Download insect transmitted plant pathogenic mollicutes, spiroplasma kunkelii

INSECT TRANSMITTED PLANT PATHOGENIC MOLLICUTES,
SPIROPLASMA KUNKELII AND
ASTER YELLOWS WITCHES' BROOM PHYTOPLASMA:
FROM STRUCTURAL GENOMICS TO FUNCTIONAL GENOMICS
DISSERTATION
Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy
in the Graduate School of The Ohio State University
By
Xiaodong Bai, M.S.
*****
The Ohio State University
2004
Dissertation Committee:
Dr. Saskia A. Hogenhout, Adviser
Approved by
Dr. David L. Denlinger
Dr. David M. Francis
Dr. Parwinder S. Grewal
Adviser
Department of Entomology
ABSTRACT
The mollicutes, Spiroplasma kunkelii and aster yellows witches' broom (AY-WB)
phytoplasma, are insect-transmitted plant pathogens. These mollicutes invade and
replicate in cells of various insect organs and tissues, and inhabit and replicate in plant
phloem tissues. They cause severe symptoms to many plant species worldwide, including
economically important crops and ornamental plants. Their fastidious nature and lack of
genetic tools have hampered the research on these plant pathogenic mollicutes.
I employed various approaches, including genome sequencing, comparative
genomics, functional genomics, and conventional molecular techniques, to study the
biology and pathogenicity mechanisms of S. kunkelii and AY-WB phytoplasma. The
partial genome of S. kunkelii and the complete genome of AY-WB phytoplasma were
sequenced. Genome annotation revealed the presence of multiple spiroplasma phage
DNA sequences in S. kunkelii and many repetitive elements in both genomes, suggestive
of frequent recombination events. The genome sequence data provide genetic basis for
the study of the biology and pathogenicity mechanisms of these organisms.
Whereas spiroplasmas and phytoplasmas are distantly related to each other, they
share the plant and insect habitats. Therefore, they may share genes involved in insect
ii
transmission and plant pathogenicity that are missing from the animal and human
pathogenic mycoplasmas. To test this hypothesis, comparative genome analysis among
mollicutes was conducted, and resulted in the identification of four genes that are present
in the genomes of all plant-pathogenic mollicutes sequenced so far, but missing from the
mycoplasmas. Another gene within both genomes might have been derived by horizontal
gene transfer between spiroplasmas and phytoplasmas.
The observation of spiroplasma surface appendages prompted the search of genes
involved in fimbriae or pili formation. Four traE gene homologs were identified as
membrane-bound ATPases in S. kunkelii M2 strain. Two homologs were localized in S.
kunkelii chromosome and two in plasmids. The presence of these homologs varied among
S. kunkelii strains of different geographical locations. The expression of the genes was
detected in culture medium and during infection of insects and plants. Adjacent
sequences of traE homologs suggest the involvement of TraE in spiroplasma conjugation
and subsequent recombination, and adhesion.
The secreted proteins of AY-WB phytoplasma are likely to directly interact with
host cell components. Hence, the AY-WB phytoplasma genome sequence was mined for
potentially secreted proteins that were further characterized by high-throughput
functional assays such as virus-based expression in Nicotiana benthamiana (tobacco) and
Lycopersicon esculentum (tomato). The in planta assay resulted in the identification of 17
candidate effector proteins. The detailed functional characterization was focused on two
phytoplasma proteins (A11 and A30) that have a nuclear localization signal (NLS), and
therefore, may be imported into plant nuclei in an importin α-dependent manner. Plant
iii
localization study with the yellow fluorescent protein fusions of these two proteins
revealed their localization in the plant nuclei and confirmed their dependence on plant
importin α for nuclear transport. Transcripts corresponding to the phytoplasma proteins
were detected in AY-WB phytoplasma-infected insects and plants by RT-PCR.
Microarrays demonstrated that phytoplasma A11 protein affected the expression profiles
of 53 tomato genes, including several transcription factors, indicating that phytoplasma
A11 protein directly or indirectly interacts with these proteins. These data are supportive
of the hypothesis that A11 is a bona fide effector protein involved in plant pathogenicity.
In summary, the research described in this dissertation resulted in the
identification of several mollicute genes that are potentially involved in insect
transmission and plant pathogenicity. It demonstrated that the genome sequencing,
comparative genomics, and functional genomics approaches allow efficient identification
and characterization of such genes in bacterial genomes. The importance of the research
lies in the application of high throughput bioinformatics, genomics and molecular
approaches in the study of agriculturally important organisms for which little
information, and molecular and diagnosis/detection tools are available. The described
research and approaches might be useful for other pathogenic mollicutes that are
recalcitrant to in vitro manipulation, detection and characterization, including the
economically important mycoplasmas that impact human health and livestock industries.
iv
Dedicated to my parents, my brother and those I love
v
ACKNOWLEDGMENTS
I wish to thank my adviser, Dr. Saskia A. Hogenhout, for her intellectual support
and encouragement that made the whole research and this dissertation possible, and for
her continuous support of my career development.
I thank my Student Advisory Committee members, Dr. David L. Denlinger, Dr.
David M. Francis, and Dr. Parwinder S. Grewal, for their advice and support of my
graduate study.
I thank Dr. Sophien Kamoun for his brilliant ideas and continuous support of my
research and for the stimulating discussions.
I am grateful to those who helped me with various experiments and techniques
during my research, especially Mr. Ian Holford for computer programming, Dr. ElDesouky Ammar and Dr. Tea Meulia for electron and confocal microscopy, Dr. Michael
M. Goodin for protein localization in plants, Dr. David M. Francis and Ms. Jorunn Bos
for microarray data analysis, Mr. Valdir Ribeiro Correa, Ms. Diane M. Hartzler, Ms.
Angela D. Strock, Ms. Miaoying Tian and Ms. Diane M. Kinney for help with the PVX
assays, Dr. Thirumala Kanneganti for assistance with virus-induced gene silencing
experiments, Mr. Edgar Huitema, Mr. Mark W. Jones, and Dr. Margaret Redinbaugh for
isotope usage, Ms. Kristen J. Willie, Ms. Janet McCormick, and Dr. Juliette Hanson for
mouse antibody production.
vi
This research is supported by The Ohio State University – Ohio Agricultural
Research and Development Center (OARDC) Research Enhancement Competitive Grant
Program, Ohio Plant Biotechnology Consortium (OPBC) and the AY-WB phytoplasma
genome-sequencing project is supported by the United States Department of Agriculture /
National Science Foundation (USDA/NSF) Microbial Genome Sequencing Program.
vii
VITA
Oct. 15, 1974................................................ Born - Daqing, P. R. China
1992-1996.................................................... B.S. Department of Biological Science and
Technology, Zhejiang University,
Hangzhou, P. R. China
1996-1999.................................................... M.S. Institute of Zoology,
Chinese Academy of Sciences,
Beijing, P. R. China
1999-2000.................................................... Researcher, Qingdao Yongsheng
Guangyuan Corporation,
Qingdao, P. R. China
2000-present ................................................ Graduate Research Associate,
Department of Entomology,
The Ohio State University, OH, USA
HONORS AND AWARDS
•
Department Fellowship, Department of Entomology, The Ohio State University –
Agricultural Research and Development Center (OARDC), OH, U.S.A. 20032004
•
OARDC Director’s Fellowship, The Ohio State University, OH, U.S.A. 20002003
•
Research Grant from OARDC Research Enhancement Competitive Grant
Program, The Ohio State University, OH, U.S.A. 2003-2004
•
American Phytopathological Society Foundation The Raymond G. Grogan Travel
Award, Milwaukee, MI, U.S.A. 2002
•
Chinese Academy of Sciences Di’Ao scholarship, Beijing, P.R. China. 1999
viii
PUBLICATIONS
Research publications
1.
Xiaodong Bai, Tatiana Fazzolari, and Saskia A. Hogenhout. 2004. Identification
and Characterization of Spiroplasma kunkelii traE genes. Gene 336(1), 81-91.
2.
Xiaodong Bai, Jianhua Zhang, Ian R. Holford, and Saskia A. Hogenhout. 2004.
Comparative genomics identifies genes shared by distantly related insect-transmitted
plant pathogenic mollicutes. FEMS Microbiology Letters 235, 249-258.
3.
Wencai Yang, Xiaodong Bai, Eileen Kabelka, Christina Eaton, Sophien Kamoun,
Esther van der Knaap, and David Francis. 2004. Discovery of single nucleotide
polymorphisms in Lycopersicon esculentum and mapping of fruit color QTL in elite
populations. Molecular Breeding 14, 21-34.
4.
El-Desouky Ammar, Dave Fulton, Xiaodong Bai, Tea Meulia and Saskia A.
Hogenhout. 2003. An attachment tip and fimbriae-like structures in plant- and insectpathogenic spiroplasmas of the class Mollicutes. Archives of Microbiology 181(2), 97105.
5.
Xiaodong Bai and Saskia A. Hogenhout. 2002. A genome sequence survey of the
mollicute corn stunt spiroplasma Spiroplasma kunkelii. FEMS Microbiology Letters
210(1), 7-17.
6.
Qiang Liu, Yan Ye, Xiaodong Bai, and Cui Ding. 2001. Genetic localization of
the synergistic factor of Pseudaletia separata granulosis virus. Acta Entomologica Sinia
44(2), 148-154.
7.
Xiaodong Bai And Cui Ding. 2000. Primary study of synergistic mechanism of
Agrotis segetum nuclear polyhedrosis virus. Chinese Journal of Applied and
Environmental Biology 6(1), 52-55.
8.
Fumian Cui, Jiaji Shi, and Xiaodong Bai. 1998. Enzymatic synthesis of
Cephradine. Wei Sheng Wu Xue Bao 38(4), 300-303.
FIELDS OF STUDY
Major Field: Entomology
Specialties: Microbial Genomics, Plant-Microbe Interactions
ix
TABLE OF CONTENTS
Page
Abstract.......................................................................................................................... ii
Dedication...................................................................................................................... v
Acknowledgments ......................................................................................................... vi
Vita................................................................................................................................. viii
List of Tables ................................................................................................................. xiii
List of Figures................................................................................................................ xv
List of Abbreviations ..................................................................................................... xvii
Chapters:
1.
Insect transmitted plant pathogenic mollicutes: A literature review ................. 1
1.1
1.2
1.3
1.4
1.5
Introduction .............................................................................................. 1
Evolution and phylogeny ......................................................................... 3
Plant symptomology................................................................................. 5
Insect transmission ................................................................................... 7
Pathogenicity mechanisms ....................................................................... 11
1.5.1 Hormonal................................................................................. 11
1.5.2 Molecular................................................................................. 13
1.6 Structure ................................................................................................... 15
1.7 Movement................................................................................................. 18
1.8 Structural genomics.................................................................................. 20
1.9 Comparative genomics............................................................................. 23
1.10 Functional genomics ................................................................................ 26
1.11 Research objectives.................................................................................. 30
1.12 Reference.................................................................................................. 31
2.
A genome sequence survey of the mollicute corn stunt spiroplasma
Spiroplasma kunkelii ........................................................................................ 47
2.1
2.2
2.3
2.4
Abstract .................................................................................................... 48
Introduction .............................................................................................. 49
Materials and Methods............................................................................. 50
Results and Discussion............................................................................. 53
x
2.5
2.6
3.
Complete genome sequences of aster yellows witches' broom (AYWB) phytoplasma and comparison with onion yellows
phytoplasma ....................................................................................................... 76
3.1
3.2
3.3
3.4
3.5
3.6
3.7
4.
Abstract .................................................................................................... 77
Introduction .............................................................................................. 78
Materials and Methods............................................................................. 80
Results ...................................................................................................... 83
Discussion ................................................................................................ 95
Acknowledgments.................................................................................... 98
References ................................................................................................ 99
Identification and characterization of traE genes of Spiroplasma
kunkelii............................................................................................................... 118
4.1
4.2
4.3
4.4
4.5
4.6
4.7
5.
Acknowledgments.................................................................................... 63
References ................................................................................................ 63
Abstract .................................................................................................... 119
Introduction .............................................................................................. 120
Materials and Methods............................................................................. 121
Results ...................................................................................................... 126
Discussion ................................................................................................ 134
Acknowledgments.................................................................................... 137
References ................................................................................................ 138
Comparative genomics identifies genes shared by distantly related
insect-transmitted plant pathogenic mollicutes ................................................. 150
5.1
5.2
5.3
5.4
5.5
5.6
5.7
Abstract .................................................................................................... 151
Introduction .............................................................................................. 152
Materials and Methods............................................................................. 154
Results ...................................................................................................... 156
Discussion ................................................................................................ 161
Acknowledgments.................................................................................... 164
References ................................................................................................ 165
xi
6.
Functional genomics identifies phytoplasmas effector proteins ....................... 175
6.1
6.2
6.3
6.4
6.5
6.6
6.7
Abstract .................................................................................................... 176
Introduction .............................................................................................. 177
Materials and Methods............................................................................. 179
Results ...................................................................................................... 184
Discussion ................................................................................................ 190
Acknowledgments.................................................................................... 193
References ................................................................................................ 193
Bibliography .................................................................................................................. 206
xii
LIST OF TABLES
Table
Page
1.1
Summary of completed mollicute genomes ...................................................... 45
2.1
Sequence tags with significant similarity (E-value ≤ 10-5) to
spiroplasma virus SpV1 and S. citri putative virulence proteins ...................... 69
2.2
Sequence tags with significant similarities (E-value ≤ 10-5) to
NCBI nr protein sequences................................................................................ 70
2.3
S. kunkelii sequence tags with similarity to rRNA genes.................................. 75
3.1
General features of the chromosomes of the aster yellows witches’
broom (AY-WB) phytoplasma and Onion yellows (OY)
phytoplasma genomes........................................................................................ 105
3.2
Comparison of the COG categories of AY-WB phytoplasma with
those of other mollicutes.................................................................................... 106
3.3
Summary of ABC transporter genes in AY-WB and OY
phytoplasma genomes........................................................................................ 107
3.4
Summary of AY-WB phytoplasmas P-type ATPase......................................... 108
3.5
Summary of redundant (either complete or incomplete) genes in
AY-WB phytoplasma genome........................................................................... 109
3.6
Summary of the secreted proteins identified in AY-WB
phytoplasma genome ......................................................................................... 111
4.1
Basic features of traE genes in S. kunkelii M2 strain........................................ 142
5.1
Four AY-WB and S. kunkelii homologues that were absent from
mycoprotdb consisting of the whole genome sequences of M.
genitalium, M. pneumoniae, U. urealyticum, M. pulmonis, M.
penetrans, and M. gallisepticum........................................................................ 169
xiii
5.2
Identities of AY-WB and S. kunkelii proteins that are more similar
to each other than to proteins in mycoprotdb .................................................... 170
6.1
Summary of PVX assay of AY-WB phytoplasma candidate
effector proteins ................................................................................................. 197
6.2
Genes that were up-regulated in PVX:A11 treated tomato plants
comparing to PVX only treated tomato plants .................................................. 199
6.3
Genes that were down-regulated in PVX:A11 treated tomato plants
comparing to PVX only treated tomato plants .................................................. 200
xiv
LIST OF FIGURES
Figure
Page
1.1
Phylogeny of mollicutes based on 16S rDNA sequences ................................. 46
3.1
Maps of the AY-WB phytoplasma plasmids..................................................... 113
3.2
The summary of AY-WB phytoplasma genome encoded
transporters and central metabolic pathways..................................................... 114
3.3
AY-WB phytoplasma genome contains more repetitive sequences,
both tandem repeats and inverted repeats, than OY phytoplasma
genome............................................................................................................... 115
3.4
The AY-WB phytoplasma genome contained many copies of
transposases genes and derivatives.................................................................... 116
3.5
The AY-WB phytoplasma genome contained one copy of complete
ATP-dependent DNA helicase and multiple copies of pseudogenes ................ 117
4.1
ClustalW alignment of the deduced protein sequences of traE1,
traE2, traE3 and traE4 of S. kunkelii strain M2................................................ 144
4.2
Phylogenetic analyses of the TraE protein sequences from S.
kunkelii M2 strain and other organisms............................................................. 145
4.3
Detection of traE sequences by Southern blot hybridization of
digested genomic DNA of S. kunkelii strains M2, CS-2B, FL-80
and PU8-17 ........................................................................................................ 146
4.4
Detection of traE sequences on chromosomal and plasmid DNA of
S. kunkelii strains M2, CS-2B, and PU8-17 ...................................................... 147
4.5
Detection of S. kunkelii spiralin gene and traE transcripts on
Northern blots of size-separated total RNA samples ........................................ 148
xv
4.6
Genetic contexts of traE genes in the genome of S. kunkelii CR23x ....................................................................................................................... 149
5.1
Algorithms employed to extract proteins that are common between
the insect-transmitted plant pathogens Aster Yellows Witches’
Broom (AY-WB) and Spiroplasma kunkelii but are absent from
five Mycoplasma spp. and Ureaplasma urealyticum ........................................ 171
5.2
Graphical representation of comparative analysis results ................................. 172
5.3
Phylogenetic analyses of proteins that are present in insecttransmitted plant pathogenic AY-WB and S. kunkelii but absent
from animal and human pathogenic mycoplasmas............................................ 173
5.4
Phylogenetic analysis for AtA (AAA type ATPase)......................................... 174
6.1
Mining of AY-WB genome sequences for putative phytoplasma
effector proteins ................................................................................................. 201
6.2
Representative plant symptoms after toothpick inoculation of
Nicotiana benthamiana leaves with transformed Agrobacterium
tumefaciens GV3101 strain ............................................................................... 202
6.3
Laser-scanning confocal microscopy images demonstrating the
subcellular localization of YFP fusions of AY-WB proteins with
NLS upon agroinfiltration into N. benthamiana leaves .................................... 203
6.4
The transportation of YFP:A11 was dependent on N. benthamiana
(Nb) importin α.................................................................................................. 204
6.5
AY-WB candidate effector proteins A11- and A30-encoding genes
were expressed during AY-WB infection to aster plants .................................. 205
6.6
AY-WB phytoplasma candidate effector proteins A11 and A30
were expressed during the infection to insects .................................................. 205
xvi
ABBREVIATIONS
[α-32P]-dCTP, alpha phosphor-32
labeled deoxy-cytosine
trisphosphate
ABC, ATP binding cassette
AP, alkaline phosphatase
ATP, adenine trisphosphate
ATPase, adenine tri-phosphatase
AYP, aster yellows phytoplasmas
AY-WB, aster yellows witches' broom
BLAST, Basic Local Alignment Search
Tool
bp, base pairs
CBPP, contagious bovine
pleuropneumonia
CBF, cmp binding factor
CCPP, contagious caprine
pleuropneumonia
DNA, deoxyribonucleic acid
EB, ethidium bromide
EBI, European Bioinformatics Institute
EMBOSS, European Molecular Biology
Open Software Suite
EST, expressed sequence tag
GAMBIT, genomic analysis and
mapping by in vitro transposition
GC, guanine and cytosine
GFP, green fluorescence protein
GGPP, geranyl geranyl pyrophosphate
HMM, hidden Markov model
IAA, indole-3-acetic acid
IgG, immunoglobulin G
IPP, isopentenyl pyrophosphate
IVET, in vivo expression technology
kb, kilobases
MBSP, maize bushy stunt phytoplasma
MLO, mycoplasma-like organism
MRFV, Maize Rayado Fino Virus
MVA, Mevalonic acid
NCBI, National Center for
Biotechnology Information
NLS, nuclear localization signal
NN, neural network
ORF, open reading frame
OY, onion yellows
PAGE, polyacrylamide gel
electrophoresis
PAUP, phylogenetic analysis using
parsimony
PBS, phosphate-buffered saline
PCR, polymerase chain reaction
PDB, The Protein Data Bank
PFGE, pulsed field gel electrophoresis
PNPase, polynucleotide phosphorylase
RADAR, Rapid Automatic Detection
and Alignment of Repeats
RNA, ribonucleic acid
RT-PCR, reverse transcription polymerase chain reaction
SDS, sodium dodecyl sulfate
STM, signature-tagged mutagenesis
TRV, tobacco rattle virus
UDP, uridine diphosphate
VIGS, virus-induced gene silencing
YFP, yellow fluorescence protein
xvii
CHAPTER 1
Insect Transmitted Plant Pathogenic Mollicutes:
A Literature Review
1.1 Introduction
The discovery of plant pathogenic mollicutes was associated with the study of
plant diseases. Phytoplasmas were first detected in plants showing dwarfing and witches'
broom symptoms, including mulberry dwarf, potato witches' broom, aster yellows, or
paulownia witches' broom (Doi et al., 1967). Phytoplasmas were initially known as
'mycoplasma-like organisms' (MLOs) because they were similar in morphology and
ultrastructure to mycoplasmas (Doi et al., 1967). The first spiroplasma, Spiroplasma citri,
was described in 1973 (Saglio et al., 1973). Since then, many more spiroplasma and
phytoplasma species have been identified and the list of plants that they infect has been
continuously growing.
While all phytoplasma species are plant pathogens, only three spiroplasma species
have been so far identified as plant pathogens. These are the citrus stubborn agent S. citri
1
(Saglio et al., 1973), the corn stunt agent S. kunkelii (Whitcomb et al., 1986), and the
periwinkle pathogen S. phoeniceum (Saillard et al., 1987). All other spiroplasma species
are commensals, symbionts or pathogens of arthropods (Gasparich, 2002). From this
point on, plant pathogenic spiroplasmas and phytoplasmas are referred to as plant
pathogenic mollicutes.
Plant pathogenic mollicutes induce severe diseases in many plant species,
resulting in significant economic losses. Much is known about the transmission biology
and ecology of plant pathogenic mollicutes. However, more has to be learned about the
biology, physiology and pathogenicity mechanisms of plant pathogenic mollicutes,
especially phytoplasmas because of their uncultivable nature. Fortunately, genome
sequencing of plant pathogenic mollicutes (Bai and Hogenhout, 2002; Bai et al., 2004b;
Liefting and Kirkpatrick, 2003; Zhao et al., 2003, 2004a, 2004b; Oshima et al., 2004; Bai
et al., in preparation) and the development of genetic tools for spiroplasmas (Foissac et
al., 1997b; Gaurivaud et al., 2000a, 2000b, 2001; Lartigue et al., 2002) greatly facilitated
the study of these pathogens.
The purpose of this literature review is to present a comprehensive overview of
the biology of plant pathogenic mollicutes. It includes the following sections: 1)
Evolution and phylogeny; 2) Plant symptomology; 3) Insect transmission; 4)
Pathogenicity mechanisms; 5) Structure; 6) Movement; 7) Structural genomics; 8)
Comparative genomics; and 9) Functional genomics. It ends with the research objectives
and the summary of the organization of the dissertation.
2
1.2 Evolution and phylogeny
Spiroplasmas and phytoplasmas belong to the Class Mollicutes that is a group of
unique bacterial organisms, characterized by small genome sizes (580 – 2,200 kb), low
GC content (23% – 40%), and the lack of cell wall (Razin et al., 1998). Based on 16S
rDNA phylogeny, mollicutes were derived from a Gram-positive bacterial ancestor in the
Clostridium linkage (Woese, 1987; Weisburg et al., 1989). The mollicutes evolved from
the Gram-positive ancestor by degenerative or reductive evolution (Razin et al., 1998;
Oshima et al., 2004). Mollicutes are divided into two major branches (Fig. 1.1): The
AAA branch with the Asteroleplasma, Anaeroplasma, and Acholeplasma species the
phytoplasmas that appear most closely related to the acholeplasmas; and the SEM branch
with the Spiroplasma, Entomoplasma, Mesoplasma, Mycoplasma, and Ureaplasma
species (Maniloff, 1996; Razin et al., 1998). Unlike members of the AAA branch, all
members of the SEM branch treat UGA as a tryptophan rather than a stop codon
(Maniloff, 1996; Razin et al., 1998). The use of UGA as a tryptophan codon has
complicated in vitro protein expression of spiroplasmas and mycoplasmas.
Spiroplasma and phytoplasmas are distantly related within the Class Mollicutes.
Spiroplasmas are closer to mycoplasmas that are notorious animal and human pathogens.
Spiroplasmas were considered early mollicutes because they generally have larger
genomes than the mycoplasmas and phytoplasmas. Phytoplasmas were placed as a
distinct monophyletic clade within the Class Mollicutes (Lim and Sears, 1992). However,
because they cannot be cultured, they have not been assigned formal species names.
3
Trivia names, usually associated with the diseases they cause, have been used to describe
the phytoplasma species. Until recently, the IRPCM (International Research Programme
on Comparative Mycoplasmology) Phytoplasma/Spiroplasma Working Team –
Phytoplasma Taxonomy Group proposed to accommodate phytoplasmas within the novel
genus 'Candidatus (Ca.) Phytoplasmas' (IRPCM Phytoplasma/Spiroplasma Working
Team – Phytoplasma Taxonomy Group, 2004). The team also assigned 'Ca. Phytoplasma'
names to different phytoplasma groups. For instance, the aster yellows phytoplasma
group is now 'Ca. phytoplasma asteris' (Lee et al., 2004).
The phytoplasma phylogeny based on 16S rRNA sequences is widely used and
mostly accepted as the standard (Razin et al., 1998). Additional sequence information,
including the conserved ribosomal protein genes (Gundersen et al., 1996), the elongation
factor EF-Tu (tuf) gene (Kamla et al., 1996; Schneider et al., 1997), the heat shock
protein gene hsp70 (Falah and Gupta, 1997), and the 16S/23S rRNA intergenic sequences
(Smart et al., 1996), have confirmed the phytoplasma phylogeny based on 16S rDNA
sequences.
It is worth mentioning the unique taxonomic status of the Mycoplasma mycoides
cluster within the Class Mollicutes. The M. mycoides cluster consists of six closelyrelated species, two of which are of particular importance, M. mycoides subsp. mycoides
SC causing contagious bovine pleuropneumonia (CBPP) and M. capricolum subsp.
capripneumoniae causing contagious caprine pleuropneumonia (CCPP) (Thiaucourt et
al., 2000). Based on the 16S rRNA phylogeny, the mycoplasma species in the M.
mycoides cluster were closer to spiroplasmas than to the mycoplasma species in hominis
4
and pneumoniae groups (Weisburg et al., 1989). Recently, the M. mycoides cluster was
shown to arise from spiroplasmas through an intermediate group of non-helical
spiroplasmal descendants, the entomoplasmas (Gasparich et al., 2004). This suggested
that the genus name of Mycoplasma is no longer suitable for the species within M.
mycoides cluster. However, because of the practical important of the species within this
group, the reclassification would have immense practical implications in diagnostic
human and veterinary medicine (Razin et al., 1998; Gasparich et al., 2004). Therefore,
the current phylogenetic classification remained. The phylogenetic status of M. mycoides
cluster has to be taken into account in the comparative genomic sequence analysis of M.
mycoides subsp. mycoides SC (small colony) complete genome (Westberg et al., 2004)
with S. kunkelii (http://www.genome.ou.edu/spiro.html) and aster yellows witches' broom
(AY-WB) phytoplasma gapped genome described in the dissertation (Chapter 5).
1.3 Plant symptomology
Plant pathogenic mollicutes have distinct plant host ranges. Spiroplasmas have a
relatively narrow or restricted plant host range. They naturally infect several plant species
including citrus, corn and periwinkle. On the other hand, phytoplasmas have a very wide
plant host range, including more than 700 plant species worldwide. The plants susceptible
to phytoplasmas include economically important vegetable crops, such as lettuce, carrot,
and celery, and ornamental plants, such as China aster and purple coneflower.
Interestingly, S. kunkelii and maize bushy stunt phytoplasma (MBSP) share the same
5
insect and plant host ranges. In fact, these two organisms, along with Maize Rayado Fino
Virus (MRFV), were often found together in diseased maize plants and hence were called
the corn stunt complex (Henriquez et al., 1996), which was transmitted by the leafhopper
species of the genus Dalbulus (Henriquez et al., 1996; Hruska and Gomez-Peralta, 1997).
Plant pathogenic mollicutes are mainly restricted to phloem tissues of plants. The
rich content of glucose and fructose within the sieve tube of the phloem tissues provides
the energy supply for plant pathogenic mollicutes (André et al., 2003). But it is also a
great challenge for these organisms as the high concentrations of sugars in phloem sap
might cause osmotic stresses to the bacteria (Purcell and Nault, 1991). Studies of S. citri
indicated that the pathogenic organisms multiply in plant hosts and translocate toward
meristems, storage organs, fruits, and other parts of plants involved in photosynthesis
(Gussie et al., 1995). Plant pathogenic mollicutes replicate within the phloem tissues to a
high titer.
Plant pathogenic mollicutes induce severe symptoms of their plant hosts. Plant
pathogenic spiroplasmas induce chlorosis, stunting with shortened length of internodes,
proliferation of ears that do not mature, and reddening. In contrast, phytoplasmas cause
more varied and severe symptoms. For instance, members of the aster yellows
phytoplasma (AYP) group, the largest group in the genus Candidatus Phytoplasma,
induce stunting and twisting, reddened or yellowish foliage, and sterile plants
(Kirkpatrick, 1989). Floral parts that are normally brightly colored may remain green,
and petals and sepals may become puckered and distorted. Secondary flower heads may
emerge from the primary flower head, become leafy, and change in color. Losses of
6
certain ornamental plantings may range from 10 to 70%. Members of the AYP group
cause dramatic yield losses of lettuce, ranging from 60 to 80% in leaf lettuces growing
area in Ohio in a certain year (Hoy et al., 1992). Symptoms caused by AYP in lettuce
include premature bolting, elongation of internodes and petioles, vein clearing,
development of axiliary shoots (witches' broom), phyllody (transformation of flowers
into leaves), virescence (greening of normally white tissue), chlorosis, necrosis, and
rosetting (Murral, 1994; Lee et al., 2000). Symptoms may vary depending on the strains,
time of infection, plant species, temperature, age and size of the plant host. Plants cannot
be cured once infected with plant pathogenic mollicutes.
1.4 Insect transmission
Plant pathogenic mollicutes are all transmitted by insect vectors, mainly phloemfeeding leafhoppers of the family Cicadellidae, planthoppers of the family Cixiidae, and
psyllids of the family Psylloidea (Harris, 1979; Tsai, 1979). Most spiroplasma species are
associated with insects but are not plant pathogens, leading to the hypothesis that plant
pathogenic mollicutes evolved from insect-inhabiting organisms (Seemüller et al., 2002).
Feeding of the infested insects on plants resultd in the transmission of the mollicutes to
plants and the mollicutes might have become plant pathogens later in evolution (Hackett
and Clark, 1989). This hypothesis seems to be supported by the fact that some insect
pathogenic spiroplasmas, such as honeybee pathogens, S. apis and S. melliferum, have a
transmission route via plant nectar (Hackett and Clark, 1989).
7
Plant pathogenic mollicutes are transmitted by insect vectors in a persistent
propagative manner (Nault, 1997; Purcell, 1982). The insects acquire the plant
pathogenic mollicute from the phloem tissue when feeding on diseased plants. The plant
pathogenic mollicutes penetrate the cell wall of the midgut portion of the intestinal tract
of the insect, and thereafter, move into and multiply in the insect hemolymph and various
insect organs. They can infect the Malpighian tubules, muscle cells and nerve cells. The
mollicutes also invade the salivary gland cells, and are introduced, with the saliva, into
the phloem tissues of healthy plants during feeding (Seemüller et al., 2002). The infection
cycle of plant pathogenic mollicutes involves insects, plants, and bacteria, which provides
an interesting model system to study the interaction and co-evolution of these three
organisms.
The interaction of the mollicute with the insects seems highly specific (Seemüller
et al., 2002). For transmission to occur, the plant pathogenic mollicutes have to penetrate
the gut and salivary gland barriers in insects. Failure to penetrate either of the barriers
resulted in failure of the entire transmission process (Fletcher et al., 1998; Foissac et al.,
1997b; Yu et al., 2000). Although the molecular mechanism of insect transmission is not
yet understood, there is evidence of the involvement of specific spiroplasma attachment
structures. S. kunkelii apparently forms fimbriae- and pili-like structures that may be
involved in attachment and virulence in insects (Özbek et al., 2003; Ammar et al., 2004).
Further, S. kunkelii have tip structures that may be important for penetration of epithelial
cells in the midgut of the leafhopper Dalbulus elimatus, as the tip structures are
associated with cup-shaped invaginations of midgut cell cells (Ammar et al., 2004).
8
Molecular and biochemical studies of S. citri resulted in the identification of an
attachment protein, P89, that was associated with attachment to insect cells of S. citri
(Fletcher et al., 1998; Yu et al., 2000). It was later renamed as SARP1 and characterized
to have a novel domain designated sarpin (Berg et al., 2001). A homolog of SARP1 in S.
kunkelii was also identified by sequence similarity searches (Bai et al., 2004a). Recently,
a solute-binding protein of an ABC transporter from S. citri was demonstrated to be
involved in insect transmission by Tn4001 transposon mutagenesis and functional
complementation (Boutareaud et al., 2004). This putative lipoprotein, Sc76, belongs to
the ABC transport family of S1_b in the ABCdb database. Deletion of the gene resulted
in a 30-fold reduction of the transmission efficiency by the leafhopper vector (Boutareaud
et al., 2004). It remains unclear which stage of the insect transmission the protein is
involved in.
Most plant pathogenic mollicutes are not transmissible transovarially (Razin et al.,
1998). However, recent PCR and electron microscopy studies revealed the presence of
AY-type phytoplasmas in the genital organs or eggs of the leafhoppers of Scaphoideus
titanus and Hishimonoides sellatiformis (Alma et al., 1997; Kawakita et al., 2000). There
has not been any indication of transovarial transmission of plant pathogenic spiroplasmas.
However, several other Spiroplasma spp., such as S. poulsonii, are transmitted to the next
generation of Drosophila and pea aphid, Acyrthosiphon pisum (Bové, 1997; Fukatsu et al.,
2001; Anbutsu and Fukatsu, 2003).
Plant pathogenic mollicutes can affect their insect vector in various ways. Xdisease phytoplasmas can reduce the lifespan of the infected leafhopper Colladonus
9
montanus to a half (Jensen, 1959). MBSP infected leafhopper species produced fewer
offsprings than healthy ones (Nault et al., 1984). Several Dalbulus species that were poor
vectors of the corn stunt agent S. kunkelii were affected adversely by S. kunkelii infection
(Madden and Nault, 1983). On the other hand, S. kunkelii is not pathogenic to its primary
vector, Dalbulus maidis. In fact, the infection of S. kunkelii improved the overwintering
ability of D. maidis (Ebbert and Nault, 1994). Another beneficial effect was observed
during the interaction of the aster leafhopper, Macrosteles quadrilineatus, and AY
phytoplasmas. The leafhoppers exposed to AY phytoplasma-infected plants lived longer
and laid more eggs than non-exposed leafhoppers (Beanland et al., 2000). Also, AY
phytoplasma infection of D. maidis increased its survival rate on a non-host plant, aster
(Purcell, 1988). These mutual beneficial effects can be explained by the prolonged
association and co-evolution of the pathogen and the insect vector (Beanland et al., 2000),
and such a relationship aids the dispersion and survival of both the plant pathogenic
mollicutes and the insect vectors.
The nature of the insect vectors directly affects the plant host range of plant
pathogenic mollicutes (Seemüller et al., 2002). C. tenellus and D. maidis are effective
vectors that can transmit both spiroplasmas and phytoplasmas (Chiykowski and Sinha
1990). The AY phytoplasmas can be transmitted by more than 30 polyphagous
leafhopper vectors to more than 300 plant species belonging to 45 families (Lee et al.,
2000). Thus, the AY phytoplasmas have low vector specificity.
10
1.5 Pathogenicity mechanisms
The lack of genetic tools and the fastidious nature of plant pathogenic mollicutes
have hampered the research progress. S. citri, the type species of the genus Spiroplasma,
has a shorter generation time than S. kunkelii, and became the primary focus of the
research on spiroplasma pathogenicity. Research on molecular pathogenicity of the
uncultivable phytoplasmas becomes possible only following the recent accumulation of
genome sequence data. The attachment, penetration and multiplication during insect
transmission and possible involvement of mollicute structures in pathogenicity are
discussed in "insect transmission" and "structure" sections, respectively. This section
focuses on the mechanisms of plant pathogenic mollicutes causing diseases to plant hosts.
1.5.1 Hormonal
In previous studies, certain factors were reported to be associated with the
development of diseases caused by plant pathogenic mollicutes (Daniels, 1983; Gabridge
et al., 1985). Factors associated with phytoplasma infection include the following:
impairment of phloem function, alteration of turnover rates of hormones, low level of
indole-3-acetic acid (IAA) oxidase activity, increase or depletion of hormone precursors,
presence of inhibitors of hormone synthesis, transport or translocation, and selective
uptake and transport of hormones (Daniels, 1979). For S. citri, the factors are: toxins with
molecular weights up to 400 (Daniels, 1979), accumulation of lactic acid (Saglio et al.,
1973), and proteolysis and arginine aminopeptidase enzymatic activity (Chang, 1998).
11
Furthermore, imbalances in hormone levels have been proposed to contribute to plant
symptoms (Chang and Lee, 1995). Some attempts have been made to evaluate the
contribution of mollicute interference with plant hormone levels to mollicute
pathogenicity (Chang, 1998). The interesting findings shed light on the pathogenicity of
plant pathogenic mollicutes.
Phytoplasmas infection may alter endogenous auxin levels in plants. The
biosynthesis of auxin is centered in young leaves and shoot tips, and the auxin product
are transported in the phloem. Phytoplasmas infection in phloem may block the transport
of auxin and reduce the concentration of the endogenous auxin (Chang, 1998).
Chlorosis in leaves of spiroplasmas- and phytoplasmas-infected plants is due to
the pigment alterations. In the infected plants, chlorophyll a, chlorophyll b, and total
chlorophyll contents are significantly lower than those in the healthy plants. The
yellowing of leaves begins with the older leaves, suggesting that the reduction in
chlorophyll is due to the destruction of these pigments in mature chloroplasts. Also in
leaf tissues, plant pathogenic mollicutes infection causes dramatic decreases of
carotenoid and anthocyanin levels several weeks after the infection (Chang, 1998).
Mevalonic acid (MVA), isopentenyl pyrophosphate (IPP), geranyl pyrophosphate,
and farnesyl pyrophosphate are key intermediates in biosynthesis of sterol. Spiroplasmas
in plants depend on plant sterols for growth (Chang, 1989). Thus, spiroplasma and
phytoplasma infection results in more consumption of these materials than in healthy
plants. IPP is also a precursor for biosynthesis of cytokinin. As a result, the competition
for IPP would upset the balance of cytokinin in infected plants. MVA is a precursor for
12
both IAA and IPP. The constant demand for IPP for sterols would also change the
balance of IAA (Chang, 1998).
MVA, IPP, and geranyl geranyl pyrophosphate (GGPP) are intermediates in
biosynthesis of carotenoids, gibberellins and chlorophyll. Since IPP is used for synthesis
of sterols for the growth of plant, spiroplasmas and phytoplasmas, there could a shortage
of IPP for cytokinin, gibberellin, chlorophyll and carotenoid biosynthesis in infected
plants (Chang, 1998).
1.5.2 Molecular
The development of the genetic tools, such as Tn4001 mutagenesis (Foissac et al.,
1997b) and pBOT1 plasmid transformation of S. citri (Renaudin et al., 1995), has greatly
facilitated the discovery of pathogenicity-related genes (Renaudin, 2002) and has resulted
in the discovery of the involvement of the fructose operon in spiroplasma pathogenicity.
The random Tn4001 insertion in S. citri genome produced a mutant GMT553, showing a
delay in the symptom appearance (Foissac et al., 1997b). Subsequent analysis localized
the Tn4001 insertion at the 5' end of the first gene of the fructose operon, fruR
(Gaurivaud et al., 2000b), which encoded an activator protein of the fructose operon
(Gaurivaud et al., 2001). Further characterization showed that the first three genes of the
fructose operon, fruR, fruA, and fruK, were all disrupted in the mutant GMT533 and the
mutant was unable to utilize fructose as a carbon or energy source (Gaurivaud et al.,
2000b). The pathogenicity and the fructose utilization were restored by pBOT-derived
plasmids carrying certain combinations of the three genes (Gaurivaud et al., 2000a).
13
However, the original mutant GMT553 could naturally revert to the wild-type phenotype
after several generations (Foissac et al., 1997b). Later, new and more stable mutants were
obtained, showing phenotypes similar to GMT553 (Gaurivaud et al., 2000a).
A hypothesis was proposed to explain the involvement of fructose utilization in
pathogenicity (Bové et al., 2003). Fructose is not abundant in plant phloem tissue. But it
is needed, along with UDP-glucose, by the companion cell for loading sucrose, the major
soluble carbohydrate in plants. Spiroplasmas utilized fructose, thus competing with the
companion cells for fructose. The depletion of fructose results in less active companion
cells, which in turn causes modified distribution of photo-biosynthesis products,
accumulation of carbohydrates in "source" leaves, and depletion of carbohydrates in
"sink" tissues (Bové et al., 2003). Consequently, the low level of carbohydrates in "sink"
tissues leads to growth impairment, whereas the high level of carbohydrates in "source"
tissues leads to chlorosis (Geigenberger et al., 1996). This hypothesis was supported by
some observations (Braun and Sinclair, 1978; Catlin et al., 1975; Lepka et al., 1999),
resulting in a novel mechanism in which sugar metabolism of pathogens interferes with
plant physiology (Bové et al., 2003). However, other mechanisms could also be involved
since fructose operon-disrupted S. citri was still pathogenic (Bové et al., 2003). In light of
these pioneered research on spiroplasmas, the attempt of elucidating phytoplasma
pathogenicity mechanisms and the availability of genome sequences of phytoplasmas
prompted the functional genomics research described in the "Functional genomics"
section in this introduction, and in more details, Chapter 6 "Functional genomics identify
phytoplasma effector proteins".
14
1.6 Structure
Plant pathogenic mollicutes are unique bacteria that do not have cell wall, which
make them naturally resistant to antibiotics inhibiting bacteria cell wall synthesis.
Spiroplasmas are also not sensitive to another antibiotic, rifampicin (Chastel and
Humphery-Smith, 1991).
Spiroplasmas have a unique helical cell shape, as implied by the name 'spiro-'.
Before spiroplasmas could be cultured in artificial media, they were thought to be
spirochetes because of their morphological resemblance. In culture media, spiroplasmas
assume a helical morphology, while in insect hosts, spiroplasma morphology may vary
from oval, spherical to helical forms (Özbek et al., 2003). The mreB gene, which has a
demonstrated role in rod shape determination of Escherichia coli (Doi et al., 1988), was
also identified in S. citri (Bové et al., 2003) and S. kunkelii (Bai et al., 2004a). The mreB
genes are also present in filamentous and helical bacteria, but not in round, spherical
bacteria or the pleiomorphic mollicutes (Bové et al., 2003). The encoded MreB proteins
are components of prokaryotic forms of actin filaments and form filamentous helical
cytoskeleton-like structures lying close to the cell surface, involved in cell-shape
determination (Jones et al., 2001; van den Ent et al., 2001).
In most, if not all spiroplasmas, spiralin is the most abundant membrane protein
and the major surface antigen (Bové et al., 2003). The nucleotide sequences of the
spiralin genes were sequenced in several spiroplasma species (Chevalier et al., 1990;
Foissac et al., 1997a). The deduced spiralin proteins contain a general amphiphilic
15
character and possess a conserved lipoprotein signal peptide (Foissac et al., 1997a; Bové
et al., 2003), suggesting the extracellular localization of these proteins. A 'carpet model'
has been proposed to explain the spiralin organization at the spiroplasmas cell surface. In
this model, spiralin exhibits two colinear domains and anchors into the outer side of the
lipid bilayer with the N-terminal lipid moiety (Castano et al., 2002). However, the
extracellular localization of spiralin has not been demonstrated yet.
Because of their surface localization, spiralin proteins might play a role in the
interaction with plant hosts and/or insect vectors. Recently, a successful trial introduced
the translation fusion of spiralin and GFP (green fluorescent protein) into S. citri using an
oriC-based targeting vector, pC55 (Lartigue et al., 2002; Duret et al., 2003). The plasmid,
containing the fusion protein, integrated into S. citri chromosome by a single-crossover
recombination at the spiralin gene. Consequently, spiralin-GFP fusion protein would be
produced and fluoresce. One mutant with disrupted spiralin gene expression could still
multiply to a high titer in plants and produce the typical symptoms. However, the
transmission efficiency of this mutant was 100 times lower than wild type (Duret et al.,
2003). Thus, the involvement of spiralin in pathogenicity was excluded. Spiralin is
needed for insect transmission of spiroplasmas; however, the precise mechanism remains
to be determined (Duret et al., 2003).
During the insect transmission process, fimbriae- and pili-like structures (Özbek
et al., 2003) and "tip structure" (Ammar et al., 2004) were observed within insect cells
and gut lumen, respectively. In order to identify the proteins responsible for the formation
of the structures, a study was performed focusing on homologs of transfer proteins (Bai et
16
al., 2004a). Four traE homologs potentially involved in bacteria conjugation were
identified from S. kunkelii CR2-3x gapped genome obtained from a public domain. The
same homologs from S. kunkelii M2 strain were cloned and sequenced, showing a 100%
match to the sequences from the CR2-3x strain. In silico studies revealed multiple
features of these traE homologs, including the presence of transmembrane domains,
ATPase domains, etc. The presence of these sequences in different strains from different
geographical locations showed variations according to the geographical isolation. One of
the four homologs was expressed during infection of insects and plants, and two
homologs appeared to have shorter transcripts than the predicted open reading frames
(ORFs). It was suspected that the shorter transcripts might have regulatory functions.
Using pulsed field gel electrophoresis (PFGE) and Southern blot hybridization, two of the
homologs were localized on the chromosome and the other two on plasmids.
Interestingly, all of the ORFs of the four traE homologs localized in a region of important
genes in the genome of the CR2-3x strain. The traE2 ORF (2.5 kb) had a transcript of
approximately 10 kb, suggesting that it is part of an operon, including mreB, the cell
shape determination gene (Jones et al., 2001; van den Ent et al., 2001). The traE3 and
traE4 genes are adjacent to the p89 gene, which is involved in attachment (Fletcher et al.,
1998; Yu et al., 2000; Berg et al., 2001). This work provides the basic knowledge for
further research to determine whether the traE genes are involved in adhesion and/or
conjugation (Bai et al., 2004a).
17
1.7 Movement
Spiroplasmas have a unique style of movement. Spiroplasmas do not have
flagella, but have internal cytoskeletons and are motile (Trachtenberg, 2004).
Spiroplasma internal cytoskeleton is a flat and membrane-bound ribbon composed of
parallel fibrils. The fibril ribbon binds to the inner side of the spiroplasma cytoplasmic
membrane, follows the shortest helical line and extends the entire length of the helix
(Charbonneau and Ghiorse, 1984; Trachtenberg et al., 2003; Williamson et al., 1984).
The structural unit of the contractile cytoskeleton is a filament comprised of pairs of a
59kDa fib (fibril) gene product (Trachtenberg, 2004; Williamson et al., 1991), while the
functional unit of the contractile cytoskeletal ribbon is a fibril comprised of an aligned
pair of filaments (Trachtenberg, 2004). The internal cytoskeletons act as a linear motor
enabling and controlling the dynamic helicity (Trachtenberg et al., 2003). The dynamics
of the elastic fibril filaments, coupled with energy producing biochemical reactions, such
as ATP hydrolysis, propagates deformations that will generate propulsive forces to drive
the swimming movement of the helical spiroplasmas (Berg, 2002; Gilad et al., 2003;
Wolgemuth et al., 2003). In addition to the fib gene, another gene from S. citri, scm1, is
involved in the motility mechanism (Jacob et al., 1997). The scm1-disrupted mutant
generated by Tn4001 insertion mutagenesis form non-diffuse, sharp-edged colonies in
contrast to fuzzy colonies of the wild type bacteria, indicating the loss of motility (Jacob
et al., 1997). The two spiroplasma cytoskeletal genes, fib and scm1, have no homologs in
18
eukaryotes and other prokaryotes (Trachtenberg, 2004), which adds more weight to the
uniqueness of spiroplasmas.
Usually, spiroplasmas exhibit a random walking motility pattern with a relatively
constant flexing frequency (Trachtenberg, 1998), which was referred to as "swimming".
The swimming velocity increases with medium viscosity (Daniels et al., 1980). In the
presence of certain attractive chemicals, spiroplasmas, as active swimmers, exhibit a
straight-line pattern with a concomitant reduction in flexing frequency (Trachtenberg,
1998). The attractants include D-fructose, D-glucose, D-maltose, sucrose, L-alanine, Laspartate, L-arginine, L-cysteine, L-glutamate, glycine, L-methionine, L-serine, etc.
Certain chemicals repel spiroplasmas, including L-histidine, L-leucine, L-phenylalanine,
L-proline, L-valine and lactic acid (Daniels et al., 1980; Trachtenberg, 1998).
In contrast to the swimming movement of spiroplasmas, their close relatives,
mycoplasmas, have a distinct gliding movement (Miyata et al., 2002; Wolgemuth et al.,
2003). Mycoplasmas are polar cells and are near spherical in shape, having a "tip
structure" at the leading end (Trachtenberg, 1998). Several mycoplasma species are
known to glide in the direction of the "tip-structure". The underlying mechanism was
unknown until the recent identification of a Gli349 protein responsible for the
cytadherence and glass binding of M. mobile (Uenoyama et al., 2004). A spike structure
was observed to protrude from mycoplasma membrane and attach to the glass surface
using a rapid-freeze-and-fracture electron microscopy technique during M. mobile gliding
(Miyata and Petersen, 2004). However, it is not clear whether Gli349 is related to the
formation of the spike structure. The gliding movement of mycoplasmas is usually slow,
19
but the clear attraction to chemicals was observed. Mycoplasmas are attracted to Dfructose, D-glucose, D-lactose, D-maltose, D-sucrose, L-arginine, and L-asparagine
(Kirchhoff, 1992).
1.8 Structural genomics
The release of the first complete genome sequences of Haemophilus influenzae
(Fleischmann et al., 1995) started a brand-new genomics era, in which whole genome
sequencing, comparative genomics, and functional genomics gradually overshadowed the
traditional research methods focusing on one or several genes. The complete microbial
genome sequence provides all genetic information about the microbe, enables highthroughput data-mining and analysis, forms the basis for bacterial phylogeny and
taxonomy, and overall, bring the microbiological research to a new high level.
Bacterial genome sequence data has been accumulating at a fast pace. The first
large-scale genome-sequencing project, initiated in 1990 by the Harvard Genome Lab in
collaboration with Heidelberg European Molecular Biology Laboratory, sequenced only
214 kb of the M. capricolum genome (Bork et al., 1995) during a 5-year period.
Nowadays, with the maturity of the whole genome shotgun (WGS) sequencing strategy
and large-scale collaboration and data-handling abilities, bacterial genome sequencing is
becoming routine.
Owing to their small genomes and clinical and agricultural importance, the
mollicutes were among the first organisms whose complete genomes were sequenced.
20
The complete genome sequence of Mycoplasma genitalium was the second released
complete genome (Fraser et al., 1995). Until August 2004, 10 mollicute genomes have
been completely sequenced (Table 1.1), spanning the genera of Mycoplasma,
Ureaplasma, Mesoplasma and Candidatus Phytoplasma.
The accumulation of mollicute genome data continues with several on-going
projects. Mycoplasma genome projects include the rodent polyarthritis pathogen M.
arthritidis and the contagious caprine pleuropneumonia (CCPP) pathogen M. capricolum.
Spiroplasmas genome projects include the citrus stubborn spiroplasma S. citri BR3-3x
strain and the corn stunt spiroplasma S. kunkelii CR2-3x strain
(http://www.genome.ou.edu/spiro.html). Survey sequencing has been published for S.
kunkelii M2 strain, a close relative of CR2-3x strain (Bai and Hogenhout, 2002) and an
85-kb genome region was reported for S. kunkelii CR2-3x strain (Zhao et al., 2003). The
proposed genome sequencing project for another mollicute, Spiroplasma melliferum, is
expected to provide information for comparative genomics of this bee pathogen with
plant pathogenic spiroplasmas.
The application of pulsed field gel electrophoresis (PFGE) in DNA separation
revealed a few interesting phytoplasma genome features. The sizes of phytoplasma
genomes vary considerably, ranging from 530 to 1,350 kb (Neimark and Kirkpatrick,
1993; Marcone et al., 1999), which are close to those of mycoplasmas species (580-1,300
kb) but smaller than the closest relatives of acholeplasmas (~ 1,600 kb) (Razin et al.,
1998). The Bermuda grass white leaf phytoplasma has a genome size of 530 kb, which is
even smaller than the genome size (580 kb) of M. genitalium that was thought to be the
21
living cell harboring the smallest genome (Mushegian and Koonin, 1996). Phytoplasmas
contain one circular double-stranded chromosomal DNA molecule (Neimark and
Kirkpatrick, 1993) and one or more short circular extrachromosomal DNAs (Lee et al.,
2000). The GC contents of phytoplasma chromosomal DNA were estimated to be
between 23 and 29% based on buoyant density centrifugation (Kollar and Seemüller,
1989) and recently obtained genome sequence data (Oshima et al., 2002; Oshima et al.,
2004). Physical maps of genomic DNA have been reported for Western X-disease
phytoplasma (Firrao et al., 1996), apple proliferation phytoplasma (Lauer and Seemüller,
2000), sweet potato little leaf phytoplasma (Marcone and Seemüller, 2001) and European
stone fruit yellows phytoplasma (Padovan et al., 2000).
Much progress has been made in phytoplasma genome sequencing efforts. So far,
complete genome sequence has been reported for OY phytoplasma (Oshima et al., 2002;
Oshima et al., 2004). Sample genome sequences have been reported for Western X (WX)
phytoplasma (Liefting and Kirkpatrick, 2003) and a complete genome-sequencing project
of WX phytoplasma is underway. Genome sequencing projects are currently ongoing for
three other phytoplasma species, aster yellows witches' broom (AY-WB) phytoplasma
(http://www.oardc.ohio-state.edu/phytoplasma), MBSP, and beet leafhopper transmitted
virescence agent (BLTVA). The complete AY-WB phytoplasma genome sequence is
available and the final annotation is underway. There are also several phytoplasmas
whose genome sequencing projects are on the priority list of the American
Phytopathological Society, including clover phyllody (CPh) phytoplasma, elm yellows
(EY) phytoplasma, and potato witches' broom (PWB) phytoplasma. These sequencing
22
projects are expected to begin soon. The genome analysis of phytoplasmas should
provide more information on genes, which is important in understanding the
pathogenicity to plant hosts and reproduction in both insect vector and plant host cells.
1.9 Comparative genomics
Comparative genomics provides an opportunity to identify the common functional
contents of genomics and study the evolutionary relationships between two or more
organisms. The whole genome comparison was first done between the first two
completely sequenced bacteria M. genitalium (Fraser et al., 1995) and H. influenzae
(Fleischmann et al., 1995), which resulted in the identification of the minimal gene
complement of a free-living cell (Mushegian and Koonin, 1996). The concept that
mycoplasmas are the smallest living cell appeared in an article published in Scientific
America in 1962 (Morowitz and Tourtellotte, 1962). The validation of this concept with
identified genes is only possible by comparative genomics. H. influenzae is a gramnegative bacterium with a 1.8 Mb genome (Fleischmann et al., 1995) and M. genitalium
is a gram-positive bacterium with a genome of 580 kb, the smallest genome sequenced so
far (Fraser et al., 1995). The genes conserved in these two very distantly related bacteria
are essential for cellular functions. The comparison resulted in 240 ORFs that are
conserved in both genomes. Considering the sequence difference of genes having the
same functions and the functional redundancy, a final set of 256 ORFs was considered
the minimal set of genes essential for a free-living cell (Mushegian and Koonin, 1996).
23
However, the living environment of the cell has to be taken into account as mycoplasmas
live a parasitic life absorbing nutrients from the hosts.
Comparative analysis of the M. pneumoniae and M. genitalium genomes
(Himmelreich et al., 1997) revealed several interesting features about these two
organisms and mollicutes in general. First, all ORFs in M. genitalium are also present in
the M. pneumoniae genome. Second, each of the two genomes has 6 segments, the orders
of which are not conserved because of translocation via homologous recombination. But
the orthologous genes within each segment are well conserved. Third, an additional 236
kb in the M. pneumoniae genome encodes ORFs in three categories, 1) 110 ORFs that are
unique to M. pneumoniae; 2) 76 ORFs that are repeated in the M. pneumoniae genome
are in single copies in the M. genitalium genome; 3) 23 ORFs encoding repetitive
sequences that were not annotated in M. genitalium. This study demonstrated, for the first
time, the usefulness of comparative genomics of closely related organisms.
The genome sequence data have steadily accumulated for plant pathogenic
mollicutes over the past several years. OY phytoplasma complete genome has been
reported (Oshima et al., 2004). S. kunkelii and AY-WB phytoplasma genome are being
sequenced and the gapped genome data are available from websites. S. kunkelii and AYWB phytoplasma are distantly related, belonging to two different branches of the
mollicutes (See 'Phylogeny and evolution' above). On the other hand, S. kunkelii and AYWB phytoplasma are both insect-transmitted plant pathogens. They both invade and
replicate in the same tissues and cells in insect vectors and plant hosts, and they both
cause physiological changes of the plant hosts. This brought up the hypothesis that S.
24
kunkelii and AY-WB phytoplasma share some common genes related to insect
transmission and/or plant pathogenicity. Further, since mycoplasmas are animal and
human pathogens and have no insect vectors, the genes shared by S. kunkelii and AY-WB
phytoplasma may not have homologs in mycoplasmas.
Based on this hypothesis, a comparative genomics study was conducted on
gapped genomes of S. kunkelii and AY-WB phytoplasma, and complete genomes of M.
genitalium, M. pneumoniae, M. pulmonis, M. gallisepticum, M. penetrans, U.
urealyticum, OY phytoplasma, and M. mycoides subsp. mycoides SC (Bai et al., 2004b).
Four deduced proteins were identified by implementation of the BLAST strategy
(Altschul et al., 1997) and designed programs, including polynucleotide phosphorylase
(PNPase), cmp-binding factor (CBF), cytosine deaminase, and YlxR protein. PNPase is
widely distributed among eukaryotic and prokaryotic organisms and was shown to be a
global regulator of virulence factors of Salmonella enterica (Clements et al., 2002). It
was speculated that PNPase could have a similar function in plant pathogenic mollicutes
(Bai et al., 2004b). Another deduced protein, CBF, could be involved in plasmid
replication as in Streptococcus aureus (Zhang et al., 1997). Plant pathogenic mollicutes
harbor plasmids containing virulence factors (Melcher et al., 1999; Oshima et al., 2002),
which implied the involvement of CBF in pathogenicity (Bai et al., 2004b).
In addition to the functional information, some evolutionary data were also
obtained from the study (Bai et al., 2004b). Four deduced proteins were shared among all
organisms included in the study. These proteins are ppGpp synthetase, HAD hydrolase,
AAA type ATPase, and P-type magnesium transport ATPase. Interestingly, they are more
25
closely related between S. kunkelii and AY-WB phytoplasma than to mycoplasmas.
Phylogenetic analysis suggested AAA type ATPase was obtained by phytoplasmas from
spiroplasmas via horizontal gene transfer. The locations of the gene within both genomes
are in insertion sequence regions (Bai et al., 2004b).
The application of comparative genomics is getting more common as more
genome sequence data become available. Nowadays, comparative genomics is practiced
together with the genome sequencing of almost all organisms, both eukaryotes and
prokaryotes. Comparative genome analysis provides useful information about the
functional implications of interesting genes and clues about evolution.
1.10 Functional genomics
The computer algorithm-assisted annotation assigns functions to genome
sequences. However, the findings are not conclusive until supported by experimental
data. Functional genomics aims to study the functions of genome sequences on the
genome level using high-throughput strategies.
Several high-throughput analysis techniques have been developed and
successfully applied to determine gene functions including in vivo expression technology
(IVET) (Mahan et al., 1993), signature-tagged mutagenesis (STM) (Walsh and Cepko,
1992), and genomic analysis and mapping by in vitro transposition (GAMBIT) (Akerley
et al., 1998). These techniques depend on the generation of noticeable phenotypes to
identify genes (Chiang et al., 1999). However, the application of these techniques to plant
26
pathogenic mollicutes is limited, if not at all applicable, because of the lack of efficient
transformation tools. So far, it is only possible for S. citri (Foissac et al., 1997b).
Because phytoplasmas cannot be cultured, functional analysis tools, including
data mining for the identification of protein candidates, in planta functional screens for
the identification of protein effectors, and studies focusing on several proteins for the
elucidation of the functions of the proteins were used and resulted in the elucidation of
several candidate virulence factors of phytoplasmas (Bai et al., in preparation). At the
time of the start of the project, the AY-WB phytoplasma genome sequence was not yet
completed. However, the gapped genome sequences have been shown to be able to give a
decent analysis result equivalent to a complete sequence (Selkove et al., 2000).
The gapped AY-WB phytoplasma genome was mined for effector proteins. The
underlying hypothesis was that the secreted proteins and membrane-bound proteins from
AY-WB phytoplasma are most likely to be involved in pathogenicity. Because
phytoplasmas are intracellular pathogens to insects and plants (Oshima et al., 2002), these
proteins have better chances to contact host cells. Proteins can be secreted via several
bacterial transport systems, including the type II Sec-dependent secretion pathway (Lai
and Kado, 2000). There are no indications of the presence of secretion pathways other
than type I and type II in the annotated genome of AY-WB phytoplasma (Bai et al., in
preparation). Proteins transported by the Sec-dependent secretion pathway have a
common feature, the presence of signal peptides (Fekkes and Driessen, 1999). Three
distinct regions comprise the N-terminal signal sequence: the charged N-terminus (nregion), the hydrophobic core (h-region), and the C-terminal cleavage domain (c-region)
27
(von Heijne, 1985). SignalP program was developed to predict the presence of signal
peptides based on algorithms of Neural Network (NN) and Hidden Markov Model
(HMM) (Nielsen et al., 1997a, 1997b). The incorporation of SignalP program, an ORF
prediction program, ORF Extractor (Bai et al., 2004b), and some perl scripts in the
research identifies 144 candidate effector proteins, which are subject to the in planta
functional analysis.
In planta assays were adapted for the functional studies of AY-WB phytoplasma.
The Potato Virus X (PVX)-based binary plant transformation vector is an effective
molecular tool for transient expression of foreign genes in a plant system. It has also been
used for virus-induced gene silencing (VIGS) in plants (Ruiz et al., 1998). Recently, the
system has been exploited as a high-throughput functional screening tool (Qutob et al.,
2002; Kamoun et al., 2002; Kamoun et al., 2003; Torto et al., 2002; Torto et al., 2003).
Using this high-throughput strategy on Nicotiana benthamiana plants, 16 phytoplasma
proteins were identified to either induce the severe necrosis symptoms by themselves or
manipulate the plant defense system resulting in the increase of PVX symptoms.
In-depth functional analysis was conducted focusing on several proteins. These
proteins were selected based on the computer-predicted presence of a nuclear localization
signal (NLS), which is specific for eukaryotic proteins. Since AY-WB phytoplasma is a
prokaryote without a nucleus, phytoplasma proteins with NLS are putative virulence
factors because they may target the nuclei of the insect and plant host cells and affect host
gene transcription. Indeed, two of the proteins (A11 and A30) were demonstrated to
28
target to N. benthamiana nuclei demonstrated by transient expression of yellow
fluorescence protein (YFP) fusion proteins in plant leaves.
The transportation of the YFP fusion proteins into plant nuclei could be via
importin-dependent pathway. Two N. benthamiana importin α homologs were identified
by data mining strategy (Kanneganti et al., in preparation). These importin α homologs
were silenced in N. benthamiana plant by VIGS via TRV (tobacco rattle virus) system
(Ratcliff et al., 2001; Dinesh-Kumar et al., 2003). The transportation of the fusion
proteins was disrupted in importin α-silence N. benthamiana plants, which suggested the
dependence of the importin pathway. The yeast two-hybrid system is employed to detect
whether the proteins directly interact with importin α of N. benthamiana.
The genes encoding the two proteins were expressed by AY-WB phytoplasma
during infection of insects and plants. Transcripts of expected sizes were detectable in
total RNA isolated from infected insects and plants. One protein A11 was produced in
FLAG-tagged format in Escherichia coli XL1-blue strain and purified by affinity
columns. Antibody against FLAG-tagged A11 was raised in mice and was used for
immuno-labeling studies. Confocal microscopy images of immuno-fluorescence-labeled
AY-WB phytoplasma-infected plant and insect tissues revealed that A11 protein is
present in plant phloem tissues and various insect tissues, including those important for
insect transmission, such as midgut and salivary gland.
29
1.11 Research objectives
The research objectives of my Ph.D. research are to identify and characterize
spiroplasma and phytoplasma genes involved in insect transmission and plant
pathogenicity using various genomic tools, including genome sequencing, sequence
annotations, comparative genomics, and functional genomics. This dissertation consists
of six chapters, including this introduction. Each chapter corresponds to one project
contributing to the research objectives. Chapter 1 is this introduction, conveying the
background knowledge and the research summary of the plant pathogenic mollicutes.
Chapter 2 is titled "A genome sequence survey of the mollicute corn stunt spiroplasma
Spiroplasma kunkelii", which was published in FEMS Microbiology Letters (Bai and
Hogenhout, 2002). It reports the survey-sequencing attempt on S. kunkelii M2 strain and
some revealed features of the genome. Chapter 3 is titled "Complete genome sequences
of aster yellows witches' broom (AY-WB) phytoplasma and comparison with onion
yellows (OY) phytoplasma". It summarizes the results from AY-WB phytoplasma
genome sequencing project, including genome data, genome annotation, metabolic
pathway reconstruction, and comparative genomics. Chapter 4 is titled "Identification
and characterization of traE genes of Spiroplasma kunkelii", which was published in
Gene (Bai et al., 2004a). It contains the gene identification from S. kunkelii gapped
genome sequences, gene sequence analysis, and characterization of the traE genes in S.
kunkelii. Chapter 5 is titled "Comparative genomics identifies genes shared by distantly
related insect-transmitted plant pathogenic mollicutes", which was published in FEMS
30
Microbiology Letter (Bai et al., 2004b). It reports the in silico comparative genomics
study employed for the identification of potential pathogenicity-related genes in plant
pathogenic mollicutes. The complete genomes of animal and human pathogenic
mycoplasmas were used in the study. Chapter 6 is titled "Functional genomics identifies
phytoplasma effector proteins". It includes the sections of the data mining for effector
proteins, high throughput functional screen, effector proteins localization and
transportation in plants, effector gene expression during infection of insects and plants,
and the function of effector proteins in plants.
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44
Organism
a
Strain
CDs
Genome
size (bp)
GC
content
(mol%)
Total
CDs as
hypothetical
proteins
n/a
Unique
hypothetical
proteins
n/a
tRNAs
rRNA
operon
GenBank
accession
number
Reference
36
1
NC_000908
Fraser et al., 1995
Himmelreich et al.,
1996
Glass et al., 2000
45
M. genitalium
G-37
580,074
32
470
CDs with
assigned
functions
n/a
M. pneumoniae
M129
816,394
40
677
333
181
163
37
1
NC_000912
U. urealyticum
(U. parvum serovar 3)
M. pulmonis
751,719
25.5
613
325
116
172
30
2
NC_002162
963,879
26.6
784
486
92
204
29
1
NC_002771
M. penetrans
ATCC
700970
UAB
CTIP
HF-2
1,358,633
25.7
1,038
n/a
n/a
n/a
29
1
NC_004432
Chambaud et al.,
2001
Sasaki et al., 2002
M. gallisepticum
R
996,422
31
742
469
150
123
33
2
NC_004829
Papazisi et al., 2003
Onion yellows
phytoplasma
(Ca. Phytoplasma
asteris)
M. mycoides subsp.
mycoides SC
M. mobile
OY-M
860,631
28
754
446
51
257
32
2
NC_005303
Oshima et al., 2004
PG1
1,211,703
24
985
59%
14%
27%
30
2
NC_005364
Westberg et al., 2004
163K
777,079
24.9
635
n/a
n/a
n/a
28
1
NC_006908
Jaffe et al., 2004
Mesoplasma florum
L1
793,224
27
683
n/a
n/a
n/a
29
n/a
NC_006055
Pending
AY-WB phytoplasma
(Ca. Phytoplasma
asteris)
AYWB
706,569
27
673
345
112
216
31
1
Pending
Pending
Table 1.1 Summary of completed mollicute genomes
a
Mycoplasma was abbreviated as M. and Ureaplasma was abbreviated as U. The species names in the parenthesis are the new species names.
n/a, no information is available.
Entomoplasma
Mesoplasma
Mycoplasma mycoides
Spiroplasm a
SEM
Ureaplasma
UG A
=T r p
Mycoplasma pneumoniae
Mollicutes
Mycoplasma hominis
Mycoplasma sualvi
Acholeplasma
W L, GL
Ca. Phytoplasma
AAA
Anaeroplasma
Asteroleplasma
Clostridium
Gram-positive bacteria
Bacillus
Fig. 1.1 Phylogeny of mollicutes based on 16S rDNA sequences. Bacillus serves as the outgroup. All
mollicutes were derived from Gram-positive bacteria of the Clostridium group and underwent reductive
evolution by loss of the cell wall (WL) and some genes (GL) (Woese, 1987; Weisburg et al., 1989; Oshima
et al., 2004). Phytoplasmas obtained a genus name, Candidatus (Ca.) Phytoplasma (Lee et al., 2004).
Members of the Mycoplasma mycoides cluster groups together with Spiroplasma, Entomoplasma, and
Mesoplasma, but not other mycoplasma species (Razin et al., 1998).
46
CHAPTER 2
A genome sequence survey of the mollicute corn stunt
spiroplasma Spiroplasma kunkelii
Xiaodong Bai, Saskia A. Hogenhout
Department of Entomology, The Ohio State University – Ohio Agricultural Research and
Development Center (OARDC), Wooster, OH 44691
47
2.1 Abstract
The mollicute corn stunt spiroplasma (Spiroplasma kunkelii) is a leafhoppertransmitted pathogen of maize. Sequencing of the ~1.6-Mb genome of S. kunkelii was
initiated to aid understanding the genetic basis of spiroplasma interactions with their plant
and leafhopper hosts. In total, 144,712 nucleotides of non-redundant, high-quality S.
kunkelii genome sequence were obtained. Sequence tags were searched against the
Mycoplasmataceae and Bacillus/Clostridium databases. Results showed that, in addition
to spiroplasma phage SpV1 DNA insertions, spiroplasma genomes harbor more purine
and amino acid biosynthesis, transcription regulation, cell envelope and DNA
transport/binding genes than Mycoplasmataceae genomes. This investigation
demonstrates that survey sequencing is an efficient procedure for gene discovery and
genome characterization. The results of the S. kunkelii sequencing project are available at
the Spiroplasma Web Page at http://www.oardc.ohio-state.edu/spiroplasma/genome.htm.
48
2.2 Introduction
The mollicute Spiroplasma kunkelii is a member of the family Spiroplasmataceae
within the order Mycoplasmatales. Spiroplasmas are primarily associated with insects
and plants in epiphytic, symbiotic or pathogenic interactions. Three spiroplasma species
evolved as plant pathogens: the citrus stubborn spiroplasma Spiroplasma citri, the corn
stunt spiroplasma (CSS) S. kunkelii, and the periwinkle yellowing spiroplasma
Spiroplasma phoeniceum. Plant-pathogenic spiroplasmas are restricted to the sieve tubes
of their plant hosts and are transmitted from plant to plant by phloem-feeding leafhoppers
in a persistent propagative manner (Nault, 1980; Purcell, 1982; Markham, 1983). CSS is
one of the most important threats to maize. Typical symptoms of CSS infection include
chlorosis, stunted plants with reduced internode length and proliferation of ears that do
not mature (Nault, 1980).
Mollicutes are thought to have diverged from a Gram-positive Clostridium-like
ancestor and differ phenotypically from other bacteria in their minute size (0.3-0.5 µm)
and lack of cell wall (Bové and Garnier, 1997; Razin et al., 1998). Genomes of
Mollicutes are smaller in size than those of most other prokaryotes as a result of
degenerative or reductive evolution. However, gene loss in spiroplasmas was not as
extensive as in other members of the class Mollicutes (Bové and Garnier, 1997; Bové,
1997). Interestingly, the spiroplasma morphology differs from that of other Mollicutes.
All members within the genus Spiroplasma have pleomorphic shapes varying from
spherical or slightly ovoid, 100-250 nm, to helical fragments that are about 120 nm in
diameter and 2-4 µm long during active growth and up to 15 µm in later stages of growth,
49
whereas members of other mollicute genera typically have a spheroidal to ovoid shape
and commonly do not have a helical elongated stage in their life cycle.
The genomes of Mycoplasma genitalium, M. pneumoniae, Ureaplasma
urealyticum and M. pulmonis of the family Mycoplasmataceae within the order
Mycoplasmatales have been sequenced to completion (Fraser et al., 1995; Himmelreich
et al., 1996; Glass et al., 2000; Chambaud et al., 2001), and close to seven full genome
sequences from the Bacillus/Clostridium group, the most closely related walled bacteria
to the class Mollicutes, are available as well. The sample-sequencing project of the S.
kunkelii genome and subsequent comparison of S. kunkelii sequence data with
Mycoplasmataceae species and Bacillus/Clostridium sequence databases as described
herein revealed interesting differences in gene content between S. kunkelii and members
of the Mycoplasmataceae.
2.3 Materials and methods
2.3.1 Selection of the S. kunkelii strain
The S. kunkelii strain CSS-M was selected for genome sequencing. The strain was
originally isolated from infected corn plants in Tlaltizapan, Mexico in 1992 (Ebbert and
Nault, 2001). It has been maintained at the Ohio Agricultural Research and Development
Center (OARDC) by serial transfers with the CSS leafhopper vector (Dalbulus maidis) as
described by Ebbert and Nault (Ebbert and Nault, 1994). CSS-M was isolated from
infected corn stems, propagated in liquid LD8A3 medium, and plated onto LD8A3 agar
plates (Lee and Davis, 1989). A culture derived from a single colony was used for
50
genomic DNA isolation. The S. kunkelii clone was transmitted to maize seedlings (Zea
mays L. ‘Early Sunglow’) by D. maidis (Nault, 1980), indicating the clone kept its
characteristics of leafhopper transmission and pathogenesis of plants.
2.3.2 Construction of S. kunkelii genomic DNA libraries
Genomic DNA was isolated from S. kunkelii using the Qiagen (Valencia, CA,
USA) Whole Genomic DNA isolation kit following the manufacturer’s procedures. The
isolated genomic DNA was used for library construction. The DNA was digested to
completion with EcoRI or HindIII, ligated into appropriately digested, phosphatasetreated pUC18, and transformed into electro-competent XL-blue Escherichia coli
(Stratagene, La Jolla, CA, USA) cells. For construction of a random sheared DNA
library, DNA was fragmented with the Hydroshear1 (GeneMachines) into pieces with a
distribution centered on 1.5 kb. Blunt-ended DNA was then ligated into pPCR-Script
Amp Sk(+) plasmid (Stratagene), and plasmid DNA was introduced into chemically
competent XL10-Gold Kan E. coli following the manufacturer’s procedures (Stratagene).
Insert-carrying plasmids were identified in transformants by detecting white colonies
after growth on X-Gal/IPTG (Sambrook et al., 1989).
2.3.3 Sequencing and sequence analysis
Colonies were grown overnight at 37°C in single wells of 96-well microtiter
plates containing 150 µl LB freeze (4 mM MgSO4, 360 mM K2HPO4, 132 mM KH2PO4,
17 mM Na-citrate, 68 mM (NH4)2SO4, 4.4% glycerol in LB, pH 7.0) and 100 µg ml-1
51
ampicillin and transferred to LB agar plates containing 100 µg ml-1 ampicillin after 18 h
using a 96-well plate replicator. The inoculated agar plates were sent to MWG-Biotech
(High Point, NC, USA) for one-pass sequencing of the inserts using the M13 forward and
reverse primers for pUC18, and T7 and T3 primers for pPCR-Script Amp Sk(+) plasmids
on an ABI377 automatic sequencer. Trace files were analyzed with the PHRED and
CROSS_MATCH algorithms of MacPhred/Phrap (Ewing et al., 1998; Ewing and Green,
1998) to translate the ABI377 chromatogram data of the sequence files into accurate
quality information for each base call and detection of plasmid sequences, respectively.
Plasmid sequences were removed from each sequence tag and high quality sequence data
(s20 phred score) were collected into a database and searched against the non-redundant
(nr) database at National Center for Biotechnology Information (NCBI) using nucleotidenucleotide BLAST (blastn) or the translating BLAST (blastx) algorithms (Altschul et al.,
1990). To screen for redundant sequence tags, the S. kunkelii sequence database was also
searched against itself with the blastn algorithm. Nucleotide sequences with significant
similarities (E-value ≤ 10-5) to sequences in the NCBI database were collected, translated
into proteins and searched against the full non-redundant protein database and nonredundant databases of Mycoplasmataceae and Bacillus/Clostridium at NCBI with the
protein-protein BLAST (blastp) algorithm. All sequence analyses were performed on
local Linux workstations.
52
2.4 Results and discussion
2.4.1 Library construction, sequencing and sequence analysis
To confirm the identity of the isolated DNA, the spiralin gene was amplified
using primers described by Foissac et al. (Foissac et al., 1997). The nucleotide sequence
of the spiralin gene amplification product was identical to that reported earlier (Foissac et
al., 1997), thus confirming the identity of the CSS-M clones of S. kunkelii (data not
shown). Insert sizes of clones from the shotgun library ranged from 0.5 to 4 kb and clones
from the EcoRI or HindIII libraries contained fragment sizes ranging from 150 bp to 10
kb.
In total, 94 inserts from the EcoRI and HindIII libraries, and 188 inserts from the
sheared DNA library were sequenced from flanking primer sites after which the
sequences were collected into a database. Low quality and cloning vector sequences were
removed from the database. The database was then searched against itself with the blastn
algorithm to analyze redundancy. Mollicute genome sequences show the presence of
highly repeated regions and spiroplasma genomes have many copies of spiroplasma
phage SpV1 DNA (Bébéar et al., 1996). Therefore, redundant sequence tags were not
assembled into contigs but within each set of redundant clones one sequence tag from the
forward and reverse direction with best phred quality scores were kept in the database
whereas others were removed. This resulted in a database of 144,712 nucleotides (396
sequence tags) of non-redundant high-quality (s20 phred score) S. kunkelii genome
sequences representing 9% of the S. kunkelii genome, based on an estimated genome size
of 1,600 kb (Bové, 1997). All sequences were deposited in the random single pass read
53
genome survey sequence database (dbGSS) of GenBank (Accession Nos. BH234783 to
BH235178).
The 396 sequence tags were searched against the complete NCBI nr database with
the blastn and blastx algorithms. The overall percentage of sequence tags with significant
similarity (E-value ≤ 10-5) to open reading frames (ORFs) in the NCBI nr database was
~40% (150/396), which is in agreement with previous findings that biological functions
can be assigned to ~50% of the ORFs in completed genome sequencing projects
(Simpson et al., 2000).
2.4.2 DNA phage sequences
Unlike the Mycoplasmataceae, spiroplasma genomes harbor many spiroplasma
phage SpV1 DNA insertions (Ye et al., 1994; Ye et al., 1995; Fraser et al., 1995; Bébéar
et al., 1996; Himmelreich et al., 1996; Glass et al., 2000; Chambaud et al., 2001). In this
survey ~5% (17/396) of the S. kunkelii sequence tag database had significant similarity to
spiroplasma virus SpV1 DNA (Table 2.1). The percentage of phage sequences in the S.
kunkelii sequence tag database is comparable to the 7% DNA phage sequences found in
the genome of the Gram-negative leafhopper-transmitted vascular plant pathogen, Xylella
fastidiosa (Simpson et al., 2000).
2.4.3 Spiroplasma-specific sequences
In total, 133 sequence tags had significant similarities to prokaryotic and/or
eukaryotic protein sequences in the NCBI nr database. Included were four sequences
54
unique to spiroplasmas with similarity to putative S. citri virulence genes encoding P123,
P58, P54, or P18 (Table 2.1) (Ye et al., 1996; Fletcher et al., 1998). These genes are part
of a 9.5-kb S. citri genome segment that is deleted from a non-transmissible line of S.
citri.
2.4.4 Comparative genome analysis
As a preliminary assessment of to what extent the S. kunkelii genome content
differs from those of Mycoplasmataceae species, sequence tags were translated into
proteins to ensure that the sequences were part of ORFs and, subsequently, the protein
sequences were searched against the Mycoplasmataceae, Bacillus/Clostridium and
complete nr protein databases of GenBank (Table 2.2). The Mycoplasmataceae database
was selected because it contains the full genome sequences of three mycoplasma and one
ureaplasma species (Fraser et al., 1995; Himmelreich et al., 1996; Glass et al., 2000;
Chambaud et al., 2001), whereas the Bacillus/Clostridium database was selected because
it contains many completed genome sequences and Bacillus/Clostridium species are
thought to be closest walled relatives to Mollicutes (Razin, 1994; Bové and Garnier,
1997). Interesting gene content differences among S. kunkelii, and Mycoplasmataceae
and Bacillus/Clostridium species are discussed below.
2.4.5 Amino acid, purine, pyrimidine, nucleoside and nucleotide metabolism
Mycoplasmataceae species lack most genes involved in de novo biosynthesis of
pyrimidines, purines and amino acids (Fraser et al., 1995; Himmelreich et al., 1996; Glass
55
et al., 2000; Chambaud et al., 2001). However, in contrast to mycoplasmas and U.
urealyticum, the S. kunkelii genome seems to harbor the nucleotide and/or amino acid
biosynthesis genes encoding adenylosuccinate lyase, adenylosuccinate synthase, GMP
synthase, deoxyguanosine kinase, and folylpolyglutamate synthase/dihydrofolate
synthetase (folC) (Table 2.2). Adenylosuccinate lyase is a tetrameric enzyme involved in
de novo synthesis of inosine monophosphate (IMP) and adenosine monophosphate
(Mantsala and Zalkin, 1992), adenylosuccinate synthase catalyzes the first step in de
novo biosynthesis of AMP (Honzatko and Fromm, 1999), and guanine monophosphate
(GMP) synthase catalyzes the last step from IMP into GMP (Mantsala and Zalkin, 1992).
Deoxyadenosine/deoxyguanosine kinase and deoxyadenosine/deoxycytidine kinase are
required, together with thymidine kinase, for deoxynucleotide synthesis in Lactobacillus
acidophilus (Ma et al., 1995). Interestingly, the deoxyguanosine kinase gene is present in
the mollicute Mycoplasma mycoides. Within the order Mycoplasmatales, M. mycoides
belongs to the Entomoplasmataceae, a family more closely related to the
Spiroplasmataceae than the Mycoplasmataceae (Bové and Garnier, 1997). The folC gene
product is essential for production of glycine, methionine, purine and thymidine (Singer
et al., 1985). These data suggest that S. kunkelii can synthesize more amino acids and
nucleotides de novo than Mycoplasmataceae species do, which is in agreement with
experimental evidence that spiroplasma culturing media are less complex than those of
the culturable mycoplasmas (Lee and Davis, 1989; Razin, 1994).
56
2.4.6 Cell envelope
The sequence data indicate that S. kunkelii harbors at least two cell envelope
biosynthesis genes that are absent from members of the Mycoplasmataceae. The gcpE
gene is involved in the acetylation of peptidoglycans and isoprenoid biosynthesis and is
broadly distributed in eubacteria and plants (Rather et al., 1997; Campos et al., 2001).
MreB is a cytoskeletal protein and forms a filamentous helical structure close to the cell
surface of eubacteria, and has an actin-like role in bacterial cell morphogenesis (Jones et
al., 2001). The clear morphological differences between spiroplasmas and
Mycoplasmataceae and our finding that the mreB gene is absent from Mycoplasmataceae
genomes but present in S. kunkelii suggest that MreB may have a critical role in the
unique helical cell structure of spiroplasmas.
2.4.7 Regulatory functions
Our sequence data show that the S. kunkelii regulatory mechanisms are more
complex that those of the Mycoplasmataceae. Three genes were identified encoding the
regulatory proteins NifR3, SinR and PNPase that were absent in the three sequenced
mycoplasmas and U. urealyticum but present in Firmicutes. NifR3 is important for the
regulation of the dormant and vegetative cell stages of the ciliate Sterkiella
histriomuscorum (Tourancheau et al., 1999). The function of NifR3 in bacteria is not
known. SinR is involved in the transition of a vegetative stage to sporulation in Bacillus
subtilis in response to nutrient depletion (Gaur et al., 1991). Spiroplasmas do not make
spores, but are extremely pleomorphic. It is tempting to speculate that NifR3 and SinR
57
may be involved in S. kunkelii cell shape regulation as a response to nutrient availability.
A third regulatory protein, polynucleotide phosphorylase (PNPase) is responsible for
mRNA decay, translation activation and transcript stabilization in B. subtilis (Wang and
Bechhofer, 1996; Oussenko and Bechhofer, 2000). The loss of PNPase is lethal for E.
coli, but affects only competence development in B. subtilis (Donovan and Kushner,
1986; Luttinger et al., 1996) and may affect competence of S. kunkelii as well. The
discovery of these regulatory factors in S. kunkelii is surprising as, thus far, members of
the Mycoplasmataceae are known to lack major regulators of gene expression (Fraser et
al., 1995; Himmelreich et al., 1996; Himmelreich et al., 1997; Weiner et al., 2000).
2.4.8 Replication
One surprising finding was that the DNA polymerase I protein of S. kunkelii did
not match the DNA polymerases of Mycoplasmataceae, whereas it had significant
similarity to the DNA polymerase I proteins of Streptococcus species (E-values: 2e-35 and
2e-32, sequence tag MEAA_B05.y, Table 2.2). Closer analysis revealed that the 193
amino acid sequence tag of S. kunkelii was similar to the C-terminal polymerase domain
of DNA polymerase I. In contrast, putative DNA polymerases I of M. genitalium
(GenBank Accession No. I64228), M. pneumoniae (S73784), U. urealyticum (C82895)
and M. pulmonis (CAC13893) are ~300 amino acids in size and consist of the N-terminal
5’-3’ exonuclease part (proofreading) part but lack the C-terminal 3’-5’ exonuclease and
polymerase domains (Klenow fragment) of the enzyme (Chambaud et al., 2001). This
finding suggests that, unlike mycoplasmas and U. urealyticum, the S. kunkelii polA gene
58
may encode the full-length DNA polymerase I protein including the proofreading and
Klenow domains similarly to that of Streptococcus pneumoniae (Lopez et al., 1989).
2.4.9 Transport and binding proteins
In contrast to Mycoplasmataceae, the S. kunkelii genome harbors at least one copy
of a traK homologue. S. kunkelii traK has the highest similarity to traK of the B.
anthracis virulence plasmid pX02.09 (Table 2.2) (Okinaka et al., 1999). This conserved
protein family binds DNA and couples plasmid to membrane proteins for transport to the
mating cell and/or are pathogenicity factors involved in transport of virulence factors to
the extracellular environment of bacteria (Errington et al., 2001; Christie and Vogel,
2000). The function of S. kunkelii TraK protein remains to be investigated.
Two S. kunkelii sequence tags (MSAC_C02.x and MSAD_C02.y, Table 2.2)
harbor sequences similar to fructose permease of the phosphoenolpyruvate:fructose
phosphotransferase system (fructose PTS). Mutagenesis of the operon encoding fructose
PTS proteins in another leafhopper-transmitted plant-pathogenic spiroplasma, S. citri,
significantly decreases plant pathogenicity (Gaurivaud et al., 2000). The most likely
explanation is that utilization of fructose in the plant sieve tubes by S. citri may interfere
with the normal physiology of the plant causing chlorosis, stunting and wilting
(Gaurivaud et al., 2000). This may be true for S. kunkelii in sieve tubes of corn plants as
well. Homologues of fructose PTS proteins were also identified in Mycoplasmataceae
and other Firmicutes (Table 2.2).
59
2.4.10 Genes in other categories
The S. kunkelii genome harbors at least one copy of a spoIIIE homologue that is
not found in the Mycoplasmataceae genome sequenced so far (sequence tag
MEAA_A06.y, Table 2.2). In B. subtilis, the spoIIIE gene product is involved in the
coordination of chromosome segregation and clearing DNA from the site of division
during septum formation (Bath et al., 2000) and, therefore, is likely to be involved in S.
kunkelii cell division. A nifU-like gene of 228 nucleotides in length was identified in this
sequencing project and harbors solely the C-terminal conserved domain containing two
conserved cysteines, whereas functional iron-sulfur cluster-binding NifU proteins contain
additional middle domains with four conserved cysteines (sequence tag MEAA_D11.x,
Table 2.2) (Ouzounis et al., 1994; Nishio and Nakai, 2000; Agar et al., 2000). Several
smaller nifU-like genes are also found in the nitrogen fixing Rhodobacter and
Azotobacter species and single gene mutagenesis studies show that they are not essential
for survival or nitrogen fixation of bacteria (Masepohl et al., 1993). The functions of
these shorter nifU-like genes are not known.
A sequence similar to the oxygen-insensitive NAD(P)H nitroreductase was found
in the S. kunkelii database (sequence tag MSAD_E03.x, Table 2.2). This enzyme
catalyzes the reduction of a variety of nitroaromatic compounds to highly toxic
metabolites (Bryant and DeLuca, 1991). Although absent from the mycoplasmas and U.
urealyticum genomes, it is found in the small (~650 kb) genome of the insect vectored
apple proliferation phytoplasma (gi405516) (Jarausch et al., 2000). It is noteworthy that
60
phytoplasmas are the only other group of Mollicutes that infect plants causing
characteristic chlorosis and stunting symptoms.
Two sequence tags have identity to the 20 kDa PsaD thiol peroxidase proteins of
Streptococcus species (Kolenbrander et al., 1994; Novak et al., 1998). Tag
MHAA_A09.x contains the N-terminal part of this protein, whereas MHAA_D09.x
harbors the C-terminal end. In S. pneumoniae, the psaD gene is located downstream from
the psa locus with the psaA, psaB and psaC genes encoding an ABC-type Mn permease
complex (Novak et al., 1998). Mutagenesis of each of four psa genes resulted in
penicillin tolerance, defective adhesion and reduced transformation efficiency of S.
pneumoniae (Novak et al., 1998). The psaA gene encodes an adhesin-like surface protein,
and psaA and psaD related genes were identified in Streptococcus sanguis, Streptococcus
parasanguis and Streptococcus gordonii (Kolenbrander et al., 1994).
Several sequence tags have identity to conserved hypothetical proteins that are
lacking from the mycoplasmas and U. urealyticum genomes sequenced thus far (Other
categories, Table 2.2). We found only one sequence tag with identity to Mollicute
sequences but not those of the Bacillus/Clostridium group (sequence tag PH_05.y, Table
2.2). The deduced protein sequence of this tag is a homologue of a hypothetical protein
encoded by a gene in the downstream region of the fibril gene region of S. citri
(Williamson et al., 1991). The fibril protein is important for the helical cell shape and
motility of spiroplasmas (Trachtenberg, 1998; Trachtenberg and Gilad, 2001) and the
gene encoding it is lacking from the genomes of the oval-shaped mycoplasmas and U.
urealyticum (Fraser et al., 1995; Himmelreich et al., 1996; Glass et al., 2000; Chambaud
61
et al., 2001). Because the hypothetical protein gene is localized near the fibril protein
gene (Williamson et al., 1991) and is unique to Mollicutes (Table 2.2), this hypothetical
protein may be an important constituent of the mollicute cytoskeleton.
2.4.11 Ribosomal RNA genes
Clones MEAA_E09 and MHAA_F02 contained part of the 16S and 23S
ribosomal RNA (rRNA) genes and the 16S-23S internal spacer with closest similarity to
rRNA gene regions from S. citri, as is expected from the S. kunkelii phylogenetic position
(Bové and Garnier, 1997) (Table 2.3). S. kunkelii rRNA genes have not been sequenced
previously.
2.4.12 Conclusions
In summary, our data show that, in addition to the large number of spiroplasma
phage DNA insertions, S. kunkelii also harbors more amino acid and nucleotide
biosynthesis, transcription regulation, cell envelope and DNA transport/binding genes
than the genomes of the Mycoplasmataceae species do. Our data also demonstrate that
genome comparisons among Mollicutes are extremely informative because of their small
genome sizes, broad host range, differences in morphology, and well-defined biology. In
addition to the already completed genome sequences of four Mycoplasmataceae species,
several genome sequence projects of Mollicutes in other families are ongoing including
those of M. mycoides and M. capricolum in the family Entomoplasmataceae
(http://www.ncbi.nlm.nih.gov/PMGifs/Genomes/bact.html), and S. kunkelii
62
(http://www.genome.ou.edu/spiro.html) and S. citri
(http://www.cwu.edu/~verheys/s.citri/). Genome comparison of species within a family,
among families within the class Mollicutes and between Mollicutes and Firmicutes
should prove extremely valuable.
2.5 Acknowledgments
The authors thank Dr. Margareth Redinbaugh for carefully reading the manuscript
and Dr. Robert Davis for help with establishing S. kunkelii in vitro cultures at the
OARDC. This research was funded by the OARDC research enhancement and
competitive grants program.
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Carraro, D.M., Carrer, H., Colauto, N.B., Colombo, C., Costa, F.F., Costa, M.C.,
Costa-Neto, C.M., Coutinho, L.L., Cristofani, M., Dias-Neto, E., Docena, C., ElDorry, H., Facincani, A.P., Ferreira, A.J., Ferreira, V.C., Ferro, J.A., Fraga, J.S.,
Franca, S.C., Franco, M.C., Frohme, M., Furlan, L.R., Garnier, M., Goldman, G.H.,
Goldman, M.H., Gomes, S.L., Gruber, A., Ho, P.L., Hoheisel, J.D., Junqueira, M.L.,
Kemper, E.L., Kitajima, J.P., Krieger, J.E., Kuramae, E.E., Laigret, F., Lambais,
M.R., Leite, L.C., Lemos, E.G., Lemos, M.V., Lopes, S.A., Lopes, C.R., Machado,
J.A., Machado, M.A., Madeira, A.M., Madeira, H.M., Marino, C.L., Marques, M.V.,
Martins, E.A., Martins, E.M., Matsukuma, A.Y., Menck, C.F., Miracca, E.C., Miyaki,
C.Y., Monteriro-Vitorello, C.B., Moon, D.H., Nagai, M.A., Nascimento, A.L., Netto,
L.E., Nhani Jr., A., Nobrega, F.G., Nunes, L.R., Oliveira, M.A., de Oliveira, M.C., de
Oliveira, R.C., Palmieri, D.A., Paris, A., Peixoto, B.R., Pereira, G.A., Pereira Jr.,
H.A., Pesquero, J.B., Quaggio, R.B., Roberto, P.G., Rodrigues, V., de, M.R.A.J., de
Rosa Jr., V.E., de Sa, R.G., Santelli, R.V.,Sawasaki, H.E., da Silva, A.C., da Silva,
A.M., da Silva, F.R., da Silva Jr., W.A., da Silveira, J.F., Silvestri, M.L., Siqueira,
W.J., de Souza, A.A., de Souza, A.P., Terenzi, M.F., Tru., D., Tsai, S.M., Tsuhako,
M.H., Vallada, H., Van Sluys, M.A., Verjovski-Almeida, S., Vettore, A.L., Zago,
67
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Tourancheau, A.B., Morin, L., Yang, T. and Perasso, R. (1999) Messenger RNA in
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Trachtenberg, S. (1998) Mollicutes - wall-less bacteria with internal cytoskeletons. J.
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Trachtenberg, S. and Gilad, R. (2001) A bacterial linear motor: cellular and molecular
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68
Identity
Spiroplasma virus SpV1 ORFs
ORF1, capsid protein
Sequence tag ID
Acc. No. of best hit
E-value
ORFa
MSAC_D10.y
MSAD_C09.y
MSAD_C12.x
MSAD_B02.x
MEAA_B07.y
MHAA_H01.y
MSAC_A12.y
MSAD_C03.y
MSAC_C02.y
MSAC_D02.y
MSAD_H02.y
MSAD_H02.x
MSAD_A08.x
MHAA_C12.y
MHAA_G02.y
MSAD_E11.x
MHAA_H07.x
9626113
1143020
9626113
1143020
1143021
9626114
1143018
1143013
9626110
9626110
P15893
U28974
9626111
P15898
1143012
U28972
1143008
2e-19
4e-94
3e-62
e-117
2e-39
3e-14
1e-14
1e-46
2e-26
2e-26
2e-42
3e-21
3e-12
3e-10
1e-09
8e-08
3e-29
S. citri putative virulence proteins
P123
P58
P54
P18
MSAD_A01.x
MHAA_A07.x
MHAA_A07.y
MHAA_F03.y
T28663
7482012
7482011
7482010
2e-61
4e-22
1e-64
2e-24
ORF3, transposase gene
ORF2
ORF4
ORF5
ORF7
ORF14
Table 2.1 Sequence tags with significant similarity (E-value ≤ 10-5) to spiroplasma virus SpV1 and S. citri
putative virulence proteins
Deduced protein sequences were searched against the non-redundant database at NCBI. Identity and
sequence tag identity (ID) are indicated and for each sequence tag the accession number (Acc. No.) and Evalue of entry with the highest similarity are listed.
69
Sequence tag ID
Identity
Amino acid biosynthesis
MEAA_A03.y
Thymidylate kinase
MSAC_C05.y
Folylpolyglutamate synthase/dihydrofolate synthetase
(folC)
MSAC_B10.x
Methionine aminopeptidase (MAP) (peptidase M)
PE_10.x
Serine hydroxymethyl transferase (glyA)
Cell envelope
MHAA_E07.x
PH_05.x
Cell shape determining protein (MreB-like protein)
GcpE protein
Fatty acid and phospholipid metabolism
MEAA_D03.x
Probable N-acetylglucosamine-6-phosphate
deacetylase
MHAA_C12.x
1-Acyl-sn-glycerol-3-phosphate acyltransferace
MHAA_H07.x
orfa
HSAD_G09.y
1-Acyl-sn-glycerol-3-phosphate acyltransferace
Sequence
length (aa)
Best entry
Mycoplasmataceae,
accession No. (E-value)
Best entry
Bacillus/Clostridium,
accession No. (E-value)
Best entry GenBank organism,
accession No. (E-value)
96
127
14089465 (5e-08)
-
2632295 (3e-12)
4930039 (3e-09)
B. subtilis, 16077096 (2e-10)
Str. pneumoniae, 15900133 (4e-09)
179
239
14089981 (3e-37)
1673936 (2e-78)
11131429 (3e-41)
12723496 (5e-84)
B. halodurans, 15612719 (1e-35)
Str. pneumoniae, 15902972 (2e-64)
172
102
-
10176363 (2e-33)
1730252 (3e-26)
B. halodurans, 15616301 (4e-07)
B. subtilis, 16079569 (1e-27)
73
14089782 (1e-06)
A69664 (4e-08)
St. aureus, 15893481 (9e-06)
112
65
117
13508038 (2e-10)
14089575 (1e-09)
13508038 (5e-22)
10174252 (0.008)
2633961 (8e-12)
2633289 (3e-05)
M. pulmonis, 15828585 (2e-06)
S. citri, 1143008 (3e-29)
M. pneumoniae, 13508038 (6e-20)
70
Cellular processes
MEAA_C12.y
MHAA_B03.x
DnaK protein (hsp 70)
GTP-binding membrane protein (LepA)
189
80
8920287 (2e-51)
6899301 (2e-17)
P45554 (5e-58)
12724067 (1e-18)
E. rhusiopathiae, 1169374 (4e-58)
La. lactis, 15673090 (2e-05)
Energy metabolism
MEAA_B07.x
MEAA_C10.x
MHAA_E06.y
MHAA_A06.y
MHAA_D08.y
Dihydrolipoamide dehydrogenase
Phosphomannomutase (PMM)
Glycerol-3-phosphate dehydrogenase
ATP synthase β chain
Fructose-biphophate aldolase
43
133
167
213
107
1674136 (1e-10)
1352196 (3e-19)
14089537 (2e-15)
14089679 (1e-94)
12044873 (2e-23)
12722900 (3e-07)
C69835 (8e-29)
1146220 (8e-27)
10176378 (1e-86)
10944298 (1e-21)
P. putida, 1706442 (7e-16)
B. halodurans, 15613669 (8e-19)
St. aureus, 15924464 (2e-24)
M. pulmois, 15828737 (1e-76)
Cl. acetobutylicum, 15894114 (2e-25)
(Continued)
Table 2.2 Sequence tags with significant similarities (E-value ≤ 10-5) to NCBI nr protein sequences
Deduced protein sequences were blastp searched against the non-redundant (nr) database and the Mycoplasmataceae and Bacillus/Clostridium protein
databases at NCBI. Sequence tag identity (ID) and deduced amino acid (aa) length are indicated and for each sequence tag accession numbers and Evalues of entries with highest similarities are listed. The organism of entry with the highest similarity is listed for the nr database search results. A.,
Aquifex; B., Bacillus; C., Chlamydia; Ca., Campylobacter; Chl., Chlorobium; Cl., Clostridium; E., Erysipelothrix; En., Enterococcus; G., Geobacillus; L.,
Listeria; La., Lactococcus; Lac., Lactobacillus; M., Mycoplasma; My., Mycobacterium; P., Pseudomonas; S., Spiroplasma; St., Staphylococcus; Str.,
Steptococcus; T., Thermotoga; U., Urealyticum; V., Vibrio; X., Xylella; Y., Yersinia; -, no significant hit and sequence is absent; ns, E-value > 10-5 but
sequence is present in genomes of one or more members of the Mycoplasmatacease or Bacillus/Clostridium group.
Table 2.2 (continued)
Sequence tag ID
Identity
Sequence
length (aa)
63
82
88
55
Best entry Mycoplasmataceae,
accession No. (E-value)
14089925 (6e-08)
14089653 (1e-07)
2146068 (1e-11)
14089932 (2e-17)
Best entry Bacillus/Clostridium,
accession No. (E-value)
7328298 (6e-06)
ns
12061042 (0.001)
8670811 (8e-16)
Best entry GenBank organism,
accession No. (E-value)
M. pulmonis, 14089925 (7e-08)
S. citri, 2384686 (3e-17)
M. pneumoniae, 13508341 (1e-04)
A. aeolicus, 6015091 (1e-14)
MHAA_F07.y
MHAA_G06.x
MSAC_G02.y
MSAD_B06.x
Transketolase
Pyruvate kinase
ATP synthase β chain precursor
Phosphopyruvate hydratase/enolase
188
123
131
106
113
144
149
89
14089558 (4e-16)
D53312 (2e-24)
14089736 (2e-29)
-
WZBSDS (2e-37)
10176653 (1e-30)
586859 (1e-08)
2636243 (4e-26)
10173982 (3e-19)
4033719 (3e-11)
12723536 (9e-33)
3483135 (2e-50)
Lac. sakei, 15727116 (1e-32)
S. citri, 1709937 (1e-49)
M. mycoides, 16040925 (7e-36)
B. subtilis, 16080759 (9e-21)
M. pirum, 1345713 (3e-22)
M. mycoides, 16040925 (2e-32)
T. maritima, 15644136 (2e-33)
X. fastidiosa, 15839020 (6e-56)
Regulatory functions
MHAA_A11.y
RNA polymerase σ factor (RpoD)
114
12045103 (2e-06)
O66381 (1e-07)
MSAC_A08.x
121
-
10172709 (1e-29)
MSAD_H07.y
Transcriptional regulator involved in
nitrogen regulation (NifR3 family)
Predicted transcription regulator SinR
Cl. acetobutylicum, 15894582
(3e-06)
B. halodurans, 15612660 (3e-28)
80
-
10174744 (3e-06)
Cl. acetobutylicum, 15894128
(3e-19)
Replication
MEAA_B05.y
MEAA_F12.x
MHAA_G05.y
DNA-directed DNA polymerase I
DNA gyrase subunit B
Chain A, helicase product complex
193
104
171
14089786 (4e-13)
14090113 (7e-11)
A32949 (3e-35)
2558946 (4e-16)
2781090 (4e-17)
MHAA_H04.x
MSAC_A09.x
MSAC_B02.y
MSAC_C06.y
MSAD_B12.y
MSAD_G04.y
DNA-directed RNA polymerase β subunit
ParA family protein
Glucose-inhibited division protein A
Cell division protein FtsH
DNA primase
ATP-dependent helicase PcrA
96
141
78
174
96
112
600226 (5e-21)
12045330 (2e-06)
14089666 (2e-22)
14090194 (2e-43)
13508092 (1e-15)
14090183 (4e-15)
12724825 (3e-19)
9968459 (4e-09)
P25812 (7e-18)
S66099 (4e-42)
664755 (6e-25)
P56255 (6e-16)
Str. pyogenes, 15674390 (2e-32)
M. capricolum, 17008093 (2e-19)
G. stearothermophilus, 9257172
(1e-18)
S. citri, 1350848 (3e-46)
S. citri, 10432498 (7e-16)
M. pulmonis, 14089666 (3e-20)
M. pulmonis, 15829250 (9e-44)
L. innocua, 16800560 (4e-24)
My. tuberculosis, 15840373 (2e-12)
Transcription antitermination factor
(NusG)
Polynucleotide phosphorylase (PNPase)
112
14089595 (1e-06)
O08386 (2e-19)
206
-
1184680 (3e-82)
Purine, pyrimidines, nucleosides, and nucleotides
MEAA_B12.x
Adenylosuccinate lysase
MEAA_C08.x
Adenylosuccinate synthetase
MEAA_D08.y
Deoxyguanosine kinase
MHAA_B06.x
Thymidine kinase
MHAA_C05.y
Cytidine deaminase
MHAA_H10.y
Deoxyguanosine kinase
MSAC_D04.y
Adenine phophoribosyltransferace
MSAC_F08.x
GMP synthetase (glutamine
amindotransferase)
71
Transcription
MHAA_E11.x
MHAA_H05.y
L. monocytogenes, 16802292 (2e07)
B. subtilis, 16078732 (4e-71)
(Continued)
Table 2.2 (continued)
Sequence tag ID
Identity
Sequence
length (aa)
MSAC_H02.y
MSAD_B09.x
DNA-directed RNA polymerase α chain
Transcription antitermination protein NusG
ATP-dependent protease (lon-protease)
50S ribosomal protein L21
Valine-tRNA ligase
Cysteinyl tRNA synthetase
50S ribosomal protein L3
50S ribosomal protein L2
Translation initiation factor 2 (InfB)
Prolyl-tRNA synthetase
Translation elongation factor G (EF-G)
Hypothetical proteins similar to Osialoglycoprotein endopeptidase
50S ribosomal protein L4
Asparaginyl-tRNA synthetase
Seryl-tRNA synthetase
50S ribosomal protein L5
30S ribosomal protein S3
30S ribosomal protein S8
50S ribosomal protein L17 (fragment)
50S ribosomal protein L19
Isoleucyl-tRNA synthetase
Threonyl-tRNA synthetase
Glutamyl-tRNA synthetase
Phenylananyl-tRNA synthetase β chain
Isoleucyl-tRNA synthetase
DNA-directed DNA polymerase (α chain)
Tryptophanyl-tRNA synthetase
Glycyl-tRNA synthetase
Heat shock protein GroEL
Ribosomal large subunit pseudouridine
synthase B
Histidyl-tRNA synthetase
Peptide chain release factor 1 (RF-1)
50S ribosomal protein L2
Glycyl-tRNA synthetase
30S ribosomal protein S17
Translation
MEAA_B02.x
MEAA_B09.x
MEAA_B09.y
MEAA_C04.y
MEAA_C06.x
MEAA_D03.y
MEAA_D09.x
MEAA_G12.y
MHAA_A08.y
MHAA_A11.x
72
MHAA_B08.y
MHAA_C07.x
MHAA_D07.y
MHAA_C09.x
MHAA_C09.y
MHAA_C11.x
MHAA_C11.y
MHAA_D12.y
MHAA_E03.y
MHAA_E05.y
MSAC_A11.x
MSAC_B10.x
MSAC_C04.y
MSAC_H02.y
MSAD_B06.y
MSAD_B12.x
MSAD_E10.x
MSAD_H08.x
PE_05.x
PE_14.y
PE_21.y
PH_04.x
PS_02.y
114
68
Best entry
Mycoplasmataceae,
accession No. (E-value)
6601578 (5e-22)
ns
Best entry
Bacillus/Clostridium,
accession No. (E-value)
12725120 (1e-18)
12725158 (5e-09)
Best entry GenBank organism, accession
No. (E-value)
M. capricolum, 629301 (1e-28)
Str. coelicolor, 1709420 (2e-06)
122
38
121
76
40
109
108
217
180
68
1674198 (8e-14)
14089744 (8e-05)
1351181 (3e-20)
1351147 (5e-12)
14090004 (4e-08)
14090000 (7e-36)
2497279 (5e-40)
14089596 (7e-63)
14089842 (5e-78)
14089531 (2e-18)
B42375 (1e-15)
12724034 (3e-08)
10175660 (9e-28)
12724882 (6e-12)
P42920 (2e-05)
P04257 (3e-36)
10175033 (3e-35)
13633967 (5e-64)
10172743 (3e-92)
1945110 (1e-19)
V. cholerae, 15641922 (1e-13)
Str. pyrogenes, 15674860 (4e-08)
St. aureus, 15927242 (2e-21)
Cl. stricklandii, 6899996 (4e-10)
M. capricolum, 132957 (2e-06)
M. capricolum, 71083 (4e-20)
M. genitalium, 12044994 (7e-49)
B. burgdorferi, 15594747 (1e-50)
B. halodurans, 15612694 (2e-80)
St. aureus, 15927624 (8e-15)
38
119
90
88
59
85
119
66
101
132
131
175
180
107
145
201
83
95
2766504 (9e-36)
14090186 (1e-28)
1361847 (8e-26)
3844757 (2e-29)
3914904 (1e-04)
14089988 (4e-17)
14089975 (8e-27)
14089881 (1e-14)
14090082 (3e-12)
13508292 (1e-38)
13508417 (4e-16)
ns
14090082 (2e-32)
6601579 (2e-25)
14090160 (7e-26)
6899491 (1e-63)
12045254 (4e-23)
14089751 (7e-04)
S24364 (3e-52)
12724857 (4e-14)
12724729 (9e-28)
4512416 (3e-33)
ns
P56209 (7e-17)
P07843 (3e-31)
10175098 (6e-20)
437916 (2e-23)
143766 (8e-42)
289282 (4e-20)
40054 (3e-06)
10175165 (3e-41)
10172773 (2e-30)
10175491 (2e-34)
4584090 (1e-30)
12723267 (5e-35)
410137 (1e-15)
M. capricolum, 132981 (2e-45)
Cl. acetobutylicum, 15896505 (1e-32)
A. aeolicus, 15605830 (7e-24)
B. halodurans, 15612709 (5e-27)
S. citri, O31161 (7e-26)
M. capricolum, 134021 (2e-23)
M. capricolum, 7674204 (1e-38)
B. halodurans, 15615041 (1e-18)
St. aureus, 1174521 (1e-24)
U. urealyticum, 13358098 (5e-45)
B. subtilis, 16077160 (2e-15)
C. pneumoniae, BAA98801.1 (4e-10)
La. sakei, 15487790 (5e-27)
M. capricolum, 629301 (1e-28)
B. halodurans, 15615433 (1e-28)
St. aureus, 15924555 (1e-55)
En. faecalis, 15625350 (1e-33)
B. subtilis, 466190 (1e-31)
220
144
195
68
85
12044885 (7e-29)
1350577 (5e-45)
14090000 (5e-66)
14089865 (1e-17)
14089993 (4e-22)
3915057 (4e-45)
S55437 (2e-44)
P04257 (5e-72)
4584090 (2e-24)
P23828 (7e-31)
L. innocua, 16800623 (2e-07)
M. capricolum, 2500137 (6e-55)
M. capricolum, 71083 (7e-77)
B. cereus, 4584090 (2e-17)
S. citri, 3122807 (3e-39)
(Continued)
Table 2.2 (continued)
Sequence tag ID
Sequence
length (aa)
Best entry
Mycoplasmataceae,
accession No. (E-value)
Best entry
Bacillus/Clostridium,
accession No. (E-value)
Best entry GenBank organism,
accession No. (E-value)
Transport and binding proteins
MEAA_F10.x
Phosphotransfereace EII (PTS system)
MEAA_G05.y
Phosphate ABC transporter, permease protein
MEAA_H04.x
ABC transporter
MSAC_A07.y
Methygalactosidase permease ATP-binding protein
MSAC_A08.y
ABC transporter, ATP-binding protein
138
177
168
229
70
14089430 (8e-14)
1361743 (4e-25)
2146659 (1e-51)
4914644 (3e-47)
12044917 (3e-15)
2633144 (6e-15)
4530449 (1e-30)
12723139 (8e-46)
12724309 (6e-47)
12724060 (1e-12)
MSAC_C02.x
Highly similar to phosphotransferace system (PTS)
fructose-specific enzyme IIABC component
ABC transporter, ATP-binding protein
Highly similar to Mg(2+) transport ATPase
87
1045736 (1e-05)
2633811 (4e-09)
M. capricolum, 530422 (9e-15)
V. vholerae, 15600843 (1e-23)
U. urealyticum, 13358103 (2e-47)
U. urealyticum, 13357571 (2e-36)
Cl. acetobutylicum, 15894109
(2e-13)
L. innocua, 16801491 (5e-07)
115
140
14089609 (2e-11)
14089568 (2e-15)
10173618 (3e-21)
12714231 (8e-31)
131
108
14090034 (7e-36)
14089827 (9e-17)
D70009 (5e-39)
S11153 (5e-31)
111
14089430 (4e-32)
66867 (2e-21)
MSAD_D03.x
MSAD_D12.y
MSAD_E06.y
MSAD_F05.x
Similar to ABC transporter (ATP-binding protein)
Similar to ABC transporter ATP-binding protein –
oligopeptide transport
Phosphotransferase system, glucose-specific IIABC
component
Oligopeptide permease (ATP-binding protein)
Transfer complex protein TrsK protein (TraK)
Cation-transporting P-ATPase
Phosphate ABC transporter, permease protein
50
75
125
96
13507956 (8e-07)
14089568 (3e-05)
13508349 (7e-14)
1420862 (2e-10)
6470167 (1e-09)
12724231 (4e-21)
4530449 (2e-20)
Str. pyogenes, 15674468 (6e-09)
B. anthracis, 6470167 (1e-07)
La. lactis, 15673239 (3e-16)
Str. pneumoniae, 15901902 (8e19)
Other categories
MEAA_A02.x
MEAA_A03.x
MEAA_A06.y
Amidase
Conserved hypothetical protein
SpoE family protein/cell division protein
62
165
141
2146059 (1e-06)
14090195 (1e-08)
-
ns
467456 (6e-04)
S09411 (2e-31)
MEAA_B03.y
MEAA_D11.x
MEAA_D12.y
Probable GTP-binding protein
Nitrogen fixation protein NifU
Predicted SAM-dependent methytransferase
56
69
145
13508214 (5e-12)
1045939 (7e-10)
1146219 (9e-17)
10176042 (6e-13)
12724027 (1e-16)
MEAA_E07.x
MEAA_E08.y
RNA-binding Sun protein
Conserved hypothetical protein
100
98
-
2633846 (4e-08)
10173873 (1e-21)
M. capricolum, 530426 (6e-10)
U. urealyticum, 13357633 (2e-10)
Str. pneumoniae, 15900761 (2e33)
B. subtilis, 1730915 (6e-15)
B. halodurans, 15615981 (7e-11)
Cl. acetobutylicum, 12724027
(1e-16)
B. subtilis, 16078637 (4e-04)
St. aureus, 10173873 (1e-21)
MSAC_D03.y
MSAC_D06.x
MSAC_F10.y
MSAD_A04.y
73
MSAD_C02.y
Identity
T. maritima, 15643786 (5e-19)
L. monocytogenes, 16804726
(5e-20)
B. subtilis, 16080207 (2e-38)
Str. pneumoniae, 15903745 (3e30)
M. pulmonis, 14089430 (4e-32)
(Continued)
Table 2.2 (continued)
Sequence tag ID
Identity
Sequence
length (aa)
112
125
56
Best entry
Mycoplasmataceae, accession
No. (E-value)
S73881 (3e-05)
7109691 (5e-06)
Best entry
Bacillus/Clostridium, accession
No. (E-value)
P54501 (1e-08)
10175125 (1e-07)
MEAA_E09.x
MEAA_E12.x
MEAA_F10.y
Hypothetical protein
199 aa conserved hypothetical protein
Similar to putative phosphoprotein phosphatase
MHAA_A09.x
Probable thiol peroxidase
77
14090123 (6e-09)
P72500 (9e-12)
MHAA_A10.y
MHAA_B12.x
Hypothetical protein
Conserved GTP-binding protein
152
124
1674179 (7e-10)
14089767 (2e-09)
2634923 (7e-13)
12724592 (2e-08)
MHAA_B12.y
tRNA δ (2) isopentenylpyrophosphate
transferase
P115-like (Mycoplasma hyorhinis) ABC
transporter ATP-binding protein
Probable thiol peroxidase
75
-
13701103 (3e-11)
Chl. tepidum, 10039641 (2e-24)
B. subtilis, P54501 (1e-08)
L. monocytogenes, 10175125
(1e-06)
Str. pneumoniae, 15901486 (1e09)
A. aeolicus, 7451802 (2e-13)
L. monocytogenes, 14089767
(2e-09)
St. aureus, 15924294 (2e-11)
168
14090129 (8e-52)
10175107 (2e-46)
M. pulmonis, 14090129 (8e-52)
56
-
P31307 (5e-07)
118
14089726 (2e-39)
2619052 (6e-09)
109
ns
9968459 (3e-12)
87
134
14089574 (3e-09)
14090092 (7e-06)
12723043 (2e-10)
13700111 (2e-24)
MHAA_G08.y
MHAA_H02.x
MSAC_A06.x
MSAC_B11.y
MSAC_C09.y
MSAC_E03.x
Acyl carrier protein phosphodiesterase (ACP
phosphodiesterase)
Partitioning or sporulation protein (ParA)
(soj protein)
Conserved hypothetical protein
Probable type I restriction enzyme restriction
chain
Exodeoxyribonuclease V (α subunit)
Conserved hypothetical protein
Conserved hypothetical protein
Hypothetical protein
Conserved hypothetical protein
Conserved hypothetical protein
Cl. acetobutylicum, 15896549
(5e-07)
M. pulmonis, 14089726 (2e-39)
84
99
195
140
174
43
14090197 (2e-07)
3845056 (6e-16)
13508006 (5e-11)
-
2635193 (4e-12)
2635763 (6e-29)
12724713 (3e-34)
12724031 (2e-19)
13027335 (5e-21)
7429432 (3e-05)
MSAC_H01.x
Conserved hypothetical protein
128
150165 (5e-09)
10175107 (3e-10)
MSAD_E03.x
MSAD_F02.y
Nitroreductase
Hypothetical protein
151
133
P75273 (2e-06)
7432647 (2e-05)
5420109 (3e-12)
MSAD_F01.x
Hypothetical 35.3 kDa protein, SLR1819
91
-
P37497 (1e-05)
MSAD_G02.x
PH_01.y
PH_05.y
Conserved hypothetical protein
BH2145 – unknown conserved protein
Hypothetical protein in fibril gene 3’ region
99
82
315
14090099 (3e-17)
12045292 (6e-14)
7328260 (8e-15)
10175035 (4e-19)
-
MHAA_C06.y
74
MHAA_D09.x
MHAA_E04.x
MHAA_F06.y
MHAA_F12.x
MHAA_G03.y
Best entry GenBank organism,
accession No. (E-value)
L. monocytogenes, 9968459
(3e-12)
Str. pyogenes, 12723043 (2e-10)
St. aureus, 15923185 (8e-27)
C. pneumoniae, 15835659 (9e-13)
St. aureus, 15923836 (2e-26)
Y. pestis, 16121243 (2e-29)
La. lactis, 12724031 (2e-19)
St. aureus, 13027335 (5e-21)
Synechocystis sp. PCC 680,
7444728 (4e-06)
Str. pneumoniae, 10175107 (3e10)
Ca. jejuni, 15792391 (2e-08)
Str. thermophilus, 5420109 (3e12)
Synechocystis sp. PCC 6803,
P73709 (2e-07)
M. pulmonis, 7328260 (8e-15)
B. halodurans, 10175035 (4e-19)
S. citri, P27712 (e-101)
Sequence tag ID
MEAA_E09.x
Identity
16S rDNA
MHAA_F02.y
16S rDNA, 16S/23S spacer region,
23S rDNA
Accession No., organism
46914, S. citri
175961, S. poulsonii
175965, S. citri
175964, S. apis
175967, S. mirum
175969¸ S. monobiae
175962, S. taiwanense
175970, S. diabroticae
175963, S. gladiatoris
175473, Entomoplasma melaleucae
46914, S. citri
E-value
0.0
0.0
0.0
0.0
0.0
0.0
e-180
e-179
e-171
e-166
0.0
4456860, Spiroplasma sp.
2707198, S. citri
5821442, M. putrefaciens
e-151
e-125
2e-19
Table 2.3 S. kunkelii sequence tags with similarity to rRNA genes
S. kunkelii sequence tags were searched against the full GenBank nr nucleotide database with the blastn
algorithm. Identities, accession numbers and organism, and E-values of the first 10 and four entries of
respectively MEAA_E09.x and MHAA_F02.y, the only sequence tags with similarities to rRNA genes, are
listed.
75
CHAPTER 3
Complete genome sequence of aster yellows witches' broom
(AY-WB) phytoplasma and comparison with
onion yellows (OY) phytoplasma
Xiaodong Bai1, Jianhua Zhang1, Kiryl Tsuckerman3, Dimitry Schevchenko3,
Eugene Goltsman3, Adam Ewing4, Sally A. Miller2, Theresa Walunas3,
John Campbell3, and Saskia A. Hogenhout1
1
Department of Entomology, 2Department of Plant Pathology, The Ohio State University
– Ohio Agricultural Research and Development Center (OARDC), Wooster, OH 44691
3
Integrated Genomics, Inc., Chicago, IL 60612
4
Department of Biology, Hiram College, Hiram, OH 44234
76
3.1 Abstract
We determined the complete genome sequence of aster yellows witches' broom
(AY-WB) phytoplasma, an intracellular bacterial plant pathogen transmitted by insects.
The AY-WB phytoplasma genome consists of a 706,569-bp single circular chromosome
and 4 plasmids with sizes of 3,972 bp, 4,009 bp, 5,104 bp, and 4,316 bp. The circular
chromosome contains 673 predicted coding sequences (CDs), one set of rRNA genes and
31 tRNA genes, and the plasmids contain 24 CDs. Among the total of 697 CDs, functions
can be assigned to 352 CDs (51%), and 232 CDs are unique to AY-WB phytoplasmas.
The AY-WB phytoplasma chromosome is 154,062-bp smaller than the OY phytoplasma
chromosome and contains 81 less CDs. However, the metabolic genes in both genomes
are mostly similar. The high numbers of paralogs are predominantly accountable for the
difference in genome size of AY-WB and OY. AY-WB phytoplasma genome contains 15
ATP-binding cassette (ABC) transporters for nutrient uptake, and the required
components for bacterial type II protein translocation. The glucose phosphotransferase
gene in the glycolysis pathway was not identified in the AY-WB phytoplasma genome
suggesting that it might not be able to utilize glucose as a carbon and energy source.
However, phytoplasmas may use malate as an alternative carbon and energy source. AYWB phytoplasma has a limited biosynthesis and energy production capacity, which is in
consistent with the parasitic life style of phytoplasmas. Multiple copies of transposase
and paralogous genes, and truncated versions of these sequences, were identified in the
AY-WB genome, suggesting that the genome is prone to recombination. Furthermore, the
AY-WB phytoplasma genome contains an incomplete type I restriction and modification
77
system making it accessible for import and integration of foreign genetic materials. A
total of 50 CDs encode soluble secreted proteins were identified that might interact with
host cell components, and hence are potential virulence factors. The AY-WB
phytoplasma genome also contains the pore-forming toxin hemolysin possibly involved
in phytoplasmas invasion of host cells. The information revealed by the genome sequence
is essential for the investigation of the biology, physiology and pathogenicity of AY-WB
phytoplasma, and is useful for understanding why phytoplasmas are not cultivable in cellfree media.
3.2 Introduction
Phytoplasmas are wall-less prokaryotes of the Class Mollicutes, unique bacteria
characterized by small genomes with low GC contents and no cell wall (Razin et al.,
1998). Phytoplasmas are insect-transmitted plant pathogens that invade and replicate in
both insect and plant cells. Phytoplasmas were derived from Gram-positive ancestors by
reductive evolution (Woese, 1987; Weisburg et al., 1989; Oshima et al., 2004). In
contrast to spiroplasmas, the other group of mollicutes that contains insect-transmitted
plant pathogens, phytoplasmas appear to have undergone more excessive genome
reductions and are likely evolutionarily late organisms (Bai et al., 2004b). Due to their
uncultivable nature and the scarce of available genetic tools, little is known about the
biology, physiology and pathogenicity of phytoplasmas.
Members of the Class Mollicutes have been the subjects of genome sequencing
efforts for years, because of their small genomes and clinical and economical importance.
78
So far, the complete genomes of 11 mollicutes have been reported, including
Mycoplasma genitalium (NC_000908, Fraser et al., 1995), M. pneumoniae (NC_000912,
Himmelreich et al., 1996), Ureaplasma urealyticum (NC_002162, Glass et al., 2000), M.
pulmonis (NC_002771, Chambaud et al., 2001), M. penetrans (NC_004432, Sasaki et al.,
2002), M. gallisepticum (NC_004829, Papazisi et al., 2003), OY phytoplasma
(NC_005303, Oshima et al., 2004), M. mycoides subsp. mycoides (NC_005364, Westberg
et al., 2004), M. mobile (NC_006908, Jaffe et al., 2004), Mesoplasma florum
(NC_006055) and M. hyopneumoniae (NC_006360, Minion et al., 2004). The genome
sequence data led to the identification of a minimal gene set for a free-living cell (Fraser
et al., 1995; Mushegian and Koonin, 1996) and other advances in mollicute research.
Specifically, the release of the first complete phytoplasma genome sequence (Oshima et
al., 2004) greatly advanced the phytoplasma research.
Aster yellows witches' broom (AY-WB) phytoplasma is a strain of aster yellows
phytoplasma, the largest group of phytoplasmas that was recently assigned a tentative
species name of Candidatus Phytoplasma asteris (Lee et al., 2004). AY-WB phytoplasma
was first described in Ohio where it causes severe damage to lettuce and China aster
(Zhang et al., 2004). Here, we report the complete genome sequence of AY-WB
phytoplasma, and comparative analysis of this genome with that of the closely related OY
phytoplasma. The analysis of AY-WB phytoplasma genome sequence data led to a better
understanding of the biology and pathogenicity mechanisms of phytoplasmas, and the
reason why phytoplasmas are not cultivable. The annotation and analysis of AY-WB
phytoplasma genome are directional for other phytoplasma genome sequencing projects
79
that are currently underway, such as the maize bushy stunt phytoplasma (MBSP)
genome-sequencing project.
3.3 Materials and Methods
3.3.1 Phytoplasma strain
The aster yellows phytoplasma (Candidatus Phytoplasma asteris) strain aster
yellows witches' broom (AY-WB) was isolated from diseased lettuce plants (Lactuca
sativa) in Ohio (Zhang et al., 2004). The AY-WB strain was maintained by serial
transmission to China aster (Callistephus chinensis) plants using aster leafhoppers
(Macrosteles quadrilineatus L.) in greenhouse and growth chambers.
3.3.2 DNA manipulation
Batches of phloem sap containing AY-WB phytoplasma were collected from
diseased lettuce plants and mixed with STE buffer (0.1 M NaCl, 10 mM Tris-HCl, 1 mM
EDTA, pH 8.0) at a ratio of 1:3 (sap/buffer). The mixture was kept on ice, divided into
1.5 ml Eppendorf tubes, and centrifuged at 9,000 rpm for 15 min at 4 °C. The pellet was
resuspended in low-melting agarose (Promega, Madison, WI) and subject to isolation
with pulsed field gel electrophoresis (PFGE) as described before (Zhang et al., 2004).
Phytoplasma genomic DNA was eluted from the gel blocks with Elutrap (Schleicher &
Schuell, Keene, NH) following the manufacturer's instruction. The genomic DNA was
ethanol precipitated following standard procedure (Sambrook et al., 1989) and
80
resuspended in deionized distilled water. The concentration of the purified DNA was
assessed using PicoGreen kit (Molecular Probes, Eugene, OR).
3.3.3 DNA sequencing and assembly
The shotgun library was constructed using 5 µg AY-WB phytoplasma genomic
DNA isolated from pulsed field gels (Zhang et al., 2004) at Integrated Genomics, Inc.
(IG) (2201 W Campbell Park, Chicago, IL 60612). The DNA was sheared into an
average of 2 kb fragments and cloned into the pGEM-3Z vector for transformation into
Escherichia coli strain DH5α. The genomic DNA library was sequenced using
MegaBACE 1000 (Amersham Biosciences, Sweden) and ABI3700 (Applied Biosystems,
Foster City, CA) sequencers. The library was sequenced to saturation at 7-fold coverage
of the AY-WB phytoplasma genome.
Primary assembly of the individual sequences were performed using the
Phred/Cross_match/Phrap package (originally developed at Washington University),
which automatically basecalls the trace data, screens out vector, removes unreliable data,
and assembles individual reads into contigs. Additional in-house-developed-tools helped
in the validation of the assembly. Manual editing of read and contig sequences, as well as
manipulations with the layout (i.e. tearing and joining of contigs, relocating reads, etc.),
was then performed with the Consed editing software.
Gaps in the assembly were covered by primer walking on gap-spanning clones.
Areas of poor consensus quality and low coverage were also improved using this method.
Sequencing oligos and templates were picked automatically using the Autofinish
81
software. We conducted four rounds of primers walking and in each round of primer
walking custom oligos were designed to further extend the sequences in regions.
3.3.4 Identification of CDs, annotation and analysis
The sequence data of AY-WB phytoplasma were uploaded into the IG database
and software suite, ERGO, for sequence annotation. CRITICA (Badger and Olsen, 1999)
and IG-proprietary tools were used for CD identification. The predicted CDs were
annotated by sequence similarity search using BLAST algorithm (Altschul et al., 1997)
against non-redundant (nr) database at the server maintained by the National Center for
Biotechnology Information (NCBI). Protein domains were analyzed by searching against
NCBI conserved domain (CD) database (Marchler-Bauer et al., 2003) and the pfam
(protein family) database (Bateman et al., 2004). Proteins were classified into COGs
(clusters of orthologous groups) at NCBI. The Kyoto Encyclopedia of Genes and
Genomes (KEGG) was used for the reconstruction of the metabolic pathways. The
assignment of enzyme commission (EC) number was according to the BRENDA
database (Schomburg et al., 2002). The repetitive sequences within the genome were
predicted using REPuter program (Kurtz and Schleiermacher, 1999) hosted at the
Bielefeld University Bioinformatics Server. The signal peptide of AY-WB phytoplasma
predicted CDs was predicted using SignalP program (version 3.0) (Bendtsen et al., 2004).
The transmembrane domains were predicted using TMHMM v2.0 program (Krogh et al.,
2001). The presence of plant nuclear localization signals (NLS) was predicted using
pSORT (Nakai and Horton, 1999) with the CDs excluding the signal peptides if present.
82
3.4 Results
3.4.1 General genome features
The genome of AY-WB phytoplasma consists of a 706,569-bp single circular
chromosome (Table 3.1) and 4 plasmids with sizes of 3,972 bp, 4,009 bp, 5,104 bp, and
4,316 bp (Fig. 3.1). The AY-WB phytoplasma chromosome has a low GC content (27%)
and 74% of the chromosome is coding regions. The AY-WB phytoplasma chromosome
contains 673 predicted coding sequences (CDs). Among the 673 CDs, functions can be
assigned to 345 CDs (51%), and 216 CDs (32%) are unique to AY-WB phytoplasmas.
The remaining 112 CDs of AY-WB phytoplasma are significantly similar to the
hypothetical proteins in databases. The four plasmids encode 24 CDs, 7 of which match
to proteins with functional annotations and 16 CDs match to hypothetical proteins. AYWB chromosome contains one ribosomal RNA operon and 31 transfer RNA genes,
whereas the OY phytoplasma chromosome contains two ribosomal RNA operons and 32
transfer RNA genes.
The AY-WB phytoplasma genome is 154,062 bp smaller and contains 81 less
CDs than the OY genome (Oshima et al., 2004). With a cutoff expectation (E) value of
10-5, AY-WB phytoplasma has 72 unique CDs (11%) and OY phytoplasma has 76 unique
CDs (10%).
3.4.2 Functional categories of the predicted CDs
The comparison of functional categories in the cluster of orthologous groups
(COGs) between AY-WB phytoplasma CDs and those of other mollicutes revealed
83
interesting features (Table 3.2). Several mollicutes genomes were selected for the
comparison. The M. genitalium genome is the smallest genome sequenced so far (Fraser
et al., 1995). The M. pneumoniae genome (Himmelreich et al., 1996) is representative of
the hominis and pneumoniae groups of mycoplasmas. M. penetrans has a slightly bigger
genome compared to other mollicutes (Sasaki et al., 2002) and it can invade host cells,
which is a characteristic similar to phytoplasmas, but not to most other mycoplasmas
(Razin et al., 1998). The M. mobile genome is the most recently reported genome (Jaffe
et al., 2004) and it has been a model for studying the sliding movement of mycoplasmas
(Piper et al., 1987; Uenoyama et al., 2004). All the above Mycoplasma species are
obligate parasites of humans or animals.
AY-WB and OY genomes harbor similar amount of genes involved in translation
and transcription. The phytoplasma genomes contain many copies of RNA polymerase
sigma factors, i.e. 14 for OY phytoplasma and 6 for AY-WB phytoplasma. In contrast, M.
genitalium genome contains only one sigma factor (Fraser et al., 1995). Even in the
genome of M. penetrans that contains the largest numbers of genes in transcription, there
is only one copy of sigma factor (Sasaki et al., 2002). Another interesting feature is that
phytoplasma genomes harbor significantly more recombination-related genes than other
mollicutes.
Both phytoplasmas encode Na+-driven drug efflux pumps and ABC multidrug
transporters, whereas other mollicutes employ only ABC transporters for drug export.
AY-WB phytoplasma contains one component of type I restriction and modification
84
system involved in defense mechanisms, whereas OY phytoplasma contains multiple
components.
Phytoplasma genomes vary in the number of genes involved in cell motility. OY
phytoplasma genome has two hypothetical proteins (PAM458 and PAM696) that are
involved in cell motility whereas AY-WB phytoplasma has none. M. penetrans (Sasaki et
al., 2002) and M. mobile (Jaffe et al., 2004) were noted for their motility and the genomes
harbor 12 and 6 cell motility genes, respectively. AY-WB phytoplasma is able to invade
and multiply in host cells (Lee et al., 2000). Thus, the phytoplasma invasion process
involves unidentified motility genes.
Phytoplasmas contain less metabolic and transport genes than other mollicutes,
suggesting that phytoplasmas are more dependent on metabolites produced by their hosts
than mycoplasmas do.
3.4.3 Metabolism
AY-WB and OY phytoplasma genomes contain similar metabolic genes. Both
phytoplasmas have most genes in the glycolysis pathway except the hexokinase gene
essential for the conversion of glucose to glucose-6-phosphate or glucose
phosphotransferase (PTS) genes (Fig. 3.2). The energy production of phytoplasmas
seems limited to the glycolysis pathway, because no genes in the pentose phosphate
pathway, tricitrate cycle (TCA), or hydrogen-driven ATP synthase genes have been
identified in either genome. Phytoplasmas seems to be able to synthesize purine
triphosphate via the salvage pathway. However, some important genes in the salvage
85
pathway are missing, such as those encoding dCMP deaminase (EC 3.5.4.12), CTP
synthase (EC 6.3.4.2) and purine-nucleoside phosphorylase (EC 2.4.2.1). Therefore, the
phytoplasma nucleotide salvage pathways are incomplete.
In both phytoplasmas, the glycolysis pathway is connected to glycolipid
metabolism by fructose bisphosphate aldolase (EC 4.1.2.13), which produces glycerone
phosphate. The glycolipid pathway leads to the formation of phosphatidylethanolamine
(PE), an essential component of the lipid bilayer of plasma membranes. The PE
biosynthesis pathway was not identified in M. genitalium (Oshima et al., 2004).
Phytoplasmas have limited biosynthesis capacities. The fructose-bisphosphatase
(EC 3.1.3.11) that converts fructose-1,6-bisphosphate into fructose-6-phosphate is absent
from both phytoplasma genomes. Therefore, phytoplasmas cannot synthesize glucose via
the gluconeogenesis pathway. Phytoplasmas can convert pyruvate into acetyl-CoA by the
pyruvate dehydrogenase complex. However, the fate of acetyl-CoA is unclear since no
downstream enzymes have been identified. One possible role of acetyl-CoA is to donate
an acetyl group to the glycolipid metabolic pathway. AY-WB phytoplasma contains
selenocysteine lyase (EC 4.4.1.16) that synthesizes selenocysteine in the selenoamino
acid biosynthesis pathway, and glutamine-dependent NAD(+) synthetase (EC 6.3.5.1)
involved in NAD biosynthesis. Both enzymes need amino acids as substrates, which
phytoplasmas cannot de novo synthesize. The phytoplasmas harbor genes involved in the
folate and tetrahydrofolate biosynthesis (Fig. 3.2) suggesting that phytoplasmas have the
ability to provide one-carbon units to important metabolic pathways, such as the nucleic
acid synthesis pathway.
86
3.4.4 Carbohydrate transport and metabolism
Phytoplasmas inhabit sugar-rich environments, such as plant phloem tissues.
Therefore, it is unexpected that the genomes of AY-WB and OY phytoplasmas harbor
significantly less amount of genes in the category of carbohydrate transport and
metabolism than their mycoplasma counterparts. Even in the 580-kb genome of M.
genitalium, 26 carbohydrate transport and metabolism genes were identified (Fraser et al.,
1995). In contrast, only 19 genes are present in the 860-kb OY phytoplasma genome
(Oshima et al., 2004) and 14 genes in the 706-kb AY-WB phytoplasma genome.
Phytoplasmas may use malate rather than glucose or sucrose as the carbon and
energy source. Malate is an intermediate metabolite in the tri-citrate (TCA) cycle, which
can be used as carbon and energy sources in many bacteria (Krom et al., 2003). Two
malate/citrate-sodium symport genes have been identified in both AY-WB (AYWB052
and AYWB438) and OY (39938772 and 39939206) phytoplasma genomes. Malate can
be converted to pyruvate by NAD-specific malic enzyme (AYWB051 and 39939207).
The usage of malate as a carbon and energy source is advantageous, because (i) malate is
readily available in the cytoplasm of insect and plant cells, where phytoplasmas reside,
and (ii) it needs fewer genes for energy production.
In addition, AY-WB and OY phytoplasmas may be able to utilize maltose as
carbon sources. A complete set of maltose transporter genes was identified in AY-WB
phytoplasma genome, including genes for the maltose transport ATP-binding protein
(malK, AYWB672), maltose transport system permease protein (malF, AYWB671;
malG, AYWB670), and maltose-binding protein (malE, AYWB669). Similarly, OY
87
phytoplasma has two copies of malK, and one copy of malF (ugpA), malG (ugpE), and
malE (ugpB). In contrast to spiroplasmas, phytoplasmas do not contain sucrose
transporters or fructose operon components. Further, the sucrose phosphorylase gene that
is important for sucrose degradation is absent from the AY-WB phytoplasma genome,
and interrupted by a premature stop codon and not functional in the OY phytoplasma
genome (Oshima et al., 2004). Therefore, phytoplasmas apparently cannot use sucrose or
fructose as carbon sources.
Whereas genes encoding maltose-degrading enzymes were not identified in the
phytoplasma genomes, the existence of maltose transport genes suggests that maltose
plays a role in phytoplasma metabolism. Phytoplasmas have to survive the high osmosis
pressure in plant phloem tissues (Lee et al., 2000). The import of carbohydrates, such as
maltose, is probably important for the maintenance of the osmotic balance. Previously,
fructose utilization was shown to affect the plant pathogenicity of spiroplasmas (Foissac
et al., 1997; Gaurivaud et al., 2000a, 2000b, 2001), however for phytoplasmas the
utilization of maltose in plants could contribute to plant pathogenicity. Indeed, the
Arabidopsis plant with 40-time elevated level of maltose were stunting with lower
chlorophyll contents (Niittylä et al., 2004).
3.4.5 ABC transporter
The genomes of both AY-WB and OY phytoplasmas harbor 15 ABC transporter
genes (Table 3.3). As evidenced by the limited metabolic capacity, phytoplasmas rely on
ABC transporters to uptake a wide variety of compounds, such as sugars, ions, peptides
88
and more complex organic molecules, from insect vectors and plant hosts. Other than
providing nutrition to phytoplasmas, ABC transporters can also participate in conjugative
DNA transfer, chemotaxis, and virulence (Detmers et al., 2001). Indeed, a solute-binding
protein Sc76 of the ABC transporter complex is involved in the insect transmission of
plant pathogenic spiroplasmas (Boutareaud et al., 2004). No Sc76 homolog was identified
in phytoplasmas; however, phytoplasmas harbor many genes of solute-binding proteins.
The largest group of phytoplasma ABC transporters is involved in transporting
dipeptide/oligopeptide and amino acids (Table 3.3). Similar to mycoplasmas, AY-WB
and OY phytoplasmas have no amino acid biosynthesis genes, making them rely on the
transport of amino acids from host cells. AY-WB phytoplasma seems to have 4 ABC
transporters for import of glutamine, arginine, methionine, and likely other amino acids,
whereas OY phytoplasmas contain 7 ABC-type amino acid transporters (Table 3.3).
However, only 3 of the 7 transporter systems contain ATPase, which was considered
essential for the transport process (Davidson and Chen, 2004). This is in consistence with
the earlier observation that the OY phytoplasma genome contains multiple redundant
copies of transporter genes (Oshima et al., 2004).
Phytoplasmas also import dipeptide/oligopeptide from host cells, as multiple
dipeptide/oligopeptide transport systems have been identified in both AY-WB and OY
phytoplasmas (Table 3.3). The imported dipeptides or oligopeptides are likely digested
by peptidases, because a gene for Xaa-His peptidase (EC 3.4.13.3) was identified in
AYWB (AYWB434) and OY (39938776). OY phytoplasma, but not AYWB, has a XaaPro aminopeptidase (E.C. 3.4.11.9, pepP, 39938731) as well.
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Phytoplasmas have ABC transporters devoted to inorganic ion uptake (Table 3.3).
Two cobalt ABC transporter systems are present in both AY-WB and OY phytoplasma
genomes. Both phytoplasmas have functional ABC systems to import cation such as zinc
and manganese ions. These cation-transport ABC transporters, together with other ion
transport P-type ATPases (see section "other transporters"), provide the inorganic ions as
nutrients or enzyme ligands.
Both AY-WB and OY phytoplasmas have systems devoted to the export of toxic
substances. AY-WB phytoplasma contains two copies of multidrug resistance ATPbinding and permease proteins, while only one copy is present in the OY phytoplasma
genome (Table 3.3). However, in another plant pathogenic mollicute, Spiroplasma
kunkelii, 7 ABC transporters conferring multidrug resistance have been identified (Zhao
et al., 2004). Plant pathogenic spiroplasmas and phytoplasmas share similar
environmental niches, and therefore likely encounter similar challenges from toxic
substances. The relatively small amount of the multidrug resistance ABC transporters in
phytoplasmas could be compensated by the three copies of norM genes encoding Na+driven multidrug efflux pumps (AYWB442, AYWB444, and AYWB653 for AY-WB;
39938766, 39938768, and 39939220 for OY) (Fig. 3.2). Spiroplasmas do not have
orthologs of Na+-driven multidrug efflux pump genes.
AY-WB and OY have ABC transporter genes (phnL) involved in lipoprotein
release (AYWB621 and 39938582, respectively). OY phytoplasma genome also contains
a second potential component (nlpA, 39938583). The function of lipoprotein release is
90
essential because the deletion of the genes in the lipoprotein translocation complex
LolCDE is lethal to E. coli (Narita et al., 2002).
AY-WB and OY phytoplasmas also contain ABC transporter systems importing
spermidine/putrescine. Spermidine and putrescine are polyamines distributed in a wide
range of organisms from bacteria to plants and animals (Tabor and Tabor, 1984), and are
likely important for phytoplasmas by providing nitrogen nutrients. The phytoplasma
import of polyamines during the infection of plants could alter the level of spermidine
and putrescine in plants and could contribute to symptom development (Walters, 2003).
3.4.6 P-type ATPase transporters
AY-WB phytoplasma genome encodes 5 P-type ATPase for the transport of
inorganic ions such as calcium, magnesium, lead, mercury, zinc, and cadmium (Table
3.4). These genes are conserved in OY phytoplasmas, suggestive of the importance of
these genes in phytoplasmas cation uptake. These inorganic ions are also important for
plant growth and development. It was reported that phytoplasma infection could
significantly affect the plant magnesium uptake (de Oliveira et al., 2002), resulting in the
accumulation of these inorganic ions and a slow growth rate of the plants. The
accumulated magnesium could serve as a nutrient source for phytoplasmas (de Oliveira et
al., 2002).
The cation transporting ATPase in phytoplasmas may play an important
physiological role in phytoplasma cells, because the deletion of cation P-ATPase resulted
in a hypersensitivity to hyperosmotic media of Synechococcus cells (Kanamaru et al.,
91
1993). Indeed, the wall-less phytoplasmas are likely osmotically more sensitive than the
walled bacteria (Razin, 1978; Razin et al., 1998). The cation P-type ATPase could also
serve as the cation efflux apparatus conferring bacterial resistance to heavy metal ions
(Nies, 2003).
3.4.7 Repetitive DNA and coding sequences
Both AY-WB phytoplasma and OY phytoplasma genomes are rich in repetitive
sequences (Fig. 3.3). AY-WB phytoplasma genome harbors more repetitive sequences
than the OY phytoplasma genome. The largest AY-WB phytoplasma repeats are the
inverted repeats of the one and only ribosomal RNA (rRNA) operon and the homologous
region consisting of 10 tRNA genes and two ABC transporter genes (artI and artM) in
the reverse direction. In contrast, OY phytoplasma has a similar repeat region consisting
of the inverted repeats of two rRNA operons. A unique feature in both AY-WB and OY
phytoplasma genomes is the repetitive regions containing dnaG (DNA primase) and
dnaB (replicative DNA helicase) genes. This organization is not present in other currently
sequenced bacteria genomes.
The OY phytoplasma genome contains two large 19 kb repetitive regions about
20 kb apart. Both regions contain 20 CDs, including one copy of uvrD (ATP-dependent
DNA helicase), 2 copies of hflB (ATP-dependent Zn protease), 2 copies of himA
(bacterial nucleoid DNA-binding protein), 2 copies of ssb (single-stranded DNA-binding
protein), 2 copies of fliA (DNA-directed RNA polymerase specialized sigma subunit) and
some hypothetical proteins. Most genes in the repetitive regions of both phytoplasma
92
genomes are involved in DNA replication and transcription. They are possibly the result
of gene duplication events.
The AY-WB phytoplasma genome harbors 4 copies of complete transposase
genes and 14 copies of transposase pseudogenes (Fig. 3.4). A pseudogene is a gene copy
that does not produce a functional, full-length protein (Vanin, 1985). Seven transposase
genes were disrupted into two or three CDs in AY-WB phytoplasma genome. Another
seven CDs have sequence similarity to only part of the complete transposase genes. The
identities between the gene sequences over the aligned regions are mostly higher than
70% and some are over 95%.
The AY-WB phytoplasma genome contains functional ATP-dependent DNA
helicase genes (uvrD) and multiple copies of uvrD pseudogenes (Fig. 3.5). uvrD gene is
involved in bacterial DNA repair (Crowley and Hanawalt, 2001). OY phytoplasma
genome harbors seven copies of uvrD genes, while AY-WB phytoplasma harbors only
two copies of uvrD genes (AYWB085 and AYWB115).
Other than the transposase and ATP-dependent DNA helicase genes, AY-WB
phytoplasma genome contains multiple copies of genes encoding DNA primase,
replicative DNA helicase, ATP-dependent Zn protease, RNA polymerase sigma factors,
thymidylate kinase, single-stranded DNA binding protein, bacterial nucleoid DNA
binding protein, site-specific DNA methylase, replication initiator protein (on plasmids),
and some hypothetical proteins (Table 3.5). Similar to the transposase genes, some of the
gene copies are pseudogenes. For instance, the pseudogenes AYWB208 and AYWB209
are 149 bp apart and highly similar to the N-terminal (83%) and C-terminal (95%) of the
93
functional site-specific DNA methylase genes (AYWB382). In addition, the AY-WB
phytoplasma genome contains large amount of repetitive sequences in non-coding
regions (data not shown). These tandem and inverted repeats are potential recombination
sites leading to the loss of gene function, gene duplication or gene deletion.
3.4.8 Virulence factors
The AY-WB phytoplasma genome harbors some known virulence factors. Two
copies of genes encoding hemolysin or derivatives (hylC, AYWB563 and tlyC,
AYWB568) have been identified in the genome. Hemolysin is an extracellular poreforming toxin that has been identified in both Gram-positive bacterial pathogen, such as
Staphylococcus aureus (Menestrina et al., 2003), and Gram-negative bacterial pathogen,
such as uropathogenic Escherichia coli (UPEC) (Emody et al., 2003). The hemolysin
gene products are possibly involved in the host cell invasion of phytoplasmas.
AY-WB phytoplasma is an intracellular pathogen whose secreted or membranebound proteins are likely involved in direct or indirect interactions with host cell
components. The AY-WB phytoplasma has a Sec-dependent (type II) protein secretion
system consisting of secA (AYWB307), secE (AYWB470), and secY (AYWB504). No
genes encoding the components of the type III or IV secretion systems have been
identified in the AY-WB phytoplasma genome. Fifty phytoplasma proteins are
potentially secreted by the Sec-dependent pathway because they contain signal peptides
as predicted with the SignalP program (Bendtsen et al., 2004) (Table 3.6). As expected, 5
solute binding protein components associated with the 5 ABC transporter systems also
94
have signal peptides. Among the other 45 potentially secreted proteins, 42 cannot be
assigned functions based on sequence similarity searches. Among them, 25 are common
in both AY-WB and OY phytoplasma genomes and the other 17 are unique to AY-WB
phytoplasma. The subcellular localization of these proteins in plant cells has implications
in their functions, and therefore other web-based software was used to look for
subcellular localization domains. Indeed, seven proteins potentially target the plant cell
nuclei based on the presence of a nuclear localization signal (NLS), suggesting that these
proteins are involved in the regulation of the replication and transcription of plant genes.
Further, three proteins target microbodies and three target chloroplasts. The remaining 29
proteins are localized in the plant cell cytoplasm.
3.5 Discussion
AY-WB phytoplasma is an intracellular pathogen that invades and replicates in
cells of insect vectors and plant hosts. Within the relatively isolated environment, the
reductive evolution was thought to occur through intrachromosomal recombination
events at repeated sequences (Achaz et al., 2002). The presence of the repetitive
sequences in the AY-WB phytoplasma genome suggests that phytoplasmas are prone to
frequent recombination events. The genome contains 4 functional transposase gene genes
and 14 transposase pseudogenes. Transposase genes are capable of moving DNA
segments to new locations within and between genomes (Rice and Baker, 2001). The
transposase pseudogenes are likely the remnants of frequent transposition events.
Furthermore, the AY-WB phytoplasma genome harbors many pseudogenes that are
95
portions of other functional genes. Therefore, AY-WB phytoplasma genome has
undergone frequent recombination and transposition events. However, the pseudogenes
might gain new functions during evolution as recently demonstrated (Hirotsune et al.,
2003).
Also present in the AY-WB phytoplasma genome are several copies of DNA
replication protein-encoding genes having no homologs in other bacteria genomes. The
functional copies of these genes are present in plasmids, suggesting that phytoplasmas
obtained these genes from other organisms. The non-functional copies (pseudogenes) of
these genes are in the chromosome, which are likely the remnants of the recombination
events between the plasmids and the chromosome. Bacteria usually use a type I
restriction and modification system to defend from outer source DNA (Murray, 2000).
However, AY-WB phytoplasma has limited modification capacity and no restriction
capacity. Furthermore, AY-WB phytoplasma contains fewer copies of the uvrD gene that
is involved in DNA repair (Crowley and Hanawalt, 2001) than OY phytoplasma. All
these suggested that the phytoplasma genomes are prone to active recombination or
transposition events.
Phytoplasmas have not been successfully cultured in cell-free media, which has
hampered the research on phytoplasmas. The complete phytoplasma genome sequences
of AY-WB and OY phytoplasmas may provide some clues about the reason causing the
failure of the culturing attempts. Based on our findings, the carbon source could be the
first limiting factor. Bacteria can transport extracellular glucose via glucose
phosphotransferase (PTS) system and such a system has been identified in many bacteria
96
species (Postma et al., 1993), including some mollicutes species such as M. genitalium
(Fraser et al., 1995). However, phytoplasmas do not have a PTS system, suggesting that
phytoplasmas may not be able to uptake extracellular glucose. Phytoplasmas have all
other enzymes in the glycolysis pathway. The culturing media supplemented with
glucose-6-phosphate but not glucose may be a solution. The second limiting factor could
be the presence of inhibitory substances in the culture media (Razin et al., 1998).
Phytoplasmas have fewer multidrug resistance ABC transporters than their cultivable
spiroplasma counterpart, suggesting that phytoplasmas may have weaker detoxication
abilities than spiroplasmas do. The possible third limiting factor is that phytoplasmas may
need anaerobic conditions to grow. It was shown that phytoplasmas grow better in nonphotosynthetic tissues that contain less oxygen than in photosynthetic tissues that are rich
in oxygen (Sears et al., 1997). The AY-WB phytoplasma genome contains no genes
dealing with the reactive oxygen species.
Phytoplasmas are insect-transmitted plant pathogens able to induce physiological
changes in plants. One aim of the genome-sequencing project is to understand the
pathogenicity mechanisms of phytoplasmas. Most of the effector proteins identified so far
are from Gram-negative bacterial plant pathogens and introduced into plant cells by
bacteria type III secretion systems. The effectors could induce defense-related
hypersensitive responses in resistant plants containing resistance genes, and diseases in
susceptible plants. For instance, the AvrBs2 protein in Xanthomonas campestris pv.
vesicatoria (Kearney and Staskawicz, 1990) could induce diseases when introduced into
susceptible plant hosts. AY-WB phytoplasma genome contains hemolysin toxins, which
97
can form pores on the plasma membranes (Menestrina et al., 2003). Phytoplasmas
hemolysin gene products are possibly involved in host cell invasion of phytoplasmas. No
other known pathogenic genes has been identified in the AY-WB phytoplasma genome.
However, potential effector proteins have been predicted in the genome based on the
notion that secreted proteins may directly or indirectly interact with host cell components.
Among the potential effector proteins, 7 were predicted to target plant cell nuclei,
suggesting that they could affect the replication or transcription of plant genes. The
effects of these genes on plants could be assessed using potato virus X-based transient
expression systems (Jones et al., 1999) and other techniques. Currently, the functional
characterization of these potential effectors is underway. The verification of the functions
by mutation and complementation is only possible after the successful culturing of
phytoplasmas.
The complete genome of AY-WB phytoplasma confirms the reductive evolution
revealed by the OY phytoplasma genome, and demonstrates that the AY-WB
phytoplasma genome has undergone frequent recombination events. Most importantly, it
provides the genetic basis of valid researches on phytoplasma pathogenicity. The further
characterization of phytoplasma genomes is expected to provide answers to the questions
about the culturing and pathogenicity of phytoplasmas.
3.6 Acknowledgments
The authors thank Melanie L. Ivy and Sophien Kamoun in the Department of
Plant Pathology at The Ohio State University - OARDC for technical support and
98
constructive discussions. The authors also thank Alla Lapidus, Nikos Kyrpides, and
Agnes Radek for their assistance in the AY-WB phytoplasma genome-sequencing
project.
This project was supported by USDA/NSF Microbial Genome Sequencing
Program, Grant Number 2002-35600-12752.
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Weisburg, W.G., Tully, J.G., Rose, D.L., Petzel, J.P., Oyaizu, H., Yang, D., Mandelco,
L., Sechrest, J., Lawrence, T.G., van Etten, J., Maniloff, J. and Woese, C.R. (1989) A
phylogenetic analysis of the mycoplasmas: basis for their classification. J. Bacteriol.
171, 6455-6467.
Westberg, J., Persson, A., Holmberg, A., Goesmann, A., Lundeberg, J., Johansson, K.E.,
Pettersson, B., and Uhlen, M. (2004) The genome sequence of Mycoplasma mycoides
subsp. mycoides SC type strain PG1T, the causative agent of contagious bovine
pleuropneumonia (CBPP). Genome Res. 14, 221-227.
Woese, C.R. (1987) Bacterial evolution. Microbiol. Rev. 51, 221-271.
Zhang, J., Hogenhout, S.A., Nault, L.R., Hoy, C.W. and Miller, S.A. (2004) Molecular
and symptom analyses of phytoplasma strains from lettuce reveal a diverse
population. Phytopathology 94, 842-849.
Zhao, Y., Wang, H., Hammond, R.W., Jomantiene, R., Liu, Q., Lin, S., Roe, B.A. and
Davis, R.E. (2004) Predicted ATP-binding cassette systems in the phytopathogenic
mollicute Spiroplasma kunkelii. Mol. Gen. Genomics 271, 325-338.
104
Length (bp)
G + C ratio
Putative protein coding sequences (CDs)
Coding region (%)
Average CDS length (bp)
CDs with functional assignmenta
CDs with matches to conserved hypothetical proteins
CDs without significant database match
Ribosomal RNA operons
tRNAs
AY-WB
706,569
27%
673
74%
779
345
112
216
1
31c
OY-M
860,631
28%
754
73%
785
446
51
257
2b
32c
Table 3.1 General features of the chromosomes of the AY-WB phytoplasma and OY phytoplasma genomes
a
Functional assignment was performed using the classification scheme from Riley (1993).
The two rRNA operons are adjacent to each other, and have an isoleucine tRNA gene in between.
c
This set of tRNAs correspond to all amino acids.
b
105
Strain
Ca.
Ca.
Mycoplasma Mycoplasma Mycoplasma Mycoplasma
Phytoplasma Phytoplasma penetrans pneumoniae genitalium
mobile
asteris
asteris
AY-WB
OY-M
HF-2
M129
G-37
163K
Genome size (bp)
706 569
Organism
G + C content (mol %)
Total number of predicted CDs
Functional categories in COGs a
860 631
1 358 633
816 394
580 074
777 079
27
28
25.7
40
32
24.9
673
754
1037
689
484
633
%
%
%
%
%
%
(-) not in COGs
259
38.5
178
23.6
289
27.9
258
37.4
99
20.5
114
18.0
J, Translation
103
15.3
104
13.8
109
10.5
102
14.8
101
20.9
106
16.7
K, Transcription
21
3.1
31
4.1
33
3.2
14
2.0
14
2.9
18
2.8
L, Replication, recombination and repair
92
13.7
147
19.5
101
9.7
43
6.2
40
8.3
64
10.1
D, Cell cycle control, mitosis and meiosis
7
1.0
18
2.4
29
2.8
5
0.7
5
1.0
7
1.1
V, Defense mechanisms
7
1.0
8
1.1
36
3.5
22
3.2
8
1.7
12
1.9
T, Signal transduction mechanisms
2
0.3
2
0.3
5
0.5
3
0.4
3
0.6
7
1.1
M, Cell wall/membrane biogenesis
6
0.9
12
1.6
17
1.6
12
1.7
12
2.5
17
2.7
N, Cell motility
0
0.0
2
0.3
12
1.2
0
0.0
0
0.0
6
0.9
U, Intracellular trafficking and secretion
6
0.9
7
0.9
33
3.2
7
1.0
6
1.2
15
2.4
O, Posttranslational modification, protein
turnover, chaperones
C, Energy production and conversion
25
3.7
51
6.8
35
3.4
20
2.9
20
4.1
22
3.5
12
1.8
16
2.1
32
3.1
20
2.9
20
4.1
28
4.4
14
2.1
19
2.5
58
5.6
37
5.4
26
5.4
46
7.3
28
4.2
40
5.3
31
3.0
24
3.5
15
3.1
21
3.3
F, Nucleotide transport and metabolism
19
2.8
24
3.2
39
3.8
21
3.0
21
4.3
21
3.3
H, Coenzyme transport and metabolism
5
0.7
9
1.2
14
1.4
14
2.0
14
2.9
16
2.5
G, Carbohydrate transport and
metabolism
E, Amino acid transport and metabolism
I, Lipid transport and metabolism
8
1.2
9
1.2
16
1.5
9
1.3
9
1.9
9
1.4
P, Inorganic ion transport and
metabolism
Q, Secondary metabolites biosynthesis,
transport and catabolism
R, General function prediction only
15
2.2
17
2.3
24
2.3
17
2.5
17
3.5
15
2.4
1
0.1
1
0.1
5
0.5
0
0.0
0
0.0
1
0.2
S, Function unknown
27
4.0
36
4.8
90
8.7
45
6.5
40
8.3
51
8.1
16
2.4
23
3.1
29
2.8
16
2.3
14
2.9
37
5.8
Table 3.2 Comparison of the COG categories of the proteins in the AY-WB phytoplasma genome with
those in other mollicutes genomes
a
COG categories of, A (RNA processing and modification), B (Chromatin structure and dynamics), Y
(Nuclear structure), Z (Cytoskeleton), and W (Extracellular structure) do not apply to mollicutes.
106
ATP-binding protein
AY-WB
Membrane protein
amino acid
glnQ (AYWB636)
AYWB637 (AYWB637)
D-methionine
amino acid (Arginine)
metN (AYWB591)
AYWB589 (AYWB589)
glnP (AYWB318)
nlpA (AYWB590)
abc (39938618)
amino acid (Glutamine)
artP (AYWB267)
artQ (AYWB268),
artM (AYWB265)
artI (AYWB266)
glnQ (39938974)
dppA (AYWB531)
dppD (39938678)
Substrate
Solute-binding protein
ATP-binding protein
OY-M
Membrane protein
Solute-binding protein
Amino acid uptake
amino acid
amino acid
glnQ (39938565),
artM (AYWB125)
amino acid
artM (39938563),
artM (39938564)
PAM134 (39938620)
artM (39938942),
artI (39938943),
artM (39938950?)
artM (39938973),
artI (39938975),
artM (39938976)
artM (39939074?)
artM (39938980?),
artM (39938981?)
artM (39939125?),
mdoB (39939127?)
nlpA (39938619)
Dipeptide/oligopeptide uptake
dipeptide or oligopeptide
107
dppF (AYWB529),
dppD (AYWB530)
dppB (AYWB532),
dppC (AYWB533)
oligopeptide
dppC (39938675),
dppB (39938676)
dppB (39938508),
PAM023 (39938509)
oppA (39938677)
malK (39939238)
ugpE (39939236),
ugpA (39939237)
ugpB (39939235)
cbiO (39938506)
cbiO (39938665)
PAM19 (39938505)
cibQ (39938666)
znuC (39938579)
znuB (39938580)
znuA (39938578)
potB (39939146),
potC (39939147)
potD (39939148)
dppD (39938511),
oppF (39938512)
PAM024 (39938510)
Sugar uptake
sugar
malK (AYWB672)
malG (AYWB670),
malF (AYWB671)
cbiO (AYWB014)
cbiO (AYWB542),
cbiO (AYWB543)
mntA (AYWB625)
cbiQ (AYWB015)
cbiQ (AYWB541)
malE (AYWB669)
Inorganic ion uptake
cobalt ion
cobalt ion
Mn/Zn ion
mntB (AYWB624),
mntB (AYWB623)
znuA (AYWB626)
Multidrug resistence
multidrug
multidrug
mdlB (AYWB028)
mdlB (AYWB029)
mdlB (39938545)
Spermidine/putrescine uptake
spermidine or putrescine
potA (AYWB095)
potB (AYWB094),
potC (AYWB093)
potD (AYWB092)
potA (39939145)
Uncharacterized
possible lipoprotein
unknown
phnL (AYWB621)
phnL (AYWB135)
Table 3.3 Summary of ABC transporter genes in AY-WB and OY phytoplasma genomes
phnL (39938582)
phnL (39939085)
nlpA (39938583)
AY-WB
OY
Gene (Length, CDs)
Possible substrate
Gene (Length, Acc. no.)
Possible substrate
mgtA (920 aa, AYWB018)
mgtA (817 aa, AYWB472)
mgtA (952 aa, AYWB535)
mgtA (892 aa, AYWB243)
zntA (666 aa, AYWB652)
cation ion
cation ion
cation ion
magnesium ion
lead, cadmium, zinc, mercury
mgtA (920 aa, 39938516)
mgtA (918 aa, 39938672)
mgtA (1056 aa, 39938738)
mgtA (892 aa, 39939071)
zntA (666 aa, 39939219)
sodium/potassium ion
calcium ion
cation
magnesium ion
cadmium ion
Table 3.4 Summary of AY-WB phytoplasmas P-type ATPase
108
CDs b
Length Alignment to
(aa) prototype c
d
d
Positives Gaps
DNA primase e
AYWB179
AYWB288
AYWB618
AYWB220
AYWB047
AYWB086
AYWB087
AYWB048
AYWB172
AYWB079
1-441/1-441
1-440/1-440
1-369/73-441
1-441/1-441
1-156/1-156
11-174/84-247
7-119/247-359
1-98/301-398
1-68/92-159
1-37/1-37
100%
97%
100%
86%
99%
96%
99%
100%
96%
100%
none
none
none
none
none
none
none
none
none
none
124
104
104
104
104
104
111
104
111
63
51
100%
99%
99%
99%
93%
84%
84%
83%
78%
86%
77%
none
none
none
none
none
none
0%
none
0%
none
1%
100%
95%
97%
92%
91%
84%
94%
97%
82%
none
none
none
none
none
1%
none
none
none
1-337/1-337
40-157/1-118
1-203/135-337
100%
96%
93%
none
none
none
1-210 / 1-210
1-210 / 1-210
1-209 / 1-210
1-209 / 1-209
1-54 / 33-86
1-62 / 70-131
100%
95%
83%
80%
86%
73%
none
none
0%
none
none
none
100%
100%
93%
none
none
none
Hypothetical protein
100%
96%
96%
none
none
none
Conserved hypothetical protein
1-124 / 1-124
1-103 / 1-103
1-103 / 1-103
1-103 / 1-103
1-103 / 1-103
1-102 / 1-102
1-104 / 1-104
1-103 / 1-103
1-103 / 1-103
2-62 / 43-103
1-51 / 1-50
768
151
173
120
89
93
56
51
69
1-768 / 1-768
1-151 / 618-768
30-156 / 635-761
1-96 / 488-583
1-87 / 394-480
2-69 / 695-761
1-56 / 1-56
1-44 / 259-302
1-59 / 373-431
337
157
203
Thymidylate kinase
AYWB074
AYWB182
AYWB285
AYWB223
AYWB154
AYWB197
210
210
209
210
59
73
Bacterial nucleoid DNA binding protein
AYWB275
AYWB193
AYWB385
122
110
83
1-122 / 1-122
1-110 /13-122
1-82 / 13-94
Bacterial nucleoid DNA binding protein
AYWB231
AYWB211
AYWB310
96
96
96
1-96 / 1-96
1-96 / 1-96
1-96 / 1-96
AYWB161
AYWB082
157
156
1-157/1-157
1-156/1-157
100%
98%
none
0%
1-224/1-224
1-224/1-224
1-149/76-224
1-77 / 124-199
100%
98%
92%
84%
none
none
none
1%
1-77 / 1-77
1-63 / 1-63
100%
88%
none
none
1-103 / 1-103
1-61 / 1-59
3-60 / 46-103
100%
86%
89%
none
3%
none
1-54 / 1-54
1-54 / 1-54
100%
100%
none
none
1-285/1-285
1-47/1-47
1-74/133-206
6-54/215-263
100%
97%
91%
73%
none
none
none
none
100%
90%
86%
91%
87%
82%
none
0%
none
none
1%
none
100%
98%
89%
80%
95%
none
none
none
none
none
1-250 / 1-250
1-208 / 42-250
1-208 / 42-250
1-166 / 42-208
1-103 / 42-145
16-131 / 1-116
15-112 / 141238
3-49 / 193-239
100%
94%
93%
93%
92%
77%
79%
none
0%
0%
0%
0%
1%
none
97%
none
1-285 / 1-285
1-274 / 1-274
1-52 / 1-52
100%
99%
95%
none
none
none
100%
87%
78%
none
0%
none
Conserved hypothetical protein
AYWB162
AYWB083
AYWB361
AYWB166
224
224
149
92
Hypothetical protein
AYWB023
AYWB158
77
63
AYWB081
AYWB158
AYWB159
103
63
60
Hypothetical protein
AYWB080
AYWB157
54
54
Hypothetical protein
AYWB186
AYWB396
AYWB395
AYWB394
285
81
75
56
Conserved hypothetical protein
ATP-dependent Zn protease
AYWB214
AYWB378
AYWB377
Positives d Gaps d
Conserved hypothetical protein
ATP-dependent Zn protease
AYWB230
AYWB350
AYWB344
AYWB351
AYWB352
AYWB071
AYWB354
AYWB353
AYWB170
Length Alignment to
(aa)
prototype c
Conserved hypothetical protein
441
441
369
441
156
175
119
98
70
50
Single-stranded DNA binding protein
pIII-b
pIV-c
pII-b
pI-e
AYWB150
AYWB274
AYWB381
AYWB194
AYWB233
AYWB384
AYWB210
CDs b
AYWB183
AYWB224
AYWB284
AYWB198
AYWB199
AYWB389
212
209
122
108
54
56
1-212/1-212
1-209/1-208
1-120/91-210
1-107/91-197
1-54/1-55
1-42/91-132
Conserved hypothetical protein
AYWB620
AYWB181
AYWB286
AYWB222
AYWB075
248
203
203
201
132
1-248 / 1-248
6-203 / 51-248
6-203 / 51-248
4-201 / 51-248
6-132 / 51-177
Conserved hypothetical protein
AYWB289
AYWB177
AYWB366
AYWB616
AYWB049
AYWB173
AYWB217
250
208
208
188
106
134
120
AYWB387
60
AYWB178
AYWB617
AYWB219
AYWB276
AYWB192
AYWB024
285
278
54
147
146
129
1-147 / 1-147
1-146 / 1-147
12-120 / 31-139
(Continued)
Table 3.5 Summary of the redundant (either functional or non-functional) genes in the AY-WB
phytoplasma genome a
109
Table 3.5 (continued)
CDs b
Length Alignment to
(aa) prototype c
Positives
d
Gaps d
Site-specific DNA methylase
AYWB382
AYWB208
AYWB209
294
125
107
1-294/1-294
1-125/14-139
1-107/188-294
CDs b
500
498
498
471
99
87
81
97
86
60
55
57
1-500/1-500
2-498/4-500
2-498/4-500
2-470/4-472
3-99/342-438
1-87/367-453
1-81/420-500
3-86/416-500
1-86/174-259
2-60/4-62
3-55/319-371
1-54/255-308
none
0%
none
100%
95%
92%
94%
96%
96%
98%
94%
88%
94%
89%
83%
none
none
none
none
none
none
none
1%
none
none
none
none
100%
95%
79%
82%
86%
94%
76%
84%
70%
none
none
none
2%
none
none
2%
none
none
100%
88%
78%
none
0%
none
pII-e
pIV-f
pI-b
pIII-f
AYWB408
208
208
201
144
120
103
90
60
63
1-208/1-208
1-208/1-208
1-199/1-199
1-143/1-145
1-111/1-111
1-83/1-83
2-89/70-159
4-60/38-94
10-63/125-178
Replication initiator protein
pII-a
pIV-a
AYWB406
a
b
c
d
e
382
375
58
1-382 / 1-382
1-363 / 1-363
15-53 / 218-256
156
156
156
138
121
1-156 / 1-156
1-156 / 1-156
1-156 / 1-156
1-138 / 1-141
1-94 / 1-94
100%
99%
96%
70%
81%
none
none
none
6%
none
1-202 / 1-202
1-202 / 1-202
100%
93%
none
none
1-277 / 1-277
23-240 / 1-218
100%
94%
none
none
1-100 / 1-100
1-100 / 1-100
100%
93%
none
none
1-197 / 1-197
1-146 / 52-197
100%
99%
none
none
1-160 / 1-160
1-160 / 1-160
1-161 / 1-159
1-50 / 1-50
100%
98%
77%
84%
none
none
2%
none
100%
91%
89%
none
none
none
100%
92%
99%
none
none
none
Conserved hypothetical protein
202
AYWB379
AYWB213
202
Hypothetical protein
277
AYWB215
AYWB376
240
Hypothetical protein
AYWB203
AYWB370
100
100
Hypothetical protein
pI-d
pIII-c
RNA polymerase sigma factor
AYWB195
AYWB362
AYWB234
AYWB383
AYWB207
AYWB357
AYWB293
AYWB045
AYWB044
Positives d Gaps d
Hypothetical protein
100%
83%
95%
Replicative DNA helicase
AYWB287
AYWB180
AYWB221
AYWB619
AYWB078
AYWB392
AYWB046
AYWB341
AYWB077
AYWB076
AYWB340
AYWB643
Length Alignment to
(aa)
prototype c
197
146
Hypothetical protein
pIV-e
pII-d
pI-c
pIII-d
160
160
161
50
Conserved hypothetical protein
AYWB240
AYWB201
AYWB200
235
53
96
1-235/1-235
1-51/1-51
1-95/133-227
Conserved hypothetical protein
AYWB227
AYWB347
AYWB346
206
149
58
1-206/1-206
1-141/1-141
1-58/149-206
The CDs in this table include the CDs on plasmids. The redundant genes include the pesudogenes.
The CDs were represented with the names of CDs in the chromosome and the plasmids. For example,
pI-a represents the first CD in plasmid I. Plasmids I, II, III, and IV are pAYWB1031, pAYWB1059,
pAYWB1063, and pAYWB1110, respectively.
The prototypes were the first CDs that were underlined in each group. The alignments were presented in
the format of start-end (CDs) / start-end (prototype).
The identities, positives and gaps were the percentage of identical amino acid residues, positive amino
acid residues and non-aligned amino acid residues in the aligned sequences between the CDs and the
prototypes. "none" stands for "no gaps". 0% means there are gaps, but the percentage is below 1%.
The annotation was according to the prototype. The CDs in the group do not necessarily have the same
annotation with the prototype since some CDs are only part of the prototypes.
110
CDs
SP a
Length
Probability (aa)
IP
MW Cleavage Annotation
(Da) positions
Localization in plant b
OY-M
orthologs
nucleus
microbody
cytoplasm
chloroplast stroma
cytoplasm
cytoplasm
cytoplasm
cytoplasm
cytoplasm
n/a
chloroplast stroma
cytoplasm
plasma membrane
cytoplasm
cytoplasm
cytoplasm
cytoplasm
cytoplasm
cytoplasm
cytoplasm
nucleus
cytoplasm
endoplasmic reticulum
n/a
39939004
39938818
39938556
39939149
n/a
39939096
n/a
n/a
n/a
39938972
39938905
39939062
39939048
39938535
n/a
39938550
n/a
n/a
39939068
n/a
39939027
39938975
cytoplasm
nucleus
nucleus
cytoplasm
cytoplasm
cytoplasm
cytoplasm
cytoplasm
microbody
cytoplasm
cytoplasm
cytoplasm
cytoplasm
nucleus
cytoplasm
nucleus
nucleus
cytoplasm
cytoplasm
39938886
39939180
39939176
n/a
n/a
39938930
39938832
39938818
n/a
n/a
n/a
39939026
39939063
n/a
39939048
39938878
39939176
39938774
39938727
cytoplasm
cytoplasm
n/a
cytoplasm
39938677
39938643
n/a
39938619
cytoplasm
microbody
chloroplast thylakoid
membrane
cytoplasm
39938578
39939176
n/a
a
AYWB022
AYWB032
AYWB033
AYWB073
AYWB091
AYWB127
AYWB145
AYWB146
AYWB148
AYWB152
AYWB169
AYWB190
AYWB204
AYWB213
AYWB225
AYWB226
AYWB230
AYWB237
AYWB238
AYWB246
AYWB259
AYWB260
AYWB266
0.988
1
1
0.98
0.994
0.999
0.813
0.547
0.973
0.688
1
0.67
0.8
0.977
0.996
0.941
0.728
0.965
0.809
1
0.98
0.655
0.611
125
135
117
186
106
206
154
198
230
61
192
264
269
202
124
230
768
192
131
199
77
259
291
10.73
7.66
10.53
11.37
8.85
8.36
9.90
9.25
9.91
10.61
5.85
9.83
4.48
7.64
9.77
8.36
9.78
7.09
10.02
9.89
10.23
7.27
9.08
15,165
15,844
13,925
21,909
12,483
23,611
17,765
23,558
27,041
7,399
22,132
31,252
31,561
23,097
14,480
26,992
89,434
22,711
15,609
23,659
9,247
31,290
32,560
32
32
31
32
53
34
45
43
45
46
35
32
31
30
33
39
27
31
32
30
31
38
23
AYWB272
AYWB278
AYWB283
AYWB297
AYWB298
AYWB332
AYWB342
AYWB343
AYWB345
AYWB369
AYWB370
AYWB372
AYWB372
AYWB373
AYWB379
AYWB390
AYWB405
AYWB436
AYWB482
1
0.947
0.812
0.806
0.541
0.989
0.98
0.981
0.919
0.766
0.793
0.973
0.936
0.999
0.982
0.596
0.789
0.889
0.552
235
715
87
165
106
285
281
65
69
117
100
130
163
121
202
149
105
401
338
9.86
8.42
10.64
10.20
8.91
9.68
9.13
6.50
10.79
9.71
9.71
11.31
9.19
10.03
8.88
10.29
10.69
10.02
9.89
28,073
83,908
10,357
19,930
12,524
33,435
33,771
7,626
8,081
13,762
11,987
15,301
19,062
14,360
23,126
17,712
12,327
46,845
37,578
40
30
34
32
32
39
31
32
39
32
32
37
32
31
30
36
34
49
29
AYWB531
AYWB564
AYWB570
AYWB590
0.617
0.657
0.718
0.816
513
311
55
348
8.37
10.22
10.30
9.31
59,420
36,578
6,229
40,170
24
41
36
42
AYWB626
AYWB642
AYWB647
0.984
0.833
0.917
380
92
268
7.95 43,443
10.70 10,887
8.98 31,443
34
32
39
AYWB669
0.75
546
9.35 64,099
33
Hypothetical protein
Conserved hypothetical protein
Conserved hypothetical protein
Conserved hypothetical protein
Hypothetical protein
Hypothetical protein
Conserved hypothetical protein
Hypothetical protein
Hypothetical protein
Hypothetical protein
Conserved hypothetical protein
Conserved hypothetical protein
Conserved hypothetical protein
Conserved hypothetical protein
Conserved hypothetical protein
Hypothetical protein
hflB, ATP-dependent Zn protease
Hypothetical protein
Hypothetical protein
Conserved hypothetical protein
Hypothetical protein
Conserved hypothetical protein
artI, ABC-type amino acid transport
system, amino acid binding protein
Conserved hypothetical protein
hflB, ATP-dependent Zn protease
Conserved hypothetical protein
Hypothetical protein
Hypothetical protein
Conserved hypothetical protein
Conserved hypothetical protein
Hypothetical protein
Hypothetical protein
Hypothetical protein
Hypothetical protein
Conserved hypothetical protein
Conserved hypothetical protein
Hypothetical protein
Conserved hypothetical protein
Conserved hypothetical protein
Conserved hypothetical protein
Conserved hypothetical protein
gpsA, glycerol 3-phosphate
dehydrogenase
dppA, dipeptide-binding protein
Conserved hypothetical protein
Hypothetical protein
nlpA, ABC-type methionine transport
system, periplasmic component
znuA, Manganese-binding protein
Conserved hypothetical protein
Hypothetical protein
malE, putative MalE
(maltose/maltodextrin-binding protein)
39939235
(Continued)
Table 3.6 Summary of the secreted proteins identified in AY-WB phytoplasma genome
111
Table 3.6 (Continued)
a
The SP (signal peptide) probability and cleavage positions in the amino acid sequences were predicted by
the SignalP program (version 3.0) (Bendtsen et al., 2004).
b
The potential localization of the proteins in plants was predicted by pSORT program (Nakai and Horton,
1999) with the proteins excluding signal peptides. Some proteins are too small for prediction after the
cleavage of signal peptides.
112
Fig. 3.1 Maps of the AY-WB phytoplasma plasmids. The sizes of the plasmids were drawn to scale. The
CDs and their orientations were indicated by arrows. CDs sharing similarities were marked in the same
color. Single stranded DNA binding protein genes (ssb, in brown) and genes encoding conserved
hypothetical proteins (in orange, navy and dark green) are present in all plasmids. pAYWB1031 and
pAYWB1063 have similar genes (rep, Sapphire blue) encoding replication initiation proteins that are
similar to germinivirus replication proteins. pAYWB1059 and pAYWB1110 have similar replication
protein-encoding genes (rep2, in black). ORF7 in pAYWB1063 (in dark gray) and ORF2 in pAYWB1110
(in light gray) have no similarity to any other proteins.
113
Fig. 3.2 The summary of the AY-WB phytoplasma genome-encoded transporters and central metabolic
pathways. The question marks (?) indicated that the substrates of the transporters were unknown. The
numbers next to the transporters indicated that copies of the same transporter in the AY-WB phytoplasma
genome. The enzyme commission (EC) number of each enzyme was indicated in the reaction the enzyme
catalyzed. Only central metabolic pathways were illustrated.
114
Fig. 3.3 AY-WB phytoplasma genome contains more repetitive sequences, both tandem repeats and
inverted repeats, than the OY phytoplasma genome. However, the repeats in the OY phytoplasma genome
are larger than those in the AY-WB phytoplasma genome. The prediction was done by using the REPuter
program (Kurtz and Schleiermacher, 1999) hosted at the Bielefeld University Bioinformatics Server. The
color coding of the size of the repetitive sequences was indicated at the bottom of each section.
115
Fig. 3.4 The AY-WB phytoplasma genome contained many copies of transposases genes and derivatives.
The ruler was in the unit of bp (base pairs). The identities, positives and gaps of the comparison of the
derivatives to the transposase gene (tra5) (in red) were indicated at right. The lengths of the CDs were
indicated in the parentheses next to the names. The numbers at the beginning and the end of the CDs
indicated the start and the end of the alignments with the transposase gene AYWB176.
116
Fig. 3.5 The AY-WB phytoplasma genome contained one copy of complete ATP-dependent DNA helicase
and multiple copies of pseudogenes. The ruler was in the unit of bp (base pairs). The identities, positives
and gaps of the comparison of the derivatives to the ATP-dependent DNA helicase (AYWB085, uvrD) (in
red) were indicated at right. The lengths of the CDs were indicated in the parentheses next to the names.
The numbers at the beginning and the end of the CDs indicated the start and the end of the alignments with
the uvrD gene AYWB085. The AYWB294-296, AYWB035-041, AYWB639-641, AYWB644-645, and
AYWB020-021 were disrupted partial ATP-dependent DNA helicase pseudogenes. Others were
pseudogenes partially aligned to the uvrD gene.
117
CHAPTER 4
Identification and characterization of traE genes of
Spiroplasma kunkelii
Xiaodong Bai, Tatiana Fazzolari and Saskia A. Hogenhout *
Department of Entomology, The Ohio State University – Ohio Agriculture and
Development Center (OARDC), Wooster, OH 44691
118
4.1 Abstract
Four traE homologs, designated traE1, traE2, traE3 and traE4, were identified
and amplified from the genome of leafhopper-transmitted corn stunt pathogen
Spiroplasma kunkelii, and predicted to encode membrane-bound ATPases. Deduced
proteins of all traE genes have 62.3% to 89.9% similarity to the conserved VirB4 domain
that are frequently components of type IV secretory pathways involved in intracellular
trafficking and secretion of DNA and proteins. In phylogenetic analysis, TraE homologs
of S. kunkelii, Mycoplasma pulmonis and M. fermentans cluster together and are more
similar to TraE proteins of Gram-positive bacteria than to those of Gram-negative
bacteria, thereby resembling the 16S rRNA phylogeny. Gene traE2 was most conserved,
whereas the presence of other three traE genes varied among S. kunkelii strains, M2, CS2B, FL-80 and PU8-17. Further, traE1 and traE2 appeared to be located on the
chromosome, and traE3 and traE4 genes on plasmids of S. kunkelii strain M2. Transcripts
of the spiralin gene and traE2 genes were detected on Northern blots containing total
RNA of S. kunkelii cultures, and S. kunkelii-infected plants and insects, in which traE2
appeared to be of a larger transcription unit. Full-length expression products of the other
traE genes were not detected. The possibility that S. kunkelii traE genes are part of
regions involved in S. kunkelii cell morphogenesis, adhesion and DNA recombination is
discussed. This is the first study providing the localization of traE genes on spiroplasma
plasmids and the expression pattern in various spiroplasma environmental niches.
119
4.2 Introduction
Spiroplasma kunkelii, the causal agent of corn stunt disease, causes economically
significant yield losses of corn on the American continent (Hruska and Gomez-Peralta,
1997). S. kunkelii belongs to the genus Spiroplasma of the Class Mollicutes, of which
members are believed to be diverged from a Gram-positive ancestor. Mollicutes
apparently underwent extensive gene loss events resulting in reduced genome sizes and
loss of peptidoglycan cell walls (Bové et al., 1989; Gasparich, 2002). S. kunkelii and two
other members of the genus Spiroplasma, S. citri and S. phoeniceum, are insecttransmitted plant pathogens that replicate in both insect vectors and plant hosts.
Spiroplasmas are mostly helical in culture media and plant phloem tissues. However, in
insects, they appear to be more often round and flask-shaped (Kwon et al., 1999; Özbek
et al., 2003).
Spiroplasmas employ various attachment structures to initiate contact with insect
cells. Spiroplasma membranes contacting the extracellular lamina of insect epithelial and
muscle cells appeared thickened (Fletcher et al., 1998; Özbek et al., 2003) and fimbriaelike appendages apparently protruding from the S. kunkelii cell surface seemed to attach
to the external laminae of insect epithelial and muscle cells (Özbek et al., 2003).
Furthermore, S. kunkelii cells appeared to be connected by pilus-like structures (Özbek et
al., 2003). These attachment structures have not been observed in other members of the
Class Mollicutes (Razin et al., 1998).
This study was aimed to identify and characterize S. kunkelii gene(s) potentially
involved in the biosynthesis of pili and/or fimbriae in the genome of S. kunkelii CR2-3x
120
and four other strains. We identified four homologs of transfer (tra) genes. traE genes
have been shown to be involved in conjugation, type IV secretion and/or cell invasion in
various bacteria (Censini et al., 1996; Lai and Kado, 2000).
4.3 Materials and Methods
4.3.1 Culturing of S. kunkelii strains
S. kunkelii strains M2 from Poza Rica, Mexico (Bai and Hogenhout, 2002; Ebbert
and Nault, 2001), CS-2B from California, FL-80 from Florida, and PU8-17 from Peru
(Lee and Davis, 1989) were cultured in LD8A3 medium as described before (Lee and
Davis, 1989). Spiroplasmas cultures were harvested at log growth phases.
4.3.2 Computational analysis
Genome sequences of S. kunkelii strain CR2-3x were obtained from the genome
sequence project website hosted by the Advanced Center for Genome Technology at
University of Oklahoma (http://www.genome.ou.edu/spiro.html). This strain was
originally isolated from leaves of a maize plant (Zea mays L.) naturally infected with S.
kunkelii in Costa Rica (Zhao et al., 2003). Putative Open Reading Frames (ORFs)
beginning with start codon ATG and ending with stop codons TAG and TAA (Bové et
al., 1989) were predicted using a Windows-based ORF extractor program
(http://www.oardc.ohio-state.edu/mcic/bioinformatics/bio_software/). Translated protein
sequences were searched against the NCBI nr database and various customized databases
consisting of genes involved in virulence, conjugation and/or pili formation using stand-
121
alone BLAST v2.0 (Altschul et al., 1997) on a local Linux workstation. The large
BLAST output text files were parsed into tab-separated formats using a Perl script and
imported into the database program FileMaker®. Contigs containing sequences of interest
were analyzed using MacVector® program. The NCBI pairwise BLAST was used for
nucleotide and amino acid comparisons. Gene codon usage was analyzed by the Chips
program (Wright, 1990) in EMBOSS. The alignment of traE genes deduced protein
sequences was produced using ClustalW (version 1.8) (Thompson et al., 1994). Protein
transmembrane domains were identified using the TMHMM2.0 program (Sonnhammer et
al., 1998) and repeats in amino acid sequences by the RADAR program (Heger and
Holm, 2000) from EBI (European Bioinformatics Institute). The SAM-T02 program
(Karplus et al., 2001) was used for domain searches. The alignment generated by
ClustalX (version 1.81) (Thompson et al., 1997) was used for phylogenetic tree
construction using PAUP* 4.0 (Swofford, 2001).
4.3.3 Genomic DNA isolation
Harvested S. kunkelii culture was pelleted at 26,900 x g at 4°C for 30 min.
Genomic DNA was extracted using the Genomic-tip 100/G kit (Qiagen, Inc., Valencia,
CA, USA) following the manufacturer’s protocol. DNA were resuspended in sterile water
and stored at -20ºC.
122
4.3.4 Amplification and sequence analysis
In order to sequence full-length traE genes designated traE1, traE2, traE3 and
traE4 from S. kunkelii strain M2, overlapping fragments were sequenced and included
upstream and downstream regions of traE genes. The following primers were used:
5'gggttaaattagatatgaaaag3' (traE1 forward1); 5'gtcctgaatagtattaattg3' (traE1 reverse1);
5'taaaaaaaattattttatagcaaatg3' (traE1 forward2); 5'gcaaaaatttaacaaaattcttc3' (traE1
reverse2); 5'cctttttgtgataaaggaaaac3' (traE2 forward1); 5'gtggatagattggataattttg3' (traE2
reverse1); 5'gtataaatgatgcgcaattttc3' (traE2 forward2); 5'gtctttccatttttcttccct3' (traE2
reverse2); 5'taagaaaaaagataaaggagac3' (traE3 forward1); 5'ttaatatgagttggtaattttgta3'
(traE3 reverse1); 5'tcataccaacaaatacagcaat3' (traE3 forward2); 5'gttcagttattttgttttactttc3'
(traE3 reverse2); 5'tagtgttttaaaacaagaaaacat3' (traE4 forward1): 5'ctttgttgtatttgttggtataat3'
(traE4 reverse1); 5'ttataccaacaaatacaacaaag3' (traE4 forward2); 5'gttcagttattttgttttacttcc3'
(traE4 reverse2). Amplification products were sequenced from both strands using 64-lane
Perkin-Elmer ABI377 Prism DNA sequencing machine and ABI BigDye Terminator
Reaction kit (Applied Biosystems, Inc., Foster City, CA, USA). Sequence quality was
assessed by MacPhred-MacPhrap. Low quality sequences (quality score < 20) were
trimmed and sequence tags were assembled using the SequencerTM (version 4.1)
software.
4.3.5 Restriction fragment analysis
Genomic DNAs (0.25 µg) were digested with AluI, EcoRV or TaqI (Promega,
Inc., Madison, WI, USA). Restriction fragments were size-separated on a 1% agarose gel
123
and transferred to BrightStar Plus Positively Charged nylon membranes (Ambion, Inc.,
Austin, TX, USA) by capillary transfer following standard procedures (Sambrook et al.,
1989). DIG-labeled probes were generated by PCR using primers of traE1 forward1 and
reverse1, traE2 forward1 and reverse1, and traE3 forward1 and reverse1 following the
protocol of PCR DIG Probe Synthesis Kit (Roche Diagnostics Co., Basel, Switzerland).
Prehybridization, hybridization, washing and detection were performed at 42°C following
the instruction for DIG Wash and Block Buffer Set (Roche Diagnostics Co.).
4.3.6 Pulsed field gel electrophoresis, Southern blotting and hybridization
Agarose sample blocks for each S. kunkelii strain were prepared from 30 ml
cultures. Bacteria were pelleted by centrifugation at 26,900x g and bacterial pellets were
used for plug preparation and pulsed field gel electrophoresis following the description in
the section for preparation of agarose embedded bacterial DNA of the CHEF-DR® III
Pulsed Field Electrophoresis Systems Instruction Manual (Bio-Rad Laboratories, Inc.,
Hercules, CA, USA). The parameters of Pulsed Field Gel Electrophoresis (PFGE) are: 60
sec of initial switch time, 120 sec of final switch time, 120º angle, 4 V cm-1 voltage
gradient, 14ºC, 20 h of run time. Pump dial set was set to 70 (~ 0.75 l min-1) to keep
circulation of running buffer. Southern blots of PFGE gels were obtained by capillary
transfer following standard procedures (Sambrook et al., 1989). PCR products of primers
described in section 4.3.5 were labeled with Random Primers DNA Labeling System
(Invitrogen, Inc., Carlsbad, CA, USA) using [α-32P]-dCTP. Hybridization was performed
124
following suggested protocol of Random Primers DNA Labeling System (Invitrogen,
Inc.).
4.3.7 Northern blot hybridization
Total RNA was isolated from S. kunkelii M2 culture, S. kunkelii M2 infected
insects and plants, and non-infected insects and plants following the instruction manual
of ToTALLY RNA Extraction Kit (Ambion, Inc.). 10 µg total RNA from insects and 1µg
total RNA from culture and plants were mixed with glyoxal load dye (Ambion, Inc.) at
1:1 (v/v) ratio. The mixture was incubated at 50°C for 30 min, followed by cooling on
ice. RNA samples were separated on 1.4% agarose gels prepared in 1 x BPTE (10 mM
PIPES, 30 mM Bis-Tris, 1 mM EDTA, pH 6.5) at 70 mV for 2 h. RNA was transferred to
BrightStar-Plus Positively Charged Nylon membrane (Ambion, Inc.) by capillary transfer
following standard procedure (Sambrook et al., 1989). RNA on blots was immobilized by
exposure to UV light for 3 min. Synthesis of [α-32P]-dCTP-labeled probes and
hybridization were performed as described in section 4.3.6.
4.3.8 Nucleotide sequence accession numbers
The nucleotide sequence data of traE1, traE2, traE3, and traE4 genes were
deposited in the GenBank database under the accession number of AY233334,
AY233335, AY23336, and AY23337, respectively.
125
4.4 Results
4.4.1 Identification of four traE homologs in S. kunkelii CR2-3x genome
The discovery of S. kunkelii fimbriae and pili (Özbek et al., 2003) prompted the
search of genes potentially involved in fimbriae formation and conjugation in the gapped
genome sequence of S. kunkelii strain CR2-3x (http://www.genome.ou.edu/spiro.html).
Computer analysis resulted in identification of four complete traE ORFs of 2,520 to
2,697 nucleotides in length with significant protein sequence similarities (E value < 1e-5)
to other traE homologs, including TrsE of Mycoplasma pulmonis (NP_326214,
Chambaud et al., 2001), four M. fermentans TraE homologs (AAN85227, AAN85273,
AAN85276, AAN85277, Calcutt et al., 2002), Lactococcus lactis TraE (NP_047296,
Doughtery et al., 1998), B. anthracis pX02.09 (NP_053164, Okinaka et al., 1999),
Staphylococcus aureus TrsE (E36891, Morton et al., 1993), Helicobacter pylori CagE
(AAF80209, Censini et al., 1996), and Agrobacterium tumefaciens VirB4 (CAA29975,
Thompson et al., 1988). Most of these TraE homologs are part of conjugation or type IV
secretion systems and some are encoded by genes on plasmids. The four S. kunkelii traE
homologs were designated traE1, traE2, traE3 and traE4.
A fifth traE-like locus spanning two adjacent ORFs (a and b) was also identified.
This locus with a total length of 972 nucleotides was considerably shorter than the four
identified S. kunkelii traE genes. Interestingly, both ORFs were most similar to TraE1 in
that the deduced protein sequence of ORFa shared 97% similarity to residues 738-828 of
the TraE1 C-terminal (Ct) region, and ORFb 92% to residues 49-163 of the TraE1 Nterminal (Nt) region. Further, the deduced protein sequence between ORFa and ORFb
126
shared 86% similarity to residues 616-735 located between Ct and Nt of TraE1. Thus,
apparently this fifth traE-like sequence underwent inversion/deletion events and was
excluded from further analysis because it is unlikely to encode a functional protein. No
additional traE homologs were identified in Blast searches of the five traE sequences
against updated S. kunkelii CR2-3x genome sequences of March 18, June 8 and October
13 of 2002, and April 26 of 2003.
4.4.2 Sequence features of S. kunkelii traE genes
Sequences of S. kunkelii M2 traE1, traE2, traE3 and traE4 PCR products showed
100% nucleotide similarities with corresponding genes of S. kunkelii CR2-3x. Sequence
features of S. kunkelii traE genes were summarized in Table 4.1. The gene lengths ranged
from 2,520 nucleotides of traE3 and traE4 to 2,697 nucleotides of traE1. The GC content
of the traE1 gene was 23%, which is identical to that of the gapped genome sequences of
S. kunkelii CR2-3x. The GC contents of traE3 and traE4 were 28%. The codon usage
(Nc) values ranged from 33.40 for traE2 to 40.85 for traE3, indicating traE2 has the
strongest codon bias and traE3 has the lowest codon bias. A stronger codon bias adds
more confidence that the ORF is more likely to be transcribed at a higher efficiency.
Pairwise BLAST analysis of deduced protein sequences of the four traE genes showed
sequence similarities of 43% to 96%, in which TraE3 and TraE4 shared the highest
similarity of 96%.
Several conserved regions were identified in the deduced protein sequences of S.
kunkelii traE genes (Fig. 4.1). The domain study program SAM-T02 (Karplus et al.,
127
2001) identified significant similarities (E value < 1e-10) to ATPase domains of PDB
entry 1e9rA, including the highly conserved DEAH box, in the C-terminal portions of
TraE1 (amino acids 513-832), TraE2 (amino acids 527-845) and TraE3 and TraE4
(amino acids 474-782). Further, three conserved transmembrane domains were predicted
in the N-terminal regions of all four proteins (Fig. 4.1). These data suggested that all S.
kunkelii M2 TraE proteins are membrane-bound ATPases, and that unlike most TraE
proteins, the C-terminal portions of approximately 800 amino acids appear to be located
extracellularly. Conserved Domain (CD) search performed at NCBI website revealed that
deduced proteins of all traE genes have 62.3% to 89.9% similarity to the conserved
VirB4 domain. Proteins containing VirB4 domains are frequently components of type IV
secretory pathways involved in intracellular trafficking and secretion (Hofreuter et al.,
2001; Li et al., 1999). Phylogenetic analysis revealed that the TraE homologs of
mollicutes cluster together and are more similar to TraE proteins of Gram-positive
bacteria than to those of Gram-negative bacteria (Fig. 4.2), thereby resembling the 16S
rRNA phylogeny (Gasparich, 2002).
4.4.3 Presence of traE1, traE2, and traE3/4 among four S. kunkelii strains
The presence of traE1, traE2, traE3 and traE4 was investigated in the four S.
kunkelii strains M2, CS-2B, FL-80 and PU8-17 by Southern blot hybridization. These
four strains were selected because of their differences in collection sites and culturing
history, and consequently may differ in the presence of traE genes. DIG-labeled probes
corresponding to the 5' halves of traE1, traE2 and traE3/4 genes (Fig. 4.3A) were
128
synthesized as described in section 4.3.5. Restriction enzymes used for genomic DNA
digestion were EcoRV, AluI, and TaqI for traE1, traE2 and traE3/4 hybridization,
respectively (Fig. 4.3A). The traE3/4 probe hybridized to both traE3 and traE4 genes
because of the high sequence similarity between them (Table 4.1). However, traE3 and
traE4 can be distinguished by TaqI digestion patterns (Fig. 4.3A).
S. kunkelii M2, CS-2B and FL-80, but not PU8-17, had at least one copy of a
traE1 homolog (Fig. 4.3A). The S. kunkelii M2 EcoRV digestion pattern matched that of
the in silico digestion pattern of S. kunkelii CR2-3x. A fourth EcoRV site was present
3,509 bp upstream of the EcoRV site in the 5' region of traE1, matching the ~ 3,500 bp
band of S. kunkelii M2, CS-2B and FL-80. However, FL-80 lacked the 900 bp EcoRVEcoRV fragment suggesting that FL-80 lacks the 900 bp EcoRV-EcoRV fragment and
strain CS-2B had an extra 2.8 kb fragment probably derived from a second copy of traE1.
S. citri seemed to harbor a traE1 homolog.
traE2 appeared to be present in the genomes of all tested S. kunkelii strains (Fig.
4.3B). A band of ~ 1,300 bp was detected in all strains and matched the in silico AluI
digestion of S. kunkelii CR2-3x because an AluI site was detected at 1,312 bp upstream of
the AluI site of position 261 in the 5’ region of traE2. The 402 and 351 bp AluI-AluI
bands were not visible in this experiment as they were too small. The traE2 probe
hybridized to one (M2) or two (CS-2B, FL80, PU8-17) bands that did not match the in
silico AluI digestion pattern of traE2. Because the hybridization signal was weaker than
that of the ~ 1,300 bp fragment, it is likely to be the result of cross-hybridization to
another traE2-like sequence that has not yet been sequenced from S. kunkelii CR2-3x.
129
M2 and FL-80 genomes harbor identical copies of traE3 and traE4, whereas CS2B seemed to contain only traE4 and PU8-17 contained neither (Fig. 4.3B). The in silico
TaqI digestion patterns of S. kunkelii M2 traE3 and traE4 matched the hybridized bands
on Southern blots. A TaqI site was located 359 bp upstream of traE3 matching the 941bp fragment (Fig. 4.3B). TaqI sites located 3,461 bp upstream and 1798 downstream of
traE4 gene explained the 3,756 bp and 2,275 bp fragments (Fig. 4.3B). Apparently, S.
kunkelii M2, FL-80 and CS-2B but not PU8-17 possesses a third homolog of traE3 and
traE4 because one hybridization band of approximately 1,500 bp did not match the
digestion patterns of traE3 or traE4 genes.
4.4.4 Localization of traE3 and traE4 genes on spiroplasma plasmids
The high variability of presence of traE genes among S. kunkelii strains gave rise
to the question whether traE genes are located on extrachromosomal DNA. Southern
blots prepared from the pulsed field and standard agarose gels were hybridized to the
traE1, traE2 and traE3/4 probes depicted in Fig. 4.3A.
Pulsed field gel profile revealed significant differences in genome sizes of M2,
CS-2B and PU8-17. The 1.6 Mb genome size of M2 (Fig. 4.4A, lane 1) is similar to the
predicted 1.6 Mb genome of CR2-3x of which 1.54 Mb has been sequenced so far. The
genomes of CS-2B (2.1 Mb) and PU8-17 (2.0 Mb) were significantly larger than that of
M2 (Fig. 4.4A, lanes 2 and 3). Hybridization profile of PFGE Southern blots with [α32
P]-dCTP labeled 16S rDNA confirmed that these large DNA fragments were
chromosomal DNA (data not shown).
130
Hybridization profiles of PFGE Southern blots showed that the traE1 probe
hybridized to chromosomal DNA of M2 and CS-2B (Fig. 4.4B), and the traE2 probe to
M2, CS-2B and PU8-17 (Fig. 4.4B), thereby confirming earlier results that PU8-17
contains a copy of traE2 but not of traE1 and demonstrating that traE1 and traE2 are
apparently located on chromosomal DNA of S. kunkelii. In contrast, the traE3/4 did not
hybridize to chromosomal DNA of strains M2 and CS-2B, but to several smaller
fragments that are likely to represent various folding confirmations of circular plasmid
DNA (Fig. 4.4B). Again, as expected the traE3/4 probe did not hybridize to PU8-17
genomic DNA. Further, M2 showed more traE3 hybridization bands than CS-2B (Fig.
4.4B, lanes 1 and 2) suggesting that traE3 and traE4 of M2 are located on different
plasmids and/or the M2 and CS-2B plasmids differ in size.
To confirm the PFGE hybridization results, plasmids were isolated from M2, CS2B and PU8-17 cultures, and size-separated on a standard agarose gel. Subsequent
hybridizations of Southern blots of these gels confirmed the hybridization patterns shown
in Fig. 4.4B (Fig. 4.4C). The traE1 and traE2 probes hybridized to residual chromosomal
DNA that was co-purified with plasmid DNA (Fig. 4.4C), whereas the traE3/4 probe
hybridized to smaller DNA fragments that appeared to be plasmids because they were
significantly smaller than the chromosomal DNA bands (Fig. 4.4C). There were two
additional smaller bands in M2 relative to CS-2B (Fig. 4.4C, compare lanes 1 and 2)
supporting the conclusion that traE3 and traE4 of strain M2 may be located on different
plasmids.
131
4.4.5 Expression of S. kunkelii M2 traE genes in culture, insects and plants
To investigate whether traE genes are expressed in S. kunkelii M2 during
infection, total RNAs isolated from S. kunkelii culture, and S. kunkelii-infected and noninfected leafhoppers and maize leaves were size-separated for Northern blot preparation.
To assess RNA quality and to investigate whether spiroplasma gene expression can be
detected in cultures and in insects and plants, Northern blots were first hybridized to a
probe prepared on the spiralin gene sequence (Foissac et al., 1997). The spiralin gene is
constitutively expressed and encodes a membrane-bound lipoprotein proposed to
determine cell shape, cell motility and viability (Beven and Wroblewski, 1997). In
contrast to 16S rDNA, the spiralin gene probe is unlikely to cross-hybridize to other
bacteria because spiralin is unique to mollicutes and highly divergent in sequence among
spiroplasmas species (Foissac et al., 1997).
Northern blot hybridizations with the spiralin gene probe showed a single
transcript of the expected size of 1.1 kb in culture and infected but not healthy RNA
samples. For unknown reason, the spiralin transcript in infected insect samples was
slightly larger than that in infected plant and culture samples (Fig. 4.5). The amounts of
detected spiralin transcript appeared to be similar in infected insects and plants RNA but
higher in cultured spiroplasmas. These results demonstrated that S. kunkelii transcripts
were detectable in cultures, insects and plants. Further, the isolated RNA was of good
quality without noticeable degradation.
Subsequently, Northern blots were hybridized to the traE probes depicted in Fig.
4.3A. No traE1 transcripts were detected (data not shown), indicating that traE1 is not
132
expressed or its expression is below detection level. In contrast, several traE2 transcripts
were detected in S. kunkelii cultures, and S. kunkelii infected insects and plants (Fig.
4.5B). An S. kunkelii transcript of ~10 kb was detected in insects, cultures and plants and
is significantly larger than traE2 ORF of 2.2 kb, suggesting that traE2 is part of a larger
transcript and my be located in an operon. A second transcript of ~3.1 kb that appeared to
be transcribed at the same level as the ~10 kb transcript was detected in insects and may
contain only the traE2 ORF (Fig. 4.5A, lane 2). However, in samples from S. kunkelii
culture and infected plants, several additional transcripts were detected, including ~4 kb
transcripts that appeared to have similar expression levels as the ~10 kb transcripts.
Interestingly, the transcript of the ~4 kb was not detected in infected insect samples
suggestive of differential regulation of traE2 transcription and flanking genes in insects
relative to cultures and plants. Further, it appeared that the overall transcription level of
the ~10 kb transcript containing traE2 in S. kunkelii culture and S. kunkelii-infected plant
samples were comparable, but is lower in S. kunkelii-infected insects (Fig. 4.5B). Thus, it
seemed that relative to the spiralin gene, transcription of S. kunkelii traE2 and
surrounding genes was upregulated in cultures and in plants, but down-regulated in
insects.
The traE3/4 Northern hybridization profile showed two transcripts of 0.7 and 0.5
kb in S. kunkelii cultures (Fig. 4.5B, lane 3). The 0.7 kb was also detected in plants (Fig.
4.5B, lane 4), whereas no hybridization signals were detected in insects (Fig. 4.5B, lane
2). The function and origin of the 0.7 and 0.5 kb transcripts that are much smaller than
the full-length traE3 and traE4 genes is unclear and awaits further investigation.
133
4.4.6 Genetic context of traE genes in S. kunkelii CR2-3x
Because the Northern blot hybridization results suggested that traE2 might be part
of larger transcripts of ~4 and ~10 kb in lengths, ORFs flanking traE2 were analyzed in
the S. kunkelii CR2-3x genome sequence (Fig. 4.6). It seemed likely that traE2 was
within a locus containing 8 genes, some of which encoded membrane proteins. The fifth
ORF within this locus encoded a homolog of MreB, which forms a filamentous helical
structure close to the cell surface of bacteria and has an actin-like role in bacterial cell
morphogenesis (Jones et al., 2001).
The genetic contexts of the other three traE genes were also investigated. This
revealed that traE1 appeared to be part of a region that contained another conjugation
gene (traK), recombination (pre), and restriction modification (dm) genes (Fig. 4.6).
Similarly, traE3 and traE4 were immediately adjacent to five ORFs, four of which
encoded proteins containing potential transmembrane domains (Fig. 4.6). Interestingly,
the traE3 and traE4 regions had similar organizations and harbored homologs of S. citri
and S. kunkelii adhesion related proteins SARP1 and SkARP1 that were approximately
89 kDa in size and contained signal peptides, repeated amino acid sequences and Cterminal transmembrane domains (Berg et al., 2001), and homologs of mob genes that
were involved in recombination and mobilization of DNA (Cabezon et al., 1997).
4.5 Discussion
We identified four traE homologs, which are 100% identical in gapped genome
sequence of S. kunkelii CR2-3x and genome of S. kunkelii M2. These genes encoded
134
proteins containing transmembrane, ATPase and VirB4 domains. TraE homologs have
been identified in some but not all mollicutes. Interestingly, the four traE homologs of
Mycoplasma fermentans are part of integrative conjugal elements (Calcutt et al., 2002)
suggesting that in mollicutes, traE homologs might also be involved in conjugation.
However, the single membrane-bound ATPase gene traE (trsE) of Mycoplasma pulmonis
(Chambaud et al., 2001) does not appear to be part of a conjugal element. Although TraE
homologs were also reported for S. citri (Laigret et al., 2000), the sequences have not
been deposited in a public database. We found an S. citri DNA fragment that hybridizes
to traE1. No traE homologs were identified in the completed genome sequences of M.
genitalium (Fraser et al., 1995), M. pneumoniae (Himmelreich et al., 1996), and
Ureaplasma urealyticum (Glass et al., 2000).
It appears that the phylogeny of TraE is similar to that of 16S rDNA sequences
(Gasparich, 2002). The most plausible explanation of this phylogeny is that several
mollicutes, such as M. genitalium, M. pneumoniae and U. urealyticum, lost their traE
genes, and that mollicutes did not acquire their traE by horizontal gene transfer from
other bacteria. Indeed, mollicutes are believed to have undergone various degrees of gene
loss events in which spiroplasmas are evolutionary early mollicutes that suffered the least
gene losses (Bai and Hogenhout, 2002; Razin et al., 1998).
Southern blot results of the presence of traE genes among S. kunkelii strains
demonstrated that traE2 is most conserved, whereas the presence of traE1, traE3 and
traE4 genes is highly variable among S. kunkelii strains. Based on presence and
restriction digestion patterns of traE, S. kunkelii M2 appeared to be most similar to S.
135
kunkelii CR2-3x, subsequently FL-80, then CS-2B and lastly PU8-17. This may be
expected based on the original collection sites of these strains. However, FL-80, CS-2B
and PU8-17 were brought into culture in 1988 and since then have not been introduced
into insects and plants (Lee and Davis, 1989), whereas M2 was brought into culture
recently (Bai and Hogenhout, 2002). Spiroplasmas undergo frequent genome
reorganization (Ye et al., 1996) including variations in plasmid content and, therefore,
CS-2B and PU8-17 may have lost the plasmids containing traE3 and/or traE4. Further,
long-term culturing of spiroplasmas results in loss of insect transmissibility (Wayadande
et al., 1993). It remains to be investigated whether CS-2B and PU8-17 can be transmitted
by leafhoppers and/or are infectious to plants.
Expression results suggested that traE2 is part of a 10 kb transcript, and genetic
context showed that traE2 is part of a locus containing several predicted membrane
proteins and mreB. Further, traE2 is located on the S. kunkelii chromosome, expressed in
insects, plants and in culture, and present in all tested S. kunkelii strains, all of which
implicated an indispensable role of traE2 in S. kunkelii. Interestingly, MreB is critical for
the rod-shaped morphology of bacteria, and as previously observed (Bai and Hogenhout,
2002), mreB is present in the helical-shaped spiroplasmas but absent in the round, oval
and flask-shaped mycoplasmas. A putative role of traE2 in S. kunkelii cell morphology
should be confirmed as soon as transformation and mutagenesis system is available for S.
kunkelii.
The genetic context of traE3 and traE4 also suggested that these genes are part of
operons that also contain homologs of the adhesin SARP1. The repeated amino acid
136
domain of SARP1 was shown to locate extracellularly, which is potentially involved in
binding of SARP1 to insect cells (Berg et al., 2001). TraE’s are frequently part of
conjugation systems and are primarily involved in pilus formation (Zatyka and Thomas,
1998) and structures that appear to be conjugation pili were observed to connect S.
kunkelii cells to each other and to insect cells (Özbek et al., 2003). Thus, genes within
tra3 and tra4 loci may be involved in the formation of attachment structures. The absence
of detectable full-length transcripts of traE3 and traE4 in Northern hybridization may be
explained by the notion that genes encoding plasmid functions such as regulation of
replication, stable maintenance in the host population and conjugation are seldom
constitutively expressed (Bingle and Thomas, 2001).
Data described herein report for the first time the detection of transcripts of
spiroplasmas grown in culture media and in their natural habitats, which are plants and
insects. It is also the first detailed description of the presence of traE genes among
Spiroplasma species. This work provides the basic knowledge for further research that
confirms whether the traE genes are involved in cell morphogenesis, adhesion,
conjugation and/or recombination.
4.6 Acknowledgments
This work was funded by The Ohio State University - Ohio Agricultural Research
and Development Center (OARDC) and Ohio Plant Biotechnology Consortium (OPBC).
The authors wish to thank Ian Holford at the Molecular and Cellular Imaging Center
(MCIC) for help with writing ORF extractor program, William Styer of Department of
137
Entomology at the OSU-OARDC for help with spiroplasma culture, insect and plant
rearing, and Dr. Sophien Kamoun of Department of Plant Pathology at the OSU-OARDC
for providing the radioactive facility. S. kunkelii gapped genome sequence was obtained
from S. kunkelii Genome Sequencing Project funded by US Department of Agriculture,
Agricultural Research Service and the authors wish to thank Dr. Robert E. Davis and
colleagues.
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142
Gene
ID
Length
(nt)
GC
content
Nc
valuea
E value of
Blastx best
hitsb
traE1
2,697
23%
34.98
traE2
2,664
26%
traE3
2,520
traE4
2,520
TraE amino acid sequence similarityc
TraE1
TraE2
TraE3
TraE4
2e-97
-
54%
48%
49%
33.40
3e-89
54%
-
43%
43%
28%
40.85
7e-57
48%
43%
-
96%
28%
40.08
7e-63
49%
43%
96%
-
Table 4.1 Basic features of traE genes in S. kunkelii M2 strain.
a
Predicted by Chips in EMBOSS. Nc value is an index of codon usage ranging from 20 (the strongest
codon bias) to 61 (the lowest codon bias).
b
Performed by local BLAST package against local NCBI nr database. The best hits of all four genes were
TrsE-like protein of M. pulmonis (NP_326214).
c
Deduced TraE amino acid sequence similarity is the percentage of positives predicted by Pairwise BLAST
program using BLOSUM62 matrix on NCBI web site.
143
Fig. 4.1 ClustalW alignment of the deduced protein sequences of traE1, traE2, traE3 and traE4 of S.
kunkelii strain M2. Three partially conserved transmembrane domains are marked, the ATPase domains
were underlined, and the conserved DEAH sequences are boxed.
144
Fig. 4.2 Phylogenetic analyses of the TraE protein sequences from S. kunkelii M2 strain and other
organisms. Numbers at the nodes indicate percentage recovery of these nodes per 1,000 bootstrap
replicates. The proteins and accession numbers are: Bacillus anthracis pX02.09 (NP_053164);
Bifidobacterium longum DJ010A HP (hypothetical protein) (ZP_00121677); Clostridium acetobutlicus
TrsE (NP_348666); Clostridium perfringens CHP (conserved hypothetical protein) (NP_150039);
Enterococcus faecalis TrsE (AAF72347); Enterococcus faecalis HP (CAC29183); Enterococcus faecium
HP (ZP_00037379); Lactococcus lactis TrsE (NP_047296); Leuconostoc mesenteroides HP
(ZP_00062942); Mycoplasma pulmonis trsE (NP_326214); Mycoplasma fermentans TraE (AAN85227);
Plasmid pIP501 ORF5 (AAA99470); Plasmid R100 TraC (NP_052960); Proteus vulgaris PBP (pilus
biogenesis protein) (NP_640173); Providencia rettgeri TraC (AAM08001); Samonella typhimurium LT2
CT (conjugative transfer) protein (NP_490573); Streptococcus agalactiae 2603VR Orf26 (NP_688287);
Streptococcus agalactiae NEM316 unknown (NP_735797); Streptococcus pneumoniae orf26
(AAG38042); Staphylococcus aureus TrsE (F36891); Sulfolobus tokodaii CHP (NP_377258); Sulfolobus
sp. HP (T31035); Thermoanaerobacter tengcongensis CHP (NP_623664); Vibrio cholerae SPA (involved
in sex pilus assembly) (AAL59681). Escherichia coli TraC (AAB61935), served as the outgroup.
145
Fig. 4.3 Detection of traE sequences by Southern blot hybridization of digested genomic DNA of S.
kunkelii strains M2, CS-2B, FL-80 and PU8-17. (A) Schematic graphs of S. kunkelii M2 traE ORFs (open
arrows) depicting the recognition sites of restriction enzymes used in genomic DNA digestions and the
positions of primers (closed arrows above traE ORFs) used for probe synthesis. (B) Southern blots
hybridizations. Genomic DNAs of S. kunkelii strains M2 (lanes 1), CS-2B (lanes 2), FL-80 (lanes 3), PU817 (lanes 4) and S. citri (lanes 5) were digested with restriction enzymes and hybridized to DIG-labeled
probes as indicated in A. The sizes of the DIG-labeled SPP1 DNA EcoRI markers (lanes M) are indicated
in base pairs at the left of panel traE1. The traE3 and traE4 restriction fragments are indicated with arrows
and fragments lengths in base pairs (bp) at the right of the panel traE3/4.
146
Fig. 4.4 Detection of traE sequences on chromosomal and plasmid DNA of S. kunkelii strains M2, CS-2B,
and PU8-17. (A) EB-stained pulsed field gel of genomic DNA. (B) Southern blots of pulsed field gels
hybridized to traE1, traE2 and traE3/4 probes. (C) Southern blots of agarose gels containing S. kunkelii
DNA isolated with Qiagen Midiprep kit (Qiagen, Inc.) hybridized to traE1, traE2, and traE3/4 probes.
Saccharomyces cerevisiae (baker’s yeast) genome was used as marker. Genomic DNA was extracted from
S. kunkelii M2 strain (lane 1), CS-2B strain (lane 2) and PU8-17 strain (lane 3) and hybridized with probes
generated by [α-32P]-dCTP labeling of PCR products (Fig. 4.3A).
147
Fig. 4.5 Detection of S. kunkelii spiralin gene and traE transcripts on Northern blots of size-separated total
RNA samples. (A) Northern blot probed with traE2. (B) Northern blot probed with traE3. Probes were
generated by [α-32P]-dCTP labeling of PCR products of the full-length PCR product of the S. kunkelii M2
spiralin gene, and primers of traE2 forward1 and reverse1 and traE3 forward1 and reverse1. Detection of
transcripts of the constitutively expressed spiralin gene was used as RNA quality and loading control, and
to compare traE expression levels. Total RNA was extracted from healthy leafhoppers (lane 1), leafhoppers
infected with S. kunkelii M2 strain (lane 2), S. kunkelii M2 culture (lane 3), plants infected with S. kunkelii
M2 strain (lane 4), and healthy plants (lane 5). Estimated sizes of bands of in A and B are marked at the left
side of blot A.
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Fig. 4.6 Genetic contexts of traE genes in the genome of S. kunkelii CR2-3x. ORFs were indicated with
open arrows and identities of ORFs with significant hits to genes in GenBank were illustrated above the
ORFs. Genes that encoded proteins with transmembrane regions are marked with an asterisk underneath the
ORF.
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CHAPTER 5
Comparative genomics identifies genes shared by distantly
related insect-transmitted plant pathogenic mollicutes
Xiaodong Bai1, Jianhua Zhang1, Ian R. Holford2 and Saskia A. Hogenhout1
1
Department of Entomology, 2Molecular and Cellular Image Center (MCIC),
The Ohio State University - OARDC, Wooster, OH 44691, U.S.A.
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5.1 Abstract
Phytoplasmas and spiroplasmas are distantly related insect-transmitted plant
pathogens within the class Mollicutes. Genome sequencing projects of phytoplasma
strain Aster Yellow Witches’ Broom (AY-WB) and Spiroplasma kunkelii are near
completion. Complete genome sequences of seven obligate animal and human pathogenic
mollicutes (Mycoplasma and Ureaplasma spp.), and OY phytoplasma have been
reported. Putative ORFs predicted from the genome sequences of AY-WB and S. kunkelii
were compared to those of the completed genomes. This resulted in identification of at
least three ORFs present in AY-WB, OY and S. kunkelii but not in the obligate animal
and human pathogenic mollicutes. Moreover, we identified ORFs that seemed more
closely related between AY-WB and S. kunkelii than to their mycoplasma counterparts.
Phylogenetic analyses using parsimony were employed to study the origin of these genes,
resulting in identification of one gene that may have undergone horizontal gene transfer.
The possible involvement of these genes in plant pathogenicity is discussed.
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5.2 Introduction
Mollicutes, characterized by small genomes and no cell wall, are believed to have
diverged from a Gram-positive bacterial ancestor in the lactobacillus group (Woese,
1997; Razin et al., 1998). Within the class Mollicutes, an early evolutionary split
occurred between the AAA (Asteroleplasma, Anaeroplasma, and Acholeplasma) branch
and the SEM (Spiroplasma, Entomoplasma, and Mycoplasma) branch, both of which
independently underwent genome reductions (Razin et al., 1998). Apparently, the
conversion of UGA from a stop codon to a tryptophan codon in the SEM branch occurred
shortly after the split of the two branches. The SEM branch contains several genera,
including Spiroplasma, Entomoplasma, Mesoplasma, Mycoplasma, and Ureaplasma.
Spiroplasmas are believed to be evolutionary early mollicutes and did not undergo as
many gene loss events as members of other genera (Razin et al., 1998).
At the start of the genomic era, mollicutes have attracted much attention because
of their small genomes and their clinical and agricultural impact. Six mollicutes genomes,
five Mycoplasma spp. and one Ureaplasma sp., have been fully sequenced, representing
obligate human and mammal pathogens of the genus Mycoplasma of the SEM branch. At
the time of preparation of this manuscript, genome sequencing projects of three other
mycoplasmas are in progress: the rodent polyarthritis pathogen Mycoplasma arthritidis,
the contagious caprine pleuropneumonia (CCPP) pathogen M. capricolum, and the
contagious bovine pleuropneumonia (CBPP) pathogen M. mycoides subsp. mycoides SC
(small colony). At the time of the revision of this manuscript, the complete genome of M.
mycoides subsp. mycoides SC, the causative agent of CBPP, was published (Westberg et
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al., 2004). Further, the Onion Yellows (OY) phytoplasma genome was completely
sequenced (Oshima et al., 2004).
Genome sequencing projects are in progress for Spiroplasma kunkelii
(http://www.genome.ou.edu/spiro.html) and Aster Yellows Witches’-Broom (AY-WB,
http://www.oardc.ohio-state.edu/phytoplasma). S. kunkelii and phytoplasmas are insecttransmitted plant pathogens that replicate in both insect vectors and plant hosts.
Interestingly, S. kunkelii and phytoplasmas are strikingly similar in their infection
patterns of insects and plants. Both are restricted to phloem tissues of plant hosts, from
which they are acquired by phloem-feeding insects, and subsequently invade and
replicate in the cells of insect gut and other tissues. Interestingly, although Spiroplasma
species and all phytoplasmas described so far share similar infection patterns and
environmental niches, they are distantly related within two branches of the class
Mollicutes. Based on phylogenies of 16S rDNA and tuf genes, membrane composition,
codon usage and metabolism (Razin et al., 1998), spiroplasmas were grouped in the SEM
branch with Mycoplasma and Ureaplasma spp. while phytoplasmas were grouped in the
AAA branch with Acholeplasma spp.
This study was initiated based on the hypothesis that genes shared by
evolutionarily divergent insect-transmitted plant pathogens but absent from obligate
human and animal pathogens are likely important for insect transmission and/or plant
pathogenicity. Using computer-assisted analysis, we have identified at least three open
reading frames (ORFs) that were present in S. kunkelii and AY-WB but absent from
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mycoplasmas. We have also identified ORFs that do not match the 16S rDNA and tuf
phylogenies. The involvement of the ORFs in pathogenicity is discussed.
5.3 Materials and Methods
5.3.1 Genome sequences
The 16 contigs totaling 695 kb of the estimated 800 kb AY-WB genome were
obtained from the phytoplasma genome sequencing project website
(http://www.oardc.ohio-state.edu/phytoplasma). The 46 contigs totaling 1.5 Mb of the
estimated 1.6 Mb S. kunkelii CR2-3x genome were obtained from the publicly accessible
S. kunkelii genome sequencing project website (http://www.genome.ou.edu/spiro.html).
Complete mycoplasma genome sequences were obtained from GenBank, including
Mycoplasma genitalium (NC_000908) (Fraser et al., 1005), M. pneumoniae
(NC_000912) (Himmelreich et al., 1996), U. urealyticum (NC_002162) (Glass et al.,
2000), M. pulmonis (NC_002771) (Chambaud et al., 2001), M. penetrans (NC_004432)
(Sasaki et al., 2002), and M. gallisepticum (NC_004829) (Papazisi et al., 2003).
5.3.2 Comparative genome analysis
Genome comparisons were conducted as illustrated in Fig. 5.1. Genome
sequences were downloaded onto a Linux workstation and used as input files for the ORF
extractor program (http://www.oardc.ohio-state.edu/mcic/bioinformatics/bio_
software/bio_ software.html#ORF). ORFs were defined as starting with ATG and ending
with in-frame TAG, TAA, or TGA for AY-WB, or TAG and TAA for S. kunkelii and all
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Mycoplasma and Ureaplasma spp. (Razin et al., 1998). ORFs longer than 90 bp were
extracted in FASTA format. Subsequently, only the longest ORF within a set of ORFs
having stop codons at the same positions was extracted. ORFs in nucleotide sequences
were translated into amino acid (aa) sequences using a Perl translation program, using
translation table 11 (bacterial code) for AY-WB and translation table 4 (Mold
mitochondria code) for all others (Benson et al., 2003; Wheeler et al., 2003). This
generated datasets AYdb for AY-WB, Skdb for S. kunkelii, and mycoprotdb for the five
Mycoplasma spp. and U. urealyticum. Subsequently, AYdb and Skdb were compared
using stand-alone BLAST (Basic Local Alignment Search Tool) package (Altschul et al.,
1997) with the expectation (E) value threshold of 10-8. Proteins having significant
similarity (E < 10-8) were extracted from AYdb to generate AY_Skdb and from Skdb to
generate Sk_AYdb. Subsequently, AY_Skdb and Sk_AYdb were compared to
mycoprotdb and proteins with non-significant hits (E > 10-8) or no hits were extracted
from AY_Skdb to generate AY_Sk-mycoprotdb and from Sk_AYdb to generate Sk_AYmycoprotdb. Proteins within AY_Sk-mycoprotdb and Sk_AY-mycoprotdb were
annotated based on sequence similarity searches against NCBI nr database and compared
manually to identify common protein sequences. Identified proteins were validated by
manual comparison with the annotated genomes of mycoplasmas and ureaplasma. After
the finish of this study, the genomes of OY phytoplasma (Oshima et al., 2004) and M.
mycoides subsp. mycoides SC strain (Westberg et al., 2004) were sequenced. The
identified proteins were searched against the annotated proteins of these organisms using
the BLAST algorithm (Altschul et al., 1997).
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Negative logistic plots of best E values for each query were generated for
searches of (i) AYdb against Skdb and mycoprotdb and (ii) Skdb against AYdb and
mycoprotdb. For comparable quantitative assessment, an E value of 0.0 was set to 10-200,
and proteins with no significant hits were assigned E values of 1000.
5.3.3 Phylogenetic analysis
Protein sequences for phylogenetic analysis were extracted from NCBI Entrez
database. Sequence alignments were produced using ClustalW (Thompson et al., 1994)
and used as inputs for phylogenetic analysis using PAUP (Phylogenetic Analysis Using
Parsimony) program (Swofford, 2001).
5.3.4 Accession numbers
AY-WB amino acid sequences identified in this study were deposited in GenBank
with the accession numbers as follows: AAA type ATPase (AtA), AY533109; cmp
binding factor (CBF), AY533110; cytosine deaminase, AY533111; hypothetical protein,
AY533112; cation transport P-ATPase, AY533113; polynucleotide phosphorylase
(PNPase), AY533114; ppGpp synthetase, AY533115; YlxR protein, AY533116.
5.4 Results
5.4.1 Extraction of ORFs
The method employed for ORF extraction resulted in more ORFs than currently
annotated in the genomes. For either S. kunkelii (1.5 Mb) or AY-WB (700 kb), the
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number of predicted ORFs was more than that estimated with the average ORF size of 1
kb (Casjens, 1998). No genome annotation is perfect. For example, re-annotation of
Mycoplasma pneumoniae resulted in more ORFs and function annotations (Dandekar et
al., 2000). We expect that we have included in our study most of the annotated ORFs
(complete or partial) in the GenBank database by using the ORF extractor program, since
most ORFs that start with alternative start codons have an in-frame ATG somewhere in
the ORF. We have verified this assessment. Of the 4,332 mycoplasma and ureaplasma
ORFs downloaded from GenBank, 991 ORFs (22.7%) start with an alternative start
codon. Of the ORFs starting with an alternative start codon, 984 ORFs (99.3 %)
contained an in-frame ATG somewhere in the ORF. Thus, only 0.7 % of the putative
ORFs starting with alternative start codons present in the GenBank database have been
excluded from the ORF extractor database. This is only 0.2 % of all the 4,332 annotated
mycoplasma and ureaplasma (i.e. members of M. pneumoniae and M. hominis groups and
Ureaplasma urealyticum) ORFs present in GenBank.
To minimize the number of false-positives produced by the method, only the
longest ORF within a set of ORFs having stop codons at the same position was extracted
for subsequent analysis. Translation of the ORFs into amino acid sequences generated
AYdb for AY-WB, Skdb for S. kunkelii, and mycoprotdb for the five Mycoplasma
species and U. urealyticum.
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5.4.2 Identification of four proteins that are present in AY-WB and S. kunkelii but absent
from mycoplasmas
Amino acid sequence similarity searches were employed to identify proteins
shared between AY-WB and S. kunkelii. AYdb and Skdb were searched against each
other using stand-alone BLAST package (Altschul et al., 1997). 290 proteins within
AYdb had significant similarity (E-value < 10-8) to proteins within Skdb, whereas 260
proteins within Skdb had significant similarity to proteins within AYdb. E (expectation)
value in BLAST search is defined as “the number of different alignments with scores
equivalent to or better than S that are expected to occur in a database search by chance”,
and it depends on the size of the search database and the scoring system (Altschul et al.,
1994). Thus, it was expected that the number of proteins with significant similarity for
the two independent searches would differ because of different database sizes.
To identify shared AY-WB and S. kunkelii proteins that are not present in animal
and human pathogenic mycoplasmas and ureaplasmas, AY_Skdb and Sk_AYdb were
searched against mycoprotdb using the blastp algorithm. Sequences that had nonsignificant similarity (E-value > 10-8) or no similarities were extracted from Skdb and
AYdb. This resulted in two datasets of AY_Sk_-mycoprotdb with 14 entries and
Sk_AY_-mycoprotdb with 7 entries.
Plotting the negative logs of the blastp E-values showed that the majority of the
predicted protein sequences shared by AY-WB and S. kunkelii had homologs in the five
Mycoplasma spp. and U. urealyticum. However, 9 AY-WB and 8 S. kunkelii proteins did
not have significant similarity to proteins in mycoprotdb (Fig. 5.2). Among these, four
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proteins present in both the AY_Sk_-mycoprotdb and Sk_AY_-mycoprotdb datasets
were analyzed because they are similar in length and have significant similarity to
proteins in NCBI nr database (closed diamonds, Fig. 5.2). The four proteins were
identified as polynucleotide phosphorylase (PNPase), cmp-binding factor (CBF), cytosine
deaminase, and YlxR protein (Table 5.1). The PNPase protein sequences of AY-WB and
S. kunkelii were 62% (452/719) similar, the CBFs 59% (138/231), cytosine deaminases
60% (86/141), and YlxR proteins 61% (46/74). To ensure that the sequences are not
present in the genomes of mycoplasmas and ureaplasmas, sequences in common between
AY-WB and S. kunkelii were searched against the mycoplasma and ureaplasma GenBank
databases. Further, the annotated protein databases of Mycoplasma spp. and U.
urealyticum were searched by keywords. Both analyses showed that no proteins for these
organisms were annotated as PNPase, CBF, cytosine deaminase, or YlxR protein. Thus,
these data suggested that these four genes are present in AY-WB and S. kunkelii but
absent from Mycoplasma spp. and U. urealyticum genomes. All these four proteins have
homologs in OY phytoplasma genome. However, all but PNPase have homologs in M.
mycoides subsp. mycoides SC strain.
5.4.3 Identification of proteins more closely related between AY-WB and S. kunkelii
Four proteins were identified from the negative logistic plots that were more
similar between AY-WB and S. kunkelii than to mycoplasmas (open circles, Fig. 5.2).
These proteins were identified as ppGpp synthetase, HAD hydrolase, AtA (AAA type
ATPase), and P-type Mg2+ transport ATPase (Table 5.2). Amino acid sequence
159
similarities between AY-WB proteins and S. kunkelii proteins were ppGpp synthetase,
59% (305/503); HAD hydrolase, 59% (449/750); AtA, 88% (362/407); and P-type Mg2+
transport ATPase, 56% (512/902). All proteins have homologs in the genomes of OY
phytoplasma and M. mycoides subsp. mycoides SC strain, except for the AtA sequence
that is lacking from OY phytoplasma.
5.4.4 Phylogenetic analysis of proteins present in AY-WB and S. kunkelii but absent from
mycoplasmas
Phylogenetic analyses were performed to investigate the origin of the proteins
identified in this study. The PNPases from AY-WB and S. kunkelii clustered with those
from the Gram-positive Bacillus and Streptococcus spp. and were clearly distinct from
those of Gram-negative bacteria (Fig. 5.3B). Thus, the PNPase phylogenetic trees are
consistent with the proposed evolutionary status of mollicutes as descendents of Grampositive bacterial ancestors (Weisburg et al., 1989; Woese, 1989). Phylogenetic analysis
of CBFs (Fig. 5.3C) resulted in a tree different from the phylogenetic tree based on 16S
rDNA sequences (Fig. 5.3A) with the CBF sequences of AY-WB and S. kunkelii
separated by CBF sequences of Gram-positive bacteria. Phylogenetic analyses of
cytosine deaminases and YlxR proteins resulted in trees with most branches having low
bootstrap values (data not shown).
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5.4.5 Phylogenetic analysis of proteins more closely related between AY-WB and S.
kunkelii
Phylogenetic analysis was employed to analyze the possible origins of the four
proteins that were more closely related between AY-WB and S. kunkelii than to
mycoplasmas. Most branches of the phylogenetic trees generated using ppGpp
synthetase, HAD hydrolase, and P-type Mg2+ transport ATPase had bootstrap values
lower than 50% (data not shown). However, bootstrap values of the phylogenetic tree
based on AtA sequences were statistically significant. Interestingly, in the AtA
phylogeny, the phytoplasma AtA sequence clustered together with the AtA sequence of
S. kunkelii in a cluster of AtA sequences of other mycoplasmas belonging to the SEM
branch (Fig. 5.4), which is different from the 16S rDNA phylogeny (Fig. 5.3A). The AtA
homolog is present in M. mycoides subsp. mycoides SC, which is also a member of the
SEM branch of mollicutes, but it is absent from the OY phytoplasma genome.
5.5 Discussion
In this study, we have identified several proteins that appear to be present in AYWB and S. kunkelii but absent from Mycoplasma spp. and U. urealyticum. These proteins
are PNPase, CBF, cytosine deaminase, and YlxR. These proteins are also present in the
genome of OY phytoplasma, another insect-transmitted plant pathogenic mollicute
closely related to AY-WB.
PNPase is an exoribonuclease belonging to the PDX family that also includes
RNase PH (Zuo and Deutscher, 2001). Most prokaryotes have PNPase homologs,
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however, thus far, none have been sequenced from mycoplasmas and Ureaplasma
urealyticum. PNPase genes are also present in the genomes of plants (Li et al., 1998) and
Drosophila (Adams et al., 2000). PNPases are highly conserved proteins that are
involved in mRNA degradation and regulation of gene expression (Carpousis, 2002).
PNPase has been shown to be a global regulator of virulence factors of Salmonella
enterica, because a single point mutation of the PNPase gene resulted in a significant
decrease in efficiency of invasion and intracellular replication of this bacterium
(Clements et al., 2002). Both AY-WB and S. kunkelii invade and replicate cells of insects
and plants (Özbek et al., 2003) and, consequently, have to adjust their gene expression
patterns continuously to different environments. In contrast, the Mycoplasma and
Ureaplasma spp. are restricted to animal hosts in which they are able to attach to and
most invade epithelial cell layers but do not appear to spread systemically throughout
their hosts (Razin et al., 1998). Thus, PNPases in plant pathogenic bacteria, AY-WB, S.
kunkelii, and OY phytoplasma, could be important for gene expression regulation
allowing adaptation to multiple environmental niches, including insect gut lumen, insect
cells, plant phloem, and plant cells. However, the involvement of PNPase in regulation of
virulence of plant pathogenic mollicutes, AY-WB and S. kunkelii, remains to be
investigated. At this time, spiroplasmas are more suitable candidates for such an
investigation, because, unlike phytoplasmas, they can be cultured (Saglio et al., 1973)
and transformed (Lartigue et al., 2002).
CBF is a protein identified in Staphylococcus aureus. It binds to the cmp
sequence, a replication enhancer identified in the pT181 plasmid of S. aureus, to
162
stimulate plasmid replication (Zhang et al., 1997). Spiroplasmas and phytoplasmas have
plasmids (Razin et al., 1998), whereas plasmids have not been reported in members of M.
pneumoniae and M. hominis groups and Ureaplasma urealyticum that do not have CBF.
Interestingly, a CBF homolog is present within the recently released complete genome of
M. mycoides subsp. mycoides SC strain (Westberg et al., 2004). Although plasmids have
not been reported in the SC type strain, plasmids are common in Mycoplasma mycoides
spp. mycoides (King and Dybvig, 1994; Djordjevic et al., 2001). It is possible that CBF is
required for regulation of plasmid replication in spiroplasmas and phytoplasmas.
Interestingly, spiroplasma and phytoplasma plasmid appear to harbor virulence factors
(Melcher et al., 1999; Oshima et al., 2002).
Cytosine deaminase is an enzyme involved in nucleotide metabolism and can
affect protein synthesis if transiently expressed in human cells (Kreuzer et al., 1996).
Thus, apparently, S. kunkelii, AY-WB and OY have an additional housekeeping gene that
is absent from other mollicutes sequenced so far. YlxR protein is expressed from the
nusA/infB operon in bacteria and proposed to be an RNA-binding protein (Osipiuk et al.,
2001).
We also identified four AY-WB and S. kunkelii ORFs that appear to be more
closely related to each other than their mycoplasma counterparts. Of these, the AtA
sequence is most interesting, because the phylogenetic tree suggests that phytoplasmas
might have obtained the AtA sequence from spiroplasmas, possibly S. kunkelii, by
horizontal gene transfer. This hypothesis is supported by additional data. First, AtA is
absent from the OY phytoplasma genome (Oshima et al., 2004). OY phytoplasmas is a
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plant pathogen in Japan where there is no occurrence of S. kunkelii. But, in the American
continent, S. kunkelii and AY-WB co-occur and share similar insect and plant host
ranges. Secondly, AtA sequences of both AY-WB and S. kunkelii are flanked by insertion
sequences that often part of mobile elements (Mahillon and Chandler, 1998). AY-WB
AtA is flanked by a truncated transposase gene at its 5’ end and an intact transposase
gene at its 3’ end, and S. kunkelii AtA is located in an IS (insertion sequence) elementrich region.
In summary, the comparative genomics study presented herein successfully
identified proteins that are common among insect-transmitted plant pathogenic
mollicutes. Further studies of these proteins may elucidate their roles in insect
transmission and plant pathogenicity.
5.6 Acknowledgments
The authors wish to thank Dr. Sophien Kamoun in the Department of Plant
Pathology, OSU-OARDC, for constructive advice; Dr. Tea Meulia for setup of the Linux
workstation and design of ORF Extractor program; and B. A. Roe, S. P. Lin, H.G. Jia,
H.M. Wu, D. Kupfer, and R. E. Davis and the Spiroplasma kunkelii Genome Sequencing
Project funded by US Department of Agriculture, Agricultural Research Service Project
Number: 1275-22000-144-02 for the S. kunkelii genome sequences.
This research was supported by OSU-OARDC Research Enhancement
Competitive Grants Program and MCIC.
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168
ID
1
2
3
4
AY-WB and S. kunkelii homologues
absent from mycoprotdb
Source
ORF ID a
Length b
AY-WB
246_1F
716
S. kunkelii
100_74F
719
AY-WB
247_200F
321
S. kunkelii
109_633F
313
AY-WB
247_187F
161
S. kunkelii
98_127R
159
AY-WB
247_205R
85
S. kunkelii
107_113R
91
Best hit against NCBI nr database
Accession #, Homology
29377522, PNPase
15902560, PNPase
27468441, cmp-binding factor 1
16078057, cmp-binding factor 1
20806575, cytosine/adenosine
deaminases
20806575, cytosine/adenosine
deaminases
541414, conserved hypothetical protein
YlxR
541414, conserved hypothetical protein
YlxR
Cellular
location c
Organism
Enterococcus faecalis
Streptococcus pneumoniae
Staphylococcus aureus
Bacillus subtilis
Thermoanaerobacter
tengcongensis
Thermoanaerobacter
tengcongensis
Bacillus subtilis
E value
1e-180
0
1e-39
3e-59
4e-26
cytoplasm
cytoplasm
cytoplasm
cytoplasm
cytoplasm
1e-16
cytoplasm
2e-14
cytoplasm
Bacillus subtilis
2e-06
cytoplasm
169
Table 5.1 Four AY-WB and S. kunkelii homologues that were absent from mycoprotdb consisting of the whole genome sequences of M. genitalium, M.
pneumoniae, U. urealyticum, M. pulmonis, M. penetrans, and M. gallisepticum.
a
ORF ID identified by ORF Extractor program.
Length of deduced amino acid sequence.
c
Cellular location was determined by pSORT (Nakai and Horton, 1999).
b
ID
1
2
Proteins shared between AY-WB
and S. kunkelii
Organism
ORF ID a
Length b
AY-WB
235_4R
414
S. kunkelii
94_78R
414
AY-WB
248_157F
889
77_20F
910
3
AY-WB
247_48R
745
4
S. kunkelii
AY-WB
106_196R
246_186F
749
528
S. kunkelii
96_41R
509
170
S. kunkelii
Best hit against NCBI nr database
Accession #, Homology
15613820, BH1257 unknown conserved
15613820, BH1257 unknown conserved
15673239, cation-transporting P-ATPase (EC
3.6.3.2)
30022224, Mg2+ transport ATPase, P type
(EC 3.6.3.2)
10443847, ppGpp synthetase
6647842, ppGpp synthetase
28378886, Hypothetical exported
protein/HAD hydrolase
401696, Hypothetical exported protein/HAD
hydrolase
Cellular
location c
Organism
Bacillus halodurans
Bacillus halodurans
Lactococcus lactis
E-value
1e-94
2e-87
2e-179
cytoplasm
cytoplasm
membrane
Bacillus cereus
0
membrane
Geobacillus
stearothermophilus
Spiroplasma citri
Lactobacillus plantarum
e-154
cytoplasm
0
1e-116
Mycoplasma mycoides
1e-92
cytoplasm
membrane
or outside
membrane
or outside
Table 5.2 Identities of AY-WB and S. kunkelii proteins that are more similar to each other than to proteins in mycoprotdb.
a
ORF ID identified by ORF Extractor program.
Length of deduced amino acid sequence.
c
Cellular location was determined by pSORT (Nakai and Horton, 1999).
b
Fig. 5.1 Algorithms employed to extract proteins that are common between the insect-transmitted plant
pathogens Aster Yellows Witches’ Broom (AY-WB) and Spiroplasma kunkelii but are absent from five
Mycoplasma spp. and Ureaplasma urealyticum. See Materials and Methods for details. Similar dataset
consists of proteins that are similar in AY-WB and S. kunkelii, while unique dataset consists of proteins that
are similar between AY-WB and S. kunkelii but absent from five Mycoplasma spp. and Ureaplasma
urealyticum. Shaded text boxes are operations with the programs indicated in parentheses. Open text boxes
are datasets either as input or output of the operations. Bacterial and mold mitochondria genetic codes are
from NCBI taxonomy databases (Benson et al., 2003; Wheeler et al., 2003).
171
Fig. 5.2 Graphical representation of comparative analysis results. A. Negative logistic plots of the top E
values of the BLAST search using AY-Skdb as query and Skdb (x-axis) and mycoprotdb (y-axis) as
databases. B. Negative logistic plots of the top E values of the BLAST search using Sk_AYdb as query and
AYdb (x-axis) and mycoprotdb (y-axis) as databases. Based on criteria described in Materials and
Methods, data points in diamonds (◆ or ◊) are proteins shared between AY-WB and S. kunkelii but absent
from five Mycoplasma spp. and Ureaplasma urealyticum, and data points in open triangles (∆) are proteins
present in AY-WB, S. kunkelii and Mycoplasma spp. and Ureaplasma urealyticum. Data points in solid
diamond (◆) are proteins having similar lengths and annotations, which are detailed in Table 5.1. Data
points in open circles (O) are AY-WB or S. kunkelii proteins that are more similar to each other than to
counterparts in five Mycoplasma spp. and U. urealyticum, which are detailed in Table 5.2.
172
Fig. 5.3 Phylogenetic analyses of proteins that are present in insect-transmitted plant pathogenic AY-WB
and S. kunkelii but absent from animal and human pathogenic mycoplasmas. Phylogenetic trees were
generated following the procedure described in Materials and Methods. Bars under the trees represent
evolutionary distances. A. Phylogenetic tree derived from 16S rDNA sequences. B. Phylogenetic tree
derived from polynucleotide phosphorylase (PNPase). C. Phylogenetic tree derived from cmp-binding
factor (CBF). Protein sequences were obtained from GenBank and aligned with ClustalW (Thompson et al.,
1994). The alignments were used for parsimony analysis in PAUP version 4.0 (Swofford, 2001). Trees
were bootstrapped 1,000 times and the bootstrap values above 50% are indicated as a percentage at the
branches. Accession numbers for protein sequences follows. (A) Acholeplasma laidlawii, M23932;
Anaeroplasma abactoclasticum, M25050; Asteroleplasma anaerobium, M22351; Bacillus subtilis,
AB042061; Mesoplasma entomophilum, AF305693; Mycoplasma capricolum, U26048; M. gallisepticum,
M22441; M. genitalium, X77334; M. hominis, AJ002268; M. mycoides, U26050; M. pulmonis, AF125582;
M. sualvi, AF412988; Streptococcus pneumoniae, AY281083; Ureaplasma urealyticum, U06098. (B)
Actinobacillus pleuropneumoniae, ZP_00134571; Bacillus halodurans, NP_243273; B. subtilis,
NP_389551; Buchnera aphidicola, NP_777952; Deinococcus radiodurans, NP_295786; Escherichia coli,
NP_312072; Haemophilus influenzae, NP_438401; Mycobacterium bovis, CAD94991; Salmonella
enterica, NP_806878; S. typhimurium, AAL22154; Shigella flexneri, NP_708965; Streptococcus
agalactiae, CAD45842; Str. mutans, NP_720625; Str. pyogenes, BAC64773; Thermotoga maritime,
NP_229146; Thermus thermophilus, CAB06341; Vibrio vulnificus, NP_935490; Xylella fastidiosa,
NP_778440; Xanthomonas axonopodis, NP_642994; Yersinia enterocolitica, CAA71697; Y. pestis,
NP_668031. (C) Bacillus subtilis, CAB12833; B. cereus, NP_830807; Clostridium perfringens,
NP_560939; C. tetani, NP_783025; Lactococcus lactis, NP_268079; Methanococcus jannaschii,
NP_247831; Staphylococcus aureus, NP_374949; Sta. Epidermidis, NP_765078; Streptococcus mutans,
NP_720807; Str. pneumoniae, NP_359386; Str. pyogenes, NP_268621;
173
Fig. 5.4 Phylogenetic analysis for AtA (AAA type ATPase). Phylogenetic trees were generated following
the procedure described in Materials and Methods. Bars under the trees stand for evolutionary distances.
Protein sequences were obtained from GenBank and aligned with ClustalW (Thompson et al., 1994). The
alignments were used for parsimony analysis in PAUP version 4.0 (Swofford, 2001). Trees were
bootstrapped 1,000 times and the bootstrap values above 50% are indicated as a percentage at the branches.
Accession numbers for protein sequences follows. Bacillus halodurans, NP_242123; B. subtilis,
NP_390631; Clostridium acetobutylicum, NP_348297; Enterococcus faecalis, NP_815655; E. faecium,
ZP_00036045; Listeria innocua, NP_470885; L. monocytogenes, NP_465039; Mycobacterium leprae,
CAA19102; Mycoplasma gallisepticum, NP_853308; M. penetrans, NP_757529; Staphylococcus aureus,
NP_646394; Streptococcus mutans, NP_722348; Str. pneumoniae, NP_346223; Ureaplasma urelyticum,
NP_078028
174
CHAPTER 6
Functional genomics identifies phytoplasma effector proteins
Xiaodong Bai1, Valdir Ribeiro Correa1, Jianhua Zhang1,
Michael M. Goodin2, Sophien Kamoun3, and Saskia A. Hogenhout1
1
Department of Entomology, The Ohio State University - OARDC, Wooster, OH 44691
2
Department of Plant Pathology, University of Kentucky, Lexington, KY 40546
3
Department of Plant Pathology, The Ohio State University – OARDC,
Wooster, OH 44691
175
6.1 Abstract
Phytoplasmas are insect-transmitted plant pathogenic bacteria that have a broad
plant host range and induce a variety of symptoms that suggest interference with plant
development. The recently completed genome of the Candidatus Phytoplasma asteris
strain aster yellows witches' broom (AY-WB) phytoplasma was mined for the presence
of genes encoding secreted proteins that are candidate effector proteins involved in
interaction with plant and insect cell components. In total, 56 genes encoded putative
secreted proteins based on the presence of an N-terminal signal peptide and lack of
transmembrane domains, and were transiently expressed in Nicotiana benthamiana
plants. The functional analysis in plants resulted in the identification of 17 putative
phytoplasma effector proteins that may directly or indirectly interact with plant
components. Five putative effector proteins contained putative nuclear localization
signals (NLSs). Yellow fluorescence protein (YFP) fusions of one protein (A11) targeted
the plant cell nuclei and of another protein (A30) the nucleoli. The genes encoding these
two proteins were expressed during phytoplasma infection of insects and plants. Further,
nuclear transport of A11 was inhibited in N. benthamiana plants in which the expression
of the gene for importin α was knocked down. Finally, transcription profiling studies
indicated that A11 differentially regulated 53 tomato genes, including several
transcription factors involved in plant development. These results supported the
hypothesis that A11 was an effector protein that manipulates plant components. This
study, for the first time, employed the combination of bioinformatics and functional
genomics to study phytoplasma pathogenesis.
176
6.2 Introduction
The aster yellows witches' broom (AY-WB) phytoplasma (Zhang et al., 2004) is a
strain of Candidatus Phytoplasma asteris (previously known as the Aster Yellows 16SrI
group) that is largest group of Candidatus Phytoplasma. Phytoplasmas belong to the
Class Mollicutes of which members are characterized by the lack of cell wall, small
genomes with low GC contents (Razin et al., 1998), and likely evolved from a Grampositive bacterial ancestor by reductive evolution (Weisburg et al., 1989; Woese, 1987).
Phytoplasmas are insect-transmitted plant pathogens and mainly reside in plant phloem
tissues. They are intracellular pathogens that invade and replicate in the cells of insect
vectors and plant hosts, and cause severe losses of crops, such as lettuce and carrot, and
ornamental plants, such as China aster. The phytoplasma-infected plants show symptoms
including phyllody (development of floral parts into leafy structures), virescence
(greening of normally white tissue), and shoot proliferation. These symptoms may be due
to the interference of phytoplasma proteins with plant hormone synthesis and utilization
(Chang, 1998). Indeed, bacteria secreted proteins are able to interfere with plant
metabolic or signaling pathways. For instance, characterized type III effector proteins
from Gram-negative plant pathogenic bacteria, such as HopPtoF from Pseudomonas
syringae pv. phaseolicola (Jackson et al., 1999), suppress plant defense-associated
hypersensitive response (HR). Some effectors were able to modify plant signal
transduction pathways (Collmer et al., 2002). Unlike many Gram-negative that are
extracellular and need type III secretion systems to cross host cell membrane for delivery
177
of effector proteins, phytoplasmas are intracellular pathogens and hence their secreted
proteins can directly interact with cellular components of plant cells.
Phytoplasmas cannot be cultured in cell-free media, and there are no available
genetic tools, making it difficult to study the biology, physiology, and pathogenicity
mechanisms of phytoplasmas. However, the recent accumulation of phytoplasma genome
sequence data provided an excellent basis for functional genomic screens to identify
phytoplasma proteins involved in pathogenesis. The genome-sequencing project of AYWB phytoplasma initiated by the Department of Entomology and the Department of Plant
Pathology at The Ohio State University in collaboration with Integrated Genomics, Inc. is
completed (Bai et al., in preparation; refer to Chapter 3 of this dissertation for details),
and was used for the study described herein. Further, the complete genome sequence of
Onion Yellows (OY) phytoplasma, another strain of Candidatus Phytoplasma asteris,
was published (Oshima et al., 2004).
Here we report, for the first time, the successful identification of phytoplasma
candidate effector proteins by a combination of bioinformatics and high throughput
functional analysis. Candidate effector proteins were predicted using computer-assisted
algorithms, and thereafter, analyzed in plants using the well-established Potato virus X
(PVX)-based transient expression system (Qutob et al., 2002) and plant localization
system using fluorescence protein fusions (Goodin et al., 2002). Seventeen proteins were
identified to induce necrosis on Nicotiana benthamiana leaves and two proteins targeted
plant cell nuclei and corresponding transcripts of these two proteins were detected in
phytoplasma-infected insects and plants. One protein was dependent on plant importin α
178
for transport into plant nuclei. It was also able to change transcription profiles of several
tomato genes, including transcription factors.
6.3 Materials and Methods
6.3.1 Bacteria and plants
Agrobacterium tumefaciens GV3101 (Holsters et al., 1980) and Escherichia coli
XL1-blue were routinely grown at 28 oC and 37 oC, respectively, in Luria-Bertani (LB)
media supplemented with appropriate antibiotics (Sambrook et al., 1989). Nicotiana
benthamiana plants were used for in planta functional assay. Lycopersicon esculentum
OH7814 was used for microarray. Inoculated or agroinfiltrated N. benthamiana and L.
esculentum plants were maintained in Biosafety Level 2 greenhouses. AY-WB
phytoplasma was maintained by serial transmission to China aster (Callistephus
chinensis) plants by aster leafhopper (Macrosteles quadrilineatus L.) in greenhouse and
growth chambers.
6.3.2 Data mining
AY-WB phytoplasma gapped genome sequence was obtained from the AY-WB
phytoplasma genome sequencing project website (http://www.oardc.ohiostate.edu/phytoplasma). Open reading frames (ORFs) were predicted using ORF extractor
program (Bai et al., 2004). The longest ORF among the set of ORFs with the same stop
codon was extracted using a perl script. Nucleotide sequences were translated into amino
acid sequences, and the presence of signal peptides in AY-WB phytoplasma amino acid
179
sequences was examined by SignalP 2.0 program (Nielsen et al., 1997). The proteins
containing signal peptides were subject to selection based on the criteria of ORF length
and lack of the transmembrane domains predicted by TMHMM2.0 program (Krogh et al.,
2001), resulting in phytoplasma candidate effector proteins. The candidate effector
proteins were analyzed by web-based BLAST (Basic Local Alignment Search Tool)
(Altschul et al., 1997) against NCBI (National Center for Biotechnology Information) nr
(non-redundant) database. The presence of nuclear localization signal (NLS) in the AYWB candidate effector proteins was predicted using ScanProsite (Gattiker et al., 2002)
and PredictNLS (Cokol et al., 2000) programs.
6.3.3 Construction of recombinant A. tumefaciens binary PVX vectors
ORFs encoding the moiety of candidate effector proteins excluding signal
peptides were PCR-amplified and cloned into the PVX vector pGR106 (Jones et al.,
1999). Gene-specific primers complementary to the 5' and 3' ends of each respective ORF
were designed to include restriction site overhangs for cloning into the pGR106 vector.
Amplification products were digested with appropriate restriction enzymes, sizefractioned and purified from 1% agarose gels using QIAprep gel extraction kit (Qiagen,
Valencia, CA). Purified products were ligated into pGR106. The resulted binary
expression constructs were electro-transformed into A. tumefaciens GV3101. The cells
were allowed to grow for 2 days at 28 oC in LB agar plates supplemented with 50 g ml-1
kanamycin. The sequences of the cloned inserts were verified by DNA sequence analysis.
Individual colonies were toothpick-inoculated onto the lower leaves of N. benthamiana
180
plants (Takken et al., 2000). The development of disease symptoms was recorded from 1
dpi (days post-inoculation) up to 21 dpi.
6.3.4 Construction of recombinant A. tumefaciens binary pGDY vectors
NLS-containing candidate effector proteins of AY-WB were fused to N-terminus
to yellow fluorescent protein (YFP) for determining the subcellular localization of
phytoplasma proteins in plant cells. To this end, ORFs encoding the moiety of NLScontaining candidate effector proteins excluding signal peptides were amplified using
gene-specific primers complementary to the 5' and 3' ends of each respective ORF and
including restriction site overhangs for cloning into the pGDY vector (Goodin et al.,
2002). Ligations were directly electro-transformed into A. tumefaciens GV3101 as
described above. Agro-infiltration of individual GV3101 colonies was conducted as
described (Goodin et al., 2002). Leaves were harvested at 48-72 h after infiltration and
examined by laser-scanning confocal microscopy using a Leica TCS SP2 filter-free
spectral confocal and multiphoton microscope.
6.3.5 Tobacco rattle virus (TRV)-mediated virus-induced gene silencing (VIGS)
To determine the dependence of YFP:A11 on N. benthamiana importin α genes
for transport into plant nuclei, the importin α genes were silenced using TRV-mediated
VIGS (Ratcliff et al., 2001; Liu et al., 2002). The importin α genes were amplified from
the N. benthamiana cDNA library (Kanneganti et al., in preparation) using gene-specific
primers designed to contain overhangs with appropriate restriction recognition sites for
181
cloning into the pTV00 vector, which allows production of the RNA2 portion of the TRV
genome. The pTV00 constructs were introduced into A. tumefaciens GV3101 by electrotransformation, and the transformed cells were propagated at 28 oC. The A. tumefaciens
GV3101 transformants were infiltrated into N. benthamiana leaves simultaneously with
A. tumefaciens GV3101 containing the pBINTRA6 vector, which allows production of
the RNA1 portion of the TRV genome (Ratcliff et al., 2001). To confirm the silencing of
N. benthamiana importin α gene, reverse transcriptase – polymerase chain reaction (RTPCR) was performed on the total RNA samples collected from the upper leaves of N.
benthamiana plants three weeks after agro-infiltration. At the same time, the matching
upper leaves of N. benthamiana plants were infiltrated with pGD constructs and, two
days later, examined by a laser scanning confocal microscope as described above. The
green fluorescence protein (GFP)-tagged AtFib1 protein that localizes to the nucleolus
(Barneche et al., 2000) was used as a negative control. The GFP:AtFib1 construct was
kindly provided by Dr. Michael Goodin at University of Kentucky.
6.3.6 RNA isolation and RT-PCR
Total RNA was isolated from leaves of healthy and AY-WB phytoplasmainfected China aster plants, and healthy and AY-WB phytoplasma-infected aster
leafhoppers following the instructions of the ToTALLY RNA kit (Ambion, Austin, TX).
Total RNA samples were treated with DNA-free kit (Ambion) to degrade residual
genomic DNA contamination.
182
RT-PCR was performed to investigate whether the genes encoding the candidate
effector proteins were expressed in AY-WB phytoplasma during the infection of insects
and plants. RT-PCR was conducted with the OneStep RT-PCR kit (Qiagen) following the
manufacturer's protocol using the gene-specific primers designed for pGDY cloning. The
thermal cycler conditions were: 1 cycle of 50 oC for 30 min, 1 cycle of 95 oC for 15 min,
30 cycle of (94 oC for 1 min, 50 oC for 1 min, and 72 oC for 1 min), 1 cycle of 72 oC for
10 min. RT-PCR products were examined in 1% agarose gel following standard
electrophoresis procedures (Sambrook et al., 1989).
6.3.9 Microarray study and data analysis
Transcription profiling of tomato genes was conducted using an oligo-based
microarray representing 15,925 tomato unigenes (http://www.tigr.org) and 12 matched
and 12 mismatched 24-mers per gene (NimbleGen Systems Inc., Madison, WI).
Cotyledons of young tomato OH7814 plants of 9 days old were toothpick-inoculated with
PVX:A11 construct (A) and PVX vector (P) constructs. Mock-inoculated (M) plants
served as controls. Plants were organized randomly in two different trays. Leaves from
one plant in each tray were collected for total RNA isolation using Trizol agents
(Invitrogen, Carlsbad, CA) following the manufacturer's instructions. For each treatment,
two replicates were prepared. The quality of total RNA was assessed by running RNA
gels and measuring the ratio of absorptions at 260 nm and 280 nm. The concentrations of
the total RNA were measured by RiboGreen kit (Invitrogen). About 20 µg good-quality
total RNA for each replicate of each treatment was sent to NimbleGen Systems Inc. for
183
labeling, hybridization and data retrieval. The experiment consisted of two replicates for
each treatment.
The hybridization data for each chip were collected from each hybridization file
into a spreadsheet file. The genes whose hybridization values were negative or zero in
any treatments were excluded from the analysis. In order to apply linear regression
algorithm to the analysis, a base 2 log transformation was conducted to bring the
hybridization data to a normal distribution.
The variations between two replicates of each treatment (M, P, and A) were assessed in a
quality control (QC) step by employing the univariate linear regression algorithm with
the confidence interval of 95% using SAS. Genes with expression values falling out the
confidence interval of 95% were considered too variable in hybridization intensities and
collected for validation of the results of the pairwise comparison. A loop design was
applied for the pairwise comparison of the means of the two replicates of each treatment
(M, P, and A) using the univariate linear regression algorithm with the confidence
interval of 99%. The genes falling out of the confidence interval of 99% were considered
differentially expressed, and were validated by taking out the genes that were collected in
the QC step.
6.4 Results
6.4.1 The AY-WB genome contained 56 candidate effector proteins.
ORFs starting with standard start codon ATG were predicted from the gapped
genome sequence of AY-WB using the ORF Extractor program, and subsequently the
184
longest ORF among the set of ORFs with the same stop codons was extracted using a perl
script, resulting in 1986 ORFs. The SignalP 2.0 software (Nielsen et al., 1997) predicted
the presence of N-terminal signal peptide sequences for 144 deduced proteins (Fig. 6.1).
These proteins were considered candidate effector proteins because they may be secreted
by the phytoplasma and interact with host factors. In total, 56 candidate effector proteins
were selected for PVX-based in planta functional analysis based on the criteria of (i)
absence of transmembrane domains to exclude membrane-bound proteins from this study
and (ii) ORF lengths between 70 bp and 2,000 bp (i.e. the minimal and maximum size
limitations of PVX). The 56 proteins (Fig. 6.1) were named A1 to A56.
The 56 candidate effector proteins were annotated by sequence similarity searches
against NCBI nr database. Fifteen proteins had no significant hits (E > 10-4) to any
proteins in the database. Most of the others have homologs in the closely related OY
phytoplasma genome. However, 32 of such proteins were annotated as “hypothetical
protein” or “conserved hypothetical protein” in the OY phytoplasma genome.
6.4.2 Transiently expressed AY-WB candidate effector proteins induced necrosis in N.
benthamiana leaves.
To test the effect of AY-WB candidate effector proteins, gene fragments
corresponding to the mature proteins were cloned into binary PVX vectors (Qutob et al.,
2002) and transiently expressed in N. benthamiana leaves via toothpick inoculation of
Agrobacterium carrying the binary PVX constructs (Takken et al., 2000). Agrobacterium
will introduce recombinant PVX vectors into plant cells, and subsequent replication of
185
the PVX will allow intracellular production and systemic spread of virions and
phytoplasma proteins in the plant. At 21 dpi, the PVX:A11 construct induced necrosis in
addition to the mosaic symptom, while the PVX:A42 construct induced the accumulation
of yet unknown substances observable under fluorescent microscope (Fig. 6.2). As
expected, N. benthamiana leaves inoculated by PVX vector only, the negative control,
showed typical mosaic symptoms of PVX, whereas the positive control, inf1 gene from
Phytophthora infestans, a eukaryotic pathogen causing potato and tomato late blight,
induced localized HR (Fig. 6.2.) (Kamoun et al., 1997). Overall, among the 37
phytoplasma genes successfully cloned and tested, 3 induced necrosis in addition to the
same mosaic symptoms with PVX and 14 induced necrosis and delayed mosaic
symptoms (Table 6.1). Thus, 17 AY-WB phytoplasma proteins alter PVX symptoms and
therefore may directly or indirectly interact with plant components.
6.4.3 AY-WB proteins A11 and A30 targeted plant cell nuclei.
Of the 56 candidate effector proteins selected for the PVX-based in planta
expression studies, 5 proteins were predicted to contain plant nuclear localization signals
(NLS) by PredictNLS (Cokol et al., 2000) and ScanProsite (Gattiker et al., 2002)
programs. Given the fact that prokaryotic phytoplasmas do not have nucleus, these
proteins (A03, A11, A22, A30, and A42) might target plant cell nuclei and affect the
transcription of certain plant genes.
To determine the subcellular localization of the five phytoplasma proteins in
plants, gene fragments corresponding to mature portions of the ORFs were cloned into
186
the pGDY vectors and transiently expressed by agroinfiltration of N. benthamiana leaves
(Goodin et al., 2002). Cloning of the A22 gene was not successful probably due to
toxicity of A22 to E. coli. Two days after the agroinfiltration, plant leaves were detached
and examined under a confocal microscope. Similar to the negative control (Fig. 6.3A),
YFP only, YFP:A03 and YFP:A42 were distributed equally between the cytoplasm and
the nucleus (data not shown). In contrast, YFP:A11 localized to the nuclei and YFP:A30
localized to the nucleoli of plant cells (Fig. 6.3A). To confirm the localization of
YFP:A30 in nucleoli, YFP:A30 was co-infiltrated with GFP-tagged AtFib1, a protein
from Arabidopsis thaliana whose localization in nucleoli was experimentally verified
(Barneche et al., 2000). Fluorescence intensity scans (Fig. 6.3B) showed the identical
intensity patterns of GFP:AtFib1 and YFP:A30 across the nucleus and nucleolus, thus
confirming the localization of the YFP:A30 protein in plant cell nucleoli.
6.4.4 Nuclear import of YFP:A11 in N. benthamiana was importin α dependent.
One of the pathways for nuclear import of proteins into plant nuclei depends on
importin α and importin β, in which importin α is responsible for recognition of NLS
and binding of the protein (Macara, 2001). Two N. benthamiana importin α gene
homologs (importin α1 and importin α2) were identified from N. benthamiana EST
(expressed sequence tag) sequences (Kanneganti et al., in preparation). To investigate
whether phytoplasma effector proteins, A11 and A30, depend on importin system for
transport into plant nuclei, the N. benthamiana importin α genes were silenced by TRVmediated VIGS (Ratcliff et al., 2001; Liu et al., 2002). Importin α-silenced plants grow
187
normally or have minor symptoms (data not shown). At two weeks after inoculation, RTPCR results demonstrated the complete silencing of importin α1 (NbImp1, Fig. 6.4A)
and partial silencing of importin α2 (NbImp2, Fig. 6.4A) relative to the constitutively
expressed plant tubulin genes. Subsequent infiltration of the N. benthamiana plants with
the pGD constructs showed that YFP:A11 protein was distributed equally between the
cytoplasm and the nucleus in importin α-silenced plants, which was different from the
nuclear localization of the YFP:A11 protein in the non-silenced control plants (Fig.
6.4B). In contrast, the GFP:AtFib1 construct was localized in plant nucleoli (Barneche et
al., 2000) in both healthy and importin α-silenced plants (Fig. 6.4B). These results
suggested that YFP:A11 was dependent on N. benthamiana importin α gene products for
the transport into plant nuclei.
6.4.5 A11 and A30 were expressed during AY-WB phytoplasma infection of plants and
insects.
Expression of the encoding genes of A11 and A30 during AY-WB phytoplasma
infection of plants and insects was tested with RT-PCR. The transcripts of A11 and A30
with the expected sizes were detected in total RNA isolated from AY-WB phytoplasmainfected plants using gene-specific primers (Fig. 6.5). The transcripts of two other NLScontaining genes, A03 and A22, were also detected, whereas a transcript of A42 was not
detectable in total RNA from AY-WB phytoplasma-infected plants (data not shown). The
RT-PCR results of AY-WB-infected leafhoppers (M. quadrilineatus) showed that A11
and A30 gene transcripts of the expected sizes were detected in total RNA samples of
188
insects that acquired AY-WB from plants 1, 2, and 3 weeks prior (Fig. 6.6). Thus, A11
and A30 genes were expressed during AY-WB phytoplasma infection of plants and
insects.
6.4.7 A11 affected the expression of tomato plant genes.
Microarray experiments were conducted to evaluate the effect of A11 protein on
plant gene expression profiles. NimbleGen chips representing 15,925 unigenes from L.
esculentum were hybridized with total RNA isolated from mock-inoculated plants and
plants inoculated with PVX only and PVX:A11 constructs. Statistical analysis of the
results of 6 hybridizations, i.e. two replicates of three treatments, revealed that 26 tomato
unigenes were up-regulated by A11 comparing to the PVX only control (Table 6.2) and
27 tomato unigenes were down-regulated by A11 comparing to the PVX only control
(Table 6.3). Up-regulated genes included those of protein kinases involved in signal
transduction and plant response to pathogen infection, such as receptor-like protein
kinase (LE04258), putative receptor-like serine-threonine protein kinase (LE08855),
leucine-rich repeat transmembrane protein kinase (LE10338), putative protein kinase
(LE11683), and putative S-receptor kinase homolog 2 precursor (LE12428). These genes
were up-regulated 3- to 7-fold in PVX-A11 inoculated plants compared to plants
inoculated with the empty PVX vector. The tomato gene that was upregulated 15.8-fold
had weak similarity to mraW methylase family protein (LE06170). Among the downregulated genes were CONSTANS-like protein (LE05848) and putative MADS-box
protein (LE11148) that are both transcription factors (An et al., 2004; Becker and
189
Theissen, 2003), and the blue copper-binding protein (LE10147) that has various
functions in the nucleus (Gruenbaum et al., 2003). Thus, these result showed that A11
can affect the expression levels of several proteins.
6.5 Discussion
In this research, we identified 17 AY-WB candidate effector proteins that induced
necrosis when expressed in plant cells. Unlike many Gram-negative bacterial pathogens
that are typically located extracellularly and have type III secretion systems (TTSS) to
deliver effector proteins into plant cells, Gram-positive bacteria apparently use the Secdependent pathway for delivery of virulence proteins as has been shown for
Streptococcus pyogenes (Rosch and Caparon, 2004). Hence, the phytoplasmas that are
related to the Gram-positive and are predominantly located intracellularly in plants cells,
probably use the Sec-dependent pathway to deliver their effector proteins. Secreted
phytoplasma proteins can then immediately interact with cell components or transported
to cell nuclei or other cell organelles.
Several candidate effector proteins of phytoplasmas, including A11 and A30,
induce cell death as evidenced by local necrotic spots when transiently expressed in
plants using the PVX-based expression system. Whereas phytoplasmas induce PR
(pathogenicity-related) proteins (Zhong and Shen, 2004), it remains to be investigated
whether phytoplasmas, and A11 and A30 induce HR. Effectors of Gram-negative
bacteria are involved in HR response. For instance, AvrPto and AvrRpt2 of P. syringae
pv. tomato (Tang et al., 1996; Leister et al., 1996) and AvrBs3 of Xanthomonas
190
campestris pv. vesicatoria (van den Ackerveken et al., 1996) could induce HR responses
in resistant plants. Alternative explanations are that phytoplasma proteins are toxic to
plant cells and induce cell death through direct interactions with plant cell components, or
indirect induction of cell death by, for example, suppression plant defense system and
subsequent increased virulence of PVX. The latter was found for Pseudomonas effector
proteins HopPtoF and AvrPphF that were shown to suppress the defense-associated HR
elicited by another bacterial effector protein (Jackson et al., 1999; Tsiamis et al., 2000).
A11 and A30 contained plant NLSs and targeted plant cell nuclei when transiently
expressed in plant cells. Because phytoplasmas are bacteria and do not have nuclei, it
seems likely that A11 and A30 have adapted to functioning in eukaryotic cells. Indeed,
A11 and A30 transcripts were detected in AY-WB-infected plants, and A11 is dependent
on importinα for nuclear import in N. benthamiana cells. Further, A11 differently
regulated 53 tomato genes. Several of the downregulated genes are transcription factors,
including the nuclear zinc finger protein CONSTANS that acts in the phloem tissue of
Arabidopsis thaliana and is involved in the regulation of Arabidopsis flowering (An et
al., 2004), and the MADS-box proteins that belong to a family of transcription factors
involved in multiple plant development processes (Becker and Theissen, 2003). A11 also
downregulated with more than 6 fold the gene of the blue copper-binding protein, a
protein similar to components of the nuclear lamina that is directly or indirectly involved
in various nuclear activities, including DNA replication and transcription, cell cycle
regulation, and cell development and differentiation (Gruenbaum et al., 2003). Thus, the
nuclear localization of A11 and the differential regulation of nuclear plant genes by A11
191
support the hypothesis that A11 is an effector protein that manipulates plant components
for efficient infection of AY-WB phytoplasma. Effector proteins of other bacteria also
target plant cell nuclei. For instance, the AvrBs3 protein of Xanthomonas contained
functional plant NLSs and targeted plant cell nuclei (Yang and Gabriel, 1995). Further,
phytoplasmas induce various interesting symptoms, including phyllody, virescence and
shoot proliferation (Zhang et al, 2004), indicative of phytoplasma interference with plant
development, and may explain why A11 interacts with plant developmental proteins.
It is not completely understood why phytoplasmas induce radical developmental
symptoms in plants. However, it has been shown that phytoplasma-infected plants
enhance the fitness of the leafhopper vectors of AY-WB by increasing the aster
leafhopper (M. quadrilineatus) lifespan and numbers of offspring (Beanland et al., 2000).
Since leafhoppers lay eggs on plant leaves, one expects that an increase in the number of
leaves per plant, as occurs in phyllody (development of floral parts into leafy structures)
and shoot proliferation, would result in an increase of leafhopper offspring. High
numbers of leafhoppers is also important for phytoplasma survival as phytoplasmas are
not seed-transmitted or passed on to next-generation leafhoppers, and therefore are
completely dependent on leafhopper transmission from plant to plant.
This study, for the first time, employed the combination of bioinformatics and
functional genomics to study phytoplasma pathogenesis, and evidently resulted in the
successful identification of candidate phytoplasma effector proteins. Because
phytoplasmas cannot be cultured, genome sequencing and subsequent mining of genome
192
sequence data for high-throughput functional analysis of phytoplasma proteins is
particularly powerful for understanding phytoplasma virulence.
6.6 Acknowledgments
The authors thank Diane M. Hartzler and Angela D. Strock in the Department of
Entomology, and Diane M. Kinney, Miaoying Tian, Edgar Huitema and Jorunn Bos in
the Department of Plant Pathology at The Ohio State University – OARDC for technical
support and constructive discussion.
This research was supported by OSU-OARDC Research Enhancement
Competitive Grant Program, Graduate Research Competition (2003-170) and
Interdisciplinary Competition (2001-052).
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196
197
Gene Length
GC
SP a Cleavage NLS c
Blast annotation
(aa) content Score
site b
Accession #
Organism
E-value
Description
A01
117
0.22
1
37
N 39938944
Onion yellows phytoplasma 7e-19 hypothetical protein
A02
161
0.26
1
41
N 39938945
Onion yellows phytoplasma 2e-12 hypothetical protein
A03
95
0.21 0.985
31
Y 39938944
Onion yellows phytoplasma 7e-08 hypothetical protein
A04
162
0.27
1
41
N 39938945
Onion yellows phytoplasma 1e-07 hypothetical protein
A05
136
0.27 0.992
32
N 39939004
Onion yellows phytoplasma 4e-49 hypothetical protein
A06
118
0.29 0.999
31
N 39938858
Onion yellows phytoplasma 5e-13 hypothetical protein
A07
71
0.34 0.523
31
N
no significant hits
A08
150
0.20 0.977
30
N 39938878
Onion yellows phytoplasma 4e-47 hypothetical protein
A09
203
0.29 0.999
30
N 39939048
Onion yellows phytoplasma 1e-69 hypothetical protein
A10
202
0.29
0.95
30
N 9621765
Peanut witches'-broom
7e-04 RNA polymerase sigma factor
phytoplasma
A11
122
0.21
1
31
Y 39939063
Onion yellows phytoplasma 6e-05 hypothetical protein
A12
252
0.22 0.998
42
N 39938647
Onion yellows phytoplasma 2e-17 hypothetical protein
A13
93
0.17 0.995
32
N 39939176
Onion yellows phytoplasma 3e-12 hypothetical protein
A14
143
0.21 0.775
32
N 39939013
Onion yellows phytoplasma 1e-48 ATP-dependent DNA helicase (partial,
13.7%)
A15
381
0.30 0.999
34
N 39938578
Onion yellows phytoplasma e-168 ABC-type Mn/Zn transport system,
periplasmic Mn/Zn-binding protein
A16
148
0.23 0.925
26
N 39939193
Onion yellows phytoplasma 1e-42 hypothetical protein
A17
209
0.22 0.999
32
N 39938539
Onion yellows phytoplasma 1e-71 hypothetical protein
A18
335
0.22 0.518
32
N 39938800
Onion yellows phytoplasma e-128 hypothetical protein
A19
187
0.25 0.979
32
N 39938857
Onion yellows phytoplasma 4e-27 hypothetical protein
A20
269
0.22 0.903
39
N
no significant hits
A21
126
0.16
0.99
32
N
no significant hits
A22
212
0.32 0.964
21
Y 39939223
Onion yellows phytoplasma 6e-98 guanylate kinase
A23
58
0.22
0.848
30
N
no significant hits
A24
A25
A26
A27
A28
A29
48
231
199
193
75
362
0.36
0.22
0.24
0.22
0.18
0.28
0.744
0.999
0.984
0.544
0.999
0.927
28
41
41
22
36
33
N
N
N
N
N
N
no significant hits
39938886
Onion yellows phytoplasma
39938886
Onion yellows phytoplasma
no significant hits
no significant hits
39938756
Onion yellows phytoplasma
2e-19
3e-13
hypothetical protein
hypothetical protein
e-172
A30
A31
106
115
0.21
0.18
0.983
0.862
34
30
Y
N
39939176
39938783
9e-11
2e-45
uncharacterized BCR, containing RmuC
domain
hypothetical protein
hypothetical protein
Onion yellows phytoplasma
Onion yellows phytoplasma
PVX assay results
Delayed mosaic with necrosis
Delayed mosaic with necrosis
Not cloned
Delayed mosaic
Delayed mosaic with necrosis
No symptoms
Mosaic same as negative control
Not cloned
Delayed mosaic
Delayed mosaic with necrosis
Delayed mosaic with necrosis
Delayed mosaic
No symptoms
Mosaic same as negative control
Not cloned
Delayed mosaic with necrosis
Delayed mosaic
Not cloned
Not cloned
Mosaic same as negative control
Delayed mosaic with necrosis
Mosaic same as negative control with
necrosis
Mosaic same as negative control with
necrosis
Delayed mosaic
Delayed mosaic
Delayed mosaic
Delayed mosaic
Not cloned
Not cloned
Delayed mosaic with necrosis
Delayed mosaic with necrosis
(Continued)
Table 6.1 Summary of PVX assays of AY-WB phytoplasma candidate effector proteins
Table 6.1 (continued)
Gene
A32
A33
A34
A35
A36
A37
A38
A39
A40
A41
A42
198
a
Length
GC
SP a Cleavage NLS c
Blast annotation
(aa) content Score
site b
Accession #
Organism
E-value
Description
514
0.25 0.855
27
N 39938677
Onion yellows phytoplasma 6e-67 ABC-type dipeptide/oligopeptide
transport system, periplasmic component
236
0.27 0.999
36
N 39938647
Onion yellows phytoplasma e-107 hypothetical protein
312
0.23 0.994
38
N 39938643
Onion yellows phytoplasma e-136 hypothetical protein
349
0.27 0.975
42
N 39938619
Onion yellows phytoplasma e-136 ABC-type uncharacterized transport
system, periplasmic component
265
0.20 0.976
28
N 39938905
Onion yellows phytoplasma 5e-91 hypothetical protein
287
0.25 0.609
24
N 39938904
Onion yellows phytoplasma e-135 hypothetical protein
91
0.19 0.599
31
N
no significant hits
203
0.28 0.996
30
N 39939048
Onion yellows phytoplasma 3e-73 hypothetical protein
114
0.23 0.939
31
N 39939028
Onion yellows phytoplasma 1e-12 hypothetical protein
132
0.21 0.635
32
N
no significant hits
78
0.12 0.978
31
Y
no significant hits
A43
A44
A45
A46
260
88
166
57
0.20
0.19
0.16
0.30
0.895
0.993
0.69
0.999
38
34
31
31
N
N
N
N
39939027
Onion yellows phytoplasma
39939176
Onion yellows phytoplasma
no significant hits
no significant hits
A47
A48
A49
A50
A51
A52
60
66
282
229
107
292
0.37
0.23
0.17
0.22
0.17
0.27
0.998
0.796
0.979
0.505
0.767
0.982
35
32
31
49
32
28
N
N
N
N
N
N
no significant hits
39938858
Onion yellows phytoplasma
39938832
Onion yellows phytoplasma
39938970
Onion yellows phytoplasma
39939027
Onion yellows phytoplasma
39938975
Onion yellows phytoplasma
A53
A54
A55
A56
221
125
270
101
0.32
0.18
0.24
0.26
0.998
0.99
0.767
0.78
35
31
31
32
N
N
N
N
39939136
Onion yellows phytoplasma
39938535
Onion yellows phytoplasma
39939062
Onion yellows phytoplasma
no significant hits
2e-22
3e-42
hypothetical protein
hypothetical protein
4e-19
e-101
7e-90
1e-09
5e-60
hypothetical protein
hypothetical protein
hypothetical protein
hypothetical protein
ABC-type amino acid transport system,
periplasmic component
hypothetical protein
hypothetical protein
hypothetical protein
2e-48
3e-29
1e-43
PVX assay results
Not cloned
Not cloned
Delayed mosaic with necrosis
No symptoms
Not cloned
Cloned but not tested
Delayed mosaic with necrosis
Not cloned
Delayed mosaic
Not cloned
Delayed mosaic, dark substance
accumulation
Not cloned
Delayed mosaic
Not cloned
Mosaic same as negative control with
necrosis
Mosaic same as negative control
Delayed mosaic with necrosis
Not cloned
Not cloned
Not cloned
Not cloned
Delayed mosaic with necrosis
Delayed mosaic with necrosis
Delayed mosaic
Delayed mosaic
Signal peptide (SP) scores were predicted by hidden Markov model (HMM) in SignalP2.0 program (Nielsen et al., 1997).
Signal peptide cleavage sites were predicted by neural network (NN) in SignalP2.0 program (Nielsen et al., 1997).
c
Nuclear localization signals (NLS) were predicted by ScanProsite (Gattiker et al., 2002) and PredictNLS (Cokol et al., 2000) programs.
b
#
Genes
199
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
LE00171
LE00473
LE02916
LE03181
LE03731
LE03884
LE04258
LE04471
LE04723
LE04949
LE06170
LE06677
LE06739
LE06800
LE07434
LE08855
LE10338
LE11457
LE11683
LE12193
LE12428
LE12485
LE13106
Fold
Difference
3.09
3.46
5.96
3.80
10.71
7.41
3.07
3.20
3.99
10.32
15.81
3.28
4.77
5.45
7.23
4.53
4.75
7.98
7.41
5.63
6.39
3.80
6.16
24
25
26
LE13392
LE13792
LE14225
3.32
5.84
6.55
Blastx
Acc. #
5669636
19256
20334373
4220541
15218215
12324199
8777368
2264373
18402561
34908910
15238896
51989474
7270437
31126772
1076611
18076583
18402209
10176726
6681335
6143896
50942589
30913024
17065936
18418072
Organism
L. esculentum
L. esculentum
L. pimpinellifolium
A. thaliana
A. thaliana
A. thaliana
A. thaliana
A. thaliana
A. thaliana
O. sativa cv. japonica
A. thaliana
N. benthamiana
A. thaliana
O. sativa cv. japonica
N. sylvestris
S. tuberosum
A. thaliana
A. thaliana
A. thaliana
A. thaliana
O. sativa cv. japonica
no significant hits
A. thaliana
S. tuberosum
A. thaliana
no significant hits
E-value
0.0
0.0
2e-92
1e-78
5e-83
2e-60
e-117
9e-83
4e-86
6e-63
2e-20
e-123
1e-32
2e-22
e-109
e-110
5e-66
e-107
9e-16
2e-06
5e-66
2e-11
1e-23
4e-66
Description
ethylene-responsive elongation factor EF-Ts precursor
heat shock protein cognate 70
cysteine protease
Rab geranylgeranyl transferase like protein
coatomer protein complex, subunit beta 2 (beta prime)
putative calmodulin-binding protein
receptor-like protein kinase
NAM (no apical meristem)-like protein
calcium-binding EF hand family protein
P0691E06.5, unknown protein
mraW methylase family protein (weak similarity)
putative RNA-dependent RNA polymerase RdRP2
invertase-like protein (weak similarity)
unknown protein, weak similarity
peroxidase (EC 1.11.1.7), anionic, precursor
putative receptor-like serine-threonine protein kinase
leucine-rich repeat transmembrane protein kinase, putative
diacylglycerol kinase ATDGK1 homolog
putative protein kinase (weak similarity)
putative translation initiation factor IF-2 (very weak similarity)
putative S-receptor kinase (EC 2.7.1.-) homolog 2 precursor
transcriptional activator DEMETER (DNA glycosylase-related protein
DME) (very weak similarity)
transmembrane protein (weak similarity)
RNA recognition motif (RRM)-containing protein
Table 6.2 Genes that are up-regulated in PVX:A11 treated tomato plants comparing to PVX only treated tomato plants
#
Genes
200
1
2
3
4
5
LE00058
LE02043
LE05848
LE06226
LE06991
Fold
Difference
-2.81
-2.01
-3.14
-2.30
-2.39
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
LE07685
LE08007
LE08458
LE08676
LE08883
LE09000
LE09282
LE10105
LE10147
LE10477
LE10529
LE10618
LE10958
LE11148
LE11151
LE11513
LE11576
LE12876
LE13274
LE13812
-2.12
-2.34
-2.58
-2.40
-2.99
-2.52
-2.15
-2.22
-6.73
-2.52
-3.61
-3.17
-10.41
-2.21
-2.99
-2.49
-4.19
-2.03
-3.31
-3.02
26
27
LE14900
LE15146
-3.55
-3.43
Acc. #
33413550
2119600
41323976
458547
50937023
Organism
L. esculentum
Flaveria pringlei
Populus deltoides
Manihot esculenta
O. sativa cv. japonica
E-value
5e-73
1e-72
4e-45
8e-10
4e-31
1362095
124119
50920507
21593012
42559164
7486957
17065916
33301670
2129630
40287494
13377782
15010628
30694193
50947193
21726980
15218042
51968886
15222119
32815941
15239451
L. esculentum
L. esculentum
O. sativa cv. japonica
A. thaliana
N. tabacum
A. thaliana
Catharanthus roseus
N. tabacum
A. thaliana
Capsicum annuum
A. thaliana
A. thaliana
A. thaliana
O. sativa cv. japonica
Solanum phureja
A. thaliana
A. thaliana
A. thaliana
A. thaliana
A. thaliana
3e-91
7e-39
e-168
e-114
e-121
4e-33
3e-85
e-147
7e-41
2e-58
8e-62
3e-57
e-115
4e-15
2e-75
3e-31
6e-36
6e-57
4e-42
2e-53
Blastx
Description
proteinase inhibitor II
glycine cleavage system protein H precursor
CONSTANS-like protein
UTP-glucose glucosyltransferase (partial)
SET-domain transcriptional regulator family-like protein
(partial)
oxidase like protein
wound-induced proteinase inhibitor I precursor
putative squalene monooxygenase
putative C-4 sterol methyl oxidase
50S ribosomal protein L3, chloroplast precursor
putative protein T13J8.190
geraniol 10-hydrooxylase
Delta-7-sterol-C5(6)-desaturase
blue copper-binding protein, 19K
putative lesion-inducing protein
fasciclin-like arabinogalactan protein 7
At1g68660/F24J5_4, unknown protein
pectate lyase family protein
putative MADS-box protein (weak similarity)
pathogenesis related protein isoform b1
zinc finger (C3HC4-type RING finger) family protein
unknown protein
MATE efflux family protein
At5g67620, unknown protein
serine/threonine protein phosphatase 2A (PP2A) regulatory
subunit B', putative
no significant hits
no significant hits
Table 6.3 Genes that are down-regulated in PVX:A11 treated tomato plants comparing to PVX only treated tomato plants
Fig. 6.1 Mining of AY-WB phytoplasma genome sequences for candidate effector proteins. From 1,986
predicted ORFs, 144 ORFs were predicted as AY-WB phytoplasma candidate effector proteins. It includes
56 AY-WB phytoplasma candidate effector proteins selected for PVX studies (Black closed diamonds), 5
AY-WB phytoplasma proteins containing NLS (Blue closed squares), and the rest (Blue open circles).
201
Fig. 6.2 Representative plant symptoms after toothpick inoculations of Nicotiana benthamiana leaves with
transformed Agrobacterium tumefaciens GV3101. Plant leaves (upper panel) were examined under the light
mode (middle panel) and the fluorescent mode (lower panel) of a fluorescence microscope. PVX:A11
induced necrotic spots (black arrows) and accumulation of fluorescent compounds (white arrows) in N.
benthamiana leaves. Plant leaves inoculated with PVX:A42 appeared healthy under the light mode.
However, fluorescent image of the leaves showed dark areas (arrowheads) that were not visible under light
mode, suggesting the accumulation of yet unknown substances. PVX and PVX:inf1 were included as
controls, and showed the typical mosaic symptom of PVX and the clear hypersensitive response induced by
Inf1.
202
Fig. 6.3 Confocal laser-scanning microscopy images demonstrating the subcellular localization of YFP
fusions of NLS-containing AY-WB phytoplasma proteins upon agroinfiltration into N. benthamiana leaves.
(A) YFP:A11 targeted the plant cell nuclei (arrow) and YFP:A30 targeted the nucleoli (arrowhead) of plant
cells. The negative (neg.) control, YFP only, was equally distributed between the cytoplasm and the
nucleus of plant cells. Bars represent 50 µm. (B) Fluorescence intensity scans showed co-localizations of
GFP:AtFib1, a functional fibrillarin homolog that targets the nucleolus (Barneche et al., 2000), and
YFP:A30. Linear fluorescence intensity scans across the nucleus (bars in confocal micrographs at left)
showed that AtFib1 and A30 had similar intensity patterns (graphs at right).
203
Fig. 6.4 The transport of YFP:A11 was dependent on N. benthamiana (Nb) importin α gene products. (A)
RT-PCR with N. benthamiana importin α1- and α2-specific primers confirmed the silencing of these two
genes in N. benthamiana plants treated with TRV constructs containing Nb importin α inserts. Noninoculated (healthy) and TRV-treated plants served as negative control. RT-PCR with N. benthamiana
tubulin gene-specific primers was included to control for equal amount of total RNA samples and RT-PCR
reaction quality. (B) Confocal microscopy study showed that YFP:A11 was not transported into plant
nuclei in N. benthamiana importin α1- and importin α2-silenced N. benthamiana plants, compared to
healthy N. benthamiana plants. The disrupted transport of YFP:A11 resulted in the equal localization of
YFP signals between plant cell cytoplasm and nuclei, which was similar to YFP only control (C). In
contrast, the nuclear transport of the GFP fusion of AtFib1 (Barneche et al., 2000) was not inhibited,
because GFP:AtFib1 was localized in plant nucleoli in both healthy and importin α-silenced plants.
Therefore, AtFib1 is transported into plant nucleoli in an importin-independent manner. Bars = 50 µm.
204
Fig. 6.5 The genes of AY-WB phytoplasma candidate effector proteins A11 and A30 were expressed
during AY-WB phytoplasma infection of China aster plants. Transcripts of both genes were detected by
RT-PCR in total RNA samples from AY-WB infected plants (iRNA) but not those from healthy plants
(hRNA). RNase-treated iRNA served as controls to test for the presence of genomic DNA contaminations
in RNA samples.
Fig. 6.6 The genes of AY-WB phytoplasma candidate effector proteins A11 and A30 were expressed in
AY-WB infected aster leafhoppers (Macrosteles quadrilineatus L.). Transcripts of both genes were
detected by RT-PCR in total RNA samples from AY-WB phytoplasma infected aster leafhoppers that were
reared on infected aster plants for 1 week (lanes 2), 2 weeks (lanes 3) and 3 weeks (lanes 4), but not in
those from the leafhoppers that did not acquire AY-WB (lanes 1). AY-WB phytoplasma 16S rDNA primers
served as controls to test for the presence of AY-WB genomic DNA contaminations in total RNA samples.
205
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