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1293 01570 7593

This is to certify that the

dissertation entitled

Investigating The Role Of An Azorhizobium caulinodans

 

DNA Binding Protein, AcBBPl, In The Expression Of
The Sesbania rostrata Leghemoglobin glhB Gene

presented by

 

Susan Yukie Fujimoto

has been accepted towards fulfillment
of the requirements for

Ph.D. degree in , Genetics

fl

 

 

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Date 5/2 0/9;Z

MSU i: an Affirmative Action/Equal Opportunity Institution 0-1277!

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f To AVOID FINES returnonorbetoredetedue.

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

MSU le An AMI-motive Action/Equel Opportunity Inetltution
Walnut

 

 

INVESTIGATING THE ROLE OF AN AZORHIZOBIUM CA ULINODANS DNA
BINDING PROTEIN, ACBBPl, IN THE EXPRESSION OF THE SESBANIA
ROSTRA TA LEGHEMOGLOBIN GLB3 GENE
B y

Susan Yukie Fujimoto

A DISSERTATION
Submitted to Michigan State University
in partial fulfillment of the requirements

for the degree of

DOCTOR OF PHILOSOPHY

Program in Genetics

1997

ABSTRACT

INVESTIGATING THE ROLE OF AN AZORHIZOBIUM CA ULINODANS DNA
BINDING PROTEIN, ACBBPI, IN THE EXPRESSION OF THE SESBANIA
ROSIRA TA LEGHEMOGLOBIN GLB3 GENE
By

Susan Yukie Fujimoto

In a screen to identify DNA binding proteins which interact with the Sesbania
rostrata leghemoglobin g1b3 (Srglb3) 5’ upstream region, a factor originating from the
symbiont of S. rostrata, Azorhizobium caulinadans was identified in extracts of S.
rostrata nodules (W elters et al., 1993). Characterization and purification of the protein
responsible for the binding activity (A. caulinodans Bacterial Binding Protein 1; AcBBPl)
in addition to the characterization of its target in the Srglb3 promoter (Bacterial Binding
Site 1; BBSl) using transgenic Lotus comiculatus plants harboring chimeric promoter-
reporter gene constructs indicated that this interaction may be important for
leghernoglobin gene expression. The gene encoding AcBBPl was isolated, sequenced,
characterized and subsequently, an AcBBPl deficient mutant was constructed. A 20%
decrease in nitrogen fixation activity was detected in nodules harboring AcBBPl deficient
bacteria using the acetylene reduction assay. Next, sections of S. rostrata stem nodules
induced by the wild-type or the AcBBPl deficient A. caulinodam strain were observed
by transmission electron microscopy. Nodules harboring the AcBBPl deficient strain
contained a greater population plant cells which were uninfected or which appeared to be

slightly delayed in the infection process when compared to its wild-type counterpart.

Immunogold labeling techniques localized AcBBPl to the bacteroid and peribacteroid
membrane. To directly test if BBPl proteins play a role in Srglb3 gene expression,
transgenic L. comiculatus plants harboring the Srglb3 promoter fused to the uidA reporter
gene were generated. In addition, the gene encoding the homolog of AcBBPl in Rhizobium
Iotr' (RIBBPI), the symbiont of L. comiculatus, was isolated, characterized and a R. [at
RlBBPl deficient strain was constructed. The R. 1011' wild-type and RlBBPl mutant
strains were inoculated onto the roots of transgenic L. corniculatus plants and assayed for
reporter gene (GUS) activity to determine what effect this protein may have on Srglb3
gene expression. No statistically significant differences between S. rostrata nodules
harboring wild-type or mutant A. caulinodans were observed. Therefore, although
AcBBPl and RlBBPl can bind to a defined region within the Srglb3 promoter in vitro, it
appears that these bacterial proteins do not significantly influence Srg1b3 gene expression

in viva.

To my parents, for their love and support.

iv

 

ACKNOWLEDGMENTS

Thanks are due to the peOple who contributed to this thesis project and enriched
my life during my time at Michigan State University. First, I would like to thank Frans J.
de Bruijn for allowing me to conduct independent research on a very interesting topic and
for having confidence in me. I also wish to thank my committee members, Thomas
Friedman, Sheng Yang He, Jonathan Walton, Lee McIntosh and Natasha Raikhel for
support and helpful advice. Research in the lab would not have been nearly as much fun
and entertaining without the antics of Krzysztof Szczyglowski, David Silver and Philipp
Kapranov. I would like to thank Kurt Stepnitz and Marlene Cameron, who provided
photographic assistance throughout my project and the numerous laboratories in the PRL
who have helped me along the way.

Finally, Iwould like to thank all the friends I have made at Michigan State for
their support throughout these years, especially the wonderful people in the de Bruijn lab
past and present who have made the lab an excellent place to work. In particular, I thank
the plant group for all of the memories; Jim Dombrowski and Maria Schneider for their
support; Judith Wopereis for her assistance, time, effort and friendship; Chris Vriezen for
his encouragement and great meals; Mary Ellen Davey and Kezar for their hospitality,
friendship, and the “Ledges”; the Szczyglowski family for their friendship and Caroline
Henein for her technical assistance. I would like to especially thank Pauline Bariola who
has been a great friend and housemate, and my parents, who have always supported and

encouraged me in all of my endeavors.

TABLE OF CONTENTS

LIST OF TABLES ........................................................................................................... ix
LIST OF FIGURES ......................................................................................................... x
CHAPTER 1
BACTERIAL-PLANT INTERACTIONS:
EVIDENCE FOR TRANSKINGDOM SIGNALING ..................................................... 1
Introduction ........................................................................................................... 2
Agrobacterium ........................................................................................... 3
Vi rD2 Protein ................................................................................ 4
Vi rE2 Protein ................................................................................. 7
Xanthomonas .................. . ......................................................................... 9
PthA and Avrb6 Proteins ............................................................. 11
AvrBsB .......................................................................................... 12
Rhizobium ................................................................................................. 14
F ixF ............................................................................................... l7
AcBBPl ........................................................................................ 18
Intercellular Transport .............................................................................. 23
Conclusions ............................................................................................... 26
References 29
CHAPTER 2
IDENTIFICATION AND CHARACTERIZATION OF THE GENE ENCODING THE
AZORHIZOBIUM CAULINODANS DNA BINDING PROTEIN, AcBBPl ................
37
Abstract ................................................................................................................ 38
Introduction.............’ ............................................................................................. 40
Materials and Methods ........................................................................................ 43
Bacterial Strains and Plasmids .................................................................. 43
Polymerase Chain Reaction ....................................................................... 43
Southern Blot Analysis ............................................................................. 44
Construction of A. caulinodans Genomic Libraries .................................. 44
DNA Sequencing and Computer Analyses ............................................... 45
Construction of the AcBBPl Deficient Strain .......................................... 45
Gel Mobility Shift Assay ......................................................................... 46
Plant Growth and Nodulation ................................................................... 47
Acetylene Reduction Assays .................................................................... 47
Northern Analysis ..................................................................................... 48

Results ................................................................................................................... 49

Identification and Cloning of the A. caulinodans AcBBPI Gene ............. 49
Analysis of the AcBBPI Gene and its Protein Product (AcBBPl) .......... 52
Creation and Characterization of an A. caulinodans AcBBPl
Deficient Strain .............................................................................. 59
Leghemoglobin (lb) Steady State mRNA Levels in S. rostrata Nodules
Harboring Wild-Type or AcBBPl Deficient Bacteria .................. 69
Discussion ............................................................................................................ 71
References ............................................................................................................ 79
CHAPTER 3
ROLE OF AcBBPl IN INFECTION AND IMMUNOLOCALIZATION OF THE
PROTEIN IN Sesbam'a rostrata NODULES ................................................................... 86
Abstract ................................................................................................................ 87
Introduction ........................................................................................................... 88
Materials and Methods ......................................................................................... 92
Transmission Electron Microscopy .......................................................... 92
Production of Anti-AcBBPl Antibodies .................................................. 92
Protein Gels and Irnmunoblot Analysis .................................................... 94
Gel Mobility Shift Assays ........................................................................ 95
Immunocytochemistry .............................................................................. 95
Results ................................................................................................................... 97
Infection of S. rostrala by an AcBBPl Deficient Mutant Strain ............. 97
Detection of AcBBPl in Cultures and in Nodules .................................... 97
Specificity of the Anti-AcBBPl Antibodies .......................................... 100
Immunolocalization of AcBBPl ............................................................. 102
Discussion .......................................................................................................... 107
References ........................................................................................................... l 12
CHAPTER 4
ANALYZING THE ROLE OF BBPl IN TRANSGENIC LOTUS
CORNICULA TUS .......................................................................................................... 115
Abstract .............................................................................................................. 116
Introduction ........................................................................................................ 1 17
Materials and Methods ...................................................................................... 121
Bacterial Strains and Plasmids Used ....................................................... 121
Southern Blot Analysis .......................................................................... 121
Construction of an R Ioti Partial Library ............................................... 122
DNA Sequencing and Computer Analyses ............................................ 122
Construction of the RlBBPl Deficient Strain ........................................ 123
Gel Mobility Shift Assay ...................................................................... 124
Plant Transformation and Nodulati on ................................................... 124
Quantification of GUS Enzymatic Activity .......................................... 125

Statistical Analysis ....................................................... - .......................... 1 25

Results ................................................................................................................ 126
Identification and Cloning of the R loti RlBBPl Gene .......................... 126
Analysis of the AcBBPl Gene and Its Protein Product (RIBBPl) ....... 126

Creation and Characterization of An R. loti RlBBPl Deficient
Strain ........................................................................................... 132
Directly Testing the Effect of BBPI on Srglb3 Promoter Activity ....... 132
Discussion .......................................................................................................... 142
References................................ ........................................................................... 146

CHAPTER 5

CONCLUSIONS ............................................................................................................ 150

LIST OF TABLES

Table 1 - Strains and Plasmids Used in This Study .......................................................... 50
Table 2 - Strains and Plasmids Used in This Study ......................................................... 127

ix

LIST OF FIGURES

Figure 1-1 - Models For Directly Influencing Plant Gene Expression By Bacterial

Proteins or DNA ....................................................................................... 5
Figure 1-2 - Structure of the Sesbam’a rostrata g1b3 Gene 5’ Upstream Region .............. 19
Figure 1-3 - Role of the BBSl Region in Srglb3 promoter activity .................................. 22
Figure 2-1 - Identification of the AcBBPl Gene in the A. caulinodans Genome ............. 51
Figure 2-2 - Primary Structure and Deduced Amino Acid Sequence of AcBBPl ............. 53

Figure 2-3 - Alignment of AcBBPl Amino Acid Sequence to Control Elements
In Type II Restriction-Modification Systems and To Immunity

Repressors of ¢105 and E. coli .................................................................. 56
Figure 2-4 - Alignment of AcBBPl With TrbA, a Regulator of Vegetative Replication

and Conjugative Transfer of the RK2 Plasmid ......................................... 58
Figure 2-5 - Comparison of the Binding Sites For C.BamI-II, AcBBPl, and the (1)105

Immunity Repressor ................................................................................. 60
Figure 2-6 - Structure of the AcBBPl ORS-425 Deletion/Insertion Mutant ................... 62
Figure 2-7 - Whole Cell Extracts Prepared From the AcBBPl Deficient Mutant Lack

Binding Activity ....................................................................................... 63
Figure 2-8 - Nitrogen Fixation Activity IS Reduced In S. rostrata Root Nodules

Induced By ORS 571-425 ........................................................................ 65
Figure 2-9- Nitrogen Fixation Activity Is Reduced In S. rostrala Stem Nodules

Induced By ORS 571-425 ........................................................................ 67

Figure 2-10 - No Differences In lb Transcripts Were Detected In S. rostrata
Nodules Harboring the Wild-Type or the AcBBPl Deficient

A. caulinodans Strain ................................................................................ 70
Figure 3-1 - S. rostrata Nodules Harboring AcBBPl Deficient Bacteria Are Delayed

In the Infection Process ............................................................................ 98
Figure 3-2 - Overexpression of AcBBPl in E. coli Using the pET System ................... 101

Figure 3-3 - Detection of AcBBPl Protein In Free Living Cultures of A. caulinodans
ORS 571 and In S. rostrata Nodules Harboring A. caulinodans

ORS 571 ................................................................................................. 103
Figure 3-4 - Characterization of AcBBPl Antibodies Using DNA Binding Assays... 104
Figure 3-5 - Immunogold Labeling Locates the AcBBPl Protein .................................. 106
Figure 4-1 - Organization of the RlBBPl Gene in the Genome of R. loti NZP

2037 and Other Bacterial Strains ........................................................... 129
Figure 4-2 - Alignment of RlBBPl With AcBBPl ....................................................... 130
Figure 4-3- Structure of the RlBBP Insertion Mutant .................................................. 133
Figure 4-4 - Whole Cell Extracts Prepared From the RIBBPI Deficient Mutant Lack

Binding Activity .................................................................................... 134
Figure 4-5 - Schematic of the LP14 Construct .............................................................. 135
Figure 4-6 - B-glucuronidase Activity in 26 Day-Old L. corniculatus Nodules

Harvested From Sandy Soil .................................................................... 138

Figure 4-7 - B-glucuronidase Activity in 26 Day-Old L. corniculatus Nodules

Harvested From Mixed Soil ................................................................... 140
Statistical Analysis ................................................................................. 125
Results ................................................................................................................ 126
Identification and Cloning of the R loti RIBBPI Gene .......................... 126
Analysis of the AcBBPl Gene and Its Protein Product (RlBBPl) ....... 126

Creation and Characterization of An R loti RIBBPI Deficient
Strain ........................................................................................... 132
Directly Testing the Effect of BBPI on Srglb3 Promoter Activity ....... 132
Discussion .......................................................................................................... 142
References ........................................................................................................... 146

CHAPTER 5

CONCLUSIONS ............................................................................................................ 150

CHAPTER]

BACTERIAL-PLANT INTERACTIONS:
EVIDENCE FOR TRANSKINGDOM SIGNALING

INTRODUCTION

Bacteria inhabit almost every niche on this earth. They have found ways to
persist where other organisms have failed, ingeniously evolving unique mechanisms to
maintain their existence. Some of these mechanisms lead to interactions with eukaryotic
organisms:

In plant biology, interactions between plants and bacteria have been studied
intensely for different reasons. Plant-pathogen interactions have been studied genetically
for quite a long time because of their agronomic impact. Since many bacteria cause disease,
which can result in catastrophic crop failure, attempts to unravel the signal transduction
pathway which mediates pathogen recognition and the activation of defense responses is
of great interest and importance. In contrast, symbiotic interactions between plants and
bacteria have somehow evolved a way to escape the defense response (Dj ordj evic et al.,
1987) and to form a relationship with a plant that is mutually beneficial. Understanding
the events leading to the establishment of such relationships is important and could have a
direct impact on the food supply of the world.

To date, some signals and commnents of these interactions are known, but much
is still a mystery. Uncovering all of the elements involved and deterrhining how
everything fits together still eludes us, although significant progress is being made to join
the gaps. One important point, which will be discussed further here, is the idea of
modulation of plant gene expression by the bacterium. This is an area where currently not

much is known. For any relationship between plants and bacteria to exist, signal exchange

between the two must occur. Different types of signals have been implicated in several
steps of the interaction between plants and bacteria. Recently, the finding that bacterial
proteins are recognized directly within plant cells in a few systems may become a more
general means of communication between microbes and plants. I will begin my discussion

with the best characterized interaction: Agrobacterium with plants.

Agrobacterium

The best studied case of a bacterium modulating plant gene expression comes from
Agrobacterium tumefaciens, the causative agent of crown gall, a disease which produces
tumors on plants. In nature, this tumorous growth occurs at wound sites and leads to the
formation of what is known as a “gall” at the soil-air junction of the plant (Kado, 1991;
Sheng and Citovsky, 1996). It is noteworthy to mention here that although
Agrobacterium is considered a pathogen, it does not kill its plant host. Within this
structure, specific compounds that Agrobacterium can utilize as a sole carbon and
nitrogen source are produced, namely the class of compounds called opines (Petit et al.,
1978). This results in the creation of a unique environment for the invading bacterium in
the plant. In recent years, this tumorous growth was shown to be the result of the
expression of genes of bacterial origin that are transferred, stably integrated and expressed
in the plant genome (see reviews by Zupan and Zambryski, 1995; Sheng and Citovsky,
1996). This lmowledge is the foundation of plant transformation and has been used

extensively to construct transgenic plants for applied and basic research. To date, this is

the only known example of stable DNA transfer between kingdoms in nature and is a

unique mechanism to modulate plant gene expressi on.

VirD2 Protein

The VirD2 protein is essential for generating the T-strand, a piece of single-
strandcd DNA (T-DNA, Transferred TEA) that is transferred to the plant cell and
integrated into the plant genome (Stachel et al., 1986). VirDZ, in concert with VirDl, is
essential in this process and is responsible for cleaving the bottom strand of the T-DNA
at the border sequences which flank it (Filichkin and Gelvin, 1993). Afier generation of
the single-stranded T-strand, the VirD2 protein remains tightly associated with the 5’ end
of the T-DNA giving it a polar nature for transport. In addition, another protein, VirE2,
binds tightly and cooperatively to this naked DNA, covering the strand to aid in
protecting it from degradation from nucleases. The T-DNA plus VirD2 and VirE2, is
termed the T-complex which is exported from the bacterium and transfered to the plant
nucleus (Howard et al., 1990; Howard et al., 1992; Zupan and Zambryski, 1995). The
VirD2 protein plays an important role not only in formation of the T-strand, but
probably also aids in importing the T-strand into the plant nucleus. Only the N-terminal
half of the VirD2 protein is required for nicking and T-strand formation (Y anofsky et al.,
1986; De Vos and Zambryski, 1989). Additionally, Agrobacterium strains containing
only the N-terminal portion of VirDZ were unable to form tumors on its host (Stachel and
Nestor, 1986; Steck et al., 1990;ng et al., 1990). Therefore it was postulated that the

C-terminal portion of VirD2 was involved in movement of the T-complex into the plant

Figure 1-1. Models for directly influencing plant gene expression by bacterial
proteins or DNA.

(A) Agrobacterium-plant interaction. The bacterial derived T-DNA is transferred to
the plant nucleus by VirD2 and VirE2 through a secretory apparatus and/or pilus
structure and integrated into the plant genome.

(B) Xanthomonas-plant interaction. The AvrBs3 protein is postulated to get into the
plant cell via a Hrp dependent secretion apparatus and/or a pilus-like structure. The
AvrBs3 protein may interact directly or indirectly with an unknown target in the plant
cytoplasm or nucleus. Eventually AvrBs3 can enter the plant nucleus and affect plant
gene expression

(C) Rhizobium-plant interaction. The AcBBPl protein gets into the plant cytoplasm
by an unknown mechanism and is somehow directed to the plant nucleus. The AcBBPl
protein is postulated to modulate plant gene expression by directly affecting transcription
by binding to the 5’ upstream region of the Srglb3 gene.

 

 

9

A. “ nucleus

  
   

Ti plasmid

 

 

 

Agrobacterium plant cell

 

 

i \ !nuc|cus
?

 

 

 

Xanthomonas plant cell

 

   
 

AcBBPl

Azorhizobium

 

 

 

plant cell

cell. This idea was tested and it was found that VirD2 is capable of directing a reporter
protein (B-glucuronidase, GUS) to the plant nucleus of tobacco or maize (Howard at al.,
1992; Citovsky et al, 1994).

VirD2 contains three stretches of baSic amino acids that may act as nuclear
localization signals. It was found by deletion analysis that the two regions found at the C-
terminus could direct chimeric reporter proteins to the plant nucleus of tobacco
protoplasts (Howard et al., 1992). These regions of basic amino acids also have similarity
to known nuclear localization signals (NLSS) found in many nuclear localized proteins
(Chelsky et al., 1989). In addition, each of these regions was mutated and tested for their
ability to direct chimeric reporter proteins to the nucleus. Both regions are necessary for
full nuclear localization (Howard et al., 1992). The absence of one of the regions leads to
partial nuclear localization of the reporter protein while the presence of both lead to strict
nuclear localization identical to fusing the full length VirD2 protein or the C—terminal half
to a reporter protein (Howard et al., 1992). Hence, VirD2 contains a bipartite NLS which

can direct it to the plant cell nucleus.

VirE2 protein

Since VirD2, the protein located at the 5’ end of the T-complex, possesses a
bipartite NLS and can deliver fusion proteins to the plant nucleus, the other component
of the T-complex, VirE2, was also tested for its ability to aid in nuclear uptake. As

mentioned earlier, VirE2 participates in forming the T-complex by coating the naked

single stranded T-DNA, thus protecting it from degradation on its way from the
bacterium to the plant cell (Citovsky et al., 1989). This idea was spawned by the fact that
the T-complex is a large protein-DNA structure (estimated to be 50,000 kDa, Howard et
al. 1992) and a single VirDZ protein may not be sufficient to direct this complex to the
nucleus.

Two regions similar to the bipartite NLS of Xenopus nucleoplasmin were
identified in VirE2 (Citovsky et al., 1992). Additionally, these sequences are conserved
among VirE2 proteins from many Agrobacterium strains identified (Hirooka et al., 1987;
Winans et al., 1987). They were tested for their role in nuclear localization. Using deletion

analysis and chimeric reporter gene constructs (B-glucuronidase, GUS), VirE2 was found

to direct fusion proteins to the nucleus of tobacco protoplasts. Akin to what was found
with VirD2, these two regions act as a bipartite NLS since constructs lacking one of the
regions does not fully localize chimeric proteins to the nucleus. Only with both sequences
present is efficient nuclear localization observed (Citovsky et al., 1992).. Additionally,
the use of transgenic tobacco plants supports the biological role of VirE2 in tumor
formation. Transgenic tobacco plants expressing the VirE2 protein are able to
“complement” an Agrobacterium virE2 mutant strain with regard to tumorigenicity.
Inoculation of this mutant strain on transgenic plants expressing the full VirE2 protein
cause tumors while inoculation of the same strain on untransformed plants do not elicit
any symptoms. Furthermore, transgenic tobacco plants expressing modified VirE2

proteins display phenotypes which positively correlated the presence of an intact

bipartite NLS and tumor formation when inoculated with the VirE2 mutant. The removal
of one portion of the bipartite NLS leads to reduced tumorigenicity while deletion of
both regions previously identified to be essential for nuclear localization completely
blocks tumor formation.

In summary, Agrobacterium represents a unique mode of transkingdom signaling
as a piece of DNA originating from the bacterium is transported to the plant nucleus and
integrated into the genome. The VirD2 and VirE2 proteins not only protect the T-strand
on its journey from the bacterial cell to the plant cell but are essential in directing the the
T-complex to the plant nucleus. Thus, this bacterium has evolved a novel mechanism to
modulate plant gene expression by stably integrating foreign genes into its host genome.
The expression of these new genes in the plant cell create and maintain an environment
suitable for its survival.

Xanthomonas

Xanthomonas comprises a genus of bacteria which cause various types of disease
on many different plants. How one pathogen can cause disease in one plant and not
another is an intensely researched subject. Recognition of the pathogen is key and crucial
to defense. When a pathogen attacks a plant, the host tries to protect itself by eliciting
defense responses which halt further invasion of the pathogen. In many instances, this
correlates with the hypersensitive response (HR) in which localized induced cell death
occurs in the host plant (Keen, 1990). It is generally believed that recognition of the
pathogen involves a gene found in the bacterium, referred to as an avirulence gene (avr),

that interacts with a resistance gene (R) found in the plant. Altemately, if no

10

corresponding R gene is found in the host plant, or no corresponding avr gene is found in
the bacterium, the pathogen will not be recognized, leading to disease of the plant. This
so-callcd “gene-for-gene” relationship defines the basis of resistance to a specific
pathogen by a particular plant (Baron and Zambryski, 1995; Staskawicz et al., 1995).

How bacterial avirulence genes confer specificity for certain hosts has remained a
mystery since-the isolation of the first avr gene over 10 years ago (Staskawicz et al.,
1984). Now over 30 bacterial avr genes have been isolated but only the mode of action of
the aer gene of Pseudomonas syringae pathovar (pv.) tomato has been described. This
avr gene encodes an enzyme which mediates the production of a low molecular weight
compound termed syringolide (Keen et al., 1990). This molecule is the specific elicitor
that is recognized by soybean plants harboring the corresponding R gene Rvg4 (Keen and
Buzzell, 1991).

In Xanthomonas, a large family of related avr genes have been identified. Recent
findings by Yang and Gabriel (1995) and Van den Ackerveken et al. (1996) suggest that
Xanthomonas avr gene products interact with components within the plant cell directly
and therefore supports the hypothesis that the avr gene product directly binds to the R
gene product or the R gene itself (Ellingboe, 1982). Structural characteristics identified in
Xanthomonas avr gene products provide more evidence for this idea. Furthermore,
Gopalan et al. (1996) showed that avr genes fi'om Pseudomonas syringae are recognized
when expressed in plant cells. This was tested by expressing the avrB gene of
Pseudomonas syringae pv. glycinea in Arabidopsis plants harboring the RPM resistance

gene. Plants expressing the AvrB protein exhibited HR in a RPM depedent fashion . This

11

provides additional support for the direct recognition of bacterial proteins in the plant
cell. The interaction of bacterial avr gene products and a currently unknown target in the
plant cell illustrates another example of transkingdom signaling In this system, host gene
expression is modulated by the direct interaction of bacterial proteins with a plant
component within the host cell. This recognition event induces a cascade of events which

trigger a defense response in the plant.

PthA and Avrb6 proteins

The pthA (pth, pathogenicity) gene of X citrr' and the avrb6 gene from X.
cwnpestris are members of a large family of avr/pth genes found in Xanthomonas. To
date, this family includes almost all avr genes described in this bacterium. All members
are very similar (at least 93%) at the nucleotide level and possess three interesting motifs
within their deduced amino acid sequence: 1) a number of tandemly arrayed leucine-rich
repeats which determine avr gene specificity, 2) a series of heptad repeats which are
similar to leucine zippers and may be involved in DNA-protein or protein-protein
interactions and 3) three putative NLSs which could direct this protein to the plant
nucleus (Herbers et al, 1992; Yang and Gabriel, 1995). The pthA and WM genes are
interesting because isogenic strains carrying either of these genes elicit different response
phenotypes on plants. Most members of the avr/pth family confer the ability to elicit the
HR on many plants but the pthA gene and avrb6 genes can specifically confer the ability
to produce hyperplasias on citrus or to elicit water-soaking in cotton, respectively

(Swarup et al., 1992; Yang et al., 1994). It was determined that the leucine—rich repeats are

12

responsible for the three phenotypes observed and that the regions flanking the leucine-
rich repeats are functionally interchangeable (Yang et al., 1994). However, the function of
the heptad repeats or the ability of these gene products to localize to the nucleus was not
tested.

Using chimeric reporter proteins (B-glucuronidase, GUS), Yang and Gabriel
(1995) determined that the C-terminal portion of the PthA and Avrb6 proteins could act
as a functional NLS(s) in plant cells. As previously mentioned, the C-terminal region of
these proteins contain three putative NLSs. However, the NLSs were not precisely
defined in this investigation nor was the presence of intact NLSs correlated with a
pathogenic phenotype. Nonetheless, these results indicate that bacterial avr/pth gene

products could act directly in the plant cell to influence plant gene expression.

AvrBs3

The AvrBs3 protein is also a member of the avr/pth family in Xanthomonas.
Unlike pthA and MM described above, the avrBs3 gene of X campestris pv. vesicatoria
(Bonas et al, 1989) specifically mediates resistance to lines of pepper- plants of the
genotype BS3. In this interaction, pathogen recognition results in the hypersensitive
response (HR, Klemet, 1982) which is characterized by localized cell death (Mittler and
Lam, 1996) that develops into a distinct area of necrosis.

Genetic data indicate that recognition of the avrBs3 gene product (Herbers et al.,

1992; Bonas et al., 1993) by resistant pepper plants is direct. As mentioned previously,

13

the leucine-rich repeats found in AvrBs3 in addition to other avr gene products in this
family determine avirulence gene specificity. Herbers et al. (1992) showed that the precise
removal of some of the leucine-rich repeats changes host specificity. In this investigation,
a four-repeat deletion in the avrBs3 gene product abolishes the ability of pepper plants
with genotype BS3 to recognize this strain as a pathogen. Instead these bacteria are now
recognized by bs3 plants (Herbers et al., 1992). Because changes in the leucine-repeat
region cause changes in specificity of pepper plant genotype, the AvrBs3 protein is
postulated to be the actual elicitor molecule that is recognized by the plant. However, the
AvrBs3 protein does not seem to be secreted from the bacterium nor does the AvrBs3
protein induce the HR when infiltrated into the intercellular spaces of pepper leaves (Van
den Ackerveken et al., 1996). Immunogold localization of the AvrBs3 protein show that it
is only present in the cytoplasm of the bacterium in culture or when the bacteria were
infiltrated into pepper leaves (Brown et al., 1993). No labeling was found in the plant cell.

Recently, Van den Ackerveken et al. (1996), tested the idea that recognition of
AvrBs3 occurs in the plant cell and investigated what role putative NLSs found in the C-
terminal end of AvrBs3 plays in this interaction. They found that when the C-terminal

end of the protein was fused to B-glucuronidase (GUS) and tested for its ability to direct

this chimeric construct to the nucleus using‘particle bombardment of onion epidermal
cells, the reporter construct was able to localize to the nucleus. Additionally, the second
and third NLS in the C-terminal end of AvrBs3 seemed to be more important for AvrBs3

activity than the first NLS.

14

The different deletion/mutation constructs were tested for their ability to elicit the
HR There was a strong correlation with nuclear localization and HR. Constructs with
mutations that did not affect AvrBs3 activity were localized to the nucleus while
mutations that resulted in a total loss or reduction of AvrBs3 activity were not able to
efficiently direct reporter gene constructs to the nucleus (Van den Ackerveken et al.,
1996).

These observations suggest that some avr genes from Xanthomonas are probably
directly recognized in the plant cell. PthA, Avrb6 and AvrBs3 are members of the same
gene family and have similar structural characteristics and all can direct chimeric reporter
proteins to the plant nucleus. These results support the idea that avr gene products may
act directly on an R gene product or the R gene itself and may facilitate the identification
of interacting factors and their locations. These results indicate that recognition of
bacterial proteins directly within plant cells are involved in pathogen recognition (Figure

1.13).

Rhizobr'um
Symbiotic nitrogen fixation is a unique example of a complex and fine tuned
interaction in which Gram-negative bacteria belonging to the genera Azorhizobium,
Bradyrhr’zobium, Rhizobr’um, and Sinorhizobium interact with legume plants to form a
novel structure, the nodule (Mylona et al, 1995; Long, 1996). Multiple signals from both
the bacterium and the plant are exchanged, which induces sets of genes in both symbiotic

partners (Nap and Bisseling, 1990; de Bnrijn and Downie, 1991; Mylona, 1995; Long,

15

1996). Plant genes which are specifically induced and expressed during symbiosis are
termed nodulin genes and are classified as “early” or “late” depending on their time of
induction (van Kammen, 1984). Early nodulin genes are associated with the infection
process and nodule ontogeny while late nodulin genes correlate with nodule function and
maintenance.

Leghernoglobins (Lbs) are late nodulins which carry oxygen to the actively
respiring, nitrogen-fixing bacteroids within the infected cells (Appleby, 1984).
Leghemoglobin (lb) gene expression in legumes is confined to the infected tissues of the
nodule and appears to be localized to the infected cells. In plants which produce
indeterminate nodules such as those elicited on alfalfa by Rhizobium melilotr‘, lb gene
expression has been detected in a single cell layer directly adjacent to the nitrogen fixation
zone. In addition, lb transcripts appear to be expressed only in infected cells of the
nodule. Furthermore, lb gene expression seems to be absent in nodules formed
spontaneously on specific alfalfa lines (Truchet et al., 1989) or induced by auxin
transport inhibitors (Hirsch et al., 1939) or by certain mutant bacterial strains (Dickstein
et al, 1988; de Bruijn et al., 1989). Since these nodules are devoid of bacteria, these
findings suggest that the presence of a bacterial signal may be important for the
expression of lb genes (Nap and Bisseling, de Billy et al., 1991; de Bruijn and Schell,
1992).

In determinate nodules, such as those elicited on soybean by Braajrhizobium
jqionicrmr, the exact location of lb transcripts and Lb proteins have not been clearly

determined. VandenBosch and Newcomb (1988) detected the Lb apoprotein in both the

16

infected and uninfected cells using irnmunochernical techniques. Further support of this
idea came from Kouchi et al. (1989) who found that although lb transcripts are mainly
detected within the infected cells of soybean nodules, low but significant amounts appear
to be present in the uninfected cells.

Chimeric lb-reporter gene constructs in transgenic legume plants have been used
successfully to study lb gene expression. The promoter regions of the soybean lbc3 gerne
and the Sesbam'a rosa'ata glb3 gene have been studied in the most detail. Both promoter
regiom can direct a nodule-specific expression pattern on reporter gernes in transgenic
alfalfa (dc Bmijn et al., 1989) and in nodules induced on Lotus comiculaus (Stougaard et
al., 1986, 1987, 1990; Szabados et al., 1990). Additionally, the work of Szabados et al.
(1990), Lauridsen et al. (1993) and Szczyglowski et al. (1994) strongly suggest that an
promoter activity is confined to the infected cells of the nodule.

Considerable progress has been made in elucidating the signals which direct early
morphogernic events in the legume host The plant produces a signal, usually a flavonoid,
which induces a set of nodulation (nod) genes in the bacterium. Subsequently, the
bacterium synthesizes a host specific signal, a lipochitooligosaccharide (nod factor),
which induces a plant developmental progarn leading to nodule formation (Hirsch, 1992;
Mylona et al., 1995; Long, 1996). Although research focused on the early stages of
nodulation has progcssed rapidly, the signals and molecular events important for
bacterial colonization or nodule functioning and mainternance remain largely urnkrnown (dc

Bruijn and Schell, 1992).

17

Although no signals have been identified in the later stages of nodule development,
much indirect evidence supports the existence of a bacterium-derived signal in the
induction of lb gene expression. It has been hypothesized that a signal originating from the
bacterium is important for the induction of lb gerne expression. This idea can be traced
back to Bruening and Wullstein (1972) who postulated that leglnernoglobin synthesis
could be irnitiated by the transfer of a bacterially produced inducer. Likewise, Truchet et
al. (1980) proposed the existence of a Rhizobr'um signal involved in the differentiation of
tlne central infected tissue of the developing nodule. de Billy et al. (1991) also suggested
that this factor may play a role in the infected cell-specific expression of the lb gerne in
alfalfa

Recent findings suggest that transkingdom signaling in symbiosis may occur.
Jabbouri et al. (1996) have identified a novel gene in Rhizobr’um NGR234 whose gerne
product has a putative NLS. In addition, Welters et al. (1993) observed that a factor
orginating from the symbiont of S. rostrata, Azorhizobium caulinodans, can bind to the S.
rash-am leghemoglobin glb3 (Srglb3) 5’ upstream region. These two pieces of evidernce are
intriguing and may illustrate the conservation of signaling mechanisms in plant-microbe
interactions in which bacterial proteins function directly in the plant cell.

FixF

Recently, a gene fixF, was isolated from the broad host range Rhizobium NGR234.
Mutations in this gene abolish the production of a novel, rhamnose-rich
lipopolysaccharide that is produced under flavonoid-induced conditions and result in a

Fix' (unable to fix nitrogen) phenotype (Jabouni et al., 1996). Upon inspection of its

18

deduced amino acid sequence, a putative NLS was found. The ability of this putative NLS
to direct a reporter protein to the plant nucleus was tested and found to localize to this
compartment (Jabbouri et al., 1996). In addition, thefixF gerne appears to be regulated by
the alternate signa factor 054. This signa factor has been shown to regulate nitrogen
fixation (m'f/fix) genes in the bacterium (Stitger et al., 1993). It will be interesting to
determine the role of this lipopolysaccharide in nitrogen fixation and to ascertain if nuclear

localization of FixF is important for nitrogen fixation.

AcBBPl

In an attempt to elucidate the signal transduction pathway leading to
leglnernoglobin (lb) gene induction, the identification of cis-acting elements within the
promoter regions of lb genes and trans-acting factors interacting with the 5’ upstream
region was pursued (Figure 1-1). In analyzing the Srglb3 promoter, a tissue-specific
element termed NICE (nodule infected cell-specific element) was iderntified. This cis-
acting element confers the tissue specificity exhibited by this promoter and can direct a
normally root enhanced promoter to become nodule infected cell specific when expressed
in transgenic L. comiculatus (Szczyglowski et al., 1994).

In addition, A/T rich elements upstream of NICE were identified. These regions,
called ATRE-BSZ“ elements, are found in multiple copies in the Srglb3 promoter, are also

highly conserved in the lbc3 and N23 promoters from soybean and in the glrny 5’

upstream region of French bean, and were found to interact specifically with DNA

19

Figure 1-2. Structure of the Sesbania rostrata glb3 gene 5’ upstream region (Taken
from Szczyglowski et al., 1994).

20

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21

binding proteins from nodules, roots, and leaves (Jensen et al., 1988; Metz et al., 1988;
Forde et al., 1990; Jacobsen et al., 1990). A/T rich elements have been identified in the
promoters of other plant genes and in some cases have been shown to act as positive
regulatory elements (Forde, 1994), although the function of these elements in the nodulin
gene promoter regions is not clear (de Bruijn and Schell, 1992; Forde, 1994). In the Srglb3
promoter, this element appears to act as a positive element (Szabados et al., 1990) but is
not essential for nodule-specific expression (Szczyglowski et al., 1994). Mutations in
ATRE-BSZ" result in diverse affects on reporter gene activity and may reflect alterations
in the interaction with specific trans-acting factors. This result is supported by Jacobsen
et al. (1990) who showed that mutations in oligonucleotides corresponding to the NT
rich elements in the soybean lbc3 promoter can change binding patterns and affinities of
three proteins NAT], NAT2 and LAT1.NAT1 and LAT] are HMG I-like proteins and
these class of proteins have been implicated in modulating chromatin structure (Jacobsen
et al., 1990). NT rich regiorns are known to serve as matrix attachment regions or
scafl‘olding attachment regions based on functional assays and may be important for the
expression of the Srglb3 gene in higher order chromatin structures (Pinaev and de Bnrijn,
unpublished results).

A factor present in extracts of stem or root nodules of S. rostrata was found to
bind specifically to a region in the Srglb3 promoter using the gel mobility shift assay. It
was demonstrated by Welters et al. (1993) that the binding activity is due to the presence
of a factor originating from Azorhizobium caulinodans, the symbiont of S. rostrata. This

factor binds to a region in the Srglb3 promoter now designated the Bacterial Binding Site 1

22

 

 

 

 

 

 

 

 

 

 

 

 

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Figure 1-3. Role of the BBS] region in Srglb3 promoter activity. The top panel
shows the Gus activity (expressed in pmol of 4-metlnylumbelliferone produced min’l mg"
of protein) of chimeric Srglb3 promoter-gas genes in transgenic L. comr'culatus plants.
The shaded columns represent Gus expression in nodules, the open columns represent
Gus expression in roots. The structure of the chimeric constructs used is shown under the
box and the position of the CAAT and TATA promoter elements by solid boxes. The
start point of transcription is indicated by a wavy arrow, labeled RNA. The position of
the 40-bp BglII linker insertion in the BBS] site is indicated by an open triangle on line 3
(Figure was taken from Welters et al., 1993).

23

(BBSl; Welters et al., 1993; Szczyglowski et al., 1994) which is located upstream of
NICE and is flanked by several A/T rich regions. A detailed characterization of the
binding activity was carried out and the developmental appearance of this complex and
the presence of Lb were positively correlated (Welters et al., 1993). The protein
responsible for this activity was purified and the N-terminal annino acid sequence
determined. Transgenic Lotus corniculatus plants harboring clnimeric Srglb3 promoter-
reporter gene fusions suggest that the BBS] may modulate Srglb3 gene expression as
disruption or deletion of this site reduces reporter gene activity (Figure 1-2; Welters et al,

1993).

Intercellular transport

In order for a microbe to enter a symbiotic or pathogenic interaction with a plant,
signal exchange between the two must occur. In Agrobacterium and Xanthomonas,
bacterial proteins can be directly recognized in the plant cell (Howard at al., 1992;
Citovsky et al., 1992; Yang and Gabriel, 1995; Van den Ackerveken et al., 1996) as these
proteins have the ability to localize to the plant nucleus. Furthermore, pathogernic
phenotypes such as tumor formation or the HR correlate positively with functional
NLSs. In symbiotic interactions, bacterial proteins also have the ability to localize to the
plant nucleus (Jabouni et al., 1996). The finding that a bacterial protein can bind to a
plant promoter is consistent with this idea (Welters et al., 1993). One central question
that has eluded scientists for some time concerns the mechanism by which these proteins

could enter the plant cells.

24

How proteins or complexes originating from the bacterium could traverse the
bacterial membranes and cell wall in addition to the plant cell wall and plasma membrane
to get into the plant cell has been enignatic for a long time. Recently, research in this area
has uncovered some interesting findings. In Agrobacterr'um, the idea that conjugation and
T-DNA transfer were sinniliar processes was first proposed following the discovery of
the T-strand (Stachel and Zambryski, 1986; Stachel et al., 1986). More recently, Less]
and Lanka (1994) extended this idea by comparing characteristics of the RP4 plasmid and
T-DNA transfer. Significant similarities in function, amino acid sequences, gene
orgarnizatiorn and physical prOperties of the trans-acting enzymes were identified. Since
then, Fullner et al. (1996) has shown that Agrobacterr’um produce pill that are proposed
to function in the conjugal transfer of the T-DNA to the plant cell. Moreover, a pilus
structure was recently identified on the surface of the bacterial pathogen Pseudomonas
syringae pv. tomato DC3000 (Roine et al., 1997) under conditions important for
pathogenic bacteria to elicit the HR in resistant plants. These two findings suggest that a
pilus structure may be one mecharnisrn by which bacterial proteins are delivered to the
plant cell.

A secretion apparatus involved in intercellular transport has also been
investigated. The genes encoded by the virB locus encode proteins that could form a
membrane associated export system (reviewed by Zambryski, 1992). The virB operon
appears to be. closely related to the Pt] operon of Bordetella pertussis which encodes
products responsible for the export of the pertussis toxin protein (Weiss, 1993). In plant-

pathogen interactions the hrc genes are conserved in the bacterial pathogens P. syringae

25

patlnovars, Erwinia, Xanthomonas, and Rabtonia (Lidell et al, 1994; Fenselau and Bonas,
1995; Huang et al., 1995; Van Gijsegem ct al., 1995; Bogandove et al., 1996) and share
sequence similarity to a set of genes found in Yersim'a spp., Shigella and Salmonella (for a
review see Van Gijesgem et al., 1993, He, 1996). All of these genes are important for the
secretion of proteins required for pathogenesis (for a review see Van Gijsegan et al.,
1993, He, 1996) and are predicted to form components of the type III secretion
apparatus. The formation of a secretion pathway illustrates another mechanism by which
virulence factors can be exported by the bacterial cell.

With the recent discovery of pilus formation on the surface of Agrobacterium and
P. syringae pv. tomato DC3000 and the identification of genes with similarity to
components of secretion systems, one can imagine how bacterial proteins could get into
the plant cell. One model for intercellular transport involves the export of virulence
factors via the secretion system alone. However, the plant cell wall would provide a
barrier for these extracellulariy localized pathogenic bacteria. Therefore, the involvement
of pili in combination with a secretory system may work together to deliver the bacterial
protein to the plant cell.

In symbiosis, bacteria have the advantage of already beingpresent in the plant cell.
However, tlret have an additional membrane, the peribacteroid membrane, which presents
another barrier that must be crossed. For the most part, not much is known about the
proteins involved in transporting metabolites from the bacteroid to the plant cell. A
dicarboxylic acid uptake mechanism exists in bacteroids (Ronson et al., 1987; Werner et

al., 1992). To date, no specialized secretion system has been identified to selectively

26

export rhizobial proteins from the bacteroid or through the peribacteroid membrane to the

plant cell cytoplasm.

Conclusions
Bacterial proteins which are recognized within the plant cell have been identified
and studied in two types of plant-pathogenic interactions and have been implicated in
symbiotic interactions. The mechanisms by which bacteria modulate host gene expression
appears to fall into two categories: the transfer of DNA to the host genome and the use of
bacterial proteins to influence host gene expression.
Agrobacten‘um is the only known example to date in which DNA originating from
a bacterium is transferred to the plant cell nucleus (Kado, 1991; Sherng and Citovsky,
1996). In this unique example, new genes are introduced into the host genome and
expressed. This transfer of DNA can be thought of as altering host gene expression as
auxin and cytokinin biosyrnthetic genes, in addition to genes which encode factors that
produce the opines, are located on the T-DNA (Kado, 1991; Zupan and Zambryski,
1995). When these genes are expressed in the host, changes in the plant developmental
program occur which ultimately lead to the formation of a gal]. (Kado, 1991; Zupan and
Zambryski, 1995).
In contrast, Xanthomonas proteins (avr gene products) and rhizobial proteins (Fix
F, AcBBPl) are recognized in the plant cell and may interact with plant proteins or DNA

directly. Avr proteins, AcBBPl and the Fix F protein could act as plant transcription

factors or repressors to turn on or turn off gene expression. It is known that avr gene

27

products contain heptad repeats which can mediate protein-DNA interactions (Yang and
Gabriel, 1995). The AcBBPl protein contains a functional DNA binding domain (W elters
et al., 1993). Alternatively, the avror fixF gene products may participate in protein-
protein interactions by interacting with a receptor yet to be identified in the plant cell.
Avr proteins from Xanthomonas contain heptad repeats and leucine-rich repeats, which
can mediate protein-protein interactions (Yang and Gabriel, 1995). AcBBPl binds DNA
and is similar to regulatory proteins from different systems. Transgenic plants carrying
chimeric Srglb3 promoter-reporter gene constructs indicate that the BBS] is important for
Srglb3 gene expression as mutation or deletion of this cis-element reduces promoter
strength (Welters et al., 1993). Hence, this protein may act to enhance Srglb3 gene
expression under specific conditions. AcBBP] is also unique in that its target is already
identified. For the Xanthomonas Avr proteins, the target within the plant cell is unknown.

Agrobacterium, Xanthomonas, and Rhizobium produce proteins which have the
ability to localize to the plant nucleus. In the plant pathogens, nuclear localization of
these proteins positively correlates with a specific pathogenic response indicating a
probable biological role. As merntioned previously, the targets of most of these proteins
are not known. In the case of avr gene products, the target may be the R gene itself ' or the
R gene product with interaction occurring in the plant cytoplasm or perhaps in the plant
nucleus. In symbiosis, the target for AcBBP] is known to be a non-A/T rich region in the
5’ upstream region of the Srglb3 gene called BBS]. The biological role of this protein in

symbiosis is the subject of my thesis.

28

Bacterial interactions with plants presents an interesting system to study.
Establishment and persistence of these relationship requires a cascade of signals which
lead to the formation of an interaction which can be detrimental or beneficial to the plant.
We are just now uncovering signals originating from the bacteria which are recognized in
the plant cell that modulate plant gene expression. As introduced in this chapter, bacterial
proteins appear to mediate plant gene expression. For the most part, thisoccurs through
the interaction of a bacterial protein signal in the plant cell.

The identification of AcBBPl, a protein produced by A. caulinodans that can bind
in vitro to the Srglb3 promoter, ”is intriguing and warrants further investigation. As
merntioned earlier in this chapter, bacterium-derived signals have been postulated to be
important for lb gene expression as many pieces of indirect evidence have been collected
over the years. My thesis work is focused on investigating the biological role of the
AcBBPl protein in the symbiosis between S. rostrata and A. caulinodans. With the
recent finding that bacterial proteins play a role in plant-pathogen interactions, perhaps

signaling of this type is more general in nature.

29

REFERENCES

Appleby C (1984) Leghemoglobin and rhizobial respiration. Annu Rev Plant Physiol
35:443-478

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CHAPTERZ

IDENTIFICATION AND CHARACTERIZATION OF THE GENE ENCODING
THE AZORHIZOBIUIW CAULHVODANS DNA BINDING PROTEIN, AcBBPl

Portiorns of this chapter will be submitted as an article to the journal Molecular Plant-
Microbe Interactions.

‘27

38

ABSTRACT

Previously, en's-acting elements in and trans-acting factors interacting with the 5’
upstream region of the Sesbama rostrata leghenoglobin glb3 (Srglb3) gene were identified
(Metz et al., 1988; Szabados ct al., 1990; Welters et al., 1993; Szczyglowski et al., 1994).
A Nodule Infected Cell Expression (NICE) cis-acting element important for expression in
infected cells of the nodule was delimited (Szczyglowski et al., 1994). DNA binding
proteins from nodule, leaf and root extracts that interacted specifically with regions in the
Srglb3 5’ upstream region were analyzed (Metz et al., 1988; Welters et al., 1993).
Interestingly, a protein originating from the infecting bacterium Azorhizobium caulinodans
(AcBBPl), could specifically interact with a cis-acting element upstream of the NICE
elenent in the Srglb3 5’ regiorn and its binding activity was characterized (Welters et al.,
1993). The gene encoding AcBBP] was isolated and the role AcBBPl may play in
symbiotic nitrogen fixation was investigated. The deduced amino acid sequence of the
AcBBPl protein consists of 72 annino acids and features a helix-tum-helix DNA binding
motif. Homology searches revealed significant similarities to other DNA binding proteins
such as the C proteins found in type II restriction-modification systems, immunity
proteins from phage 4’ 105 and E. coli, and a regulator of vegetative replication and
conjugative transfer of the RK2 plasmid. Arr AcBBPl deficient mutant was created via

reverse genetics and used to nodulate S. rostrata plants. This strain was found to be

nodnrlation proficient (N od+), but S. rostrata nodules induced by this strain were

39

observed to have a small reduction in nitrogen fixation activity. Total leghenoglobin
steady state mRNA levels were identical in S. rostrata nodules harboring wild-type or

AcBBP] deficient bacteria.

40

INTRODUCTION

The induction of nitrogen fixing stem or root nodules on legume plants by bacteria
belonging to the genera Azorhizobium, Bradyrhizobium, Sinorhizobium and Rhizobium
involves the carefully orchestrated exchange of signals involved in the induction of sets of
genes in both the plant and the bacterium (Nap and Bisseling, 1990; de Bruijn and
Downie; 1991, Verma, 1992; Mylona et al., 1995; Long, 1996). The genes that are
expressed in this newly formed plant organ are referred to as nodulin genes and are
usually divided into two classes, early and late, depending on their time of induction.
Early nodulin genes correlate with the infection process and nodule formation while the
late nodulin genes encode proteins that are involved in the firnctioning and maintenance of
the nodule (Nap and Bisseling, 1990; Vance and Heichel, 1991; Hirsch, 1992, Mylona et
al., 1995). The latter class includes genes encoding enzymes involved in specific

biochennical pathways such as the 7 subunit of glutamine synthetase involved in ammonia

assimilation (Lara et al., 1983, Bennett et al., 1989, Boron and Legocki, 1993), sucrose
syntlnase (Tlnummler and Verna, 1987), peribacteroid membrane proteins (F ortin et al.,
1985, 1987; Miao and Verma, 1993) and leghemoglobins (Brisson et al., 1982).
Leghemoglobin (Lb), an abundant and essential protein within the nodule, is a late
nodulin involved in facilitating oxygen diffusion to the actively respiring bacteroids within
the infected tissue of the nodule. For the most part, leghemoglobin (lb) gene expression

has been shown to be nodule-specific and mainly confined to the infected cells of the

41

nodule (Stougaard et al., 1986, 1987, 1990; de Billy et al., 1991; de Bnrijn and Schell,
1992; Szczyglowski et al., 1994). Alfalfa nodules formed spontaneously on roots of
specific alfalfa lines (Truchet et al., 1989) and those induced by auxin transport inhibitors
(Hirsch et al., 1989) or by specific mutant rhizobial strains (Dickstein et al., 1988; de
Bruijn et al., 1989; de Bruijn and Schell, 1992) do not express lb or other late nodulin
genes, and contrasts the expression of some early nodulin genes such as £3de and
Enadlz which are clearly induced.

Chimeic lb-reporter gene constructs and transgenic plants have been extensively
used to identify the cis-acting elements responsible for the infected cell-specific
expression of lb genes in the nodule. The promoter regions of the lbc3 gene from soybean
and the glb3 gene from Sesbam'a rostrata have been shown to be responsible for nodule-
specific expression in indeterminate nodules induced on transgenic alfalfa (de Bruijn et al.,
1989) and in determinate nodules induced on Lotus corniculatus (Stougaard et al., 1986,
1987, 1990; Szabados et al., 1990; Szczyglowski et al., 1994). More specifically, b
promoter activity has been reported to be predominantly localized to the infected cells
(Szabados et al., 1990; Lauridsen et al., 1993; Szczyglowski et al., 1994). Generally,
nodules or nodule-like structures that are devoid of intracellular rhizobia appear to be
lacking lb gene expression, snrggesting that the physical presence of rhizobia in the plant
cell cytoplasm may be required for late nodulin gene induction (de Bruijn et al., 1989; de
Bruijn and Schell, 1992).

Cis-acting DNA elements within the lb promoter regions of the soybean lbc3 and

S. rostrata glb3 genes have been delimited and characterized by mutation analyses. These

42

investigations lead to the identification of the grgan-specific glenent (OSE) in the lbc3 and
the godule infected gel] gxpression (NICE) element in the Srglb3 5’ upstream regions of
these genes which are responsible for the observed infected cell specific gene expression
patterns (Stougaard et al., 1987; de Bnrijn et al., 1989; Stougaard et al., 1990; Szabados et
al., 1990; Rarnlov et al., 1993; Szczyglowski et al., 1994). In order to identify
components of the signal transduction pathway responsible for this infected cell-specific
expression, gel retardation assays were used to identify DNA binding proteins which
interact with the 5’ upstream regions of lb genes (Jensen et al., 1988; Metz et al., 1988;
Jacobsen et al., 1990; Forde et al., 1990; Jensen et al., 1991; Welters et al., 1993). In the
Srglb3 promoter region, a factor derived from the infecting bacterium Azorhizobium
caulinodans strain ORS 571 (AcBBPl) was identified which could bind specifically to a
region about 700 bp upstream from the start of transcription of the Srglb3 gene (Bacterial
Binding Site 1; BBS]; Welters et al., 1993). A detailed characterization of the BBS] and
complex formation was carried out. The protein responsible for the major peak of DNA
binding activity was purified and its N terminal amino acid sequence was determined
(AcBBPl; Welters et al., 1993).

The gene encoding AcBBP] was cloned, its deduced gene product was
characterized, an AcBBPI mutant strain was constructed and the effect of this mutation

on symbiotic nitrogen fixation in S. rostrata nodules was analyzed.

43

MATERIALS AND NIETHODS

Bacterial Strains and Plasmids

The bacterial strains and plasmids used in this study are described in Table 1.

Polymerase Chain Reaction

Degenerate oligonucleotide primers were designed to amplify an 80-bp stretch of
DNA which encoded the first 27 amino acids of the AcBBPl protein that were
previously determined by N-terrninal sequencing (Welters et al., 1993). Primer 1 was
designed to correspond to the N terminal portion of AcBBPl (5’
ATGGACITATGCIGGIAAA/GCITTIGT 3 2). In addition, two degenerate primers were
designed to correspond to amino acids 20-27 of the AcBBPl peptide sequence: Primer 2;
(5’ GTCTGCIGACA/GTCCITTCTGCIGGT 3’); Primer 3; (5'
GTC/TTGIACA/GTCCITTCGITIGT 3 ’). The reaction mixture contained (final
concentrations): 1x Taq DNA polymerase buffer (Promega; Madison, WI), 2.5 units Taq
DNA polymerase (Promega), 2.5 mM dNTPs, 50 pmol of primers, 100 ng of A.

callinodans genomic DNA in a final volume of 25 u]. The PCR reaction was carried out

using a DNA Thermocycler (model 430, Perkin Elmer, Foster City, CA). The following
PCR conditions we'e used: 94°C , 2 nnin; 31°C, 1 min; 45°C, 3 min with 35 cycles
followed by a 10 min extension cycle at 45°C. The resnrlting fragnents were blunt-ended

using the Klenow fragnent of DNA polymerase I (Boehringer Mannheim; Indianapolis,

44

IN) and purified through an 8% acrylamide gel. The purified fragnents were cloned into

the SmaI-digested vector pK18 (Pridmore, 1987).

Southern Blot Analysis

Total bacterial genomic DNA was isolated as described by Meade et al. (1989).
Ten microgams of A. caulinodans total genomic DNA was completely digested with
restriction enzymes, separated on a 0.8% agarose gel, and transferred to a nylon filter
(Hybond-N; Amersham, Adington Heights, IL) according to standard procedures
(Sambrook, 1989). Membranes were prehybridized and hybridized in a solution
containing 4 X SSC, 5 X Denhardt’s solution (Sambrook, 1989), 0.5% (w/v) SDS and 100
ug/ml denatured sheared salmon sperm DNA at 65°C. The membranes were washed once
for 15 nrin in 4 X SSC, 0.1% (w/v) SDS; twice for 15 min in 1 X SSC, 0.1% (w/v) SDS,
and twice for 15 min in 0.5 X SSC, 0.1% (w/v) SDS at 65°C. The 80-bp PCR amplified

probe was and labeled with [‘y’zP]ATP (Sarnbrook et al., 1989), while the full length
AcBBPl gene probe was labeled with [or-”PldATP using a random primer kit

(Boehringer Mannheim) following the manufacturer’s instructions.

Construction of A. caulinodans Partial Genomic Libraries
Total genomic DNA from A. caulinodans ORS 571 was digested with EcoRI or

PstI and 10 pg of DNA were separated on a 0.8% preparative agarose gel. A one

centimeter area in the size range of the fragnents found to hybridize with the AcBBPl

45

PCR probe was excised and the DNA isolated using a phenol freeze-thaw method. The
purified fragnents were cloned into pK18 (Pridmore, 1987) digested with EcoRI or PstI.
Positive colonies were identified using standard colony hybridization methods (Sarnbrook

et al. 1989) using the 80-bp PCR fragnent as a probe (see above).

DNA Sequencing and Computer Analyses

A plasmid carrying a 6-kb EcoRI fiagnent hybridizing with the AcBBPl probe
was mapped using single or multiple enzyme digests. The smallest fragnent containing
the AcBBPl gene was cloned and the nucleotide sequence determined (pSF27). DNA
sequencing was performed using a Sequenase 7-Deaza-dGTP DNA sequencing kit
(United States Biochemical, Cleveland, OH), according to the manufacturer’s instructions.
Computer analysis of DNA sequences was carried out using Sequencher 2.] (Gene Codes
Corp., Ann Arbor, MI) and Squd (Applied Biosystems) software. Multiple sequence
alignment was done using pile up in the GCG (Genetics Computer Group, Madison, WI)
progarn and Squu 1.01 (Garvarr Institute of Medical Research; Sydney, Australia).
Analysis of predicted protein sequences was performed using PHDsec (Rost and Sander,
1993; 1994). Homology searches were performed using the BLAST algorithm (Altschul et

al., 1990).

Construction of the AcBBPl Deficient Strain
A plasmid carrying a 14-kb PstI fragnent hybridizing with the AcBBP] probe

was mapped using single or multiple enzyme digests. The location of the AcBBPl gene

46

was delimited using Southern blot analysis. A 2.4-kb SacII fragnent harboring the
AcBBPl locus was cloned into the Bluescript SK' vector (pSF354) and used for gene
replacement. An internal StyI fragnent which contained the entire AcBBP] coding region
in addition to 74 bp (upstream) and 176 bp (downstream) of flanking DNA sequences
(see Figure 2-1A) was excised and rendered blunt-ended using the Klenow fragnent of
DNA polymerase I (Boelnringer Mannheim). A blunt-ended PstI fragnent canying the
kanamycin cassette from pUC4K (Phannacia Biotech; Uppsala, Sweden) replaced the
native Sty] fiagnent creating a deletion/insertion mutation (pSF354del). The entire
pSF3 54del plasmid was cloned into the PM site of the broad host range vector pLAFR 3
(Staskawicz et al, 1987) and mobilized from E. coli to A. caulinodans ORS 571 using the
helper plasmid pRK2013 (Ditta et al., 1980). Transconjugants were selected as described

by Pawlowski et al. (1987) on plates supplemented with tetracycline (Tc) 10 ug/ml,
carbernicillin (Cb) 500 ug/ml, and kanarnycin (Km) 200 rig/ml. A plasmid (pPHlJI)

incompatible with the donor plasmid was introduced to selected transconjugants to force

replacement of the mutated AcBBPl gene (de Bruijn, 1987).

Ge] Mobility Shift Assay
Extracts of A. caulinodans ORS 571 and the mutant ORS 571-425 strains were
prepared as described by Welters et al. (1993). An 80-bp fragment of the Srglb3 promoter

containing the BBS] (fragnent 5’203) was used in all reactions under conditions

47

previously described (Welters et al., 1993). All reaction mixtures were resolved on 8%

acrylamide gels.

Plant Growth and Nodulation

S. rostrata seeds were surface-sterilized and germinated as described by
Pawlowski et al. (1987). Seedlings were grown in autoclaved soil
(sandzvermiculitezMetroMix, 53:1) in growth chambers with 75% RH, 18-hr light,
28°C/6-hr dark, 22°C regime for 5 weeks. For in vitro plant nodulation assays, the
sterilized seeds were put directly onto B+D medium slants solidified with 1.5% aw
(Broughton and Dilworth, 1971) and the tubes were maintained under artificial light at
21°C (16 hr day). Plants grown in soil or on agar slants were inoculated with a two-day
old culture of A. caulinodam ORS 571 or ORS 571-425 grown in the presence of
antibiotics but washed with sterile water to remove antibiotics and residual medium. The
pelleted cells were resuspended in sterile water and one ml of this suspension was
inoculated onto the roots of one-week old S. rostrata seedlings. For stem inoculations, the
cultures were generated as described above and applied to five-week old stems of S.

rostrata plants using a sterile cotton applicator.

Acetylene Reduction Assays
To determine the nitrogen fixation activity in S. rostrata root or stem nodules the
acetylene reduction assay was performed. Root or stem nodules induced on S. rostrara

plants were excised, placed into vaccutainer tubes and sealed. One milliliter of acetylene

48

was injected into the tubes (~8% of the final gas phase) as described by Pawlowski et al.
(1987) with the following modifications. Samples were drawn every 15 min and injected
into a gas chromatograph (V arian model 3700, Sunnyvale, CA). The data were expressed

as nmol of ethylene produced/g of fresh weight.

Northern Analysis
Total RNA from 5-week old nodules was isolated using the hot phenol method
(Verwoerd et al., 1989) except that the extraction bufl‘er was as described by Hall et al.

(1978). Ten ug of total RNA was separated through 1.2% agarose-formaldehyde gels in
MOPS buffer, as described in Sambrook et al. (1989) and transferred to 0.22um

nitrocellulose membranes (NitroPlus, Micron Separations, Inc; Westboro, MA).
Prehybridization and hybridization reactions were canied out using a solution composed
of 0.5M phosphate buffer pH 7.2, 7% (w/v) SDS and 1% (w/v) BSA (Fraction V; Sigma,
St. Louis, M0) at 65°C, according to Church and Gilbert (1984). Filters were washed
twice for 15 min in l X SSC, 0.1% SDS and once for 15 min in 0.5 X SSC, 0.1% SDS at
65°C. Probes were labeled using the random primer kit (Boehringer Mannheim), according

to the manufacturer’s instructions.

49

RESULTS

Identification and Cloning of the A. caulinodans AcBBPI Gene

Using a reverse genetics approach, the gene encoding AcBBPl from A
caulinadans strain ORS 571 was isolated. Degenerate oligonucleotide primers
corresponding to the N terminal amino acid sequence of AcBBPl previously determined
(Welters et al., 1993), were used to amplify a DNA fragment encoding this 27 amino acid
region using the polymerase chain reaction (PCR). The DNA sequence of the resulting 80-
bp PCR product was determined and found to encode the expected 27 amino acid
peptide. The PCR fragment was used as a probe to determine the AcBBPI gene copy
number in ORS 571. Southem blot analysis of A. caulinodms genomic DNA digested
with Ecoar, BamHI, HindlII, and P311 revealed single hybridizing bands, suggesting that
the AcBBPl gene is not reiterated (Figure 2-1). This analysis revealed that the AcBBPl
gene was located on a 6-kb EcoRI fragment. To isolate the complete AcBBPI locus, a
partial EcoRI library of ORS 571 genomic DNA was constructed, enriching for fragments
of ~6 kb and screened via colony hybridization using the 80-bp AcBBPI PCR product as
the probe. The recombinant plasmids from several hybridizing colonies were isolated,
mapped with several restriction enzymes and re-hybridized with the AcBBPI probe.
These analyses revealed that the AcBBPI gene was located near the border of the 6-kb
fragment and would not be suitable for future experiments (e.g. gene replacement).
Therefore, a partial Pstl library of ORS 571 genomic DNA was constructed, enriching for

fragments ~14 kb and screened using the method previously described. A plasmid

50

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51

23.1 kb—)
9.41 kb—)
6.55 kb—>

4.36 kb—)

2.32 kb—)
2.03 kb—)

 

Figure 2-1. Identification of the AcBBPI gene in the A. caulinodans genome. Single
hybridizing bands were detected when a genomic Southern blot was probed with the 80-
bp AcBBPI PCR fragment. Ten micrograms of total genomic DNA from A. caulinodans
was digested with: Lane 1, EcoRI; Lane 2, BamI-II; Lane 3 EcoRI+BamHI; Lane 4,
HindIII; Lane 5, P311; Lane 6, HindIII+PstI.

52

(pSF318) containing the AcBBPl gene within the 14-kb fragment was restriction
mapped. Within this 14-kb Pstl fragment, a 2.4-kb SacII fiagment that contained the
AcBBPI gene centrally located was identified, subsequently subcloned into the SaclI site
of pBluescript KS“ (pSF 3 54), and used in further studies. Based on the restriction map of
pSF3 54, an internal 470 bp StyI fragment contained the AcBBPI gene. The StyI fragment
was blunt-ended, cloned into the SmaI site of pK18, and the DNA sequence of several
inserts showing hybridization with the AcBBPl probe was determined. Plasmid pSty7L
with a 470 bp insert was found to contain an Open reading frame (ORF) encoding a
polypeptide with 100% sequence identity to the N—terminal 27 amino acid sequence of

AcBBPl determined by Welters et al. (1993).

Analysis of the AcBBPI Gene and Its Protein Product (AcBBPl) .

The AcBBPI gene consists of 210 bp and encodes a 72 amino acid protein with a
predicted molecular mass of 9 kDa (Figure 2-2A) and a pI of about 9.5. This molecular
mass correlates well with the AcBBPl protein mass determined by Welters et al. (1993)
using biochemical methods (9-10 kDa). Two regions of the AcBBPl protein were found
to contain stretches of basic amino acid residues, which may indicate putative nuclear
localization signals (NLSs; Garcia-Bustos et al., 1991; Raikhel, 1992; Figure 2-1A
highlighted in red and green). DNA binding helix-tum-helix motifs were found in the
central portion of AcBBPl (residues 22-41; Figure 2-2B) as well as the C-tenninus of the
protein (residues 51-70; Figure 2-2C). Both of these regions contain conserved amino acid

residues important for the formation of the DNA binding domain (Pabo and Sauer, 1984).

53

Figure 2-2. Primary structure and deduced amino acid sequence of AcBBPl.

(A) Deduced amino acid residues are shown in one-letter notation below the nucleotide
sequence of the SM-Styl fiagment harboring AcBBPI. Underlined residues match the
protein sequence obtained previously (Welters et al., 1993). Residues highlighted in red or
in green may encode a nuclear localization signal (NLS).

(B) Alignment of a AcBBPl helix-tum-helix DNA binding motif. AcBBPl (this work),
Lac Rep, 1 Rep, 2. Cro (lactose repressor, 2. repressor, k Cro protein; Pabo and Sauer,
1984). Residues highlighted in red represent residues thought to be important for
maintaining structure (position 5 usually A, position 9 usually G, position 15 usually I or
V). Green highlights identical residues while pale green denotes conservative substitutions
in the alignment. '

(C) Aligrunent of the second putative helix-tum-helix DNA binding motif in AcBBPl
with C proteins, immunity repressor proteins and a regulator of conjugal transfer and
vegetative replication of the RK2 plasmid. AcBBPl (this work), control elements: BgIIIC,
Anton et al., (1996); BamHIC, Nathan and Brooks (1988); MunIC, Siksnys et al., (1994);
NBC, Tao et al. (1991); SmaIC, Heidemann et al. (1989); EcoRV, Bougueleret et al.
(1984); Imrep MOS, Cully and Garro (1985); Dhaese et al. (1985); Imrep Ecoli, Aiba et
al. (1996), TrbA, Jagura-Burdzy et al. (1992). Red shading indicates those residues
conserved in helix-tum-helix motifs while great shading denotes identical residues and
pale green shading indicates conservative substitutions. Alignment was done using the
method of Karlin and Brendel, (1994). Residues were grouped as follows: M, L, I, V, F,
G,A,W;H,Q,N,P;C,S,T,Y;R,K,E,D.

54

StyI

CCTRIXIECANMKXQHCCTNNKmflmCTHEMEHGAEMMKHYEGA

GGGCGTAAATTGCGATCCGTCTGCGCATGGATATGCGCAAGCTGGTCG
M ID 14 It P: L. V

GCCGGAACTTCGCGCGCCTGCGTCAGGAGAAGGGCCTGACACAGGAGG
G> R Ii F' A. R 1; Pl 0 E I< Ci L 'T C) E

ACGTACAGACGCGATCCGGCTTCAGCCAGCAGTACATCAGCGGGCTCG
L2 y Q T R 13 (3 F' S Q Q Y I S (3 I;

AACGCGGCCGGCGCAATCCCACTGTCATCACGCTCTATGAACTGGCAC
E IR (3 R. R IQ F’ T \f I 'T I; Y IE I; A

IWECGCHIXXKHHRGCCNKHUWEGCTTGEKKXKKHGAGQHHUWKB
Q It L <3 V’ S I! E IE I; V 1% At D (3 K

ACTGAACCCCGGAGGGGAAGCACCGGCCGAAACCGCCCGATGCGTTGC
D *

CGGCTCGCCCGCCGCGCCGGCCGCGAAGGGGGCGACCAAATCAGCGAA
AAGGTNPHKXEHCCTOGflflyflxflflCAGGTBREKXXKHGEAHGEHZ
(XHKIEHCGTOTRXHUHECCCNflXXHflflCTOOTRKS

StyI

1 5 10 15 20
AcBBPl 22 QIDVQTRBOISQQYISOL8841
BglIIC 22 QIKLAIISRLDRTYIOOVIR 41
BarrHIC 25 QIKLAI’ICDLERTYISDIIR“
MunIC 25 QIBIAAQIDLDRTYYSBIBI 43
PvuIIC 27 QIBLADLVGIBRTYIOBIIR‘B
SmaI 30 QIQLAIISGLHRTYIOSVIR49
EcoRV 42 QOEVAKALGRPQBYISRIEQ 61
PhilOS 18 QVQLAIKANLSRBYLADIERTI
ImRepEC 23 QRRAAILSGLTHBAISTIIQ 42
TrbA 21333LSIRAGVSIBPL8DLTIIO
H e 1 i x —-—turn--- H e 1 i x

1 S 10 15 20
ACBBPl 51 LY! HQ GLVSBII VRADO 70
LacRep 6 LYD AIY GVSY QT VBRVAI 25
lRep 33 QIBVRDRNGMOI80VOALII 52
kCro 16 QTKTARDLGVYQSAIIKAIH 35

Helix---turn---Helix

48
96

144

192

240

288

336

384
432
470

55

Secondary structure analysis of AcBBPl using the PHD-sec program (Rost and Sander,
1993; 1994) revealed the presence of two regions containing two adjacent helical
structures separated by a loop or turn with a high probability score (7-9 on a scale from
0-9; Figure 2-2 B, C).

A sequence similarity search was carried out using the BLAST algorithm (Altschul
et al., 1990). This analysis revealed significant similarity of AcBBPl to regulatory
proteins. The most significant matches were found to be control elements in several type
II restriction-modification (RM) systems (Bougueleret et al., 1984; Nathan and Brooks,
1988; Heidemann et al., 1989; Tao ct al., 1991; Siksnys et al., 1994; Anton et al., 1996) ,

to an immunity repressor protein from the Bacillus subtilis phage c105 (Cully and Garro,

1985; Dhaese et al., 1985), a randomly sequenced Kohara plasmid of E. coli (Aiba et al.,
1996) and a regulator of vegetative replication and conjugal transfer of the RK2 plasmid
(Jagura-Burdzy et al., 1992; Figure 2-3 and 2-4). AcBBPl shares identical and similar
amino acid residues with the C proteins throughout the amino acid sequence. AcBBPl
also shares similar residues in the N terminal regions of the repressor proteins and the
replication/conjugal transfer regulator. Identity within helical domains thought to be
important for the helix-tum-helix DNA binding motif was observed among the amino acid
sequences in all classes of proteins (Tao et al., 1991; Dhaese et al., 1985; Van Kaer et al.,
1987;1agura-Burdzy et al., 1992).

The binding site for CflamI-II, AcBBPl (J. Brooks, personal communication;

Welters et al., 1993) and the operator sequence to which the (>105 immunity repressor

56

Legend for Figure 2-3. Alignment of AcBBPl amino acid sequence to control
elements in type II restriction-modification systems and to immunity repressors of
@105 and E. coli. AcBBPl (this work), control elements: BglIIC, Anton et al. (1996);
BamHIC, Nathan and Brooks (1988); MunIC, Siksnys et al. (1994); PvuIIC, Tao et al.
(1991); SmaIC, Heidemann et al. (1989); EcoRV, Bougueleret et al. (1984); Imrep 4:105,
Cully and Garro (1985); Dhaese et al. (1985); Imrep Ecoli, Aiba et al. (1996). Boxed
residues represent identity while shaded areas represent similarity to the AcBBPl
sequence. Identities were barred if 50% of the aligned sequences contained the same
residue. Sequences were aligned using the algorithm of Karlin and Brendel (1994).
Residues were grouped as follows: M, L, I, V, F, G, A, W; H, Q, N, P; C, S, T, Y; R, K,
E, D. ’

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binds were compared (Van Kaer et al., 1987; Van Kaer et al., 1988). Although these three
types of binding sites do not share a high degree of similarity with each other, short

stretches of identical nucleotide residues were observed in these binding sites (Figure 2-5).

Creation and Characterization of An A. caulinodans AcBBPl Deficient Strain

In an attempt to define the function of AcBBPl, an ORS 571 strain lacking the
ability to synthesize AcBBPl was constructed (ORS 571-425). A kanamycin resistance
(Km') cassette derived from pUC4K was used to replace a 470 bp StyI fragment harboring
the AcBBPI gene in addition to 74 bp (upstream) and 176 bp (downstream) of flanking
region carried on plasmid pSF3 54. The resulting deletion/substitution mutation was used
to replace the wild type gene via gene replacement (de Bruijn, 1987). The structure of the
mutated AcBBPI locus in strain ORS 571-425 was confirmed by Southern blot analysis
(see Figure 2-6). Whole cell extracts were prepared from the wild-type ORS 571 strain
and the ORS 571-425 mutant and used in gel retardation assays using the fragment of the
S. rostrata glb3 5’ upstream region, harboring the binding site for AcBBPl (BBSl;
fragment 5’203; Welters et al., 1993; see Figure 2-7) as target DNA. No binding activity
could be observed with extracts prepared from strain ORS 571-425 (Figure 2-7),
confirming that the insertion/deletion mutation abolishes AcBBPl production.

To further examine the phenotype of the AcBBPl deficient mutant strain, the

growth rate in the free living state was monitored. Cultures of the A. caulinodans wild

type or the AcBBPl deficient strain were grown in the presence of Cb 250 rig/ml until

60

C . BamHI TGTAAQTTATAGTCTGTAGCCTATAGTCTACT
Ac BBPl QCTATA CATAC TTTATGTGATATCC

* * 3k * *
q>1 o 5 oR CTTGTATTTCCGTC

Figure 2-5. Comparison of the binding sites for C.BamHl, AcBBPl and the (1)105
immunity repressor. Underlined nucleotides show identity between AeBBPl and
C.BamHI while * indicates identity between ACBBPI and the immunity repressor from
(1)105. The red shading denotes identity between C.BamHI and the binding site of the
immunity repressor.

61

late log phase. The cultures were inoculated into fresh TY medium (Beringer, 1974)

supplemented with Cb 250 ug/ml at a 1:1000 dilution. One milliliter samples were taken

and measured every four hours until the cultures reached stationary phase. No differences
in growth rate were observed (data not shown).

The AeBBPl deficient mutant strain was also examined for dinitorgen dependent
growth characteristics (N if phenotype) under reduced oxygen conditions with or without
the presence of ammonium as described by Pawlowski et al. (1987). No differences in
growth or colony morphology were observed (data not shown).

The effect of the AcBBPl mutation on nodulation and symbiotic nitrogen fixation
(Nod and Fix phenotype) was also determined. For this purpose, sterile seedlings of S.
rostrata were inoculated on the stems or on the roots with ORS 571 or ORS 571-425.
The AcBBPl deficient mutant strain was found to be capable of inducing both stem and
root nodules on its host plant (Nod+). To determine the relative nitrogen fixation levels in
wild type versus AcBBPl deficient nodules, acetylene reduction assays were performed.
The results of three independent root nodule experiments are shown in Figure 2-8.

In the first experiment, 13 plants were assayed for nitrogen fixation. The acetylene
reduction (nitrogen fixation) activity of 8 independent root nodule samples harboring the
AcBBPl deficient strain were compared to 5 root nodule samples from control plants. A
~16% reduction in activity was observed (Figure 2-8 A). In the second trial, 18 plants
were assayed. The nitrogen fixation activity of 13 independent nodule samples induced

by the AcBBPl deficient strain were compared to 5 samples from control plants. A

62

23.1 kb

9.41 kb

6.55 kb

4.36 kb

2.32 kb
2.03 kb

 

Figure 2-6. Structure of the AcBBPl ORS-4-25 deletion/insertion mutant. Lane 1,
SacII fragment containing AcBBPI ; Lane 2, SacII without SryI-Styl fragment+kan
cassette, Lane 3, plasmid pSF354 digested with SacII, Lane 4, ORS 571 genomic DNA
digested with PstI, Lane 5, ORS 571 genomic DNA digested with SacII, Lane 6-7
genomic DNA digested with SaclI from transconjugants 346, 425. Plasmid DNA or 10
ug of total genomic DNA isolated from the above strains were digested as noted, separated
on a 0.8% agarose gel, transferred to a nylon membrane and hybridized with the entire wild
type SacII fragment as probe. *=gene replacement.

63

 

 

 

 

 

(-694) (-653) (—48)
rs a". ;'7 Srglb3
|_l
5'203
ATG

 

Figure 2-7. Whole cell extracts prepared from the AcBBPl deficient mutant lack
binding activity. Extracts prepared from ORS 571 and ORS 571-425 were tested for
DNA binding activity using the BBS] as target DNA. (A) Schematic of the Srglb3 5’
upstream region. The arrow indicates the start point of transcription. The fragment 5’203
(shaded in red) containing the 8881, was used as the target fragment in gel mobility shift
assays. (B) Lane 1, extracts prepared from wild-type ORS 571 (high salt extraction),
Lane 2, ORS 571 (low salt extract), Lane 3, extracts prepared from ORS 571-425 (high
salt extraction) Lane 4, ORS 571-425 (low salt extraction), Lane 5 control (wild-type
extract isolated previously). The free fragment (F, 5’203) is indicated with an arrowhead.

64

~36% reduction in activity was apparent (Figure 2-8 B). In the final trial, 34 plants were
tested. The acetylene reduction activity of 18 root nodule samples induced by ORS 571-
425 were compared to 16 samples from control plants. A 40% reduction in activity was
detected (Figure 2-8 C). The overall nitrogen fixation activity is presented for each trial
(Figure 2-8D)

S. rostrata stem nodules were also tested for nitrogen fixation activity. The results
of three independent stem nodule experiments are presented in Figure 2-9. In the first
experiment, 24 plants were tested. The acetylene reduction activities of 13 independent
nodule samples harboring the mutant strain were compared to 11 nodule samples
harvested from control plants. A 45% reduction in activity was observed. In the second
experiment, 32 independent plants were assayed. Equal numbers of nodules induced by
the AcBBPl deficient strain were compared to nodules harboring the wild type strain. A
~16% reduction was detected in this trial. Finally, 13 plants were assayed for nitrogen
fixation activity. Five independent nodule samples induced by ORS 571-425 were
compared to 8 control samples. A 45% difference in acetylene reduction activity was
observed. Thus it appears that the AcBBPI mutant strain induces nodules on S. rostrata
plants that display an approximately ~16% reduction in nitrogen fixation activity,
suggesting that AcBBPl does play a role in symbiotic nitrogen fixation, but is clearly not

essential.

65

Figure 2-8. Nitrogen fixation activity is reduced in S. rostrata root nodules induced
by ORS 571-425. S. rostrata root nodules were tested for nitrogen fixation activity by the
acetylene reduction assay. (A) Trial 1; (B) Trial 2; (C) Trial 3; (D) Average nitrogen
fixation activity. Light bars = ORS 571; dark bars = ORS 571-425. All results are
expressed as nmol ethylene produced/g fresh weight/hr.

66

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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52 m CON rMs NAU.I U W9 m U .
s - oov n a w n a m
_ is I N M m. oom N M m _
,_ i 80 5 6 A
r com 000—

 

 

 

cmew m it... m— _ _ cmem Int... < w

ii Jilii . It: x 511.1 - Iii.

67

Figure 2-9. Nitrogen fixation activity is reduced in S. rostrata stem nodules induced
by ORS 571-425. S. rostrata stem nodules were tested for nitrogen fixation activity by the
acetylene reduction assay. (A) Trial 1; (B) Trial 2; (C) Trial 3; (D) Average nitrogen
fixation activity. Light bars = ORS 571; dark bars = ORS 571-425. All results are
expressed as nmol ethylene produced/g fresh weight/hr.

68

 

.2; 3363 0:50: Eon—.88:—

flflz U
WELL 000—.
oomw

3.3.3 I

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O
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O

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I. 11 i 1 ill it'lL r1 _

69

Leghemoglobin (lb) Steady State mRNA Levels in S. rostrata Nodules Harboring
Wild Type or AcBBPl Deficient Bacteria

The observed effect of the AcBBPl mutation on symbiotic nitrogen fixation can be
caused at various stages of the nodulation/nitrogen fixation process (see also Chapter 3 of
this thesis). Since the target of the AcBBPl protein is located in the Srglb3 5’ upstream
region and a role for AcBBPl in infected cell specific expression of lb genes had been
postulated (Welters et al., 1993), the relative levels of steady-state lb mRNAs in nodules
elicited by ORS 571 and ORS 571-425 were examined. Total RNA from S-week old
nodules induced by wild-type A. caulinodans or the AcBBPl deficient strain was
hybridized with the S. rostrata leghemoglobin probe Srclbl (Metz et al., 1988). High
steady state levels of lb mRNA were detected both in nodules induced by the wild-type
and in those induced by the mutant strain (Figure 2-10). Quantification of the lb gene
hybridization signal versus the 18S rRNA gene control failed to reveal significant
differences in lb mRNA levels between the two types of nodules, suggesting that the
~20% reduction of nitrogen fixation activity in the nodules harboring the mutant strain

does not directly correlate with a reduction in total lb mRNA levels.

7O

     

 

A
ORS 571 ORS 571-425
lb —> n -
.5. a a
B

 

Abundance of lb mRNA Derived
From Nodules Harboring ORS 571
and ORS 571-425

 

 
  

—L
0.014.an

 

level

 

 

‘
ru ‘ - -'
‘1 ‘ '9, r s:
1 t
I K "r
. _. . . L
l

ORS 571 ORS 571-
425

 

 

 

 

Relative mRNA

 

 

 

 

 

Figure 2-10. Northern blot analysis of leghemoglobin gene expression. Ten
micrograms of total RNA from 5 week old S. rostrata stem nodules harboring wild-type
or AcBBPl deficient bacteria was isolated and hybridized with radiolabeled Srclbl, a
CDNA encoding a leghemoglobin from S. rostrata. as a probe (Metz et al., 1988). The blot
was stripped and re-hybridized with the 18S rRNA gene from rice as a loading control.

71

DISCUSSION

In this chapter, the gene encoding the A. caulinodans ORS 571 AcBBPl DNA
binding protein that was previously shown to interact with a portion of the 5’ upstream
region of the Srglb3 gene was isolated and characterized. This protein (AcBBPl) was
identified during a screen for DNA-binding proteins (trans-acting factors) interacting with
cis-acting elements in the 5’ upstream region of a leghemoglobin gene from the legume
host of A. caulinodans, namely S. rostrata (Srglb3; Welters et al., 1993). The binding site
for AcBBPl in the promoter region of the S. rostrata glb3 gene (BBSl), was found to
play a role in Srglb3 promoter activity since a BBSl insertion mutation displayed a
substantially reduced level of Srglb3-uidA reporter gene expression in transgenic legume
plants (Welters et al., 1993).

It was surprising that the AcBBPl protein was of bacterial origin and that it could
interact with a plant gene promoter, however this is not an entirely new idea.
Leghemoglobin gene expression appears to be infected cell specific. In the case of
indeterminate nodules, strong evidence has been presented in support of this idea as lb
transcripts in alfalfa nodules are only found in infected cells (de Billy et al., 1991). In
determinate nodules, although the precise localization of lb transcripts is not clear,
transgenic L. corniculatus plants carrying chimeric lb promoter-reporter gene constructs
appear to only be expressed in the infected cells (Szabados et al., 1990; Lauridsen et al.,

1993; Ramlov et al., 1993; Szczyglowski etal., 1994).

72

lb genes are not expressed in alfalfa nodules that do not contain intracellular
rhizobia such as those which spontaneously form on the roots of specific alfalfa lines
(Truchet et al., 1989), or induced by the treatment of auxin transport inhibitors (Hirsch et
al. 1989) or by particular mutant strains of rhizobia (Dickstein et al., 1988; de Bruijn et
al., 1989). These data have suggested that a rhizobial factor produced by the intracellular
bacteria or related to the release of bacteria from the infection thread may be essential for
activation of lb genes (Nap and Bisseling, 1990; de Billy et al., 1991; Dickstein et al.,
1991; de Bruijn and Schell, 1992). Furthermore, Wullstein and Bruening (1972) showed
that rhizobial metabolites could be transferred to the nuclei of clever nodule cells and
hypothesized that rhizobia may induce clover cells to begin leghemoglobin synthesis by
the transfer of a bacterial produced inducer. Truchet et al. (.1980) proposed the existence
of a non-diffusable Rhizobium-related signal involved in the differentiation of the central
infected tissue of the nodules and de Billy et al. (1991) postulated that such a factor may
play a role in infected cell specific expression.

The observation that the A. caulinodans AcBBPl protein could bind to a
leghemoglobin promoter suggest that a bacterial protein is transported out of the cell and
is directed to the plant nucleus. The concept of bacterial-plant transkingdom signaling is
not a completely novel one. In the Agrobacterium-plant interaction, the transfer of the T-
DNA from the bacterium to the plant cell is mediated by two discrete DNA binding
proteins, VirD2 and VirE2. These two proteins direct and protect the T-DNA strand on
its journey from Agrobacterium to the plant cell. Both VirD2 and VirE2 contain bipartite

NLSs (Howard et al., 1992; Citovsky et al., 1992) and the presence of an intact NLS

73

correlates with the ability of this bacterium to form tumors. Recently, avirulence proteins
have been shown to be recognized directly in the plant cell. Yang and Gabriel (1995) first
indicated that Xanthomonas avr/pth gene products contained NLSs and could be directed
to the plant nucleus, suggesting that these proteins are deposited in the plant cell.
F urthermorc, Van den Ackerveken et al. (1996) showed that the AvrBs3 protein contains
a functional NLS and that a fimctional targeting signal correlates positively with HR. In
symbiosis, the fixF gene product could direct chimeric reporter gene constructs to the
plant nucleus. So far this is the only example of a rhizobial protein in which a functional
NLS has been tested using chimeric NLS-reporter gene constructs. This observation
indicates that bacterial proteins may be important in establishing or maintaining
symbiosis (Jabbouri et al., 1996).

To further investigate what role the A. caulinodans AcBBPl protein may play in
Srglb3 gene expression, the gene encoding this factor was cloned via a reverse genetics
approach using oligonucleotide primers designed to the amino acid sequence previously
determined by Welters et al. (1993). Upon inspection of the amino acid sequence
encoding this protein, putative NLSs and two regions with similarities to helix-trun-helix
DNA binding motifs were identified (Figure 2-2). Both helix-turn-helix regions contain
conserved amino acid residues that are important for the formation of the DNA binding
domain. Secondary structure analysis using the PHD-Sec computer program (Rost and
Sander, 1993; 1994) predicted two adjacent helical structures separated by a loop or turn

with fairly high probabilities (7 to 9 on a scale of 0-9).

74

Small patches of basic residues that may serve to direct this protein to the plant
nucleus were detected. Although, no consensus sequences for NLSs are known in any
system (Garcia-Bustos et al., 1991; Raikhel, 1992), a general feature consistent among
these targeting sequences is the presence of clusters of basic residues. The basic R-K
residues at position 4 and 5 followed by a patch of residues in which 3 out of 6 are basic
R-L-R-Q-E-E could act as a bipartite NLS. The second patch of basic residues G-L-E-R-
G-R-R—N-P-T has some similarities to a previously identified NLS of the regulatory
protein R from maize (NLS-M; Figure 2-2). NLS-M in combination with one other NLS
found in the R protein was necessary for efficient transport of chimeric NLS-reporter
gene constructs to the nucleus (Shieh et al., 1993). NLS-M and the second putative NLS
in AcBBPl (highlighted in green) are similar in sequence and both are localized to the N-
terrninal portion of the helix-turn-helix DNA binding motif. The MyoDl regulatory
protein was found to contain an NLS in the helix-turn-helix motif although the precise
location within the domain was not delimited (Tapscott et al., 1988). The observation
that the transcription factors MyoDl and R have NLSs within the DNA binding motif
presents an interesting correlation. The NLS may have evolved to co-localize with the
essential DNA binding domain.

A sequence similarity search using the BLAST algorithm (Altschul et al., 1990)
revealed that AcBBPl was similar to a class of control elements found in type II
restriction-modification systems: EcoRV (Bougueleret et al., 1984), BamHI (Nathan and
Brooks, 1988), SmaI (Heidemann et al., 1989), Pqu (Tao et al., 1991), BgIII (Anton et

al., 1996), MunI (Siksnys ct al., 1994), Eco721 (Rimseliene et al., 1995); an immmunity

75

region in the B. subtilis phage $105 (Cully and Garro, 1985; Dhaese et al., 1985), a

randomly sequenced ORF from Ecoli (Aiba et al., 1996), and a regulator of vegetative
replication and conjugal transfer of the plasmid RK2 (Jagura-Burdzy et al., 1992). All of
these proteins contain putative helix-turn-helix motifs which are fairly conserved between
these proteins (Pabo and Sauer, 1984; Tao et al., 1991; Figure 2-2 B, C).

The most significant match to AcBBPl was to the Bacillus subtilis BglII control
gene product (Anton et al., 1996). C genes have been identified in some type II
restriction-modification (RM) systems but not all. C genes encode proteins that have
been implicated to activate and/or repress expression of the essential restriction
endonuclease (R) and modification methylase (M) genes (Nathan and Brooks, 1989; Tao
et al., 1991; Tao and Blumenthal, 1992; Ives et al., 1992; Ives et al., 1995). These control
genes were found as small ORFs upstream of or in close proximity to the B gene (Tao et
al., 1991). The best studied C genes are bamHIC and pvuIIC from the BamHI and Pqu
RM systems respectively. ORFs similar to these have been identified in a number of
bacterial RM systems but currently, no functions for the rest of these genes have been
assigned. All C genes or putative homologs share a few common characteristics: 1) the
deduced amino acid sequence of all members identified thus far encode small proteins of
about 9-10 kDa and 2) a helix-tum-helix DNA binding motif is located in the central
region of the protein and fairly conserved between all members. The AcBBPl protein

exhibits both characteristics.

76

The AcBBPl protein shares amino acid sequence similarity with an immunity

repressor from the phage $105 (Cully and Garro, 1985; Dhaese et al., 1985) and a

randomly sequenced Kohara plasmid from E. coli (Aiba et al., 1986) proposed to have a
similar function. Both of these amino acid sequences contain helix-turn-helix DNA binding
motifs at the N-terminus. The AcBBPl sequence contains similar amino acid residues to
the immunity repressor proteins throughout the sequence. The immunity repressor from

$105 was found to be involved in the regulation of lysogeny and superinfection (Cully

and Garro, 1985, Dhaese et al., 1985). It is known that phage encoded repressors are
required for lysogeny in response to infection, so that lytic genes are not expressed.
These repressors are also responsible for the immunity of secondary infection by the
same or closely related phage termed “superinfection”.

The binding site for C.BamHI, AcBBPl and the $105 immunity repressor have

been determined (Figure 2-4; Van Kaer et al, 1987; Van Kaer et al, 1988; Welters et al.,
1993; J. Brooks, personal communication). All three sites share some common
nucleotides that may be important for protein recognition.

The AcBBPl protein shares amino acid sequence similarity to a vegetative
replication/conjugal transfer trans-acting factor of the RK2 plasmid. Conjugal transfer has
been implicated as a means to transfer bacterial factors to the plant cell. It was
hypothesized by Stachel and Zambryski in 1986 that conjugal transfer may be a
mechanism which Agrobacterium uses to mobilize the T-complex from the bacterium to

the plant. The identification of surface appendages or pili on Agrobacterium tumefaciens

77

(virE-dependent pilus; Fullner et al., 1996) involved in T-DNA transfer and more
recently the Hrp pilus (Reine et al., 1997) produced by Pseudomonas syringae pv.
tomato suggest that this mechanism may be involved in delivering bacterial factors to the
plant cell.

The phenotype of the AcBBPl deficient strain under free-living and nitrogen
fixing conditions was monitored. No difference in growth rate was observed under either
of these conditions. The ORS 571-425 mutant strain was tested for nodule formation on
S. rostrata. Nodules were induced on both the stems and roots of S. rostrata indicating a
(N od”) phenotype. To determine the relative nitrogen fixation levels in nodules harboring
wild-type versus the AcBBPl deficient mutant, acetylene reduction assays were
performed. In three separate trials, a total of 65 root nodule samples were tested. In all
cases a ~20% reduction in total nitrogen fixation was detected by the acetylene reduction
assay. In three separate S. rostrata stem nodule trials, a total of 69 samples were tested
for nitrogen fixation levels and a 15-20% reduction was observed in nodules harboring the
mutant strain. Since a difference in total nitrogen fixation ability was detected in nodules
induced by the AcBBPl deficient strain, the leghemoglobin (lb) steady state mRN A levels
in nodules harboring the wild-type or mutant strain were compared. Total RNA isolated
from 5-week old S. rostrata stem nodules harboring the wild-type or mutant strain was
hybridized with the S. rostrata leghemoglobin probe Srclbl. Quantification of the lb gene
hybridization signal versus a loading control (188) failed to reveal any significant

differences in these two types of nodules indicating that the reduction of nitrogen fixation

78

in nodules harboring mutant bacteria does not directly correlate with a reduction in lb
mRN A levels.

Since lb genes comprise a small gene family of highly homologous members (~95-
97% identical) in S. rostrata, it is not too surprising that transcripts remained unchanged
in the two types of nodules tested. The glb3 gene product represents a minor form of
leghemoglobin in S. rostrata (Bogusz et al., 1987). Additionally, northern blot analysis
can only assess the global lb mRNA levels in the plant rather than the expression pattern
of a particular member because gene specific probes are almost impossible to obtain.
Therefore, small changes in Srglb3 gene expression could be masked in northern blots.
Experiments to directly investigate the role of AcBBPl in Srglb3 gene expression will be

addressed in Chapter 4 of this thesis.

79

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Tao T, Blumenthal RM (1992) Sequence and characterization of pvuIIR, the Pqu
endonuclease gene, and of pvuIIC, its regulatory gene. J Bacteriol 174:3395-3398

Tao T, Bourne JC, Blumenthal RM (1991) A family of regulatory genes associated with
type II restriction-modification systems. J Bacteriol 173:1367-1375

Thummler F, Verma DPS (1987) Nodulin-lOO of soybean is the subunit of sucrose
synthetase regulated by the availabllty of free heme in nodules. J Biol Chem
262:14730-14736

Truchet G, Barker DG, Camut S, de Billy F, Vasse J, Huguet T (1989) Alfalfa nodulation
in the absence of Rhizobium. Mol Gen Genet 219:65-68

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866

85

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Plant Cell 4:373-382.

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Welters P, Metz B, Felix G, Palme K, Szczyglowski K, de Bruijn FJ (1993) Interaction of
a rhizobial DNA-binding protein with the promoter region of a plant leghemoglobin
gene. Plant Physiol 102: 1095-1 107

CHAPTER3

ROLE OF AcBBPl IN INFECTION AND IMMUNOLOCALIZATION
OF THE PROTEIN IN Sesbania rostrata NODULES

Portions of this chapter will be submitted as an article to the journal Molecular Plant-
Microbe Interactions.

86

87

ABSTRACT

As discussed in Chapter 2, a gene encoding a bacterial DNA binding protein that
can bind to a specific region in the Srglb3 promoter was isolated and characterized. A
microscopical study was initiated to determine if any structural differences between
nodules harboring the wild type or the AcBBPl deficient mutant existed. Using
transmission electron microscopy, it appeared that nodules harboring bacteria deficient in
the production of AcBBPl were delayed in the infection process. Fewer infected plant
cells were found in the central infected zone of the nodule when compared to the wild
type. In addition, antibodies raised against AcBBPl were generated. These antibodies
compete with A. caulinodans extracts for complex formation in gel mobility shift assays
and therefore probably recognizes an epitope that interferes with DNA binding. Western
blot analysis detected the presence of the AcBBPl protein only in whole cell extracts or
in Sesbania rostrata stem nodule extracts harboring wild type Azorhizobium caulinodans.
Extracts prepared from the AcBBPl deficient mutant or S. rostrata stem nodules
harboring these mutant bacteria do not express the AcBBPl protein. Localization of
AcBBPl in nodules was attempted using irnmunogold labeling techniques. The colloidal
gold marker was located within the bacteroids and in the peribacteroid membrane and was

not detected in the plant cytoplasm or nucleus.

88

INTRODUCTION

Leghemoglobin (Lb) is a late nodulin which functions to facilitate oxygen diffirsion
to the actively respiring bacteroids within the infected cells of the nodule (Appleby,
1984). In the tropical legume Sesbania rostrata, a small family of leghemoglobin genes
exists. Seven distinct leghemoglobin components have been identified in S. rostrata and
can be distinguished by their biochemical properties and primary protein sequence
(Bogusz et al., 1987).

The expression of lb genes appears to be confined to the infected cells of the
nodule as chimeric leghemoglobin promoter-reporter gene fusions seem to only be
expressed in these cell types (Stougaard et al., 1986, Stougaard et al., 1987; de Bruijn et
al., 1989; Stougaard et al., 1990; Szabados et al., 1990; de Bruijn and Schell, 1992; Ramlov
et al., 1993; Szczyglowski et al., 1994). Cis-acting elements that are essential for tissue
specific expression of leghemoglobin genes have been identified (Stougaard et al, 1986;
1987; 1990; Szabados et al., 1990; Ramlov et al., 1993; Szczyglowski et al., 1994). In
particular, the tissue-specific NICE (Nodule Infected Cell Expression) element found in
the Srglb3 5’ upstream region was delimited and could confer a nodule-specific expression
pattern on a normally root-enhanced promoter (Szczyglowski et al., 1994). To
complement the cis analysis of leghemoglobin promoters, trans-acting factors interacting
with these 5’ upstream regions were identified using gel mobility shift assays (Jensen et

al., 1988; Metz et al., 1988; Jacobsen et al., 1990; Welters et al., 1993).

89

In a search to find factors that could bind to the promoter region of the Srglb3
gene, Welters et al. (1993) observed DNA binding activity in S. rostrata nodule extracts
which could specifically interact with a portion of the Srglb3 5’ upstream region. (BBSl).
Interestingly, the factor responsible for the binding activity was derived from the
symbiont of S. rostrata, Azorhizobium caulinodans (AcBBPl). The AcBBPl protein in
addition to the BBSl within the Srglb3 promoter was characterized Welters et al., 1993).
The analysis of the BBSl in transgenic Lotus corniculatus plants using chimeric
promoter-reporter constructs suggested that the 8881 was important for modulating
levels of Srglb3 promoter activity but was clearly not essential.

The identification of a bacterial protein which has the ability to bind in vitro to a
plant gene promoter is very intriguing and if proven to be biologically significant, would
support the concept of transkingdom signaling that was described in plant-pathogenic
interactions (Howard et al., 1992; Citovsky et al., 1992; Yang and Gabriel, 1995; Van den
Ackerveken et al., 1996). Targeting motifs have been identified in a few bacterial proteins
which have the ability to be delivered to the plant nucleus. These reports, which involve
Agrobacterium tumefaciens and some members of the Xanthomonas genus and plants, not
only showed that bacterial proteins were able to direct chimeric reporter gene constructs
to the plant nucleus but also is supported by a correlation with the presence of a nuclear
localization signal (NLS) and a pathogenic phenotype (Howard et al., 1992; Citovsky et
al., 1992; Van den Ackerveken, 1996).

Proteins can be directed to the nucleus of a cell by several mechanisms. In some

cases, a nuclear localization signal (NLS) found within the amino acid sequence can target

90

a protein to this compartment. Although there are no consensus sequences for NLSs in
any system, the majority are characterized by short stretches of basic amino acids and
involve cellular components which actively transport the protein through the nuclear pore
(Garcia-Bustos et al, 1991; Dingwall and Laskey, 1991; Raikhel, 1992). However, it is
also believed that proteins smaller than 40-60 kDa may passively enter the nucleus by
simple diffusion (Dingwall and Laskey, 1986). Furthermore, proteins could be shuttled or
ride “piggyback” into the nucleus by interacting with another NLS containing protein in
the cytoplasm (Kang et al., 1994). As mentioned previously, the AcBBPl amino acid
sequence contains patches of basic residues which could act as NLSs. However, this
protein is also very small (9 kDa) and could easily diffuse through the nuclear pore
complex and not be actively transported.

The gene encoding AcBBPl was cloned and characterized. To assess the role of
AcBBPl in symbiosis, an A. caulinodans strain unable to produce this protein was
constructed. Growth in the free-living state and under a reduced oxygen environment was
tested and no significant differences were observed. However, when nitrogen fixation
activity was assayed, S. rostrata nodules induced by the AcBBPl deficient strain
displayed a ~20% % reduction in activity. The steady state level of lb mRNA in S.
rostrata stem nodules was monitored. Quantification using a densitometer indicated no
difference in lb expression between the wild-type or mutant nodules.

Determining the ultrastructure of a tissue in combination with the subcellular
location of a protein can greatly aid in deducing this protein’s biological role. Therefore S.

rostrata stem nodules harboring wild-type or AcBBPl-deficient bacteria were analyzed

91

under the transmission electron microscope. In addition the subcellular localization of
AcBBPl in nodules was attempted.

One of the goals of this study was to investigate whether or not this bacterial
protein plays a role in symbiosis. In an attempt to address this question, a study at the
cellular level was initiated. Data obtained by microscopy suggest that the infection of
plant cells within nodules harboring the AcBBPl-deficient mutant is delayed. Polyclonal
antibodies raised against AcBBPl interfere with the DNA-binding domain, and appear to
be specific. Localization studies using the colloidal gold marker indicate that AcBBPl is
localized in the bacteroids and the region surrounding the bacteroids of S. rostrata nodules

and does not appear to be present in any other compartment.

92

MATERIALS AND METHODS

Transmission electron microscopy

Five week old S. rostrata stem nodule slices were prepared for microscopy as
described by Subba-Rao et al. (1995). In brief, the nodule slices were fixed in a solution
containing 4% glutaraldehyde and 1% formaldehyde in 50 mM Na Cacodylate buffer for 2
hours under vacuum. After brief washing in 50 mM Na Cacodylate buffer, the samples
were post fixed in 1% osmium tetroxide for 2 hours at room temperature. After
dehydration through a graded acetone series, the samples were infiltrated with Spurr’s
resin (Electron Microscopy Sciences; Fort Washington, PA). Infiltrated samples were

positioned in molds and polymerized in a 60°C oven for 24 hours. Thin sections (1-2 um)

were fixed to glass slides by briefly passing them over a flame. Subsequently, the samples
were stained with toluidine blue to visualize the tissue integrity under the light
microscope. Ultra thin (90 nm) sections were mounted on flamed copper grids and stained
with 1% uranyl acetate and 16.6% lead citrate. The sections were carbon coated and

examined under a transmission microscope (Model CMlO, Phillips, New Jersey).

Production of Anti-AcBBPl Antibodies
In order to overexpress AcBBPl in E. coli, it was necessary to place the AcBBPl
coding region in the proper reading flame of an E. coli expression vector (pET 22b’;

Novagen, Madison, WI) so that the protein would be translated correctly. To facilitate

93

insertion of the target gene into pET 22b+, the AcBBPl gene was modified using PCR to
generate an Na'eI site at the 5’ end and an XhoI restriction site at the 3’ end. Plasmid DNA
from pSF354 was used in the modification/amplification reaction. Insertion of the target
gene into the NdeI site of the vector allowed translation of the recombinant protein to
begin with the ATG found in AcBBPI gene. Furthermore, the XhoI site not only made
insertion of the AcBBPI gene into the expression vector easy, it also mutated the native
stop codon in the AcBBPI gene so that a 6X histidine (His) tag was translationally fused
to the C-terminus of the recombinant AcBBPl protein. This 6X His tag serves as an
affinity tag for one-step purification by metal chelation chromatography (His Bind Resin,
Novagen) which can facilitate the purification of the recombinant protein. To confirm that
the target gene was in the proper reading frame, the nucleotide sequence of the construct
was determined. This plasmid was introduced into E. coli strain BL21(DE3) (Novagen)
via electrotransforrnation (Sambrook etal., 1989) and the T7 RNA polymerase gene was
induced with 1 mM IPTG to express the AcBBPI gene. Induced cells were collected by
centrifugation, resuspended in 50 mM Tris pH 7.5, 2 mM EDTA, and sonicated at 5
second intervals every 20 seconds for about 2 minutes until slightly viscous. The extract
was purified over a nickel column as the 6X tag binds to divalent cations (N iz’)
immobilized on the His Bind resin and then eluted. The recombinant protein was firrther
purified by separation on SDS-PAGE and visualized by equilibration in 0.19 M Tris-HCl
followed by immersion in 0.3 M CuClz (Lee et a1. 1987). The band corresponding to the

His-tagged AcBBPl protein was electroeluted from the gel. The purity of the

recombinant protein was monitored by SDS-PAGE and Coomassie blue staining. The

94

protein was concentrated using Centricon-IO units (Amicon, Beverly, MA) in phosphate-
buffered saline (PBS, Sambrook et al. 1989). The adjuvant Titerrnax (Cthx Corporation,
Norcross, GA) was used to irnmunize rabbits following the instructions in the
manufacturer’s manual. Anti-AcBBPl serum was affinity purified using Afiigel 10 (Bio

Rad, Hercules, CA), according to the manufacturer's instructions.

Protein Gels and Immunoblot Analysis

Cultures of bacterial strains expressing the AcBBPl protein were grown to
saturation, pelleted, and resuspended in 50 mM Tris pH 7.6. Protein extracts from plant
tissues were obtained by grinding them in liquid nitrogen with a mortar and pestle and
boiling the mixture for 10 min in protein extraction buffer (100 mM Tris, pH 6.8, 5%

SDS, 0.5% B-mercaptoethanol). For western blot analysis, 50 ug of total proteins from

bacterial cells or plant tissues were separated by SDS-PAGE (Laemmli, 1970) using 18%

acrylamide gels, and electroblotted overnight onto 0.45 pm nitrocellulose membranes

(Protran BA 85, Schleicher and Schuell; Keene, NH) in Towbin buffer (Towbin et al.,
1989). The blocking, binding, and washing steps were performed using a solution
containing 1% BSA (w/v), 0.05% Tween 20, 10 mM Tris-HCl, pH 7.4, 150 mM NaCl
(VandenBosch, 1991). AcBBPl serum was used at a 1:1000 dilution. Antibody detection
was achieved using goat anti-rabbit antibodies conjugated to alkaline phosphatase
(Kirkegaard and Perry Laboratories, Gaithersburg, MD) and developed using nitroblue

tetrazolium (NBT, United States Biochemical; Cleveland, OH) and 5-choloro-4-bromo-3-

95

indoyl phosphate (BCIP, United States Biochemical), as described by Harlow and Lane

(1988)

Gel Mobility Shift Assays
Extracts of ORS 571 bacteria were prepared as described by Welters et al. (1993).
A fragment of the Srglb3 promoter containing the BBSl (fragment 5’203) was end labeled

with [‘y-32P]ATP using T4 polynucleotide kinase (Gibco BRL, Grand Island, NY), as

described by Sambrook et al. (1989). Binding reactions were carried out as described
previously (Welters etal., 1993). Antisera raised against AcBBPl, pre-immune serum, or
purified anti-AcBBPl antibodies were included in the binding reactions at the

concentrations described in the legends of the appropriate figures.

Immunocytochemistry

Sixteen day old S. rostrata stem nodules were sliced and fixed in a solution
containing 4% formaldehyde, 1% glutaraldehyde, in 50 mM potassium phosphate buffer
pH 7.2 and vacuum infiltrated for two hours at room temperature. The samples were
rinsed twice for 15 min in 50 mM potassium phosphate buffer (pH 7.2) and dehydrated
in a graded ethanol series (10%, 30%, 50%, 70%, 3x 100%, 15 min per step, 3 hours to
overnight in 100% ethanol). Dehydrated samples were infiltrated with London Resin
White (Electron Microscopy Sciences; Fort Washington, PA) and polymerized in

aluminum weighing dishes (Fisher, St. Louis, M0) at 58°C. Ultra-thin sections were

96

prepared using an Ultracut E Microtome (Reichert-Jung, Vienna, Austria) and mounted
on Pioloform-coated 300 mesh nickel grids (Ted Pella, Inc., Redding, CA).

The grids with samples were incubated in a blocking solution (10 mM Tris-HCl
pH 7.4, 150 mM NaCl, .05% Tween 20, 1% BSA) twice for 30 min and then incubated in
a solution of purified AcBBPl antibody and blocking solution (1:1) for 1 hr at room
temperature. After incubation, the grids were washed in 1 X TBS and put into blocking
buffer again for 30 min. The grids were then incubated in blocking solution with the
secondary antibody (protein A conjugated to 15 nm colloidal gold, Ted Pella, Inc.,
Redding, CA) diluted 1:50. The grids were rinsed with 1X TBS followed by a brief rinse
in water. The samples were stained with a solution of uranyl acetate and potassium
permanganate (4:1 saturated uranyl acetate:l% potassium permanganate solution) for 5
min, thoroughly rinsed in water and dried. Sections were viewed under a transmission

electron microscope (Model CMlO, Phillips, New Jersey).

97

RESULTS

Infection of S. rostrata by an AcBBPl Deficient Mutant Strain

Nodules harboring the wild-type A. caulinodans strain ORS 571 or the AcBBPl
deficient mutant ORS 571-425 were examined by transmission electron microscopy. S.
rostrata cells harboring wild-type ORS 571 bacteria were found to be densely packed
with bacteroids. Up to ten bacteroids were enclosed within the peribacteroid membrane
(symbiosome) in infected plant cells (Figure 3-1). In nodules induced by the AcBBPl
deficient strain, the infected cells were found to be less densely packed with bacteroids
(Figure 3-1 B, C, D). Uninfected cells and recently infected cells could easily be detected
(Figure 3-1 B, C). The cytoplasm of individual infected cells was found to be more
prominent in the cells harboring the AcBBPl deficient strain than in cells harboring the
wild-type strain. Moreover, the majority of symbiosomes contained a single bacteroid
encased by a peribacteroid membrane. No other ultrastructural differences could be

detected in nodules harboring the AcBBPl deficient mutant versus the wild-type strain.

Detection of AcBBPl in Cultures and in Nodules

The full length AcBBPl protein was synthesized in E. coli, using a pET-based
vector system which adds a 6X Histidine tag to facilitate purification of the recombinant
protein. A recombinant protein of expected molecular weight was found to accumulate in
the soluble fraction of E. coli cells harboring the plasmid pET 22b’/Ac4 following

induction (Figure 3-2).

98

Figure 3-1. S. rostrata nodules harboring AcBBPl deficient bacteria are delayed in
the infection process. (A) Overview of a plant cell infected with wild-type A. caulinodans
ORS 571 (B) Overview of a plant cell infected with mutant A. caulinodans ORS 571-425
(C) A magnified view of an infection thread and an (D) uninfected cell near other infected
cells within a nodule harboring mutant bacteria. For A, B bar = 211m. For C, D bar = 1

um.

99

 

100

This recombinant protein was purified over a Ni2+ column and further purified by
SDS-PAGE. The AcBBPl protein was electroeluted from the gel and used to raise
polyclonal antibodies. Western blot analysis of total protein extracts fi'om bacterial
cultures or S. rostrata nodules induced by the wild-type or AcBBPl deficient strain of A.
caulinodans was performed (Figure 3-3). A single band of about 9 kDa was consistently
detected in extracts of wild-type ORS 571 bacteria and in extracts from nodules induced
by the wild-type strain (Figure 3-3). The protein band detected in nodule extracts was
usually very faint and diffuse. This may be due to the low abundance of the protein or to
difficulties in liberating the protein from the bacteroid/symbiosomes. The diffuse nature
of the band may be due to large amounts of Lb protein found in the nodule which
obscures the separation of the proteins. No cross-reacting AcBBPl protein could be
detected in extracts prepared from AcBBPl deficient bacteria or nodules induced by this
mutant strain (Figure 3-3). These results support and extend our previous observations
(see Chapter 2) to the absence of the AcBBPl protein in free living cultures of mutant

bacteria or in nodules induced by strain ORS 571-425.

Specificity of the Anti-AcBBPl Antibodies

To provide further evidence that the anti-AcBBPl antibodies specifically
recognize AcBBPl, antisera and purified anti-AcBBPl antibodies were incorporated in
gel mobility shift assays using the AcBBPl binding site as the target, as described by
Boulanger et al. (1987) and L’Etoile et a1. (1994). A “supershift” effect can be observed if

the antibody recognizes its corresponding protein in the extract. The protein-DNA

101

kDa l 2 3 4 5 6 7 8

97.4-

.
i

b)
p—s
O
I
.7-

-7- .

~‘

\‘a .~

 

Figure 3-2. Overexpression of AcBBPl in E. coli using the pET system. Production of
AcBBPl protein after Lane 1, 0 hours; Lane 2, 1 hour; Lane 3, 2 hours; Lane 4, 3 hours
after inducing cultures with 1 mM IPTG. A control strain harboring the vector alone Lane
5, 0 hr, Lane 6, 1 hr; Lane 7, 2 hr; Lane 8, 3 hr after inducing cultures under the same
conditions yield no accumulation of protein of expected molecular weight.

102

complex will be shifted to a higher molecular weight and migrate slower in the gel due to
the formation of a complex with the antibody. Competition rather than a super shift was
observed when antiserum or purified antibodies generated to AcBBPl were incorporated
into the DNA binding assays (Figure 3-4). This observation can be explained by the fact
that the antibodies generated specifically recognize an epitope on the AcBBPl protein
which interferes with the DNA binding domain. The DNA binding domain may be
directly recognized or may be sterically hindered by another epitope. The competition
event observed was concentration dependent and appears to be specific as complex
formation was inhibited as more antiserum or purified antibodies were added. The
addition of four times as much pre-immune serum did not affect complex formation. This
result provides further evidence that the antibodies generated are specific to the AcBBPl

protein.

Immunolocalization of AcBBPl

Before the S. rostrata nodule samples were fixed and embedded in resin, the
fixative was tested to determine if the formaldehyde or glutaraldehyde destroyed or
modified the antigen located on the AcBBPl protein. Therefore, western blots were
briefly incubated in the fixative which contained 4% formaldehyde, 1% glutaraldehyde in
50 mM potassium phosphate buffer, or in solutions of formaldehyde or glutaraldehyde
alone and developed to determine if this treatment would affect antigenicity

(VandenBosch, 1991). No masking of AcBBPl detection was observed (data not shown).

103

kDa l 2 3 4 5
14.5-
_
6.5-

Figure 3-3. Detection of AcBBPl protein in free living cultures of A. caulinodans
ORS 571 and in S. rostrata nodules harboring A. caulinodans ORS 571. Lane 1.
recombinant AcBBPl; Lane 2 and 3, extract prepared from A. caulinodans ORS 571 and
A. caulinodans ORS 571-425 respectively; Lanes 4 and 5, total protein extracted from S.
rostrata nodules harboring wild-type or mutant bacteria respectively. Western blots were
probed with anti-AcBBPl serum.

104

(-694) (-653) (-48)

F hm“ _. Srglb3

 

 

 

 

 

5'203
ATG

F —+ U:::*“:

Figure 3-4. Characterization of AcBBPl antibodies using DNA binding assays.
AcBBP] antibodies were incubated with extracts prepared from ORS 57] and were tested
for DNA binding activity using the BBS] as target DNA. (A) Schematic of the Srglb3 5’
upstream region. The arrow indicates the start point oftranscription. The fragment 5’203
(shaded in red) containing the BBS], was used as the target fragment in gel mobility shift
assays. (B) ORS 57] extracts were incubated with the following: Lane 1, no additions;
Lanes 2-4, 0.005 pg, 0.025 pg, 0.05 pg affinity purified AcBBPl antibody respectively;
Lanes 5-7, 0.05 pg, 0.0], 0.1 pg AcBBPl antisera respectively; Lane 8, 0.4 pg pre-
immunc serum. The free fragment(F, 5’203) is indicated with an arrowhead.

105

The anti-AcBBPl antibodies characterized earlier were used to imrnunolocalize
AcBBPl at the subcellular level. Slices of S. rostrata stem nodules induced by the wild-
type strain ORS 571, were fixed, infiltrated with resin and labeled with the colloidal gold
marker as described in the Materials and Methods. Labeling of AcBBPl with colloidal
gold was observed predominantly in the bacteroids and the region surrounding them
(Figure 3-5). This result is not too surprising since AcBBPl is of bacterial origin.
Background levels or no labeling was detected in the plant cell cytoplasm within the
infected cell or in an uninfected cell bordering the infected zone. Although the

concentration of particles was not very high, the labeling appears to be specific.

106

 

Figure 3-5. The colloidal gold marker locates the AcBBPl protein. (A) Bacteroids
within an infected cell of a S. rostrata nodule harboring A. caulinodans ORS 571. (B) A
peripheral uninfected cell in the same sample. Bars = 300 nm.

107

DISCUSSION

In this chapter, S. rostrata nodules induced by wild-type A. caulinodans and the
AcBBPl deficient mutant were compared at the ultrastructural level. It was shown in
Chapter 2 that nitrogen fixation activity was about 20% lower in nodules harboring the
AcBBPl deficient strain. To investigate this reduction in nitrogen fixation a microscopical
study was canied out to determine if any differences in the ultrastructure of nodules
induced by the A. caulinodans mutant existed. Using transmission electron microscopy
sections of S. rostrata nodules induced by the wild-type or AcBBPl deficient strain were
observed. The plant cells of nodules induced by mutant bacteria appear to be slightly
delayed in the infection process. Structures associated with the infection process such as
infection threads were visible. Uninfected plant cells were also observed. The number of
bacteroids within the infected cell also appeared to be reduced when compared to the
nodules colonized by wild-type bacteria. Figure 3-1 suggest that there is a difference in
the number of bacteroids encased within the peribacteroid membrane (symbiosome). It
can be observed that approximately one or two bacteroids are surrounded by a
peribacteroid membrane in plant cells infected with the AcBBPl deficient strain. In
contrast, the S. rostrata nodules harboring the wild-type A. caulinodans were densely
packed with bacteria with virtually no plant cytoplasm visible.

It was also of interest to localize the AcBBPl protein subcellularly in the infected
plant cell. The deduced amino acid sequence of AcBBPl has stretches of basic residues

which may be involved in targeting this protein to the plant nucleus. On the other hand,

108

AcBBPl is a small protein of about 9 kDa and could passively diffuse through the nuclear
pore complex and gain access to the plant nucleus. To localize this bacterial derived
protein, antibodies specific to AcBBPl were produced. The AcBBPl protein was
overexpressed in E. coli (pET system, Novagen, Madison, WI) and purified over a nickel
column using the 6X His tag that was fused to the recombinant protein. The AcBBPl
recombinant protein was further purified by electroelution and injected to rabbits.

A few lines of evidence indicated that antibodies generated were specific to
AcBBPl. First, these antibodies recognized the recombinant AcBBPl protein. In
addition, a unique band of about 9 kDa was detected in extracts prepared from free living
cultures of A. caulinodans or extracts obtained from nodules harboring wild-type bacteria.
The band observed in nodule extracts is usually diffuse and is not very abundant. This
may indicate that the protein is not synthesized in large quantities in the bacteroid or that
the protein is difficult to extract from the nodule tissue. The bacteroid is very
membranous and may entrap the protein making detection a little difficult. The band may
also be obscured by all the soluble proteins found in nodule extracts such as
leghemoglobin which may affect the way the protein sample separates on the gel. The
difference in the molecular weight of the recombinant protein and the native protein is
most likely due to the 6X histidine tag that was fused to AcBBPl for purification
purposes. Second, gel mobility shift assays also support the idea that these antibodies are
specific to AcBBPl since incorporating the immune serum or purified antibodies to the
binding reactions lead to competition of complex formation. Pre-immune serum however,

was unable to compete for complex formation. These two pieces of evidence suggest that

109

the antibodies that were generated do in fact specifically recognize the AcBBPl protein.
The binding studies also indicate that the epitope to which the antibodies are generated
affect DNA binding since competition rather than a super shift was observed.

Because no established protocols were available for immunolocalization of
proteins in this tissue, a great deal of experimentation with fixation and infiltration was
necessary. The dehydration step was extremely important for good fixation of S. rostrata
nodule tissues. A graded ethanol series with smaller steps in addition to long incubation
periods in 100% ethanol was important (see Materials and Methods) for retaining
structure. These small modifications made a difference.

Gold particles were detected in the bacteroids of the infected cells, most often
within the bacteroid or within the symbiosome. The observed labeling pattern in the
region surrounding the bacteroid may reflect the secretion of the protein into this region or
may be a fixation or sectioning artifact. Labeling in the bacteroids was not very intense
and may be due to the presence of a large population of bacteroids within a section which
could titrate out the antibody. Alternatively, it may reflect the in vivo concentration of
the protein within the bacteroid. Nothing is known about the abundance of this protein in
this differentiated state. The colloidal gold marker was not detected in the plant nucleus or
other plant organelles or compartments.

NLS-reporter protein fusions are classic methods to determine if a protein can be
targeted to the nucleus. Recently, Avrb6, PthA, AvrBs3 and FixF proteins were shown to
have frmctional NLSs which could direct fusion proteins into the plant cell nucleus (Yang

and Gabriel, 1995; Van den Ackerveken et al., 1996, Jabbouri et al., 1996). Similar

110

experiments using the uidA reporter gene fused to entire AcBBPI gene were tested in
onion cells. In this assay, the chimeric construct is precipitated onto gold particles and
bombarded into onion epidermal layers. Localization of the chimeric protein can be
observed using the histochemical Gus activity assay (Jefferson, 1987). AcBBPl did not
localize to the nucleus of onion cells (data not shown). This result may indicate that
AcBBPl does not have an NLS. Alternatively, this result could be due to the addition of

the B-glucuronidase protein to AcBBPl. The reporter protein could mask or sterically

hinder the putative NLSs previously identified in the deduced amino acid sequence (Shieh
et al., 1993). On the other hand, this protein may not need an NLS to enter the plant
nucleus since it is so small (Dingwall and Laskey, 1986). The addition of the reporter gene
may prevent the protein from freely diffusing through the nuclear pore or interacting with
other cellular components (Kang et al., 1994) which could possibly direct it to the
nucleus.

In conclusion, differences were observed in nodules induced by the A. caulinodans
wild-type and AcBBPl deficient strain. The plant cells inhabited by l the mutant strain
appear to be delayed in the infection process as infection threads and uninfected cells
were clearly visible in the central portion of the nodule. Immunogold localization showed
that AcBBPl is localized to the bacteroid and within the symbiosome. Unfortunately,
this location does not provide much evidence as to the role this protein may play in
Srglb3 gene regulation. AcBBPl was not localized to the nucleus using irnmunochemical

methods or by the chimeric NLS-reporter gene system. Since the AcBBPl protein is

111

small, it may simply diffuse into the nucleus or may be shuttled in by another nuclear
localized protein. On the other hand, AcBBPl could be present in the plant nucleus but is
undetectable by the antibodies that were generated. To test the hypothesis that AcBBPl
plays a role in lb gene expression, transgenic plants harboring the Srglb3 promoter-
reporter construct will be used. Nodules induced on these transgenic plants by a BBPl
deficient R. loti strain will be monitored for reporter protein activity and compared to the
nodules induced by the wild-type strain. These experiments will be discussed in Chapter

4 of this thesis.

112

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CHAPTER4

ANALYZING THE ROLE OF BBPl IN TRAN SCENIC
LOTUS CORNIC ULA T US

Portions of this chapter will be submitted as an article to the journal Molecular Plant-
Microbe Interactions.

115

116

ABSTRACT

The gene from Rhizobium loti which is similar to the AcBBPI (Bacterial Binding
Protein) gene from Azorhizobium caulinodans was isolated. The sequence of the R. loti
gene (RlBBPl) is 63% identical to the AcBBPI gene at the nucleotide level. At the amino
acid level, the two BBP] proteins are 78% identical and 85% similar. Putative nuclear
localization signals and helix-turn-helix DNA binding domains are conserved. Factors
present in extracts prepared from R. loti interacted with the same region of the 5’
upstream region of the Sesbania rostrata leghemoglobin glb3 gene to which AcBBPl
binds (BBSl; Welters et al., 1993). An R loti strain deficient in the production of the
RlBBPl protein was constructed by inserting a kanamycin resistance cassette into the
RlBBPl gene. Extracts prepared from this mutant strain did not form any complexes with
the BBS] in gel mobility shift assays. The interaction between RIBBPl and the BBSl
region within the Srglb3 promoter was further examined by monitoring the activity of
Srglb3 promoter-uidA reporter gene fusions in transgenic Lotus corniculatus plants.
Transgenic L. corniculatus plants were inoculated with the wild-type or the RlBBP]
deficient strain. Gus enzymatic activity within nodules was quantified using a
fluorometric assay. No statistically significant differences in Gus expression between
nodules induced by the wild-type and the RIBBPI deficient strains could be observed,

raising doubt that BBP] is involved in Srglb3 gene expression.

117

INTRODUCTION

Symbiotic nitrogen fixation is a unique example of a complex and highly regulated
biological process which occurs in a novel plant structure, the nodule. A complex and
fine-tuned exchange of signals between bacteria belonging to the genus Azorhizobium,
Bradyrhizobium, Rhizobium or Sinorhizobium and legume plants leads to the formation of
a plant organ in which atmospheric nitrogen is converted into ammonium (Nap and
Bisseling, 1990; de Bruijn and Schell, 1992; Mylona et al., 1995; Long, 1996). A
collection of genes encoding nodule-specific proteins, collectively known as nodulin genes
(van Kammen, 1984) have been isolated and characterized from many species of legumes.
These genes have been classified as early or late depending on their developmental
appearance (Nap and Bisseling, 1990). Early nodulin genes correlate with the infection
process and nodule ontogeny while late nodulin genes are involved in nodule functioning
and maintenance.

Nodulin gene expression depends on direct or indirect signals originating from both
the plant and symbiotic bacteria in addition to the tissue or cell specific receptiveness in
the plant. One line of research to elucidate the signal transduction pathway involved in
nodulin gene induction has focused on the identification of cis-acting elements within the
promoters of these genes and trans-acting factors which interact with these regions (de
Bruijn and Schell, 1992).

Leghemoglobin (lb) genes belong to a class of highly studied late nodulin genes and

encode proteins which function to facilitate oxygen diffusion to the actively respiring

118

bacteroids of the nodule. Functional analysis of two leghemoglobin genes have defined cis-
acting elements important for specific expression in the infected cells of nodules
(Stougaard et al., 1986, 1987; Szabados et al., 1990; Ramlov et al., 1933 and
Szczyglowski et al., 1994). The apparent expression of the lb promoter in the infected
cells of transgenic Lotus corniculatus nodules suggest that the physical presence of
bacteria may be essential for expression of this gene (de Bruijn and Schell, 1992).

To complement this work, several DNA binding proteins which interact with the
promoters of these genes have been identified (Jensen et al., 1988; Metz et al., 1988;
Welters et al., 1993), although none of these factors have been directly implicated in
modulating gene expression. Welters and co-workers (1993) showed that a protein of
bacterial origin (AcBBPl) was able to bind to the leghemoglobin Srglb3 5’ upstream
region and that disruption or deletion of its binding site, the Bacterial Binding Site 1
(BBSl), decreased expression of a chimeric promoter-reporter gene construct in Lotus
corniculatus. This finding provided a possible explanation for the infected cell specific
expression pattern of the Srglb3 gene and warranted more investigation.

The gene encoding AcBBPl from Azorhizobium caulinodans AcBBPl was cloned
and characterized, and an AcBBPl deficient strain was constructed. No obvious
differences in growth of the mutant strain under free living or reduced oxygen conditions
were observed. The AcBBPl deficient strain was able to induce nodules on S. rostrata
plants that were able to fix nitrogen (N od+, Fix+). However, the nitrogen fixation activity
was ~20% lower in the mutant strain as assayed by acetylene reduction. The

ultrastructure of nodules harboring this mutant revealed that plant cells appeared to be at

119

a different stage of the infection process. Less bacteroids appeared to be encased within
the peribacteroid membrane and provides additional evidence for delay in the infection
process.

Welters et al. (1993) showed that a factor similar to AcBBPl exists in nodules of
L. corniculatus and in free living extracts of its symbiont, Rhizobium loti strain NZP 2037
(Pankhurst et al., 1986). This factor binds to the identical site (BBSl) in the S. rostrata
promoter as the AcBBPl protein. It was therefore postulated, that these two factors play
similar roles in their respective interactions. To address the biological significance of these
proteins in leghemoglobin gene expression, a heterologous transgenic plant system was
used which had previously been employed to study lb promoter activation (Stougaard et
al., 1986; 1987; 1990; Szabados et al., 1990; Ramlov et al., 1993; Welters et al., 1993;

Szczyglowski et al., 1994).

To directly test the effect of BBPl on glb3 promoter activity, the homolog of
AcBBPl inR. loti (RlBBPl) was identified. The corresponding gene is 63% identical at
the nucleotide level, and its protein product is 78% identical and 85% similar at the
deduced amino acid level. RIBBPl binds in vitro to the same target in the 5’ upstream
region of the Srglb3 promoter as AcBBPl. A RlBBPl deficient mutant was constructed
by insertional mutagenesis and gel mobility shift assays confirmed the loss of complex
formation in extracts of this mutant. The wild type and RIBBPl deficient strain were
inoculated on the roots of transgenic L. corniculatus plants harboring the Srglb3 5’

upstream region fused to the uidA reporter gene and nodules of the resulting transgenic

120

plants were monitored for GUS reporter gene activity. No differences in GUS activity
levels were detected in nodules harboring the wild-type or BBPl deficient mutant strain

suggesting that the BBPl protein is not involved in Srglb3 gene expression.

121

MATERIALS AND METHODS

Bacterial Strains and Plasmids used

The bacterial strains and plasmids used in this study are described in Table 2.

Southern Blot Analysis

Total bacterial genomic DNA was isolated as described by Meade et al. (1989).
Ten ug of total genomic DNA was completely digested with restriction enzymes,
separated on 0.8% agarose gels, and transferred to nylon filters (Hybond-N; Amersham,
Arlington Heights, IL) according to standard procedures (Sambrook, 1989). Membranes
were prehybridized and hybridized in a solution containing 4 X SSC, 5 X Denhardt’s

solution (Sambrook, 1989), 0.5% SDS (w/v) and 100 pg/ml denatured sheared salmon

sperm DNA at 65°C. The membranes were washed once for 15 min in 4 X SSC, 0.1%
SDS (w/v), twice for 15 min in 1 X SSC, 0.1% SDS (w/v), and twice for 15 min in 0.5 X
SSC, 0.1% SDS (w/v) at 65°C. The full length AcBBPl gene probe was labeled with [0t-

32P]dATP using a random primer kit (Boehringer Mannheim) following the

manufacturer’s instructions.

 

122

Construction of an R. loti Partial Library
Total genomic DNA from R. loti NZP 2037 (Pankhurst et al., 1986) was digested

with PstI and 10 pg of DNA was separated on a 0.8% preparative agarose gel. A one

centimeter area in the size range of the fragments found to hybridize with the AcBBPI
gene probe were excised and the DNA isolated using a phenol freeze-thaw method. The
purified fragments were cloned into pBluescript KS' (Stratagene, La Jolla, CA) digested
with PstI. Positive colonies were identified using a pooled plasmid mini preparation
method. Ten individual colonies were grown in LB media with the appropriate antibiotics,
pooled together and the plasmid DNA isolated (Sambrook et 1., 1989). Southern blot
analysis was carried out as described in the previous section using the AcBBPI gene as the
probe. Plasmid DNA was isolated from individual cultures of positive hybridizing pools
and were screened again using Southern analysis. Out of ten pools of ten colonies each

(100 colonies screened), two positive hybridizing strains were found.

DNA Sequencing and Computer Analyses

A plasmid carrying a 6-kb fragment hybridizing with the AcBBPl probe was
mapped using single or multiple enzyme digests. The smallest fi'agment containing the
RlBBPl gene was cloned and the nucleotide sequence determined (pCla3). DNA
sequencing was performed using a Sequenase 7-Deaza-dGTP DNA sequencing kit
(United States Biochemical, Cleveland, OH), according to the manufacturer’s instructions.

Computer analysis of DNA sequences was carried out using Sequencher 2.1 (Gene Codes

 

123

Corp., Ann Arbor, MI) and Squd (Applied Biosystems) software. Multiple sequence
alignment using Pileup in the GCG (Genetics Computer Group, Madison, WI) program
and Squu 1.01 (Garvan Institute of Medical Research; Sydney, Australia). Analysis of
predicted protein sequences was performed using PHDsec (Rost and Sander, 1993; 1994).

Homology searches were performed using the BLAST algorithm (Altschul et al., 1990).

Construction of the RlBBPl Deficient Strain

A plasmid carrying a 6-kb PstI fragment hybridizing with the AcBBPl gene probe
was mapped using single or multiple enzyme digest. The location of the RlBBPl gene
was delimited using Southern blot analysis. The 6-kb PstI fragment harboring the RIBBPl
locus was cloned into the pBluescript KS' vector (pSF99C) and used for gene
replacement. A BamHI digested fragment carrying the kanamycin cassette from pUC4K
(Phannacia Biotech; Uppsala, Sweden) was introduced into the unique BglII site 80-bp
from the beginning of the RIBBPI ORF (pSF99CKm). The fragment containing the
disrupted RlBBPl gene was liberated from plasmid pSF99CKm and cloned into the PstI
site of the suicide vector pSup202 (Simon et al., 1983). The vector was mobilized from E.
coli to NZP 2037 using the helper plasmid pRK2013 (Ditta et al., 1980). Transconjugants
were selected as described by Simon et al. (1983) on S-20 medium (Chua et al., 1985)

supplemented with tetracycline (Te) 10 pg/ml and kanamycin (Km) 200 pg/ml initially

and were later scored for the lack of growth on media containing Tc which confirmed gene

replacement into the genome.

124

Gel Mobility Shift Assay

Extracts of R. loti NZP 2037 and the mutant NZP 2037-11 strains were prepared
as described by Welters et al., (1993). The fragment of the Srglb3 promoter containing the
BBSl (fragment 5’203) was used in all reactions under conditions previously described

(Welters et al., 1993).

Plant Transformation and Nodulation

Transgenic L. corniculatus plants cv Rodeo were generated according to
Szczyglowski et al. (1994). Binary vectors carrying the 5’ upstream region of Srglb3
fused to the reporter gene uidA (LP14, Szczyglowski et al., 1994)) were transferred to
Agrobacterium rhizogenes strain A4 (Tempe and Casse-Delbart, 1989) using the freeze
thaw method (Hofgen and Willrnitzer, 1988). After plants were regenerated, multiple
cuttings from a plant with average expression levels were propagated and allowed to root
in sterile “sandy “soil (3:1 sand:metromix) or “mixed” soil (3:3:1:1 sand:mediurn
vermiculite:metromiszrabidopsis mix) for two weeks in growth chambers with an 16-hr
light (24°C) and 8-hr dark (18°C) cycle. Plants were inoculated with 1ml of a two-day old
culture of NZP 2037 or NZP 2037-11 grown in the presence of antibiotics but washed
with sterile water to remove antibiotics and residual medium. The pelleted cells were
resuspended in sterile water and 1 ml of this suspension was inoculated onto the roots of
transgenic L. corniculatus plants. Twenty-eight days after infection, all nodules from each

plant with the exception of four were harvested and frozen in liquid nitrogen until

125

analyzed. The remaining four nodules were used to resolute bacteria from the nodule

(Pawlowski et al., 1987) to ensure that no cross contamination took place.

Quantification of GUS enzymatic activity

GUS enzymatic activity was quantified using the fluorescence assay (Jefferson,
1987; Jefferson et al., 1987) with a fluorescence spectrophotometer (Model F-2000,
Hitachi). Enzymatic activity was expressed as picomoles of 4-methylumbelliferone
produced per minute per milligram of protein. Protein concentration of the extract was

determined using the Bradford assay using BSA as a standard (Bradford, 1976).

Statistical Analysis

Each set of data was analyzed using the non-parametric Mann Whitney test.
Friedman’s test was used to determine if data from different trials could be pooled. The
pooled data was subsequently analyzed using the Mann Whitney test (Gardener and de

Bruijn, 1997).

126

RESULTS

Identification and Cloning of the R. loti RlBBPl Gene

The gene encoding RlBBPl from R. loti strain NZP 2037 was isolated to directly
test the effect of BBPl on Srglb3 promoter activity. The AcBBPI gene from A.
caulinodans was used as a probe to identify any sequences cross hybridizing in the R. loti
genome. Southern blot analysis of R. loti NZP 2037 genomic DNA digested with BamI-II,
EcoRI, HindIII, and PstI revealed single hybridizing bands, suggesting that the RlBBPl
gene is unique (Figure 4-1). This analysis revealed that the RlBBPl gene was located on a
~6-kb PstI fragment. Single hybridizing bands were observed in closely related strains of
R loti (NZP 2235, NZP 2234, and PN184) genomic DNA but not in Rhizobium meliloti
1021 or two E coli strains (Figure 4-1B). To isolate the complete RIBBPI locus, a partial
PstI library of R. loti NZP 2037 was constructed, enriching for fragments of ~6 kb and
screened via a pooled plasmid mini preparation method using the AcBBPl gene as a
probe. This screening procedure yielded two positive clones. The recombinant plasmids
from one colony was isolated, mapped with several restriction enzymes and re-
hybridized with the AcBBPI gene probe (pSF99C). The RlBBPl gene was located on a

~500 bp ClaI-EcoRI fragment and its nucleotide sequence was determined.

Analysis of the AcBBPl Gene and Its Protein Product (RIBBPl)
The gene encoding RlBBPl consists of 201 bp and is 63% similar to the AcBBPI

gene at the nucleotide level. The BBPl proteins are about 78% identical and about 85%

127

 

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similar at the amino acid level. The RIBBPI protein retains the two regions found to
contain stretches of basic amino acid residues which may act as nuclear localization
signals (NLSs; Garcia-Bustos et al., 1991; Raikhel, 1992; Figure 4-2). The DNA binding
helix-tum-helix motifs in the central portion of RIBBPI (residues 22-41) as well as the C-
terminus of the protein (residues 51-70) that were identified in AcBBPl are also retained.
Secondary structure analysis of RIBBPl using the PHD-sec program (Rost and Sander,
1993; 1994) predict the presence of regions containing two adjacent helical structures
separated by a loop or turn with a high probability (7-9 on a scale from 0-9).

The RlBBPl amino acid sequence aligns well with the control elements in several
type II restriction-modification (RM) systems (Bougueleret et al., 1984; Nathan and
Brooks, 1988; Heidemann et al., 1989; Tao et al., 1991; Siksnys et al., 1994; Anton et al.,

1996), immunity repressor proteins from phage (1:105 and E coli (Cully and Garro, 1985;

Dhaese et al., 1985; Aiba et al., 1996) and the regulator of vegetative replication and
conjugal transfer of the plasmid RK2 (J agura-Burdzy et al., 1992) previously identified to
have significant similarity to AcBBPl. Identity within the helical domains thought to be
important for the helix-tum-helix DNA binding motif in these proteins was also
conserved in RlBBPl (Tao et al., 1991; Dhaese et al., 1985; Van Kaer et al., 1987; Jagura-

Burdzy et al., 1992).

129

A B

123456 12345678910
-23.1 kb -

-- ~ 0
-9.41 kb -

a
-6.55 kb - .u .
-r/I
// -4.36 kb -
b

-2.32 kb -
-2.03 kb -

Figure 4-1. Organization of the RIBBPI gene in the genome of R. loti NZP 2037
and other bacterial strains. (A) Ten micrograms of R. loti NZP 2037 genomic DNA was
completely digested with restriction enzymes Lane 1 positive control: ORS 571 digested
with PstI; Lane 2, blank; Lane 3, BamHI; Lane 4, EcoRI; Lane 5, HindlII; Lane 6, P511
separated on a .8% agarose gel and probed with [a—32P]dATP labeled AcBBPl DNA. (B)
Southern blot hybridization of genomic DNA. Lanes 1, 3 were digested with PstI. Lanes
4-10 were digested with EcoRI. Lane 1, ORS 571; Lane 2, blank; Lane 3, ORS 571-425;
Lane 4, R. loti NZP 203 7; Lane 5 NZP 2235; Lane 6, NZP 2234; Lane 7, PN184; Lane
8, R. meliloti 1021; Lane 9, E. coli strain INFoz’; Lane 10 E. coli strain HBlOl.

130

Figure 4-2. Alignment of RIBBPI with AcBBPl. (A) Nucleotide sequence; asterisks (*)
indicates identity, (B) Amino acid sequence; asterisks (*) indicates identity, semi-colon (2)
indicates conservative substitutions.

A

131

ACBBPl ATGGATATGCGCAAGCTGGTCGGCCGGAACTTCGCGCGCCTGCGT
*************** ******* ** ** * a *
RlBBPl ATGGATATGCGCAAGTTGGTCGGACGAAATGCACGCAGGATCAGG
ACBBPl CAGGAGAAGGGCCTGACACAGGAGGACGTACAGACGCGATCCGGC
** ** *** **** ****** * * ******
RlBBPl GAGAAGGCCGGCTTGACGCAGGAGCAGCTTGCCGAGATCTCCGGC
ACBBPl TTCAGCCAGCAGTACATCAGCGGGCTCGAACGCGGCCGGCGCAAT
************************** ** ** * **
RlBBPl TTCAGCCAGCAGTACATCAGCGGGCTGGAGAAGGGTAAAAGGAAC
AcBBPl CCCACTGTCATCACGCTCTATGAACTGGCACAGGCGCTGGGGTTA
***** ** ****** ************** * ** k *
RIBBPl CCCACCATCGTCACGCTTTATGAACTGGCACAAGCCCTCCGTGTC
ACBBPl CGCCACGAAGAGCTTGTTCGCGCTGACGGCAAGGACTGA
* ** ** ** ** ** ***** *
RlBBPl AGTCATATCGATCTGGTGCGACCTGACTGA
B
ACBBPl MDMRKLVGRNFARLRQEKGLTQEDVQTRSGFSQQYISGLE
**********: *:* : ***** : ************
RlBBPl MDMRKLVGRNARRIREKAGLTQEQLAEISGFSQQYISGLE
ACBBPl RGRRNPTVITLYELAQALGVSHEELVRADGKD
* ****::********* *** :***:*
RlBBPl KGKRNPTIVTLYELAQALRVSHIDLVRPD

45

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90

135
135

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210
201

40
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69

 

132

Creation and Characterization of An R. loti RlBBPl Deficient Strain

To directly determine if BBP1 proteins play a role to modulate Srglb3 gene
expression, an R. loti NZP 2037 strain lacking the ability to synthesize RIBBPl was
constructed (NZP 2037-11). A kanamycin resistance (Km') cassette derived from
pUC4K was inserted into the unique BgIII site 80 bp from the beginning of the ORF. The
resulting insertion mutation was used to replace the wild-type gene via gene replacement
(Simon et al., 1983). The structure of the mutated RIBBPl locus in strain NZP 2037-11
was confirmed by Southern blot analysis (see Figure 4-3). Whole cell extracts were
prepared from the wild-type NZP 2037 strain and the NZP 2037-11 mutant and used in
gel retardation assays using the fragment of the S. rostrata glb3 5’ upstream region,
harboring the binding site (BBSl; fragment 5’203; Welters et al., 1993: see Figure 4-4) as
target DNA. No binding activity could be observed with extracts of the RIBBPI deficient

strain (Figure 4-4) confirming that the mutation created abolishes RIBBPI production.

Directly Testing the Effect of BBP1 on Srglb3 Promoter Activity

To directly test the effect of BBP1 on Srglb3 promoter activity, transgenic L.
corniculatus plants harboring the Srglb3 promoter fused to the uidA reporter gene were
produced as described by Szczyglowski et al. (1994). The resulting plants were tested for
tissue specific reporter gene (GUS) activity. All plants had detectable GUS activity in the
infected cells of nodules (data not shown). One plant with average GUS activity of ~600
pmol methylumbelliferone (MU) per minute per mg protein was chosen for further

study.

 

133

*
123456789

23.1 kb -

9.41 kb -
6.55 kb -

4.36 kb -

2.32 kb -
2.03 kb -

 

Figure 4-3— Structure of the RIBBPl insertion mutant. All lanes were digested with
PstI. Lane 1, NZP 2037 genomic DNA; Lane 2, NZP 2235 genomic DNA; Lane 3
blank; Lane 4, transconjugant 1; Lane 5; transconjugant 8; Lane 6, transconjugant 9;
Lane 7, transconjugant 10; Lane 8, transconjugant 11; Lane 9, P511 fragment containing
the inserted kanamycin cassette. The entire PstI-Pstl fragment containing the RIBBPl gene
(see Methods and Materials) was used as a probe. Asterisk (*) denotes the strain selected
for use in future studies as the RlBBPl deficient mutant.

I34

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5'203
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Figure 4-4. Whole cell extracts prepared from the RlBBPl deficient mutant lack
binding activity. Extracts prepared from NZP 2037 or NZP 2037-11 were tested for
DNA binding activity using the BBS] as target DNA. (A) Schematic of the Srglb3 5’
upstream region. The arrow indicates the start point of transcription. The fragment 5’203
(shaded in red) containing the 8881, was used as the target fragment in gel mobility shift
assays. (B) Lane 1, extracts prepared from wild type ORS 57], Lane 2, NZP 2037 Lane
3, extracts prepared from NZP 2037—1 1.

135

The effect of the RIBBPI mutation on nodulation and symbiotic nitrogen fixation
was determined. Vegetatively propagated transgenic L. corniculatus plants harboring
chimeric Srglb3 promoter-uidA fusions were inoculated with either the wild-type or R. loti
strain lacking the ability to produce RIBBPI in a sandy soil or a mixed soil. The RlBBPl
deficient mutant strain was capable of inducing root nodules on its host plant (Nod+). In
order to determine the Srglb3 promoter activity in nodules harboring wild-type or
RIBBPl deficient bacteria, GUS activity assays were performed as described by
Szczyglowski et a1. (1994) using the fluorescence assay (Jefferson, 1987). The results of
three separate GUS activity assays in sandy soil were pooled and presented in Figure 4-5.

Thirty-six plants grown in the sandy soil were assayed for GUS activity. The
GUS activity in nodules harvested from eighteen transgenic L. corniculatus plants
inoculated with the RlBBPl deficient strain was compared to the GUS activity in nodules
induced by the wild-type strain. The GUS activity was measured and the non-parametric
Mann-Whitney statistical test was applied. No statistically significant difi‘erence in GUS
activity was detected between the two treatments suggesting that BBP1 proteins do not
play a role in Srglb3 gene expression (Figure 4—6).

Twenty-two plants grown in mixed soil were assayed for GUS activity. Nodules
harvested from eleven plants inoculated with the R. loti mutant strain were compared to
nodules harvested from eleven plants inoculated with the wild-type strain. The GUS
activity was measured and the non-parametric Mann-Whitney statistical test was

applied. No statistically significant difference in GUS activity was detected between the

136

 

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137

two treatments which suggest that BBP1 proteins do not play a role in Srglb3 expression

(Figure 4-6).

138

Figure 4-6. B-glucuronidase activity in 26 day-old L. corniculatus nodules harvested
from sandy soil. In three independent experiments, transgenic L. corniculatus harboring
the LP14 construct (Srglb3 promoter fused to uidA) were nodulated with the wild-type
or the RIBBPI deficient R. loti mutant. Light bars = wild-type, dark bars = mutant. All
results are expressed as pmol MU/min/mg protein. Non-parametric statistics indicate that
there is no significant difference between treatments.

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Figure 4-7. B-glucuronidase activity in 26 day-old L. corniculatus nodules harvested
from mixed soil. In three independent experiments, transgenic L. corniculatus harboring
the LPl4 construct (Srglb3 promoter fused to uidA) were nodulated with the wild-type
or the RIBBPI deficient R. loti mutant. Light bars = wild-type, dark bars = mutant. All
results are expressed as pmol MU/min/mg protein. Non-parametric statistics indicate that
there is no significant difference between treatments.

141

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142

DISCUSSION

In this chapter, the gene encoding the Rhizobium loti RIBBPl DNA binding
protein which is homologous to the AcBBPl protein from Azorhizobium caulinodans
was isolated and characterized. This protein was previously shown to bind to the 5’
upstream region of the Srglb3 gene and was implicated to play a role in Srglb3 gene
expression (BBSl, Welters et al., 1993).

Since previous indirect evidence indicated that lb gene expression may require a
bacterial signal, the observation that a protein originating from the symbiotic bacterium
could bind to a region of an Ib gene was intriguing. Efforts to define the role of AcBBPl in
Srglb3 gene expression are described eariier in this thesis. Unfortunately, a clear answer
was not obtained. Therefore, a system using transgenic plants canying the Srglb3
promoter fused to the uidA reporter gene was used to directly test what role BBP1 plays
in Srglb3 gene expression. In addition, the homologous gene from R. loti was isolated and
characterized and an RIBBPl deficient mutant constructed.

To investigate what role BBP1 proteins play in Srglb3 gene expression, the gene
encoding the R. loti RIBBPI protein was cloned using the A. caulinodans AcBBPI gene as
a probe. Upon inspection of the nucleotide and amino acid sequence of the two proteins,
the RIBBPl gene is 63% identical to the AcBBPI gene at the nucleotide level while the two
BBP1 proteins are 78% identical and 85% similar to each other at the amino acid level.
Putative NLSs such as the NLS identified in the R protein is conserved in RIBBPI (Shieh

et al., 1993). In addition, the helix-turn-helix DNA binding domains are also conserved

143

spatially and in amino acid content. Secondary structure analysis using the PHD-Sec
computer program (Rost and Sander, 1993; 1994) predicted two adjacent helical
structures separated by a loop or turn with fairly high probabilities (7 to 9 on a scale of O-
9).

The RlBBPl sequence also aligns well with the class of control elements found in
type II restriction-modification systems (Bougueleret et al., 1984; Nathan and Brooks,
1988; Heidemann et al., 1989; Tao et al., 1991; Anton et al., 1996), to the two immunity
repressor proteins previously mentioned (Cully and Garro, 195; Dhaese et al., 1985; Aiba
et al., 1996) and to the TrbA protein which is a regulator of vegetative replication and
conjugal transfer of the RK2 plasmid. (Jagura-Burdzy et al., 1992). The most significant
sequence similarities were to the C proteins which are found in some type II restriction-
modification systems. The overall sequence similarity at the nucleotide and amino acid
level is striking when comparing the BBP1 proteins and the C proteins.

The phenotype of the RIBBPI deficient strain under free living conditions was
monitored. No difference in growth rate was observed. The NZP 2037-11 mutant strain
was tested for nodule formation on L. corniculatus. Nodules were induced on the roots
indicating a (Nod+) phenotype. Gel mobility shift assays in which the BBSl site in the
Srglb3 promoter region was incubated with extracts prepared from the wild-type or
RIBBPl deficient strain, confirmed that the binding activity was abolished in the mutant
strain.

Transgenic L. corniculatus plants were inoculated with a two-day culture of NZP

2037 or NZP 2037-11. The bacterial cultures were pelleted and washed with sterile water

144

to remove residual medium and antibiotics. Three independent experiments were carried
out in sandy soil. A total of 36 plants were assayed for GUS activity and the data
analyzed using the non-parametric Mann-Whitney statistical test. In the mixed soil,
nodules from two experiments involving a total of 22 plants were assayed for GUS
activity and the data analyzed using the Mann-Whitney non-parametric test. No
statistically significant differences were detected in the two treatments. However, in
almost all cases, nodules harboring RIBBPI deficient mutants appear to have less activity.
From these GUS activity data, it appears that BBPs are not involved in Srglb3 gene
expression.

Since AcBBPl does not seem to play a role in Srglb3 gene expression, does this
bacterial protein have a function in the plant? It seems clear that the B38] in the 5’
upstream region of the Srglb3 gene may play a role in modulating leghemoglobin gene
expression. Welters et al. (1993) investigated the importance of the binding site (BBSl).
Insertional mutagenesis and deletion of the site reduces promoter activity. This suggests
that more than one factor can bind to this site in viva. Disruption of this site could affect
the binding of a trans-acting factor that has not yet been identified. Perhaps one of the
other two binding activities obtained by Welters et al. (1993) could produce the results
observed. The question that was addressed in this chapter investigated the interaction of a
DNA binding protein and its in vitro target within the Srglb3 promoter. In this case, it
appears that AcBBPl fortuitously binds to this region in vitro and does not appear to
affect leghem0globin expression. Although the hypothesis that bacterial proteins are

involved in Ib gene expression has not been proven, bacterial proteins could still play a

145

role in symbiotic plant gene expression. The recent identification of the bacterial protein
FixF, which contains functional NLSs is a candidate for interkingdom signaling in the
Rhizobium-legume plant symbiosis (Jabbouri et al., 1996). Although no targets have been
identified, mutations within the fixF gene lead to a Fix’ phenotype.

What role does RIBBPI and AcBBPl play in their respective bacterium? This is a
mystery. The BBP1 proteins have similarities to bacterial regulators, both activators and
repressors. The most logical guess is that the BBP1 proteins may be related to the C
proteins found in some type II restriction-modification (RM) systems. The features of
the BBP1 proteins are strikingly similar to the class of C proteins already identified. One
kb of DNA flanking the AcBBPl gene has been sequenced, but no matches have been
found in the databases. This suggests that the BBP1 proteins are novel and possess a
function which is still unknown. Alternatively, these proteins may have a function
homologous to the C proteins but were unable to be detected in the flanking sequences.
The organization of the locus may be different from the type 11 RM systems identified
thus far or similarities in the primary protein sequence are below the level to be scored as

significant in a database search.

 

146

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Cully DF, Garro AJ (1985) Nucleotide sequence of the imnmunity region of Bacillus
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Dhaese P, Seurinck J, De Smet B, Van Montagu M (1985) Nucleotide sequence and
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Dreyfus FL, Dommergues YR (1981) Nitrogen-fixing nodules induced by Rhizobium on
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Jefferson R (1987) Assaying chimeric genes in plants: The GUS gene fusion system.
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CHAPTERS

CONCLUSIONS

150

 

151

The observation that a bacterial protein, AcBBPl, from Azorhizobium
caulinodans binds to a region in the Sesbania rostrata glb3 leghemoglobin promoter
(Welters et al., 1993) was an intriguing finding. If proven biologically significant, this
observation would provide the first piece of direct evidence that bacterial proteins are
involved in leghemoglobin (lb) gene expression. Since the early 1970’s, many have
speculated that a signal from the symbiotic bacterium was necessary for the induction of
lb genes. The interaction between AcBBPl and its target in the Srglb3 promoter (BBSl)
was a nice system to investigate this idea. The goal of this thesis project was to test the
hypothesis that bacterial proteins are involved in lb gene expression. To begin the
investigation, the gene encoding AcBBPl was cloned. The deduced amino acid sequence
revealed two putative nuclear localization signals and two helix-tum-helix DNA binding
motifs in the central and C-terminal end. The AcBBPl protein shares sequence similarity
with a number of regulatory proteins. The best match was to a class of control proteins
found in some type II restriction-modification systems. To address the biological
significance of the AcBBPl protein in Srglb3 gene expression, a deletionfrnsertion mutant
lacking the ability to produce AcBBPl was constructed. Gel mobility shifi assays and
western blot analysis confirmed that the protein was not produced. The wild-type and
mutant A. caulinodans strain was inoculated onto the stems and roots of S. rostrata. The
nitrogen fixation ability of nodules induced by the wild-type and mutant bacteria was
determined using the acetylene reduction assay. On average, the nodules induced by the
AcBBPl deficient strain fixed about 20% less nitrogen than its wild-type counterpart.

Since a difference in nitrogen fixation was observed, total leghemoglobin mRNA steady

 

152

state levels were monitored. The expression levels of leghemoglobin in nodules harboring
wild-type or mutant bacteria were identical suggesting that the AcBBPl protein does not
significantly alter the total leghemoglobin levels in the nodules induced by the mutant
strain.

These observations led to the next phase of this thesis project, in which
experiments designed to further elucidate potential roles of the AcBBPl protein were
initiated. Transmission electron microscopy revealed that plant cells harboring wild-type
or mutant bacteria were at different stages of the infection process. The plant cells
harboring mutants were delayed in the infection process as more uninfected cells were
present in the nodule tissue. In addition, only one or a few bacteroids were encased by a
peribacteroid membrane while up to ten wild-type bacteroids were surrounded by this
membrane. Antibodies specific to AcBBPl were produced and used to localize this
protein to the bacteroid and peribacteroid membrane. This was not an unexpected result
as this protein is of bacterial origin. The AcBBPl protein was not found in the uninfected
cells or in the nucleus of infected cells. This suggests that the protein may not be directed
to the plant nucleus or at least can not be detected in this compartment.

It was previously shown that a homologous factor in R loti extracts could bind to
the BBSl in the 5’ upstream region of the Srglb3 gene (Welters et al., 1993). To directly
monitor what role the BBP1 proteins play in Srglb3 gene expression, two tools were
developed. First, transgenic Lotus corniculatus plants harboring the 5’ upstream region of
the Srglb3 gene fused to the reporter gene uicbl were made. Second, an R. loti mutant

deficient in the production of BBP1 was created. The AcBBPl homolog in R. loti

 

 

153

(RlBBPl) was cloned. The RIBBPI gene is 63% identical to AcBBPI at the nucleotide
level and the two proteins are 78% identical and 85% similar at the amino acid level. An
RlBBPl deficient mutant was constructed by insertional mutagenesis and was used to
nodulate the transgenic L. corniculatus plants harboring the chimeric promoter-reporter
gene construct. Nodules harboring wild-type or RIBBPl deficient bacteria were assayed
for GUS activity and no statistically significant differences were detected. Therefore, our
hypothesis that bacterial proteins are involved in lb gene expression can not be
substantiated at this time.

The investigation of this interesting phenomenon was undertaken and the goals set
forth were accomplished. The biological significance of the interaction between AcBBPl
and its target in the Srglb3 promoter was explored and it appears that this bacterial DNA
binding protein does not modulate Srglb3 gene expression. However, examples in other
plant-bacterial systems implicate that bacterial components (i.e. proteins or DNA) to be
necessary to obtain specific plant responses. As more knowledge is gained in this area,

the concept of transkingdom signaling may become more universal.

~

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