/C16:3/ 2,3 (w-1)OH C18 to C26 R. tropici Phaseolus, Sulfate, hydrogen C18:1 3 Leucaena, Medicago, Macroptilium R. fredii Soybean 2-O-methyl- C18:1 1-3 fucose/fucose R. NGR 234 Very broad 2-O-methyl- C18:1/C18:0 2,3 host range fucose/3-S- methyl-fucose/3 or 4 acetyl-methyl- fucose R. leguminosarum Pea, vetch Hydrogen 18:1/C18:4 2,3 bv. viciae R. leguminosarum Clover Acetyl C18:1/C18:4 2,3 bv. trifolii R. etli Phaseolus -O-acetyl-L-fucose A. caulinodans Sesbania Arainose/hydrogen C18:1/C18:0 2,3 fucose

a The host plants are the plants on which the various Nod signals were found to be biologically active. R1 represents the position of various modifications made on the reducing end of the molecule (see Fig. 1-5). R5 represents the position of the fatty acyl chain at the non-reducing end (see Fig. 1-5). n indicates the length of the oligo-chitin chain. In the cases where n = 2, 3, this indicates that both tetrameric and pentameric Nod signals are produced by the specific strain of Rhizobium, Note that modifications are also made to several Nod signals produced by individual strains of Rhizobium that are not covered in the table.

1.9 Plant response to LCOs

The plant flavonoids that signal the nod genes to be transcribed cause chemotaxis

of the Rhizobia to the rhizosphere and root hairs as well as division and cell growth

(Phillips 1992). When legume roots are exposed to nod factors (LCOs) they exhibit

various responses in the root epidermal cells, cortex and pericycle (Phillips 1992,

Schmidt et al. 1988). Most Rhizobial species do not excrete a single LCO molecule, but

rather an array of LCOs that may interact cooperatively to induce the plant response

(Minami et al. 1996).

The first plant morphological response observed when the Rhizobia colonize the

root is root hair curling. The root hairs curl into a Shepard’s crook with the Rhizobia concentrated in the curl of the hook (Figure 1-7B) (Turgeon and Bauer 1985, Hirsch

1992, Goormachtig et al. 2004). Isolated nod factors have been shown to induce root hair

deformation and curling in concentrations as low as 10-12 M (Lerouge et al. 1990, Spaink

et al. 1991, Price et al. 1992, Schultze et al. 1992).

Once the Rhizobia are confined in the crook of the curled root hair, they begin to

hydrolyze the root hair cell wall and form an invagination in the plasma membrane. A

new tubular structure forms as the invagination grows, called the infection thread. The

bacteria will migrate down the infection thread and enter the cortical cells of the root

(Figure 1-7C) (Turgeon and Bauer 1985, van Spronsen et al. 1994, Hirsch 1992, Kijne et

al. 1992, Goormachtig et al. 2004).

Figure 1-7. The first three steps involved in initiation of nodulation. A) The Rhizobia congregate at the tip of the root hair based on a flavonoid signal from the host. B) The root hair begins to grow and curl. C) An infection thread forms and the bacteria enter and migrate to the root cortical cells (Figure courtesy of Judith Kipe-Nolt).

As the infection thread grows, the cortical cells begin to differentiate and divide growing into the nodule primordia. Nodules that are initiated by an infection thread will form in the inner cortex of the root. The infection thread will transverse the outer cortex

cells and the bacteria will bud through the plasma membrane and enter the nodule

primordia; the young nodule with growing and dividing cortical cells (Figure 1-8) (van

Brussel et al. 1992, Hirsch 1992, Goormachtig et al. 2004).

1.10 Plant recognition

Legumes are able to distinguish among strains or species of rhizobium in their

root environment. Many researchers believe that it is the specific LCO repertoire that is

excreted by the Rhizobia that determine the host range. Though isolated LCOs have the

ability to induce morphological changes in the plant host, such as root hair curling, they

are not able to elicit formation of a complete nodule (Long 1996). It appears that other

plant recognition mechanisms are in place that continue the development of the nodule

(van Spronsen et al. 1994, Vijn et al. 1995).

Much of the research in host plant recognition systems has been done on alfalfa,

pea and clover, but not beans. Dazzo and Hubbell (1975) first derived the lectin-

recognition hypothesis that states there are sites of nodule initiation on the legume root

that contain lectins which cross-react with, and bind, carbohydrates on the surface of the

appropriate Rhizobium strain. Evidence to support this was seen in clover and soybean

(nodulated by R. trifolii and R. japonicum respectfully) nodulation where the ability of

rhizobium strains to bind to the roots of particular legumes is correlated with the ability

of the strains to nodulate these legumes. (Bohlool and Schmidt 1974).

Figure 1-8. Last three steps in nodulation initiation. A) The bacteria enter the root cortical cells. B) The bacteria begin to bud through the root plasma membrane C) The bacteroids are formed and begin to colonize root cells. They signal the plant to grow and divide (photo courtesy of Judith Kipe-Nolt).

Clover roots bind capsulated cells of R. trifolii (Dazzo and Brill, 1979) with

FITC-labeled capsular polysaccharide (Dazzo and Brill, 1977) and antibody to clover

seed lectin (Dazzo et al. 1978). In each case binding is most intense in the region of the

root hair zone. The binding is inhibited by the sugar which is the antigenic determinant

of the bacterial polysaccharide or the host lectin (Dazzo and Brill 1977). Intergeneric

hybrids of Azotobacter and Rhizobium, which express R. trifolii genes for specific

binding with clover lectins, also have the ability to bind to clover root hairs (Dazzo and

Brill 1979, Bishop et al. 1977).

These results support the fact that there are LCO receptors in clover roots and

these receptors are lectins. But there is also research that contradicts these findings;

cultivars of P. vulgaris and Glycine max have been identified which appear to be

defective in lectin production but still nodulate (Chen and Phillips 1976, Law and

Strejdom 1977, and Dazzo and Hubbell 1975b).

The lectin-recognition hypothesis currently offers no explanation for the

competitive differences that exist between Rhizobium strains having essentially similar

external polysaccharides. There have been lectin receptors identified that seem to be

essential in clovers, but none have been found in beans. In fact, lectins seem non-

essential in nodulation of beans, suggesting that if there are LCO receptors in bean roots,

they are not lectins. However, if the lectin hypothesis is valid, it could explain how some

environmental factors affect nodule initiation.

1.11 Nodule Formation

As stated earlier, LCOs seem to play a major role in host specification and initial

plant response (Long 1996, Hungria and Long 1997, Long 2001, Mathesius 2003). LCOs

signal the plant root hairs to begin to curl allowing for Rhizobial colonization in the

Shepard’s crook. Once inside, the bacteria begin to weaken the plant cell wall and form a

tubular indentation (Verma et al. 1992, van Spronsen et al. 1994, Kijne 1992, Cohn et al.

1998). The bacteria migrate down the tube until it reaches the nodule primordium. The

nodule primordium is an area in the inner cortex of dividing cells (Verma et al. 1992, Rae

et al. 1992, Kijne 1992, Cohn et al. 1998).

Once the bacteria arrive at the nodule primordium, the inner most end of the

infection thread does not have a cell wall, just a plasma membrane. The bacteria will

push through the plant plasma membrane, or bud through, creating a bacterium with two

membranes, the outer membrane consistent with the host plasma membrane, termed a

peribacteroid membrane (Dart 1975). The endocytosis of the bacteria creates a new cell

organelle termed a bacteroid, with a typical gram-negative cell wall within the double

membrane (Figure 1-9). The bacteroids appear to signal the plant and bacteria to undergo

rapid cell division. The rapid root growth creates an initial tumor growth, or nodule primordial (Verma et a. 1992, Rae et al. 1992, Bassett et al. 1977, Perotto et al. 1994,

Cohn et al. 1996).

Once the bacteroids are formed they continually signal the plant to locally grow

and divide, creating the final completed nodule. Legumes create two distinct nodule

types; 1) Determinate nodules which do not form a persistent meristem, and therefore

Figure 1-9. Plant cell with bacteroids formed and inhabiting the cell (photo courtesy of Dr. Art Trese).

remain spherical-shaped (Figure 1-10) and 2) Indeterminate nodules which contain a

persistent meristem and result in cylindrical-shaped nodules and are multi-lobed. Both

form approximately 2 weeks after exposure to Rhizobia and determine the nodule shape

and size. Both nodule types are formed by the same invasion route, the formation of an

infection thread. Pea (Pisum sativum) plants form indeterminant nodules, while soybeans

(Glycine max) and beans (P. vulgaris) form determinant nodules (Turgeon and Bauer

1985, Pawloski et al. 1996, Cohn et al. 1998, Hirsch 1992).

Legume species vary with the number of root hairs that become infected, such as

clover that can range from 20-100 nodules per plant, while P. vulgaris have anywhere from 20-150 nodules per plant. Many host cultivars that control differences in nodule number have now been found in a number of species, including Trifolium subterraneum

(Nutman 1967) and Phaseolus vulgaris (Graham 1973).

When the nodule is fully formed, the bacteroids grow to approximately twice their

original size and begin fixing nitrogen. The enzyme the Rhizobia use is called nitrogenase and is actually a multi-enzyme complex termed a Nitrogenase complex. It consists of two protein components, which in turn consist of multiple subunits. One subunit, dinitrogenase reductase, or iron (Fe) protein, functions to reduce the second subunit dinitrogenase or molybdenum-iron (Mo-Fe) protein. The Mo-Fe protein reduces nitrogen gas to ammonia. The process requires 16 ATP to “fix” one molecule of nitrogen

(N2) (Verma et al. 1992).

The nitrogenase complex is susceptible to oxygen (O2) in that it will disassociate

and not function in the presence of oxygen. To compensate for this the plant produces a

Figure 1-10. Pink nodules formed on P. vulgaris. Note the pink color and the spherical shape (photo courtesy of Dr. Art Trese).

compound called leghemaglobin. This hemoglobin-like protein carries O2 into the nodule

but keeps it away from the nitrogenase in the bacteroids. It is reddish-pink in color and is

the determining characteristic of a successful, fully active root nodule (Figure 1-10)

(Verma et al. 1992). The biological nitrogen-fixation system is roughly twice as efficient

as the Haber-Bosch chemical process (Kim and Rees 1994). Ammonia produced by the

activity of nitrogenase is usually synthesized into other organic compounds prior to

transport to the plant host.

1.12 Nitrogen Cycle

The nitrogen cycle is one of the most important elemental cycles on earth.

Nitrogen makes up approximately 78% of the earth's atmosphere and is essential to all

life as it is a component of protein, DNA and vitamins. The nitrogen cycle is considered

a mineral cycle because although our atmosphere is made of gaseous nitrogen, or dinitrogen (N2), it can only be assimilated into organisms in mineral form. Dinitrogen is

inert and unavailable to living organisms unless it is "fixed" or converted into a

biologically assessable form (Atlas 1993).

The nitrogen cycle involves many organisms and five necessary steps that are;

1) Nitrogen Fixation, 2) Ammonium Assimilation (Immobilization) 3) Ammonification

(mineralization), 4) Nitrification and 5) Nitrate Reduction (Figure 1-11).

Nitrogen fixation involves converting inert dinitrogen into a reduced form of nitrogen,

ammonia (NH3) (Equation 1). Microbes, and small amounts from lightening, are

responsible for fixing atmospheric nitrogen with 65% of the annual N2 fixed coming from

terrestrial environments, including natural and managed agricultural systems (Table 1-2).

Figure 1-11: The nitrogen cycle. The figure depicts the nitrogen cycle including assimilation, decompositions, nitrogen fixation, ammonification, nitrification, and denitrification (http://www.physicalgeography.net/fundamentals/9s.html).

Table 1-2. Rates of nitrogen fixation from natural systems.

N2-fixing system Nitrogen fixation (kg N/ hectare/year)

Rhizobium-legume 200-300

Anabaena-Azolla 100-120

Cyanobacteria-moss 30-40

Rhizosphere associations 2-25

Free-living 1-2

Humans have developed an industrial process, the Haber-Bosch process, to fix nitrogen

for fertilizers that is now responsible for 15% of total nitrogen fixed (Myrold 1998,

Tiedje 1988, Santos et al. 2000).

- Equation 1: N2 + 8H+ + 8e + 16 ATP = 2NH3 + H2 + 16ADP + 16 Pi

A variety of organisms can fix atmospheric nitrogen in nature, including and most importantly Rhizobia sp. Bacteria such as Azotobacter, Beijerinckia, Azospirillum, and

Clostridium are all free-living and can fix atmospheric nitrogen, but in much smaller

amounts than the rhizobium-legume symbiosis (Table 1-2) (Myrold 1998, Tiedje 1988).

The rhizobium-legume symbiosis is responsible for the majority of nitrogen fixed

and made available to living organisms. The bacteria, in the family Rhizobiaceae

collectively Rhizobia, live inside the plant host and actively fix atmospheric nitrogen.

Any excess ammonium is directly available for plant uptake. This relationship is

beneficial because it takes advantage of the energy supply from the plant. A constant

energy supply helps the bacteria with the high-energy demand needed to carry out the

nitrogen fixing reaction. This can also be seen with cyanobacteria that are photosynthetic

and therefore have access to more energy. These aquatic organisms fix nitrogen at one to

two orders of magnitude higher than free-living microbes (Myrold 1998, Tiedje 1988,

Santos et al. 2000, West et al. 2001).

The end product of N fixation is ammonia (NH3), which is quickly ionized to

+ ammonium ions (NH4 ). This form of nitrogen can be easily assimilated into living

organisms. The process of nitrogen assimilation is known as ammonium assimilation or

immobilization. Organisms prefer to take up nitrogen in this form and quickly convert it

into amino acids and proteins. The reverse of ammonium assimilation is ammonification

or ammonium mineralization where ammonia is released into the soil or environment

from dead or decaying biological matter (Myrold 1998, Tiedje 1988, Santos et al. 2000).

In the environment it will be broken down into nitrate. This is a two step process of

ammonium becoming oxidized into nitrite by one type of organism and then nitrite is transferred into nitrate by other organisms. The first step is carried out by predominantly aerobic chemoautotrophic microbes such as Nitrosomonas sp. (Equation 2). The second step in the reaction is the oxidation of nitrite to nitrate (Equation 3) that is carried out by other aerobic chemoautotrophs such as Nitrobacter sp (Myrold 1998, Tiedje 1988. Santos et al. 2000).

+ + - + Equation 2: NH4 + O2 + 2H → NH2OH + H2O → NO2 + 5H

- - Equation 3: NO2 + 0.5O2 → NO3

These two organisms, nitrifiers, are generally found together in the environment.

Because of this, nitrite does not often accumulate in the soil.

Once nitrate is formed, it also has a variety of fates. It can be leached into

groundwater and surface water or taken up by organisms and incorporated into biomass.

Once nitrate is absorbed it will be immediately converted back to ammonium. This is

done by a process called assimilatory nitrate reduction or nitrate immobilization. A third option for nitrate is that it is used by certain microorganisms as a terminal electron acceptor. This can be done in one of two ways; certain microbes carry out 1)

Dissimilatory nitrate reduction to ammonium, where ammonium is the final product, or

2) Denitrification, where a mixture of N2 gas and N2O is released (Myrold 1998, Tiedje

1988, Santos et al. 2000).

The cycle is then complete. The human practice of industrially fixing atmospheric nitrogen has potentially detrimental effects on the environment. Increase use of nitrogen fertilizer causes an increase in nitrate and nitrite run-off from leaching into the groundwater, which causes algal blooms and potential dead zones. Furthermore, this practice is not sustainable unless some renewable source of abundant energy becomes available (Myrold 1998, Tiedje 1988, Santos et al. 2000).

1.13 Nitrogen fixation by Rhizobia

A wide variety of prokaryotes, including Rhizobia, have the ability to carry out

nitrogen fixation, the reduction of nitrogen gas into ammonia (Young 1992). The genes

involved in nitrogen fixation are highly conserved and all organisms catalyze the

reduction of N2 to NH3 by the same enzyme, nitrogenase (Bishop and Premakumar

1992). The reduction of N2 is highly endothermic requiring 16 ATP for every molecule

of N2 (Hill 1992).

The activity of nitrogenase is inhibited by oxygen, but oxygen is a primary product of cellular respiration and the production of ATP. This causes an oxygen paradox within the root nodule. Research has shown that this problem has been solved by the symbiotic relationship and its structural component. The nodule, an outgrowth of plant material, when fully developed has a poorly permeable layer just beneath the nodule epidermis which prevents excess O2 from entering. Oxygen concentration inside an actively fixing nodule is as low as 5-30 μM as compared to 250μM under aerobic cellular

conditions (Appleby 1984). Leghaemoglobin (Lb), a protein, is responsible for binding

oxygen and is detected in root nodules just before nitrogenase activity appears. It buffers

free oxygen and facilitates O2 diffusion to the actively respiring bacteroids. The protein

has a red color which can be seen in nodules, the red color can be used to distinguish

nodules with bacteroids that are actively fixing nitrogen (Appleby 1983).

1.14 Mutants that restrict nodulation

Mutants that affect nodulation have been found in a variety of legumes including

alfalfa (Medicago truncatula) soybeans(Glycine max) and peanuts (Arachis hypogea)

(Beneban et al. 1995 Williams and Lynch 1954, Nambiar et al. 1982).

Soybeans (G. max) are one of the more important legume crops in the world and

therefore is one of the most studied. Trese (1994) characterized a McCall soybean that

prevented nodulation with a strain of R. fredii. The gene controlling this phenotype is a

single dominant gene. Two recessive genes have also been characterized from soybeans

that affect nodulation. One, rj1, is a spontaneous mutant that restricts nodulation by a

majority of Rhizobia, while permitting a select few to nodulate the host (Williams and

Lynch 1954, Pueppke and Payne 1987). A second is an EMS mutant that restricts

nodulation, rj6 (Mathews et al. 1987).

Mutants such as red clover (Trifolium pretense) have been isolated that

completely restrict nodulation. The recessive allele was mutated and found to condition a

complete lack of nodulation. The gene operates in conjunction with a cytoplasmic factor

(Nutman 1949). In this trait, root hairs are formed and do curl, but rhizobium strains

appear incapable of penetrating them. (Nutman 1949).

In the peanut (A. hypogea), Nambiar et al. (1982) identified two recessive

mutations controlling the lack of nodulation, but they found that in some crosses with plants having one or other of the genes, a few very large nodules formed. The mechanism has not been explained.

Purdom and Trese characterized a mutant cowpea (Vigna unguiculata) that carries

a recessive allele, cpi, that expresses the formation of non-N2 fixing nodules when

inoculated with Rhizobia (1995).

Common bean (P. vulgaris) mutants have been shown to phenotypically express supernodulation or completely restrict nodulation (Pedalino et al. 1992, Park and Buttery

1989, Park and Buttery 1994). But the common bean is the only legume with determinate nodules in which a recessive mutation causes delayed nodule development independent of the strain used as inoculum (Pedalino and Kipe-Nolt 1993).

1.15 The mutant P. vulgaris

A spontaneous P. vulgaris mutant was isolated in Dr. Art Trese’s lab that exhibits strain

specific restriction of nodulation by Rhizobial species. The mutant was isolated from a

commercial seed supply TopCrop ™ wild-type U.S. bush beans. Backcrossing

determined a new allele that is recessive and has the ability to restrict nodulation in a

strain specific manner. These findings are novel in P. vulgaris and legume genetics.

Any strains with the ability to nodulate the mutant P. vulgaris are termed

“overcoming” and were found to be R. leguminosarum USDA 9017, R. etli USDA 9032,

and R. leguminosarum USDA 9041. Any strains that cannot nodulate the mutant bean

are termed “excluded”. The strain is R. leguminosarum USDA 2669 will be used in most

of the experiments to represent the excluded strains.

1.16 Objective

The legume rhizobium symbiosis is not only important to the organisms involved,

but also to the animals, including humans, which depend on legumes for their caloric and

protein nutrition source. Currently, over 80% of the tropical and subtropical human

populations depend on plants to provide their necessary dietary protein, with legumes

being the primary source (Golley et al. 1992).

The demand for food and protein is on the rise and is only expected to grow. The

earth’s population is expected to reach 9 billion people, almost double the current

population, by 2050 (Bockman et al. 1990, Vance 2001). Scientists and agriculturalists

can only calculate an estimated increase in the demand for food over the next 40 years.

Currently, nitrogen is a limiting nutrient in agricultural systems (Newbould 1989).

Cropping systems have required an increase of nitrogenous fertilizer application for the past 50 years currently needing 90 Tg nitrogen per year (Newbould 1989, Waggoner

1994, Vance 2001). Besides the economic and energy costs of industrial nitrogen fixation, carried out by the Haber-Bosch process, the use of nitrogen fertilizer brings about other problems. With an increased use of nitrogen fertilizer more nitrogen oxides

(greenhouse gases) are released into the atmosphere, causing an imbalance in the global

- nitrogen cycle, scientists also see an increase in toxic NO3 leaching into groundwater and

a depletion of nonrenewable resources (Kinzig and Socolow 1994). Finding a more

53 sustainable system to obtain nitrogen would be beneficial to the environment and human health (Bohlool et al. 1992, Vance and Graham 1995).

Naturally occurring nitrogen fixing systems reduce the need for N fertilizers.

These natural systems produce nitrogen that is bound in organic matter, therefore it is not as volatile and susceptible to leaching which results in a reduction in soil chemical transformations and toxic nitrogen oxides in the atmosphere and groundwater.

Understanding and/or exploiting this natural biological relationship will help humans reduce their detrimental impact on the environment. There are a wide variety of free living bacteria and endosymbiotic relationships responsible for the total biological nitrogen fixed annually, but the legume-Rhizobia symbiosis accounts for nearly 80% of all biologically fixed nitrogen (Vance 1996). Legumes therefore contribute 25-35% of worldwide protein which is equivalent to 90 Tg N/ year. It would cost $30 billion per year to obtain the same amount using the Haber-Bosch process (Kinzig and Socolow

1994).

The current demand for nitrogen and costs of industrially producing available nitrogen show that improving the natural symbiosis should be a common goal. Phaseolus vulgaris (common bean) is second to only to Glycine max (soybean) in the world as a staple crop, providing protein for nearly 300 million people. However, compared to other legume crops, P. vulgaris obtains the least amount of nitrogen per ha per season (Table 1-

3). Thus improving the P.vulgaris-Rhizobial symbiosis should be of great interest to scientists and agriculturalists.

The mutant P. vulgaris isolated in Dr. Trese's lab selectively restricts nodulation.

This host plant is somehow differentiating between strains of Rhizobia, all of which are able to nodulate the wild-type P. vulgaris. This mutant can not only serve as a model to learn more about how host legumes recognize symbiotic bacteria, but could also potentially serve as a basis for symbiotic improvement and therefore agricultural improvement. Currently, Rhizobia that have the ability to fix nitrogen at a much higher rate cannot compete for nodule occupancy against native Rhizobia with higher competition efficiency (Mavingui et al. 1997, Savka and Farrand 1997, Yates et al. 2003,

Denisona and Kiersa 2004). This dissertation attempts to examine the mutant bean more closely to determine how this host is selecting its endosymbiont, and also explore the possibility of utilizing the mutant host's selectivity as a way to improve the efficiency of the natural symbiotic relationship and its possible use in agriculture.

-1 Table 1-3. N2 fixed ha per season median value (kg) by a variety of legumes. Calculated or adapted from Heichel (1987), Nutman (1976) and Peoples and Craswell (1992).

-1 Legume species N2 fixed ha per season median value (kg) Pisum sativum 72

Glycine max 120

Arachis hypogaea 114

Phaseolus vulgaris 65

Vigna angularis 80

Vicia faba 151

Lupinus angustifolius 170

Lens culinaris 100

Chapter 2 Rhizobial Screening to Examine Nodulation Capacity of Nod- Mutant Phaseolus vulgaris

Summary:

During routine inoculations, with a variety of Rhizobial strains, of wild-type P. vulgaris; a P. vulgaris phenotype arose without any nodules on the roots. In further investigations it was found that this specific P. vulgaris biovar exhibited a strain specific restriction of nodulation among the 5 species of Rhizobia known to form a symbiosis with P. vulgaris. This experiment was set up to examine what strains specifically were restricted and to determine if there was any correlation to species designation. This was carried out in three experiments; first, a general inoculation; second, an inoculation of wild-type and mutant P. vulgaris with their roots in close proximity and third, inoculation with the addition of naringenin, an inducer of nodulation. Forty strains of Rhizobium were inoculated onto the mutant bean plant, resulting in nodule formation on the mutant plant by three strains, USDA 9017, USDA 9032 and USDA 9041 (overcoming strains) while the remaining 37 strains did not form functional nodules (excluded strains).

Addition of wild-type exudates as well as the plant flavonoid naringenin, in conjunction

with excluded strains, did not induce nodulation on the mutant host by any of these

strains.

Introduction

Formation of nodules on beans involves the highly regulated exchange of signals between the plant host and the soil bacteria. During nodule formation, the bacteria enter the root hair, migrate to the root cortical cells, and then bud through the plasma membrane entering the cells and becoming bacteroids, membrane bound bacteria that live inside the plant root cell. The bacteroids use plant energy and nutrients to survive while fixing atmospheric nitrogen for their own use. The process of nitrogen fixation results in production of excess ammonia that becomes available for the plant. This process

(nodulation) results in the formation of a plant nodule where the bacteria reside and fix atmospheric nitrogen. The bacteria apparently enter the root without inducing plant defense responses, suggesting the bacteria employ mechanisms that either are not recognized by the plant or are recognized by the plant as a benefit (Iannetta et al. 1997).

Symbiosis is initiated and regulated by chemical signals that are exchanged

between the plant host and the soil bacteria (Hirsch 1999, Hungria and Stacey 1997).

The plant host secretes many chemicals into the soil rhizosphere including flavonoids.

Plant flavonoids are assimilated into Rhizobia where they bind with nodD gene products,

nodD proteins. NodD is constitutively expressed in Rhizobia sp. and is involved in the

symbiotic nodulation process. Once flavonoids bind to the nodD protein, the resulting complex upregulates expression of other common nod genes which encode the enzymes to manufacture plant signaling molecules, lipo-chitin oligosaccharides (LCOs) (Peters et al. 1986, Bassam et al. 1988, Phillips 1992). The down regulation of these genes in the absence of flavonoids is believed to be a survival strategy based on the assumption that

there is an energy cost in expressing nod genes (Mulligan and Long 1985, Peters et al.

1986, Phillips 1992).

Chemical mutagenesis resulted in the discovery of four P. vulgaris genes involved

in nodulation. Two phenotypes were seen among these four gene mutations; 1)

nodulation is eliminated, or nodules are ineffective or 2) the plant loses control over

nodulation and exceptional numbers of nodules develop (Pedalino et al. 1992, Park and

Buttery 1989, Park and Buttery 1994).

Two P.vulgaris supernodulating mutants were identified when wild-type plants were treated with ethyl methyl sulfonate. Back-crossing resulted in the discovery of a single recessive gene (nts/nod) that controlled the Nitrate-tolerant supernodulation

(NTSN) characteristic in both mutants. This induced mutation permits abundant nodulation in the presence of high nitrogen and is determined by the genotype of the shoot (Park and Buttery, 1989). Other mutants have been identified, all induced and resulting in non-nodulating P. vulgaris. NOD125, a non-nodulating mutant, was identified using ethyl methyl sulfonate mutation. Further investigation resulted in identification of a single recessive gene (nnd/ sym-1) that is determined by the genotype of the root. The specific characteristic is only expressed in the roots and results in failure of nodulation and consequently, yellowing of leaves and abscission of the first leaves

(Pedalino et al. 1992). Two more genes were identified by ethyl methyl sulfonate

mutagenesis resulting in ineffectively nodulating P. vulgaris. The genes nie and nnd-2 were both identified by Park and Buttery (1994) and mutations in these genes result in ineffective nodulation and loss of older leaves.

During routine inoculation experiments of P. vulgaris in Art Trese's lab, a host

plant, cultivar Top Crop™, was identified that had failed to nodulate following

inoculation with Rhizobium leguminosarum strain USDA 2669. Further investigation

showed that in some instances the mutant did form functioning nodules when inoculated

with different strains of Rhizobia sp. This type of phenotype is novel when compared to

earlier mutations for two reasons; first, it occurred spontaneously without chemical

mutagenesis, and second, it is a phenotype that shows selectivity, consistently allowing

only specific strains to form a symbiosis while blocking other strains. All previously

identified phenotypes resulted in either abolished control over nodule number or altered

the ability to develop a functional nodule. These are recessive, loss of function

mutations. Unpublished research in Dr. Trese’s lab demonstrated that the allele

responsible for selective nodulation is recessive, and is not allelic with any of the other

nodulation mutants identified in P. vulgaris.

The following experiment was designed to more extensively examine the

nodulation capacity of the mutant P.vulgaris. The first objective was to examine forty representative strains of Rhizobia, from the 5 Rhizobium species known to nodulate

P.vulgaris, by screening for their ability to nodulate the mutant bean (Table 2-1).

The homozygous recessive host will not recognize the LCO necessary to induce

symbiotic interaction with most Rhizobia that nodulate P. vulgaris. The mutant may in

fact be missing the correct flavonoid signal necessary to induce nodulation by the excluded strains. The second objective was to test this hypothesis using two strategies; 1)

Table 2-1. Rhizobium strains used in this study. The table includes the USDA designations along with any synonyms found in the literature.

Strain Synonym Species USDA CE3 "effective" R. leguminosarum USDA H2C 2, 3IIb2 R. leguminosarum USDA Kim5 Kim5 R. etli USDA CFN3 3, 3IIb3 R. leguminosarum USDA 10 R. etli USDA 51 C-051 R. tropici USDA 123 H123 R. leguminosarum USDA 227 PRC 183 R. leguminosarum USDA 234 NGR234 R. tropici USDA 2370T C-12-C13 R. leguminosarum USDA 2669 * semia 0476, RCR 3610, R. leguminosarum 127K14 USDA 2670 semia 0490 R. leguminosarum USDA 2671 RCR 3644, CIAT 905, R. leguminosarum 127K17 USDA 2673 semia 0491 R. leguminosarum USDA 2676 CIAT 166 R. tropici USDA 2680 Kim5 R. etli USDA 2696 127K51, Phas 61 R. leguminosarum USDA 2713 127K77, Phas 115, R. leguminosarum 2281, USDA 2739 CIAT 632 R. etli USDA 3622 TAL169, 176, A22 R. leguminosarum USDA 3456 semia 6032 R. leguminosarum USDA 3516 3A4 R. leguminosarum USDA 9003 BR 836 R. legumionsarum USDA 9005 BR 842 R. leguminosarum USDA 9008 BR 847 R. leguminosarum USDA 9017 BR 860 R. leguminosarum USDA 9023 BR 10043 R. leguminosarum USDA 9028 C-05-35, 51 R. tropici USDA 9030T CIAT 899, 2744 R. tropici USDA 9032T CFN 42, 2940, Viking I R. etli USDA 9039 CFN 299, 9029 R. etli USDA 9041 TAL 182 R. leguminosarum R602T USDA 2918 R. gallicum PhI21 R. gallicum PhD12 R. gallicum H152T R. giardinii H251 R. giardinii Ro52 R. giardinii Ro67 R. giardinii Ro65 R. giardinii

T : Type strain of each species *: Type strain for laboratory, Ohio University

nodulation of the mutant by excluded strains while grown in conjunction and contact with

wild-type host plants to test if wild-type exudates could contribute to nodulation of the

mutant host and 2) at the time of inoculation, excluded strains will be supplemented with naringenin, a common nodulation inducing flavonoid. Isolated wild-type exudates have

been shown to induce nodulation in non-nodulating legumes varieties (Sprent 1994),

while naringenin has been shown to induce nodulation at a concentration of 1.5 μM in legumes such as alfalfa (Cocking 2003). The only identified spontaneous mutations involving nodulation affect the symbiosis after formation of the nodule primordia and nodules, this suggests the mutations do not involve the compounds and mechanisms involved in initiation of nodulation. All the mutations result in some form of nodule structure whether an effective or ineffective nodule. The strain selective mutant isolated

in Art Trese’s lab does not form any nodules, ineffective or otherwise, and therefore it is

expected that the mutation occurs during initiation of the symbiosis. Wild-type exudates

in proximity to the mutant P. vulgaris roots as well as the addition of the plant flavonoids

and nodulation inducer naringenin will induce nodulation of the excluded strains.

Materials and Methods

Growth of Phaseolus vulgaris

Top Crop P. vulgaris seeds were sterilized in 95% ethanol for 3 minutes and then

in 50% bleach for 1 minute. The seeds were then rinsed for 1 minute in tap water. Seeds

were placed in sterile 6 inch azalea pots filled with vermiculite and watered. The pots

were placed in a 30ºC incubator for 24-48 hours until germination. Seedlings were then individually transferred to 6" azalea pots for inoculation.

Growth of Rhizobium species

The 40 strains (Table 2-1) were grown on petri dishes with MAG media (Cole and Elkan

1973)). A single isolated colony was obtained and placed in 100ml of liquid MAG media 24-48 hours in a 30ºC shaker, until they reached log phase growth.

Strains were obtained from the USDA Beltsville Rhizobial Collection or directly from

Dr. Laguerre's laboratory in France.

Inoculation of P. vulgaris

Six mutant seeds and 6 wild-type seeds were inoculated with each strain from

Table 2-1. Each pot was inoculated with 98 ml of tap-water, 2 ml of Jensen's reagent

(50X) and 2 ml of liquid media with one of the forty strains. Jensen's reagent (50X) was made with 10g of CaHPO4, 2g K2HPO4, 2g MgSO4 · 7H2O, 2g NaCl, and 2.6g Fe-EDTA.

After 4 weeks of growth in the greenhouse, roots were examined for successful nodulation. The presence of pink nodules was considered a sign of positive nodulation and active nitrogen fixation.

Inoculation of Nod- mutant with wild type exudates in proximity

Due to the complexity of isolating all wild-type exudates in the presence of a

Rhizobial species, this experiment was set up to grow mutant host P. vulgaris in the same pot as well as the same growth pouch with the wild-type plant. The two plant types were grown in proximity to allow for their roots to come in contact with each other while they grew and formed nodules. Three pots were inoculated with each of the 40 strains (Table

2-1). The experiment was carried out 3 times, once with the plants grown in 6" azalea pots and arranged in a crisscross pattern (Fig. 2-1). This allows for the production of any

Fig. 2.1: Arrangement of wild-type and mutant P. vulgaris plants in 6 " azalea pots. W = wild-type and M = mutant.

M W

W M

wild-type exudates to induce nodulation onto the mutant plant. Each pot was inoculated with 98 ml of tap-water, 2 ml of Jensen's reagent (50X) and 2 ml of liquid media with one of 10 strains that were found in the previous experiment to not have the ability to nodulate the mutant P. vulgaris. To promote more intimate interaction between mutant and wild type root systems, mutant plants were also grown in growth pouches; a single mutant seedling was flanked with two wild-type seedlings. Inoculation of the growth pouches included bacteria suspended in 15 ml tap water, 0.3 ml Jensen's reagent (50X), and 1 ml of liquid MAG media with one of 10 strains that were found in the previous experiment to not have the ability to nodulate the mutant P. vulgaris .

Plant growth

After inoculation with selected strains, the plants in the pots were grown in the

greenhouse for 4 weeks. Each pot was watered twice weekly. Plants grown in growth

pouches were placed in a growth chamber for 3 weeks and watered daily.

Inoculation with Naringenin

Germination of bean seeds was carried out as described above. Individual mutant

P. vulgaris plants were placed in a growth pouch and inoculated with each of the ten

strains identical to the above experiment. One ml of naringenin (1.5μM) was added at

the point of inoculation to each growth pouch. Plants were allowed to grow in the growth

chamber for 3 weeks with daily watering. This experiment was repeated 3 times.

Harvest and identification of positive inoculation.

Plants were harvested, roots were washed in tubs individually and total nodules were

counted per plant. The presence of pink nodules was scored as a positive result for

nodulation while any white or underdeveloped nodules were not scored as positive

nodulation.

Results

Strain specificity test

After 4 weeks of growth in the 6” azalea pot, wild type plants developed normal nodulation patterns exhibiting extensive nodulation, green healthy leaves, and pink

(actively fixing nitrogen) nodules with all 40 strains of Rhizobia. The mutant plant lacked nodulation and showed clear signs of nitrogen deficiency, exhibiting chlorotic leaves, with all but three strains (Fig. 2-2); USDA 9017 R. leguminosarum, USDA 9032

R. etli and USDA 9041 R. leguminosarum (Table 2-2).

Wild-type exudate proximity experiment

Wild-type plants and mutant plants were grown together in 6” azalea pots and

inoculated with excluded Rhizobia strains. All wild-type plants formed nodules while

none of the mutant hosts formed nodules (Table 2-3). When grown in the growth

pouches and inoculated with excluded strains, none of the mutant plants formed nodules

while the 100% of the wild-type plants developed nodules (Table 2-3).

Naringenin addition experiment

The addition of naringenin did not induce nodulation on the mutant P. vulgaris

when inoculated with the non-nodulating strains (Table 2-4). All wild-type controls

formed nodules when inoculated with excluded strains and naringenin.

Figure 2-2. Chlorotic leaves of P. vulgaris from lack of nodulation. The plant on the left is a nodulated wild-type P. vulgaris and the plant on the right is a wild-type P. vulgaris without nodulation.

Table 2-2. Inoculation results of 40 strains of Rhizobia on wild-type and mutant P. vulgaris. Positive designation identifies pink nodule formation (>50 nodules on root system). Negative designation identifies strains that did not form a successful symbiosis with the host plant (0 pink nodules formed). Results were identical when carried out in 6” azalea pots.

Strain Wild-type Mutant USDA CE3 + - USDA H2C + - USDA Kim5 + - USDA CFN3 + - USDA 10 + - USDA 51 + - USDA 123 + - USDA 227 + - USDA 234 + - USDA 2370 + - USDA 2669 + - USDA 2670 + - USDA 2671 + - USDA 2673 + - USDA 2676 + - USDA 2680 + - USDA 2696 + - USDA 2713 + - USDA 2739 + - USDA 3622 + - USDA 3456 + - USDA 3516 + - USDA 9003 + - USDA 9005 + - USDA 9008 + - USDA 9017 + + USDA 9023 + - USDA 9028 + - USDA 9030 + - USDA 9032 + + USDA 9039 + - USDA 9041 + + R602 + - PhI21 + - PhD12 + - H152 + - H251 + - Ro52 + - Ro67 + - Ro65 + -

Table 2-3. Inoculation success of excluded Rhizobia strains on the mutant P. vulgaris when grown in proximity to wild-type host roots in a single pot. Positive designation identifies pink nodule formation (>50 nodules on root system). Negative designation identifies strains that did not form a successful symbiosis with the host plant (0 pink nodules formed).

Strain Mutant USDA 2669 - R. leguminosarum USDA CE3 - R. leguminosarum USDA 9017 + R. leguminosarum USDA 9041 + R. leguminosarum USDA Kim 5 - R. etli USDA 2673 - R. etli USDA 9032 + R. etli USDA 2676 - R. tropici USDA 9030 - R. tropici R602 - R. gallicum PhI21 - R. gallicum H150 - R. giardinii Ro52 - R. giardinii

Table 2-4. Inoculation success of excluded Rhizobia strains on the mutant P. vulgaris with the addition of the plant flavonoid naringenin. Positive designation identifies pink nodule formation (>50 nodules on root system). Negative designation identifies strains that did not form a successful symbiosis with the host plant (0 pink nodules formed).

Discussion

Three strains, of two species, are able to form a successful and functional nodule

on the mutant Phaseolus vulgaris (Table 2-2). Positive nodulation was determined by the

presence of at least 50 pink nodules. The pink color is indicative of the presence of

leghemaglobin, a chemical produced during active nitrogen fixation. R. leguminosarum

USDA 9017 and USDA 9041 along with R. etli USDA 9032 all have the ability to form a symbiosis with the mutant P. vulgaris. None of the remaining 37 strains of Rhizobia were able to form any pink nodules on the mutant P. vulgaris. The three strains that are

able to nodulate the mutant bean will be referred to as overcoming strains, while remaining strains are referred to as excluded. Rhizobia are classified into their species based on their symbiont host (Chapter 1). The three strains that are able to overcome the restriction are found within the five species known to nodulate P. vulgaris and are members of two species, R. leguminosarum and R. etli. These results show that whatever

the genetic basis of overcoming strains might be it is found in more than a single species

of bean compatible Rhizobia. The restricted phenotype (excluded strains) is found in all

five species of bean compatible Rhizobia. These results suggest that the phylogeny of

Rhizobia should be examined for their relationship in 16S rRNA rather than a basis in

host range.

This novel mutation presents a unique combination of traits: a recessive gene and strain specific nodulation. The restriction occurs at the initiation of nodulation perhaps

resulting from an alteration of the plant’s flavonoid profile, thus signaling only specific

nod genes effectively. This hypothesis was tested by setting up two experiments; 1) mixed root system in close proximity and 2) addition of naringenin. The mix root system

created a situation where the wild-type root exudates might compensate for any lack of

flavonoids produced by the mutant, while the addition of naringenin would accomplished

the same but with an exogenous flavonoid which is known to induce initiation of

nodulation in non-nodulating P. vulgaris (Cocking 2003).

The addition of wild-type root systems in proximity and resulting non-nodulation

of the mutant and concurrent nodulation of the wild-type suggest that the bacteria are

responding to a chemical signal released by the wild-type roots, but are still not able to

nodulate the mutant. While grown in the pot, the wild type roots were occasionally close to the mutant root system, potentially providing enough flavonoid signal to initiate nodulation. However, not even one nodule was detected on the mutant root systems in these experiments. To increase the physical contact, and intimacy, of the wild-type and mutant root systems they were grown together in growth pouches, where roots grew side- by-side within millimeters of each other and in contact with each other. Again, the juxtaposition of the two root systems did not compensate the mutant plants for the putative compound or product that may be lacking in their interaction with excluded strains.

Naringenin is a specific plant flavonoid that has been used historically to induce early nodulation responses on non-nodulating P. vulgaris hosts in the absence of plant flavonoids. Addition of this compound would potentially compensate for any lack of signal missing from the mutant P. vulgaris. A positive control experiment for the effect

of naringenin was not available (Cocking 2003).

The mutant P. vulgaris, now termed Nod- mutant, is not lacking the correct

signals to initiate nodulation with at least three strains. However, excluded strains, even

with some exposure to a wild-type signal, or naringenin, cannot form a symbiosis on the

mutant bean. Since three strains of Rhizobia can, in fact, form a symbiosis on the mutant,

the data indicate that the recessive mutation is blocking the formation of the symbiosis at

some other point during initiation.

Initiation of nodulation can be broken down into four steps; 1) plant production

and secretion of flavonoids, 2) Rhizobial perception of flavonoid signal and consequent induction of nod genes and production of LCO signal, 3) perception of LCO signal by plant and 4) root hair curling, infection thread formation leading to nodule formation

(Brewin et al. 1992). Although a direct conclusion cannot be made about the timing of the restriction from these results, they do provide a direction for continued research.

Since the excluded strains have responded to wild-type exudates as to successfully form a

symbiosis with the wild-type P. vulgaris, it can be concluded that the mutation affects the

nodulation process at some point after step 2 either in step 3 or step 4, Rhizobial

perception of flavonoids signal and consequent induction of nod genes and production of

LCO signal.

These results stimulated further research on the possible difference in LCO signal

from the excluded and overcoming strains based on the fact that the mutant plant could

not be forced to form nodules with the excluded strains with either the addition of

naringenin or wild-type plant root exudates.

Chapter 3 Phenotypic, Developmental and Morphological Differences Between the Nodulation of the Nod- Phaseolus vulgaris and the Wild-type.

Summary

The Nod- mutant P. vulgaris appears to have one phenotypic trait, selective

restriction of nodulation that distinguishes it from the wild-type; however, the apparent

phenotype may not be the only characteristic affected by the mutation. It is possible that

the mutation also causes latent or deleterious affects, such as delayed nodulation, poor

nitrogen fixation, or stunted growth. This experiment examines general phenotypic

characteristics of the wild-type bean plant and compares these characteristics to the Nod- mutant to assess other possible phenotypes. Both the wild type and Nod- mutant plants

were examined for height, weight, percent nitrogen, and nodule number. The Nod-

mutant P. vulgaris was found to have no deleterious phenotypic characteristics when

compared to the wild-type host. The Nod- mutant was similar in plant height, dry weight,

nodule number and % nitrogen when inoculated with one of the three overcoming strains,

USDA 9017, USDA 9032 and USDA 9041, as compared to the wild-type when

inoculated with the same three strains or an excluded strain, USDA 2669. The Nod-

mutant was not shown to express any deleterious phenotypes other than strain specific

restriction of nodulation. This suggests that the recessive mutation in the P. vulgaris only

affects the nodulation capacity of the host plant.

Introduction

Common bean (Phaseolus vulgaris) is one of the major leguminous crops grown in the world. As a crop, beans provide protein and necessary calories to over 300 million people worldwide. Vigorous scientific efforts are directed to improve yield and nitrogen accumulation through breeding, natural selection and genetic engineering (Babiker et al.

1995).

Research that has focused on identifying Rhizobial strains with high nitrogen

fixing capabilities have not resulted in improved yields in field trials (Carter et al. 1995).

When highly efficient nitrogen fixing strains are inoculated in field trials, the native soil

strains out-compete for nodule occupancy. These results have changed the direction of

research, encouraging scientists to isolate more competitive strains first and then

concentrate on selecting for higher nitrogen fixing capabilities (Yates et al. 2003,

Denisona and Kiersa 2004, Savka and Farrand 1997, Mavingui et al. 1997, Hungria et al.

1991, Winarno and Lie 1979, Simms et al. 2006).

To gain a better understanding of the symbiosis and competition of nodulation of

P. vulgaris, scientists have focused on the plant host genes involved in recognition necessary for successful nodulation (Brockwell 1980, Carter et al. 1995, Silsbury 1991).

As stated in the introduction, only 4 genes have been found that are involved in control and regulation of host plants P. vulgaris control over nodulation. All of these genes result in either supernodulation, i.e. uncontrolled nodulation, complete restriction of nodulation, or ineffective nodulation (Park and Buttery 1994, Pedalino et al. 1992).

Ineffective nodulation is defined as a formation of a small, white nodule that does not reduce N2 (Nutman 1965, Vance et al. 1981).

A P.vulgaris spontaneous recessive mutant (Nod-) was isolated in Dr. Art Trese's

lab that incurred strain specific host selectiveness during nodulation. The mutant restricts

nodulation by all but three strains of Rhizobia, R. leguminosarum USDA 9017 and

USDA 9041, and R. etli USDA 9032. This type of mutation, one that will selectively restrict nodulation in vitro and is recessive, has not been identified previously and may be used as a model in understanding what mechanisms the host plant uses for nodulation selection. When backcrossed, the mutation was found to be a single recessive gene.

Previous work done by Art T. Trese, showed that the restrictive bean when backcrossed

with its parents resulted in the wild-type phenotype, showing that the mutation was

recessive. The bean mutant described in my research may provide an alternative strategy;

select or engineer a highly efficient overcoming strain and couple that with a bean

cultivar bred to be homozygous recessive for the strain selective non-nodulation allele.

This strategy would only be practical if there is no deleterious effect associated with the

mutation.

Spontaneous mutations occur in all living organisms resulting in silent or

detectable phenotypes. Currently, the only known phenotypic expression of the Nod- P. vulgaris is a selective restriction of nodulation. This experiment focuses other phenotypic characteristics that may be expressed by the mutant. The Nod- mutant was

examined for phenotypic responses when inoculated with the three overcoming strains,

USDA 9017, USDA 9032, USDA 9041 and one restricted strain, USDA 2669. The objective was to determine what, if any, phenotypic differences are associated with the

Nod- mutant. Morphological differences including height, dry weight, nodule number, and nitrogen content were all compared in wild-type and Nod- mutant P. vulgaris.

Materials and Methods

Growth of Plants and Inoculation

Thirty Nod - mutant seeds and 30 wild-type seeds were sterilized and planted in

Cone-tainers™ with vermiculite. Surface sterilization was carried out by immersing seeds for 3 minutes in 95% ethanol, followed by 1 minute of 50% bleach, and 1 minute of

a water rinse. The seeds were planted in 6” azalea pots and allowed to germinate in a 30º

C incubator for 48 hours.

Rhizobium sp. strains included in this study were lab type strain USDA 2669,

USDA 9017, USDA 9032 and USDA 9041. All strains were obtained from USDA

Rhizobium Culture Collection in Beltsville, Maryland. Strains were grown in 100 ml

Tryptone yeast (TY) media overnight at 30º C. TY media is composed of 10 g of

tryptone, 5 g of yeast extract and 5 g of sodium chloride. The 100 ml cultures were quantified on a spectrometer (1 absorbance unit/ml at 260 nm) for turbidity, and each culture was then diluted to equal concentrations of approximately 3 million cells/ ml.

Each strain was inoculated at 1ml per plant on 30 plants of Nod- and wild-type. Plants

were grown in growth chambers at 37º C with 8 hours of light and 16 hours of dark.

Plants were harvested after 5 weeks of growth and measured for height, weight,

nodule number and nitrogen content. Each plant was measured for height and then dried

by clipping at the hypocotyl and placing it in paper bags in a forced-air dryer at 70ºC for

48 hours. Plant dry weight was recorded as well as nitrogen accumulation. Nodule

number was enumerated; only pink, actively fixing nodules were included. Controls of

non-inoculated samples were compared to inoculated samples in all analyses. The

experiment was repeated three times, and results were combined.

Analysis of nitrogen content: Elementar C:N Analyzer

After drying, each sample was ground using a mortar and pestle and analyzed for

C:N ratio. The samples were weighed to 20.0 mg and placed in a Vario EL C:N

Analyzer. One blank was used to purge the system and a second was used to stabilize the

Carbon and Nitrogen Peak. Three more ACE standards were used to standardize the machine (approximately 5.0 mg). The EL C:N analyzer provides C:N ration, % C and %

Thirty individuals from each treatment were examined in duplicate for nitrogen

content. The response variable, percent nitrogen, was compared for the mutant and wild-

type plants when inoculated with each of the four strains, USDA 2669, USDA 9017,

USDA 9032 and USDA 9041 using analysis of variance (ANOVA) (Zar 1999). The

significant differences were compared using the Bonferonni Post Hoc test by using the

NCSS program for statistical analysis (Hintze 1999).

Results

There were no significant differences between the mutant P. vulgaris and wild-

type based on plant height and plant weight. (Figures 3-1 and 3-2). The uninoculated

wild-type was not significantly taller than the uninoculated Nod- mutant (Figure 3-1, P >

0.05). When the wild-type is inoculated with any strain, its height increases (18 cm to

31.0 cm with USDA 2669, 34.3 cm with USDA 9017, 29.9 cm with USDA 9032 and

28.8 cm with USDA 9041), with no significant difference between any of the inoculant

strains (Figure 3-1, P > 0.05). When the Nod- mutant is inoculated with the non-

nodulating USDA 2669, there is a significant decrease in plant height from the wild-type;

19.7 cm to 31.0 cm respectively (Figure 3-1, P ≤ 0.05).

45 c c 40 c c c c b,c 35

30 a,b a,b 25 a Wild-type Mutant 20 Height (cm) Height 15

0 none 2669 9017 9032 9041 Inoculant

Figure 3-1. Mean heights of both wild-type and mutant P. vulgaris with and without inoculation. Bars with different lowercase letters represent significant differences (P ≤ 0.05, N = 30).

0.60 b b b b b 0.50 b b

a 0.40

a a Wild-type 0.30 Mutant

Average weight (g) 0.20

0.10

0.00 none 2669 9017 9032 9041 Inoculant

Figure 3-2. Mean weight of wild-type and mutant P. vulgaris with and without inoculation. Bars with different lowercase letters represent significant difference (P ≤ 0.05, N = 30).

The uninoculated wild-type and Nod- host plants did not differ significantly in

their dry weight, 0.252 g and 0.235 g respectively (Figure 3-2, P > 0.05). When the wild-

type is inoculated with any strain its weight increases significantly with no significant

difference between inoculant strains (Figure 3-2, P > 0.05). However, the Nod- mutant weighs significantly less than the wild-type when inoculated with USDA 2669 (Figure 3-

2, P ≤ 0.05).

There was a significant difference seen in the nitrogen percentage accumulated and the number of nodules between the wild-type and mutant with inoculation by USDA

2669, USDA 9017, USDA 9032 and USDA 9041 (Figures 3-3 and 3-4). There was significant difference in nitrogen accumulated in the wild-type host versus the mutant host when inoculated with USDA 2669 (Figure 3-3, P ≤ 0.05). The uninoculated wild- type, uninoculated Nod- mutant and the Nod- mutant inoculated with USDA 2669 did not

differ in nitrogen accumulation (Figure 3-3, P > 0.05). The inoculated wild-type did not

differ in nitrogen accumulated from the Nod- mutant when the mutant was inoculated

with one of the three overcoming strains, USDA 9017, USDA 9032, and USDA 9041

(Figure 3-3, P > 0.05).

When both the wild-type and Nod- mutant are not inoculated, neither formed

effective nodules (Figure 3-4, P > 0.05). When the wild-type was inoculated with any of

the four strains, there was formation of nodules, with slightly less nodules forming on the wild-type inoculated with USDA 9032 (Figure 3-3, P ≤ 0.05). In terms of number of nodules formed, the Nod- mutant was not distinguishable from the wild type.

7 b b b 6 b b b b a,b 5 a, b a 4 Wild-type Mutant 3 % Nitrogen

0 none 2669 9017 9032 9041 Inoculant

Figure 3-3. Percent nitrogen isolated from wild-type and mutant P. vulgaris with and without inoculation. Bars with different lowercase letters represent significant difference (P ≤ 0.05, N = 30).

90 b b b 80 b,c b,c

b,c 70 c 60

50 Wild-type Mutant 40

Average Number of Nodules of Number Average 20

10 aa a 0 none 2669 9017 9032 9041 Inoculant

Figure 3-4. Number of nodules formed on wild-type and mutant P. vulgaris, with and without inoculation. Bars with different lowercase letters represent significant difference (P ≤ 0.05, N = 30).

Discussion

The Nod- mutant is equivalent to the wild-type when inoculated with any of the

three overcoming strains. When the Nod- mutant is inoculated with a restricted strain,

USDA 2669, it behaves as if it was not inoculated at all. These results suggest that there

are no deleterious effects of the Nod- mutant besides restricted nodulation and the indirect

phenotypes that result from that restriction, i.e. reduced height, weight, % nitrogen accumulated and number of nodules. Therefore, the mutation remains potentially useful as a means to manipulate nodule occupancy in the field The non-inoculated Nod- mutant

P. vulgaris did not have any deleterious affects other than restricted nodulation. The height, shoot weight, and percent nitrogen are not significantly different from the non- inoculated wild-type (Figures 3-1, 3-2, and 3-3). The average plant height for the wild- type is 18.8 cm with the Nod- being slightly less at 16.3, but not significantly less (Figure

3-1, P ≤ 0.05). The Nod- mutant plant weight was not significantly different from the

wild-type either; 0.0252 g and 0.235 g respectively (Figure 3-2, P ≤ 0.05), as is the

amount of nitrogen accumulated without nodules, 3.08% and 3.67% respectively (Figure

3-3, P ≤ 0.05). This shows us that as a plant developing without inoculation the mutant

does not show any deleterious growth habits.

When the Nod- mutant is inoculated with an excluded strain, USDA 2669, it

exhibits growth habits as though it was not inoculated. The plant height; 19.7 cm,

weight; 0.280 g and % nitrogen accumulated; 4.02% did not differ from the uninoculated

wild-type and Nod- mutant (Figures 3-1, 3-2 and 3-3, P ≤ 0.05).

Formation of nodules was not significant on the Nod- mutant when inoculated with USDA 2669, an excluded strain. It did appear that a few nodules formed, but these were small and when compared to uninoculated plants, show no significant increase in nodule number (Figure 3-4, P ≤ 0.05).

The Nod- mutant did not show any deleterious phenotypes when it was nodulated,

compared to the wild-type (Figures 3-1, 3-2, 3-3, 3-4, P ≤ 0.05). The Nod- mutant plant

height increased with inoculation with overcoming strains USDA 9017, USDA 9032 and

USDA 9041 to 35.0 cm, 31.9 cm and 33.9 cm respectively, equal to the USDA 2669

inoculated wild-type (Figure 3-1, P ≤ 0.05). Likewise, the Nod- mutant did not differ in

plant height when inoculated with the three overcoming strains compared to the wild-type

when inoculated with the overcoming strains (Figure 3-1, P ≤ 0.05).

The Nod- mutant did not show any difference in weight from the wild-type when inoculated with any of the three overcoming strains nor did the Nod- mutant show any

significant difference in dry weight from the non-inoculated wild-type and non-

inoculated Nod- mutant (Figure 3-2, P ≤ 0.05). The Nod- mutant was significantly lighter

when inoculated with the restricted USDA 2669 than the wild-type when inoculated with

USDA 2669. The Nod- mutant weighs the same as a non-inoculated host (Figure 3-2, P ≤

0.05).

The Nod - mutant accumulated the same % nitrogen when inoculated with the

three overcoming strains as did the wild-type when inoculated with USDA 2669, USDA

9017, USDA 9032 and USDA 9041. When the Nod- mutant was inoculated with the

87 restricted USDA 2669 it accumulated the same amount of nitrogen as a plant that was not inoculated, both wild-type and Nod- mutant (Figure 3-3, P ≤ 0.05).

The number of nodules that formed on the Nod- mutant was equal to the number of nodules on the wild-type when they were inoculated with USDA 9017 and USDA

9041 (Figure 3-4, P ≤ 0.05). The number of nodules formed on the Nod- mutant when inoculated with USDA 9017 and USDA 9041 was also equal to the number of nodules that formed on the wild-type when it was inoculated with USDA 2669. The number of nodules only differed when the wild-type and Nod- mutant were inoculated with USDA

9032. The number of nodules formed when both the wild-type and Nod- mutant are inoculated with USDA 9032 is significantly less then when the host is inoculated with any of the other strains. USDA 9032 still forms a successful symbiosis resulting in an average of 55.8 nodules per plant (wild-type) and 59.9 nodules per plant (Nod- mutant)

(Figure 3-4, P ≤ 0.05). These results suggest that USDA 9032 is successful in nodulating the Nod- mutant, but USDA 9032 will not form as many nodules as the other two strains.

These results suggest that the Nod- mutant can be used in the field and in agricultural settings. Because of the mutant's height, weight, nodule number and nitrogen fixed it can be assumed that the fruit yield will be equivalent to the wild-type (not tested).

The mutant plant forms a symbiotic relationship that yields equivalent nitrogen and nodule number showing that it is equivalent to the wild-type. The Nod- mutant may be the answer to the problem of native soil Rhizobia out-competing inoculant strains. As stated in the introduction, the nitrogen fixation efficiency of the P. vulgaris/ rhizobium symbiosis results in a low amount of nitrogen being fixed, approximately 40% of

available nitrogen. Scientists have focused their research on more competitive strains because highly efficient nitrogen fixing strains of Rhizobia isolated showed low nodule occupancy due to the fact that they are not able to out-compete native Rhizobia for nodule occupancy (Yates et al. 2003, Denisona and Kiersa 2004, Savka and Farrand

1997, Mavingui et al. 1997, Winarno and Lie 1979, Simms et al. 2006).

The Nod- mutant may be used to solve this problem. The mutant is able to

distinguish between different strains of Rhizobia and therefore may be able to do the

same in the field. If the Nod- mutant can pick and choose between Rhizobia based on

their individual profile than the mutant may be able to pick out the three overcoming

strains of Rhizobia that could be better equipped to fix nitrogen.

Chapter 4 Mechanism of Nodulation in Nod- P. vulgaris.

Summary

Rhizobia form a symbiosis with beans by first colonizing the root hair and then by initiating formation of an infection thread. The first indication of infection thread formation is root hair elongation and tip curling (Newcomb 1976, Kijne 1992, Brewin

1991). Alternatively, some Rhizobia nodulate through root openings such as lateral root emergence points, as in the case of peanuts (Sprent and Raven 1992) or through root wounds, as seen in the aquatic legume Neptunia natans (Chandler et al. 1982, de Faria et al. 1988, Subba-Rao et al. 1995). The objective of this experiment was to determine if the overcoming strains are forming the symbiosis on the Nod- mutant using the same mechanism typical of wild-type P. vulgaris or if they were employing a different method.

When inoculated onto the Nod- mutant, the overcoming strains induced root hair curling and infection thread formation. A second objective was to determine at which point the

Nod- mutant host restricted nodulation by an excluded strain. It was determined that the excluded strains produced no root hair curling or infection thread formation on the Nod- mutant host. A third objective of this experiment was to examine if piggybacking (Long

1989) of excluded strains is possible when an infection thread is formed on the Nod- mutant by an overcoming strain. Nod- mutants were inoculated with overcoming strain

USDA 9032 and excluded strain USDA 2669 concurrently. When inoculated onto the

Nod- mutant, infection threads formed, which contained the overcoming strain, USDA

9032, while the excluded strain, USDA 2669, was restricted from entering the infection thread.

Introduction

The symbiosis between Rhizobia and legumes is controlled and initiated by chemical signals that are exchanged between the host plant and the bacteria (Fisher and

Long 1992). Initiation of the relationship is dependent on these signals being expressed and detected by the organisms involved. Once initiation is complete, bacteria reside in the plant nodule as endosymbiotic bacteroids that actively fix atmospheric nitrogen.

In the P. vulgaris host, the initiation process of nodulation can be simplified into

four necessary steps; 1) Host plant flavonoid production and release into the rhizosphere,

2) Flavonoid perception by the bacteria, 3) Bacterial production of lipo-chitin

oligosaccharides (LCO) that is positively regulated by flavonoid perception and the

resulting release of the LCOs into the rhizosphere and 4) Host plant perception and

physical response to the bacterial LCOs includes root hair curling and root cell division

(Brewin et al. 1992). The curled root hair is the location where an infection thread forms,

an indentation that evolves into a long tube, where the symbiotic bacteria enter and

migrate to the host root cells and bud through their plasma membrane resulting in a

functional symbiosis.

Peanuts (Arachis hypogaea) differ from other legumes in their mechanism of

nodule formation. Infection results from intracellular penetration through the epidermis,

occurring at the point of emergence of the lateral root, and is termed "crack entry". Cell

divisions are induced in root tissue, but there is no formation of infection threads (Sprent

and Raven 1992). Nodule formation by this method is also seen in Stylosanthes sp. and

Mimosa scabrella, while the aquatic legume Neptunia natans, a plant that does not have

root hairs forms nodules by bacteria entering wounds on the roots (Chandler et al. 1982,

de Faria et al. 1988, Subba-Rao et al. 1995).

There are 5 species known to nodulate P. vulgaris; Rhizobium leguminosarum, R.

etli, R. tropici, R. gallicum, and R. giardinii and among them, only three strains are able

to circumvent the described mutation and form a symbiosis with the Nod- mutant P.

vulgaris. All five species typically form a symbiosis with beans by initiating infection

thread formation, as described above. This experiment first attempts to examine the

method by which the three overcoming strains form a symbiosis with the Nod- mutant P.

vulgaris. It is known that other legumes form a successful symbiosis with Rhizobium by

other methods, i.e. peanuts, and it is possible that these three specific strains employ a

different method to successfully nodulate the Nod- mutant host. This experiment first determined whether the overcoming strains induce root hair curling and infection thread

formation. The three overcoming strains do form efficient nitrogen fixing nodules that

are in equal numbers to the wild-type, therefore it was expected that the three overcoming

strains induce root hair curling and infection thread formation.

Second; the experiment set out to determine at what point the excluded Rhizobia

are blocked from forming a symbiosis with the mutant host P. vulgaris by examining if

restricted Rhizobia are able to signal the host, which will be determined by the presence

or absence of root hair curling and infection thread formation. We have seen from

previous research that when the Nod- mutant is inoculated with an excluded strain no

nodules form, not even small non nitrogen fixing nodules, therefore it was expected that

the excluded strains are restricted by the Nod- mutant at some point during initiation

before the formation of infection threads.

Third; this experiment set out to screen for a theorized phenomenon called

“Piggybacking”, a hypothesized phenomenon that states that once an infection thread is

formed on the root hair, it acts as an unrestricted tunnel allowing any adjacent bacteria,

even strains that did not initiate formation of the infection thread, to enter and become

part of the nodule (Long 1989). It was believed that if an overcoming strain can initiate

infection thread formation on the Nod- mutant, than any excluded strains would be able to

enter that infection thread and become part of the mature nodule.

In order to make it possible to distinguish strains within an infection thread, four strains of Rhizobia were transformed with a plasmid (pHC60) containing the gene that encodes the green fluorescent protein (GFP), a protein that glows green under UV radiation (Ardourel et al. 1994, Cheng and Walker 1998). The GFP gene is isolated from

Aequorea victoria, a Pacific Northwest jellyfish that flashes the color in order to scare off any potential predators (Sinicropi et al. 2004). This gene has been used as a reporter gene in numerous molecular and cellular research projects over the past 30 years and its presence in organisms is not detrimental to their normal function (Novoselova et al.

2005). Rhizobium leguminosarum USDA 9041 and USDA 2669 and Rhizobium etli

USDA 9032 and USDA 9017 were all transformed with the plasmid pHC60 containing the GFP gene in order to visualize the Rhizobia on the root hairs and within an infection thread.

Materials and Methods

GFP transformed strains

Four strains of Rhizobia sp. were transformed with the plasmid pHC60 containing

the green fluorescent protein gene (GFP). Rhizobium leguminosarum USDA 2669

(excluded), USDA 9017 and USDA 9042 as well as Rhizobium etli USDA 9032 were

transformed by triparental mating. Escherichia coli containing the helper plasmid pM16

was grown on MAG plates with tetracycline (20μg/ml) at 37º C overnight. E. coli with the construct plasmid (pHC60) containing GFP was also grown on LB media with kanamycin (20μg/ml) overnight (Ardourel et al. 1994, Cheng and Walker 1998). Each

Rhizobial strain was grown at 30º C for 48 hours. A 50 μl aliquot of each of the three

bacterial strains was then spread on a Tryptone yeast (TY) (Chapter 2, materials and

methods) agar plate containing tetracyclin and kanamycin (20μg/ml). Rhizobial strains

are tolerant of tetracycline, but would only grow on kanamycin plates if they were

successfully transformed with pHC60. Surviving strains were examined using a light

microscope and UV radiation to check for fluorescence. Transformed strains were

designated with GFP preceding the strain number, e.g. GFP-2669.

Growth of bacteria

Before inoculation, each strain of Rhizobium was taken from the plate culture and inoculated in 100 ml of liquid TY media with tetracyclin (20μg/ml) overnight at 30º C.

Determination of overcoming and excluded strain’s ability to induce root hair curling

Ten nod - and 10 wild type seeds were sterilized and planted in 6" azalea pots

containing vermiculite. After germination, seeds were transferred to growth pouches and

the location of their root tips were marked with a Sharpie marker. The plants were

inoculated with equal volumes (v/v, 109 cells ml-1) of either the newly transformed

overcoming strain GFP-9032 or the excluded strain GFP-USDA 2669. Plants were placed

in a growth chamber set at 30º C with 8 hours dark and 16 hours light. After 5 days of

growth, roots were removed and ten 1 cm sections were cut from the roots of each

inoculated plant. The sections were removed from the root starting at the above-

mentioned mark and below. This ensured that the root tissue examined had developed

root hairs subsequent to inoculation. The sections were placed on a slide and microscopically examined for root hair curling. The slides were labeled and then covered with tape over the label to ensure a "blind" study.

Examination of overcoming and excluded strain’s ability to induce infection thread formation.

Seeds were sterilized and placed in 6-inch azalea pots with water and vermiculite

and placed in a growth chamber at 30º C. After two days each germinated seed was

transferred to a growth pouch. Inoculation by bacteria was done once seedlings were

transferred and the root growth was marked with a sharpie. Ten nod - and 10 wild type

seeds were inoculated with each of GFP-2669 and GFP-9032 strains. Plants were set in a

growth chamber set at 24º C with 8 hours dark and 16 hours light. After 5 days of growth, roots were removed and ten 1 cm sections were cut from the roots of each

inoculated plant from areas above and below the aforementioned mark. The sections were placed on a slide and examined for infection threads.

Piggybacking

Ten Nod- and 10 wild-type seeds were sterilized and planted in 6" azalea pots.

After 2 days in a 30º C incubator, the germinated seeds were transferred to growth

pouches. Seeds were inoculated with USDA 9017, USDA 9032 and USDA 9041 independently. Concurrently, the same plants were inoculated with GFP-USDA 2669 in mixed ratios of 1:1 (v/v, 109 cells ml-1). The plants were placed in a growth chamber for

5 days at 30º C, and root sections were examined for infection threads containing any

GFP-bacteria as described above.

Results

GFP transformation of all four strains was successful. Individual bacteria are

easily visible and distinguishable because they appear green under UV light (Fig. 4-1).

Root hair curling and infection thread formation by overcoming strains inoculated onto Nod- mutant P. vulgaris.

Uninoculated controls showed no root hair curling or infection thread formation

(Figure 4-2, Table 4-1). Wild-type P. vulgaris exhibits root hair curling and infection

thread formation when inoculated with strain USDA 2669 (Figure 4-3, Table 4-1). The

overcoming strain, USDA 9032, induced root hair curling and infection thread formation

on both the wild-type and Nod- mutant P. vulgaris (Figure 4-4, Table 4-1).

Figure 4-1. GFP transformed USDA 9032. The bacteria were inoculated onto wild-type P. vulgaris and the picture was taken under UV two days after inoculation (100X).

Table 4-1. Mean number of infection threads found on 1 cm lengths of roots isolated from both the wild-type and mutant P. vulgaris.

Inoculum Wild-type P. vulgaris Nod- mutant P. vulgaris Non inoculated 0 0 USDA 9032 11.16 2.50 USDA 2669 10.00 0

Figure 4-2. Root hairs on uninoculated P. vulgaris A) Wild-type P. vulgaris and B) Nod- mutant P. vulgaris. Note linear root hairs without curling (100X).

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Figure 4-3. Wild-type P. vulgaris response to inoculation with USDA 2669. A) Root hair curling in response to USDA 2669 B) Infection thread formation with GFP-USDA 2669 (100X).

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Figure 4-4. Root hairs of inoculated P. vulgaris. (A) Nod- mutant when inoculated with USDA 9032, 2 dai.AB (B) mutant P. vulgaris inoculated with GFP-USDA 9032, 5 dai (days after inoculation) (100X).

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Root hair curling and infection thread formation in Nod- mutant when inoculated with excluded USDA 2669.

When the mutant P. vulgaris was inoculated with excluded strain USDA 2669, the root hairs did not respond and begin to curl, nor did any infection threads form (Figure 4-5 and Table 4-1).

Piggybacking

The Nod- mutant exhibited root hair curling and infection thread formation when inoculated with USDA 9032 and GFP-USDA 2669 simultaneously. The infection threads did not contain any glowing bacteria and therefore contained no USDA 2669 bacteria (Table 4-2, Figure 4-6).

Discussion BA BA The overcoming strain, USDA 9032, did induce root hair curling and infection threads on the mutant P.vulgaris (Fig 4-4, Table 4-1). These results confirm that the overcoming strains do indeed form the symbiosis with the mutant host employing the same mechanism used to nodulate the wild-type host. The experiment was repeated with overcoming strains USDA 9017 and USDA 9041 resulting in root hair curling and infection thread formation as with USDA 9032.

In Figure 4-4, one can see root hair curling when the Nod- mutant is inoculated with USDA 9032. This same response is seen when USDA 9032 and USDA 2669 nodulate the wild-type (Figure 4-3 and 4-4). The number of infection threads was also examined (Table 4-1). The results suggest that the Nod- mutant P. vulgaris develops fewer infection threads, even with the overcoming strain USDA 9032, but it still

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Figure 4-5. Nod- mutant P. vulgaris inoculated with excluded USDA 2669 A) Root hairs of Nod- mutant P. vulgaris when inoculated with USDA 2669 B) Absence of infection threads after Nod- mutant P. vulgaris was inoculated with GFP-USDA 2669 (100X).

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Table 4-2. Mean number of infection threads per 1 cm section on plants inoculated with GFP transformed bacteria. Mutant and wild-type P.vulgaris was inoculated with USDA 9032 and GFP-USDA 2669 concurrently with mixed ratios of 1:1 (v/v, 109 cells ml-1).

Average number of infection threads containing GFP expressing Rhizobia Wild-type P. vulgaris 13 Mutant P. vulgaris 0

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12 a a

USDA 2669 6 USDA 9032

c 4

Mean number of infection threads 2

b 0 WT M Host plant

Figure 4-6. Mean number of infection threads found on roots of wild-type and Nod- mutant P. vulgaris when inoculated with the same volume of USDA 2669 or USDA 9032. Mean number of infection threads per 1 cm sample taken from roots. Each root had 10 samples taken from it. Letters that are similar represent no significant difference (P > 0.02) and letters that are different represent a significant difference (P ≤ 0.05, N = 100). WT represents Wild-type P. vulgaris while M represents the Nod- mutant P. vulgaris.

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allows for significantly more infection threads than when inoculated with USDA 2669,

an excluded strain (Table 4-1, Figure 4-3).

Because it was only select strains, three to be exact, that have the ability to

nodulate the Nod- mutant it was hypothesized that these overcoming strains may be using

an alternate route to form the symbiosis. The overcoming stains that nodulate the mutant

P. vulgaris may only have that ability because of some unknown mechanism in the

symbiotic relationship. Other legumes such as A. hypogaea (peanuts), Stylosanthes sp.

and M. scabrella do not use infection threads to form their symbioses, and resulting

nodules are initiated by Rhizobia penetrating the root epidermis at the point of emergence

of the lateral root (Sprent and Raven 1992). Also N. natans form nodules from infection

of Rhizobia in open wounds (Chandler et al. 1982, de Faria et al. 1988, Subba-Rao et al.

1995). Therefore, it is possible that the three overcoming strains have evolved an

alternative method to nodulate bean plants. The results of this experiment suggest that

this is not the case. The three strains formed infection threads when in contact with the

Nod- mutant P. vulgaris and the mean number of infection threads formed were not significantly different from the mean number of infection threads formed on the wild- type host. The mean number of nodules formed by the three overcoming strains was not significantly different from the wild-type which indicates that the infection threads seen during the initiation phase are indeed what leads to the formation of nodules on the Nod- mutant P. vulgaris.

Many strains of Rhizobia are excluded from nodulating the Nod- mutant P.

vulgaris. This experiment allowed us to examine the point at which the nodulation of the

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Nod- mutant is restricting certain Rhizobia strains. USDA 2669 did not form any infection threads, and did not cause root hair curling on the mutant P. vulgaris (Figure 4-

5, Table 4-1). Therefore, it can concluded that when the mutant P. vulgaris is inoculated with the excluded strains both root hair curling and infection thread formation are absent.

In Figure 4-2 root hairs do not curl when the host plant has not been exposed to any Rhizobia. One can see the same reaction when the mutant P. vulgaris is inoculated with USDA 2669 (Figure 4-3). USDA 2669 does induce root hair curling on the wild- type host, supporting earlier results that the strain does in fact recognize plant flavonoids and respond to them by forming LCOs. These LCOs then signal the plant to begin root hair curling. The excluded strain USDA 2669 did not induce root hair curling and did not form any infection threads on the mutant P. vulgaris (Fig 4-3 and Table 4-1).

Together these two experiments suggest that the Nod- P. vulgaris is behaving as the wild-type during initiation of nodulation, while maintaining a selective characteristic that is somehow choosing between strains of Rhizobia that nodulate the host. This selective restriction is strain specific and is occurring at some point prior to root hair elongation and curling, as well as prior to infection thread formation. Referring to the four basic steps (Introduction) necessary to form a symbiosis the Nod- mutation is blocking nodulation prior to step 4) Host plant perception and physical response to the bacterial LCOs includes root hair curling and root cell division. Based on previous research, we can surmise that the mutant P. vulgaris is in fact releasing flavonoids that those flavonoids are recognized by at least the overcoming strains and they are in fact turning on LCO production (Chapter 2). This raises the possibility that the LCO structure

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and profile of the excluded strains as well as the overcoming strains are the determining

factors in strain selection.

Research has provided, through chemical mutagenesis, a variety of host legumes

with the ability to restrict nodulation, but many of these result in the formation of

ineffective nodules as opposed to a restriction at the initiation point (Benaben et al. 1995,

Weiser et al. 1990). Benaben et al. isolated TE7, after chemical mutagenesis with

ethylmethane sulfonate, a M. truncatula species that forms two types of ineffective

nodules on the same root system. The first type of nodules is small and round and

completely void of any bacteria. The second type is elongated nodules with bacteria

inside that never form bacteroids (Benaben et. al 1995). In addition, Weiser et al. (1990)

isolated a mutant soybean (G. max) that forms small inefficient nodules with USDA 31.

Both of these examples show that a mutation in the legume genome can prevent

formation of efficient nitrogen fixing nodules, but after the nodule is formed. The Nod- mutant P. vulgaris in this paper is novel in that the nodulation restriction occurs prior to nodule formation.

No “piggybacking’ was observed following inoculation of the Nod- mutant with

both USDA 9032 and GFP-USDA 2669. Infection threads were formed, presumably by

USDA 9032 but none had any fluorescent bacteria present inside them (Table 4-2).

Therefore, when an infection thread was formed by USDA 9032, USDA 2669 was still

excluded and therefore, the excluded strains did not "piggyback" inside the infection

thread (Table 4-2). Piggybacking was not observed when both USDA 9032 and GFP-

USDA 2669 were inoculated onto the wild-type host. These results indicate that although

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Long (1989) observed this phenomenon, it does not occur with this specific cultivar of P.

vulgaris. Long (1989) concluded in her experiments that in many cases certain strains of

Rhizobia are able to enter an infection thread on a root hair that the strain itself did not

signal to form. The research showed that this is possible for Rhizobia in the rhizosphere

and vicinity of the growing infection thread. It was hypothesized that because of this

research it was possible for USDA 2669 to piggyback with an overcoming strain. This

experiment shows that this was not the case.

From this it can be concluded that the Nod- mutant will remain selective during

the nodulation process. If this mutant could be used in agricultural settings, it is likely

that the plant can select strain specific Rhizobia, form the symbiosis while preventing unwanted Rhizobial strains from entering into the nodulation process and therefore the

resulting nodule.

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Chapter 5 LCOs Derived from Overcoming and Excluded Rhizobial Strains

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Summary

Initiation of the symbiosis between P. vulgaris and Rhizobia sp. is only partially

understood. Scientists have accepted that a variety of plant flavonoids act as

transcriptional activators to nod genes in the bacteria by binding to nodD gene products and upregulating the structural nod genes. Nod genes encode the lipo-chitin oligosaccharides (LCOs) which are extracellular signal molecules that are able to induce the deformation of root hairs and initiation of nodule organogenesis. LCOs are believed to determine host range based on their structural modifications. Researchers have shown different LCO modifications in different Rhizobial species, but specifically have not shown their exact role in nodulation of various host P. vulgaris.

This experiment attempts to define the role LCOs play in nodulation of the P.

vulgaris mutant. LCOs were isolated from overcoming and excluded strains using high

performance liquid chromatography. These isolated LCOs were examined for their

biological activity on both wild-type and mutant P. vulgaris. Biological activity was assessed by examining the host roots for root hair curling and infection thread formation.

LCOs were isolated from the three overcoming strains, USDA 9017, USDA 9032 and USDA 9041, as well as from the restricted strain USDA 2669. The isolated LCOs were all able to induce root hair curling on the wild-type P. vulgaris. Only the three overcoming strains' LCOs were able to induce root hair curling on the Nod- mutant.

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Introduction

A successful symbiotic relationship between P. vulgaris and Rhizobia sp. is

specific and dependent on reciprocal signal exchange between the symbiotic partners

(Fisher and Long 1992). Specific plant flavonoids are excreted into the rhizosphere,

there soil Rhizobia detect and take up the flavonoids that act as co-transcriptional activators with nodD gene products to induce Rhizobial nodulation (nod) genes. The nod genes encode lipo-chitin oligosaccharides (LCOs), which are excreted into the surrounding rhizosphere. The mechanism is not known, but LCOs are believed to signal the host plant to begin the nodulation process by recognition of the specific LCO signal and responding by root hair curling (Lopez-Lara et al. 1995).

Scientists have determined that the flavonoids produced by the host plant bind to

the product of the constitutive nodD gene in the endosymbiont and they work together as

transcription factors that upregulate the transcription of common nod genes; NodA, NodB,

and NodC. The nodABC genes are essential for nodulation in that they are responsible

for producing the basic lipo-chitin oligosaccharide (LCO) backbone consisting of three to

five β-1,4 linked glucosamine residues bearing an amide bound fatty acyl residue. If any

of the three genes are mutated or deleted, the LCO cannot be formed or exported to signal

the plant to begin to have root hair curling and deformation. These genes are

homologous across almost all rhizobium species (high degree of sequence and functional

homology) (John et al. 1993, Geremia et al. 1994, Bladergroen and Spaink 1998, Denarie

et al. 1996, Downie et al. 1998).

LCOs are believed to determine host specificity (Denarie et al. 1992, Denarie et

al. 1996, Folch-Mallol et al. 1996, Cardenas et al. 1996, Johnston 1998, Perret et al.

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2000). Different Rhizobial species produce characteristic nod factor structures with

chemical substitutions on the reducing and non-reducing sugars, and variations in the

structure of the acyl chain. Some Rhizobia are very promiscuous in that they are able to

form nodules on a wide range of legume genera (Michiels et al. 1998). This can be seen

in the Rhizobium strain NGR234, which has a wide host range, including common beans,

alfalfa, and clovers. Other Rhizobium species and strains are very specific such as

Bradyrhizobium japonicum, which is species specific for soybeans (Glycine max).

Structural variations influencing the biological activity of the LCOs are sulfation of the

reducing terminal residue (Roche et al., 1991) variations in the degree of unsaturation of

the fatty acid (Spaink et al. 1991, Truchet et al. 1991), O-acetylation of the non-reducing

terminal residue (Spaink et al. 1991) and variations in the length of the oligosaccharide

backbone (Schultze et al. 1994)

When a plant detects LCOs it responds as follows; first the zone of root hair

growth responds, all different tissues of this responsive region, including the epidermal

and cortical cells respond to LCOs. Nod factors have been shown to elicit epidermal

responses, such as root hair deformation or induction of certain nodulation genes, at pico-

or even femtomolar concentrations. The response seen in epidermal cells, such as

cortical cell division, that are in direct contact with LCOs is so rapid and specific that they seem to be prime candidates for perception of LCOs (Denarie et al. 1992, Denarie et al. 1996, Long 1996, Phillips 1992, Schmidt et al. 1988).

Scientists have not determined how Phaseolus sp. detect and recognize LCOs but

research has been done on related plants. Plants in both Trifolieae and Vicieae, e.g.

Medicago, Pisum and Vicia, have two different receptors that recognize 3-5 linked N-

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acetyl glucosamines and chitin fragments respectively (Recourt et al. 1991). Other

research has shown that LCOs will rapidly insert into membranes without receptors, and studies with fluorescently labeled molecules suggest that they can transfer either into

membranes vesicles or directly into plant roots, probably by a mechanism involving

desorption of monomers. However, LCOs appear unable to flip-flop between membrane

leaflets and therefore cannot spontaneously enter the plant cell without a specific

reception and transport mechanism. Moreover, the physiochemical properties of LCOs

imply that their initial perception is at or outside the cell membrane and might involve

recognition of LCOs either from the aqueous rhizosphere or from the plant or bacterial

membranes (Long 2001).

When examining the legume roots as having a recognition system, past studies

have found that legume roots contain a variety of enzymatic activities that can degrade nod factors, some of which preferentially cleave certain LCOs depending on their substitutions. It has been suggested that the preferential hydrolysis of certain nod factors might contribute to the specificity of nod factor perception (Denarie et al. 1996,

Fujisheige et al. 2006), although it is unlikely that this occurs rapidly enough to influence many nod factor responses. The precise role of these products in normal nodulation remains undefined.

If LCOs are indeed responsible for host range, then it can be hypothesized that the

LCO signal is what is preferentially determining the host range of the mutant P. vulgaris.

Isolated LCOs have been shown to induce deformation of root hairs, the formation of pre-infection threads, and the division of root cortical cells (Carlson 1993, Lerouge 1990,

Lopez-Lara et al. 1995). In order to assess this hypothesis this experiment attempts to

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isolate the LCOs produced by Rhizobium sp. USDA 2669, USDA 9017, USDA 9032, and

USDA 9041 using High performance liquid chromatography (HPLC) after LCO induction by the addition of the plant flavonoid naringenin. The isolated LCOs were

examined for their biological activity with the mutant P. vulgaris. The roots were

examined for root hair curling and infection thread formation.

Materials and Methods

Isolation of LCOs

Bacterial cultures (USDA 2669, USDA 9017, USDA 9032, and USDA 9041)

were grown in 100 ml TY (chapter 2 materials and methods) culture overnight at 30º C

with continuous shaking. Each 100 ml culture was inoculated into 1 L of TY media and

incubated at 30º C overnight in a shaker. Naringenin was added at a concentration of

0.15 mM to induce LCO production (Cocking 2003). The next day, 500ml of 1-butanol

was added and the mixture was replaced on the shaker overnight at 30º C. After 24 hours

the flask was taken out of the shaker and left to sit on the bench top for several hours

while the two phases separate.

The butanolic phases was removed and placed in a fresh beaker. The butanolic

phase was vacuum dried at 60º C. The residue was redissolved in 5 ml acetonitrile and

filtered through a C18 cartridge from Resprep. The solution was vacuum dried at 60º C

and redissolved in 1% CHAPS to a total of 25ml. This solution was used at 0.1%

CHAPS for HPLC and biological assays (Cardenas et al. 1995, Soria-Diaz et al. 2003).

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High Performance Liquid Chromatography (HPLC)

The isolation of LCOs on a Pharmacia SuperPac Pep-S column (5mm, 4 X 250 mm)

was accomplished with the following protocol (Cardenas et al. 1995, Soria-Diaz et al.

2003);

a. 5 min of isocratic elution with 30% acetonitrile

b. 30 min of isocratic elution with 40% acetonitrile

c. 15 minutes of isocratic elution with 50% acetonitrile

d. linear gradient in 10 min from 50-100% acetonitrile

HPLC elution was performed at a flow rate of 0.7 ml min –1 and the eluent was monitored

at 206 nm. All fractions were kept for biological activity assay.

The experiment was repeated three times at Ohio University plus an additional repetition at the Complex Carbohydrate Research Center (CCRC) in Athens, Georgia.

Biological activity of LCOs

Isolated LCOs from the previous experiment were used as inoculant on the wild-

type and mutant P. vulgaris. Host seeds were surface sterilized and placed in 6" azalea

pots with vermiculite at 30º C to stimulate germination. After germination

(approximately 48 hours), seedlings were transferred to growth pouches.

LCOs were inoculated onto the growth pouches at a concentration of 0.1%

CHAPS, to represent the average amount of LCOs produced during biological Rhizobial

nodulation. Root hair curling and infection thread formation was examined

microscopically 2, 3 and 5 days after inoculation. One centimeter pieces of the roots

were placed on a labeled slide and examined for root hair curling or infection thread

formation. The label was then covered with tape to ensure a blind study.

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Results

Figure 5-1 shows the baseline peak profile for naringenin alone. Lipo-chitin

oligosaccharides were produced and isolated from all four strains, USDA 2669, USDA

9017, USDA 9032 and USDA 9041 (Figures 5-2, 5-3, 5-4, and 5-5). All four HPLC

profiles showed similarity to LCOs isolated from other species, e.g. R. giardinii bv.

giardinii (Figure 5-6). Each bacterial culture produced at least 30 products after being

exposed to naringenin (Figure 5-2, 5-3, 5-4, and 5-5).

The profiles differed in number and location of peaks. Figure 5-2 shows the

LCO profile for USDA 2669 after exposure to naringenin. USDA 2669 produced new

products in response to naringenin that eluted at 5-8 min (minutes), 15 min, 18 min, 21

min and 22-25, with an overall increase in peak strength and height in peaks 17 through

25. USDA 9017 also showed chemicals produced in response to naringenin with new

peaks at 6 min, 21 min, 22 min and 24 min (Figure 5-3). When exposed to naringenin,

USDA 9032 produced two new peaks at 8 min 30 sec (seconds) and at 17 min 30 sec

(Figure 5-4). When exposed to naringenin, USDA 9041 produced new products as seen

in Figure 5-5 at 5 min, 9-11 min, 14-15 min with an overall increase in the intensity and

height of peaks 22-26 min.

Plant response to isolated LCOs

Plants were examined for root hair curling at 2, 3 and 5 days after addition of crude

extract (daa). Isolated LCOs from USDA 2669 induced root hair curling on the wild-type

P. vulgaris, but did not induce root hair curling on the mutant P. vulgaris (Figure 5-6,

Table 5-1). Isolated LCOs from USDA 9017, USDA 9032 and USDA

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Figure 5-1. Baseline of HPLC with naringenin.

119

Figure 5-2. HPLC of USDA 2669 with and without naringenin induction.

120

Figure 5-3. HPLC of USDA 9017 with and without naringenin induction. A) USDA 9017 exudates without naringenin. B) USDA 9017 exudates after induction with naringenin.

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Figure 5-4. HPLC of USDA 9032 with and without naringenin induction. A) USDA 9032 exudates without naringenin. B) USDA 9032 exudates after induction with naringenin.

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Figure 5-5. HPLC of USDA 9041 with and without naringenin induction. A) USDA 9041 exudates without naringenin. B) USDA 9041 exudates after induction with naringenin.

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A B

C D

Figure 5-6. Root hair response to inoculation with LCOs isolated from HPLC after induction with naringenin. A) mutant P. vulgaris inoculated with LCOs from USDA 2669 B) mutant P. vulgaris inoculated with LCOs from USDA 9017, C) mutant P. vulgaris when inoculated with LCOs from USDA 9032 and D) mutant P. vulgaris response when inoculated with LCOs from USDA 9041.

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Table 5-1. Curled root hairs seen in LCO inoculated P. vulgaris 2, 3, and 5 days after addition of crude extract (daa).

Treatment Root hair curling days after inoculation (dai)______Wild-type P. vulgaris Mutant P. vulgaris 2 daa 3 daa 5 daa 2 daa 3 daa 5 daa LCOs USDA 9017 - + + - + + LCOs USDA 9032 + + + - + + LCOs USDA 9041 - + + - + + LCO;s USDA 2669 + + + - - - No inoculum ------

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9042, the overcoming strains, induced root hair deformation and curling on both the wild- type and mutant P. vulgaris (Table 5-1 and Figure 5-7).

Discussion

All three overcoming strains, USDA 9017, USDA 9032 and USDA 9041, produced butanol extractable products when exposed to naringenin (Figures 5-3, 5-4, 5-5

and 5-6). The chemicals isolated using HPLC were added onto both wild-type and

mutant P. vulgaris. Both plant types responded to the inoculant by exhibiting root hair

curling, the response typically seen when initiating a symbiosis (Figure 5-7 and Table 5-

1). In a few of the curled root hairs, structures that appear to be infection threads were

seen (Figure 5-6 D). The butanol soluble products show similar HPLC patterns to LCOs

isolated in previous experiments (Figure 5-7, Soria-Dias et al. 2003). Further research

must be done in order to examine the exact structure of these chemicals, but it can be

concluded that these are LCOs and that they are responsible for signaling both the mutant

and wild-type P.vulgaris to initiate the nodulation process. Scientists at the Complex

Carbohydrate Research Center in Athens, Georgia confirmed LCO HPLC profile.

The restricted strain USDA 2669 was exposed to naringenin and the products

were collected and isolated. The results of the HPLC (figure 5-2) show similarity to LCO

products of other Rhizobia sp (Figure 5-7). The butanol soluble products from USDA

2669 induced root hair curling on the wild-type P. vulgaris host suggesting that in the isolated chemicals, there are indeed the necessary signaling products present (Figure 5-6,

Table 5-1). Consistent with the lack of nodulation of root hair infection thread formation,

isolated LCOs from USDA 2669 did not induce root hair curling when inoculated onto

the mutant P. vulgaris (Figure 5-6, Table 5-1). This suggests that these chemicals are the

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Figure 5-7. HPLC profile of extracts from R. giardinii bv. giardinii H152 (Soria- Dias et al. 2003). The researchers carried out an n-butanol extract from a culture of R. giardinii bv. giardinii H152. F denotes "Fraction" used for analysis.

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determining factor in the root hair curling step necessary for initiation of nodulation and

that some aspect of the LCO complex produced by USDA 2669 lacks the needed

structure to elicit a response from the mutant host plants.

The HPLC did not conclusively identify all of the chemicals present, but we can make conclusions from the response of the plant to those isolated chemicals. First, the

HPLC profiles show the Rhizobial products without the presence of naringenin, a plant

flavonoid that stimulates LCO production. Once naringenin was added, the HPLC profile

of USDA 2669, USDA 9017, USDA 9032, USDA 9041 all change and due to their

similarities to other LCO profiles, we can assume that all four strains do produce LCOs.

(Michiels et al. 1998). In addition, all four strains’ exudates are able to elicit a response by the wild-type host.

The products that are exuded by the four strains when exposed to naringenin are

stimulating a plant response. When the products from USDA 2669, USDA 9017, USDA

9032 and USDA 9041 are inoculated onto wild-type the plants' root hairs grew and curled

(Figure 5-6). When the products exuded by the four strains were inoculated onto the

mutant P. vulgaris, root hair curling only occurred when the mutant was inoculated with

one of the three overcoming strains. Exudates were also collected from bacteria that

were not induced with naringenin, these exudates were inoculated onto both wild-type

and Nod- hosts and resulted in no root hair curling. These findings suggest that the

isolated compounds are LCOs based on research that has shown that isolated LCOs are

sufficient in signaling a plant response (Aguilar et al. 2006, Cardenas et al. 1995, Soria-

Diaz et al. 2003, Soria-Diaz et al. 2006) as well as the findings that LCOs determine host

128

range in a variety of legumes (Vargas et al. 1990, Roche et al., 1991, Spaink et al. 1991,

Truchet et al. 1991, Schultze et al. 1992).

Although it is not conclusive that the signaling molecules are LCOs, this

experiment suggests that the exudates are necessary for nodulation of the Nod- mutant P.

vulgaris. These exudates signal the plant to begin the symbiosis and are therefore an

essential part of the nodulation process. This experiment also leads us to another

discovery. The Nod- mutant is known to restrict nodulation. Previous experiments show

that excluded strains do not form nodules and they do not cause root hair curling or

infection thread formation. This experiment shows that the excluded strains do indeed

respond to plant flavonoids, i.e. naringenin, and produce an LCO signal. This signal is

able to induce wild-type plant symbioses, but not the Nod- mutant. This would suggest

that the Nod- mutant is selecting the Rhizobial strains based on their LCO fingerprint and therefore, it is the LCO fingerprint that determines host range in the bean rhizobium symbiosis. Additionally, LCOs are the chemical signals responsible for host specificity

(Denarie et al. 1992, Denarie et al. 1996, Folch-Mallol et al. 1996, Cardenas et al. 1996,

Johnston 1998, Perret et al. 2000, Michiels et al. 1998, Roche et al. 1991, Spaink et al.

1991, Truchet et al. 1991, Schultze et al. 1994).

Wild-type plants perceive and respond to USDA 2669; however the Nod- mutant

cannot detect or respond to USDA 2669, but still retains the ability to respond to LCOs of a few strains. The salient result of this experiment is not the exact identity of the LCOs but rather the presence of those LCOs in the presence of naringenin. Hypothetically, the

recessive Nod- mutant could lack the correct flavonoid signal that would induce production of LCOs from USDA 2669 and therefore prevent nodulation. This

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experiment shows that USDA 2669 does indeed produce LCOs, but they are not able to

induce early symbiosis specific responses in the mutant host and therefore the Nod- host has the correct receptors, the restricted strains do not have the correct signal.

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Chapter 6 Competitiveness of the Overcoming Strains

131

Summary

Nitrogen fixation in beans is highly inefficient. Many scientists have focused on

developing a super nitrogen fixing strain of Rhizobia, but have had poor field trials resulting in little or no nodule occupancy by these selected strains. The reason appears to be that the super nitrogen fixing strains do not have the ability to compete with native

Rhizobia in a soil environment, in the field. The Nod- mutant bean has the ability to form

nodules with only specific strains of Rhizobia and it may be possible to engineer one of

the overcoming strains such that it provides enhanced nitrogen fixation. When such a

strain is coupled with host plants that are homozygous for the nodulation-restrictive

allele, a very high level of nodule occupancy by the elite strain might be achieved even in

field soils with a resident population of bean compatible Rhizobia.

This study was performed to determine if an overcoming strain could compete with other strains for nodule occupancy of the wild-type and mutant P. vulgaris. For ease

of identification, overcoming strains USDA 9017, USDA 9032 and USDA 9041 were

transformed with the green fluorescent protein (GFP). Wild-type and mutant P. vulgaris

were inoculated with a transformed overcoming strain and the excluded strain USDA

2669. After 3-4 weeks, roots were examined for nodules containing fluorescent bacteria

(overcoming strain) or non-fluorescent bacteria (USDA 2669). Results indicate nearly

100% nodule occupancy of fluorescent Rhizobia (USDA 9017, USDA 9032 and USDA

9041) in nodules of the nodulation-restrictive host.

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Introduction

The nitrogen fixation (nif) genes are found on the same plasmid as the nodulation

genes (nod) suggesting that the characteristics are inherited together and therefore affect

the survivability of the species where they are maintained (Hombrecher 1981). Even

though nodulation capacity and nitrogen fixation capacity are found together in the

Rhizobial genome, success in one aspect does not correlate with success in the other.

Some Rhizobial strains are more efficient at fixing nitrogen, but not as efficient at competing for nodule occupancy, while other strains are efficient at nodulation and yet

are not as efficient at fixing nitrogen. When compared with soybeans, which are 90% efficient at fixing nitrogen, the Rhizobia symbiosis with beans is inefficient, fixing 30% of available nitrogen (Michiels et al. 1998).

Recognition of the importance and need of sustainable agriculture is driving the

increase in utilization of intercropping, crop rotation and no-tillage agriculture. These

practices are dependent on the presence of legumes with high nitrogen fixing capabilities

(Hungria 1994, Phillips 1992). But, as stated above, one of the most important legume

crops, beans, has low nitrogen fixing capabilities in field conditions (Michiels et al.

1998).

Success in isolating Rhizobial strains that are highly efficient in fixing nitrogen

failed to translate into improved nitrogen fixation in field trials. The super nitrogen

fixing strains did not occupy a large percentage of nodules and did not increase the

amount of nitrogen accumulated. Nodulation of economically important crops by

superior inoculant strains seems to be limited by highly competitive soil resident strains

(Hopper et al. 1995, Kapulnik et al. 1987, Hungria and Phillips 1993, Nishi et al. 1996).

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The Rhizobia may be employing a survival mechanism that focuses on out-

competing other strains for nodule occupancy while sacrificing nitrogen-fixing capability.

Legumes, such as beans, determine nodule occupancy based on the symbiont at initiation of nodulation, not after the infection is complete. Once the bacteria are established and a nodule is formed, the plant will control the number of nodules, not the occupancy or identity of the Rhizobial endosymbiont, and therefore the host plant cannot differentiate between poor nitrogen fixers and efficient nitrogen fixers after the nodule is formed. The host plant determines the number of nodules based on nitrogen demand, not on the identity of the Rhizobia. Rhizobial occupancy is determined by the LCO signal produced at initiation of nodulation independently of nitrogen fixing capability (Roche et. al 1991).

The process of nodulation can be separated into two distinct actions; 1) Initiation of nodulation and 2) Development of the nodule. The nodulation control mechanisms employed by P. vulgaris, along with other legumes, only takes place after the symbiosis is formed. P. vulgaris cannot choose at the initiation of nodulation which strains of

Rhizobia, within their natural symbionts, are going to nodulate its roots (Amarger 1981).

Only after the symbiosis is initiated, P. vulgaris will block nodulation if N supply is sufficient (Day et al. 1989, Rolfe and Gresshoff 1980). The plant host uses ethylene signals to regulate the number of nodules that form and to block formation of new nodules (Singleton and Stockinger 1983). The plant host responds by senescing any nodules by acid hydrolysis, limiting carbon supply or limiting oxygen supply (Udvardi and Kahn 1993). Little is known of the mechanisms of how the host plant will detect amount of nitrogen and regulate the number of nodules.

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Researchers in the field of nodulation biology, when given the tool of

recombinant DNA technology, set out a goal of engineering a super strain of Rhizobium

that would not only be able to fix high amounts of nitrogen but also nodulate a wide

variety of plants from an array of families.

After years of research, results showed that their goals needed to shift and focus

specifically on producing super nodulating and highly efficient nitrogen fixing strains for a given host. As researchers selected genetically engineered super bacteria, they began inoculating them in the field. In beans (Phaseolus vulgaris L.), inoculation of the super

Rhizobium strains generally failed to increase yields because the inoculant strains could not compete with native, or resident strains and therefore could not occupy a sufficient portion of the nodules (Moxley et al. 1986, Ramos and Boddey 1987, Thies et al. 1991,

Wolff et al. 1991).

These findings led scientists in an alternate direction, competition (Aguiler et al.

2001, Drevon et al. 2001, Hungria et al. 2000). Super nodulating and nitrogen fixing

Rhizobia were of no use if they could not out-compete native strains and inefficient strains. Vlassak et al. (1996) began looking at intrinsic competitiveness of Rhizobial strains and found two highly competitive strains, KIM5 and TAL182. These results showed that both strains occupied a majority of the nodules in field trials of monoxenic environments. The studies also showed that the competitiveness was greatly influenced by the environment. Therefore, one strain would only be competitive in a single environment and not competitive in a variety of environmental conditions. These results were inconsistent with the hypothesis that one or a few genes could be isolated that would confer high competitive ability on elite strains.

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Both KIM5 and TAL182 were examined further and found to be competitive in very specific conditions. KIM5 would be competitive in high temperatures and high aluminum toxicity, but more sensitive than other strains to a low pH and high concentrations of tannins and phytoalexins in the surrounding soil (Wolff et al 1991).

Alternatively, TAL182 shows a drastic drop in competitiveness when inoculated in unlimed soils (Frey and Blum 1994).

R. tropici as a species is highly competitive in warm weather tropics, when inoculated into field soils that are acidic and have high concentrations of metal. Vlassak et al. (1996) found two strains of R. tropici IIB that seemed to stand out as highly competitive strains among all the R. tropici strains, CIAT899 and F98.5. Initial studies showed that both strains showed persistence and competitiveness in the field, but they also demonstrated that both subgroups disappeared from the nodule population over time and were replaced by a Rhizobial population which consisted mainly of other bean- nodulating species such as R. etli, R. leguminosarum bv. phaseoli or R. tropici IIA. From this field experiment it was not clear whether the tested R. tropici IIB strains had disappeared from the soil population because of their poor adaptation and survival in the prevailing soil conditions, or whether they were still present in the field, but showed a poor competitiveness for nodule occupancy compared to the developed indigenous bean- nodulating population (Vlassak et al. 1996). Nodule occupancy was examined both early

(when intrinsic competitiveness of the strains is most prevalent) and at a later plant growth stage (when environmental factors become important as well). The experiment showed that in monoxenic conditions, both R. tropici IIB strains proved more competitive than the R. etli strains at early harvest (13 days after inoculation). At the later harvest (27

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days after inoculation), the high competitiveness of R. tropici IIB strain CIAT899 was still prevalent, while both treatments with R. tropici IIB strain F98.5 demonstrated a significantly lower nodule occupancy than was measured than at early harvest. In the soil core microcosms, the lower competitiveness of R. tropici IIB strain F98.5 was evident at early harvest (16 days after emergence) as well as at the later harvest (37 days after emergence). R. tropici IIB strain CIAT899 was still competitive in the soil core microcosms at early harvest, but lost part of this competitiveness at a later stage of the plant growth, which is demonstrated by the significantly lower nodule occupancy for R. tropici IIB strain CIAT899 on bean plants 37 days after emergence. Finally, this experiment indicates that the inoculated R. tropici IIB strains CIAT899 and F98.5 possess a good intrinsic competitiveness declines at a later plant growth stage and in multiple year, field soil conditions. The poor saprophytic competence of R. tropici IIB strain

CIAT899 was further demonstrated by its poor survival in soil core microcosms after bean harvest. Other Rhizobial isolates from field plots that had been repeatedly inoculated with R. tropici IIB strain CIAT899, showed higher nodule occupancy compared to R. tropici IIB strain CIAT899, and this higher competitiveness exhibited by the field isolates might be an additional reason for the poor performance of the inoculant strain R. tropici IIB strain CIAT899 in the field study. Plots with and without a history of bean production revealed after 3-year bean cultivation an almost completely different population that also significantly differed in competitiveness (Vlassak et al. 1996).

As stated in the introduction, three strains, USDA 9017, USDA 9032 and USDA

9041, which are found in two different species, R. leguminosarum and R. etli, can nodulate the Nod- P. vulgaris. This experiment had two objectives; 1) to determine if any

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of the overcoming strains would out-compete the restricted strains when both were

inoculated on the wild-type and 2) to determine if the mutant plant would exhibit equivalent nodule occupancy when inoculated with a single overcoming strain versus an overcoming strain in combination with a restricted strain. The competitiveness of the three overcoming strains was examined against the restricted strain, USDA 2669, on both mutant and wild-type host P. vulgaris. The experiment combined two strains of

Rhizobia, pairwise, to see if the mutant can select out the overcoming strain, from the mix and develop abundant nodulation. The bacteria were inoculated in combinations as follows; USDA 2669 + USDA 9017, USDA 2669 + USDA 9032, and USDA 2669 +

USDA 9041.

Materials and Methods

Nodule occupancy in growth chambers and under greenhouse conditions

P. vulgaris wild-type and mutant seeds were sterilized, as described previously

(Chapter 2), and grown in vermiculite in Cone-tainers™ for 48 hours. Seedlings were

provided with water and grown in 30º C incubators. After germination plants were

inoculated with equal amounts of Rhizobia, approximately 2 million cells/ ml, determined by spectrometer at 690 nm and placed in a growth chamber with 8 hours of light and 16 hours of dark. Non-inoculated plants were used as controls as well as plants inoculated with a single strain. In order to clearly identify which strain was occupying any given nodule, the growth chamber experiments were carried out with GFP transformed USDA

2669 being compared to non transformed USDA 9017, USDA 9032 and USDA 9041.

Each of the transformed overcoming strains, USDA 9017, USDA 9032 and USDA 9041 was compared to USDA 2669.

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The experiment was repeated in the greenhouse with the same inoculation

strategies using Cone-tainers™ and plastic 5 gallon buckets with temperatures averaging

28.5/23.4 C day/night. In this experiment USDA 9017, USDA 9032 and USDA 9041

were transformed with GFP. Plants were harvested 28 days after inoculation and examined for root nodule occupancy.

Identification of Rhizobia was done by using GFP-strains of Rhizobia. Each

combination included one strain that was transformed with GFP and one that was not.

All nodules that formed on the plants were collected and placed on glass slides and

viewed under a fluorescent microscope. Strain identity for each nodule was determined

by the presence or absence of fluorescence of the intact nodules (Figure 6-1).

Statistical Analyses

Statistical analyses were done using NCSS. Tukey's two-way T-test was performed and

one-way ANOVA's were performed to test for statistical differences in nodule number (P

≤ 0.5).

Results

All strains tested were able to nodulate the wild-type host successfully with equal

amounts of nodules forming (Figure 6-2). When the mutant bean was inoculated with

the excluded strain USDA 2669 no nodules formed (Figure 6-2). USDA 9032 showed a

slight drop in number of nodules formed when inoculated onto the wild-type and mutant

host, but still formed a significant amount of nodules (Figure 6-2).

Figure 6-2 shows the significant absence of nodules on the mutant host when

inoculated with USDA 2669. The overcoming strains show a lower nodule formation

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Figure 6-1. Three nodules under UV light. The nodules flanking the center nodule are formed by GFP-transformed strains. The nodule in the center was formed by non-GFP transformed strain.

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90 b b b 80 b

b b b 70

50 Wild-type Mutant 40

30 Mean number of nodules of number Mean 20

10 aa a

0 Control 2669 9017 9032 9041 Inoculant

Figure 6-2. Mean number of nodules formed on both wild-type and mutant P. vulgaris when inoculated with individual Rhizobium strains. The graph shows the average number of nodules found on both the wild-type and the mutant P. vulgaris host 28 dai (days after inoculation). All values represent an equal amount of inoculant with 30 replicates. Columns with the same letter represent no statistical difference (Tukey, P ≤ 0.05, N = 30). Control represents non inoculated wild-type and mutant P. vulgaris.

141 when inoculated onto the mutant host, but statistically there was not a significant drop in nodule number (Figure 6-2, P ≤ 0.05).

In the competition experiment, USDA 9017 and USDA 9041 occupied a greater amount of nodules then USDA 2669 occupying 88.8 and 83.2% of the nodules respectively. While USDA 2669 occupied more nodules when in competition with

USDA 9032, residing in 66% of the nodules (Figure 6-3, 6-4), results that have not been seen in previous experiments.

The competition experiment on the mutant host showed that again, USDA 9017 and USDA 9041 occupied 94 and 83.5% of the nodules formed (Figure 6-3, 6-4). When in competition with USDA 9032, USDA 2669 again occupied 66% of the nodules on the mutant host (Figure 6-3, 6-4).

Graphical analysis shows how both USDA 9017 and USDA 9041 out-compete

USDA 2669 for nodule occupancy (Figure 6-3, 6-4). A significant drop in the overall nodule number formed on the mutant is also apparent when the overcoming strain is co- inoculated with the excluded strain (Figure 6-3, 6-4).

Discussion

All four strains, USDA 2669, USDA 9017, USDA 9032 and USDA 9041, were able to form nodules on the wild-type bean. Although it seems that USDA 9032 did form slightly fewer nodules on the wild-type, the lower number of nodules did not differ significantly (Figure 6-2). The three overcoming strains, USDA 9017, USDA 9032 and

USDA 9041, were able to form nodules on the mutant bean in numbers that are not significantly different from the wild-type nodule formation. USDA 2669 did not form nodules on the mutant host when inoculated singly.

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c 80 b 70

b 60

50 d Competitor 40 GFP-USDA 2669 30 Number of nodules of Number 20 e e 10 e aa a 0

M T T M M l W WT 2 tro 7 1 3 n 1 32 W 4 669 0 0 0 2 9017 M 9 9041 M Co 2669 WT 9 9 90 Control WT Competing strain and host type

Figure 6-3. Nodule occupancy of both wild-type (WT) and Nod- mutant (M)P. vulgaris. This graph shows the response of the wild-type and Nod- mutant bean to inoculation with three Rhizobium strains in mixed ratios of 1:1 (v/v, 109 cells ml-1) with GFP-2669. All values represent the mean of 30 replicates with the letters indicating statistically equivalent total nodule number (Tukey, P ≤ 0.05, N = 30). The control represents non- inoculated wild-type and Nod- mutant P. vulgaris.

143

80 b b c 70

60 d

50 GFP-Competitor 40 USDA 2669 30 Number of nodules of Number 20 e 10 e e a a a 0

T T M M

W W 9 7 1 N 9 7 6 1 4 6 1 6 0 6 0 2 9 9032 M 90 Control M 2 9 9032 WT 9041 WT Control WT Competing strain and host type

Figure 6-4. Nodule Occupancy of both wild-type (WT) and Nod- mutant (M) P. vulgaris. This graph shows the response of the wild-type and Nod- mutant bean to inoculation with three Rhizobium strains in mixed ratios of 1:1 (v/v, 109 cells ml-1) with USDA 2669. In this repeat of the above experiment, the competing strains, USDA 9017, USDA 9032 and USDA 9041 are transformed with GFP. All values represent the mean of 30 replicates with the letters indicating statistically equivalent total nodule number (Tukey, P ≤ 0.05, N = 30). The control represents non-inoculated wild-type and Nod- mutant P. vulgaris.

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Examining the competition of the overcoming strains versus the restricted strain,

we see that USDA 9017 was able to occupy 88% of the nodules on the wild-type,

showing its ability, in this experiment, to out-compete USDA 2669. USDA 9041 was

also more competitive than USDA 2669 for nodule occupancy on the wild-type

comprising 84% of the nodules. USDA 2669 was more successful at establishing nodule

occupancy on the wild-type host than USDA 9032 establishing residency in 63% of the

nodules formed (Figure 6-3 and 6-4).

The overcoming strains, USDA 9017 and USDA 9041 increased nodule

occupancy on the mutant P. vulgaris, occupying 96% and 88% of the nodules

respectively. However, USDA 9032 only occupied 32% of the nodules formed on the

mutant P. vulgaris (Figure 6-3 and 6-4). When USDA 2669 is inoculated onto the

mutant, it is not able to form any nodules. It was expected that in combination with

overcoming strains all the nodules formed would be occupied by overcoming strains.

This seems to be the case when inoculating the mutant with USDA 9017 and USDA

9041, but not with USDA 9032.

An unexpected observation arose during the competition experiment. All three

overcoming strains are able to form nodules on the Nod- mutant at a capacity equal to

nodulation of the wild type host. When these strains are inoculated with USDA 2669 on

the wild-type plant, the number of nodules formed drops dramatically (Figure 6-2, 6-3,

and 6-4). Each Nod- mutant forms only a few nodules per plant resulting in low levels of

fixed nitrogen and lowered plant health. Inoculation of USDA 2669 individually on the

Nod- mutant results in no nodulation, but it was not believed to have an effect on the nodulation by the overcoming strains. The presence of USDA 2669 and a second

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inoculum does not reduce nodule numbers of the wild-type plant. These results suggest

that the restriction of nodulation that occurs when USDA 2669 is inoculated onto the

Nod- mutant is impressed upon the overcoming strains.

It is possible that the plasmid carrying the GFP gene is not stable in Rhizobia for

the 4 weeks of the experiment. Other research has shown that GFP is stable, but the

results of this experiment indicate that over time the excluded strain, USDA 2669,

develops the ability to nodulate Nod- P. vulgaris. It is possible that only the overcoming

strains nodulate the Nod- mutant, but overtime they lose their color and therefore appear

to not have GFP and were incorrectly identified as the excluded strain. This would be

remedied by using other GFP alternatives such as RFP (red fluorescent protein) and YFP

(yellow fluorescent protein) which are red and yellow colors.

Field studies of nodulation occupancy have shown that a variety of Rhizobium

species may infect a single host bean (Vlassak et al. 1996, Wolff et al. 1991, Frey and

Blum 1994). These studies suggest that although one strain or species may be more

competitive than another, they do not influence the entire host nodulation. Therefore it

can be expected that the Nod- mutant would respond to a variety of inoculums similarly.

Although, research by Catford et al. (2003) suggests that the presence of initial nod

factors from one strain can influence the nodule occupancy by other strains. The researchers determined that the host plant can autoregulate nodule occupancy based on

feedback inhibition. Catford et al. (2003) showed that when legume root hairs were first

treated with nod factors and then inoculated with Rhizobia they failed to nodulate. A

second experiment by Winarno and Lie (1979), showed similar results when examing

nodulation of pea (P. sativum). During competition experiments they found that in the

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presence of a non-nodulating strain, a nodulating strain will be inhibited from forming a

symbiosis. They concluded that although there is variation in the competitive ability of

Rhizobium strains, that competitive ability is not related to the strain’s ability to nodulate

a host plant. Their results indicate that there is a 24 hour period after inoculation when

competition takes place. The suppression of nodulation by the presence of USDA 2669

is supported by Catford et al. (2003) as well as by Winaro and Lie (1979).

USDA 2669 could be inhibiting nodulation by blocking the formation of infection threads or by producing chemicals that block the host receptors that prevent any other

Rhizobia from forming a symbiosis even if they have the innate capability to do so.

It is not known whether the impediment of nodulation is global or local. Since

both strains of Rhizobia were inoculated at the same time, if the blockage of nodulation

was temporary due to USDA 2669, then the overcoming strains may be able to nodulate

the Nod- mutant after the USDA 2669 restriction wears off.

This question could be answered with a split root system. A future experiment

would study inoculations with two different strains of Rhizobia, an excluded and overcoming strain onto a single plant that has been separated into two root systems. One could conclude from the results whether or not the USDA 2669 restriction is local or global by examining the separate roots for nodule formation.

A follow up experiment could examine if the USDA 2669 nodulation block was

temporal. If one inoculates the Nod- mutant with USDA 2669 and 24 and 48 hours later

inoculates the plant with one of the overcoming strains and nodules formed, this would

mean that the restriction is local and temporary. USDA 2669 may be blocking specific receptors only found on the Nod- mutant that are specific to the overcoming strains’

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signals, or USDA 2669 may be producing chemicals that interact with signals released from the overcoming strains during initiation of nodulation.

If during the experiment, USDA 2669 shuts off nodulation of the Nod- mutant completely and permanently, then one may be observing a system control mechanism where the host plant is responding systemically to the USDA 2669 signal.

Either of aformentioned outcomes would be novel and informative with respect to the host plant, in this case P. vulgaris, responds to the signal produced by its symbiont.

This experiment has shown that while specific strains of Rhizobia are more competitive for nodule occupancy, the complete control of nodule occupancy is not clearly understood. Future research should be done to understand how the Nod- mutant is responding to the signals produced by the Rhizobia that can nodulate it as well by

Rhizobia that cannot nodulate the mutant.

A second follow-up experiment would be to use alternatives to GFP. Scientists have developed GFP with different wavelengths resulting in red and yellow colors (RFP and YFP respectively). When carrying out a competition experiment, each different strain could be transformed with a different color. This would accomplish two goals; 1) it would address the stability of the GFP in the Rhizobia and 2) it would clearly identify each strain. If at the end of the experiment the host plant was nodulated by Rhizobia that were not colored, one could assume that the plasmids carrying the GFP genes were not stable.

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Chapter 7 Tn5 mutagenesis of USDA 2669

149

Summary

This experiment was carried out to determine the identity of the genes involved in

the restriction of the excluded strains by the Nod- mutant P. vulgaris. Chapter 6

suggested that the excluded strains could be producing an additional chemical or alternate

LCO that is preventing them from forming the symbiosis with the mutant host. Tn5

random mutagenesis was carried out on USDA 2669 to determine if nodulation of the

Nod- mutant could be induced when inoculated singly onto the Nod- host. The

mutagenesis resulted in approximately 12,000 clones that were used as inoculum on wild-

type and Nod- mutant P. vulgaris. None of the 12,000 mutagenic products of USDA

2669 had the ability to nodulate the Nod- mutant P. vulgaris, while nodules formed on the

wild-type P. vulgaris. This suggests that USDA 2669, as well as other excluded strains, lacks the correct signals to nodulate the mutant P. vulgaris, rather than produces an additional chemical that blocks its nodulation of the mutant. These results indicate that the lack of correct signals is the impetus for the inhibition of nodulation with the Nod- mutant.

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Introduction

It has been shown that USDA 2669 is excluded by the mutant P. vulgaris at some point during initiation before root hair curling and infection thread formation and after the Rhizobia has detected the plant flavonoids exudate. The excluded strain can successfully nodulate the wild-type host, therefore it can detect the plant flavonoid signal, turns on its nod genes and produce lipo-chitin oligosaccharides (LCOs). The mutant host does not respond to USDA 2669's LCO signal, as no root hair curling and no infection thread formation occurs.

At this point, it is difficult to know exactly why the USDA 2669 as well as 46 other strains was excluded, while three strains have the ability to nodulate the mutant. It is possible that the three strains lack a product that allows them to be misidentified.

Because they are in the minority, it may be that all other strains have a complete genome, while these three strains have mutated in some way as to prevent them from being restricted. The chemical may cause nodulation restriction, including, but not limited to being recognized by the plant as an indicator of a pathogenic organism or being recognized by the plant as an incorrect LCO signal.

The objective of this experiment is to determine the identity of the gene involved in USDA 2669’s specific exclusion of nodulation by the Nod- mutant P. vulgaris. An experiment on competition showed that nodulation of the mutant P. vulgaris is significantly repressed in the presence of USDA 2669, whether or not an overcoming strain is present, therefore it is hypothesized that the excluded strains such as USDA

2669, are producing an additional compound that prevents them from forming the

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symbiosis as well as preventing any overcoming strains from forming the symbiosis as

well.

To examine possible genes involved in the exclusion of nodulation by USDA

2669 Tn5 insertion mutagenesis was used coupled with successful nodulation of the

mutant host P. vulgaris. A transposable element mutation, using Tn5, was set up to

examine the above hypothesis. Tn5 was used to randomly mutate USDA 2669's genome

to determine if nodulation of the mutant P. vulgaris can be induced.

Materials and Methods

Introduction of Tn5 into R. leguminosarum strain USDA 2669 was accomplished

by conjugative transfer of the plasmid pSUP1011 (Simon et al. 1983). The plasmid was

mobilized from E. coli and transferred to R. leguminosarum via triparental mating (Ditta

et al. 1980) with the helper plasmid pRK2073 (Leong et al. 1982). Equal volumes (0.1

ml) of donor and recipient cells were mixed and suspensions were spread onto MAG

agar. The plates were incubated at 30º C for 4 days. Bacteria from the plates were then

suspended in 4 ml MAG broth containing 0.01% Tween 80. Suspensions were vortexed

until cell aggregates were removed and 0.1 ml aliquots were spread onto MAG agar.

Streptomycin and kanamycin were added to select for R. leguminosarum transconjugants

containing Tn5. Tn5 encodes resistance to kanamycin and streptomycin in various

species of Rhizobium (Putnoky et al. 1983, Selvaraj and Iyer 1984). Chloramphenicol

(30 mg/ml) was added to counter-select against E. coli donor and helper strains. R.

leguminosarum is resistant to chloramphenicol at this low concentration. The plates were inoculated for 8 days at 30º C.

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Inoculation of wild-type and mutant P. vulgaris with 12,000 Tn5 mutated USDA 2669 conjugates.

The 12,000 colonies were suspended in water and Jensen's reagent. Ten wild-

type and 10 mutant P. vulgaris seeds were surface sterilized as described previously

(Chapter 2, Materials and Methods). The seeds were place in vermiculite and set to

germinate for 48 hours in a 30º C incubator. Each plant was placed in a new 6" azalea

pot and inoculated with the Tn5 mutated USDA 2669 colonies. The plants were placed

in the greenhouse and watered for 28 days. Plants were then observed for nodule

formation.

Seeds (P. vulgaris) were surface sterilized and germinated as described

previously. A total of 12,000 separate Km/Sm-resistant colonies of USDA 2669 were

inoculated (1 ml of log phase culture) onto 10 Nod- mutant P. vulgaris plants. Plants

were examined for nodule formation 4 weeks after inoculation.

Results

Ten Nod- mutant plants and 10 wild-type plants were examined for nodule formation 28 days after inoculation. The Tn5 mutated USDA 2669 did not induce any nodule formation on the Nod- restrictive mutant P.vulgaris while forming nodules on the

wild-type plant at an average of 15 (Table 7-1).

Discussion

It was hypothesized that the excluded strains (USDA 2669) are producing a

chemical or signal that is preventing the nodulation of the Nod- mutant P. vulgaris.

Random Tn5 mutagenesis on USDA 2669 resulted in the mutated strain keeping its

restricted phenotype. This suggests that there is not a gene product that the restricted

strains produce that would be responsible for the restriction.

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When USDA 2669 was mutated, it resulted in approximately 12,000 clones. The

Rhizobial genome has approximately 6,000 genes, therefore 12,000 mutagenic events

suggests that a wide variety of genes in the Rhizobial genome were mutated. Tn5 mutagenesis was carried out to see if by mutating a specific sequence, we could block the

transcription and production of a gene product that could be involved in the restriction of

nodulation by the mutant P. vulgaris. None of the mutated USDA 2669 Rhizobial strains

could nodulate the mutant, while a few (ave. 15) still maintained the ability to nodulate

the wild-type bean plant. This suggests that by mutating USDA 2669 we did not wipe

out nodulation capacity completely, but we also did not induce nodulation of the mutant

bean. The restricted strain, USDA 2669, does not seem to be producing an additional

product that is preventing it from nodulation the mutant bean.

Although this experiment seems inconclusive on its own, it does provide support

for other experiments. The experimentation with the mutant P. vulgaris up to this point

suggests that the three overcoming strains, USDA 9017, USDA 9032, and USDA 9041

are producing an exclusive LCO profile that distinguishes them from other Rhizobia in

their genus and within their species. Their specific LCO profile, not the existence of

inhibitors or chemicals, it what provides them with the ability to nodulate a mutant host

that would in most cases restrict the symbiosis with Rhizobia. It is, this distinct LCO

profile produced by all Rhizobia that determine their host range. The mutant P. vulgaris

is, perhaps, more specific in the LCO profile necessary to initiate nodulation.

While the excluded strain not only prevents nodulation when inoculated singly

onto the Nod- mutant, it also prevents nodulation in the presence of overcoming strains.

Although the mechanism is unknown, the restriction of nodulation by the excluded

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Table 7-1. Mean number of nodules that formed on the wild-type and mutant P. vulgaris after inoculation with Tn5 mutated USDA 2669. Average number of nodules was determined by counting nodules on the 10 plants of each type (N=10).

P. vulgaris type inoculated with Tn5 Mean number of nodules formed USDA 2669 Wild-type 15

Mutant 0

155 strain affects the Nod-‘s ability to form a symbiosis with Rhizobia with the correct signal.

Although these results indicate some conclusions, it must be remembered that the results were negative and therefore they cannot be completely accepted. As stated in the previous chapter, further research should investigate temporal restriction of nodulation as well as local vs. entire nodulation restriction.

156

Chapter 8 Conclusions

157

Conclusions

The common bean (P.vulgaris) is one of the most important leguminous crops in the world. It provides over 300 million people with a source of food and protein.

Humans and animals all benefit from beans and their nutritional value because of their symbiotic relationship with soil bacteria Rhizobia (Fred et al. 1932, Maier and Triplett

1996). This relationship benefits both organisms, the host legume and the bacteria, by supplying each with the necessary substances for life. The plant host provides a secure niche and environment to harbor the bacteria even providing energy in the form of sugar, while the bacteria actively fix atmospheric nitrogen making excess nitrogen solely available to the host plant. The relationship is highly regulated by both participating organisms in the form of chemicals that are encoded and excreted by the bacteria and the host.

Scientists have recognized the importance of this crop as well as its symbiotic relationship and have therefore studied it extensively. The research focused on two areas;

1) Examining if the beneficial symbiosis could be extended to include non legumes such as corn or wheat and 2) Examining if the current legume/Rhizobia symbiosis could be improved upon. The first area was quickly abandoned when research demonstrated the complexity of the highly controlled and regulated symbiosis.

Therefore, science has focused on improving the established symbiotic relationship between legumes and Rhizobia. It has been stated that the nitrogen fixing efficiency of Rhizobia with beans is very low, around 30%. Many scientists have tried to improve this efficiency by one of two methods, either by focusing on the control and

158

regulation of the symbiotic relationship or by engineering or choosing highly efficient N2

fixing Rhizobial strains that could be used as inoculum in an agricultural setting.

The Nod- mutant P. vulgaris expresses a phenotype of a strain specific selection

of nodulation. With further investigation it was found that this Nod- mutant restricts

nodulation by all but 3 strains tested. These three strains, R. leguminosarum USDA

9017, R. etli 9032 and R. leguminosarum USDA 9041, named “overcoming” strains have

been shown to form a symbiosis with the mutant P. vulgaris. These three strains

represent two species of the five total that can nodulate beans. A wild-type P. vulgaris

has the ability to form symbioses with hundreds of strains within the 5 species in the

genus Rhizobia. The mutant can differentiate between strains within the 5 species. This

selection shows that the host plant is able to differentiate between strains, therefore there

is a selection during initiation and formation of the symbiosis.

This mutant with its specific phenotype was used as a tool to examine two aspects

of the legume/rhizobium symbiosis; 1) Understand the legume/rhizobium symbiosis on a

different level and 2) Examine a new Nod- mutant that could be used in an agricultural

setting. The Nod- mutant not only provides a model to understand the symbiotic relationship, but it also serves as a potential use in an agricultural setting.

To carry out the first objective we must first examine what scientists know.

Currently, it is known that when legumes, including P. vulgaris, are planted in the soil they release a variety of chemicals including flavonoids. Many of these chemicals act as signals that stimulate Rhizobial species in the rhizosphere to reproduce and migrate to the root of the growing plant. Among other functions, flavonoids then act as co- transcriptional regulators of nod genes found in the genome of the Rhizobia sp. The plant

159 flavonoids, along with a constitutive nodD gene product, upregulate other nod genes that encode LCOs or nod factors. These nod factors are exuded into the rhizosphere where they signal the host plant’s root hairs to elongate and form an infection thread whereas the Rhizobia can enter and infect the plant. Once infection or initiation of nodulation is complete a nodule is formed. A nodule is a tumor like growth that is considered a plant organ once it is mature; the Rhizobia reside inside and fix atmospheric nitrogen that becomes available to the plant.

Many scientists have focused on understanding this relationship by examining the identity of flavonoids exuded by the host plant or by characterizing the Rhizobial species that nodulate a specific host while still more research focuses on the identity and structure of the bacterial signal that is exuded into the rhizosphere to signal the plant. Much of this information has been determined but what scientists do not know is the identity of the signal exuded from the bacterial symbiont as well as how they are recognized and perceived by the plant host. The Nod- mutant P. vulgaris that exhibits a strain specific restriction of nodulation is an ideal model to examine these questions closer.

This dissertation has shown that the mutant prevents nodulation by the excluded strains before root hair curling takes place and after the plant excretes the flavonoid signal. Initiation of nodulation can be broken down into 4 basic steps; 1) Excretion of flavonoid signal from the host plant, 2) Flavonoid perception by the endosymbiont, 3)

Flavonoid and nodD gene product transcriptional activation of endosymbiont nod genes that encode lipo-chitin oligosaccharides which are secreted into the rhizosphere, and 4)

Host plant perception of the LCO signal and concurrent development of the nodule. This

160 research has shown that the mutant blocks nodulation sometime after step 3 and before step 4.

This has been shown by first demonstrating that the overcoming strains do respond to signals secreted from the Nod- host. There is a Nod- mutant host response when inoculated with the overcoming strains. In Chapter 5, when naringenin is added to the overcoming strains, they produce an array of chemicals, one, at least, of which is stimulating the root hair curling of the Nod- mutant host (Chapter 5). The chemical fingerprint produced when exposed to naringenin, has shown similiarity to LCO profiles seen with other Rhizobia sp. Although their identity is not known, their HPLC profile and previous research that supports naringenins stimulation of LCO production, suggest that the chemicals are indeed LCOs. Even without knowing their identity, these chemicals are the necessary signals to initiate nodulation of the mutant P. vulgaris (Chapter 4 and

Chapter 5).

Additionally, in chapter 2, it was shown that excluded strains do not nodulate even with naringenin present. But, from Chapter 5, we see that in the presence of naringenin overcoming and excluded strains are producing chemicals that stimulate nodulation of wild-type plants. Therefore, it has been shown that excluded strains are producing LCOs but it is the Nod- mutant plant that is not responding to them.

Secondly, the Nod- mutant is forming fully functional nodules when inoculated with one of the three overcoming strains and it forms these nodules by means of an infection thread (Chapter 4). The restricted strains, sampled as USDA 2669, do respond to wild-type plants and nodulate them by infection thread formation. Once these strains are exposed to wild-type exudates or naringenin, they produce an LCO signal (Chapter 5)

161

but this signal is not sufficient in inducing root hair curling and infection thread

formation on the mutant P. vulgaris (Chapter 2, Chapter 5). The chemicals produced by

USDA 2669 when exposed to wild-type exudates and naringenin do not give the strain

the ability to correctly signal the mutant plant and they cannot nodulate the mutant P.

vulgaris. The mutant P. vulgaris is restricting nodulation at the point of initiation and is

perhaps doing so by responding to the LCO profile produced by the individual strains of

Rhizobia.

This dissertation also supports the claim that LCOs are the bacterial signal that

stimulates nodulation of the host plant as well as determines the host plant’s range. It is

seen above that there are chemicals produced from the overcoming strains as well as from

the excluded strains that are necessary to form the symbiosis with P. vulgaris, or not form

the symbiosis, as the case may be. Chapter 5 shows that these necessary chemicals have

LCO similarities. More support is seen in the random mutation of the USDA 2669

genome. Tn5 mutagenesis of the USDA 2669 resulted in the strain retaining its restricted

status. The results suggest that it is not a chemical or pathogenic mechanism that is

restricting nodulation by these specific strains, but rather their lack of the correct LCO

profile. The strains that are restricted are not being recognized by the mutant P. vulgaris

shown by the mutants lack of response to the individual bacteria and their exudate profile

when exposed to wild-type exudates and to naringenin.

Therefore, this dissertation shows that the Nod- mutant P. vulgaris selectively

restricts nodulation by specific strains of Rhizobia during its response to the

endosymbiont LCO signal. The mutant successfully produces a flavonoid chemical

profile, which the soil bacteria respond to by producing LCOs. The mutant will then

162

select strains for nodulation based on a specific LCO signal, only seen in three strains,

USDA 9017, USDA 9032 and USDA 9041.

These results suggest that the Nod- mutant could be used in an agricultural setting.

Scientists have been trying to isolate and engineer highly efficient N2 fixing bacteria.

Once highly efficient nitrogen fixers are isolated or created, they are inoculated into the

field. Research has shown that inoculum strains cannot compete for nodule occupancy

with native strains. Over the past two decades, a number of strategies have been

proposed to enhance the ability of root nodule bacteria to provide increased amounts of

fixed N to its legume host in the already established symbiosis. Many scientists have not

only hunted down super nitrogen fixing strains (Weiser et al. 1990, Hungria et al. 2000,

Aguilar et al. 2001, Drevon et al. 2001) but have engineered strains that can out compete.

Iniguez et al. (2004) isolated a plasmid with Trifolitoxin-producing genes that could be

transformed into P. vulgaris symbionts. This chemical would prevent nodule formation

by any Rhizobia in the rhizosphere other than the ones carrying the plasmid. Some

scientists have tried antibiotic enhancement, where highly N2-fixing efficient Rhizobia

carry a plasmid to produce antiobiotics. These antibiotics are released into the rhizosphere, killing and inhibiting any bacteria that are not carrying the correct plasmid

(Robleto et al. 1998). Other scientists use gene amplification of nodulation genes in

order to improve competition (Flores et al. 1993, Romero et al. 1991).

The mutant bean plant shows no other deleterious phenotypic responses besides

the altered nodulation pattern. The mutant plant grows and forms leaves and nodules as

does the wild-type plant (Chapter 3). The mutant grows to a similar size and weight and

forms nodules in the same manner as the wild-type, by first signaling the bacteria and

163

then responding to the LCO signal by curling its root hairs. It does not form the

symbiosis through wounds or junctions in the root, instead forms infection threads

(Chapter 4). Because of these reasons the Nod- mutant plant seems like a perfect model

for agriculture. The mutant selectively restricts nodulation and therefore it is possible

that this bean will have the ability to pick and choose beneficial Rhizobia over non-

beneficial Rhizobia at the point of initiation, before establishment of inefficient nitrogen

fixers. As it turns out, this doesn't seem to be the case. As seen in Chapter 6, the nodule

occupancy of the mutant bean tends to be predominantly overcoming strains. But

something else seems to be happening. The overall nodulation of the mutant when

inoculated with an overcoming strain in combination with an excluded strain shows some

unexpected results. The overall number of nodules formed drops significantly suggesting

the mutant will respond to the excluded strain and restrict nodulation whether or not an overcoming strain is present.

These results indicate that the excluded strains are inducing a plant defense

response that is non-specific. It could also suggest that the plant receptors are responding

to the excluded strains and therefore turning off before the overcoming strains have the

chance to signal the mutant host. There is a possibility that over time the overcoming

strains would be able to establish nodule occupancy, but this is not known. The restricted

strain is preventing nodulation of the mutant P. vulgaris, not only of itself, but also of any other Rhizobia including the overcoming strains. The question that needs to be answered, is the restriction of nodulation by USDA 2669 permanent or temporary?

A. tumefaciens is a related bacteria to Rhizobia that causes disease in plants, could

there be a link to the signaling mechanisms between these organisms? It is possible that

164

the mechanism used by the host plant when responding to Rhizobia species is similar and

related to the host plant's defense mechanisms. Similar or homologous receptors could

be involved that not only detect beneficial bacterial signals, but pathogenic ones as well.

The mutant P. vulgaris could have its pathogenic receptors confused with its receptors

involved in nodulation, thereby restricting nodulation from typical Rhizobial species.

The fact that three, a small percentage of overall Rhizobial strains, have the ability to circumvent the mutation and nodulate the Nod- P. vulgaris, shows that they may be the exception, not the rule, and they have the slight difference that makes them unique.

The mutation brings up many more questions that need to be examined. The three

overcoming strains seem to have similar LCO products, suggesting similar genetic make-

up that is different from the excluded strain USDA 2669. The nod genes which encode the LCO structure seem to play a major role and should be examined further. This mutant bean has served as a model to examine specificity of nodulation and will continue to do so in the future. It shows that even within host specificity, there is more specific selection going on at the molecular level. Rhizobial species in the 5 genera are classified based on their host range, simply, can they nodulate or can’t they nodulate the putative host. The specificity of the nodulation process seems to end here, with certain genera being able to nodulate a specific species of plant, while others cannot. This Nod- mutant

P. vulgaris shows a different story. Within the 5 species that can nodulate the P. vulgaris plant, we see further division, strain specific division. Nodulation of a host plant is not done at the species level; it is done at the strain level.

Currently, I am advising an undergraduate senior research student, at Denison

University, who is working on two projects to continue this research. One project is

165

studying the nodulation of the Nod- mutant with a split root inoculation system. We hope

to examine the excluded strain’s control and role in blocking further nodulation of the

Nod- mutant. The second project is designed to create a cosmid library of the overcoming

strain USDA 9041, this experiment aims to categorize the genes found in an overcoming

strain and then insert them into an excluded strain to determine if nodulation of the Nod-

mutant can be induced. Rhizobia are currently classified by their host range and therefore

by which host exudates they recognize. Although, this research shows that the Rhizobia

differ in their LCO profile and that character may be a more correct classification.

Research has shown that the LCO profile is what determines host range, not the plant

flavonoids produced by the host. The five species of Rhizobia that nodulate P. vulgaris are placed in that designation because of their ability to form a symbiosis with the same host. This dissertation has shown that among these 5 species, this is not the case. Within

the species, strains produce different LCOs that change their host range. Therefore, I am

also carrying out a 16s rRNA phylogeny of Rhizobial strains including the three

overcoming strains to examine their molecular relationship.

166

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