Contents
Acknowledgements1
Program2
Plenary Sessions
5
5
Plenary Session I: Nodule Development and Physiology
Plenary Session II: Agricultural Applications
12
Plenary Session III: Evolution of Rhizobia, Legumes, and Symbioses
18
Plenary Session IV: Physiology and Genetics of the Bacteria
21
Plenary Session V: Physiology and Genetics of the Plant Host
26
Plenary Session VI: Genomic Approaches to Symbiosis
29
Plenary Session VII: Associative Fixation, Non-Legumes, & Endophytes
35
Poster Session
38
List of Conference Attendees
93
Acknowledgements
Sponsors
The 22nd North American Symbiotic Nitrogen Fixation Conference gratefully acknowledges the kind support of:
Novozymes Biologicals, Inc., The Samuel Roberts Noble Foundation, the United Soybean Board, the USDA
National Institute of Food and Agriculture, the University of Minnesota Biotechnology Institute, College
of Biological Sciences, and College of Food, Agricultural and Natural Resource Sciences.
Organizing Committee
Mike Sadowsky, University of Minnesota
Ford Denison, University of Minnesota
Carroll Vance, University of Minnesota
Advisory Committee
Jean-Michael Ané, University of Wisconsin
Woo-Suk Chang, University of Texas, Arlington
Brian Driscoll, McGill University
Georgina Hernández Delgado, Universidad Nacional Autónoma de México (UNAM)
Ann M. Hirsch, University of California, Los Angeles
Marc Libault, The University of Oklahoma
Esperanza Martínez-Romero, Universidad Nacional Autónoma de México (UNAM)
Gyaneshwar Prasad, University of Wisconsin, Milwaukee
Janine Sherrier, University of Delaware
Ellen Simms, University of California, Berkeley
Gary Stacey, University of Missouri
Krzysztof Szczyglowski, Agriculture and Agri-Food Canada
Turlough Finan, McMaster University
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Program
Sunday July 14, 2013
Registration
Welcoming Addresses and Opening Session (7:00-8:00 PM)
Opening Remarks, M. J. Sadowsky, University of Minnesota (7:00-7:10 PM)
Keynote Address, Gary Stacey, University of Missouri (7:15-8:00 PM)
Reception/Mixer (8:00-9:30 PM)
Monday July 15, 2013
Breakfast (7:30-8:30 AM)
Plenary Session I: Nodule Development and Physiology (8:30 AM–12:00 PM)
Chairperson: Michael Udvardi
Michael Udvardi, Samuel Roberts Noble Foundation (8:30-9:00 AM)
David Emerich, University of Missouri (9:00-9:30 AM)
Janine Sherrier, University of Delaware (9:30-10:00 AM)
Coffee Break (10:00-10:30 AM)
Senthil Subramanian, South Dakota State University (10:30-11:00 AM)
Federico Sánchez Rodriguez, Universidad Nacional Autónoma de México (UNAM) (11:00-11:30 AM)
Muthusubramanian Venkateshwaran, University of Wisconsin, Madison (11:30 AM-12:00 PM)
Lunch on your own (12:00-1:30 PM)
Plenary Session II: Agricultural Applications (1:30-5:00 PM)
Chairperson: Felix Dakora
Diane Knight, University of Saskatchewan (1:30-2:00 PM)
Donald Smith, McGill University (2:00-2:30 PM)
Felix Dakora, Tshwane University (2:30-3:00 PM)
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Coffee Break (3:00-3:30 PM)
Yaowei Kang, Novozymes Biologicals, Inc. (3:30-4:00 PM)
Larry Purcell, University of Arkansas (4:00-4:30 PM)
Paul Woomer, N2 Africa Program (4:30-5:00 PM)
Posters (5:00-7:00 PM)
Presenters stand by their posters
Dinner on your own
Tuesday July 16, 2013
Breakfast (7:30-8:30 AM)
Plenary Session III: Evolution of Rhizobia, Legumes, and Symbioses (8:30-10:00 AM)
Chairperson: Janine Sherrier
Esperanza Martínez-Romero, Universidad Nacional Autónoma de México (UNAM), Cuernavaca (8:30-9:00 AM)
Ellen Simms, University of California, Berkeley (9:00-9:30 AM)
Pierre-Marc Delaux, University of Wisconsin, Madison (9:30-10:00 AM)
Coffee Break (10:00-10:30 AM)
Plenary Session IV: Physiology and Genetics of the Bacteria (10:30 AM-2:30 PM)
Chairperson: Mark O’Brian
Hauke Hennecke, ETH, Switzerland (10:30-11:00 AM)
Mark O’Brian, SUNY Buffalo (11:00-11:30 AM)
Lunch on your own (11:30 AM–1:00 PM)
Ivan Oresnick, University of Manitoba (1:00 AM-1:30 PM)
Kathryn Jones, Florida State University (1:30-2:00 PM)
Silvia Rossbach, Western Michigan University (2:00-2:30 PM)
Coffee Break (2:30-3:00 PM)
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Plenary Session V: Physiology and Genetics of the Plant Host (3:00-4:30 PM)
Chairperson: Krzysztof Szczglowski
Krzysztof Szczglowski, Agriculture and Agri-Food Canada (3:00-3:30 PM)
Jeanne Harris, University of Vermont (3:30-4:00 PM)
Catalina Pislariu, Samuel Roberts Noble Foundation (4:00-4:30 PM)
Assemble for bus trip to River Boat (5:00 PM)
Banquet (6:30 PM)
Wednesday July 17, 2013
Breakfast (7:30-8:30 AM)
Plenary Session VI: Genomic Approaches to Symbiosis (8:30-11:50 AM)
Chairperson: Ann Hirsch
Turlough Finan, McMaster University (8:30-9:00 AM)
Ann Hirsch, University of California, Los Angeles (9:00-9:30 AM)
Svetlana N. Yurgel, Washington State University (9:30-10:00 AM)
Coffee Break (10:00-10:20 AM)
Nevin Young, University of Minnesota (10:20-10:50 AM)
Georgina Hernández Delgado, Universidad Nacional Autónoma de México (UNAM) (10:50-11:20 AM)
Salehin Mohammad, University of North Texas (11:20-11:50 AM)
Lunch on your own (11:50 AM–1:00 PM)
Plenary Session VII: Associative Fixation, Non-Legumes, & Endophytes (1:00-2:30 PM)
Chairperson: Louis Tisa
K. Minamisawa, Tohuku University, Japan (1:00-1:30 PM)
Louis Tisa, University New Hampshire (1:30-2:00 PM)
Michelle R. Lum, Loyola Marymount University (2:00-2:30 PM)
Coffee Break (2:30-3:00 PM)
Closing Remarks (3:00 PM)
M. J. Sadowsky, University of Minnesota
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Plenary Session I: Nodule Development and Physiology
Take it out of the closet…your ‘old BNF’ is fashionable again!
Gary Stacey
Divisions of Biochemistry and Plant Science, C.S. Bond Life Science Center,
National Center for Soybean Biotechnology, University of Missouri, Columbia, MO, USA
Since the declaration of its official discovery in the late 1800s, biological nitrogen fixation has held the
fascination of the plant and microbial research communities. However, one could argue that the modern era
of biological nitrogen fixation research had its beginnings in the energy crisis of the early 1970s and the
hype that surrounding claims that BNF could provide a major solution to the high cost of fertilizer, derived
from fossil fuels. This is certainly the first time that I heard claims that symbiotic BNF could ultimately be
transferred from legumes to non-legumes, such as corn. The hype of the 70s died down somewhat but was
reinvigorated in the 1980’s when a number of high profile biotech companies became major players in the
field. Clearly, through the full history of BNF, scientific progress has been steady and the field has advanced
significantly with many major discoveries. However, it would seem that, for most of the past few decades,
BNF has not garnered the same attention in public forums and within industry that it did in these periods
in the 70s and 80s. However, in recent years, certainly, the last 5 years, it would appear that ‘Happy Days’
are again here with a number of popular articles appearing about BNF, industry showing increasing interest
and major philanthropic organizations investing. The long held “Holy Grail’ of transferring BNF from
legumes to non-legumes is again being discussed and, more importantly, projects funded. The ‘old BNF’ is
fashionable again. My presentation will cover the following questions: To what do we owe this apparently
new found excitement in BNF? Is the new excitement justified? Are we in a new period of ‘over hype’ or are
exciting new, revolutionary advancements near at hand? What areas of BNF justify this level of attention
and where will the new advancements arise? Will the future be exemplified, as in the past, by a steady and
exciting increase in our basic understanding of BNF or are we in addition entering an era where our basic
discoveries will see real-world application? The talk will cover my own personal views on these questions
and, therefore, the hoped for outcome will be to stimulate further discussion throughout the remainder of
the meeting.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Plenary Session I: Nodule Development and Physiology
Medicago reverse genetics: functional characterization of
regulatory and transporter genes in nitrogen-fixing nodules
Michael Udvardi1, Senjuti Sinharoy1, Igor Kryvoruchko1, Catalina Pislariu1, Ivone Torres-Jerez1,
Kaustav Bandyopadhyay1, Mingyi Wang1, Mark Taylor1, Shulan Zhang1, Xiaofei Cheng1,
Jiangqi Wen1, Jin Nakashima1, Kirankumar Mysore1, Pascal Ratet2, Attila Kereszt3,
Eva Kondorosi2,3, and Vagner A Benedito4.
1. Samuel Roberts Noble Foundation, Ardmore, OK, USA;
2. ISV-CNRS, Gif sur Yvette, France;
3. Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary;
4. West Virginia Universi- ty, USA; [email protected]
Several key resources have now been developed for functional genomics in Medicago truncatula, including
the genome sequence, Gene Expression Atlas (MtGEA), and Tnt1-insertion mutant populations. Currently,
the Tnt1-mutant population consists of 21,700 lines encompassing approx. 500,000 Tnt1-insertions in
the genome. Efforts are underway to sequence genomic DNA adjacent to as many Tnt1-insertions as
possible: currently we have 44,000 of these so-called Flanking Sequence Tags (FSTs) in a searchabledatabase (http://medicago-mutant.noble.org) and plan to have 200,000 FSTs by the end of 2014.
We have taken a reverse-genetics approach, using the Tnt1-insertion mutant population, to determine the
functions of selected transcription factors (TFs) and transporters that are induced during nodule development.
One of the TFs, a C2H2 family member that we named Regulator of Symbiosome Differentiation (MtRSD)
is nodule-specific, controls symbiosome maturation after bacterial endocytosis from infection threads, and
is essential for symbiotic nitrogen fixation (SNF). Transcriptome analysis of an rsd mutant identified eleven
genes as potential targets of MtRSD repression. MtRSD protein was found to interact physically with the
promoter of one of these genes, MtVAMP721a, which encodes a vesicle-associated membrane protein.
Thus, MtRSD may control symbiosome development, in part, by repressing transcription of MtVAMP721a
and modifying vesicle- trafficking in nodule cells.
Twenty transporter genes are being investigated using reverse-genetics and mutants in several of these
are defective in SNF. Among seven transporters potentially involved in metal homeostasis, a putative
iron-citrate transporter of the MATE family was localized to symbiosome membrane, which is of special
interest to us.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Plenary Session I: Nodule Development and Physiology
Progressions of Bradyrhizobium japonicum
bacteroids into senescence.
David W. Emerich, Sooyoung Frank, and Kent N. Strodtman
University of Missouri, Columbia, MO
Bradyrhizobium japonicum bacteroids were isolated from soybean nodules at intervals up to 95 days
after planting and examined by transcriptomic and proteomic analyses. Over this time the bacteroids
underwent a global shift in gene expression patterns, although the total RNA quantity and quality remain
steady. Four common patterns of transcription were found, with one of the patterns displaying active gene
expression at 95 days after planting, which was during late senescence. During senescence bacteroids lose
their symbiotic characteristics and re-acquire functions of free-living bacteria. Among the genes found to
be expressed late were those of flagellar and pili biosynthesis. Electron microscopic analysis of nodules
55 days and older revealed the appearance of fibrillar appendages, demonstrating that post-symbiotic
B. japonicum has the ability to alter its structure. Proteomic analysis suggested the post-symbiotic form of
B. japonicum was accumulating and hydrolyzing peptides from the decaying plant nodules cells. Both global
analyses identified dioxygenase activities, which bioinformatics analysis suggested were localized to the
periplasm. A proteomic analysis of the periplasmic fractions verified the location of these enzyme activities,
in particular a protocatechuate dioxygenase, which was shown by mutational analysis to be necessary for
symbiotic functioning. Protocatechuate dioxygenase was among a number of enzymes, which continued
to express activity through senescence. A number of soybean proteins were identified in the periplasm
proteome including Histone H2A and lipoxygenase. Transmission electron microscopy using gold-labeled
antibodies against these two plant proteins was found to be predominately associated with the bacteroid
surface consistent with a periplasmic location.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Plenary Session I: Nodule Development and Physiology
Deep-Tissue Imaging Reveals Extensive Symplastic
Transport Network in Nitrogen Fixing Root Nodules
Cherish Skeen, Jeffry Caplan, and D. Janine Sherrier.
University of Delaware, Delaware Biotechnology Institute, Department of Plant and Soil Sciences, Newark, NJ
In legume-rhizobium symbiosis, plant cells divide and differentiate to accommodate the specialized
functions necessary for successful symbiosis. The extracellular matrix within the central zone of the nodule
undergoes extensive modification during organ development. However, it can be difficult to study the
extracellular interface between two cells within the central tissue, since they are buried under layers of the
uninfected nodule periphery and classic approach to section and optically reconstruct these tissues is labor
intensive and often results in the loss of image resolution. In this study, our goal was elucidate the potential
symplastic connectivity between cells in the central zone and to evaluate changes in the symplastic transport
architecture during cellular development in pea (Pisum sativum) nodules using whole or bisected nodules.
We developed and applied a novel tissue clearing method to whole root nodules, and in combination with
cellular markers for extracellular components, and analysed changes in the cell wall architecture occurring
throughout each developmental stage using three-dimensional modeling.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Plenary Session I: Nodule Development and Physiology
microRNA160 regulates auxin and cytokinin
sensitivities during soybean nodule development
Marie Turner, Narasimha Rao Nizampatnama, Mathieu Baron, Stéphanie Coppin, Suresh Damodaran,
Sajag Adhikari, Shivaram Poigai Arunachalam, Oliver Yu, and Senthil Subramanian*
* South Dakota State University, Department of Plant Science
Symbiotic root nodules in leguminous plants result from interaction between the plant and nitrogenfixing rhizobia bacteria. There are two major types of legume nodules, determinate and indeterminate.
Determinate nodules do not have a persistent meristem while indeterminate nodules have a persistent
meristem. The plant hormone auxin is thought to play a role in the development of both these types of
nodules. However, inhibition of rootward auxin transport at the site of nodule initiation is crucial for the
development of indeterminate nodules, but not determinate nodules. Using the synthetic auxin-responsive
DR5 promoter in soybean, we show that there is relatively low auxin activity during determinate nodule
initiation and that it is restricted to the nodule periphery subsequently during development. To examine
if and what role auxin plays in determinate nodule development, we generated soybean composite
plants with altered sensitivity to auxin. We over- expressed microRNA393 to silence the auxin receptor
gene family and these roots were hyposensitive to auxin. These roots nodulated normally suggesting
that minimal/reduced auxin signaling is sufficient for determinate nodule development. We overexpressed microRNA160 to silence a set of repressor ARF transcription factors and these roots were
hypersensitive to auxin. These roots nodulated poorly suggesting that auxin hypersensitivity inhibits
nodule development. To understand the mechanism of auxin action, we examined cytokinin sensitivity
in these roots. These roots were also hyposensitive to cytokinin, and had attenuated expression of
key nodulation-associated transcription factors known to be regulated by cytokinin. We propose a regulatory
feedback loop involving auxin and cytokinin during nodulation.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Plenary Session I: Nodule Development and Physiology
Rethinking the role of trehalose in legume-rhizobia interactions
Aarón Barraza, Juan Elias Olivares, Georgina Estrada-Navarrete, Xochitl Alvarado-Affantranger,
Carmen Quinto, and Federico Sánchez.
Departamento de Biología Molecular de Plantas, Instituto de Biotecnología, Universidad Nacional Autónoma de México.
Av. Universidad # 2001, Col. Chamilpa. C.P.: 62210, Cuernavaca, Morelos, México.
It has been reported that modification of trehalose metabolism contributes to the regulation of the
symbiotic interaction between legumes and Rhizobium, (Schluepmann and Paul, 2009; Suarez et al.,
2008). Herein, it is described that trehalose nodule content was increased by 78%, in transgenic common
bean nodulated hairy roots, by interference RNA (RNAi) for the trehalase (PvTRE1) gene. Increased
accumulation of trehalose also caused that the number of R. etli CFUs derived from these transgenic
nodules augmented one order of magnitude with a 71% enhanced effect on nitrogenase activity. These
dramatic changes had no apparent detrimental effect on the rest of the non-transgenic organs (Barraza
et al., 2013). By using this same system, a heterologous trehalose-6-phosphate synthase gene from
Rhizobium etli (ReTPS), also increased trehalose content (50%). However, bacteroid viability (50%
less) and nitrogenase activity (20% levels) were negatively affected. Interestingly, a positive systemic
effect was observed on the upper part of the plant (non-transgenic organs), where the leaf area (34%)
and relative water content (6%) were increased. The main difference in this case was that trehalose
and trehalose-6-phosphate (T6P) levels were both increased. Altogether these results indicate that
the trehalose/ trehalose-6-phosphate ratio in nodules has a profound effect on bacteroid viability,
nitrogen fixation levels and the establishment of a systemic sink-and-source relationship within
the nodulated plant.
References:
Barraza A, Estrada-Navarrete G, Rodriguez-Alegria ME, Lopez-Munguia A, Merino E, Quinto C, Sánchez F. 2013. Down-regulation
of PvTRE1 enhances nodule biomass and bacteroid number in the common bean. New Phytologist. 197(1): 194-206
Schluepmann H, Paul M. 2009. Trehalose metabolites in Arabidopsis – elusive, active and central. In: Last R, Chang C,
Jander G, Kliebenstein D, McClung R, Millar H, Torii K, Wagner D, eds. The Arabidopsis book. Washington, DC, USA:
American Society of Plant Biologists, 1-17
Suárez R, Wong A, Ramírez M, Barraza A, Orozco MdC, Cevallos MA, Lara M, Hernández G, Iturriaga G. 2008.
Improvement of Drought Tolerance and Grain Yield in Common Bean by Overexpressing Trehalose-6- Phosphate
Synthase in Rhizobia. Molecular Plant-Microbe Interactions 21(7): 958-966.
Acknowledgments:
This work was partially supported by grants from the Consejo Nacional de Ciencia y Tecnología (CONACYT),
México No. 0083324 and No. 177744, and DGAPA No. IN 214909-3.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Plenary Session I: Nodule Development and Physiology
Regulation of the mevalonate pathway by symbiotic
receptor-like kinases and its role in early symbiotic signaling
Muthusubramanian Venkateshwaran1, Dhileepkumar Jayaraman1, Brendan K. Riely2,
Estibaliz Lar- rainzar2, Douglas R. Cook2 and Jean-Michel Ané1
1. Department of Agronomy, University of Wisconsin Madison
2. Department of Plant Pathology, University of California Davis
HMGRs (3-hydroxy-3-methylglutaryl coenzyme A reductases) are key enzymes in the mevalonate
pathway controlling isoprenoid biosynthesis. Surprisingly, one of these enzymes (HMGR1) was found
to interact with the symbiotic receptor-like kinase DMI2 and is required for legume nodulation in the
model legume Medicago truncatula (Kevei et al., 2007). Using split-ubiquitin assays, interactions between
HMGR1 and two other symbiotic receptor-like kinases, NFP and LYK3, were found. In vitro kinase
assays revealed that HMGR1 is phosphorylated by DMI2 but not by NFP or LYK3. Mass spectrometry
was used to characterize phosphorylation sites in the linker region of HMGR1, a region which is highly
variable between different HMGR isoforms. Enzymatic assays revealed that HMGR1 activity is affected
by interaction with DMI2. Mimicking phosphorylation by serine to aspartic acid substitutions at the
phosphorylation sites also affected HMGR1 enzymatic activity. HMGR1-silenced roots were impaired
for nuclear calcium spiking and symbiotic gene expression. Reciprocally, application of mevalonate,
the product of HMGR1 activity, was sufficient to induce calcium spiking and symbiotic gene expression
in wild-type and HMGR1-silenced roots. Mevalonate was able to induce nuclear calcium spiking
and ENOD11 expression in dmi2 but not in dmi1 mutants. These results indicate that HMGR1 plays
an early role in the symbiotic signaling cascade after DMI2 and before the nuclear cation channel
DMI1 (Venkateshwaran et al., 2013). We hypothesize that HMGR1 connects signaling events at the plasma
membrane level to nuclear ones by generating second messengers controlling downstream symbiotic
signaling.
References
Kevei Z, Lougnon G, Mergaert P, Horvath GV, Kereszt A, Jayaraman D, Zaman N, Marcel F, Regulski K, Kiss GB, Kondorosi A,
Endre G, Kondorosi E, Ané JM (2007) 3-hydroxy-3-methylglutaryl coenzyme A reductase1 interacts with NORK and is crucial for
nodulation in Medicago truncatula. Plant Cell 19: 3974-3989
Venkateshwaran M, Volkening JD, Sussman MR, Ane JM (2013) Symbiosis and the social network of higher plants. Current opinion
in plant biology 16: 118-127
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Plenary Session II: Agricultural Applications
Using stable isotope techniques to investigate below-ground
nitrogen and carbon contributions from annual grain legumes
J. Diane Knight, Melissa M. Arcand and Richard E. Farrell
Department of Soil Science University of Saskatchewan, Saskatoon, SK Canda
Crop diversification across the Canadian Prairies has led to the wide-spread inclusion of annual grain
legumes (pulse crops) in crop rotations. A typical rotation will include a pulse crop once every three or
four years. Recognizing the N (and non-N) benefits that pulse crops convey to subsequent non-legume
crops in rotation, producers are now questioning whether they can increase the frequency of pulses in
rotations. Using a variety of 15N- and 13C-labeling studies we are quantifying the below-ground (roots,
nodules & root exudates) contribution of pea, lentil and chickpea to soil N and C pools, plant N uptake
and N- and C- cycling processes in a variety of crop rotation sequences. Traditional root recovery
protocols have grossly underestimated these below-ground contributions. Pulse species, frequency of
pulses in rotation, as well as the impact of species composition of non-pulse crops in rotation are all
being investigated. This talk will discuss the results of several recently completed and on-going studies.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Plenary Session II: Agricultural Applications
N2-fixation, Signal Exchange and the Phytomicrobiome
Donald L. Smith and Sowmyalakshmi Subramanian
The group of rhizobacteria that consistently associate with plant roots constitute the rhizosphere portion
of the phytomicrobiome. The first step in establishment of the legume-rhizobia nitrogen fixing symbiosis
is the exchange of signal compounds (flavonoids from the plants and lipo-chitooligosaccharides (LCOs)
from rhizobia). We have shown that low root zone temperatures can disrupt this signaling and that using
plant-to- microbe signals can activate the rhizobial node genes, predisposing the rhizobia for infection and
nodulation. In the course of evaluating this approach we discovered that this treatment also caused soybean
plants to emerge sooner. Testing demonstrated that the LCOs were responsible for this condition. In addition,
we found that a small protein, thuricin 17, made by Bacillus thuringiensis NEB 17, produced similar growth
stimulation. However, these effects could be inconsistent and we were eventually able to demonstrate that
they were greater under stress conditions (low temperature, drought, salt). We were also able to demonstrate
spray application of LCOs caused changes in the expression level of stress response genes of soybean.
More recently we have taken a label-free proteomics approach and have demonstrated that the proteome of
soybean germinating seeds is also clearly affected by application of these signal compounds in that there
is a meaningful increase in the presence of stress related proteins. Work with Arabidopsis has confirmed
the effects of these signals in non-legume plants, with regard to effects in the presence of salt and drought
stress, and protein production patterns.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Plenary Session II: Agricultural Applications
N2 fixation, N contribution, and water-use efficiency in
Bambara groundnut (Vigna subterranea L. Verdc) plants
sampled from farmers’ fields in South Africa
Felix D. Dakora, Keletso C. Mohale and Alphonsus K. Belane
Tshwane University of Technology, South Africa
Bambara groundnut (Vigna subterranea L. Verdc) is the second most important indigenous food legume
in Africa, yet there is very little information on it as a crop. The aim of this study was to evaluate plant
growth, N2 fixation, N contribution and plant water relations in Bambara groundnut grown in farmers’
fields in South Africa. Plant shoots plus pods were sampled from 26 farmers’ fields, and processed for
15N and 13C isotopic analysis using mass spectrometry. The data revealed marked (p≤0.05) differences in
plant dry matter yield, δ15N, the proportion of N derived from symbiotic fixation (%Ndfa), and amounts
of N-fixed. Bambara groundnut plants obtained between 33 and 98% (mean = 72%) of their N nutrition
from symbiotic fixation, and contributed about 4 to 200 kg N-fixed.ha-1 (mean = 102 kg N-fixed.ha-1). Plant
density correlated positively with %N (r = 0.31***), δ15N (r = 0.126***) and amount of N-fixed (r =
0.15*), indicating that where high %Ndfa values yielded little symbiotic N, this was due to low plant density
rather than poor symbiotic functioning. The δ13C of plant shoots (a measure of water-use efficiency in C3
plants) ranged from -28.01‰ to -26.20‰ for the 26 sites, indicating differences in plant water relations
between the Bambara groundnut landraces. The significantly positive correlation obtained between δ13C
and N-fixed (r = 0.15*) or δ13C and N content (r = 0.14*) suggests a functional relationship between
water-use efficiency and N2 fixation, just as the significant correlations between δ15N and dry matter yield
(r = 0.24***), N-fixed and dry matter weight (r = 0.76**), N content and dry matter yield (r = 0.99*), as
well as N-fixed and C content (r = 0.76**) also indicate a functional relationship between N2 fixation and
photosynthesis. Furthermore, the positive correlation between δ13C and dry matter yield (r = 0.14*), and/
or δ13C and C content (r = 0.15*), implies a functional link between water-use efficiency and plant growth.
Taken together, the data suggest that improved water-use efficiencyin Bambara groundnut appeared to have
metabolically enhanced plant growth, symbiotic functioning, and photosynthetic activity, in the same way
that N2 fixation stimulated photosynthesis.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Plenary Session II: Agricultural Applications
Method Development for Bradyrhizobium japonicum
competitiveness screening
Yaowei Kang, Jessica Smith, and Shawn Semones
Novozymes Biologicals Inc.
Maximum benefit of N2 fixation by soybean often requires the inclusion of selected strains of Bradyrhizobium
japonicum as seed inoculants. The inoculant strain must be effective in its ability to fix N2 with the cultivar
concerned and possess the ability to compete for nodulation of the plant with other strains of rhizobia
that might be present in the soil. Strain competitiveness is influenced by the genetic diversity of both
symbiotic partners (Triplett and Sadowsky 1992) as well as the soil environment in which nodulation
occurs. Determination of strain competitiveness was previously seldom undertaken in inoculant selection
programmes because of technical difficulties. Earlier techniques relied mainly on antibiotic-resistant
mutants or immunological methods to identify the inoculant strain. This type of technique only becomes
practical when it is used for dealing with a few strains. It could not be used for screening for a more
competitive strain from hundreds or thousands of strains. Mutation for antibiotic resistance often resulted
in lowered effectiveness and competitiveness of rhizobia containing these markers while immunological
methods were often limited by the presence of cross- reactive antibodies. Both methods were arduous,
expensive and identifications could only be made from small, statistically inadequate samples of the large
numbers of nodules formed. A new method for identifying inoculant rhizobia was tried, based on transfer
of the gusA marker gene coding for the enzyme b-glucuronidase (GUS) into the desired strain (Wilson et
al. 1995). This has helped in the isolation and selection of marked strains equally competitive with the wild
type (Streit et al.1995; Sessitch et al. 1997). As nodules containing the marked strain are identified by color
formation after reaction with a suitable substrate, accurate typing of large numbers of nodules and accurate
determination of competitive ability is possible. Because soybean plants possess a high demand of N, poor
N2-fixation performance leads to depletion of soil N-fertility. This problem was observed worldwide, also
with other legume crops, and is referred to as the problem of competition for nodulation (Sadowsky &
Graham, 1999). This problem has been addressed in recent decades using different approaches, but with
little success. Use of genetically engineered rhizobial strains was proposed (Toro, 1996), but application
of this approach for a commercial usage is nearly impossible due to tough regulation laws. Therefore,
it is essential for a commercial company to develop a new method that can be used for screening the
competiveness of B. japonicum strain from large number of isolates. Here we report a PCR-based screening
method that allows us to quickly screen thousands of strains in a short time period. Based on DNA sequence
analysis of a commercial strain, a very unique set of primers has been identified for the strain. A PCR based
high-throughput screening protocol for competitiveness of a strain in situ has been developed. This method
allows us to differentiate the nodules infected from a commercial strain and an unknown new isolate when
both of them were co-inoculated into soybean at equal ratio. More than two thousand nodule isolates have
been screened for their nodulation ability and their nitrogen fixation ability against a commercial strain and
a few strains with more effectiveness and competitiveness have been identified. Those new isolates were
found to increase by over 30% nodulation capability against a commercial strain.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Plenary Session II: Agricultural Applications
Prolonged nitrogen fixation in soybean during drought –
physiologic and genetic characterizations.
Larry C. Purcell1, Alejandro Bolton2, Andy C King1, Jeffery Ray3, Felix B. Fritschi4,
Arun Prabhu Dhanapal4, Sadal Hwang2 and Perry B. Cregan5
1. University of Arkansas, Fayetteville, AR
2. D
epartment of Crop, Soil, and Environmental Sciences, University of Arkansas, Fayetteville, AR
3. U
SDA-ARS, Stoneville, MS
4. University of Missouri, Columbia, MO, Division of Plant Sciences, University of Missouri, Columbia, MO
5. Soybean Genomics and Improvement Lab, USDA-ARS, Beltsville, MD
Nitrogen fixation in legumes provides important economic advantages for crop production by eliminating
the cost of nitrogen fertilizer, but the extreme sensitivity of nitrogen fixation to drought in soybean makes the
crop particularly vulnerable to drought. Over 20 years ago, the genotype Jackson was identified as having
prolonged nitrogen fixation during the early stages of drought. This early research determined that during
drought, ureides accumulate throughout the plant. The genotype Jackson, however, had considerably lower
concentrations of ureides under drought than did drought-sensitive genotypes. Further research demonstrated
that genotypes with relatively low concentrations of ureides or N under water-replete conditions were able to
continue nitrogen fixation at relatively low soil-moisture contents compared with genotypes with high shoot
N concentration that began to decrease nitrogen fixation rates at considerably higher soil-moisture contents.
Recently, we identified quantitative trait loci (QTLs) for ureide and N concentration in a recombinant
inbred population developed from a cross between KS4895 (drought sensitive for nitrogen fixation) and
Jackson (drought tolerant for nitrogen fixation). Lines from this population with extremes for ureide and
N concentration were evaluated in field experiments for their response to drought. Lines with low ureide
or N concentrations under well-watered conditions had higher rates of nitrogen fixation and higher yield
when exposed to a severe drought than those lines with high ureide or N concentrations. Under well watered
conditions, however, nitrogen fixation and yield increased as the ureide or N concentrations increased. In
this population it appears that low shoot N concentration may be beneficial in prolonging nitrogen fixation
under drought conditions but that under water-replete conditions, lines with low shoot N concentration
may have lower nitrogen fixation rates and yield. A key question is whether the benefits of low shoot N
concentration can be separated from the adverse effects under water-replete conditions. A genome-wide
association study (GWAS) has been used to identify alleles that might be associated with nitrogen or ureides
in soybean other than those found in the KS4895 x Jackson population. A set of 373 diverse, maturity
group IV accessions were phenotyped in four environments for ureide and N concentrations and genotyped
with approximately 12,000 single nucleotide polymorphisms. Results from the GWAS confirmed several
of the QTLs found in the KS4895 x Jackson population and identified other putative QTLs that can be
incorporated into breeding strategies for increasing drought tolerance of nitrogen fixation.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Plenary Session II: Agricultural Applications
N2Africa: Delivering BNF Technologies to African
Small-Scale Farmers
Paul L. Woomer, Nancy K. Karanja, and Ken E. Giller
N2Africa is “Putting Nitrogen Fixation to Work for Smallholder Farmers in Africa”. It is a large-scale
program focused upon increasing grain legume production and its symbiotic N2-fixation in smallholder
farming systems of sub-Saharan Africa, directed toward four major grain legumes (bean, cowpea,
groundnut and soybean) in eight countries (D.R. Congo, Ghana, Kenya, Malawi, Mozambique, Nigeria,
Rwanda and Zimbabwe). The program conducts strategic research in legume agronomy and rhizobiology
that backstops BNF technology dissemination activities targeting 225,000 poor households over four years.
Its partners include international research centers, universities, international NGOs, national research and
extension organizations, farmer associations and private sector input producers and grain legume buyers.
As this effort is quite diverse, this abstract focuses upon rhizobiology activities in Kenya as an example
of its mechanisms and achievements. Rhizobiology studies are conducted at the University of Nairobi
MIRCEN Laboratory and through the field activities of 26 grassroot groups in west Kenya. These actions
include bio-prospecting native rhizobia, greenhouse and field evaluation of these isolates, working with
the MEA Fertilizer Company the producers of BIOFIX® inoculants to assure product quality and assisting
the Ministry of Agriculture to frame bio-fertilizer legislation. To date, 387 isolates were collected from
20 genera in 14 different ecological zones, 85% belonging to the tribe Phaseoleae. Isolates were tested
against inoculant industry standard for bean (CIAT 899) and soybean (USDA 110) and six consistently
outperform them. Collaborative efforts with inoculant production include testing the effects of carrier
material selection, storage condition and different quality assessment approaches. Peat and sugar cane
factory filter mud carriers perform well while vermiculite, coconut coir and charcoal do not. Refrigeration
extends soybean inoculant shelf life by 30%. Recent tests of BIOFIX® ranged between 1.1 and 6.2 x 109
rhizobia g-1 using the YMA drop plate technique. Evaluation using MPN procedures in the greenhouse yield
lower, and more variable results. Soybean response to inoculation with BIOFIX® under farmer conditions
increases yield by +260 kg ha-1 worth $129, nodulation by +11 plant-1, crown nodulation by +44% and red
interior pigmentation by +58%. The partial return ratio to inoculant adoption is 9.2. To date, improved
grain legume and inoculation technologies were introduced to 50,127 Kenyan households with an adoption
ratio of approximately 70%. On another front, inoculant standards of >109 rhizobia and <106 contaminants
were included within Kenya’s draft Biofertilizer Act, will likely be adopted into law and N2Africa plans
to assist producers to meet and monitor these thresholds. The activπities in Kenya are representative of
the program’s larger impact on BNF technologies across Africa, with additional attention directed toward
laboratory and skills development, and pilot inoculant production in many other countries.
For more information on our approaches and achievements, visit the program’s website at www.n2africa.org.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Plenary Session III: Evolution of Rhizobia, Legumes, and Symbioses
Rhizospheric lifestyle of rhizobia and the buffet hypothesis
E. Ormeño Orrillo, M. A. Rogel, M. G. López-Guerrero, J. Althabegoiti, J. Martínez Romero, I. Toledo,
M. Rosenblueth, L. Servín, V. González, and Esperanza Martínez-Romero.
Genomic Science Center, UNAM, Cuernavaca, Mor., México
Rhizobia are successful colonizers of legume and non-legume rhizospheres and may promote plant growth.
Transcriptomics is being used to determine the genes expressed in natural habitats such as the rhizosphere.
The many uptake transporters induced in rhizobia (1) suggested that there are many different nutrients
available at the rhizosphere, that may be considered as a buffet in which distinct microbes have different
nutrient preferences that allow a complex community to be established (2). The role of rhizobia and many
other microbes in the degradation of soil or plant derived substances has been emphasized and compared
to gut microbial functions (3). Degrading capabilities of the great diversity of substances at the rhizosphere
may be encoded in the large genomes of rhizospheric bacteria and many unknown genes could participate
in the process. Additionally, we have remarked that degradation of natural soil and plant products is poorly
understood and not well analyzed in phenotypic studies in taxonomy, instead we discussed that a genomic
approach would be useful. Additionally we proposed a speciation hypothesis driven by divergence on
nutrient usage (4).
Thanks to PAPIIT grant IN205412.
References:
1. López-Guerrero, et al. 2012. Plasmid 68:149-158.
2. López-Guerrero et al. 2013. Front. Plant Science 4:188. doi: 10.3389/fpls.2013.00188
3. Ramírez-Puebla et al. 2013. Appl. Environ. Microbiol. 79:2-9.
4. Ormeño-Orrillo & Martínez-Romero 2013. Syst. Appl. Microbiol. 36:145-147.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Plenary Session III: Evolution of Rhizobia, Legumes, and Symbioses
Effect of resource enrichment on mutualist
intergenerational feedbacks
Ellen L. Simms and Stephanie S. Porter
Department of Integrative Biology, University of California, Berkeley, CA
A survey of nodules collected from the natural community occupying Bodega Dunes in Sonoma County,
California, found that hosts in the genus Acmispon interact with a genetically narrow range of symbionts
in the genus Bradyrhizobium, whereas hosts in the genus Lupinus interact with a genetically broader group
in Bradyrhizobium. Single-strain inoculation of a host from each genus with a broad range of genotypes from
the community indicated that, although both hosts can nodulate the entire range of genotypes, Acmispon
strigosus is a specialist that benefits only from the genetically narrow group of Bradyrhizobium with
which it normally interacts. In contrast, Lupinus bicolor benefits from most tested genotypes. As predicted
by generalist-specialist theory, L. bicolor receives less benefit from its broad range of symbionts than
A. strigosus does from its narrow range of symbionts. This specialist generalist trade-off suggested that
hosts might produce positive intraspecific cross-generational feedbacks via the rhizobium community.
This talk will present the results of a two-generation greenhouse experiment designed to test this hypothesis
at two different levels of external nitrogen.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Plenary Session III: Evolution of Rhizobia, Legumes, and Symbioses
Origin, evolution and loss of the plant –
microbe symbiotic ‘toolkit’
Pierre-Marc Delaux, Patrick P. Edger ,Kranthi Varala, Christophe Roux, Tomoaki Nishiyama, Hiroyuki
Sekimoto, J. Chris Pires, Gloria M. Coruzzi, and Jean-Michel Ané
Department of Agronomy, University of Wisconsin Madison, Madison, WI
Rhizobia are able to form a nitrogen-fixing symbiosis with legumes whereas arbuscular mycorrhizal fungi
associate with most of land plants. Despite this host-range difference, these two microbial symbioses
share striking similarities in their initiation. In both cases, symbiotic microbes perceive signals from root
exudates and produce lipochitooligosaccharidic signals. These microbial signals are then perceived by the
host plant leading to the regulation of symbiotic gene expression, and allowing the colonization process.
Because the arbuscular mycorrhizal symbiosis is much more ancient than the rhizobia – legume one,
it has been proposed that this symbiotic ‘toolkit’ was first used for interactions between primitive land
plants and arbuscular mycorrhizal fungi, and later co-opted for interactions with rhizobia. If this symbiotic
‘toolkit’ is now well described in legumes, little is known about its evolution and conservation within
the plant lineage. Using a comprehensive phylogenetic analysis of these components in the plant lineage
together with transcriptomic, physiologic and biochemical approaches, we characterized the stepwise
appearance of this symbiotic pathway, with some components predating the first land plants whereas
some others appeared rather recently in flowering plants1. In addition, we found that independent losses
of the arbuscular mycorrhizal symbiosis in at least five flowering plant lineages are systematically
correlated with the loss of the entire ‘toolkit’. Using this correlation and a bioinformatic comparison
of sequenced angiosperm genomes and transcriptomes, we identified new candidate genes potentially
required for symbiotic associations.
1. Delaux et al. Evolution of the plant – microbe symbiotic ‘toolkit’. 2013. Trends Plant Sci. 6: 298-304.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Plenary Session IV: Physiology and Genetics of the Bacteria
Regulation and assembly of cytochrome
oxidases in Bradyrhizobium japonicum
Hennecke, H., Bonnet, M., Kurz, M., Gruetter, M., Serventi, F., Abicht, H., Youard, Z., Mohorko, E.,
Fischer, H.M., and Glockshuber, R.
ETH Zürich, Institut f. Mikrobiologie, Zurich, Switzerland
The natural habitat of Bradyrhizobium japonicum is either the soil (free-living) or the root-nodule
of legume hosts (symbiotic). In symbiosis, B. japonicum fixes N2. Energy conservation occurs strictly
by aerobic or anaerobic respiration. The genome encodes at least seven different terminal oxidases
for respiration with O2. Cytochrome aa3 is the predominant oxygen reductase in the free-living state,
whereas a high-affinity cbb3-type cytochrome oxidase is essential for micro-oxic respiration in the
endosymbiotic state. Expression of genes for the latter is strongly induced in micro-oxia. The regulators
involved respond positively to low oxygen concentrations (via FixLJ) and negatively to reactive oxygen
species (via FixK2). The 1.7Å-resolution crystal structure of the FixK2-DNA complex has revealed
a mechanism by which oxidation might inhibit transcription activation by FixK2. Several protein
factors (mostly copper chaperones) have been identified that play a role in the biogenesis of the hemecopper aa3- and cbb3-type cytochrome oxidases. Our focus is currently on three proteins functioning
in the periplasm: TlpA, ScoI, and PcuC. While ScoI and PcuC bind copper, the TlpA protein acts as a
reductant for ScoI and probably also for subunit II of cytochrome aa3. Expression of the pcuC gene is
induced by copper limitation suggesting that it is also involved in copper acquisition by B. japonicum.
A tentative model of copper trafficking in B. japonicum is proposed.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Plenary Session IV: Physiology and Genetics of the Bacteria
Control of nutritional metal homeostasis
in Bradyrhizobium japonicum
Mark R. O’Brian
Department of Biochemistry, State University of New York at Buffalo, Buffalo, New York
We initiated a project focused on understanding how cells sense and acquire nutritional manganese
from the environment, and its function in cellular processes. Recent studies of Mn2+ transport mutants
suggest that manganese is not essential in E. coli and other bacteria unless they are under oxidative stress.
However, a manganese transport mutant (mntH) of Bradyrhizobium japonicum has a severe growth
phenotype under unstressed conditions. We are currently identifying cellular processes that require this
metal to understand why it is an essential nutrient. The prevailing model of bacterial membrane function
predicts that the outer membrane is permeable to divalent manganese and most small solutes due to pores
with limited selectivity based primarily on size. We challenged this model by the identification of MnoP,
an outer membrane protein expressed specifically under manganese limitation. MnoP acts as a selective
channel to facilitate the tranlocation of Mn2+ into the periplasm. An mnoP mutant is defective in high
affinity Mn2+ transport into cells, and has a severe growth phenotype under manganese limitation.
We suggest that the outer membrane is a barrier to divalent metal ions that requires a selective channel
to meet the nutritional needs of the cell. We found previously that manganese affects the cellular level
of Irr, a transcriptional regulator that controls the iron stimulon. Despite this, only some genes within the
Irr regulon are affected by manganese. This has led to identifying novel mechanisms for the regulation of
gene expression.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Plenary Session IV: Physiology and Genetics of the Bacteria
Carbon Catabolism and Competition for Nodule Occupancy
Ivan J. Oresnik
Department of Microbiology, University of Manitoba, Winnipeg, Manitoba Canada
Competition for nodule occupancy is a complex phenotype that covers multiple phases of the symbiotic
process. Knowledge about what is catabolized in the rhizosphere or during the infection process is limited.
We are interested in how carbon catabolism affects competition for nodule occupancy. Work in our lab has
been focused on two carbon compounds; rhamnose and galactose.
Rhamnose is not found in high concentrations in the rhizosphere, yet both R. leguminosarum and
S. meliloti rhamnose catabolic mutants are uncompetitive nodule occupancy. Although we have not been
able to elucidate why they are uncompetitive, biochemical characterization of the R. leguminosarum
ABC rhamnose transporter has shown that this transporter is novel in that it is dependent on the sugar
kinase (RhaK) for transport activity. By using linker-scanning mutagenesis we have been able to genetically
separate the sugar kinase activity of RhaK from its ability to affect transport.
In contrast galactose is found in relatively high abundance in the alfalfa rhizosphere. Galactose is catabolized
through the De Ley-Doudoroff pathway S. meliloti. Although annotation suggests that genes that encode
the enzymes necessary for this pathway are found at one locus, only 3 of these are necessary. Galactose
transport appears to be carried out by a previously described arabinose transporter that is highly induced
by root exudate. Mutants unable to catabolize galactose were found to be more competitive for nodule
occupancy. Our more recent work suggests that the inability to catabolize galactose is correlated with an
altered expression of exopolysaccharide biosynthesis genes.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Plenary Session IV: Physiology and Genetics of the Bacteria
The role of the bacterial polysaccharide succinoglycan in
Medicago host plant invasion by Sinorhizobium meliloti.
Kathryn M. Jones, Hajeewaka C. Mendis, and Clothilde Queiroux
Department of Biological Science, Florida State University, Biology Unit I, Tallahassee, Fl
The invasion of legume plant roots by nitrogen-fixing rhizobia requires that multiple signals be exchanged
between these symbiotic partners. Curling of root hairs occurs in response to rhizobia-produced Nod factor.
Rhizobia colonize the roots of most legumes through tubules called infection threads that originate from
bacterial microcolonies enclosed by these curled root hairs. Infection threads are progressive ingrowths
of plant cell membrane containing a matrix of bacterial exopolysaccharides (EPSs) and plant cell wall
material. To successfully induce infection thread formation on the host Medicago truncatula, Sinorhizobium
meliloti. 1021 must be able to produce the acidic EPS succinoglycan (reviewed in [1]). Succinoglycan is
composed of octasaccharide repeating units, modified with succinyl, acetyl and pyruvyl substituents [1].
These oligosaccharide chains are produced in a mixture of high molecular weight (HMW) and low MW
(LMW) forms [1]. Over 15 genes are involved in the S. meliloti succinoglycan biosynthesis and modification
pathway [1]. We have recently determined that increased succinoglycan production by S. meliloti (due to
exoY overexpression) results in increased symbiotic productivity with M. truncatula [2]. We have also
found that production of LMW succinoglycan by glycanase cleavage (e.g. ExoK) increases the efficiency of
the symbiotic interaction with M. truncatula, but is not absolutely required for a successful symbiosis [3].
In contrast, the presence of the acidic succinyl group on succinoglycan (added by exoH-encoded succinyl
transferase) is essential for establishing the symbiosis. This requirement for the succinyl group appears to
be independent of the effect of this modification on succinoglycan molecular weight distribution.
References:
1. J
ones, K.M., H. Kobayashi, B.W. Davies, M.E. Taga, G.C. Walker, How rhizobial symbionts invade plants: the
Sinorhizobium-Medicago model. Nat Rev Microbiol, 2007. 5(8): p. 619-33.
2. J
ones, K.M., Increased production of the exopolysaccharide succinoglycan enhances Sinorhizobium meliloti 1021 symbiosis
with the host plant Medicago truncatula. J Bacteriol, 2012. 194(16): p. 4322-31.
3. Mendis, H.C., C. Queiroux, T.E. Brewer, O.M. Davis, B.K. Washburn, K.M. Jones, The succinoglycan endoglycanase encoded by
exoK is required for efficient symbiosis of Sinorhizobium meliloti. 1021 with the host plants Medicago truncatula and
Medicago sativa (alfalfa). MPMI, 2013. accepted.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Plenary Session IV: Physiology and Genetics of the Bacteria
Rhizobial inositol catabolism and flavonoid-inducible
efflux systems contribute to efficient nitrogen fixing symbioses
Silvia Rossbach1, Petra Kohler1, Mary Thwaites1, Olivia Walser1, Kati Kunze2, Susann Albert2,
Susanne Zehner2, and Michael Göttfert2
1. Department of Biological Sciences, Western Michigan University
2. Institute of Genetics, TU Dresden, Germany
This talk will discuss two metabolic features of rhizobia that enhance symbiotic nitrogen fixation: i.) the
ability to catabolize inositol derivatives and ii.) the presence of efflux pump genes that are inducible by
plant-derived flavonoids. The different stereoisomers myo-, scyllo- and D-chiro-inositol can serve as sole
carbon sources for Sinorhizobium meliloti., the endosymbiont of alfalfa. The idhA and the iolABCDE
genes were necessary for the degradation of myo- and D-chiro-inositol, whereas the iolY gene was shown
to be essential for growth with scyllo-inositol (Kohler et al., AEM 76:7972). We also identified genes
involved in the catabolism of the methylated inositol derivative, pinitol. Mutants with insertions in the
iol genes were shown to be deficient in their ability to compete with the wild type in co-challenge plant
inoculation assays. In addition to the well- known flavonoid-inducible rhizobial nod genes, transcriptomic
studies have identified luteolin-inducible efflux pump genes in S. meliloti (smc03167/smc03168; Barnett et
al., PNAS 101:16636; Capela et al., AEM 71:4910). Using reporter gene fusions, we found that these genes
are induced by luteolin, apigenin and naringenin. The TetR-like regulator encoded by the adjacently located
smc03169 gene was cloned and purified. DNA-shift assays showed that the regulatory protein binds to the
upstream region of the efflux pump genes. The phenotype of mutants with insertions in this region support
the notion that not only the ability to catabolize a diverse array of carbon sources promote successful plantmicrobe interactions, but also the ability to withstand potential harmful plant-derived metabolites.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Plenary Session V: Physiology and Genetics of the Plant Host
Cytokinin receptors: footprints along the evolutionary
path to symbiotic root nodule formation
Mark Held1,2#, Hongwei Hou1, Mandana Miri1,2, Christian Huynh1, Loretta Ross1, Shushei Sato3,
Satoshi Tabata3, Jillian Perry4, Trevor Wang4, and Krzysztof Szczyglowski1,2
1. A
griculture and Agri-Food Canada, Southern Crop Protection and Food Research Centre, London, Ontario, N5V 4T3 Canada
2. Department of Biology, University of Western Ontario, London, Ontario, N6A 5BF Canada
3. Kazusa DNA Research Institute, 2-6-7 Kazusa-Kamatari, Kisarazu, Chiba, 292-0818 Japan
4. John Innes Centre, Norwich Research Park Colney, Norwich NR4 7UH, United Kingdom
#. Current address: Biotechnology Institute, University of Minnesota, Saint Paul, MN 55108.
Analysis of loss-of-function and gain-of-function alleles of the Lhk1 cytokinin receptor gene showed
that it is required and also sufficient for nodule organogenesis in Lotus japonicus (Murray et al., 2007;
Tirichine et al., 2007; Hackmann et al., 2012). The L. japonicus mutant carrying the loss-of function
lhk1-1 allele is hyperinfected by Mesorhizobium loti, with infection threads heavily present in segments
of the root epidermis and cortex in the initial absence of nodule organogenesis (Murray et al., 2007).
At a later time-point following inoculation, lhk1-1 develops a limited number of nodules (Murray et
al., 2007), suggesting the presence of an Lhk1-independent signalling mechanism for nodule formation.
We have tested a hypothesis that other cytokinin receptors function in at least a partially redundant manner
with LHK1 to mediate nodule organogenesis in L. japonicus. We show that L. japonicus contains a small
family of four cytokinin receptor genes, which all respond to M. loti infection. A model for the cytokinin
receptor-dependent signalling will be discussed. In this model, we postulate that LHK1 exerts a unique
function in the root epidermis but works partially redundantly with LHK1A and LHK3 within the root
cortex to mediate cell divisions for nodule primordia formation.
J. Murray et al., Science 315, 101 (2007).
L. Tirichine et al., Science 315, 104 (2007)
A. Hackmann et al., MPMI 24, 1385 (2011)
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Plenary Session V: Physiology and Genetics of the Plant Host
Shedding light on an underground problem:
light regulation of symbiotic nodule development
Yucan Zhang, Beck Powers, Jim Weller, and Jeanne Harris
Plant Biology Department, University of Vermont, Burlington, VT
The formation of symbiotic root nodules has become embedded within plant regulatory pathways in
legumes, so that nodule development is coordinated with other developmental processes. Because plants
are continuously developing, their development is strongly influenced by environmental conditions, and
one of the most important is light. We found that Red, Blue and Far Red (FR) light regulate the formation
of nitrogen-fixing nodules in multiple legume species: Red light stimulates nodulation and FR and Blue
light inhibit nodulation. Interestingly, Red/FR and Blue signals intersect with nodulation signaling at
different points in the pathway. Red and FR light regulate gene expression in both the epidermal and
cortical pathways, whereas Blue light inhibits only cortical gene expression, not epidermal. We have
identified a gene, GIRAFFE, that is required for Red/FR signaling during photomorphogenesis that is also
required for Red/FR regulation of nodulation. The giraffe mutant has a deletion in a heme oxygenase gene,
homologous to Arabidopsis HY1 and pea PCD1 that cosegregates with the Red-light-insensitive nodulation
phenotype. We found that GIRAFFE gene function is required in the shoot to regulate nodulation in the
root in response to Red light, indicating the requirement for a shoot-to-root signal. Interestingly, ethylene
signaling is required for FR and Blue light inhibition of nodulation, but not for Red light stimulation. Gene
expression analysis reveals antagonistic regulation of ABA synthetic and degradative enzymes by Red and
Blue light, thus providing a mechanism for regulation of the nodulation signaling pathway by different
wavelengths of light.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Plenary Session V: Physiology and Genetics of the Plant Host
Dissection of symbiotic nitrogen fixation using a tobacco
retrotransposon Tnt1-insertion mutant collection in
Medicago truncatula
Catalina Pislariu1, Senjuti Sinharoy1, Igor Kryvoruchko1, Ivone Torres-Jerez1, Yuhong Tang1,
Mingyi Wang1, Jiangqi Wen1, Xiaofei Cheng1, Mark Taylor1, Shulan Zhang1, Jin Nakashima1,
Elison Blancaflor1, Rujin Chen1, Kirankumar Mysore1, Vagner Benedito2, Jeremy Murray3,
Gyles Oldroyd3, Pascal Ratet4, and Michael Udvardi1
1.The Samuel Roberts Noble Foundation, Plant Biology Division, Ardmore, OK, USA;
2. West Virginia University, Morgantown, WV, USA;
3. J
ohn Innes Center, Norwich, UK;
4. Institut des Sciences du Végétal, CNRS Gif-sur-Yvette, France;
[email protected]
Many plant genes are expressed during nodule development and symbiotic nitrogen fixation (SNF) but
few of these have been functionally characterized. Noble Foundation has developed a genetics resource
for Medicago truncatula using tobacco retrotransposon (Tnt1)-mutagenesis [1, 2]. By sequencing genomic
regions adjacent to sites of Tnt1 integration in lines with symbiotic phenotypes, novel genes required for
symbiosis can be identified efficiently.
From a Tnt1-insertion mutant population of M. truncatula ecotype R108 we isolated 179 mutants impaired
at different stages of nodule development and SNF during the interaction with Sinorhizobium meliloti. [3].
In this collection we identified 39 alleles of known symbiotic genes. While some symbiotic mutants may be
accounted for by insertions in known but untested genes, this collection is a valuable resource for cloning
novel symbiotic genes. Some of these mutants will be described in detail. The phenotype in one of the
Fix- mutants co- segregates with Tnt1 insertion in a member of the PLAT (Polycystin-1; Lipoxygenase,
Alpha-Toxin domain or LH2 (Lipoxygenase Homology 2) superfamily, which was named the Nodulationspecific PLAT-domain gene, NPD1, and will be presented in more detail.
A spatially-resolved transcriptome of Medicago nodules will also be presented. The expression patterns
described for genes in different nodule zones are similar to a virtual in situ hybridization catalog.
This information is useful to link nodule zone-specific gene expression with molecular processes necessary
for nodule development and SNF, and is a good resource to target genes of interest for functional
characterization via reverse-genetics.
1. d
’Erfurth, I., et al., Efficient transposition of the Tnt1 tobacco retrotransposon in the model legume Medicago truncatula.
Plant Journal, 2003. 34(1): p. 95-106.
2. Tadege, M., et al., Large-scale insertional mutagenesis using the Tnt1 retrotransposon in the model legume Medicago truncatula.
Plant Journal, 2008. 54(2): p. 335-347.
3. Pislariu, C.I., et al., A Medicago truncatula tobacco retrotransposon insertion mutant collection with defects in nodule
development and symbiotic nitrogen fixation. Plant Physiology, 2012. 159(4): p. 1686-99.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Plenary Session VI: Genomic Approaches to Symbiosis
Toxin and antitoxin systems of Sinorhizobium meliloti.
Branka Milunovic, George diCenzo, Richard A. Morton and Turlough M. Finan
Dept. of Biology, McMaster University, Hamilton, Ontario, Canada L8S 4K1
In Type II toxin-antitoxin (TA) systems, the activity of toxin protein is inhibited through a physical
interaction with an antitoxin protein. Antitoxins proteins are less stable than toxins and therefore the loss of
these addiction modules, and particularly loss of antitoxin genes is toxic to cells. The biological functions
of toxin-antitoxin (TA) gene pairs is controversial and this may reflect different roles for different systems
in the same organism. The roles of TA systems in root-nodule bacteria has a particular fascination as these
bacteria persist in soil as free-living cells for long periods and they also establish a symbiosis and live within
nodules that eventually senesce. Informatic analysis of the genome of the alfalfa symbiont, Sinorhizobium
meliloti. identified 210 TA associated genes and 93 of these are located on the 1.4 Mb pSymA and the
1.7 Mb pSymB megaplasmids. Here we describe and discuss the construction of over eighty strains in
which defined regions of pSymA or pSymB have been deleted. Of TA systems on the megaplasmids, we
identified only four TA loci, whose deletion resulted in a cessation of cell growth or decrease in cell viability.
We present the identification of these loci and the analysis of their expression relative to the other systems
TA genes in S. meliloti. We will also discuss symbiotic and other phenotypes uncovered during the analysis
of the megaplasmid deletion mutants.
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Plenary Session VI: Genomic Approaches to Symbiosis
Nitrogen-fixing, Symbiotic Burkholderia Species Lack
Typical Strategies Required in Mammalian Pathogenesis
Annette A. Angus, Christina M. Agapakis, Stephanie Fong, Shaila Yerrapragada, Paulina Estrada
de los Santos, Paul Yang, Nannie Song, Stephanie Kano, Jésus Caballero-Mellado,
Sergio M. de Faria, Felix D. Dakora, George Weinstock, and Ann M. Hirsch
UCLA Department of Molecular, Cell and Developmental Biology
Burkholderia is a diverse and dynamic genus, containing species that are pathogens as well as
species that form complex symbiosis with plants. Pathogenic strains, such as B. pseudomallei and
B. mallei, can cause serious disease in mammals, while other Burkholderia strains are opportunistic
pathogens, infecting humans or animals with a compromised immune system. Although some of the
opportunistic Burkholderia pathogens are known to promote plant growth and even fix nitrogen, the
risk of opportunistic infection has not only resulted in a restriction of their use, but has also limited the
application of non-pathogenic, symbiotic species, several of which nodulate legume roots. However,
recent phylogenetic analyses have demonstrated that Burkholderia species separate into distinct sublineages, suggesting the possibility for safe use of certain symbiotic species in agricultural contexts.
A number of environmental strains that promote plant growth or degrade xenobiotics are also included in
the symbiotic sub-lineage. Many of these species have the potential to enhance agriculture in areas where
fertilizers are not readily available and may serve in the future as inocula for crops growing in soils impacted
by climate change. In this paper, we address the pathogenic potential of the symbiotic Burkholderia strains
using bioinformatics and functional tests.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Plenary Session VI: Genomic Approaches to Symbiosis
Global proteome analysis of Sinorhizobium medicae–
Medicago truncatula Jemalong A17 symbiosis:
new approach and new insights
Svetlana N. Yurgel1, Yi, Qu3, Jennifer Rice1, Mary Lipton3, and Michael L. Kahn1,2.
1. Institute of Biological Chemistry, Washington State University, Pullman, WA, USA
2. School of Molecular Biosciences, Washington State University, Pullman, WA, USA
3. Pacific Northwest National Laboratory, Richland, WA, USA
We employed tandem mass spectrometry (MS/MS) to analyze the proteomes of M. truncatula A17 and
S. medicae strain WSM419 in free-living and symbiotic tissues. A total of 1872 distinct proteins were
identified in the free-living and symbiotic proteomes of S. medicae. 608 proteins displayed a strong lifestyle specialization, 666 proteins were similarly represented in both proteomes, and 598 were found either
in free-living or in symbiotic proteomes but at such low abundance that quantitative comparisons were not
statistically significant. The data obtained from the proteome analysis are consistent with current models of
global changes in bacteroid metabolism. Additionally, our data raise the question of the importance of plant
derived sugars in symbiosis and show that, while most of the enzymes involved in Glycolysis/Gluconeogenesis
and the pentose phosphate pathway are present in nodules, the low amount of these enzymes in nodules
indicates that these pathways are much less active in symbiosis. On the other hand, sugar transport appears
to be highly active in symbiosis, as indicated by the presence of numerous periplasmic sugar binding
proteins. Our data also indicate active bacterial stress responses in the S. medicae–M. truncatula symbiosis,
especially regarding oxidative stress. We identified a number of lifestyle-specific WSM419 proteins that are
not encoded in S. meliloti Rm1021. Understanding the roles of these proteins in the interaction between
WSM419 and M. truncatula might clarify the factors affecting the relative ineffectiveness of S. meliloti
Rm1021 in symbiosis with Mt A17.
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Plenary Session VI: Genomic Approaches to Symbiosis
Exploring Nodulation through Genome-wide
Association Studies of Medicago truncatula
Nevin Dale Young and the Medicago truncatula Hapmap Consortium
Derpartment of Plant Pathology, University of Minnesota, St. Paul, MN
Genome-wide association studies (GWAS) make it possible to explore the architecture of complex traits
and pinpoint genes that play causative roles. The Medicago Hapmap Consortium has re-sequenced 330
diverse accessions of M. truncatula and related taxa to discover more than 6,000,000 single nucleotide
polymorphisms (SNPs) suitable for GWAS. Analysis of this SNP dataset provides insight into: genome
sequence diversity (nucleotide diversity estimates range from 4 to 6 per kb, comparable to Arabidopsis),
recombination (centromeric, high gene density and NBS-LRR regions exhibit higher rates of recombination)
and linkage disequilibrium (LD) (declines to background within 10 kb, much shorter than cultivated
soybean). A total of 226 accessions from the Medicago diversity panel were assayed for developmental and
nodulation phenotypes and then compared statistically to the SNP dataset using the program TASSEL after
accounting for population structure (Stanton-Geddes et al, PLoS ONE 8: e65688). The highest scoring 200
SNPs for each trait (with non-Bonferroni corrected probability < 10-6) were examined in detail. Flowering
time and trichome density phenotypes each exhibited a single major peak, pinpointing strongly supported
candidate genes (Arabidopsis FD ortholog for flowering time; Petunia UNSHAVEN MADS-box ortholog
for trichome density). In the case of nodulation, nodule number and strain occupancy were assayed. For
these traits, high scoring SNPs tagged loci throughout the genome including a modest number of peaks
consisting of SNPs in high LD. Various nodulation- related genes were tagged by significant SNPs within or
flanking their coding sequences (including CaML3, NFP, SERK2, others). SNPs associated with nodulation
by GWAS were also significantly over-represented in genes expressed primarily in nodules. Some, including
the most significant peaks in the nodulation GWAS, are located in genes that play roles in DNA repair,
ubiquination, or as molecular chaperones. Additional high scoring SNPs tag nodule-upregulated genes with
no known molecular function. In parallel work, we also sequenced 46 Sinorhizobium strains and these data,
along with plant genome analyses, provide insight into this important symbiosis.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Plenary Session VI: Genomic Approaches to Symbiosis
The nodule transcriptome of common bean (Phaseolus vulgaris)
Georgina Hernández, Jamie A. O’Rourke, Luis P. Íñiguez, Fengli Fu, Bruna Bucciarelli, Susan S. Miller,
Scott A. Jackson, Philip E. McClean and Carroll P. Vance
Center for Genomic Sciences (CCG). National University of Mexico (UNAM). Mexico
Phaseolus vulgaris (common bean) comprises approximately 50% of the grain legumes consumed worldwide
and is important as a primary source of dietary protein. Combining the genomic resources of the P. vulgaris
genome sequence and predicted gene calls with the Illumina-RNAseq platform we have measured the gene
expression patterns from seven tissues (24 RNA samples) of P. vulgaris cv. Negro Jamapa. Nine RNA
samples were collected from root and nodule tissue: young (pre-fixing) and mature (N-fixing, SNF) nodules
elicited by Rhizobium tropici, ineffective nodules elicited by R. giardinii, roots associated with each type
of nodules and root samples from plants provided with complete nitrogen solution. We identified 245
nodule specific genes, 21 of these were homologous to soybean nodule genes and included transcription
factors (TF) and transporters highlighting the importance of regulating gene expression and the exchange
of materials between nodules and roots. Effective vs. ineffective nodules showed 2,953 differentially
expressed genes; gene ontology (GO) statistics revealed eight over-represented GO categories related to
defense and oxidation/reduction processes. The 2,932 genes differentially expressed between pre-fixing
nodules and N-fixing nodules are important in both nodule development and the establishment of SNF;
these include a suite of genes previously identified in other rhizobia–legume symbioses. Genes for both
purine and ureide biosynthesis, essential processes for common bean nodule function, were highly and
specifically up-regulated in effective nodules; 404 genes were coexpressed with these. Analysis of samples
from SNF vs. nitrate-fed plants identified 2,970 genes with expression patterns that appear to be directly
dependent on the source of available N. Our assembled data will be publicly available in The Common
Bean Gene Expression Atlas Database, so researchers can query gene expression profiles.
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Plenary Session VI: Genomic Approaches to Symbiosis
Hormone and nutrient regulation of nodule and lateral
root development through NIP/LATD in the model
legume Medicago truncatula
Mohammad Salehin1, Rammyani Bagchi1, Ying-Sheng Huang1 and Rebecca Dickstein1
1. Department of Biological Sciences, University of North Texas, Denton, TX
Medicago truncatula NIP/LATD is required for symbiotic nitrogen fixing root nodule development and
for normal root architecture: nip/latd mutants have defects in these processes. NIP/LATD encodes a
member of the diverse NRT1/PTR family of transporters (Yendrek at al. 2010. Plant J. 62: 100; Harris
& Dickstein. 2010. Plant Signal & Behav 5: 1365). Evidence points to NIP/LATD functioning as a
high-affinity nitrate transporter: its expression confers high-affinity nitrate transport to Xenopus laevis
oocytes and restores chlorate – a toxic nitrate mimic – susceptibility to the Arabidopsis chl1-5 mutant.
However, the weakest nip/latd mutant allele also displays high-affinity nitrate transport in oocytes and
Atchl1-5 chlorate resistance rescue, suggesting that NIP/LATD has another function (Bagchi et al. 2012.
Plant Physiol 160:906; Salehin et al. 2013. Plant Signal & Behav 8:1). Recent experiments hint at this
second NIP/LATD function in hormone modulation. Constitutive expression of NIP/LATD in several
species including M. truncatula results in plants with a growth phenotype. Using a synthetic auxin reporter,
we observed the presence of higher auxin concentrations in both a nip/latd mutant and in plants constitutively
expressing NIP/LATD compared to wild-type plants. Previous experiments showed NIP/LATD expression
is hormone modulated and the NIP/LATD promoter is active in root and nodule meristems (Yendrek at al.
2010. Plant J. 62: 100) as well as the vasculature. Two potential binding sites for an auxin response factor
(ARF) were found in the NIP/LATD promoter. Chromatin immunoprecipitation- qPCR confirmed that an
ARF attaches to these binding sites. Mutating the binding sites increases NIP/LATD expression, suggesting
a mechanism for auxin repression of NIP/LATD. Consistent with these results, constitutive expression of
an ARF in wild-type plants partially phenocopies nip/latd mutants’ phenotypes. Current research is aimed
at understanding the relationship between NIP/LATD expression and hormone homeostasis.
This work was supported by NSF IOS-0923756 to RD.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Plenary Session VII: Associative Fixation, Non-Legumes, & Endophytes
Soybean symbiosis with Bradyrhizobium japonicum:
Evolution and global warming
Kiwamu Minamisawa
Tohoku University, Katahira, Aoba-ku, Sendai, Japan
During phylogenetic analyses of bradyrhizobia, we want to address how “non-symbiotic” soil bradyrhizobia
evolved to highly sophisticated, symbiotic bradyrhizobia. Bradyrhizobium oligotrophicum S58T is a
nitrogen- fixing bacterium from paddy soil. S58 genome comprised single chromosome lacking nod genes
and symbiosis island. It was similar to those of stem-nodulating bradyrhizobia ORS278 in terms of ndv and
nif/fix genes. Soil- dwelling B. oligotrophicum formed effective nitrogen-fixing nodules to Aeschynomene
indica. Thus, Nod factor- independent symbiosis is common in nodABC- and symbiosis island-lacking
strains within the photosynthetic bradyrhizobia (1). As most evolved symbiotic bradyrhizobia, we
nominated B. japonicum USDA122 that are incompatible with Rj2-soybean. rhcJ mutant for the type III
protein secretion system (T3SS) failed to secrete typical effectors and gained the ability to nodulate Rj2soybean (2). Thus, the effectors secreted via T3SS trigger incompatibility between both partners. Finally,
I will introduce a Bradyrhizobium story to prevent global warming. Nitrous oxide (N2O) is a greenhouse
gas from agricultural soils. In soybean ecosystems, organic N inside nodules is mineralized to N2O, which
is then emitted into atmosphere or is further reduced by N2O reductase (encoded by nosZ). Pure culture
and pot experiments showed lower N2O emission by nosZ+ and nosZ++ strains than by nosZ_ strains.
Post-harvest N2O emission from soils can be mitigated by inoculation of nosZ+ and nosZ++ strains at a
field scale (3).
References
1. O
kubo et al. 2013. Appl. Environ. Microbiol. 79: 2542. 2) Tsukui et al. 2012. Appl. Environ. Microbiol. 79: 1048. 3)
Itakura et al. 2013. Nature Climate Change 3: 208.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Plenary Session VII: Associative Fixation, Non-Legumes, & Endophytes
Genome-guided approaches toward elucidating the
signaling mechanisms in the actinorhizal symbiosis.
Sergio Svistoonoff1, Nicholas Beauchemin2, Virginie Vaissayre1, Claudine Franche1, Didier Bogusz1,
and Louis S. Tisa2
1. Equipe Rhizogenèse, UMR DIADE, IRD, 911 avenue Agropolis, 34394, Montpellier Cedex 5, France
2. Dept. of Molecular, Cellular and Biomedical Sciences, University of New Hampshire, USA
Frankia forms nitrogen-fixing symbioses with 8 different Angiosperm families, commonly known as
actinorhizal plants. Symbiotic interactions between Frankia and the host plant are not well understood and
very little is known about the initial molecular interactions in the rhizosphere. The nature of the chemical
signals exchanged between the two partners of actinorhizal symbioses is still unknown and is a focus of our
studies. Due to the absence of genetic tools for Frankia, we have also pursued new genomic approaches
toward studying these bacteria. Eleven Frankia genomes have sequenced providing opportunities to use
bioinformatics approaches and other new technologies. A correlation between genome size and plant host
range was suggested from these data. Larger genome had broader host ranges. The absence of obvious
nodulation genes similar to those found in Rhizobia genomes suggests that the actinorhizal symbiosis uses
novel signal compounds during the infection process. Analysis of the Frankia genomes also demonstrated
the presence of unexpected numbers of secondary metabolite gene clusters and potential novel natural
products as candidates. Besides comparative genomics approaches to identify key genes, we have used
other genome-guided approaches to search for marker genes of symbiotic interaction to identify symbiotic
signals emitted by actinorhizal plant roots. A molecule present in root exudates from Casuarina glauca
plants induced molecular and physiological changes in Frankia including the ability to establish root
nodules on the host plant significantly earlier than untreated cells. The presence of an extracellular signaling
molecule(s) produced by the exudates-treated Frankia was identified by the use of a bioassay with transgenic
C. gluaca plants and specific genetic markers in the nodulation pathway. These results provide support and
insight on the hypothesis of chemical signaling between actinorhizal host plant and Frankia.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Plenary Session VII: Associative Fixation, Non-Legumes, & Endophytes
Functional genomics of nitrogen-fixing,
plant-associative Burkholderia sp.
Michelle R. Lum, Tommy Nguyen, Michael Onofre, and Salma Soltani
Department of Biology, Loyola Marymount University, Los Angeles, CA
The Burkholderia genus contains plant and human pathogens, but a number of the species isolated
have been found to be beneficial to plants and associate with them in the rhizosphere or as endophytes. We
are focusing on two nitrogen-fixing species of Burkholderia, B. unamae and B. tuberum. B. unamae was
originally isolated from maize and has plant growth promoting effects on various plants including tomato
(Caballero-Mellado et al., 2004). B. tuberum is a nodulating species, a beta-rhizobia, originally isolated
from Aspalathus carnosa nodules (Vandamme et al., 2002). To better understand how these bacteria engage
in a beneficial interaction with plants, and in particular how this compares to alpha-rhizobia, we are using
functional genomics methods to mutagenize specific genes and to target processes we hypothesize will
alter the ability of these species to interact with the host plant. We screened transposon-tagged libraries
for alterations in motility and exopolysaccharide production by B. unamae and additionally screened
B. tuberum mutants for nodulation defects. Analysis of a mutant in flhC, a transcriptional regulator of
flagella biosynthesis, suggests that motility and exopolysaccharide production are coordinately regulated in
B. unamae. A B. tuberum pilA mutant has alterations in nodulation.
We are analyzing several other mutants as well, including some in genes previously implicated in nodulation
by alpha-rhizobia.
Caballero-Mellado, J., Martínez-Aguilar, L., Paredes-Valdez, G., Estrada-de los Santos, P. 2004. Burkholderia unamae sp. nov.,
an N2-fixing rhizospheric and endophytic species. Int. J. Syst. Evol. Microbiol. 54: 1165-1172.
Vandamme, P., Goris, J., Chen, W.M., De Vos, P., Willems, A. 2002. Burkholderia tuberum sp. nov. and
Burkholderia phymatum sp. nov., Nodulate the Roots of Tropical Legumes. Syst. Appl. Microbiol. 25: 507-512.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Poster Presentation 1
Analysis of the genomic structure of Rhizobium etli, symbiotic
and non-symbiotic bacteria, associated to common bean plants
Phaseolus vulgaris.
Olga María Pérez Carrascal, María Soledad Juárez, and Víctor González
Centro de Ciencias Genómicas, UNAM, Cuernavaca, Morelos, Mexico
Bacteria of the genera Rhizobium are commonly isolated from nitrogen fixing nodules in the roots of
leguminous plants. Rhizobia population and comparative genomic studies have been conducted mostly
between strains isolated from nitrogen-fixing nodules and different geographical origins. A comprehensive
study about genomic structure of rhizobium associated with legumes must include at least three kinds of
populations: symbiotic (isolated nodule), symbiotic (isolated from the rhizosphere), and non-symbiotic
(isolated from the rhizosphere). Five common bean plants (Phaseolus vulgaris L. “Black Veracruz”) were
harvested from a field in Tepoztlan, Morelos; symbiotic and non-symbiotic isolates were obtained from five
nodules by plant and from the rhizosphere soil. 124 isolates were evaluated by growth properties, molecular
gene markers from the symbiotic plasmid and the chromosome, and plasmid profiles. Phylogenetic
reconstruction using dnaB and glyA indicate that all the isolates (both from nodule and rhizosphere)
belong to Rhizobium etli bv. phaseoli. Plasmid patterns vary even in the closest genotypes, and are more
diverse among isolates from rhizosphere than those recovered from nodule. Nonetheless, some rhizosphere
isolates lack of the symbiotic plasmid. To understand the fine scale evolutionary process behind the genetic
structure of this R. etli population, we are carrying out whole genome sequencing of several symbiotic and
non-symbiotic isolates. Genomic comparisons will allow infer the rate of mutation, recombination, and
horizontal transfer in a sympatric R. etli population at short divergence scales.
Acknowledgements
This work is supported by CONACYT CB-131499
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Poster Presentation 2
Poolseq analysis of Rhizobium Leguminosarum
genotype selection by the legume plant host
Jorrín, Beatriz1 and Imperial, Juan 1,2*
1. Centro de Biotecnología y Genómica de Plantas (UPM-INIA), Madrid, Spain
2. Consejo Superior de Investigaciones Científicas (CSIC), Spain.
* [email protected]
Rhizobium leguminosarum bv. viciae establishes highly specific nitrogen-fixing symbioses with different
legume genera (Pisum, Lens, Lathyrus and Vicia). Classic studies using trap plants provided evidence
that, given a choice, specific hosts select specific genotypes of rhizobia which are, apparently, particularly
adapted to that host (Mutch & Young, 2004; Louvrier et al, 1996).
We have applied a Pool-Seq approach (Kofler et al, 2011), to study plant host selection of genotypes
from the available rhizobial genomic diversity present in a well-characterized agricultural soil (INRA
Bretennieres). Plants of Pisum sativum, Lens culinaris, Vicia sativa and V. faba were employed as traps.
We pooled 100 nodules from each host, and the pooled DNAs were sequenced (BGI-Hong Kong; Illumina
Hi-seq 2000, 180 bp PE libraries, 100 bp reads, 12 Mreads). Reads were quality filtered with FastQC and
Trimmomatic. Filtered reads were mapped with Bowtie2 using Rhizobium leguminosarum bv. viciae 3841
as reference genome. Single Nucleotide Polymorphisms (SNPs) were called with VarScan. Results were
visualized with SeqMonk and IGV.
Our results confirm, at the genomic level, previous observations regarding plant selection of specific
genotypes. We expect that further, ongoing comparative studies on differential Pool-Seq sequences will
identify specific gene components of the plant-selected genotypes.
ACKNOWLEDGMENTS
We thank Gisèle Laguerre for encouragement, discussions and soil samples. Supported by the Microgen Project
(Consolider-Ingenio 2010, CSB2009-00006, MCINN, Spain) to JI.
REFERENCES
Mutch L.A. & Young J.P. 2004. Mol Ecol. 13: 2435
Louvrier P. et al. 1996. Appl Environ Microbiol. 62:4202
Kofler, R., et al. 2011. Bioinformatics, 27:3435–3436
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Poster Presentation 3
A new species of Bradyrhizobium endosymbiont
of Lupinus Mariae-Josephae, an endemic lupine
of basic-lime soils in Eastern Spain
David Durán1, Luis Rey1, Tomás Ruiz-Argüeso1 and Juan Imperial 1,2
1. C
entro de Biotecnología y Genómica de Plantas (CBGP) and Departamento de Bio- tecnología (ETSI Agrónomos),
Campus de Montegancedo, Universidad Politécnica de Madrid, 28223 Pozuelo de Alarcón (Madrid), Spain
2. CSIC, Spain
Lupinus mariae-josephae is a recently described species of lupine endemic of basic-lime soils of Eastern
Spain. Bacterial strains isolated from nitrogen-fixing nodules of this lupine species have been characterized
by genetic, phenotypic and symbiotic approaches. Analysis of their 16S rRNA genes placed them in
the Bradyrhizobium genus within a group closely related (99.1% identity) to Bradyrhizobium elkanii
USDA76T, B.pachyrhizi PAC48T, B. jicamae PAC68T and B. lablabi CCBAU 23086T. These results were
consistent with those obtained from phylogenetic analysis of three concatenated housekeeping genes, recA,
atpD and glnII, and from overall genomic identities calculated as Average Nucleotide Identity (ANI) among
strains using draft genomic sequences obtained for relevant strains. While L. mariae-josephae strains
M1 and M3 showed a 98,4% ANI, they were significantly distant (<93% ANI) from type strains of their
closest species (B. lablabi CCBAU23086T and B. jicamae PAC68TT). These results suggest that the
L. mariae-josephae strains represent a new Bradyrhizobium species. This assumption was also supported
by whole-cell matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (WC-MALDITOF-MS) analysis of proteomic patterns of the same strains. The symbiotic nodC, nodA and nifH genes
from strains nodulating L. mariae-josephae were again phylogenetically related to those from B. lablabi
CCBAU 23086T, but divergent from those of strains that nodulate other lupine species. Consequently,
based on the genetic, genomic, proteomic and phenotypic data presented in this study, L. mariae-josephae
nodulating strains should be grouped into a new species of the Bradyrhizobium genus.
Supported by FBBVA (BIOCON08-078) and Comunidad de Madrid (Microambiente S2009/AMB-1551) to TRA and by MICINN
(CSD2009-00006 and CGL2011-26932) to JI
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Poster Presentation 4
MTNRAMP1 mediates iron import in rhizobia-infected
Medicago truncatula cells
Benjamín Rodríguez-Haas1, Lydia Finney2, Igor Kryvoruchko3, Pablo González-Melendi1,
Michael Udvardi3, Juan Imperial1,4, and Manuel González-Guerrero1*
1. C
entro de Biotecnología y Genómica de Plantas (CBGP) Universidad Politécnica de Madrid. Campus de Montegancedo,
Pozuelo de Alarcón, 28223 Madrid, Spain.
2. Biosciences Division. Advanced Photon Source, Argonne National Laboratory Argonne, IL 60439
3. The Samuel Roberts Noble Foundation, Plant Biology Division, Ardmore, Oklahoma, 73401
4. Consejo Superior de Investigaciones Científicas (CSIC). Madrid, Spain
* [email protected]
Iron is an essential cofactor for key proteins involved in symbiotic nitrogen fixation (nitrogenase,
leghemoglobin, etc…). This nutrient must be provided by the host plant through at least one of these
possible paths: i) increased metal uptake by the nodule epidermis, ii) increased mobilization of pre-existing
iron reserves in the nodule, or iii) long-distance transport by the vasculature. Using synchrotron-based
X-ray fluorescence, we have determined that the third possibility is at least the most important. Iron is
delivered by the vasculature and released in the apoplast of zone II in M. truncatula nodules. The metal is
then transported through the plasma membrane of the rhizobia-infected cells. Subsequently, iron crosses
the peribacteroid membrane to be used in the synthesis of nitrogenase and other bacteroid ferroproteins.
Consistent with this model, studies of the nodule metallotranscriptome revealed the upregulation of a
number of metal transporters, among them the Nramp family member MtNramp1. MtNramp1 is an iron
and manganese importer, localized in the plasma membrane of rhizobial-infected cells in zone II of the
nodule. This makes it a likely candidate to being the transporter responsible for introducing into the cell
the iron that will be used to synthesize metalloproteins such as nitrogenase. Supporting this hypothesis is
the observation that knock-down plants on MtNramp1 have a 60% reduction of nitrogenase activity when
compared to the wild type. Future work will be directed to further validating this phenotype by studying
changes in iron distribution as a consequence of changes in the expression levels of MtNramp1.
Supported by RYC-2010-06363, APS General User Proposal 26208, and Marie Curie IIRC MENOMED (all to MGG).
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Poster Presentation 5
The autoregulation of nodulation in Medicago truncatula:
a network of signals
Elise L. Schnabel, Tessema K. Kassaw, Ashley D. Crook, and Julia A. Frugoli
The Department of Genetics and Biochemistry, Clemson University, Clemson, SC
Legumes form a symbiotic interaction with rhizobia to obtain fixed nitrogen in exchange for providing
carbon to the bacteria. Because the process is energetically costly to the plant, the formation of nitrogenfixing nodules in legumes is tightly controlled by a long-distance signaling system in which nodulating
roots signal to shoot tissues to suppress further nodulation. We have published several mutants in Medicago
truncatula that lack the ability to regulate nodule number from the shoot (sunn and lss) or from the root
(rdn1) and here present evidence that the genes act in the same pathway to regulate nodule number. Using
a published split root system, we are able to show a second nodule regulatory event occurring 10 days
later than the first that is unaffected in hypernodulation mutants. In parallel with identifying the timing and
nature of autoregulatory signals, we report a suppressor of the sunn, rdn1 and the lss lesions, a mutation
in the MtCRE1 cytokinin receptor. Unlike Mtcre1 alleles identified by others, this suppressor allele has
almost no effect on nodule number alone; rather its effect on nodulation is observed only in plants with
disruptions of the SUNN pathway (rdn1, sunn and lss mutants). The combination of these findings, our work
on interacting partners of the SUNN kinase, and overexpression of CLE peptides allows us to incorporate
all of these findings into a general signal transduction pathway for nodule number regulation that includes
hormonal signals, SUNN, LSS, RDN1 and CLE peptides.
Supported by NSF award #IOS1146014.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Poster Presentation 6
Gene coexpression analysis in common bean nodules
Luis P. Iñiguez, Jamie A. O’Rourke, Carroll P. Vance, and Georgina Hernández
Center for Genomic Sciences (CCG). National University of Mexico (UNAM). Mexico
The genomic era has generated a great amount of data where useful information needs to be pulled out.
One clear example is the data from high throughput sequencing, especially transcriptome sequences (RNAseq). A whole transcriptome analysis on Phaseolus vulgaris was done for 24 samples from seven tissues
including nodules and roots tissues. This work allowed knowing which genes were expressed in a particular
condition and how was their expression level. The pattern of expression among conditions of a gene with
certain function could be shared with other genes with unknown function which may be relevant for the
same biologicol process or molecular function. Based on the P. vulgaris RNA-seq data we designed an
algorithm to identify the pattern of expression for a gene and to search others genes with a similar pattern;
these were named coexpressed genes. The purine biosynthesis, a fundamental process for common bean
nodules function, shows a very high and specific nodule expression, but the genes from this pathway should
not be the only highly expressed in nodules. A coexpression analysis was performed based on the purine
biosynthesis and it revealed 404 genes with a similar expression pattern. Gene Onthology (GO) statistics on
this list showed known processes relevant in the nodules, such as IMP biosynthesis, amino acid biosynthesis
and of course purine biosynthesis. Around 50% of the purine biosynthesis coexpressed genes were annotated
and the rest have an unknown function based on their expression pattern one can hypothesize that these are
relevant for common bean mature nodule function. The information from our gene coexpression analysis
could be the basis for future experimental research on common bean – rhizobia symbiosis.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Poster Presentation 7
Functional analysis of the Sinorhizobium meliloti megaplasmids
George C. diCenzo, Allyson M. MacLean, Branislava Milunovic, and Turlough M. Finan
Department of Biology, McMaster University, Hamilton, ON, Canada
As with other rhizobia, the genome of Sinorhizobium meliloti is divided and consists of the 3.65 Mb
chromosome and the 1.35 Mb pSymA and 1.68 Mb pSymB megaplasmids. Using a large-scale genome
reduction strategy, we examined the contribution of the megaplasmids to the free-living phenotype of
the bacterium. Using a combination of growth studies and a phenotype microarray analysis (Biolog), we
suggest that while the megaplasmids contribute greatly to the metabolic diversity of this organism, they
encode few functions that are of significant value during free-living growth in bulk soil. Instead, they are
likely to provide a niche specific fitness advantage.
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Poster Presentation 8
Forward and reverse genetic analysis of nodule
development and symbiotic nitrogen fixation in
Medicago truncatula using Tnt1 insertion mutants
Vijaykumar Veerappan1, Khem Kadel1, Naudin Alexis1, Ashley Scott1, Catalina Pislariu2,
Senjuti Sinharoy2, Pascal Ratet3, Michael Udvardi2, and Rebecca Dickstein1
1. Department of Biological Sciences, University of North Texas, Denton, TX 76203, USA.
2. The Samuel Roberts Noble Foundation, Plant Biology Division, Ardmore, OK, 73401, USA.
3. Institut des Sciences du Végétal, Centre National de la Recherche Scientifique, 91198 Gif sur Yvette Cedex, France.
Legumes are unique compared to other plants because they form a symbiotic relationship with soil rhizobia
to capture and use atmospheric nitrogen as a source of bioavailable nitrogen fertilizer via symbiotic nitrogen
fixation. To further understand the molecular and genetic mechanisms underlying the legume-rhizobia
symbiosis, we are undertaking both forward and reverse genetic approaches. In a forward genetic screen,
we isolated seven Medicago truncatula Tnt1 insertion mutants in the R108 ecotype that are defective in
nodule development and symbiotic nitrogen fixation in response to Sinorhizobium meliloti. Those mutants
include ones that have Nod+Fix- nodules with brown pigments, those that have Nod+Fix- white nodules,
and some that have Nod+/- phenotype. To identify the causal insertions, we are undertaking two strategies:
the first one is to identify Tnt1 insertions from each mutant by TAIL-PCR and the second one is to develop
mapping populations by outcrossing mutants in the R108 ecotype to alternative ecotypes A17 and A20.
Genes responsible for the mutant phenotypes will be identified by either co-segregation analysis of mutant
phenotypes with the Tnt1 insertions using BC1F2 population or by positional cloning. We previously
characterized a serine-threonine protein kinase gene MtIRE (Pislariu et al. 2007) which belongs to AGC
protein kinase family. MtIRE is uniquely expressed in the invasion zone of Medicago truncatula nodules
(Pislariu et al. 2007). To elucidate the role of MtIRE in nodule organogenesis and symbiotic nitrogen
fixation, we are currently characterizing four different Mtire Tnt1 insertion alleles. In a complementary
approach, we are also characterizing the ectopic overexpression of MtIRE cDNA driven by the Arabidopsis
EF1a promoter. All these efforts will expand our knowledge of the molecular mechanisms underlying
legume-rhizobia symbiosis.
We gratefully acknowledge support from NSF IOS-1127155 to MU and RD.
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Poster Presentation 9
Functional assessment of MtNIP/LATD’s
biological/biochemical activities.
Rammyani Bagchi1, Mohammad Salehin1, Ying-Sheng Huang1, and Rebecca Dickstein1
1. Department of Biological Sciences, University of North Texas, Denton, TX
Our lab studies the nitrogen-fixing symbiosis between legume Medicago truncatula and Sinorhizobium
meliloti. using genetic, molecular and biochemical approaches. Mutants with defects in the MtNIP/LATD
gene respond to S. meliloti by producing abnormal nodules with numerous aberrant infection threads
and very rare release of rhizobia into host plant cells. Mtnip/latd mutants have an abnormal defense-like
response in root nodules and a defective lateral root development (Veereshlingam et al. 2004. Plant Physiol.
136:3692; Bright et al. 2005. MPMI 18:521; Teillet et al. 2008. MPMI 21:535). The MtNIP/LATD gene
was cloned using a positional approach. Phylogenetic analysis showed that MtNIP/LATD encodes a protein
belonging to the NRT1(PTR) family of nitrate and peptide transporters (Yendrek et al. 2010. Plant J 62:100;
Tsay et al. 2007. FEBS Lett 581:2290). Experiments with Mtnip/latd mutants demonstrated a defective
root architecture response associated with low (250 μM) external nitrate concentration rather than high (5
mM) nitrate concentration. This suggested that the mutants could have defective nitrate transport. To test if
MtNIP/LATD is a nitrate transporter, Xenopus laevis oocytes (Bagchi et al. 2012. Plant Physiol. 160:906)
and Arabidopsis chl1-5 were used as expression systems. Heterologous expression of MtNIP/LATD in
Xenopus laevis oocytes and Atchl1-5 lines conferred on them the ability to take up nitrate from the external
media with high affinity, showing that MtNIP/LATD is a nitrate transporter. X laevis expressing either
mutant allele Mtnip-1 or Mtlatd were unable to transport nitrate. However oocytes, expressing less severe
mutant allele Mtnip-3 were able to transport nitrate suggesting another role for MtNIP/LATD besides high
affinity nitrate transport (Bagchi et al. 2012. Plant Physiol. 160:906). It has been shown previously that
MtNIP/LATD level is regulated by hormones (Yendrek et al. 2010. Plant J 62:100). ABA added externally
rescues the lateral root defect in Mtlatd mutants (Liang et al. 2007. Devel Biol 304:297). External addition
of ABA or IAA down-regulates MtNIP/LATD transcript levels whereas BAP up- regulates MtNIP/LATD
levels. Preliminary experiments done in our lab using an auxin responsive promoter- reporter construct
shows that auxin levels in the vasculature are higher in Mtnip-1 compared to WT. Similarly using a cytokinin
responsive promoter-reporter construct, we found that cytokinin levels are lower in the mutant compared to
WT. Recent reports show that Arabidopsis NRT1(PTR) proteins have substrate specificities beyond nitrate
and small peptides. Experiments are being performed to see if the MtNIP/LATD protein transports anything
other than nitrate.
We gratefully acknowledge support from NSF IOS-0923756 to RD.
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Poster Presentation 10
Optimized Agrobacterium-mediated
transformation of Leucaena leucocephala
Jadd Correia*, Dung Pham, Michael Honda, Lee Chaille and Dulal Borthakur
*Department of Molecular Biosciences and Bioengineering, University of Hawaii-Manoa, Honolulu, HI
Leucaena leucocephala, (leucaena) is a fast-growing leguminous tree with many positive applications.
This research aims to improve forageability of leucaena by reducing the concentration of the toxic
amino acid mimosine. Our approach to accomplish this was through transforming leucaena with genes
for mimosine degradation from Rhizobium. The main focus of this current work was improvement of
existing transformation protocols by overcoming problems with tissue regeneration and DNA transfer.
Experimental trials indicated three key limiting factors: (i) prduction of phenolic exudate on explant,
(ii) accumulation of necrotic material at explant cut surface, and (iii) inefficient rooting as a hindrance
for regeneration. We hypothesized that transformation and regeneration efficiency could be enhanced
to 5-7% by overcoming these three specific barriers. Reduced production of phenolic exudate was
accomplished by introducing 10% activated charcoal to adsorb the phenolics released into the growth
media. Introduction of a cell recovery phase and supplementation of the medium with 10% activated
charcoal prevented development of necrotic cell material. Root induction was improved by introducing
an elongation period enabling explants to elongate shoots prior to root induction.
In addition, leucaena root formation was inhibited by excess light. To overcome this problem, activated
charcoal was utilized as a darkening media agent for improved root induction. Our results established
that prevention of necrotic cell death coupled with timely induction of a healthy root system through
darkened media can improve transformation frequency of leucaena. PCR analysis has confirmed the
presence of the transgenes in multiple transformants. This research contributes towards the development of
mimosine-free leucaena and also improving transformation protocol for woody legumes.
This research was supported by the National Science Foundation award CBET 08-27057.
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Poster Presentation 11
Biochemistry of Rhizobium and Leucaena leucocephala
enzymes for catabolism of mimosine.
Vishal S. Negi, Jon-Paul Bingham, Qing X. Li, and Dulal Borthakur*
*Department of Molecular Biosci ences and Bioengineering, University of Hawaii-Manoa, Honolulu, HI
The tree-legume Leucaena leucocephala (leucaena) contains a large amount of a toxic non-protein aromatic
amino acid, mimosine. Leacaena foliage and its root nodule symbiont Rhizobium TAL1145 have enzymes
for degradation of mimosine. We have identified one Rhizobium enzyme and one leucaena enzyme that
have very similar catalytic and kinetic properties but have dissimilar amino acid sequences. The amino
acid sequences of both enzymes show similarities with several aminotransferases and lyases. Recombinant
enzymes were prepared by expressing the Rhizobium gene midD and leucaena cDNA encoding a putative
aminotransferase/lyase in E. coli, and the purified enzymes were assayed for mimosine degradation. The
HPLC/UV chromatograms of the major degradation products from both enzymes were found identical to
that of 3-hydroxy-4-pyridone (3H4P), which was further verified by ESI-MS/MS. Both enzymes require
pyridoxal 5’-phosphate (PLP) for activity but not α-keto acid and therefore they are not an aminotransferase.
In addition to 3H4P, pyruvate and ammonia were the other degradation products of mimosine by both
enzymes. The dependence of the enzymes on PLP, and production of 3H4P with the release of ammonia
indicate that they are C-N lyases. The Km values of the Rhizobium and leucaena enzymes were 1.27x104 and 1.16x10-4 moles, respectively. The presence of other aromatic amino acids, including L-tyrosine,
L-phenylalanine and L-tryptophan, in the reaction did not show any competitive inhibition of both
enzymes. The isolation of these genes for mimosine degradation and the biochemical characterization of
the recombinant enzymes will be useful in developing transgenic leucaena with reduced mimosine content
in the future.
This research was supported by the National Science Foundation Award No. CBET 08-27057.
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Poster Presentation 12
Testing systemic effects of NaCl on nodulation
in Medicago truncatula
Sanhita Chakraborty and Jeanne M. Harris
Plant Biology Department, University of Vermont, Burlington, VT
Soil salinity is a major agricultural concern worldwide as excess NaCl in the form of salt stress is
inhibitory to nodulation in legumes. Another major regulator of nodulation, nitrate (NO3-), is widely
studied and is known to have inhibitory effects on nodulation, both locally and systemically. The local
effects of salinity on nodulation are well documented but its systemic effects have not been studied
yet. We aim to find out whether there is any systemic effect of salt stress on nodulation in Medicago
truncatula. We use the genotype A17, and employ a split root system using compartmentalized growth
pouches. We pretreat plant roots with 100 mM NaCl and inoculate them with Sinorhizobium meliloti
(Rm1021). To study the systemic effects, one half of the root of one plant in one compartment is
pretreated with NaCl while the remaining half in the other compartment is inoculated. This design is
distinct from our local treatment, in which we inoculate the roots in the same compartment pretreated
with NaCl. Through this experimental approach, we hope to understand the systemic role of NaCl signaling
in nodulation.
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Poster Presentation 13
miRNAS in common bean (Phaseolus vulgaris) nodules
Bárbara Nova-Franco, Luis P. Íñiguez, Alfonso Leíja, José Luis Reyes, Lourdes Girard,
and Georgina Hernández.
Center for Genomic Sciences (CCG). National University of Mexico (UNAM). Mexico
Common bean (Phaseolus vulgaris), the most important legume for human consumption. MicroRNAs
(miRNAs) (21-22 nt) are post-transcriptional regulators involved in relevant processes such as plant
organ development. Our group is interested in investigating roles of miRNAs in common bean.
Valdes-Lopez et al. (2010) reported the miRNA expression profile in common bean nodules through
miRNA- macroarray approach. We analized the recently released P. vulgaris genome sequence
(www.phytozome.net) to identifed genes coding for miRNAs that were previously detected in common
bean nodules. The expression of selected conserved and legume miRNAs in common bean nodules was
confirmed by qRT-PCR. In the miRNA-macroarray analysis (Valdes-Lopez et al., 2010) miR172 was the
only miRNA detected exclusively in nodules and not in roots or leaves, therefore we are analyzing its
posible role in the symbiosis. Four isoforms of miR172 (miR172a, miR172c, miR172f and miR172e)
are coded in the bean genome. We have confirmed (Northern blot and qRT-PCR) the nodule enhanced
miR172 expression as compared to other common bean organs. The target gene of miR172 in several
plants including common bean is the AP2 transcription factor (Arenas-Huertero et al., 2009). The bean
genome coded for 201 AP2 genes; bioinformatics and expression analysis confirmed that two AP2 genes:
Phvul.005G138300.1 and Phvul.011G071100.1 are targets of miR172. Our goal is to define the role of
miR172 and its target PvAP2 in the common bean-rhizobia symbiosis.
Arenas-Huertero C. et al. (2009) Plant Mol Biol. 70(4), 385-401
Valdés-López, O. et al. (2010) New Phytol 187, 805-818
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Poster Presentation 14
Thigmomorphogenesis: physiological and morphological
responses to mechanical stimuli in Acacia koa
Kazue Ishihara, Brad Porter, Eric Lee, Isabel Rushanaedy, and Dulal Borthakur
Department of Molecular Biosciences and Bioengineering, University of Hawaii-Manoa, Honolulu, HI
Acacia koa (koa) is a leguminous timber tree endemic to the Hawaiian Islands. Because of the high value
of koa wood, understanding of the factors influencing wood quality is crucial. The objectives of this project
were to investigate changes in morphology and gene expression induced by mechanical stimuli in koa.
Based on published reports on characteristics of genes involved in wood development, we hypothesize that
some touch- inducible genes are involved in wood development in koa. Koa seedlings were gently bent in
four cardinal directions daily for six months, after which morphological alternations were quantified. To
identify touch- inducible genes, microarray analysis was designed for the comparison of gene expression in
untouched and touched koa seedlings. For this analysis, 4,000 cDNA sequences were selected on the basis
of similarities with genes that might be related to wood formation and development. Touched plants had
increased stem diameter, epidermal outgrowths, anthocyanin, root-shoot ratio, and decreased stem length,
root weight, and leaf size. Among the 4,000 genes tested, the expression of over 60 genes was elevated more
than twofold by touch. Some of the most up-regulated expression was that of zinc-finger protein, ethyleneresponsive-element binding protein, WRKY transcription factor, protein phosphatase 2C, disease resistance
proteins, and MAP kinase. These results suggest that touch may alter kinase signaling, disease resistance,
and transcriptional responses, which may lead to morphological changes. This project contributes toward
identifying genes regulating wood formation and development in koa. This research is supported by the
McIntire-Stennis Cooperative Forestry Program.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Poster Presentation 15
Role of heme oxygenase in regulating expression
of ROS-generating enzymes in Medicago truncatula
Parna Ghosh and Jeanne Harris
Plant Biology Department, University of Vermont, Burlington, VT
Heme Oxygenase (HO) is an enzyme universally found in animals, plants and microbes. In plants, the
role of heme oxygenase in the synthesis of the phytochrome chromophore is well recognized and has
been extensively studied; however its role in regulating reactive oxygen species (ROS) in plants is just
beginning to be explored, particularly in legumes. Legumes interact with Rhizobium bacteria to form
symbiotic nitrogen fixing nodules. ROS plays an important role in the development of roots as well as
symbiotic nodules. In the model legume Medicago truncatula, ROS in the root is regulated in part by
the LATD/NIP gene. The M. truncatula giraffe mutant has a deletion that removes the entire HO coding
sequence. We are testing the role of M. truncatula GIRAFFE HO in regulating expression of LATD/NIPregulated ROS genes such as RBOHA, RBOHC, Cu/Zn SOD and a cell wall peroxidase (PRX2). We
found that in roots, the wild-type function of GIRAFFE is to upregulate expression of RBOHA, in contrast
to LATD/NIP, which downregulates it. We find that LATD/ NIP does not regulate GIRAFFE expression.
We are currently investigating the role of GIRAFFE in regulating nodule senescence since the expression
of GIRAFFE HO is highest in a senescing nodule. At present, with changing climatic conditions and
exposure to various environmental stresses that can alter ROS homeostasis, characterizing the role of
GIRAFFE in the antioxidant machinery of legumes can be useful in improving crop productivity and for
enhancing soil fertility.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Poster Presentation 16
A Phaseolus vulgaris Rboh gene is required for rhizobial
infection, bacteroid development and nitrogen fixation
Arthikala, M.K., Montiel, J., Nava, N., Sánchez-López, R., Cárdenas, L., Santana, O., and Quinto, C.
Departamento de Biología Molecular de Plantas, Instituto de Biotecnología, Universidad Nacional Autóno- ma de México, UNAM,
Apartado Postal 510-3, Cuernavaca, Morelos 62271, México.
RBOHs (Respiratory Burst Oxidase Homologs) are plant membrane proteins that catalyze oxygen reduction
to produce superoxide, a form of reactive oxygen species (ROS). ROS generation by RBOHs activity
is essential in diverse developmental processes and adaptation to (a) biotic stresses in plants (Torres,
2010), albeit their role in symbiotic associations is poorly understood. This prompted us to explore the
role of RBOHs in the P. vulgaris (common bean)-Rhizobium symbiosis. The Phytozome database, was
searched and nine Rbohs were found in the P. vulgaris genome: PvRbohA-PvRbohI. RT-qPCR assays
revealed the differential expression patterns of these genes in P. vulgaris organs, being the PvRbohB the
most abundantly expressed gene in roots and nodules (Montiel et al., 2012). Herein, the role of RbohB
during the symbiotic interaction between P. vulgaris and Rhizobium tropici was assessed by RNAi and
over-expression approaches using a hairy root system. The results obtained revealed that in common beans,
RbohB-dependant ROS production is required for infection thread progression, bacteroid growth and
development and also for nitrogen fixation.
References
Torres, M. 2010. ROS in biotic interactions. Physiol Plantarum 138: 414-429.
Montiel, J., Nava, N., Cárdenas, L., Sánchez-López, R., Arthikala, M.K., Santana, O., Sánchez, F., and Quinto, C. 2012. A Phaseolus vulgaris NADPH-OXIDASE genes is required for root infection by rhizobia. Plant and Cell Physiology, 53(10): 1751-1767
Work supported by Consejo Nacional de Ciencia y Tecnologìa (CB-2010-153718) to C.Q. and a post-doctoral fellowship (17656) to
A.M.K.Poster
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Poster Presentation 17
The analysis of cross-talk between nitrogen and
phosphate stress responses in Sinorhizobium meliloti
Kelly Hagberg1,2, Svetlana N. Yurgel1, Jennifer Rice1, Monika Mulder1, and Michael L. Kahn1,2.
1. Institute of Biological Chemistry
2. School of Molecular Biosciences, Washington State University, Pullman, WA
Sinorhizobium meliloti are soil-dwelling bacteria that can establish a symbiotic relationship with Medicago
plants. Bacteria have several response pathways to deal with environmental stresses like nitrogen or
phosphorous limitation. The Nitrogen Stress Response (NSR) in S. meliloti involves a signaling cascade
initiated by GlnD. Under nitrogen limitation, GlnD modifies the PII proteins, GlnB and GlnK, which then
activate the NtrB/NtrC two component regulatory system. A Rm1021 strain lacking the PII proteins had
severe growth impairment in free-living conditions. Slow growth could be suppressed by a mutation in the
phoB gene, which codes for the phosphate stress response response regulator. This phenotype prompted
us to examine the interaction between phosphate and nitrogen stress responses. Using the PII and/or phoB
mutant strains, we altered nitrogen and phosphate levels to examine how the responses are interacting and
adapting to nutrient limitations. Previously, it was shown that Glutamine Synthetase I (GSI, glnA) activity
and GSII (glnII) expression are regulated by nitrogen stress. We found that expression of glnA and glnII
was down-regulated by phosphate stress. GSs are major enzymes induced by an active NSR. We found that
GS activity was decreased when phosphate was limited, regardless of the nitrogen status and demonstrated
that phosphate stress affects components of the NSR that are known to be regulated by nitrogen conditions.
We also showed that a mutation in the C-terminal domain of PhoB affected GS gene expression, enzyme
activity, and protein levels.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Poster Presentation 18
Characterizing the role of nucleoporins
in plant-microbe symbiosis
Audrey Wiley, Muthusubramanian Venkateshwaran, Pierre-Marc Delaux, Haruko Imaizumi-Anraku,
Marisa Otegui, and Jean-Michel Ané
Department of Agronomy, University of Wisconsin-Madison, Madison, WI
Nucleoporin proteins (NUPs) are part the Nuclear Pore Complex which is responsible for selective nucleocytoplasmic trafficking of macromolecules. While some NUPs are involved in general cellular processes
such as the trafficking of transcription factors and messenger RNAs, three of them (NUP133, NUP85 and
NENA) have been found to play a unique role in the signaling pathway leading to the establishment of
plant-microbe symbioses in the model legume, Lotus japonicus. We hypothesize that NUP133, NUP85
and NENA are tightly linked to the normal functioning of certain ion channels which are localized to the
nuclear envelope and required for symbiotic associations in legumes. We are analyzing the function of
NUP133, NUP85 and NENA in the trafficking of these symbiosis-related ion channels (LjCASTOR and
LjPOLLUX from Lotus japonicus and MtDMI1 from Medicago truncatula) on the nuclear membranes.
By characterizing the precise role of NUPs in this process, we hope to better understand mechanisms that
control the localization of nuclear envelope proteins in plants and how they affect specific physiological
responses of plants to their environment.
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Poster Presentation 19
Comparative and population genomics of Sinorhizobium spp.
Brendan Epstein, Masayuki Sugawara, Nevin Young, Peter Tiffin, and Michael Sadowsky
Biotechnololgy Institute, Univertsity of Minnesota, St. Paul, MN
Sinorhizobium is among the most well studied members of nitrogen-fixing root nodule bacteria and
contributes substantial amounts of fixed nitrogen to the biosphere. While the alfalfa symbiont Sinorhizobium
meliloti RM1021 was one of the first rhizobial strains to be completely sequenced, little information is
available about the genomes of this large and diverse species group. Here we report the draft assembly and
annotation of 48 strains of Sinorhizobium comprising five genospecies. While S. meliloti and S. medicae
are taxonomically related, they displayed different nodulation patterns on diverse Medicago host plants,
and have differences in gene content including genes involved in conjugation, organic sulfur utilization,
Nod-factor and polysaccharide biosynthesis, denitrification, and Type III, IV, and VI secretion systems.
Nucleotide diversity also varies among and within species: S. medicae has much less diversity than S.
meliloti, and the chromosomes of both species have less diversity than the plasmids. Population genetic
methods revealed evidence for a selective sweep over half the S. meliloti chromosome, as well as 82 genes
with a signature of recent adaptive evolution, including several genes that are likely to affect symbiosis with
the plant host.
Sugawara et al.: Genome Biology 2013, 14:R17
Epstein et al.: PLoS Genetics 2012, 8:e1002868
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Poster Presentation 20
Identification of multiple domains in the MtENOD8
protein that target it to the symbiosome
Matthew Meckfessel and Rebecca Dickstein
Department of Biological Sciences, University of North Texas, Denton, TX
Symbiotic nitrogen fixation occurs in nodules, specialized organs on the roots of legumes. Within nodules,
host plant cells are infected with rhizobia found within a plant-derived membrane, forming a novel organelle,
the symbiosome. In Medicago truncatula, the symbiosome consists of the symbiosome membrane, a single
rhizobium, and the soluble space between them, the symbiosome space. The symbiosome space is enriched
with plant-derived proteins, including the MtENOD8 protein. MtENOD8 was fused, in whole and in part,
to GFP and transformed into M. truncatula using the hairy root system. We found that the MtENOD8
protein contains at least three targeting domains, including its N-terminal signal peptide (SP), capable of
localizing GFP to the symbiosome. When ectopically expressed in non-nodulated root tissue, fluorescence
from MtENOD8- SP- GFP is found in the vacuole. During a nodulation time-course, there is a nodulespecific re-direction MtENOD8-SP-GFP from the vacuole to punctate intermediates and subsequently to
symbiosomes. The re-direction of MtENOD8-SP-GFP from the vacuole to punctate intermediates precedes
intracellular rhizobial infection. Experiments with Medicago nodulation mutants showed that MtNIP/
LATD, encoding a transporter, but not MtDNF1, encoding a signal peptidase complex subunit, is required
for re-direction of MtENOD8-SP- GFP from the vacuoles to punctate intermediates in nodules.
Support was provided by NSF IOS-0923756 and the UNT Faculty Research Program to Rebecca Dickstein.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Poster Presentation 21
Using a suppressor mutation strategy to study cell envelope
gene interactions within Rhizobium leguminosarum.
K.D. Neudorf, E.M. Vanderlinde, and C.K. Yost
Department of Biology, University of Regina, Regina, Saskatchewan, CanadaGram-negative bacteria
have an outer membrane which serves as a protective barrier against environmental stresses and as
an interface during host-microbe interactions. Mutations that result in structural defects in the outer
membrane often increase a cell’s sensitivity to various stressors and impair host-microbe interactions.
We are studying an uncharacterized four gene operon and investigating its role in cell envelope function in
Rhizobium leguminosarum. The operon is highly conserved among the alpha-proteobacteria, and consists
of a moxR-like AAA+ ATPase (RL3499), a hypothetical protein (RL3500), and two large transmembrane
proteins (RL3501 and RL3502). Mutation of the operon results in sensitivity to membrane disruptors,
an inability to grow on glycine or peptide rich media, and an increase in nodule number on pea plants.
Furthermore, RL3499- RL3502 mutant cells are enlarged, circular and distorted when exposed to glycine
media or peptide-rich complex media. We have used a suppressor mutation strategy and subsequent
isolation of suppressor mutants that regain the ability to grow on glycine or peptide rich media to identify
genes that interact with the RL3499- RL3502 operon. We have isolated suppressor strains that were
able to grow on glycine or peptide rich media and have regained wild-type rod shaped cell morphology.
Transposon mutagenesis was performed in an attempt to identify candidate genes associated with the
suppressor phenotype. This screen led to the isolation of a mutant where the Tn5 insertion had disrupted an
uncharacterized tetratricopeptide repeat containing protein (RL0936) that is also highly conserved in the
alpha-proteobacteria. Mutation of RL0936 results in a decrease in both biofilm formation and desiccation
tolerance, and increased sensitivity to the antibiotics erythromycin and tetracycline, relative to the wildtype strain. Transcriptional fusions using beta-glucuronidase assays revealed that RL0936 is up-regulated in
both the RL3499-RL3502 mutants and the suppressor strains. These results suggest that a newly identified
gene network exists between the RL3499-RL3502 operon and RL0936, and also provides new insight to
the biological function of genes of unknown function.
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Poster Presentation 22
GmFWL1, a regulator of soybean nodulation,
encodes a membrane raft-associated protein.
Marc Libault1,2, Zhenzhen Qiao1, Laurent Brechenmacher2, Gregory W. Strout3, Charlie Jones2,
Scott D. Russell3 and Gary Stacey2
1. Department of Microbiology and Plant Biology, University of Oklahoma, Norman, OK 73019, USA.
2. D
ivision of Plant Sciences, National Center for Soybean Biotechnology, C.S. Bond Life Sciences Center,
University of Missouri-Columbia, Columbia, MO 65211, USA.
3. Samuel Roberts Noble Electron Microscopy Laboratory, University of Oklahoma, Norman, OK 73019, USA.
Membrane rafts are plasma membrane sub-regions enriched in sterol and sphingolipids and exhibiting
a different proteome from the rest of the plasma membrane. Several studies using the model legume
Medicago truncatula highlighted the contribution of membrane raft-associated proteins in regulating
legume nodulation [1]. We are presenting new evidence for the role of membrane rafts in regulating legume
nodulation through the biochemical, molecular and sub-cellular characterization of GmFWL1 (FW2.2-like
1), a soybean protein specifically expressed in response to Bradyrhizobium japonicum inoculation and
regulating soybean nodulation [2]
To characterize the molecular function of GmFWL1, we initiated our analysis by identifying the
protein partners of GmFWL1 in soybean nodules. Expressing the HA-tagged GmFWL1 protein, we coimmunoprecipitated the GmFWL1 protein partners including many membrane raft-associated proteins,
such as prohibitins and flotillins. The direct interactions between three prohibitins and GmFWL1 were
confirmed in tobacco leaves using the split-luciferase assay. Additional experiments suggested that FWL1
might anchor a network of prohibitins to the plasma membrane. Confirming our biochemical analysis, we
identified a M. truncatula homolog of GmFWL1 from the detergent-resistant, plasma membrane fraction
of Medicago roots [3].
To definitively characterize GmFWL1 as a membrane raft-associated protein, we are using the ryofracture
method and transmission electron microscopy on soybean nodules expressing HA-tagged FWL1 proteins
under the control of the native GmFWL1 promoter. Preliminary observations confirmed the plasma
membrane location of GmFWL1 supporting the accessibility of the tag to the antibody. This work will
provide experimental evidence for of the role of membrane raft proteins in soybean nodulation.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Poster Presentation 23
RNAseq to compare hormone biosynthesis and
signaling pathways during lateral root and nodule
development in Soybean.
Sajag Adhikari1, Suresh Damodaran1, Brian Moore2, and Senthil Subramanian1
1. Department of Plant Science,
2. University Networking Systems & Services, South Dakota State University, Brookings, SD
Lateral roots and symbiotic nodules are two important root lateral organs in legumes and play crucial
roles in nutrient acquisition. Lateral roots initiate in response to developmental and environmental clues
where as root nodules arise from symbiotic interactions between legume roots and rhizobia bacteria. Both
these organs arise through postembryonic organogenesis processes. Lateral roots arise from pericycle
founder cells present adjacent to protoxylem poles. In contrast, root nodules arise from cortex cells in the
majority of leguminous plants. In addition, lateral roots have a central vasculature while nodules have
peripheral vasculature. Though these two organs differ in their structure and origin, it has been speculated
that development of nodule might have co-opted some of the functions of lateral root development. We
dissected lateral root and nodule tissues and examined global gene expression using RNAseq to identify
key regulatory pathways active in these tissues. Illumina, single end, 50nt, directional libraries (with three
biological replicates) were prepared for emerging lateral root (ELR), young lateral root (LR), emerging
nodule (EN), mature nodule (MN) and root segments above below each organ as control for respective
samples. Specifically, we present results from our analysis of the above RNAseq data showing the
regulation of the biosynthesis and signaling pathways associated with the two major plant hormones auxin
and cytokinin. Our results are in agreement with functional assays from our lab which indicated that auxin
action might be tightly regulated during nodule development.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Poster Presentation 24
Heterologous expression of rgtF from Mesorhizobium loti in
Rhizobium etli causes the synthesis of lipid A with α-(1,1)-GalA
Artur Muszynski, Dusty B. Brown, and Russell W. Carlson
Complex Carbohydrate Research Center, University of Georgia, Athens, GA
Mesorhizobium loti is a nitrogen-fixing endosymbiont of Lotus japonicus. An unusual α-(1,1)galacturonosyl (GalA) lipid A modification has been reported in the LPS of a number of bacteria
including the stalk forming Caulobacter crescentus, the hyperthermophillic Aquifex aeolicus and the
nitrogen fixing Azospirillum lipoferum, M. huakui and M. loti. We report the structure of the lipid A of
the sequenced M. loti MAFF303099 not previously described, and the identity of the lipid A α-(1,1)-GalA
transferase (GalAT) gene, which we named rgtF. We demonstrated that M. loti lipid A is a fatty acylated
β-(1,6)-2,3-diamino-2,3-dideoxyglucosamine (DAG) disaccharide that is substituted with phosphate at
the 4’ position and an α-(1,1)-GalA at the reducing end, similar to that reported for M. huakuii.
We predicted candidate rgtF genes in bacterial species known to produce lipid A with α-(1,1)-GalA, cloned
the M. loti MAFF303099 rgtF gene into an expression plasmid and introduced that plasmid into agriculturally
important Rhizobium etli bv. phaseoli strains that do not contain α-(1,1)-GalA or the rgtF gene. MALDITOF MS analysis combined with chemical and NMR studies revealed that these rgtF complemented strains
expressed lipid A substituted with an additional α-(1,1)-GalA.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Poster Presentation 25
Legume and non-legume extracts influence
swarming behavior in Rhizobium leguminosarum
Dinah D. Tambalo, Elizabeth M. Vanderlinde, Shawn Robinson, Anupama Halmillawewa,
Michael F. Hynes, and Christopher K. Yost
Department of Biology, University of Regina, Regina, Saskatchewan, Canada
Plants are known to secrete chemical compounds that can change the behavior of rhizosphere inhabiting
bacteria, thus we investigated the effects of host legume seed exudates (SE) and extracts from a bryophyte
(Physomitrella patens) on the swarming behavior of Rhizobium leguminosarum. Faba SE inhibited
swarming of R. leguminosarum VF39, whereas lentil SE and moss extract enhanced swarming, while
pea SE had no effect. Swimming motility was not significantly impacted indicating that the observed
effects are specific to swarming motility. Since swarming is characterized by up-regulation of flagellin
synthesis, we investigated the effect of the extracts on the expression of the major flagellin gene,
flaA and on flagellation of swarmer cells. Exposure to lentil SE and the moss extract increased flaA expression
2-fold while faba SE decreased expression 3-fold, suggesting that flagellin gene regulation may play a
role in the altered swarming behavior. Transmission electron microscopy of swarmer cells corroborates
the results from the transcriptional assays. The moss extract’s promotive effect was reduced in a moss
strigolactone-deficient mutant (Ppccd∆8), suggesting that the plant hormone strigolactone may be involved
in activating swarming motility in R. leguminosarum. Overall, this study demonstrated the ability of
legume and non-legume extracts to influence swarming in R. leguminosarum. The ability of legume SE
to influence rhizobial swarming could have effects on root colonization and host infection while the
promotive effect of moss extract suggests the potential for signalling exchange between rhizobia and
ancestors of vascular plants.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Poster Presentation 26
A galactose catabolic mutant of Sinorhizobium
meliloti exhibits early production of succinoglycan
Barney A. Geddes and Ivan J. Oresnik
Department of Microbiology, University of Manitoba, Winnipeg, MB, Canada
The inability of rhizobial inoculums to compete for nodule occupancy with indigenous strains can limit
the ability of commercial inocula to increase legume crop yields. During the investigation of galactose
catabolism in Sinorhizobium meliloti, we demonstrated that a galactose mutant (SRmD304) was more
competitive for nodule occupancy than wild type (Rm1021). Production of the symbiotically active
exopolysaccharide succinoglycan by S. meliloti Rm1021 is essential for successful invasion of the host
plant. In this study we observed visual effects on colony mucoidy in SRmD304 when grown on minimal
medium in the presence of galactose that suggested effects on exopolysaccharide production. We observed
unique plate phenotypes using the carbohydrate stains calcofluor, congo red and aniline blue. However,
under these conditions SRmD304 was able to acidify its environment drastically more than Rm1021.
Dye phenotypes were abolished in strongly buffered medium suggesting that either pH was affecting the
binding properties of these stains, or that pH changes within the local environment resulted in changes
to carbohydrate production in S. meliloti. To address this we quantified succinoglycan production over
time in liquid culture. We have shown that increased acidification of the medium was accompanied by
earlier production of the symbiotic exopolysaccharide succinoglycan in SRmD304. This production is also
correlated with increased expression of succinoglycan biosynthetic genes. Taken together these suggest that
increased competition for nodule occupancy in a galactose catabolic mutant may be mediated by increased
succinoglycan production in the galactose rich environment near the plant root.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Poster Presentation 27
Ability to catabolise rhamnose is a determinant in competition
for nodule occupancy in both Rhizobium leguminosarum and
Sinorhizobium meliloti
Damien Rivers and Ivan J. Oresnik
Department of Microbiology, University of Manitoba, Winnipeg, MB, Canada
Rhizobium leguminosarum strains unable to utilize rhamnose as a sole carbon source are less competitive for
nodule occupancy. To determine if the ability to use rhamnose as a sole carbon source affects competition
for nodule occupancy in Sinorhizobium meliloti Tn5 mutants unable to use rhamnose as a sole carbon source
were isolated. S. meliloti mutations affecting rhamnose utilization were found in two operons syntenous to
those of R. leguminosarum.
Although the S. meliloti Tn5 mutants were complemented using the R. leguminosarum cosmid that
contains the entire wild-type rhamnose catabolic locus, complementation did not occur if the cosmids
carried Tn5 insertions within the locus. Through a series of heterologous complementation experiments,
enzyme assays, gene fusion experiments, and transport experiments we show that the S. meliloti regulator,
RhaR, is dominant to the R. leguminosarum regulator. The data also supports the hypothesis that the
R. leguminosarum kinase is capable of directly phosphorylating rhamnose and rhamnulose whereas the
S. meliloti kinase does not have any activity using rhamnose as a substrate.
When competition for nodule occupancy experiments were carried out it was found that S. meliloti mutants
unable to use rhamnose as a sole carbon source were uncompetitive with respect to the wild-type. This
suggests that the ability to utilize rhamnose may be a general determinant in competition for nodule
occupancy.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Poster Presentation 28
The isolation of bacteriocin producing
Bradyrhizobia from field soil in Manitoba
Harry Yudistira, MacLean G. Kohlmeier, and Ivan J. Oresnik
Department of Microbiology, University of Manitoba, Winnipeg, MB, Canada
The ability of Bradyrhizobium to produce bacteriocin has been previously reported, but the characterization
of this trait has not been carried out in a systematic manner. The goal of this work was to isolate Bradyrhizobia
from Manitoba soils to determine if they are capable of producing bacteriocin(s), and to evaluate whether
bacteriocin production is a determinant that can affect competition for nodule occupancy.
Bradyrhizobia were isolated from Manitoba field soil using nodule trapping experiments. Since large
endemic populations of Bradyrhizobia are not present in Manitoba soils only 2 distinct isolates were found.
Sequencing of the V3 region of 16S ribosomal DNA identified these as Bradyrhizobium japonicum (FN1)
and Bradyrhizobium yuanmingense (BM1). These strains, as well as other collected Bradyrhizobia, were
tested for their ability to produce bacteriocins. The results showed that at least 6 different bacteriocin or
bacteriocin- like zones of inhibition could be detected. One strain, (FN1), was chosen for further study.
Initial studies show that FN1, when competed against a bacteriocin sensitive strain, was more competitive
nodule occupancy when inoculated onto soybean. This data suggests that the production of bacteriocin
appears to be correlated with the competitiveness for nodule occupancy. To determine if the production of
bacteriocin directly affects the competition for nodule occupancy we are planning on constructing isogenic
strains unable to produce bacteriocin. To facilitate construction we have sequenced the strain FN1 and are
searching for candidate genes.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Poster Presentation 29
Characterization of a putative Mg2+ efflux
protein in Sinorhizobium meliloti
Justin Hawkins and Ivan J. Oresnik
Department of Microbiology, University of Manitoba, Winnipeg, MB, Canada
Sinorhizobium meliloti is capable of producing at least two types of exopolysaccharide (EPS); EPS I,
succinoglycan; and EPS II, galactoglucan. EPS plays an important role in the physiology of a bacterial cell.
In S. meliloti low molecular weight EPS has been shown to play a role in plant-microbe interactions. It has
also been shown to play a role in survival in a medium that contains high ion concentrations. It has been
previously shown that high concentrations of Mg2+ or K+ were capable of suppressing the mucoid (EPS
II) phenotype associated with an expR+ derivative of Rm1021.
To determine how Mg2+ affects expression of genes involved with EPS biosynthesis an expR+ derivative
of Rm1021 was mutagenized with Tn5 and plated onto medium containing high concentrations of Mg2+
and screening for mucoid variants. The mutants isolated consisted of strains carrying Tn5 insertions in
exoX, emmB, phoC, as well as SMc00722. SMc00722 is annotated as a hypothetical transmembrane
protein that is conserved in the proteobacteria, and contains a site with weak homology to the magnesium
binding domain of CorA. Characterization of SMc00722 in a Rm1021 background showed that the
increased mucoidy was due to EPS-I. In addition to increased EPS biosynthesis, strains carrying
mutations in SMc00722 showed increased biofilm production and were more sensitive to elevated
Mg2+ concentrations. In addition we show that strains carrying a SMc00722 have elevated cytoplasmic
Mg2+ concentrations. Taken together the data is consistent with the hypothesis that SMc0722 may be a
involved in Mg2+ efflux from the cell.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Poster Presentation 30
Genetic characterization of a suppressor mutation that
allows Sinorhizobium meliloti Rm1021 to utilize xylitol
MacLean G. Kohlmeier, Barney A. Geddes, Jacqueline Donogh, and Ivan J. Oresnik
Department of Microbiology, University of Manitoba, Winnipeg, MB, Canada
Metabolic pathways allowing for the degradation of xylitol are not common among bacteria. During the
investigation of erythritol, adonitol and L-arabitol catabolism in S. meliloti Rm1021, it was observed that
S. meliloti is not capable of efficient growth using xylitol as a sole carbon source. However, spontaneous
suppressor mutants that were capable of utilizing xylitol arose at a frequency of 10-8. The goal of this work
was to identify the mechanism that permitted xylitol utilization in these suppressor mutants of Rm1021.
One of these mutants, SRmD268, was further characterized in this work.
A typical strategy for polyol utilization is the formation of a keto-sugar, followed by phosphorylation
before entering central metabolism. The reduction of xylitol leads to the formation of xylulose. The
introduction of xylB (xylulose kinase) mutation into SRmD268 by transduction abolished the ability
to grow on xylitol, suggesting the pentitol is catabolized via a traditional pathway. The mutation that
permitted growth on xylitol was localized by transduction and mutagenesis to a chromosomal region
encompassing a putative operon SMc02021-SMc01990. Candidate xylitol dehydrogenases within this
operon are currently being characterized by deletion and overexpression analysis. The region is also being
sequenced to identify the molecular nature of the mutation.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Poster Presentation 31
New insights into rhizobial plasmid organization
and function from the Rhizobium leguminosarum
biovar viciae strain VF39 genome sequence
Michael F. Hynes, Cynthia B. Yip, and Hao Ding
Department of Biological Sciences, University of Calgary. Calgary, AB, Canada
Plasmids in the rhizobia can constitute up to 45% of the genome of certain strains of the genera Sinorhizobium
and Rhizobium. In addition to encoding genes important for symbiosis, plasmids carry catabolic and other
genes that contribute to the competitive fitness of the strain. R. leguminosarum strain VF39, orginally
isolated from faba bean, carries six plasmids (130 to 900kb), and was the first strain for which derivatives
cured of each individual plasmid were obtained (1). We have recently completed the draft genome of VF39,
which contains >700 kb of DNA not found in strain 3841. Most of this DNA is associated with plasmids,
much of it coming from the two smallest plasmids, pRleVF39a and pRleVF39b. pRleVF39a has a repABC
system similar to one of two located on pRL7JI in 3841. It carries a complete set of transfer genes of Type
I (2), which is functional despite lacking a traI gene. It also carries tyrosinase genes involved in melanin
production that are highly related to orthologous genes in Nitrobacter hamburgensis, but not found in other
rhizobial genomes. pRleVF39b encodes a restriction system, along with a previously unstudied type of
conjugative transfer system belonging to Type 4 (3). pRleVF39b replication is dependent on a repABC
operon corresponding to a second repABC gene set present on pRL7JI.
1. Hynes MF & McGregor NF (1990) Mol Microbiol 4:567-574
2. Ding H & Hynes MF (2009) Can J Microbiol 55:917-927
3. Ding H, Yip CB, Hynes MF (2013) J Bacteriol 195:328-3392013-06-12 09:47:27
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Poster Presentation 32
Ectopic expression of microRNA160 inhibits symbiotic
nodule initiation and development in soybean
Narasimha Rao Nizampatnam1, Marie Turner1, Oliver Yu2, and Senthil Subramanian1
1. Plant Science Department, South Dakota State University, Brookings, SD
2. Donald Danforth Plant Science Center, 975 North Warson Road, St Louis, MO
Abstract Legume nodules are specialized root lateral organs that result from symbiotic interaction between
the plant and a compatible group of nitrogen-fixing soil bacteria collectively termed rhizobia. The role
of auxin in root nodule development is not completely understood. We examined auxin-inducible gene
expression during nodule development in soybean composite plants using the synthetic auxin-responsive
DR5 promoter. We show that there is relatively low auxin activity during nodule initiation and that it
is restricted to the nodule periphery subsequently. To examine if and what role auxin plays during
nodule development, we generated soybean composite plants with altered sensitivity to auxin. We overexpressed microRNA393 to silence the auxin receptor gene family and these roots were hyposensitive
to auxin. These roots nodulated normally suggesting that minimal/reduced auxin signaling is sufficient
for nodule development. We overexpressed microRNA160 to silence a set of repressor ARF transcription
factors and these roots were hypersensitive to auxin. These roots nodulated poorly suggesting that auxin
hypersensitivity inhibits nodule development. To understand the mechanism of auxin action, we examined
cytokinin sensitivity in these roots. These roots were also hyposensitive to cytokinin, and had attenuated
expression of key nodulation-associated transcription factors known to be regulated by cytokinin. Thus,
over expression of miR160 resulted in hypersensitivity to auxin and hyposensitivity to cytokinin led to
inhibition of nodules.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Poster Presentation 33
Accessory plasmid-dependent induction of late-stage
incompatibility in the Medicago-Sinorhizobium symbiosis
Paul A. Price, Houston R. Tanner, and Joel S. Griffitts
Department of Microbiology & Molecular Biology, Brigham Young University, Provo, UT
Legume-Rhizobium pairs are often observed that are able to nodulate but fail to achieve a productive nitrogenfixing symbiosis at later stages of nodule development. In the well-characterized Medicago-Sinorhizobium
symbiosis, some natural isolates of Sinorhizobium are able to nodulate and productively fix nitrogen on a
wide variety of Medicago species, whereas other isolates display a host-restricted phenotype and are only
able to fix nitrogen on specific species or even specific ecotypes of Medicago. Upon analyzing natural
variation in the USDA Sinorhizobium collection, we observed that certain Sinorhizobium isolates harbor
large accessory plasmids that restrict nitrogen fixation on certain hosts. However, the loss of these plasmids
restores nitrogen fixation on previously restricted hosts. We used a plasmid-specific random transposon
mutagenesis strategy to identify the gene(s) on these plasmids that are responsible for host-specific nitrogen
fixation defects. Of the four plasmids tested thus far, the restriction associated with one of these four
plasmids is caused by an M16 Zn-dependent metalloprotease. On the remaining three plasmids, a two-gene
cluster consisting of an orphan LuxR regulator and an orphan two-component response regulator appears
to be responsible for the host-range restriction phenotype. Interestingly, these studies have also revealed an
alternative replication origin on three of the four plasmids that appears to be involved in bacteroid-specific
plasmid replication. Studies are currently underway to determine the precise mechanisms by which these
genes are able to restrict nitrogen fixation on specific host plants.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Poster Presentation 34
The quorum sensing regulator ExpR directly regulates
symbiotically important Flp pili in Sinorhizobium meliloti.
Hardik Zatakia, Cassandra Nelson, Anjali Sharma, and Birgit Scharf
Department of Biological Sciences, Virginia Tech, Blacksburg, Virginia
Type IVb pili (Tfpb) are mostly studied in enteropathogens and have diverse functions in bacterial
aggregation, attachment, and microcolony formation. The Sinorhizobium meliloti. genome contains two pil
gene clusters; one on the chromosome (pil1) and the other on the pSymA megaplasmid (pil2), both of which
code for the genes required for Tfpb synthesis. pilA1 in the pil1 gene cluster encodes the putative pilin
subunit belonging to the flp family of pilins. To establish the role of Tfpb in the symbiotic interaction of
S. meliloti and its host Medicago sativa, we conducted competitive nodulation assays. The S. meliloti pilA1
deletion strain was about 30% deficient in nodulation as compared to the wild type. Transcriptional reporter
gene assays illustrated that the expression of pilA1 peaks at early stationary phase and is repressed by
ExpR. ExpR is a LuxR-type transcriptional regulator and part of the quorum sensing system in S. meliloti. It
regulates exopolysaccharides production and motility in S. meliloti. Direct binding of AHL-activated ExpR
to the pilA1 promoter region was confirmed with electrophoretic mobility shift assays. A 28-bp protected
region on the promoter was defined using DNase I footprinting analyses. This protected region comprised a
17-bp sequence that matches the consensus sequence for ExpR binding (Charoenpanich, Meyer et al. 2013).
Thus, our studies show that S. meliloti coordinates temporal expression of Type IVb pili with other cellular
processes namely exopolysaccharide synthesis and motility for optimal interaction with its host.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Poster Presentation 35
Identification of transcription factors functioning
in root ABA signaling mediated by MtLATD/NIP
by high-throughput qRT-PCR
Chang Zhang, Ivone Torres-Jerez, Michael K. Udvardi, and Jeanne M. Harris
Plant Biology Department, University of Vermont, Burlington, VT
The Medicago truncatula nitrate transporter LATD/NIP (NPF1.7) plays an important role in maintaining
root and nodule meristem function and in mediating ABA signaling. In order to identify transcription
factors that function in the ABA-LATD/NIP signaling pathway, we performed a large-scale transcription
factor (TF) gene expression profiling by qRT-PCR on wild-type and latd roots grown with or without
ABA. We found that the set of TFs regulated by ABA differs in wild-type and latd mutants with only
42 out of 192 genes (22%) regulated by ABA in both genotypes. Conversely, we found that the genes
misexpressed in latd mutants change almost completely in the presence of exogenous ABA. The only gene
whose expression is altered by ABA in both genotypes and by the latd mutation under both ABA treatments
is the Nodulation Signaling Pathway2 (NSP2) gene. We show that NSP2 and preMIR171h, which encodes
a miRNA that regulates NSP2 expression, are both regulated by nitrate, suggesting a role for NSP2 in root
nitrate responses. preMIR171h is also regulated by ABA and LATD, indicating that NSP2 is regulated
by ABA at both the transcriptional and post-transcriptional level, and that LATD/NIP is required for this
process. We are currently trying to determine how NSP2 is involved in ABA-LATD/NIP-mediated root
elongation, and to characterize the function of LATD/NIP in the crosstalk between ABA and cytokinin in
the regulation of NSP2.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Poster Presentation 36
Polyhydroxybutyrate accumulation in
Rhizobium etli bacteroids depends on a phasin
protein whose synthesis is controlled by NifA.
Dale Noel, Sihui Yang, and Laura Muck
Department of Biological Science, Marquette University, Milwaukee, WI
Poly-3-hydroxybutyrate (PHB) is a lipid polymer whose synthesis stores excess carbon and reducing
power within various bacteria. Proteins called phasins co-fractionate with PHB granules isolated from
lysed bacteria. The abundance of these proteins suggests that they may coat the granules to prevent
deleterious PHB-metabolite interactions in bacterial cytoplasm, but the necessity and function of phasins
are still not understood. In 2003 Jahn et al. (MPMI 16:65-73), reported a very abundant protein termed
BacS in bacteroids of Rhizobium etli CFN42 on host Phaseolus vulgaris. Its synthesis could be induced
ex planta at low oxygen concentrations dependent on NifA, the transcriptional regulator of nitrogenase
and certain other bacteroid proteins. However, no function was discovered for BacS, and its elimination
by mutation caused no obvious problems in symbiosis. BLAST searches of the protein sequence database
a few years later led to the hypothesis that BacS is a phasin. The following observations in the present
study support that hypothesis. Mutant bacteroids that lacked all three bacS genes accumulated almost
no PHB. A simple method developed for PHB isolation highly enriched both BacS and PHB from
bacteroid lysates. NifA-mutant bacteroids lacked both BacS and PHB. These observations support
the prior suggestion by Wang et al. (J. Bacteriol. 189: 9050–9056 [2007]) that phasins are required for
PHB accumulation. Furthermore, the regulation of bacS expression suggests that it is to the advantage of R.
etli to coordinate PHB synthesis with the bacteroid state or other activities controlled by NifA and oxygen
concentration.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Poster Presentation 37
Biosynthesis of UDP-N-acetyl-D-quinovosamine,
a precursor to the biosynthesis of the symbiotically
important O antigen in Rhizobium etli CE3
Tiezheng Li, Evgenii L. Kovrigin, and K. Dale Noel
Department of Biological Science, Marquette University, Milwaukee, WI
The rare sugar N-acetyl-D-quinovosamine (QuiNAc) in lipopolysaccharide (LPS) has been correlated with
successful infection of Rhizobium bacteria on legume hosts. The structure of Rhizobium etli CE3 O-antigen
indicates that D-QuiNAc is likely to be the first sugar added in O-antigen synthesis. All examined strains of
R. etli and R. leguminosarum harbor on different plasmids a short cluster of O-antigen genes. We propose
that two of those genes in R. etli CE3, wreU and wreV, in association with a third gene (wreQ) located
on the chromosome, specify the synthesis of UDP-D-QuiNAc and the first step of O-antigen synthesis.
Therefore, the presence of D-QuiNAc and its position in LPS may be a conserved feature of the genus
Rhizobium. In our model, wreV and wreQ encode a dehydratase and a putative reductase which together
catalyze the synthesis of UDP-D-QuiNAc from UDP-D-GlcNAc in a two-step manner. wreU encodes a
putative glycosyltransferase which we think initiates O-antigen synthesis by adding phospho-QuiNAc
to bactoprenol-phosphate. To test this model, we first attempted to synthesize UDP-D-QuiNAc in vitro
with purified enzymes. WreQ was overexpressed as histidine-tagged protein and purified by nickel affinity
chromatography. A coupled enzyme reaction was carried out containing substrate UDP-D-GlcNAc, a wellstudied homolog of WreV, and WreQ. The final product was UDP-D-QuiNAc, as confirmed by GC-MS
and NMR. The synthesized UDP-D-QuiNAc is being used as a substrate for the predicted WreU reaction in
vitro, and it can be applied to the studies of other polysaccharides that contain D-QuiNAc.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Poster Presentation 38
Rhizobium etli CFN42 utilizes a cytochrome O-type
quinol oxidase to adapt to lower oxygen concentrations
Zac Lunak and Dale Noel
Department of Biological Science, Marquette University, Milwaukee, WI
Rhizobia, like other bacteria, respire aerobically through a variety of terminal oxidases. It has been well
established rhizobia utilize a high-affinity cbb3 oxidase at low oxygen concentrations, such as during the
symbiosis with Phaseolus vulgaris. Little is known about how other oxidases are integrated into aerobic
physiology. As a start to understanding the roles of other oxidases in rhizobia, Rhizobium etli mutants
deficient in each of the terminal oxidases were analyzed for their ability to grow at high (21%) and low
oxygen concentrations (1% and 0.1%). When transferred from high to low oxygen conditions, a Cyomutant, defective in the “cytochrome o”-type quinol oxidase, had greatly delayed growth compared with
other oxidase mutants and the wild type. In the symbiosis with Phaseolus vulgaris, the nitrogenase activity
and staining of bacterial content of Cyo- nodules was significantly lower compared to nodules harboring
the wild type during early nodule maturation (7-8 days post inoculation). As nodules matured, differences
due to Cyo- were no longer observed. From our observations, it is hypothesized that Cyo is utilized in
intermediate oxygen concentrations acting as a “bridge oxidase” between low and high-affinity cytochrome
C oxidases. During the symbiosis, it is hypothesized Cyo is utilized during infection when rhizobia are
forced to adapt to decreasing oxygen concentrations.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Poster Presentation 39
Rhizobium sp. IRBG74 utilizes common mechanisms for
endophytic colonization of Sesbania cannabina and rice.
Shubhajit Mitra, Arijit Mukherjee, Matthew Crook, Euan James, Michael Sadowsky, Jean-Michel Ane,
and Prasad Gyaneshwar
Department of Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee, WI
Rhizobium sp. IRBG74 is the first known nitrogen-fixing symbiont in the Agrobacterium/Rhizobium clade
that nodulates the aquatic legume Sesbania sp. and is also a growth promoting endophyte of wetland rice.
Using a strain marked with GUS and GFP, we show that Rhizobium sp. IRBG74 is attracted towards rice
roots and this chemotaxis is abolished by glucose or dicarboxylic acids, but not by ammonium. Rhizobium
sp. IRBG74 not only formed extensive cell aggregation and biofilms on the surface of rice roots, but
also penetrated into rice roots and was localized within intercellular spaces and in the xylem of roots and
stems. Transposon-induced mutants in rffB or in thiQ showed significantly reduced external and internal
colonization of rice, as well as defects in nodulation of S. cannabina. Analysis of LPS from rffB mutant
suggested the absence of the O-antigen. External addition of LPS from the wild-type strain complemented
the rice colonization but not S. cannabina nodulation defects of the rffB mutant. We also report observations
that (i) Rhizobium sp. IRBG74 showed significantly less colonization of rice mutant lacking OsDM13, a
gene involved in mycorrhizal colonization of rice; and (ii) Inoculation of rice with Rhizobium sp. IRBG74
induced the expression of mycorrhizal signaling pathway. Taken together, these results indicate that
Rhizobium sp. IRBG74 is a novel rice endophyte that likely utilizes lipo-chitooligosaccharide mediated
signaling to colonize its host-legume and rice and thus may have promising characteristics for engineering
more efficient nitrogen fixation in cereals. To further characterize this unique association between rhizobia
and rice, we have determined the genome sequence of Rhizobium sp. IRBG74 which is composed of a
circular chromosome, a linear chromosome, and a symbiotic plasmid, pIRBG74a.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Poster Presentation 40
Characterization of rhizobial symbionts of invasive and native
Mimosa spp in India
Shubhajit Mitra, Hukam Gehlot, Nisha Tak, Wen-Ming Chen, Janet Sprent, Peter Young, Euan James,
and Prasad Gyaneshwar.
Department of Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee, WI
Indian subcontinent contains two endemic Mimosa species (M. hamata and M. himalayana) and one
invasive (M. pudica) species thus representing a unique situation to examine the role of rhizobial symbionts
in plant invasion. In this study rhizobial symbionts from these three Mimosa spp growing in different
parts of India were isolated and characterized based on their 16S rDNA, nifH and nodA sequences. the
symbionts of M. hamata and M. himalayana (from Rajasthan, NW India) were very similar to Ensifer
saheli bv. acacieae. However, their nifH sequences were more similar to E. kostiensis. Their nodA
sequences were distinct from any known rhizobial species, but showed some similarities to those from
E. arboris (95%) and E. kostiensis (91%). In contrast to these native species, the invasive M. pudica
was predominantly nodulated by Cupriavidus taiwanensis in the northern, western and central parts
of India, and by Burkholderia spp. in the eastern and north-eastern parts. All strains tested effectively
nodulated their original hosts, but the symbionts of the native species could not nodulate M. pudica,
and the M. pudica symbionts either failed to nodulate or formed ineffective nodules on the native
Mimosa spp. These results suggest that the native species of Mimosa in India are nodulated by
different and unique alpha-rhizobial microsymbionts, and that they appear not to share symbionts
with the invasive species M. pudica, which are mostly beta-rhizobial.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Poster Presentation 41
Early stages of symbiosis between Sinorhizobium meliloti
and Medicago sativa involves chemotaxis to seed derived
amino acids
Benjamin A. Webb, Sherry Hildreth, Aziz Traore, Bahareh Behkam, Richard Helm, and Birgit E. Scharf
Department of Biological Sciences, Virginia Tech, Blacksburg, Virginia
Host specificity for Sinorhizobium meliloti. is determined mainly at the site of root hair infection, involving
the exchange of host plant secreted flavonoids and rhizobial derived nodulation factors [1,2]. In this study,
we investigated chemotactic signals preceding the established line of communication with Medicago sativa
(alfalfa) and demonstrated that S. meliloti is chemotactically attracted toward the seed exudate of alfalfa.
The importance of amino acids in exudate detection was substantiated by the analysis of Methyl accepting
Chemotaxis Protein U (McpU), one of the eight chemoreceptors that S. meliloti requires for chemotaxis.
Previous studies from our lab revealed that McpU is important for chemotaxis toward a wide range of
nutrient attractants [3]. Here, we present that McpU plays a major role in chemotaxis toward alfalfa seed
exudate and in particular, the exudate component, proline. Behavioral and biochemical analyses confirmed
that the response of S. meliloti to proline is mediated via direct binding to the periplasmic region of McpU
and involves two conserved aspartate residues. Quantitative mass spectrum analyses of proline present in
seed exudate has allowed us to model the diffusion of proline from a germinating seed into an aqueous
spermosphere. The model revealed that proline concentrations present in the spermosphere are sufficient to
elicit a chemotactic response at a distance of one cm from germinating seeds. These results demonstrate that
McpU has a positive role in sensing components of host seed exudate, suggesting that chemotaxis toward
the germinating seed is a key step in the establishment of the endosymbiosis.
References
1. G
oedhart J, Bono JJ, Gadella TW, Jr.: Rapid colorimetric quantification of lipo-chitooligosaccharides from Mesorhizobium loti
and Sinorhizobium meliloti.. Mol. Plant Microbe Interact. 2002, 15(9):859-865.
2.Phillips DA, Tsai SM: Flavonoids as plant signals to rhizosphere microbes. Mycorrhiza 1992, 1(2):55-58.
3. M
eier VM, Muschler P, Scharf BE: Functional analysis of nine putative chemoreceptor proteins in Sinorhizobium meliloti.
J. Bacteriol. 2007, 189(5):1816-1826.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Poster Presentation 42
Link between motility and transcriptional regulation
of the polyhydroxybutyrate cycle in Sinorhizobium meliloti.
Maya D’Alessio and Trevor Charles
University of Waterloo, Waterloo, N2L 4X7 Canada
Sinorhizobium meliloti. produces polyhydroxybutyrate (PHB) under conditions of nutrient imbalance.
The major PHB cycle enzymes and their corresponding genes have been identified, but their regulatory
system remains unknown. A Tn5 insertion mutant showing increased phbC and decreased bdhA
expression underwent sequence analysis and the transposon was found to be in motB, a gene from the
mot operon, which comprises motB, motC and fliK, and is known to be involved in flagellar synthesis and
regulation. In Helicobacter pylori, fliK affects the regulation of RpoN, the σ54 sigma factor responsible
for the regulation of Class II flagellar genes and nitrogen utilization. Findings suggest that there is a
specific and local fliK dependent feedback on RpoN-mediated regulation (1). We hypothesize that the
Tn5 insertion in motB causes a polar effect on the mot operon, resulting in the observed phenotype.
To address this hypothesis, the open reading frames of fliK, motB and both motB and motC were separately
cloned into the expression vector, pSRKGm (2). The expression constructs were then introduced into the
parental strain and the Tn5 mutant containing a lacZ fusion to either phbC or bdhA. These strains were
induced with 0.4 mM IPTG and assayed for β-galactosidase activity. Early results show that the presence of
fliK does not restore the wild type phenotype, but in fact increases the severity of the mutant phenotype. The
strain containing both the motB and motC open reading frames did, however, complement the phenotype,
returning expression of bdhA and phbC to normal levels.
1. F. P. Douillard, K. A. Ryan, J. Hinds, P. W. O’Toole, Effect of FliK mutation on the transcriptional activity of the 54 sigma
factor RpoN in Helicobacter pylori, Microbiology 155, 1901-1911 (2009).
2. S. R. Khan, J. Gaines, R. M. Roop, S. K. Farrand, Broad-Host-Range Expression Vectors with Tightly Regulated
Promoters and Their Use To Examine the Influence of TraR and TraM Expression on Ti Plasmid Quorum Sensing,
Applied and Environmental Microbiology 74, 5053-5062 (2008).
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Poster Presentation 43
Lotus japonicus AMP1 and HAR1 act
synergistically to regulated root architecture.
Alexandre Tromas1, Chong S. Kim1,2, Mark Held 1,#, Takuya Suzaki3, Bogumil Karas1,¶, Shusei Sato4,
Satoshi Tabata4, Masayoshi Kawaguchi3, and Krzysztof Szczyglowski1,2
1. Agriculture and Agri-Food Canada, Southern Crop Protection and Food Research Centre, London, ON, N5V4T3, Canada
2. University of Western Ontario, Department of Biology, London, ON, N6A5B7, Canada
3. National Institute for Basic Biology, Division of Symbiotic Systems, Aichi 444-8585, Japan.
4. Kazusa DNA Research Institute, Kisarazu, Chiba 292-0812, Japan
#. Current address: University of Minnesota, Department of Biochemistry, Saint Paul, MN 55108, USA
¶. Current address: J. Craig Venter Institute, San Diego, CA, 92121, USA
Deleterious mutations in the L. japonicus HYPERNODULATION ABERRANT ROOT FORMATION
1 (HAR1) locus lead to hypernodulation and hypermycorrhization phenotypes but also restrict root
length and significantly increase root branching of un-inoculated har1-1 mutant plants. HAR1 is a
central regulator of symbiotic and non-symbiotic root development in L. japonicus. A search for genetic
suppressors of the har1-1 phenotype lead to the identification of a root branching hypermorph, called
L. japonicus cluster root-like1 (crl1; so named for its superficial resemblance to genuine cluster roots).
Instead of wild-type root architecture, crl1 forms one large cluster of short rootlets with limited growth
capacity. Genetic analyses have shown that the crl1 root phenotype is determined by two independently
segregating recessive mutations, har1-1 and Ljamp1-1. We show that the L. japonicas AMP1 gene
encodes a predicted homologue of the Arabidopsis ALTERED MERISTEM PROGRAM 1 protein.
As in Arabidopsis, the Ljamp1-1 mutation has a pleiotropic effect on L. japonicus as reflected by
increased cotyledon number, low fertility and short and highly branched shoots and roots. Thus,
root architecture seems to be regulated by a synergistic action between HAR1 and LjAMP1 and
the simultaneous impairment of these two genes results in reduced apical dominance. Although the
Ljamp1 single mutant root phenotype resembles har1-1, the Ljamp1 mutation does not affect the
symbiotic properties of L. japonicus Gifu, which is unlike an allelic Ljamp1 mutation in L. japonicus
MG20 (Suzaki et al., 2013). We are currently testing the root architecture and nodulation phenotype
of two new Gifu null amp1 mutants.
T. Suzaki et al., Development 140, 353 (2013)
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Poster Presentation 44
Characterization of the nod genes required for the
colonization of different hosts by Rhizobium sp. IRBG74
Matthew B. Crook1, Audrey K. Wiley, Arijit Mukherjee, Shubhajit Mitra, Pierre-Marc Delaux,
Michael J. Sadowsky, Prasad Gyaneshwar, and Jean-Michel Ané
1. Department of Agronomy, University of Wisconsin-Madison, Madison, WI
Rhizobium sp. IRBG74 is capable of nodulating and fixing nitrogen in association with several legume
species of the genus Sesbania. It has also been found to colonize rice as an epiphyte and an endophyte
and promote rice growth through auxin production, though nitrogen is not fixed in this non-legume host.
More recently, we have shown that colonization of rice by Rhizobium sp. IRBG74 requires components
of the common symbiosis pathway also required for colonization by arbuscular mycorrhizal fungi.
Furthermore, a nodA mutant of Rhizobium sp. IRBG74 is incapable of colonizing rice roots. Taken
together, these results suggest that the ability of Rhizobium sp. IRBG74 to colonize rice is dependent
on the production of nodA-dependent signals activating the mycorrhizal signaling pathway. In order to
identify nod genes possessed by Rhizobium sp. IRBG74, as well as to facilitate genetic analysis of this
organism, we sequenced the genome of Rhizobium sp. IRBG74. The genome proved to be similar to those
of Agrobacterium tumefaciens C58 and Agrobacterium sp. H13-3, possessing a circular chromosome and
a linear chromosome. In addition, Rhizobium sp. IRBG74 possesses a symbiosis plasmid (pIRBG74a)
which contains a large number of nod, nif, and fix genes which are required for nitrogen fixation within
legume hosts. The completed genome of Rhizobium sp. IRBG74 allows us now to analyze the role of
specific nod genes in the symbiotic association with two distinct hosts, a legume (Sesbania bispinosa)
and a cereal (rice).
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Poster Presentation 45
Redundant role of cytokinin receptors during symbiotic
root nodule organogenesis in Lotus japonicus
Mandana Miri1,2, Mark Held1,2 #, Hongwei Hou1, Christian Huynh1, Loretta Ross1,
Shushei Sato3, Satoshi Tabata3, Jillian Perry4, Trevor Wang4, and Krzysztof Szczyglowski1,2
1. Agriculture and Agri-Food Canada, SCPFRC, 1391 Sandford Street, London, Ontario, Canada N5V 4T3.
2. University of Western Ontario, Department of Biology, London, Ontario, Canada N6A 5BF
3. Kazusa DNA Research Institute, 2-6-7 Kazusa-Kamatari, Kisarazu, Chiba, 292-0818 Japan
4. John Innes Centre, Norwich Research Park Colney, Norwich NR4 7UH, United Kingdom
#. Current address: Biotechnology Institute, University of Minnesota, Saint Paul, MN55108.
Cytokinins are essential plant hormones that control many aspects of plant development. They participate
in responses to endogenous cues and play an important role as messengers for external stimuli related to
diverse biotic and abiotic conditions. During beneficial root symbiosis between legumes and nitrogenfixing rhizobia, cytokinin signaling constitutes the key endogenous signal for the nodule structure
organogenesis. In Lotus japonicus, activation of the LHK1 histidine kinase cytokinin receptor is required
and also sufficient for this process to occur [1, 2]. However, the fact that some nodulation still occurs
in lhk1-1 loss-of-function mutants indicates the presence of an LHK1-independent signaling pathway.
In this study, we have tested a hypothesis that additional L. japonicus cytokinin receptors, such as LHK1A,
LHK2 and LHK3, might function in at least a partially redundant manner to mediate nodule formation.
We show that unlike lhk1-1, the triple mutant plant carrying deleterious mutations in all three cytokinin
receptor genes, Lhk1, Lhk1A and Lhk3, does not form nodules. On the other hand, ectopic application
of cytokinin to wild-type roots increases the steady-state level of all of the cytokinin receptor mRNAs
but fails to do so in the lhk1-1 mutant. Based on the obtained data, a working model is presented in
which LHK1 has a unique function in the root epidermis but acts partially redundantly with LHK1A
and LHK3 in the root cortex to mediate differentiation of nodules in L. japonicus.
1. Murray, J. et al., Science 315, 101 (2007).
2. Tirichine, L. et al., Science 315, 104 (2007).
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Poster Presentation 46
Dissection of sensor kinase structural elements responsible
for modulating transmembrane signaling in response to
a periplasmic regulator protein
Ryan D. VanYperen, Taylor S. Orton, and Joel. S. Griffitts
Department of Microbiology & Molecular Biology, Brigham Young University, Provo, UT
The FeuP/Q two-component system has been shown to be required for symbiotic nitrogen fixation in
Sinorhizobium meliloti. Little is known about how two-component systems recognize external signal
information and transfer this information across the membrane into the cell. The Sinorhizobium meliloti
FeuPQ(N) two-component system is a convenient study system, since the small periplasmic protein FeuN
can be thought of as a genetically encoded external cue. Genetic experiments suggest the sensor kinase FeuQ
may both add and remove phosphate from the response regulator FeuP. Phosphorylated FeuP is required for
downstream gene transcription. FeuN somehow negatively regulates FeuPQ signaling, presumably through
its interaction with FeuQ in the periplasm. Here we present an analysis of FeuQ mutants displaying aberrant
signaling phenotypes. “Locked-negative” mutants actively inhibit downstream gene activation, even in the
absence of the negative regulator FeuN. “Locked-positive” mutants fail to negatively regulate downstream
gene activation, even in the presence of FeuN. Alterations leading to locked-negative and locked-positive
phenotypes cluster to similar regions of the FeuQ, namely the N-terminal periplasmic region, the HAMP
domain, and the active catalytic (AC) domain. Locked-negative mutations that cluster in the periplasmic
domain of FeuQ suggest a specific surface that interacts with FeuN and/or communicates information about
this interaction through transmembrane helices to the AC domain. We hypothesize that the periplasmic
interaction between FeuN and FeuQ initiates a conformational change in FeuQ, resulting in a net increase
in phosphatase activity in the cytoplasm that inactivates FeuP.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Poster Presentation 47
MIR171 family microRNAs negatively regulate soybean
nodulation by targeting GRAS transcription factors
Md Shakhawat Hossain, Zhe Yan, and Gary Stacey
National Center for Soybean Biotechnology, Divisions of Plant Science and Biochemistry,
University of Missouri, Columbia, USA.
In plants, miRNAs (21-24 nucleotides in length) negatively control the expression of endogenous target
genes through transcript cleavage or translational inhibition and play significant roles during growth and
development. Legume plants have the ability to form a specialized organ, the nodule, through interaction
with nitrogen fixing, soil bacteria (i.e., rhizobia). In order to better understand the regulatory roles of
miRNAs in the legume nodulation processes, we characterized gma-miR171-1 and gma-miR171-2 and
their target genes in soybean. These two miRNAs were identified from Bradyrhizobium japonicum
infected soybean root hairs (a single cell model) and stripped roots (i.e., roots with root hairs removed)
by Illumina Solexa sequencing. Although the mature miRNA sequences of these miRNAs are identical,
their precursors are divergent. Strong, ectopic expression of gma-miR171-1 and gma-miR171-2 in
soybean roots resulted in a significant reduction of nodule formation after B. japonicum inoculation.
Paired Analysis of RNA Ends (PARE) sequencing identified the target genes of these two miRNAs. Both
gma-miR171-1 and gma-miR171-2 target a GRAS superfamily transcription factor (TF): gma-miR171-1
targets GRAS-1 TF, while gma-miR171-2 targets GRAS-2 TF. RNA- seq transcriptomic analysis
revealed that GmGRAS-2 is expressed during the very early stages of root hair-B. japonicum interaction,
while GmGRAS-1 is expressed at later stages of nodule development. RNAi silencing of GmGRAS-1
significantly decreased nodule numbers, consistent with the results from gma-miR171-1 overexpression.
Moreover, our result suggests that gma-miR171-1 and gma-miR171-2 mediated regulation of GRAS-1
and GRAS-2 might influence expression of the Nodule Inception (NIN) and Early Nodulin 40 (ENOD40)
genes during the B. japonicum-soybean nodulation process.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Poster Presentation 48
Characterization of a Bradyrhizobium japonicum
P450 involved in gibberellin biosynthesis.
Ariana Marcassa and Trevor Charles.
Department of Biology, University of Waterloo, Waterloo, ON, Canada
The symbiosis between B. japonicum and soybean is of tremendous agricultural importance. While much
is known about this interaction, the role of bacterially-derived gibberellin (GA) in this relationship is not
well understood. Additionally, although the biosynthesis of GA in fungi and plants has been thoroughly
characterized, the bacterial pathway has not yet been completely elucidated at the molecular level. The
gibberellin biosynthesis operon in B. japonicum is known, (Tully and Keister, 1993) but only two of the
corresponding proteins have been experimentally characterized (Morrone et al., 2009). In an attempt to
further understand the genetics of the pathway in B. japonicum, we have focused on cloning and expressing
the pathway’s three cytochrome P450 enzymes, coupled with precise chromosomal deletions of the open
reading frames. Overexpressing and purifying the gene products from Escherichia coli followed by study of
the substrate specificity and kinetics of the enzymes will provide insight into the catalytic capabilities of the
pathway and generate new knowledge about the biosynthesis of gibberellin in bacteria. These data may also
contribute to a better understanding of the role that bacterial gibberellins play in the symbiotic relationship
between B. japonicum and soybean.
Morrone D., Chambers J., Lowry L., Kim G., Anterola A., Bender K., Peters R. J. 2009. Gibberellin biosynthesis in bacteria:
separate ent-copalyl diphosphate and ent-kaurene synthases in Bradyrhizobium japonicum. FEBS Lett. 583:475-480.
Tully R. E., Keister D. L. 1993. Cloning and mutagenesis of a cytochrome P-450 locus from Bradyrhizobium japonicum
that is expressed anaerobically and symbiotically. Appl. Environ. Microb. 59:4136-4142.
Tully R. E., van Berkum P., Lovins K. W., Keister D. L. 1998. Identification and sequencing of a cytochrome P450 gene cluster from
Bradyrhizobium japonicum. Biochem. Biophys. Acta. 1398:243-255.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Poster Presentation 49
Transcriptome analysis and identification of
stress resistance genes in Leucaena leucocephala
Dung T. Pham, Brad W. Porter, and Dulal Borthakur.
Department of Molecular Biosciences & Bioengineering, University of Hawaii-Manoa, Honolulu, HI
Leucaena leucocephala (leucaena) is a leguminous tree adapted to the semi-arid tropics that provides a
valuable source of livestock forage and fuel to people living on marginal lands world-wide. To explore
the molecular basis of leucaena’s drought tolerance, insect and pathogen resistance, nitrogen fixation,
and mimosine biosynthesis, total RNA was extracted from the leaves and roots of two-month-old plants
to construct cDNA libraries for Illumina transcriptome sequence analysis. The resulting leaf and root
transcriptomes were compared for gene candidates with potentially differential expression and screened for
stress response genes previously identified by interspecies suppression subtractive hybridization of leucaena
and Acacia confuse.The homology of 3.4 million contigs was determined using the BLASTX algorithm to
compare sequence to the NCBI non-redundant protein sequences (nr) database. Genes were then classified
into three groups: (i) leaf specific, (ii) root specific, and (iii) those with leaf and root expression. Within
these categories genes associated with drought tolerance, insect and pathogen resistance, and nitrogen
fixation were identified. As a step toward isolating potential genes for mimosine biosynthesis, 3337, 6036,
and 1371 sequences were identified that showed homology with various ligases, synthases and synthetases,
respectively. Quantitative PCR is currently underway to confirm differential expression under a range
of conditions. With this sequence now in hand, candidate markers may be identified to begin screening
populations, with the goal of improving leucaena across a range diverse environments around the world.
This research was supported by NSF Grant No. CBET 08-27057 and HATCH Grant No. HAW00551-H
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Poster Presentation 50
An initial characterization of competition associated
phenotypes in two indigenous strains of Rhizobium
leguminosarum bv. viciae
Benjamin J. Perry, Elizabeth M. Vanderlinde, and Christopher K. Yost
Department of Biology, University of Regina, Regina, SK, Canada
The effectiveness of Rhizobium leguminosarum bv. viciae (Rlv) strains as commercial inoculants may be
limited by the strains inability to outcompete indigenous Rlv strains present in the rhizosphere. The focus of
this research was to characterize two novel indigenous strains of Rlv (CG3 and CG9) isolated from soil with
a history of Pisum sativum cultivation. Characterization examined selected phenotypic traits considered to
be important for competitiveness in the soil environment. The CG3 strain had a relatively low number of
plasmids, a relatively high metabolic diversity, and an inability to swarm on swarming agar. CG3 formed
nodules on P. sativum roots, but the nodules appeared to be inefficient in nitrogen fixation as plant growth
was stunted and leaves were chlorotic. The CG9 strain was observed to have a relatively high number of
plasmids, a slightly lower metabolic diversity than CG3, and was efficient at swarming. Plants inoculated
with CG9 formed healthy nodules with no symptoms of nitrogen deficiency. Presently, a transposon
insertion identification approach using next generation sequencing is being developed, in conjunction with
inter-strain competition assays, as a high through-put method to identify genetic determinants correlated
with fitness in the rhizosphere.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Poster Presentation 51
A proteomic approach to lipo-chitooligosaccharide and
thuricin 17 effects on soybean salt stress at 48h germination
Sowmya Subramanian, Emily Ricci, Alfred Souleimanov, and Donald L. Smith
Department of Plant Sceinces, McGill University, Macdonald Campus, Ste. Anne de Bellevue, Quebec, Canada
Salt stress is an important abiotic stressor affecting crop growth and productivity. Of the 20% of agricultural
land available globally, 50% of the cropland is estimated by the United Nations Environment Program
(The UNEP) to be salt-stressed (Yan, 2008). Increased soil salinity has profound effects on seed germination
and germinating seedlings as they are frequently confronted with much higher salinities than vigorously
growing plants, because germination usually occurs in surface soils, which is the site of soluble salt
accumulation (Dabuxilatu and Ikeda, 2005). Soybean exposed to 40 mM NaCl is significantly affected
while an exposure to 80 mM NaCl is often lethal (Sobhanian et al., 2010). When treated with bacterial
signal compounds lipo- chito-oligosaccharide and thuricin 17, the soybean seeds (Absolute RR) responded
positively to salt stress of up to 150 mM NaCl. Shotgun proteomics of unstressed and 100 mM NaCl stressed
seeds (48h) revealed many hypothetical, predicted and unknown proteins. Vegetative storage protein A,
ferritin, isocitrase, importin subunits, malate dehydrogenase, allergen Gly m Bd 28k, subtilisin like protease,
lipoxygenase L5, oleosin isoform, alcohol dehydrogenase, aquaporin TIP-type alpha-like proteins were
observed in unstressed seeds, while the stressed group showed the presence of serine-carboxypeptidases,
heat shock 70kDa, lea-protein precursor, thio-redoxin like protein, glutathione transferase, respiratory burst
oxidase homolog and superoxide dismutase mitochondria-like. All these suggest that the germinating seeds
partition their proteome based on stress, the specificity that plays a crucial role in organ maturation and
transition from one stage to another in the plants life cycle, the understanding of which is of fundamental
importance in agriculture especially to global food production.
References
1. Y
an L (2008) Effect of salt stress on seed germination and seedling growth of three salinity plants.
Pakistan Journal of Biological Sciences,11: 1268-1272.
2. D
abuxilatu MI, Ikeda M. (2005) Distribution of K, Na and Cl in root and leaf cells of soybean and cucumber plants
grown under salinity conditions. Soil Science Plant Nutrition, 51:1053-7.
3. S
obhanian H, Razavizadeh R, Nanjo Y, Ehsanpour AA, Jazii FR, Motamed N, Komatsu S (2010) Proteome analysis
of soybean leaves, hypocotyls and roots under salt stress. Proteome science, 8:1-15.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Poster Presentation 52
Transcriptional responses of Bradyrhizobium
japonicum to environmental factors
Woo-Suk Chang, Anchana Thaweethawakorn, Hae-In Lee, Andrew J. Donati, and Jeong-Min Jeon
Department of Biology, University of Texas, Arlington, Texas
Before and during the nodulation process, the soybean symbiont Bradyrhizobium japonicum frequently
encounters fluctuation of environmental factors such as osmotic pressure, low nutrients, heat, low pH,
desiccation, and the oxidative burst produced by the host plant as a defense mechanism. A DNA microarray
was employed to reveal transcription profiles of B. japonicum to these environmental factors. Treatment
with 50 mM NaCl activated stress-inducible genes, but repressed genes involved in chemotaxis and motility.
Increased expression was seen for genes involved in translation, motility, and cell envelope synthesis in
nutrient rich conditions. We also examined differential gene expression between free-living cells and
bacteroids. A number of nif, fix, and hup genes were up-regulated in bacteroids. H2O2 and paraquat were
used as sources of the oxidative stress. Interestingly, aceA encoding isocitrate lyase (ICL) was upregulated
across of many factors tested, specifically highly induced under desiccation and oxidative stress. We
evaluated the physiological and functional role of ICL in the B. japonicum-soybean symbiosis. carQ
encoding extracytoplasmic fuction (ECF) sigma factor also showed high expression under many stressful
conditions. We constructed a carQ knock-out mutant to identify the role of carQ in physiological responses
of B. japonicum to stressful conditions. The findings in this study will provide an insight into how ICL and
the ECF sigma factor are regulated in B. japonicum to deal with environmental stresses.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Poster Presentation 53
Sinorhizobium melilot root hair invasion requires
signal exchange
Shari Walcott, Mary Ellen Heavner, Adrien Claude, and Hai-Ping Cheng
Biological Sciences Department, Lehman College, The City University of New York, Bronx, NY
Sinorhizobium meliloti.-alfalfa nitrogen fixing symbiosis, like all other nitrogen fixing symbiosis, takes
place in soil, where billions of other soil bacteria compete for a chance to get inside the plant roots for a
“free meal”. To ensure the entry of their own symbiotic partner into root nodules, S. meliloti and alfalfa
have to recognize each other through signal exchanges at different points during the establishment of the
symbiosis. The first such signal exchange is the exchange of alfalfa flavonoids and S. meliloti Nod factor.
The successful recognition leads to the formation of curled alfalfa root hairs with S. meliloti trapped in the
middle. The next step of the symbiosis is the formation of infection threats inside curled root hairs, which
can be blocked by either bacterial or plant mutations. S. meliloti mutants that fail to produce the right
structure and amount of exopolysaccharide, succinoglycan, can not elicit the formation of infection threads.
The production of succinoglycan is regulated by the S. meliloti ExoR-ExoS/ChvI (RSI) signal transduction
pathway. Our recent analyses of the RSI pathway suggest that ExoR could function as a periplasmic sensor
to detect yet to be identified plant signals in regulating the production of succinoglycan. These new findings
and previously published results suggest that the formation of infection thread requires an independent set
of signal exchange between S. meliloti and alfalfa.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Poster Presentation 54
Regulation of soybean HD-ZIPIII genes
during root lateral organ development
Suresh Damodaran and Senthil Subramanian
South Dakota State University, Brookings, SD
Class III homeodomain leucine zipper (HD-ZIPIII) proteins are a group of plant-specific transcriptional
factors (TF) that are known to play key role in plant developmental process. Based on reports in Arabidopsis
and other plants, expression and activity of this TF is tightly regulated. But the regulatory mechanism during
nodule development of soybean in symbiosis with rhizobia is unknown. We examined transcriptional, posttranscriptional and post-translational regulation of two GmHD-ZIPIII (GmHD-ZIPIII-1 & GmHD-ZIPIII-2)
during nodule development. Promoter:tdTomato constructs revealed gene expression in root tips, root
vasculature, emerging lateral roots, emerging nodules and vasculature of mature nodules. HD-ZIPIIIs are
regulated in a post-transcriptional manner by miR166 in many plant species. Consistently, we also validated
miR166-mediated cleavage of all 12 soybean GmHD-ZIPIIIs by miR166. Examination of miR166 activity
using a miR166 sensor suggested that it spatially restricts GmHD-ZIPIII expression to the metaxylem
and epidermis in the roots. In mature nodules, GmHD-ZIPIII expression is restricted to the basal part of
nodule vasculature. Since Arabidopsis HD-ZIPIII proteins are regulated post-translation by ZPR proteins,
we examined the interaction of GmHD-ZIPIII-1 & 2 proteins with all eight predicted GmZPR proteins
using yeast-two-hybrid assay. Both GmHD-ZIPIII genes showed positive interaction only with ZPR3b,
ZPR3c and ZPR3d but quantitative assay based on β-GAL activity indicated that ZPR3d - GmHD-ZIPIII-2
pair had the strongest interaction. Examination of ZPR3b, 3c & 3d expression using promoter:tdTomato
constructs indicated that each ZPR might act in distinct nodule zones. These observations suggested that
GmHD-ZIPIII is prone to multiple regulatory mechanisms during root lateral organ development.
The 22nd North American Symbiotic Nitrogen Fixation Conference
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Poster Presentation 55
Origin, evolution and loss of the
plant – microbe symbiotic ‘toolkit’
Pierre-Marc Delaux, Patrick P. Edger, Kranthi Varala, Christophe Roux, Tomoaki Nishiyama,
Hiroyuki Sekimoto, J. Chris Pires, Gloria M. Coruzzi, and Jean-Michel Ané
Department of Agronomy, University of Wisconsin Madison, Madison, WI
Rhizobia are able to form a nitrogen-fixing symbiosis with legumes whereas arbuscular mycorrhizal fungi
associate with most of land plants. Despite this host-range difference, these two microbial symbioses
share striking similarities in their initiation. In both cases, symbiotic microbes perceive signals from
root exudates and produce lipochitooligosaccharidic signals. These microbial signals are then perceived
by the host plant leading to the regulation of symbiotic gene expression, and allowing the colonization
process. Because the arbuscular mycorrhizal symbiosis is much more ancient than the rhizobia – legume
one, it has been proposed that this symbiotic ‘toolkit’ was first used for interactions between primitive
land plants and arbuscular mycorrhizal fungi, and later co-opted for interactions with rhizobia. If this
symbiotic ‘toolkit’ is now well described in legumes, little is known about its evolution and conservation
within the plant lineage. Using a comprehensive phylogenetic analysis of these components in the plant
lineage together with transcriptomic, physiologic and biochemical approaches, we characterized the
stepwise appearance of this symbiotic pathway, with some components predating the first land plants
whereas some others appeared rather recently in flowering plants1. In addition, we found that independent
losses of the arbuscular mycorrhizal symbiosis in at least five flowering plant lineages are systematically
correlated with the loss of the entire ‘toolkit’. Using this correlation and a bioinformatic comparison
of sequenced angiosperm genomes and transcriptomes, we identified new candidate genes potentially
required for symbiotic associations.
1. Delaux et al. Evolution of the plant – microbe symbiotic ‘toolkit’. 2013. Trends Plant Sci. 6: 298-304.
The 22nd North American Symbiotic Nitrogen Fixation Conference
92
Conference Attendees
Sajag Adhikari
George Dicenzo
Kelly Hagberg
Rammyani Bagchi
Rebecca Dickstein
Jeanne Harris
Dulal Borthakur
David Emerich
Fawzy Hashem
Sanhita Chakraborty
Brendan Epstein
Justin Hawkins
Woo-Suk Chang
Richard Farrell
Hauke Hennecke
Haiping Cheng
Turlough Finan
South Dakota State University
[email protected]
University of North Texas
[email protected]
University of Hawaii-Manoa
[email protected]
The University of Vermont
[email protected]
University of Texas-Arlington
[email protected]
City University of New York
Lehman College
[email protected]
Jadd Correia
University of Hawaii-Manoa
[email protected]
Matthew Crook
University of Wisconsin, Madison
[email protected]
Felix Dakora
Tshwane University
[email protected]
Maya D’Alessio
McMaster University
[email protected]
University of North Texas
[email protected]
University of Missouri
[email protected]
University of Minnesota
[email protected]
University of Saskatchewan
[email protected]
McMaster University
[email protected]
Julia Frugoli
Clemson University
[email protected]
Barney Geddes
University of Manitoba
[email protected]
Parna Ghosh
University of Vermont
[email protected]
Lourdes Girard
University of Waterloo
[email protected]
Centro De Ciencias
Genómicas, UNAM
[email protected]
Suresh Damodaran
Victor Gonzalez
South Dakota State University
[email protected]
Pierre-Marc Delaux
University of Wisconsin, Madison
[email protected]
Washington State University
[email protected]
University of Vermont
[email protected]
University of Maryland, Eastern Shore
[email protected]
University of Manitoba
[email protected]
ETH, Institute of Microbiology
Zurich, Switzerland
[email protected]
Georgina Hernández Delgado
Universidad Nacional
Autónoma De México
[email protected]
Ann Hirsch
University of California, Los Angeles
[email protected]
Heinz Hoben
MSI Bio-Next
[email protected]
Michael Honda
University of Hawaii-Manoa
[email protected]
Md Shakhawat Hossain
University of Missouri
[email protected]
Centro De Ciencias
Genómicas, UNAM
[email protected]
Michael Hynes
Joel Griffitts
Juan Imperial
Brigham Young University
[email protected]
The 22nd North American Symbiotic Nitrogen Fixation Conference
University of Calgary
[email protected]
Universidad Politécnica De Madrid
[email protected]
93
Conference Attendees
Luis Iniguez Rabago
Universidad Nacional
Autónoma De México
[email protected]
Kazue Ishihara
University of Hawaii-Manoa
[email protected]
Kathryn Jones
Florida State University
[email protected]
Yaowei Kang
Novozymes Biologicals, Inc.
[email protected]
J. Diane Knight
University of Saskatchewan
[email protected]
Maclean Kohlmeir
University of Manitoba
[email protected]
Tiezheng Li
Marquette University
[email protected]
Marc Libault
University of Oklahoma
[email protected]
Michelle Lum
Loyola Marymount University
[email protected]
Zachary Lunak
Marquette University
[email protected]
Ariana Marcassa
University of Waterloo
[email protected]
Esperanza Martínez-Romero
Universidad Nacional
Autónoma De México
[email protected]
Kiwamu Minamisawa
Ivan Oresnik
Mandana Miri
Onur Oztas
Tohoku University
[email protected]
Agriculture And Agri-Food
Canada/University of
Western Ontario
[email protected]
Shubhajit Mitra
University of Wisconsin-Milwaukee
[email protected]
Salehin Mohammad
University of North Texas
[email protected]
Artur Muszynski
Complex Carbohydrate
Research Center,
University Of Georgia
[email protected]
Kara Neudorf
University of Regina
[email protected]
Narasimha Rao Nizampatnam
South Dakota State University
[email protected]
Dale Noel
Marquette University
[email protected]
Scott Norris
Bio-Next, Inc.
[email protected]
Bárbara Nova Franco
Centro De Ciencias
Genómicas, UNAM
[email protected]
Mark O’Brian
State University of New York, Buffalo
[email protected]
The 22nd North American Symbiotic Nitrogen Fixation Conference
University of Manitoba
[email protected]
University of Massachusetts Amherst
[email protected]
Olga María Pérez Carrascal
Universidad Nacional
Autónoma De México
[email protected]
Benjamin Perry
University of Regina
[email protected]
Dung Pham
University of Hawaii-Manoa
[email protected]
Catalina Pislariu
The Samuel Roberts
Noble Foundation
[email protected]
Gyaneshwar Prasad
University of Wisconsin Milwaukee
[email protected]
Paul Price
Brigham Young University
[email protected]
Larry Purcell
University of Arkansas
[email protected]
Carmen Quinto
Universidad Nacional
Autónoma De México
[email protected]
Damien Rivers
University of Manitoba
[email protected]
Silvia Rossbach
Western Michigan University
[email protected]
94
Conference Attendees
M. J. Sadowsky
University of Minnesota
[email protected]
Mohammad Salehin
University of North Texas
[email protected]
Federico Sánchez Rodriguez
Instituto De Biotecnologia-UNAM
[email protected]
Birgit Scharf
Virginia Tech
[email protected]
Janine Sherrier
University of Delaware
[email protected]
Ellen Simms
University of California, Berkeley
[email protected]
Donald Smith
McGill University
[email protected]
Stewart Smith
Novozymes Biologicals, Inc.
[email protected]
Gary Stacey
University of Missouri
[email protected]
Senthil Subramanian
South Dakota State University
[email protected]
Sowmyalakshmi Subramanian
Muthusubramania
Venkateshwaran
University of Wisconsin Madison
[email protected]
McGill University
sowmyalakshmi.subramanian@
mail.mcgill.ca
Benjamin Webb
Krzysztof Szczyglowski
Audrey Wiley
Dinah Tambalo
Paul Woomer
Louis Tisa
Nevin Young
Alexandre Tromas
Harry Yudistira
Michael Udvardi
Svetlana N. Yurgel
Agriculture And Agri-Food Canada
krzyszt [email protected]
University of Regina
[email protected]
University of New Hampshire
[email protected]
Agriculture And Agri-Food Canada
[email protected]
Samuel Roberts Noble Foundation
[email protected]
Ryan Vanyperen
Brigham Young University
[email protected]
Vijaykumar Veerappan
University of North Texas
[email protected]
The 22nd North American Symbiotic Nitrogen Fixation Conference
Virginia Tech
[email protected]
University of Wisconsin Madison
[email protected]
N2africa
[email protected]
University of Minnesota
[email protected]
University of Manitoba
[email protected]
Washington State University
Institute of Biological Chemistry
[email protected]
Hardik Zatakia
Virginia Tech
[email protected]
ChangZhang
University of Vermont
[email protected]
95